Applying a Real-Time PCR Assay for Histoplasma capsulatum to Clinically Relevant Formalin-Fixed Paraffin-Embedded Human Tissue

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Koepsell, Scott A.; Hinrichs, Steven H.

2012-01-01

A real-time PCR assay to detect Histoplasma capsulatum in formalin-fixed, paraffin-embedded (FFPE) tissue is described. The assay had an analytical sensitivity of 6 pg/μl of fungal DNA, analytical specificity of 100%, and clinical sensitivity of 88.9%. This proof-of-concept study may aid in the diagnosis of histoplasmosis from FFPE tissue. PMID:22855519

Virus characterization and discovery in formalin-fixed paraffin-embedded tissues.

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Bodewes, Rogier; van Run, Peter R W A; Schürch, Anita C; Koopmans, Marion P G; Osterhaus, Albert D M E; Baumgärtner, Wolfgang; Kuiken, Thijs; Smits, Saskia L

2015-03-01

Detection and characterization of novel viruses is hampered frequently by the lack of properly stored materials. Especially for the retrospective identification of viruses responsible for past disease outbreaks, often only formalin-fixed paraffin-embedded (FFPE) tissue samples are available. Although FFPE tissues can be used to detect known viral sequences, the application of FFPE tissues for detection of novel viruses is currently unclear. In the present study it was shown that sequence-independent amplification in combination with next-generation sequencing can be used to detect sequences of known and unknown viruses, although with relatively low sensitivity. These findings indicate that this technique could be useful for detecting novel viral sequences in FFPE tissues collected from humans and animals with disease of unknown origin, when other samples are not available. In addition, application of this method to FFPE tissues allows to correlate with the presence of histopathological changes in the corresponding tissue sections. Copyright © 2015 Elsevier B.V. All rights reserved.

A single simple procedure for dewaxing, hydration and heat-induced epitope retrieval (HIER for immunohistochemistry in formalin fixed paraffin-embedded tissue

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I.M.S. Paulsen

2015-11-01

Full Text Available Heat-induced epitope retrieval (HIER is widely used for immunohistochemistry on formalin fixed paraffin-embedded tissue and includes temperatures well above the melting point of paraffin. We therefore tested whether traditional xylene-based removal of paraffin is required on sections from paraffin-embedded tissue, when HIER is performed by vigorous boiling in 10 mM Tris/0.5 mM EGTA-buffer (pH=9. Immunohistochemical results using HIER with or without prior dewaxing in xylene were evaluated using 7 primary antibodies targeting proteins located in the cytosol, intracellular vesicles and plasma membrane. No effect of omitting prior dewaxing was observed on staining pattern. Semiquantitative analysis did not show HIER to influence the intensity of labelling consistently. Consequently, quantification of immune labelling intensity using fluorescent secondary antibodies was performed at 5 dilutions of primary antibody with and without prior dewaxing in xylene. No effect of omitting prior dewaxing on signal intensity was detectable indicating similar immunoreactivity in dewaxed and non-dewaxed sections. The intensity of staining the nucleus with the DNA-stain ToPro3 was similarly unaffected by omission of dewaxing in xylene. In conclusion, the HIER procedure described and tested can be used as a single procedure enabling dewaxing, hydration and epitope retrieval for immunohistochemistry in formalin fixed paraffin-embedded tissue.

Proteome stability analysis of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human colon mucosal biopsies

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Bennike, Tue Bjerg; Kastaniegaard, Kenneth; Padurariu, Simona

2016-01-01

Large repositories of well characterized RNAlater preserved samples and formalin-fixed, paraffin-embedded samples have been generated worldwide. However, the impact on the proteome of the preservation methods remain poorly described. Therefore, we analyzed the impact on the proteome of preserving...... throughput gel free quantitative proteomics. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002029....

Clinical relevance of DNA microarray analyses using archival formalin-fixed paraffin-embedded breast cancer specimens

International Nuclear Information System (INIS)

Sadi, Al Muktafi; Wang, Dong-Yu; Youngson, Bruce J; Miller, Naomi; Boerner, Scott; Done, Susan J; Leong, Wey L

2011-01-01

The ability of gene profiling to predict treatment response and prognosis in breast cancers has been demonstrated in many studies using DNA microarray analyses on RNA from fresh frozen tumor specimens. In certain clinical and research situations, performing such analyses on archival formalin fixed paraffin-embedded (FFPE) surgical specimens would be advantageous as large libraries of such specimens with long-term follow-up data are widely available. However, FFPE tissue processing can cause fragmentation and chemical modifications of the RNA. A number of recent technical advances have been reported to overcome these issues. Our current study evaluates whether or not the technology is ready for clinical applications. A modified RNA extraction method and a recent DNA microarray technique, cDNA-mediated annealing, selection, extension and ligation (DASL, Illumina Inc) were evaluated. The gene profiles generated from FFPE specimens were compared to those obtained from paired fresh fine needle aspiration biopsies (FNAB) of 25 breast cancers of different clinical subtypes (based on ER and Her2/neu status). Selected RNA levels were validated using RT-qPCR, and two public databases were used to demonstrate the prognostic significance of the gene profiles generated from FFPE specimens. Compared to FNAB, RNA isolated from FFPE samples was relatively more degraded, nonetheless, over 80% of the RNA samples were deemed suitable for subsequent DASL assay. Despite a higher noise level, a set of genes from FFPE specimens correlated very well with the gene profiles obtained from FNAB, and could differentiate breast cancer subtypes. Expression levels of these genes were validated using RT-qPCR. Finally, for the first time we correlated gene expression profiles from FFPE samples to survival using two independent microarray databases. Specifically, over-expression of ANLN and KIF2C, and under-expression of MAPT strongly correlated with poor outcomes in breast cancer patients. We

Reliable PCR quantitation of estrogen, progesterone and ERBB2 receptor mRNA from formalin-fixed, paraffin-embedded tissue is independent of prior macro-dissection

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Tramm, Trine; Hennig, Guido; Kyndi, Marianne

2013-01-01

Gene expression analysis on messenger RNA (mRNA) purified from formalin-fixed, paraffin-embedded tissue is increasingly used for research purposes. Tissue heterogeneity may question specificity and interpretation of results from mRNA isolated from a whole slide section, and thresholds for minimal...... tumor content in the paraffin block or macrodissection are used to avoid contamination from non-neoplastic tissue. The aim was to test if mRNA from tissue surrounding breast cancer affected quantification of estrogen receptor α (ESR1), progesterone receptor (PGR) and human epidermal growth factor...... receptor 2 (ERBB2), by comparing gene expression from whole slide and tumor-enriched sections, and correlating gene expression from whole slide sections with corresponding immunohistochemistry. Gene expression, based on mRNA extracted from a training set (36 paraffin blocks) and two validation sets (133...

A method to evaluate genome-wide methylation in archival formalin-fixed, paraffin-embedded ovarian epithelial cells.

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Qiling Li

Full Text Available The use of DNA from archival formalin and paraffin embedded (FFPE tissue for genetic and epigenetic analyses may be problematic, since the DNA is often degraded and only limited amounts may be available. Thus, it is currently not known whether genome-wide methylation can be reliably assessed in DNA from archival FFPE tissue.Ovarian tissues, which were obtained and formalin-fixed and paraffin-embedded in either 1999 or 2011, were sectioned and stained with hematoxylin-eosin (H&E.Epithelial cells were captured by laser micro dissection, and their DNA subjected to whole genomic bisulfite conversion, whole genomic polymerase chain reaction (PCR amplification, and purification. Sequencing and software analyses were performed to identify the extent of genomic methylation. We observed that 31.7% of sequence reads from the DNA in the 1999 archival FFPE tissue, and 70.6% of the reads from the 2011 sample, could be matched with the genome. Methylation rates of CpG on the Watson and Crick strands were 32.2% and 45.5%, respectively, in the 1999 sample, and 65.1% and 42.7% in the 2011 sample.We have developed an efficient method that allows DNA methylation to be assessed in archival FFPE tissue samples.

Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma

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Gjerdrum, Lise Mette; Sorensen, Boe Sandahl; Kjeldsen, Eigil

2004-01-01

We studied the feasibility of using real-time quantitative PCR to determine HER-2 DNA amplification and mRNA expression in microdissected formalin-fixed, paraffin-embedded breast tumors and compared this with standard immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods...... tumors as being amplified. Interestingly, all these scored 2+ with the HercepTest, but were negative using FISH. We believe that real-time quantitative PCR analysis of HER-2 DNA amplification following microdissection represents a useful supplementary or perhaps even an alternative technique...

Influence of parasite density and sample storage time on the reliability of Entamoeba histolytica-specific PCR from formalin-fixed and paraffin-embedded tissues.

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Frickmann, Hagen; Tenner-Racz, Klara; Eggert, Petra; Schwarz, Norbert G; Poppert, Sven; Tannich, Egbert; Hagen, Ralf M

2013-12-01

We report on the reliability of polymerase chain reaction (PCR) for the detection of Entamoeba histolytica from formalin-fixed, paraffin-embedded tissue in comparison with microscopy and have determined predictors that may influence PCR results. E. histolytica-specific and Entamoeba dispar-specific real-time PCR and microscopy from adjacent histologic sections were performed using a collection of formalin-fixed, paraffin-embedded tissue specimens obtained from patients with invasive amebiasis. Specimens had been collected during the previous 4 decades. Association of sample age, parasite density, and reliability of PCR was analyzed. E. histolytica PCR was positive in 20 of 34 biopsies (58.8%); 2 of these 20 were microscopically negative for amebae in neighboring tissue sections. PCR was negative in 9 samples with visible amebae in neighboring sections and in 5 samples without visible parasites in neighboring sections. PCR was negative in all specimens that were older than 3 decades. Low parasite counts and sample ages older than 20 years were predictors for false-negative PCR results. All samples were negative for E. dispar DNA. PCR is suitable for the detection of E. histolytica in formalin-fixed, paraffin-embedded tissue samples that are younger than 2 decades and that contain intermediate to high parasite numbers. Negative results in older samples were due to progressive degradation of DNA over time as indicated by control PCRs targeting the human 18S rRNA gene. Moreover, our findings support previous suggestions that only E. histolytica but not E. dispar is responsible for invasive amebiasis.

Automated Extraction of Formalin-Fixed, Paraffin-Embedded Tissue for High-Risk Human Papillomavirus Testing of Head and Neck Squamous Cell Carcinomas Using the Roche Cobas 4800 System.

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Kerr, Darcy A; Sweeney, Brenda; Arpin, Ronald N; Ring, Melissa; Pitman, Martha B; Wilbur, David C; Faquin, William C

2016-08-01

-Testing for high-risk human papillomavirus (HR-HPV) in head and neck squamous cell carcinomas (HNSCCs) is important for both prognostication and clinical management. Several testing platforms are available for HR-HPV; however, effective alternative automated approaches are needed. -To assess the performance of the automated Roche cobas 4800 HPV real-time polymerase chain reaction-based system on formalin-fixed, paraffin-embedded HNSCC specimens and compare results with standard methods of in situ hybridization (ISH) and p16 immunohistochemistry. -Formalin-fixed, paraffin-embedded samples of HNSCC were collected from archival specimens in the Department of Pathology, Massachusetts General Hospital (Boston), and prepared using the automated system by deparaffinization and dehydration followed by tissue lysis. Samples were integrated into routine cervical cytology testing runs by cobas. Corresponding formalin-fixed, paraffin-embedded samples were evaluated for HR-HPV by ISH and p16 by immunohistochemistry. Discrepant cases were adjudicated by polymerase chain reaction. -Sixty-two HNSCC samples were analyzed using the automated cobas system, ISH, and immunohistochemistry. Fifty-two percent (n = 32 of 62) of formalin-fixed, paraffin-embedded tumors were positive for HR-HPV by cobas. Eighty-eight percent (n = 28 of 32) of cases were the HPV 16 subtype and 12% (n = 4 of 32) were other HR-HPV subtypes. Corresponding testing with ISH was concordant in 92% (n = 57 of 62) of cases. Compared with the adjudication polymerase chain reaction standard, there were 3 false-positive cases by cobas. -Concordance in HNSCC HR-HPV status between cobas and ISH was more than 90%. The cobas demonstrated a sensitivity of 100% and a specificity of 91% for detection of HR-HPV. Advantages favoring cobas include its automation, cost efficiency, objective results, and ease of performance.

Detection of Streptococcus suis by in situ hybridization, indirect immunofluorescence, and peroxidase-antiperoxidase assays in formalin-fixed, paraffin-embedded tissue sections from pigs

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Boye, Mette; Feenstra, Anne Avlund; Tegtmeier, Conny

2000-01-01

and the immunohistochemical methods were used for detection of S. suis in formalin-fixed, paraffin-embedded tissue sections of brain, endocardium, and lung from pigs infected with S. suis. The methods developed were able to detect single cells of S. suis in situ in the respective samples, whereas no signal was observed from...

Multicenter Evaluation of a Novel Automated Rapid Detection System of BRAF Status in Formalin-Fixed, Paraffin-Embedded Tissues.

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Schiefer, Ana-Iris; Parlow, Laura; Gabler, Lisa; Mesteri, Ildiko; Koperek, Oskar; von Deimling, Andreas; Streubel, Berthold; Preusser, Matthias; Lehmann, Annika; Kellner, Udo; Pauwels, Patrick; Lambin, Suzan; Dietel, Manfred; Hummel, Michael; Klauschen, Frederick; Birner, Peter; Möbs, Markus

2016-05-01

The mutated BRAF oncogene represents a therapeutic target in malignant melanoma. Because BRAF mutations are also involved in the pathogenesis of other human malignancies, the use of specific BRAF inhibitors might also be extended to other diseases in the future. A prerequisite for the clinical application of BRAF inhibitors is the reliable detection of activating BRAF mutations in routine histopathological samples. In a multicenter approach, we evaluated a novel and fully automated PCR-based system (Idylla) capable of detecting BRAF V600 mutations in formalin-fixed, paraffin-embedded tissue within 90 minutes with high sensitivity. We analyzed a total of 436 samples with the Idylla system. Valid results were obtained in 421 cases (96.56%). Its performance was compared with conventional methods (pyrosequencing or Sanger sequencing). Concordant results were obtained in 406 cases (96.90%). Reanalysis of eight discordant samples by next-generation sequencing and/or pyrosequencing with newly extracted DNA and the BRAF RGQ Kit confirmed the Idylla result in seven cases, resulting in an overall agreement of 98.57%. In conclusion, the Idylla system is a highly reliable and sensitive platform for detection of BRAF V600 mutations in formalin-fixed, paraffin-embedded material, providing an efficient alternative to conventional diagnostic methods, particularly for routine diagnostics laboratories with limited experience in molecular pathology. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Profiling cancer gene mutations in clinical formalin-fixed, paraffin-embedded colorectal tumor specimens using targeted next-generation sequencing.

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Zhang, Liangxuan; Chen, Liangjing; Sah, Sachin; Latham, Gary J; Patel, Rajesh; Song, Qinghua; Koeppen, Hartmut; Tam, Rachel; Schleifman, Erica; Mashhedi, Haider; Chalasani, Sreedevi; Fu, Ling; Sumiyoshi, Teiko; Raja, Rajiv; Forrest, William; Hampton, Garret M; Lackner, Mark R; Hegde, Priti; Jia, Shidong

2014-04-01

The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic "hotspot" regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology. We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls. Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed "true-positive" gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent "false-positive" calls in clinically druggable oncogenes such as PIK3CA. AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent "false-positive" variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making.

Optimization of Single- and Dual-Color Immunofluorescence Protocols for Formalin-Fixed, Paraffin-Embedded Archival Tissues.

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Kajimura, Junko; Ito, Reiko; Manley, Nancy R; Hale, Laura P

2016-02-01

Performance of immunofluorescence staining on archival formalin-fixed paraffin-embedded human tissues is generally not considered to be feasible, primarily due to problems with tissue quality and autofluorescence. We report the development and application of procedures that allowed for the study of a unique archive of thymus tissues derived from autopsies of individuals exposed to atomic bomb radiation in Hiroshima, Japan in 1945. Multiple independent treatments were used to minimize autofluorescence and maximize fluorescent antibody signals. Treatments with NH3/EtOH and Sudan Black B were particularly useful in decreasing autofluorescent moieties present in the tissue. Deconvolution microscopy was used to further enhance the signal-to-noise ratios. Together, these techniques provide high-quality single- and dual-color fluorescent images with low background and high contrast from paraffin blocks of thymus tissue that were prepared up to 60 years ago. The resulting high-quality images allow the application of a variety of image analyses to thymus tissues that previously were not accessible. Whereas the procedures presented remain to be tested for other tissue types and archival conditions, the approach described may facilitate greater utilization of older paraffin block archives for modern immunofluorescence studies. © 2016 The Histochemical Society.

Molecular Markers for Prostate Cancer in Formalin-Fixed Paraffin-Embedded Tissues

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Tamara Sequeiros

2013-01-01

Full Text Available Prostate cancer (PCa is the most frequently diagnosed type of cancer in developed countries. The decisive method of diagnosis is based on the results of biopsies, morphologically evaluated to determine the presence or absence of cancer. Although this approach leads to a confident diagnosis in most cases, it can be improved by using the molecular markers present in the tissue. Both miRNAs and proteins are considered excellent candidates for biomarkers in formalin-fixed paraffin-embedded (FFPE tissues, due to their stability over long periods of time. In the last few years, a concerted effort has been made to develop the necessary tools for their reliable measurement in these types of samples. Furthermore, the use of these kinds of markers may also help in establishing tumor grade and aggressiveness, as well as predicting the possible outcomes in each particular case for the different treatments available. This would aid clinicians in the decision-making process. In this review, we attempt to summarize and discuss the potential use of microRNA and protein profiles in FFPE tissue samples as markers to better predict PCa diagnosis, progression, and response to therapy.

Matrix-comparative genomic hybridization from multicenter formalin-fixed paraffin-embedded colorectal cancer tissue blocks

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Köhne Claus-Henning

2007-04-01

Full Text Available Abstract Background The identification of genomic signatures of colorectal cancer for risk stratification requires the study of large series of cancer patients with an extensive clinical follow-up. Multicentric clinical studies represent an ideal source of well documented archived material for this type of analyses. Methods To verify if this material is technically suitable to perform matrix-CGH, we performed a pilot study using macrodissected 29 formalin-fixed, paraffin-embedded tissue samples collected within the framework of the EORTC-GI/PETACC-2 trial for colorectal cancer. The scientific aim was to identify prognostic genomic signatures differentiating locally restricted (UICC stages II-III from systemically advanced (UICC stage IV colorectal tumours. Results The majority of archived tissue samples collected in the different centers was suitable to perform matrix-CGH. 5/7 advanced tumours displayed 13q-gain and 18q-loss. In locally restricted tumours, only 6/12 tumours showed a gain on 13q and 7/12 tumours showed a loss on 18q. Interphase-FISH and high-resolution array-mapping of the gain on 13q confirmed the validity of the array-data and narrowed the chromosomal interval containing potential oncogenes. Conclusion Archival, paraffin-embedded tissue samples collected in multicentric clinical trials are suitable for matrix-CGH analyses and allow the identification of prognostic signatures and aberrations harbouring potential new oncogenes.

Droplet digital polymerase chain reaction detection of HER2 amplification in formalin fixed paraffin embedded breast and gastric carcinoma samples.

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Zhu, Yazhen; Lu, Dan; Lira, Maruja E; Xu, Qing; Du, Yunzhi; Xiong, Jianghong; Mao, Mao; Chung, Hyun Cheol; Zheng, Guangjuan

2016-04-01

Human epidermal growth factor receptor 2 (HER2) is a key driver of tumorigenesis, and over-expression as a result of HER2 gene amplification has been observed in a number of solid tumors. Recently HER2 has become an important biomarker for the monoclonal antibody treatment of HER2-positive metastatic breast and advanced gastric cancer. The HER2 targeting antibody trastuzumab treatment requires accurate measurement of HER2 levels for proper diagnosis. Droplet digital PCR (ddPCR) with highly direct, precise and absolute nucleic acid quantification could be used to detect HER2 amplification levels. Our objective was to evaluate a robust, accurate and less subjective application of ddPCR for HER2 amplification levels and test the assay performance in clinical formalin-fixed paraffin-embedded (FFPE) breast and gastric carcinoma samples. Genomic DNA from HER2 amplified cell line SK-BR-3 was used to set up the ddPCR assays. The copy number of HER2 was compared to the chromosome 17 centromere reference gene (CEP17), expressed as HER2:CEP17 ratio. Genomic DNAs of FFPE specimens from 145 Asian patients with breast and gastric carcinomas were assayed using both standard methods, immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH), and ddPCR. Based on 145 clinical breast and gastric carcinoma cases, our study demonstrated a high concordance of ddPCR results to FISH and IHC. In breast cancer specimens, the ddPCR results had high concordance with FISH and IHC defined HER2 status with a sensitivity of 90.9% (30/33) and a specificity of 100% (77/77). In gastric cancer specimens that were concordant in both FISH and IHC, our assay was 95.5% concordant with FISH and IHC (21/22). ddPCR has the advantage of automation and also allows levels of HER2 amplification to be easily evaluated in large numbers of samples, and presents a potential option to define HER2 status. Copyright © 2015 Elsevier Inc. All rights reserved.

A methodological study of genome-wide DNA methylation analyses using matched archival formalin-fixed paraffin embedded and fresh frozen breast tumors.

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Espinal, Allyson C; Wang, Dan; Yan, Li; Liu, Song; Tang, Li; Hu, Qiang; Morrison, Carl D; Ambrosone, Christine B; Higgins, Michael J; Sucheston-Campbell, Lara E

2017-02-28

DNA from archival formalin-fixed and paraffin embedded (FFPE) tissue is an invaluable resource for genome-wide methylation studies although concerns about poor quality may limit its use. In this study, we compared DNA methylation profiles of breast tumors using DNA from fresh-frozen (FF) tissues and three types of matched FFPE samples. For 9/10 patients, correlation and unsupervised clustering analysis revealed that the FF and FFPE samples were consistently correlated with each other and clustered into distinct subgroups. Greater than 84% of the top 100 loci previously shown to differentiate ER+ and ER- tumors in FF tissues were also FFPE DML. Weighted Correlation Gene Network Analyses (WCGNA) grouped the DML loci into 16 modules in FF tissue, with ~85% of the module membership preserved across tissue types. Restored FFPE and matched FF samples were profiled using the Illumina Infinium HumanMethylation450K platform. Methylation levels (β-values) across all loci and the top 100 loci previously shown to differentiate tumors by estrogen receptor status (ER+ or ER-) in a larger FF study, were compared between matched FF and FFPE samples using Pearson's correlation, hierarchical clustering and WCGNA. Positive predictive values and sensitivity levels for detecting differentially methylated loci (DML) in FF samples were calculated in an independent FFPE cohort. FFPE breast tumors samples show lower overall detection of DMLs versus FF, however FFPE and FF DMLs compare favorably. These results support the emerging consensus that the 450K platform can be employed to investigate epigenetics in large sets of archival FFPE tissues.

Genetic Characterization of Echinococcus granulosus from a Large Number of Formalin-Fixed, Paraffin-Embedded Tissue Samples of Human Isolates in Iran

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Rostami, Sima; Torbaghan, Shams Shariat; Dabiri, Shahriar; Babaei, Zahra; Mohammadi, Mohammad Ali; Sharbatkhori, Mitra; Harandi, Majid Fasihi

2015-01-01

Cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus, presents an important medical and veterinary problem globally, including that in Iran. Different genotypes of E. granulosus have been reported from human isolates worldwide. This study identifies the genotype of the parasite responsible for human hydatidosis in three provinces of Iran using formalin-fixed paraffin-embedded tissue samples. In this study, 200 formalin-fixed paraffin-embedded tissue samples from human CE cases were collected from Alborz, Tehran, and Kerman provinces. Polymerase chain reaction amplification and sequencing of the partial mitochondrial cytochrome c oxidase subunit 1 gene were performed for genetic characterization of the samples. Phylogenetic analysis of the isolates from this study and reference sequences of different genotypes was done using a maximum likelihood method. In total, 54.4%, 0.8%, 1%, and 40.8% of the samples were identified as the G1, G2, G3, and G6 genotypes, respectively. The findings of the current study confirm the G1 genotype (sheep strain) to be the most prevalent genotype involved in human CE cases in Iran and indicates the high prevalence of the G6 genotype with a high infectivity for humans. Furthermore, this study illustrates the first documented human CE case in Iran infected with the G2 genotype. PMID:25535316

Analysis of formalin-fixed, paraffin-embedded (FFPE) tissue via proteomic techniques and misconceptions of antigen retrieval.

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O'Rourke, Matthew B; Padula, Matthew P

2016-01-01

Since emerging in the late 19(th) century, formaldehyde fixation has become a standard method for preservation of tissues from clinical samples. The advantage of formaldehyde fixation is that fixed tissues can be stored at room temperature for decades without concern for degradation. This has led to the generation of huge tissue banks containing thousands of clinically significant samples. Here we review techniques for proteomic analysis of formalin-fixed, paraffin-embedded (FFPE) tissue samples with a specific focus on the methods used to extract and break formaldehyde crosslinks. We also discuss an error-of-interpretation associated with the technique known as "antigen retrieval." We have discovered that this term has been mistakenly applied to two disparate molecular techniques; therefore, we argue that a terminology change is needed to ensure accurate reporting of experimental results. Finally, we suggest that more investigation is required to fully understand the process of formaldehyde fixation and its subsequent reversal.

DNA degrades during storage in formalin-fixed and paraffin-embedded tissue blocks.

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Guyard, Alice; Boyez, Alice; Pujals, Anaïs; Robe, Cyrielle; Tran Van Nhieu, Jeanne; Allory, Yves; Moroch, Julien; Georges, Odette; Fournet, Jean-Christophe; Zafrani, Elie-Serge; Leroy, Karen

2017-10-01

Formalin-fixed paraffin-embedded (FFPE) tissue blocks are widely used to identify clinically actionable molecular alterations or perform retrospective molecular studies. Our goal was to quantify degradation of DNA occurring during mid to long-term storage of samples in usual conditions. We selected 46 FFPE samples of surgically resected carcinomas of lung, colon, and urothelial tract, of which DNA had been previously extracted. We performed a second DNA extraction on the same blocks under identical conditions after a median period of storage of 5.5 years. Quantitation of DNA by fluorimetry showed a 53% decrease in DNA quantity after storage. Quantitative PCR (qPCR) targeting KRAS exon 2 showed delayed amplification of DNA extracted after storage in all samples but one. The qPCR/fluorimetry quantification ratio decreased from 56 to 15% after storage (p DNA analyzable by qPCR represented only 11% of the amount obtained at first extraction. Maximal length of amplifiable DNA fragments assessed with a multiplex PCR was reduced in DNA extracted from stored tissue, indicating that DNA fragmentation had increased in the paraffin blocks during storage. Next-generation sequencing was performed on 12 samples and showed a mean 3.3-fold decrease in library yield and a mean 4.5-fold increase in the number of single-nucleotide variants detected after storage. In conclusion, we observed significant degradation of DNA extracted from the same FFPE block after 4 to 6 years of storage. Better preservation strategies should be considered for storage of FFPE biopsy specimens.

Revealing the Molecular Portrait of Triple Negative Breast Tumors in an Understudied Population through Omics Analysis of Formalin-Fixed and Paraffin-Embedded Tissues.

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Vaca-Paniagua, Felipe; Alvarez-Gomez, Rosa María; Maldonado-Martínez, Hector Aquiles; Pérez-Plasencia, Carlos; Fragoso-Ontiveros, Veronica; Lasa-Gonsebatt, Federico; Herrera, Luis Alonso; Cantú, David; Bargallo-Rocha, Enrique; Mohar, Alejandro; Durand, Geoffroy; Forey, Nathalie; Voegele, Catherine; Vallée, Maxime; Le Calvez-Kelm, Florence; McKay, James; Ardin, Maude; Villar, Stéphanie; Zavadil, Jiri; Olivier, Magali

2015-01-01

Triple negative breast cancer (TNBC), defined by the lack of expression of the estrogen receptor, progesterone receptor and human epidermal receptor 2, is an aggressive form of breast cancer that is more prevalent in certain populations, in particular in low- and middle-income regions. The detailed molecular features of TNBC in these regions remain unexplored as samples are mostly accessible as formalin-fixed paraffin embedded (FFPE) archived tissues, a challenging material for advanced genomic and transcriptomic studies. Using dedicated reagents and analysis pipelines, we performed whole exome sequencing and miRNA and mRNA profiling of 12 FFPE tumor tissues collected from pathological archives in Mexico. Sequencing analyses of the tumor tissues and their blood pairs identified TP53 and RB1 genes as the most frequently mutated genes, with a somatic mutation load of 1.7 mutations/exome Mb on average. Transcriptional analyses revealed an overexpression of growth-promoting signals (EGFR, PDGFR, VEGF, PIK3CA, FOXM1), a repression of cell cycle control pathways (TP53, RB1), a deregulation of DNA-repair pathways, and alterations in epigenetic modifiers through miRNA:mRNA network de-regulation. The molecular programs identified were typical of those described in basal-like tumors in other populations. This work demonstrates the feasibility of using archived clinical samples for advanced integrated genomics analyses. It thus opens up opportunities for investigating molecular features of tumors from regions where only FFPE tissues are available, allowing retrospective studies on the search for treatment strategies or on the exploration of the geographic diversity of breast cancer.

Proteomic workflow for analysis of archival formalin-fixed and paraffin-embedded clinical samples to a depth of 10 000 proteins.

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Wiśniewski, Jacek R; Duś, Kamila; Mann, Matthias

2013-04-01

Archival formalin-fixed and paraffin-embedded clinical samples represent a very diverse source of material for proteomic investigation of diseases, often with follow-up patient information. Here, we describe an analytical workflow for analysis of laser-capture microdissected formalin-fixed and paraffin-embedded samples that allows studying proteomes to a depth of 10 000 proteins per sample. The workflow involves lysis of tissue in SDS-containing buffer, detergent removal, and consecutive digestion of the proteins with two enzymes by the multienzyme digestion filter-aided sample preparation method. Resulting peptides are fractionated by pipette-tip based strong anion exchange into six fractions and analyzed by LC-MS/MS on a bench top quadrupole Orbitrap mass spectrometer. Analysis of the data using the MaxQuant software resulted in the identification of 9502 ± 28 protein groups per a 110 nL sample of microdissected cells from human colonic adenoma. This depth of proteome analysis enables systemic insights into the organization of the adenoma cells and an estimation of the abundances of known biomarkers. It also allows the identification of proteins expressed from tumor suppressors, oncogenes, and other key players in the development and progression of the colorectal cancer. Our proteomic platform can be used for quantitative comparisons between samples representing different stages of diseases and thus can be applied to the discovery of biomarkers or drug targets. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Complete solubilization of formalin-fixed, paraffin-embedded tissue may improve proteomic studies.

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Shi, Shan-Rong; Taylor, Clive R; Fowler, Carol B; Mason, Jeffrey T

2013-04-01

Tissue-based proteomic approaches (tissue proteomics) are essential for discovering and evaluating biomarkers for personalized medicine. In any proteomics study, the most critical issue is sample extraction and preparation. This problem is especially difficult when recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, improving and standardizing protein extraction from FFPE tissue is a critical need because of the millions of archival FFPE tissues available in tissue banks worldwide. Recent progress in the application of heat-induced antigen retrieval principles for protein extraction from FFPE tissue has resulted in a number of published FFPE tissue proteomics studies. However, there is currently no consensus on the optimal protocol for protein extraction from FFPE tissue or accepted standards for quantitative evaluation of the extracts. Standardization is critical to ensure the accurate evaluation of FFPE protein extracts by proteomic methods such as reverse phase protein arrays, which is now in clinical use. In our view, complete solubilization of FFPE tissue samples is the best way to achieve the goal of standardizing the recovery of proteins from FFPE tissues. However, further studies are recommended to develop standardized protein extraction methods to ensure quantitative and qualitative reproducibility in the recovery of proteins from FFPE tissues. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multi-Center Evaluation of the Fully Automated PCR-Based Idylla™ KRAS Mutation Assay for Rapid KRAS Mutation Status Determination on Formalin-Fixed Paraffin-Embedded Tissue of Human Colorectal Cancer

DEFF Research Database (Denmark)

Solassol, Jérôme; Vendrell, Julie; Märkl, Bruno

2016-01-01

, was assessed on archived formalin-fixed paraffin-embedded (FFPE) tissue sections by comparing its results with the results previously obtained by routine reference approaches for KRAS genotyping. In case of discordance, samples were assessed further by additional methods. Among the 374 colorectal cancer FFPE...

Comparative investigations of T cell receptor gamma gene rearrangements in frozen and formalin-fixed paraffin wax-embedded tissues by capillary electrophoresis

DEFF Research Database (Denmark)

Christensen, M; Funder, A D; Bendix, K

2006-01-01

AIM: To compare clonal T cell receptor gamma (TCRgamma) gene rearrangements in frozen and formalin-fixed paraffin wax-embedded (FFPE) tissue, using capillary electrophoresis for use in diagnostics, as T cell lymphomas may be difficult to diagnose by conventional methods.METHODS: The DNA for PCR......% for patient specimens and the specificity 100%. The junctional region between the Vgamma and Jgamma segments was specific for each patient.CONCLUSIONS: Capillary electrophoresis of PCR products from frozen and FFPE tissue is suitable for detecting clonal TCRgamma gene rearrangements. It is important, however...

Impact of pre-analytical factors on the proteomic analysis of formalin-fixed paraffin-embedded tissue.

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Thompson, Seonaid M; Craven, Rachel A; Nirmalan, Niroshini J; Harnden, Patricia; Selby, Peter J; Banks, Rosamonde E

2013-04-01

Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a tremendous potential resource for biomarker discovery, with large numbers of samples in hospital pathology departments and links to clinical information. However, the cross-linking of proteins and nucleic acids by formalin fixation has hampered analysis and proteomic studies have been restricted to using frozen tissue, which is more limited in availability as it needs to be collected specifically for research. This means that rare disease subtypes cannot be studied easily. Recently, improved extraction techniques have enabled analysis of FFPE tissue by a number of proteomic techniques. As with all clinical samples, pre-analytical factors are likely to impact on the results obtained, although overlooked in many studies. The aim of this review is to discuss the various pre-analytical factors, which include warm and cold ischaemic time, size of sample, fixation duration and temperature, tissue processing conditions, length of storage of archival tissue and storage conditions, and to review the studies that have considered these factors in more detail. In those areas where investigations are few or non-existent, illustrative examples of the possible importance of specific factors have been drawn from studies using frozen tissue or from immunohistochemical studies of FFPE tissue. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-throughput sequencing and copy number variation detection using formalin fixed embedded tissue in metastatic gastric cancer.

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Seokhwi Kim

Full Text Available In the era of targeted therapy, mutation profiling of cancer is a crucial aspect of making therapeutic decisions. To characterize cancer at a molecular level, the use of formalin-fixed paraffin-embedded tissue is important. We tested the Ion AmpliSeq Cancer Hotspot Panel v2 and nCounter Copy Number Variation Assay in 89 formalin-fixed paraffin-embedded gastric cancer samples to determine whether they are applicable in archival clinical samples for personalized targeted therapies. We validated the results with Sanger sequencing, real-time quantitative PCR, fluorescence in situ hybridization and immunohistochemistry. Frequently detected somatic mutations included TP53 (28.17%, APC (10.1%, PIK3CA (5.6%, KRAS (4.5%, SMO (3.4%, STK11 (3.4%, CDKN2A (3.4% and SMAD4 (3.4%. Amplifications of HER2, CCNE1, MYC, KRAS and EGFR genes were observed in 8 (8.9%, 4 (4.5%, 2 (2.2%, 1 (1.1% and 1 (1.1% cases, respectively. In the cases with amplification, fluorescence in situ hybridization for HER2 verified gene amplification and immunohistochemistry for HER2, EGFR and CCNE1 verified the overexpression of proteins in tumor cells. In conclusion, we successfully performed semiconductor-based sequencing and nCounter copy number variation analyses in formalin-fixed paraffin-embedded gastric cancer samples. High-throughput screening in archival clinical samples enables faster, more accurate and cost-effective detection of hotspot mutations or amplification in genes.

Molecular identification of Coccidioides immitis in formalin-fixed, paraffin-embedded (FFPE) tissues from a Colombian patient.

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Canteros, Cristina E; Vélez H, Alejandro; Toranzo, Adriana I; Suárez-Alvarez, Roberto; Tobón O, Ángela; Jimenez A, María del Pilar; Restrepo M, Ángela

2015-06-01

Coccidioides immitis and C. posadasii are the etiologic agents of coccidioidomycosis, an endemic fungal disease of the Americas. In Colombia, this mycosis is uncommon, and only five cases, two of them imported, have been documented.By means of DNA sequencing, C. immitis was identified in formalin-fixed, paraffin-embedded archival tissues samples from the 5th Colombian patient diagnosed in 1997. The patient was born in Pinto, Department of Magdalena, and had never visited other geographic regions, a reason to consider that the mycosis had been acquired locally.This species is primarily found in California although it has been occasionally reported in other geographic areas such as Mexico and Brazil. This is the first indigenous report of C. immitis-associated coccidioidomycosis in a Colombian patient. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.

Development of a monoclonal antibody that specifically detects tissue inhibitor of metalloproteinase-4 (TIMP-4) in formalin-fixed, paraffin-embedded human tissues.

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Donover, P Scott; Wojciechowski, Brian S; Thirumaran, Rajesh; Zemba-Palko, Vlasta; Prendergast, George C; Wallon, U Margaretha

2010-08-01

Overexpression of the extracellular metalloproteinase inhibitor TIMP-4 in estrogen receptor-negative breast cancers was found recently to be associated with a poor prognosis for survival. To pursue exploration of the theranostic applications of TIMP-4, specific antibodies with favorable properties for immunohistochemical use and other clinical assays are needed. Here we report the characterization of a monoclonal antibody (clone 9:4-7) specific for full-length human TIMP-4 with suitable qualities. The antibody was determined to be an IgG(2b) immunoglobulin. In enzyme-linked immunosorbent assay (ELISA) and immunoblotting assays, it did not exhibit any detectable crossreactivity with recombinant forms of the other human TIMPs 1, 2, and 3. In contrast, the antibody displayed high specificity and sensitivity for TIMP-4 including in formalin-fixed and paraffin-embedded specimens of human breast specimens. An analysis of tissue microarrays of human cancer and corresponding normal tissues revealed specific staining patterns with excellent signal-to-noise ratios. This study documents TIMP-4 monoclonal antibody clone 9:4-7 as an effective tool for preclinical and clinical investigations. Published 2010 Wiley-Liss, Inc.

Multiplex polymerase chain reaction for the detection of high-risk-human papillomavirus types in formalin-fixed paraffin-embedded cervical tissues

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Mini P Singh

2017-01-01

Full Text Available Detecting high-risk-human papillomavirus (HPV types has become an integral part of the cervical cancer screening programmes. This study aimed to develop a multiplex polymerase chain reaction (PCR for identification of HPV types 16 and 18 along with the beta globin gene in formalin-fixed and paraffin-embedded cervical biopsy specimens. A total of 59 samples from patients with cervical abnormalities were tested. HPV 16 positivity was 50% in cervical cancers and 52.9% in cervical intraepithelial neoplasia. Our multiplex PCR protocol can be used as a simple and cost-effective tool for high-risk-HPV detection in cervical cancer screening programmes.

Differential N-glycan patterns identified in lung adenocarcinoma by N-glycan profiling of formalin-fixed paraffin-embedded (FFPE) tissue sections.

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Wang, Xiaoning; Deng, Zaian; Huang, Chuncui; Zhu, Tong; Lou, Jiatao; Wang, Lin; Li, Yan

2018-02-10

N-glycan profiling is a powerful approach for analyzing the functional relationship between N-glycosylation and cancer. Current methods rely on either serum or fresh tissue samples; however, N-glycan patterns may differ between serum and tissue, as the proteins of serum originate from a variety of tissues. Furthermore, fresh tissue samples are difficult to ship and store. Here, we used a profiling method based on formalin-fixed paraffin-embedded (FFPE) tissue sections from lung adenocarcinoma patients. We found that our method was highly reproducible. We identified 58 N-glycan compositions from lung adenocarcinoma FFPE samples, 51 of which were further used for MS n -based structure prediction. We show that high mannose type N-glycans are upregulated, while sialylated N-glycans are downregulated in our FFPE lung adenocarcinoma samples, compared to the control samples. Our receiver operating characteristic (ROC) curve analysis shows that high mannose type and sialylated N-glycans are useful discriminators to distinguish between lung adenocarcinoma and control tissue. Together, our results indicate that expression levels of specific N-glycans correlate well with lung adenocarcinoma, and strongly suggest that our FFPE-based method will be useful for N-glycan profiling of cancer tissues. Glycosylation is one of the most important post-translational protein modifications, and is associated with several physiopathological processes, including carcinogenesis. In this study, we tested the feasibility of using formalin-fixed paraffin-embedded (FFPE) tissue sections to identify changes in N-glycan patterns and identified the differentially expressed N-glycans of lung adenocarcinoma. Our study shows that the FFPE-based N-glycan profiling method is useful for clinical diagnosis as well as identification of potential biomarkers, and our data expand current knowledge of differential N-glycan patterns of lung adenocarcinoma. Copyright © 2017 Elsevier B.V. All rights reserved.

Improved protein extraction and protein identification from archival formalin-fixed paraffin-embedded human aortas.

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Fu, Zongming; Yan, Kun; Rosenberg, Avraham; Jin, Zhicheng; Crain, Barbara; Athas, Grace; Heide, Richard S Vander; Howard, Timothy; Everett, Allen D; Herrington, David; Van Eyk, Jennifer E

2013-04-01

Evaluate combination of heat and elevated pressure to enhance protein extraction and quality of formalin-fixed (FF), and FF paraffin-embedded (FFPE) aorta for proteomics. Proteins were extracted from fresh frozen aorta at room temperature (RT). FF and FFPE aortas (3 months and 15 years) were extracted at RT, heat alone, or a combination of heat and high pressure. Protein yields were compared, and digested peptides from the extracts were analyzed with MS. Combined heat and elevated pressure increased protein yield from human FF or FFPE aorta compared to matched tissues with heat alone (1.5-fold) or at RT (8.3-fold), resulting in more proteins identified and with more sequence coverage. The length of storage did adversely affect the quality of proteins from FF tissue. For long-term storage, aorta was preserved better with FFPE than FF alone. Periostin and MGF-E8 were demonstrated suitable for MRM assays from FFPE aorta. Combination of heat and high pressure is an effective method to extract proteins from FFPE aorta for downstream proteomics. This method opens the possibility for use of archival and often rare FFPE aortas and possibly other tissues available to proteomics for biomarker discovery and quantification. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

MicroRNA expression profiles of multiple system atrophy from formalin-fixed paraffin-embedded samples.

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Wakabayashi, Koichi; Mori, Fumiaki; Kakita, Akiyoshi; Takahashi, Hitoshi; Tanaka, Shinya; Utsumi, Jun; Sasaki, Hidenao

2016-12-02

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression. Recently, we have shown that informative miRNA data can be derived from archived formalin-fixed paraffin-embedded (FFPE) samples from postmortem cases of amyotrophic lateral sclerosis and normal controls. miRNA analysis has now been performed on FFPE samples from affected brain regions in patients with multiple system atrophy (MSA) and the same areas in neurologically normal controls. We evaluated 50 samples from patients with MSA (n=13) and controls (n=13). Twenty-six samples were selected for miRNA analysis on the basis of the criteria reported previously: (i) a formalin fixation time of less than 4 weeks, (ii) a total RNA yield per sample of more than 500ng, and (iii) sufficient quality of the RNA electrophoresis pattern. These included 11 cases of MSA and 5 controls. Thus, the success rate for analysis of RNA from FFPE samples was 52% (26 of 50). For MSA, a total of 395 and 383 miRNAs were identified in the pons and cerebellum, respectively; 5 were up-regulated and 33 were down-regulated in the pons and 5 were up-regulated and 18 were down-regulated in the cerebellum. Several miRNAs down-regulated in the pons (miR-129-2-3p and miR-129-5p) and cerebellum (miR-129-2-3p, miR-129-5p and miR-132-3p) had already been identified in frozen cerebellum from MSA patients. These findings suggest that archived FFPE postmortem samples can be a valuable source for miRNA profiling in MSA. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Optimization of Glial Fibrillary Acidic Protein Immunoreactivity in Formalin-fixed, Paraffin-Embedded Guinea Pig Brain Sections

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2003-09-01

fixed, paraffin-embedded guinea pig brain sections using a variety of commercially available GFAP antibody clones. Of the 7 clones tested for cross...determining neuropathological consequences in the guinea pig following exposure to chemical warfare nerve agent.

MALDI imaging mass spectrometry profiling of N-glycans in formalin-fixed paraffin embedded clinical tissue blocks and tissue microarrays.

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Powers, Thomas W; Neely, Benjamin A; Shao, Yuan; Tang, Huiyuan; Troyer, Dean A; Mehta, Anand S; Haab, Brian B; Drake, Richard R

2014-01-01

A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers.

Rate of Clinical Complete Response for 1 Year or More in Bone-Metastatic Breast Cancer after Comprehensive Treatments including Autologous Formalin-Fixed Tumor Vaccine.

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Kuranishi, Fumito; Imaoka, Yuki; Sumi, Yuusuke; Uemae, Yoji; Yasuda-Kurihara, Hiroko; Ishihara, Takeshi; Miyazaki, Tsubasa; Ohno, Tadao

2018-01-01

No effective treatment has been developed for bone-metastatic breast cancer. We found 3 cases with clinical complete response (cCR) of the bone metastasis and longer overall survival of the retrospectively examined cohort treated comprehensively including autologous formalin-fixed tumor vaccine (AFTV). AFTV was prepared individually for each patient from their own formalin-fixed and paraffin-embedded breast cancer tissues. Three patients maintained cCR status of the bone metastasis for 17 months or more. Rate of cCR for 1 year or more appeared to be 15% (3/20) after comprehensive treatments including AFTV. The median overall survival time (60.0 months) and the 3- to 8-year survival rates after diagnosis of bone metastasis were greater than those of historical control cohorts in Japan (1988-2002) and in the nationwide population-based cohort study of Denmark (1999-2007). Bone-metastatic breast cancer may be curable after comprehensive treatments including AFTV, although larger scale clinical trial is required.

Protein extraction from methanol fixed paraffin embedded tissue blocks: A new possibility using cell blocks

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Kokkat, Theresa J.; McGarvey, Diane; Patel, Miral S.; Tieniber, Andrew D.; LiVolsi, Virginia A.; Baloch, Zubair W.

2013-01-01

Background: Methanol fixed and paraffin embedded (MFPE) cellblocks are an essential cytology preparation. However, MFPE cellblocks often contain limited material and their relatively small size has caused them to be overlooked in biomarker discovery. Advances in the field of molecular biotechnology have made it possible to extract proteins from formalin fixed and paraffin embedded (FFPE) tissue blocks. In contrast, there are no established methods for extracting proteins from MFPE cellblocks. We investigated commonly available CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate) buffer, as well as two commercially available Qiagen® kits and compared their effectiveness on MFPE tissue for protein yields. Materials and Methods: MFPE blocks were made by Cellient™ automated system using human tissue specimens from normal and malignant specimens collected in ThinPrep™ Vials. Protein was extracted from Cellient-methanol fixed and paraffin embedded blocks with CHAPS buffer method as well as FFPE and Mammalian Qiagen® kits. Results: Comparison of protein yields demonstrated the effectiveness of various protein extraction methods on MFPE cellblocks. Conclusion: In the current era of minimally invasive techniques to obtain minimal amount of tissue for diagnostic and prognostic purposes, the use of commercial and lab made buffer on low weight MFPE scrapings obtained by Cellient® processor opens new possibilities for protein biomarker research. PMID:24403950

Integrative analysis of copy number and gene expression in breast cancer using formalin-fixed paraffin-embedded core biopsy tissue: a feasibility study.

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Iddawela, Mahesh; Rueda, Oscar; Eremin, Jenny; Eremin, Oleg; Cowley, Jed; Earl, Helena M; Caldas, Carlos

2017-07-11

An absence of reliable molecular markers has hampered individualised breast cancer treatments, and a major limitation for translational research is the lack of fresh tissue. There are, however, abundant banks of formalin-fixed paraffin-embedded (FFPE) tissue. This study evaluated two platforms available for the analysis of DNA copy number and gene expression using FFPE samples. The cDNA-mediated annealing, selection, extension, and ligation assay (DASL™) has been developed for gene expression analysis and the Molecular Inversion Probes assay (Oncoscan™), were used for copy number analysis using FFPE tissues. Gene expression and copy number were evaluated in core-biopsy samples from patients with breast cancer undergoing neoadjuvant chemotherapy (NAC). Forty-three core-biopsies were evaluated and characteristic copy number changes in breast cancers, gains in 1q, 8q, 11q, 17q and 20q and losses in 6q, 8p, 13q and 16q, were confirmed. Regions that frequently exhibited gains in tumours showing a pathological complete response (pCR) to NAC were 1q (55%), 8q (40%) and 17q (40%), whereas 11q11 (37%) gain was the most frequent change in non-pCR tumours. Gains associated with poor survival were 11q13 (62%), 8q24 (54%) and 20q (47%). Gene expression assessed by DASL correlated with immunohistochemistry (IHC) analysis for oestrogen receptor (ER) [area under the curve (AUC) = 0.95], progesterone receptor (PR)(AUC = 0.90) and human epidermal growth factor type-2 receptor (HER-2) (AUC = 0.96). Differential expression analysis between ER+ and ER- cancers identified over-expression of TTF1, LAF-4 and C-MYB (p ≤ 0.05), and between pCR vs non-pCRs, over-expression of CXCL9, AREG, B-MYB and under-expression of ABCG2. This study was an integrative analysis of copy number and gene expression using FFPE core biopsies and showed that molecular marker data from FFPE tissues were consistent with those in previous studies using fresh-frozen samples. FFPE tissue can provide

Label-free protein profiling of formalin-fixed paraffin-embedded (FFPE) heart tissue reveals immediate mitochondrial impairment after ionising radiation.

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Azimzadeh, Omid; Scherthan, Harry; Yentrapalli, Ramesh; Barjaktarovic, Zarko; Ueffing, Marius; Conrad, Marcus; Neff, Frauke; Calzada-Wack, Julia; Aubele, Michaela; Buske, Christian; Atkinson, Michael J; Hauck, Stefanie M; Tapio, Soile

2012-04-18

Qualitative proteome profiling of formalin-fixed, paraffin-embedded (FFPE) tissue is advancing the field of clinical proteomics. However, quantitative proteome analysis of FFPE tissue is hampered by the lack of an efficient labelling method. The usage of conventional protein labelling on FFPE tissue has turned out to be inefficient. Classical labelling targets lysine residues that are blocked by the formalin treatment. The aim of this study was to establish a quantitative proteomics analysis of FFPE tissue by combining the label-free approach with optimised protein extraction and separation conditions. As a model system we used FFPE heart tissue of control and exposed C57BL/6 mice after total body irradiation using a gamma ray dose of 3 gray. We identified 32 deregulated proteins (p≤0.05) in irradiated hearts 24h after the exposure. The proteomics data were further evaluated and validated by bioinformatics and immunoblotting investigation. In good agreement with our previous results using fresh-frozen tissue, the analysis indicated radiation-induced alterations in three main biological pathways: respiratory chain, lipid metabolism and pyruvate metabolism. The label-free approach enables the quantitative measurement of radiation-induced alterations in FFPE tissue and facilitates retrospective biomarker identification using clinical archives. Copyright © 2012 Elsevier B.V. All rights reserved.

STR typing of formalin-fixed paraffin embedded (FFPE) aborted foetal tissue in criminal paternity cases.

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Reshef, Ayeleth; Barash, Mark; Voskoboinik, Lev; Brauner, Paul; Gafny, Roni

2011-03-01

Sexual assault or rape cases occasionally result in unwanted pregnancies. In almost all such cases the foetus is aborted. A forensic laboratory may receive the foetus, the placenta, or paraffin embedded abortion material for paternity testing. Obtaining a foetal profile DNA from a foetus or placenta may not be successful due to the age or condition of the tissue. Moreover, maternal contamination of placental material will invariably result in a mixed DNA profile. However, the use of properly screened abortion material from paraffin blocks will almost always result in obtaining a foetal DNA profile. Furthermore, foetal tissue fixed in paraffin blocks does not require special conditions for submission and storage as required to preserve fresh foetal or placental tissue. As hospitals routinely prepare foetal tissue in paraffin blocks, which should be readily obtainable by forensic laboratories, these samples would appear to be the preferred choice for paternity testing. 2010 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.

Elevated pressure improves the extraction and identification of proteins recovered from formalin-fixed, paraffin-embedded tissue surrogates.

Directory of Open Access Journals (Sweden)

Carol B Fowler

2010-12-01

Full Text Available Proteomic studies of formalin-fixed paraffin-embedded (FFPE tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing.In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%. Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates.These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form suitable for proteomic analysis.

Elevated Pressure Improves the Extraction and Identification of Proteins Recovered from Formalin-Fixed, Paraffin-Embedded Tissue Surrogates

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Fowler, Carol B.; Chesnick, Ingrid E.; Moore, Cedric D.; O'Leary, Timothy J.; Mason, Jeffrey T.

2010-01-01

Background Proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing. Principal Findings In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%). Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis) of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates. Conclusions These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form

Antigen retrieval prior to on-tissue digestion of formalin-fixed paraffin-embedded tumour tissue sections yields oxidation of proline residues.

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Djidja, Marie-Claude; Claude, Emmanuelle; Scriven, Peter; Allen, David W; Carolan, Vikki A; Clench, Malcolm R

2017-07-01

MALDI-mass spectrometry imaging (MALDI-MSI) has been shown to allow the study of protein distribution and identification directly within formalin-fixed paraffin-embedded (FFPE) tissue sections. However, direct protein identification from tissue sections remains challenging due to signal interferences and/or existing post-translational or other chemical modifications. The use of antigen retrieval (AR) has been demonstrated for unlocking proteins prior to in situ enzymatic digestion and MALDI-MSI analysis of FFPE tissue sections. In the work reported here, the identification of proline oxidation, which may occur when performing the AR protocol, is described. This facilitated and considerably increased the number of identified peptides when adding proline oxidation as a variable modification to the MASCOT search criteria. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann. Copyright © 2016 Elsevier B.V. All rights reserved.

Detection of c-myc amplification in formalin-fixed paraffin-embedded tumor tissue by chromogenic in situ hybridization (CISH).

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Todorović-Raković, Nataša

2013-01-01

In situ hybridization (ISH) allows evaluation of genetic abnormalities, such as changes in chromosome number, chromosome translocations or gene amplifications, by hybridization of tagged DNA (or RNA) probes with complementary DNA (or RNA) sequences in interphase nuclei of target tissue. However, chromogenic in situ hybridization (CISH) is also applicable to formalin-fixed, paraffin-embedded (FFPE) tissues, besides metaphase chromosome spreads. CISH is similar to fluorescent in situ hybridization (FISH) regarding pretreatments and hybridization protocols but differs in the way of visualization. Indeed, CISH signal detection is similar to that used in immunohistochemistry, making use of a peroxidase-based chromogenic reaction instead of fluorescent dyes. In particular, tagged DNA probes are indirectly detected using an enzyme-conjugated antibody targeting the tags. The enzymatic reaction of the chromogenic substrate leads to the formation of strong permanent brown signals that can be visualized by bright-field microscopy at 40 × magnification. The advantage of CISH is that it allows the simultaneous observation of gene amplification and tissue morphology and the slides can be stored for a long time.

Molecular glycopathology by capillary electrophoresis: Analysis of the N-glycome of formalin-fixed paraffin-embedded mouse tissue samples.

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Donczo, Boglarka; Szarka, Mate; Tovari, Jozsef; Ostoros, Gyorgyi; Csanky, Eszter; Guttman, Andras

2017-06-01

Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze endoglycosidase released and fluorophore-labeled N-glycans from formalin-fixed paraffin-embedded (FFPE) mouse tissue samples of lung, brain, heart, spleen, liver, kidney and intestine. The FFPE samples were first deparaffinized followed by solubilization and glycoprotein retrieval. PNGase F mediated release of the N-linked oligosaccharides was followed by labeling with aminopyrene trisulfonate. After CE-LIF glycoprofiling of the FFPE mouse tissues, the N-glycan pool of the lung specimen was subject to further investigation by exoglycosidase array based carbohydrate sequencing. Structural assignment of the oligosaccharides was accomplished by the help of the GUcal software and the associated database, based on the mobility shifts after treatments with the corresponding exoglycosidase reaction mixtures. Sixteen major N-linked carbohydrate structures were sequenced from the mouse lung FFPE tissue glycome and identified, as high mannose (3) neutral biantennary (3) sialylated monoantennary (1) and sialylated bianennary (9) oligosaccharides. Two of these latter ones also possessed alpha(1-3) linked galactose residues. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rate of Clinical Complete Response for 1 Year or More in Bone-Metastatic Breast Cancer after Comprehensive Treatments including Autologous Formalin-Fixed Tumor Vaccine

Directory of Open Access Journals (Sweden)

Fumito Kuranishi

2018-01-01

Full Text Available Introduction. No effective treatment has been developed for bone-metastatic breast cancer. We found 3 cases with clinical complete response (cCR of the bone metastasis and longer overall survival of the retrospectively examined cohort treated comprehensively including autologous formalin-fixed tumor vaccine (AFTV. Patients and Methods. AFTV was prepared individually for each patient from their own formalin-fixed and paraffin-embedded breast cancer tissues. Results. Three patients maintained cCR status of the bone metastasis for 17 months or more. Rate of cCR for 1 year or more appeared to be 15% (3/20 after comprehensive treatments including AFTV. The median overall survival time (60.0 months and the 3- to 8-year survival rates after diagnosis of bone metastasis were greater than those of historical control cohorts in Japan (1988–2002 and in the nationwide population-based cohort study of Denmark (1999–2007. Conclusion. Bone-metastatic breast cancer may be curable after comprehensive treatments including AFTV, although larger scale clinical trial is required.

Detection of a putative novel adenovirus by PCR amplification, sequencing and phylogenetic characterisation of two gene fragments from formalin-fixed paraffin-embedded tissues of a cat diagnosed with disseminated adenovirus disease.

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Lakatos, Béla; Hornyák, Ákos; Demeter, Zoltán; Forgách, Petra; Kennedy, Frances; Rusvai, Miklós

2017-12-01

Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in formalin-fixed paraffin-embedded tissue samples of a cat that had suffered from disseminated adenovirus infection. The identity of the amplified products from the hexon and DNA-dependent DNA polymerase genes was confirmed by DNA sequencing. The sequences were clearly distinguishable from corresponding hexon and polymerase sequences of other mastadenoviruses, including human adenoviruses. These results suggest the possible existence of a distinct feline adenovirus.

The effect of aging of formalin-fixed paraffin-embedded tissues on the in situ hybridization and immunohistochemistry signals in cervical lesions.

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Nuovo, Allison J; Garofalo, Michela; Mikhail, Alexandria; Nicol, Alcina F; Vianna-Andrade, Cecilia; Nuovo, Gerard J

2013-09-01

Formalin-fixed, paraffin-embedded tissues are widely used in biomedical research but little is known about the effect of the age of the block or unstained slides on the in situ hybridization or immunohistochemistry signal. We compared the in situ-based and immunohistochemistry-based signals for cervical intraepithelial neoplasia samples that ranged from 0 to 15 years of age. There was a progressive and statistically significant decrease in the strength of the p16 signal when comparing tissues prepared from recent unstained slides (0 to 1 y old, mean score of 92%) to those of intermediate age (5 to 7 y old, mean score of 49%) to old unstained slides (cut 13 to 15 y ago, mean score of 10%). Equivalent, progressive, and significant decreases in the intensity of the signals for microRNAs, CD45, and human papillomavirus DNA were seen in tissues stored on slides from 5 to 7 years and 13 to 15 years, respectively. However, the diminution of signal was much less, although still statistically significant, if the sections from the 13- to 15-year-old paraffin blocks were prepared in 2012. The data likely does not represent degradation of the targets as extraction of several microRNA from the old blocks showed no detectable degradation, despite the markedly weakened in situ hybridization signal. It is concluded that in situ-based signal for DNA, microRNAs, and proteins in paraffin-embedded tissues are significantly reduced over time, especially when stored long term on glass slides which, in turn, can lead to a significant underestimation of the amount and presence of the nucleic acid or protein target.

HaloPlex Targeted Resequencing for Mutation Detection in Clinical Formalin-Fixed, Paraffin-Embedded Tumor Samples.

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Moens, Lotte N J; Falk-Sörqvist, Elin; Ljungström, Viktor; Mattsson, Johanna; Sundström, Magnus; La Fleur, Linnéa; Mathot, Lucy; Micke, Patrick; Nilsson, Mats; Botling, Johan

2015-11-01

In recent years, the advent of massively parallel next-generation sequencing technologies has enabled substantial advances in the study of human diseases. Combined with targeted DNA enrichment methods, high sequence coverage can be obtained for different genes simultaneously at a reduced cost per sample, creating unique opportunities for clinical cancer diagnostics. However, the formalin-fixed, paraffin-embedded (FFPE) process of tissue samples, routinely used in pathology departments, results in DNA fragmentation and nucleotide modifications that introduce a number of technical challenges for downstream biomolecular analyses. We evaluated the HaloPlex target enrichment system for somatic mutation detection in 80 tissue fractions derived from 20 clinical cancer cases with paired tumor and normal tissue available in both FFPE and fresh-frozen format. Several modifications to the standard method were introduced, including a reduced target fragment length and two strand capturing. We found that FFPE material can be used for HaloPlex-based target enrichment and next-generation sequencing, even when starting from small amounts of DNA. By specifically capturing both strands for each target fragment, we were able to reduce the number of false-positive errors caused by FFPE-induced artifacts and lower the detection limit for somatic mutations. We believe that the HaloPlex method presented here will be broadly applicable as a tool for somatic mutation detection in clinical cancer settings. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Implementation of formalin-fixed, paraffin-embedded cell line pellets as high-quality process controls in quality assessment programs for KRAS mutation analysis

DEFF Research Database (Denmark)

Dijkstra, Jeroen R; Opdam, Frank J M; Boonyaratanakornkit, Jerry

2013-01-01

. We assessed a novel synthetic control for formalin-fixed, paraffin-embedded (FFPE) tumor samples in a blind study conducted within nine laboratories across Europe. We show that FFPE material can, at least in part, mimic clinical samples and we demonstrate this control to be a valuable tool....... For a testing laboratory to become accredited to perform such tests, it is essential that they perform reliability testing, but it has not previously been possible to perform this kind of testing on the complete workflow on a large scale without compromising reproducibility or the mimicry of the control sample...... receptor (EGFR)-targeted therapy with increased progression-free survival only if the tumor does not carry a mutation in KRAS. Many different analytical platforms, both those commercially available and those developed in house, have been used within pathology laboratories to assess KRAS mutational status...

Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies

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R Nada

2016-01-01

Full Text Available Renal biopsy interpretation requires histopathology, direct immunofluorescence (DIF and electron microscopy. Formalin-fixed, paraffin-embedded tissue (FFPE sent for light microscopy can be used for DIF after antigen retrieval. However, complement staining has not been satisfactory. We standardized DIF using proteinase-K for antigen retrieval in FFPE renal biopsies. A pilot study was conducted on known cases of membranous glomerulonephritis (MGN, membranoproliferative type-1 (MPGN-1, immunoglobulin A nephropathy (IgAN, and anti-glomerular basement disease (anti-GBM. Immunofluorescence panel included fluorescein isothiocyanate (FITC conjugated IgG, IgA, IgM, complements (C3 and C1q, light chains (kappa, lambda and fibrinogen antibodies. After standardization of the technique, 75 renal biopsies and 43 autopsies cases were stained. Out of 43 autopsy cases, immune-complex mediated glomerulonephritis (GN was confirmed in 18 cases (Lupus nephritis-11, IgAN-6, MGN-1, complement-mediated dense deposit disease (DDD-1 and monoclonal diseases in 4 cases (amyloidosis-3, cast nephropathy-1. Immune-mediated injury was excluded in 17 cases (focal segmental glomerulosclerosis -3, crescentic GN-6 [pauci-immune-3, anti-GBM-3], thrombotic microangiopathy-5, atherosclerosis-3. Renal biopsies (n-75 where inadequate or no frozen sample was available; this technique classified 52 mesangiocapillary pattern as MPGN type-1-46, DDD-2 and (C3GN-4. Others were diagnosed as IgAN-3, lupus nephritis-2, MGN-4, diffuse proliferative glomerulonephritis (DPGN-1, Non-IC crescentic GN-1, monoclonal diseases-3. In nine cases, DIF on FFPE tissue could not help in making diagnosis. Proteinase-K enzymatic digestion of FFPE renal biopsies can unmask complements (both C3 and C1q in immune-complexes mediated and complement-mediated diseases. This method showed good results on autopsy tissues archived for as long as 15 years.

Whole-genome gene expression profiling of formalin-fixed, paraffin-embedded tissue samples.

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Craig April

2009-12-01

Full Text Available We have developed a gene expression assay (Whole-Genome DASL, capable of generating whole-genome gene expression profiles from degraded samples such as formalin-fixed, paraffin-embedded (FFPE specimens.We demonstrated a similar level of sensitivity in gene detection between matched fresh-frozen (FF and FFPE samples, with the number and overlap of probes detected in the FFPE samples being approximately 88% and 95% of that in the corresponding FF samples, respectively; 74% of the differentially expressed probes overlapped between the FF and FFPE pairs. The WG-DASL assay is also able to detect 1.3-1.5 and 1.5-2 -fold changes in intact and FFPE samples, respectively. The dynamic range for the assay is approximately 3 logs. Comparing the WG-DASL assay with an in vitro transcription-based labeling method yielded fold-change correlations of R(2 approximately 0.83, while fold-change comparisons with quantitative RT-PCR assays yielded R(2 approximately 0.86 and R(2 approximately 0.55 for intact and FFPE samples, respectively. Additionally, the WG-DASL assay yielded high self-correlations (R(2>0.98 with low intact RNA inputs ranging from 1 ng to 100 ng; reproducible expression profiles were also obtained with 250 pg total RNA (R(2 approximately 0.92, with approximately 71% of the probes detected in 100 ng total RNA also detected at the 250 pg level. When FFPE samples were assayed, 1 ng total RNA yielded self-correlations of R(2 approximately 0.80, while still maintaining a correlation of R(2 approximately 0.75 with standard FFPE inputs (200 ng.Taken together, these results show that WG-DASL assay provides a reliable platform for genome-wide expression profiling in archived materials. It also possesses utility within clinical settings where only limited quantities of samples may be available (e.g. microdissected material or when minimally invasive procedures are performed (e.g. biopsied specimens.

Optimization and analysis of a quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples

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Reis Patricia P

2010-06-01

Full Text Available Abstract Background MicroRNAs (miRs are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE tissue. Results Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p 35, we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p Conclusion Our study thus demonstrates the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies.

A Comparison of RNA-Seq Results from Paired Formalin-Fixed Paraffin-Embedded and Fresh-Frozen Glioblastoma Tissue Samples.

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Anna Esteve-Codina

Full Text Available The molecular classification of glioblastoma (GBM based on gene expression might better explain outcome and response to treatment than clinical factors. Whole transcriptome sequencing using next-generation sequencing platforms is rapidly becoming accepted as a tool for measuring gene expression for both research and clinical use. Fresh frozen (FF tissue specimens of GBM are difficult to obtain since tumor tissue obtained at surgery is often scarce and necrotic and diagnosis is prioritized over freezing. After diagnosis, leftover tissue is usually stored as formalin-fixed paraffin-embedded (FFPE tissue. However, RNA from FFPE tissues is usually degraded, which could hamper gene expression analysis. We compared RNA-Seq data obtained from matched pairs of FF and FFPE GBM specimens. Only three FFPE out of eleven FFPE-FF matched samples yielded informative results. Several quality-control measurements showed that RNA from FFPE samples was highly degraded but maintained transcriptomic similarities to RNA from FF samples. Certain issues regarding mutation analysis and subtype prediction were detected. Nevertheless, our results suggest that RNA-Seq of FFPE GBM specimens provides reliable gene expression data that can be used in molecular studies of GBM if the RNA is sufficiently preserved.

Numerical and structural genomic aberrations are reliably detectable in tissue microarrays of formalin-fixed paraffin-embedded tumor samples by fluorescence in-situ hybridization.

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Heike Horn

Full Text Available Few data are available regarding the reliability of fluorescence in-situ hybridization (FISH, especially for chromosomal deletions, in high-throughput settings using tissue microarrays (TMAs. We performed a comprehensive FISH study for the detection of chromosomal translocations and deletions in formalin-fixed and paraffin-embedded (FFPE tumor specimens arranged in TMA format. We analyzed 46 B-cell lymphoma (B-NHL specimens with known karyotypes for translocations of IGH-, BCL2-, BCL6- and MYC-genes. Locus-specific DNA probes were used for the detection of deletions in chromosome bands 6q21 and 9p21 in 62 follicular lymphomas (FL and six malignant mesothelioma (MM samples, respectively. To test for aberrant signals generated by truncation of nuclei following sectioning of FFPE tissue samples, cell line dilutions with 9p21-deletions were embedded into paraffin blocks. The overall TMA hybridization efficiency was 94%. FISH results regarding translocations matched karyotyping data in 93%. As for chromosomal deletions, sectioning artefacts occurred in 17% to 25% of cells, suggesting that the proportion of cells showing deletions should exceed 25% to be reliably detectable. In conclusion, FISH represents a robust tool for the detection of structural as well as numerical aberrations in FFPE tissue samples in a TMA-based high-throughput setting, when rigorous cut-off values and appropriate controls are maintained, and, of note, was superior to quantitative PCR approaches.

A rapid technique for analysis of formalin-fixed, paraffin-embedded tissues by fluorescent in situ hybridization with alpha-satellite probes

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Nilce Barril

1998-12-01

Full Text Available We describe a rapid procedure for preparing archival tissues for interphase FISH analysis. The present protocol differs from others previously described because it allows the obtention of nuclei in satisfactory number and quality without using special equipments, adhesive-treated slides or solutions for chromatin decondensation. The method is of low cost and useful for retrospective analyses of formalin-fixed, paraffin-embedded samples.Descrevemos aqui um procedimento rápido para obtenção de núcleos interfásicos a partir de amostras arquivadas que podem ser utilizados para análise citogenética através da técnica de FISH. Este procedimento difere de outros previamente descritos porque permite a obtenção de núcleos em número e qualidade satisfatórios sem a utilização de equipamentos ou lâminas especiais e soluções para descondensação da cromatina. O método é de baixo custo e possibilita estudos retrospectivos de tecidos fixados em formol e emblocados em parafina.

Scores for standardization of on-tissue digestion of formalin-fixed paraffin-embedded tissue in MALDI-MS imaging.

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Erich, Katrin; Sammour, Denis A; Marx, Alexander; Hopf, Carsten

2017-07-01

On-slide digestion of formalin-fixed and paraffin-embedded human biopsy tissue followed by mass spectrometry imaging of resulting peptides may have the potential to become an additional analytical modality in future ePathology. Multiple workflows have been described for dewaxing, antigen retrieval, digestion and imaging in the past decade. However, little is known about suitable statistical scores for method comparison and systematic workflow standardization required for development of processes that would be robust enough to be compatible with clinical routine. To define scores for homogeneity of tissue processing and imaging as well as inter-day repeatability for five different processing methods, we used human liver and gastrointestinal stromal tumor tissue, both judged by an expert pathologist to be >98% histologically homogeneous. For mean spectra-based as well as pixel-wise data analysis, we propose the coefficient of determination R 2 , the natural fold-change (natFC) value and the digest efficiency DE% as readily accessible scores. Moreover, we introduce two scores derived from principal component analysis, the variance of the mean absolute deviation, MAD, and the interclass overlap, J overlap , as computational scores that may help to avoid user bias during future workflow development. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular genotyping of Echinococcus granulosus using formalin-fixed paraffin-embedded preparations from human isolates in unusual tissue sites.

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Hizem, A; M'rad, S; Oudni-M'rad, M; Mestiri, S; Hammedi, F; Mezhoud, H; Zakhama, A; Mokni, M; Babba, H

2016-07-01

Cystic echinococcosis (CE) caused by Echinococcus granulosus remains a serious problem worldwide for issues relating to public health and the economy. The most predominantly affected sites are the liver and the lungs, but other organs such as the heart, the spleen and the peritoneum can also be infected. Access to cysts from uncommon sites has limited genomic and molecular investigations. In the present study, genotypes of E. granulosus sensu lato were identified from formalin-fixed paraffin-embedded tissues (FF-PETs) implicated in human CE. Tissue samples were obtained from 57 patients with histologically confirmed CE. DNA samples were analysed using Egss 1 polymerase chain reaction (PCR) specific to the mitochondrial 12S rRNA gene of E. granulosus sensu stricto. All cysts were typed as E. granulosus sensu stricto with up to 35% of the liver and 16.6% of lungs being the most frequently infected, and up to 48.4% of samples being from rare sites. No correlation was found between cyst site and either the gender or the age of patients. This study demonstrates the possibility of exploiting atypical cysts using FF-PET samples and highlights the predominance of E. granulosus sensu stricto species in the Tunisian population, even in unusual infection sites.

Evaluation of three methods of DNA extraction from paraffin-embedded material for the amplification of genomic DNA by means of the PCR technique

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MESQUITA Ricardo Alves

2001-01-01

Full Text Available There are several protocols reported in the literature for the extraction of genomic DNA from formalin-fixed paraffin-embedded samples. Genomic DNA is utilized in molecular analyses, including PCR. This study compares three different methods for the extraction of genomic DNA from formalin-fixed paraffin-embedded (inflammatory fibrous hyperplasia and non-formalin-fixed (normal oral mucosa samples: phenol with enzymatic digestion, and silica with and without enzymatic digestion. The amplification of DNA by means of the PCR technique was carried out with primers for the exon 7 of human keratin type 14. Amplicons were analyzed by means of electrophoresis in an 8% polyacrylamide gel with 5% glycerol, followed by silver-staining visualization. The phenol/enzymatic digestion and the silica/enzymatic digestion methods provided amplicons from both tissue samples. The method described is a potential aid in the establishment of the histopathologic diagnosis and in retrospective studies with archival paraffin-embedded samples.

The influence of DNA degradation in formalin-fixed, paraffin-embedded (FFPE) tissue on locus-specific methylation assessment by MS-HRM.

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Daugaard, Iben; Kjeldsen, Tina E; Hager, Henrik; Hansen, Lise Lotte; Wojdacz, Tomasz K

2015-12-01

Readily accessible formalin-fixed paraffin embedded (FFPE) tissues are a highly valuable source of genetic material for molecular analyses in both research and in vitro diagnostics but frequently genetic material in those samples is highly degraded. With locus-specific methylation changes being widely investigated for use as biomarkers in various aspects of clinical disease management, we aimed to evaluate to what extent standard laboratory procedures can approximate the quality of the DNA extracted from FFPE samples prior to methylation analyses. DNA quality in 107 FFPE non-small cell lung cancer (NSCLC) samples was evaluated using spectrophotometry and gel electrophoresis. Subsequently, the quality assessment results were correlated with the results of locus specific methylation assessment with methylation sensitive high resolution melting (MS-HRM). The correlation of template quality with PCR amplification performance and HRM based methylation detection indicated a significant influence of DNA quality on PCR amplification but not on methylation assessment. In conclusion, standard laboratory procedures fairly well approximate DNA degradation of FFPE samples and DNA degradation does not seem to considerably affect locus-specific methylation assessment by MS-HRM. Copyright © 2015 Elsevier Inc. All rights reserved.

MicroRNA expression in melanocytic nevi: the usefulness of formalin-fixed, paraffin-embedded material for miRNA microarray profiling.

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Glud, Martin; Klausen, Mikkel; Gniadecki, Robert; Rossing, Maria; Hastrup, Nina; Nielsen, Finn C; Drzewiecki, Krzysztof T

2009-05-01

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate cellular differentiation, proliferation, and apoptosis. MiRNAs are expressed in a developmentally regulated and tissue-specific manner. Aberrant expression may contribute to pathological processes such as cancer, and miRNA may therefore serve as biomarkers that may be useful in a clinical environment for diagnosis of various diseases. Most miRNA profiling studies have used fresh tissue samples. However, in some types of cancer, including malignant melanoma, fresh material is difficult to obtain from primary tumors, and most surgical specimens are formalin fixed and paraffin embedded (FFPE). To explore whether FFPE material would be suitable for miRNA profiling in melanocytic lesions, we compared miRNA expression patterns in FFPE versus fresh frozen samples, obtained from 15 human melanocytic nevi. Out of microarray data, we identified 84 miRNAs that were expressed in both types of samples and represented an miRNA profile of melanocytic nevi. Our results showed a high correlation in miRNA expression (Spearman r-value of 0.80) between paired FFPE and fresh frozen material. The data were further validated by quantitative RT-PCR. In conclusion, FFPE specimens of melanocytic lesions are suitable as a source for miRNA microarray profiling.

High Quality Genomic Copy Number Data from Archival Formalin-Fixed Paraffin-Embedded Leiomyosarcoma: Optimisation of Universal Linkage System Labelling

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Salawu, Abdulazeez; Ul-Hassan, Aliya; Hammond, David; Fernando, Malee; Reed, Malcolm; Sisley, Karen

2012-01-01

Most soft tissue sarcomas are characterized by genetic instability and frequent genomic copy number aberrations that are not subtype-specific. Oligonucleotide microarray-based Comparative Genomic Hybridisation (array CGH) is an important technique used to map genome-wide copy number aberrations, but the traditional requirement for high-quality DNA typically obtained from fresh tissue has limited its use in sarcomas. Although large archives of Formalin-fixed Paraffin-embedded (FFPE) tumour samples are available for research, the degradative effects of formalin on DNA from these tissues has made labelling and analysis by array CGH technically challenging. The Universal Linkage System (ULS) may be used for a one-step chemical labelling of such degraded DNA. We have optimised the ULS labelling protocol to perform aCGH on archived FFPE leiomyosarcoma tissues using the 180k Agilent platform. Preservation age of samples ranged from a few months to seventeen years and the DNA showed a wide range of degradation (when visualised on agarose gels). Consistently high DNA labelling efficiency and low microarray probe-to-probe variation (as measured by the derivative log ratio spread) was seen. Comparison of paired fresh and FFPE samples from identical tumours showed good correlation of CNAs detected. Furthermore, the ability to macro-dissect FFPE samples permitted the detection of CNAs that were masked in fresh tissue. Aberrations were visually confirmed using Fluorescence in situ Hybridisation. These results suggest that archival FFPE tissue, with its relative abundance and attendant clinical data may be used for effective mapping for genomic copy number aberrations in such rare tumours as leiomyosarcoma and potentially unravel clues to tumour origins, progression and ultimately, targeted treatment. PMID:23209738

Integrated and convenient procedure for protein extraction from formalin-fixed, paraffin-embedded tissues for LC-MS/MS analysis.

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Lai, Xianyin; Schneider, Bryan P

2014-11-01

Because fresh-frozen tissue samples associated with long-term clinical data and of rare diseases are often unobtainable at the present time, formalin-fixed paraffin-embedded (FFPE) tissue samples are considered a highly valuable resource for researchers. However, protein extraction from FFPE tissues faces challenges of deparaffinization and cross-link reversion. Current procedures for protein extraction from FFPE tissue require separate steps and toxic solvents, resulting in inconvenience in protein extraction. To overcome these limitations, an integrated method was developed using nontoxic solvents in four types of FFPE tissues. The average amount of proteins from three replicates of bladder, kidney, liver, and lung FFPE tissues were 442.6, 728.9, 736.4, and 694.7 μg with CVs of 7.5, 5.8, 2.4, and 4.5%, respectively. Proteomic analysis showed that 348, 417, 607, and 304 unique proteins were identified and quantified without specification of isoform by a least two peptides from bladder, kidney, liver, and lung FFPE tissue samples, respectively. The analysis of individual protein CV demonstrated that 97-99% of the proteins were quantified with a CV ≤ 30%, verifying the reproducibility of the integrated protein extraction method. In summary, the developed method is high-yield, reproducible, convenient, simple, low cost, nonvolatile, nonflammable, and nontoxic. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A new classification method for MALDI imaging mass spectrometry data acquired on formalin-fixed paraffin-embedded tissue samples.

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Boskamp, Tobias; Lachmund, Delf; Oetjen, Janina; Cordero Hernandez, Yovany; Trede, Dennis; Maass, Peter; Casadonte, Rita; Kriegsmann, Jörg; Warth, Arne; Dienemann, Hendrik; Weichert, Wilko; Kriegsmann, Mark

2017-07-01

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) shows a high potential for applications in histopathological diagnosis, and in particular for supporting tumor typing and subtyping. The development of such applications requires the extraction of spectral fingerprints that are relevant for the given tissue and the identification of biomarkers associated with these spectral patterns. We propose a novel data analysis method based on the extraction of characteristic spectral patterns (CSPs) that allow automated generation of classification models for spectral data. Formalin-fixed paraffin embedded (FFPE) tissue samples from N=445 patients assembled on 12 tissue microarrays were analyzed. The method was applied to discriminate primary lung and pancreatic cancer, as well as adenocarcinoma and squamous cell carcinoma of the lung. A classification accuracy of 100% and 82.8%, resp., could be achieved on core level, assessed by cross-validation. The method outperformed the more conventional classification method based on the extraction of individual m/z values in the first application, while achieving a comparable accuracy in the second. LC-MS/MS peptide identification demonstrated that the spectral features present in selected CSPs correspond to peptides relevant for the respective classification. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann. Copyright © 2016 Elsevier B.V. All rights reserved.

Application of the FICTION technique for the simultaneous detection of immunophenotype and chromosomal abnormalities in routinely fixed, paraffin wax embedded bone marrow trephines

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Korać, P; Jones, M; Dominis, M; Kušec, R; Mason, D Y; Banham, A H; Ventura, R A

2005-01-01

The use of interphase fluorescence in situ hybridisation (FISH) to study cytogenetic abnormalities in routinely fixed paraffin wax embedded tissue has become commonplace over the past decade. However, very few studies have applied FISH to routinely fixed bone marrow trephines (BMTs). This may be because of the acid based decalcification methods that are commonly used during the processing of BMTs, which may adversely affect the suitability of the sample for FISH analysis. For the first time, this report describes the simultaneous application of FISH and immunofluorescent staining (the FICTION technique) to formalin fixed, EDTA decalcified and paraffin wax embedded BMTs. This technique allows the direct correlation of genetic abnormalities to immunophenotype, and therefore will be particularly useful for the identification of genetic abnormalities in specific tumour cells present in BMTs. The application of this to routine clinical practice will assist diagnosis and the detection of minimal residual disease. PMID:16311361

Utility of the Roche Cobas 4800 for detection of high-risk human papillomavirus in formalin-fixed paraffin-embedded oropharyngeal squamous cell carcinoma.

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Pettus, Jason R; Wilson, Terri L; Steinmetz, Heather B; Lefferts, Joel A; Tafe, Laura J

2017-02-01

Clinical laboratories are expected to reliably identify human papilloma virus (HPV) associated oropharyngeal squamous cell carcinoma (OPSCC) for prognostic and potential therapeutic applications. In addition to surrogate p16 immunohistochemistry (IHC) testing, DNA-based HPV-specific testing strategies are widely utilized. Recognizing the efficiency of the Roche Cobas 4800 platform for testing gynecological cytology specimens for high-risk HPV, we elected to evaluate the potential utility of this platform for testing formalin-fixed paraffin-embedded (FFPE) OPSCC tissue. Using the Roche Linear Array assay for comparison, we tested twenty-eight samples (16 primary OPSCC, 2 lymph node metastases from primary OPSCC, 1 oral tongue carcinoma, 3 benign squamous papillomas, and 3 non-oropharyngeal carcinoma tissues). Excluding two invalid results, the Roche Cobas 4800 testing resulted in excellent inter-assay concordance (25/26, 96.2%) and 100% concordance for HPV-16/HPV-18 positive samples. This data suggests that the Roche Cobas 4800 platform may be a cost-effective method for testing OPSCC FFPE tissues in a clinical molecular pathology laboratory setting. Copyright © 2016 Elsevier Inc. All rights reserved.

Bisulfite-Based DNA Methylation Analysis from Recent and Archived Formalin-Fixed, Paraffin Embedded Colorectal Tissue Samples.

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Kalmár, Alexandra; Péterfia, Bálint; Hollósi, Péter; Wichmann, Barnabás; Bodor, András; Patai, Árpád V; Schöller, Andrea; Krenács, Tibor; Tulassay, Zsolt; Molnár, Béla

2015-09-01

We aimed to test the applicability of formalin-fixed and paraffin-embedded (FFPE) tissue samples for gene specific DNA methylation analysis after using two commercially available DNA isolation kits. Genomic DNA was isolated from 5 colorectal adenocarcinomas and 5 normal adjacent tissues from "recent", collected within 6 months, and "archived", collected more than 5 years ago, FFPE tissues using either High Pure FFPET DNA Isolation kit or QIAamp DNA FFPE Tissue kit. DNA methylation analysis of MAL, SFRP1 and SFRP2 genes, known to be hypermethylated in CRC, was performed using methylation-sensitive high resolution melting (MS-HRM) analysis and sequencing. QIAamp (Q) method resulted in slightly higher recovery in archived (HP: 1.22 ± 3.18 μg DNA; Q: 3.00 ± 4.04 μg DNA) and significantly (p < 0.05) higher recovery in recent samples compared to High Pure method (HP) (HP: 4.10 ± 2.91 μg DNA; Q: 11.51 ± 7.50 μg DNA). Both OD260/280 and OD260/230 ratios were lower, but still high in the High Pure isolated archived and recent samples compared to those isolated with QIAamp. Identical DNA methylation patterns were detected for all 3 genes tested by MS-HRM with both isolation kits in the recent group. However, despite of higher DNA recovery in QIAamp slightly more reproducible methylation results were obtained from High Pure isolated archived samples. Sequencing confirmed DNA hypermethylation in CRCs. In conclusion, reproducible DNA methylation patterns were obtained from recent samples using both isolation kits. However, long term storage may affect the reliability of the results leading to moderate differences between the efficiency of isolation kits.

[Use of archival formalin-fixed, paraffin-embedded (FFPE) tissue samples for molecular genetic analysis in diffuse large B-cell lymphoma (DLBCL)].

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Jarošová, Marie; Kučerová, Jana; Flodr, Patrik; Mikešová, Michaela; Procházka, Vít; Papajík, Tomáš

2014-04-01

The currently valid molecular genetic subclassification of patients with diffuse large B-cell lymphoma (DLBCL) into three prognostic subgroups based on expression profiling has been the objective of numerous genetic studies. In routine clinical practice, however, expression profiling technology remains unavailable for the most of centers. Apart from the technology, in some cases molecular genetic laboratories have problems obtaining high-quality material, i.e. fresh tissues, for RNA isolation to determine gene expression. One possibility is to determine the gene expression from RNA obtained by isolation from formalin-fixed, paraffin-embedded (FFPE) tissue. This pilot study aimed at isolating RNA from FFPE in patients diagnosed with DLBCL and verifying the potential use of such RNA for the expression analysis of 7 selected genes. Although the study showed that it is possible to isolate RNA and determine the expression of the selected genes from archival material, the values of relative expression of some genes in the set were too variable to be used for unambiguous prognostic classification. It was confirmed that retrospective analyses of selected genes may be performed with sufficient material obtained, and that properly archived blocks may be used for molecular biology analyses even after 8 years.

KLC1-ALK: a novel fusion in lung cancer identified using a formalin-fixed paraffin-embedded tissue only.

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Yuki Togashi

Full Text Available The promising results of anaplastic lymphoma kinase (ALK inhibitors have changed the significance of ALK fusions in several types of cancer. These fusions are no longer mere research targets or diagnostic markers, but they are now directly linked to the therapeutic benefit of patients. However, most available tumor tissues in clinical settings are formalin-fixed and paraffin-embedded (FFPE, and this significantly limits detailed genetic studies in many clinical cases. Although recent technical improvements have allowed the analysis of some known mutations in FFPE tissues, identifying unknown fusion genes by using only FFPE tissues remains difficult. We developed a 5'-rapid amplification of cDNA ends-based system optimized for FFPE tissues and evaluated this system on a lung cancer tissue with ALK rearrangement and without the 2 known ALK fusions EML4-ALK and KIF5B-ALK. With this system, we successfully identified a novel ALK fusion, KLC1-ALK. The result was confirmed by reverse transcription-polymerase chain reaction and fluorescence in situ hybridization. Then, we synthesized the putative full-length cDNA of KLC1-ALK and demonstrated the transforming potential of the fusion kinase with assays using mouse 3T3 cells. To the best of our knowledge, KLC1-ALK is the first novel oncogenic fusion identified using only FFPE tissues. This finding will broaden the potential value of archival FFPE tissues and provide further biological and clinical insights into ALK-positive lung cancer.

Clinical Usefulness of a One-Tube Nested Reverse Transcription Quantitative Polymerase Chain Reaction Assay for Evaluating Human Epidermal Growth Factor Receptor 2 mRNA Overexpression in Formalin-Fixed and Paraffin-Embedded Breast Cancer Tissue Samples.

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Wang, Hye-Young; Ahn, Sungwoo; Park, Sunyoung; Kim, SeungIl; Lee, Hyeyoung

2017-01-01

Currently, the two main methods used to analyze human epidermal growth factor receptor 2 (HER2) amplification or overexpression have a limited accuracy and high costs. These limitations can be overcome by the development of complementary quantitative methods. In this study, we analyzed HER2 mRNA expression in clinical formalin-fixed and paraffin-embedded (FFPE) samples using a one-tube nested reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay. We measured expression relative to 3 reference genes and compared the results to those obtained by conventional immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) assays with 226 FFPE breast cancer tissue samples. The one-tube nested RT-qPCR assay proved to be highly sensitive and specific based on comparisons with IHC (96.9 and 97.7%, respectively) and FISH (92.4 and 92.9%, respectively) obtained with the validation set. Comparisons with clinicopathological data revealed significant associations between HER2 overexpression and TNM stage (p < 0.01), histological type (p < 0.01), ER status (p < 0.001), PR status (p < 0.05), HER2 status (p < 0.001), and molecular subtypes (p < 0.001). Based on these findings, our one-tube nested RT-qPCR assay is a potentially useful and complementary screening tool for the detection of HER2 mRNA overexpression. © 2016 S. Karger AG, Basel.

A Comparison of Fresh Frozen vs. Formalin-Fixed, Paraffin-Embedded Specimens of Canine Mammary Tumors via Branched-DNA Assay

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Florenza Lüder Ripoli

2016-05-01

Full Text Available Mammary neoplasms are the tumors most affecting female dogs and women. Formalin-fixed, paraffin-embedded (FFPE tissues are an invaluable source of archived biological material. Fresh frozen (FF tissue is considered ideal for gene expression analysis. However, strategies based on FFPE material offer several advantages. Branched-DNA assays permit a reliable and fast workflow when analyzing gene expression. The aim of this study was to assess the comparability of the branched-DNA assay when analyzing certain gene expression patterns between FF and FFPE samples in canine mammary tumors. RNA was isolated from 109 FFPE samples and from 93 FF samples of different canine mammary tissues. Sixteen (16 target genes (Tp53; Myc; HMGA1; Pik3ca; Mcl1; MAPK3; FOXO3; PTEN; GATA4; PFDN5; HMGB1; MAPK1; BRCA2; BRCA1; HMGA2; and Her2 were analyzed via branched-DNA assay (b-DNA. ACTB, GAPDH, and HPRT1 were used as data normalizers. Overall, the relative gene expression of the two different origins of samples showed an agreement of 63%. Still, care should be taken, as FFPE specimens showed lower expression of the analyzed targets when compared to FF samples. The fact that the gene expression in FFPE proved to be lower than in FF specimens is likely to have been caused by the effect of storage time. ACTB had the best performance as a data normalizer.

Evaluation of two methods DNA extraction from formalin-fixed, paraffin-embedded tissues on non-optimal conditions

International Nuclear Information System (INIS)

Bustamante, Javier Andres; Astudillo, Miryam; Pazos, Alvaro Jairo; Bravo, Luis Eduardo

2011-01-01

Paraffin wax embedded tissues are an invaluable material for retrospective studies requiring the application of molecular analysis. Multiple methods are available to extract DNA from these kinds of samples. However, the most common methods are slow and the reagents often contribute to the fragmentation of genetic material. In order to optimize the procedure, two methods for DNA extraction from paraffin embedded tissue non-optimal conditions were used. 47 blocks containing paraffin-embedded biopsies of pleura, lung and pericardium from 24 patients (66.6% males) older than 18 years, with biopsy proven chronic granulomatous inflammation referred to the department of pathology at University Hospital of Valle between 2002 and 2007 were selected. Each sample was subjected to 10 cuts and was to two methods of DNA extraction: 1. conventional and 2. QIAamp - DNA mini kit. The efficiency of the extracted DNA was assessed by spectrophotometry and PCR amplification of a fragment of the housekeeping gene GAPDH. The concentration of DNA samples extracted by the conventional method was of 65.52 ng/Mu l ± 11.47 (mean ± SE) and the 260/280 absorbance ratio ranged between 0.52 and 2.30 the average concentration of DNA of the samples extracted by the commercial method was 60.89 ng/Mu l ± 6.02 (mean ± SE), with an absorbance that fluctuated between 0 and 2.64. The DNA obtained was amplified by PCR, of 47 samples extracted by methods, 25 and 23 respectively the GAPDH gene amplified successfully. The methods used to obtain DNA showed similar performance, highlighting the potential utility of both extraction methods for the retrospective studies from paraffin embedded tissues in unsuitable conditions.

RNA analysis of inner ear cells from formalin fixed paraffin embedded (FFPE) archival human temporal bone section using laser microdissection--a technical report.

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Kimura, Yurika; Kubo, Sachiho; Koda, Hiroko; Shigemoto, Kazuhiro; Sawabe, Motoji; Kitamura, Ken

2013-08-01

Molecular analysis using archival human inner ear specimens is challenging because of the anatomical complexity, long-term fixation, and decalcification. However, this method may provide great benefit for elucidation of otological diseases. Here, we extracted mRNA for RT-PCR from tissues dissected from archival FFPE human inner ears by laser microdissection. Three human temporal bones obtained at autopsy were fixed in formalin, decalcified by EDTA, and embedded in paraffin. The samples were isolated into spiral ligaments, outer hair cells, spiral ganglion cells, and stria vascularis by laser microdissection. RNA was extracted and heat-treated in 10 mM citrate buffer to remove the formalin-derived modification. To identify the sites where COCH and SLC26A5 mRNA were expressed, semi-nested RT-PCR was performed. We also examined how long COCH mRNA could be amplified by semi-nested RT-PCR in archival temporal bone. COCH was expressed in the spiral ligament and stria vascularis. However, SLC26A5 was expressed only in outer hair cells. The maximum base length of COCH mRNA amplified by RT-PCR was 98 bp in 1 case and 123 bp in 2 cases. We detected COCH and SLC26A5 mRNA in specific structures and cells of the inner ear from archival human temporal bone. Our innovative method using laser microdissection and semi-nested RT-PCR should advance future RNA study of human inner ear diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

Trypanosoma cruzi Necrotizing Meningoencephalitis in a Venezuelan HIV+-AIDS Patient: Pathological Diagnosis Confirmed by PCR Using Formalin-Fixed- and Paraffin-Embedded-Tissues

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Marcello Salvatore Rossi Spadafora

2014-01-01

Full Text Available Coinfections with human immunodeficiency virus (HIV and infectious agents have been recognized since the early 90s. In the central nervous system (CNS of HIV+ patients, parasitic protozoans like Toxoplasma gondii have been described as responsible for the space occupying lesions (SOL developed. However, the involvement of Trypanosoma cruzi is also described but appears to be less frequent in acquired immunodeficiency syndrome (AIDS and transplant recipients, associated with necrotizing myocarditis and neurological symptoms related to the occurrence of necrotizing pseudotumoral encephalitis (NPE and meningoencephalitis (NME. The present work aims to present a Venezuelan case of NME associated with the coinfection of HIV and a T. cruzi-like trypanosomatid as well as its evolution and diagnosis by histopathological techniques, electron microscopy, and PCR methods using formalin-fixed- (FF- and paraffin-embedded- (PE- tissues. Postmortem cytological studies of leptomeninges imprints reveal the presence of trypomastigotes of Trypanosoma sp. Histopathological and electron microscopy studies allowed us to identify an amastigote stage and to reject the involvement of other opportunistic microorganisms as the etiological agent of the SOL. The definitive confirmation of T. cruzi as the etiological agent was achieved by PCR suggesting that the NME by T. cruzi was due to a reactivation of Chagas’ disease.

Trypanosoma cruzi Necrotizing Meningoencephalitis in a Venezuelan HIV+-AIDS Patient: Pathological Diagnosis Confirmed by PCR Using Formalin-Fixed- and Paraffin-Embedded-Tissues

Science.gov (United States)

Rossi Spadafora, Marcello Salvatore; Céspedes, Ghislaine; Romero, Sandra; Fuentes, Isabel; Boada-Sucre, Alpidio A.; Cañavate, Carmen; Flores-Chávez, María

2014-01-01

Coinfections with human immunodeficiency virus (HIV) and infectious agents have been recognized since the early 90s. In the central nervous system (CNS) of HIV+ patients, parasitic protozoans like Toxoplasma gondii have been described as responsible for the space occupying lesions (SOL) developed. However, the involvement of Trypanosoma cruzi is also described but appears to be less frequent in acquired immunodeficiency syndrome (AIDS) and transplant recipients, associated with necrotizing myocarditis and neurological symptoms related to the occurrence of necrotizing pseudotumoral encephalitis (NPE) and meningoencephalitis (NME). The present work aims to present a Venezuelan case of NME associated with the coinfection of HIV and a T. cruzi-like trypanosomatid as well as its evolution and diagnosis by histopathological techniques, electron microscopy, and PCR methods using formalin-fixed- (FF-) and paraffin-embedded- (PE-) tissues. Postmortem cytological studies of leptomeninges imprints reveal the presence of trypomastigotes of Trypanosoma sp. Histopathological and electron microscopy studies allowed us to identify an amastigote stage and to reject the involvement of other opportunistic microorganisms as the etiological agent of the SOL. The definitive confirmation of T. cruzi as the etiological agent was achieved by PCR suggesting that the NME by T. cruzi was due to a reactivation of Chagas' disease. PMID:25763312

[Histopathological Diagnosis of Invasive Fungal Infections in Formalin-Fixed and Paraffin-Embedded Tissues in Conjunction with Molecular Methods].

Science.gov (United States)

Shinozaki, Minoru; Tochigi, Naobumi; Sadamoto, Sota; Yamagata Murayama, Somay; Wakayama, Megumi; Nemoto, Tetsuo

2018-01-01

The main objective of this study was to evaluate the relationship between histopathology, polymerase chain reaction (PCR), and in situ hybridization (ISH) for the identification of causative fungi in formalin-fixed and paraffin-embedded (FFPE) tissue specimens. Since pathogenic fungi in tissue specimens can be difficult to identify morphologically, PCR and ISH have been usually employed as auxiliary procedures. However, little comparison has been made on the sensitivity and specificity of PCR and ISH using FFPE specimens. Therefore, to compare and clarify the reproducibility and usefulness of PCR and ISH as auxiliary procedures for histological identification, we performed histopathological review, PCR assays, and ISH to identify pathogenic fungi in 59 FFPE tissue specimens obtained from 49 autopsies. The following are the main findings for this retrospective review: i) even for cases classified as "mold not otherwise specified" (MNOS), two cases could be identified as Aspergillus species by molecular methods; ii) all cases classified as non-zygomycetes mold (NZM) were Aspergillus species and were not identified by molecular methods as other fungi; iii) all 3 cases classified as zygomycetes mold (ZM) could be identified by molecular methods as Mucorales; iv) except for 1 case identified by molecular methods as Trichosporon spp., 5 cases were originally identified as dimorphic yeast (DY). As a measure of nucleic acid integrity, PCR and ISH successfully detected human and fungal nucleic acids in approximately 60% of the specimens. Detection of Aspergillus DNA by nested PCR assay and by ISH against the A. fumigatus ALP gene were similarly sensitive and significant (pmolecular methods such as ISH and PCR on FFPE specimens with pathological diagnosis should improve diagnostic accuracy of fungal infection.

Improved method for extraction and detection of Helicobacter pylori DNA in formalin-fixed paraffin embedded gastric biopsies using laser micro-dissection.

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Loayza, María Fernanda; Villavicencio, Fernando Xavier; Santander, Stephanie Carolina; Baldeón, Manuel; Ponce, Lourdes Karina; Salvador, Iván; Vivar Díaz, Nicolás

2015-01-01

To assess the molecular events exerted by Helicobacter pylori interacting directly with gastric epithelial cells, an improved procedure for microbial DNA isolation from stained hematoxilin-eosin gastric biopsies was developed based on laser micro-dissection (LM) [1]. Few articles have described the use of LM to select and detect H. pylori genome from formalin-fixed paraffin embedded gastric tissue [2]. To improve the yield and quality of DNA isolated from H. pylori contacting intestinal epithelial cells, the following conditions were established after modification of the QIAamp DNA Micro kit. •Use of at least 25 cut sections of 10-20 μm of diameter and 3 μm thick with more than 10 bacteria in each cut.•Lysis with 30 μL of tissue lysis buffer and 20 μL of proteinase K (PK) with the tube in an upside-down position.•The use of thin purification columns with 35 μL of elution buffer. The mean of DNA concentration obtained from 25 LM cut sections was 1.94± 0 .16 ng/μL, and it was efficiently amplified with qPCR in a Bio Rad iCycler instrument. The LM can improve the sample selection and DNA extraction for molecular analysis of H. pylori associated with human gastric epithelium.

Multiplexed color-coded probe-based gene expression assessment for clinical molecular diagnostics in formalin-fixed paraffin-embedded human renal allograft tissue.

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Adam, Benjamin; Afzali, Bahman; Dominy, Katherine M; Chapman, Erin; Gill, Reeda; Hidalgo, Luis G; Roufosse, Candice; Sis, Banu; Mengel, Michael

2016-03-01

Histopathologic diagnoses in transplantation can be improved with molecular testing. Preferably, molecular diagnostics should fit into standard-of-care workflows for transplant biopsies, that is, formalin-fixed paraffin-embedded (FFPE) processing. The NanoString(®) gene expression platform has recently been shown to work with FFPE samples. We aimed to evaluate its methodological robustness and feasibility for gene expression studies in human FFPE renal allograft samples. A literature-derived antibody-mediated rejection (ABMR) 34-gene set, comprised of endothelial, NK cell, and inflammation transcripts, was analyzed in different retrospective biopsy cohorts and showed potential to molecularly discriminate ABMR cases, including FFPE samples. NanoString(®) results were reproducible across a range of RNA input quantities (r = 0.998), with different operators (r = 0.998), and between different reagent lots (r = 0.983). There was moderate correlation between NanoString(®) with FFPE tissue and quantitative reverse transcription polymerase chain reaction (qRT-PCR) with corresponding dedicated fresh-stabilized tissue (r = 0.487). Better overall correlation with histology was observed with NanoString(®) (r = 0.354) than with qRT-PCR (r = 0.146). Our results demonstrate the feasibility of multiplexed gene expression quantification from FFPE renal allograft tissue. This represents a method for prospective and retrospective validation of molecular diagnostics and its adoption in clinical transplantation pathology. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Amplification of chromosomal translocation junctions from paraffin-embedded tissues of follicular lymphoma patients

International Nuclear Information System (INIS)

Nambiar, Mridula; Raghavan, Sathees C; Choudhary, Bibha; Rao, Clementina R

2008-01-01

Follicular lymphoma is associated with the t(14;18) translocation, which is one of the most common chromosomal translocations in cancer. Generally, tissues from such patients are preserved as formalin-fixed and paraffin-embedded samples. Most of the time, retrieving the molecular information from such samples is hampered due to quality of preservation, extraction procedures and reaction conditions. In the present study, we isolate the chromosomal DNA from the paraffin-embedded nodal tissues of lymphoma patients and use a highly sensitive nested PCR approach to detect t(14;18) translocation. Our studies show that despite the sheared DNA obtained, appropriate modification of PCR reaction conditions can help in obtaining the desired amplifications. The DNA extraction protocol from paraffin-embedded nodal tissues and modifications in the PCR conditions are discussed. This study would contribute to the successful use of archival tissue samples in obtaining valuable information for cancer research

Amplification of chromosomal translocation junctions from paraffin-embedded tissues of follicular lymphoma patients

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Nambiar, Mridula; Raghavan, Sathees C [Department of Biochemistry, Indian Institute of Science, Bangalore-560 012 (India); Choudhary, Bibha [Manipal Institute of Regenerative Medicine, Manipal University, Bangalore-560 071 (India); Rao, Clementina R [Department of Pathology, Kidwai Memorial Institute of Oncology, Bangalore-560 029 (India)], E-mail: sathees@biochem.iisc.ernet.in

2008-09-01

Follicular lymphoma is associated with the t(14;18) translocation, which is one of the most common chromosomal translocations in cancer. Generally, tissues from such patients are preserved as formalin-fixed and paraffin-embedded samples. Most of the time, retrieving the molecular information from such samples is hampered due to quality of preservation, extraction procedures and reaction conditions. In the present study, we isolate the chromosomal DNA from the paraffin-embedded nodal tissues of lymphoma patients and use a highly sensitive nested PCR approach to detect t(14;18) translocation. Our studies show that despite the sheared DNA obtained, appropriate modification of PCR reaction conditions can help in obtaining the desired amplifications. The DNA extraction protocol from paraffin-embedded nodal tissues and modifications in the PCR conditions are discussed. This study would contribute to the successful use of archival tissue samples in obtaining valuable information for cancer research.

Gelatin in situ zymography on fixed, paraffin-embedded tissue: zinc and ethanol fixation preserve enzyme activity.

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Hadler-Olsen, Elin; Kanapathippillai, Premasany; Berg, Eli; Svineng, Gunbjørg; Winberg, Jan-Olof; Uhlin-Hansen, Lars

2010-01-01

In situ zymography is a method for the detection and localization of enzymatic activity in tissue sections. This method is used with frozen sections because routine fixation of tissue in neutral-buffered formalin inhibits enzyme activity. However, frozen sections present with poor tissue morphology, making precise localization of enzymatic activity difficult to determine. Ethanol- and zinc-buffered fixative (ZBF) are known to preserve both morphological and functional properties of the tissue well, but it has not previously been shown that these fixatives preserve enzyme activity. In the present study, we show that in situ zymography can be performed on ethanol- and ZBF-fixed paraffin-embedded tissue. Compared with snap-frozen tissue, ethanol- and ZBF-fixed tissue showed stronger signals and superior morphology, allowing for a much more precise detection of gelatinolytic activity. Gelatinolytic enzymes could also be extracted from both ethanol- and ZBF-fixed tissue. The yield, as analyzed by SDS-PAGE gelatin zymography and Western blotting, was influenced by the composition of the extraction buffer, but was generally lower than that obtained from unfixed tissue.

The Genomic Grade Assay Compared With Ki67 to Determine Risk of Distant Breast Cancer Recurrence

DEFF Research Database (Denmark)

Ignatiadis, Michail; Azim, Hatem A; Desmedt, Christine

2016-01-01

Importance: The Genomic Grade Index (GGI) was previously developed, evaluated on frozen tissue, and shown to be prognostic in early breast cancer. To test the GGI in formalin-fixed, paraffin-embedded breast cancer tumors, a quantitative reverse transcriptase polymerase chain reaction assay...

Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: comparative evaluation with immunohistochemical findings and histological features

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Paula R. Almeida

2012-08-01

Full Text Available The diagnosis of Mycoplasma hyopneumoniae infection is often performed through histopathology, immunohistochemistry (IHC and polymerase chain reaction (PCR or a combination of these techniques. PCR can be performed on samples using several conservation methods, including swabs, frozen tissue or formalin-fixed and paraffin-embedded (FFPE tissue. However, the formalin fixation process often inhibits DNA amplification. To evaluate whether M. hyopneumoniae DNA could be recovered from FFPE tissues, 15 lungs with cranioventral consolidation lesions were collected in a slaughterhouse from swine bred in herds with respiratory disease. Bronchial swabs and fresh lung tissue were collected, and a fragment of the corresponding lung section was placed in neutral buffered formalin for 48 hours. A PCR assay was performed to compare FFPE tissue samples with samples that were only refrigerated (bronchial swabs or frozen (tissue pieces. M. hyopneumoniae was detected by PCR in all 15 samples of the swab and frozen tissue, while it was detected in only 11 of the 15 FFPE samples. Histological features of M. hyopneumoniae infection were presented in 11 cases and 7 of these samples stained positive in IHC. Concordance between the histological features and detection results was observed in 13 of the FFPE tissue samples. PCR was the most sensitive technique. Comparison of different sample conservation methods indicated that it is possible to detect M. hyopneumoniae from FFPE tissue. It is important to conduct further research using archived material because the efficiency of PCR could be compromised under these conditions.

RT-PCR amplification of RNA extracted from formalin-fixed, paraffin-embedded oral cancer sections: analysis of p53 pathway.

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Tachibana, Masatsugu; Shinagawa, Yasuhiro; Kawamata, Hitoshi; Omotehara, Fumie; Horiuchi, Hideki; Ohkura, Yasuo; Kubota, Keiichi; Imai, Yutaka; Fujibayashi, Takashi; Fujimori, Takahiro

2003-01-01

We present a new approach towards the detection of the mRNAs in formalin-fixed, paraffin-embedded samples using a reverse transcriptase (RT)-polymerase chain reaction (PCR). The total RNAs were extracted from 10-micron-thick sections and were reverse-transcribed, then the RT-products were subjected to PCR amplification of GAPDH mRNA for screening the mRNA degradation. Next, nested PCR was performed for examining the expression of p53-related genes, p21WAF1, MDM2, p33ING1 and p14ARF. GAPDH mRNA expression was detectable in 12 out of 21 oral squamous cell carcinoma (SCC) samples. p21WAF1 mRNA expression was detectable in 5 out of 12 SCC samples, MDM2 mRNA expression was detectable in 5 our of 12 SCC samples and p33ING1 mRNA expression was detectable in 6 out of 12 SCC samples. However, the expression of p14ARF mRNA was not detectable in any of the samples. Seven out of 12 oral SCC samples showed abnormal nuclear accumulation of p53 protein by immunohistochemical staining, whereas 5 out of 12 oral SCCs showed negative staining for p53 protein. Of of p33ING1 mRNA. One of these was a verrucous carcinoma in which the p53 gene products might be inactivated by the oncoprotein E6 of human papilloma virus. Thus, the p53 tumor suppressor pathway was disrupted in most oral SCCs at the cellular levels, due to either an abnormality in p53 itself or loss of expression of p53 regulatory factors. This method would assist in making diagnosis, determining therapeutic strategy and predicting the prognosis of various cancers including oral SCCs.

Pre-Analytical Considerations for Successful Next-Generation Sequencing (NGS: Challenges and Opportunities for Formalin-Fixed and Paraffin-Embedded Tumor Tissue (FFPE Samples

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Gladys Arreaza

2016-09-01

Full Text Available In cancer drug discovery, it is important to investigate the genetic determinants of response or resistance to cancer therapy as well as factors that contribute to adverse events in the course of clinical trials. Despite the emergence of new technologies and the ability to measure more diverse analytes (e.g., circulating tumor cell (CTC, circulating tumor DNA (ctDNA, etc., tumor tissue is still the most common and reliable source for biomarker investigation. Because of its worldwide use and ability to preserve samples for many decades at ambient temperature, formalin-fixed, paraffin-embedded tumor tissue (FFPE is likely to be the preferred choice for tissue preservation in clinical practice for the foreseeable future. Multiple analyses are routinely performed on the same FFPE samples (such as Immunohistochemistry (IHC, in situ hybridization, RNAseq, DNAseq, TILseq, Methyl-Seq, etc.. Thus, specimen prioritization and optimization of the isolation of analytes is critical to ensure successful completion of each assay. FFPE is notorious for producing suboptimal DNA quality and low DNA yield. However, commercial vendors tend to request higher DNA sample mass than what is actually required for downstream assays, which restricts the breadth of biomarker work that can be performed. We evaluated multiple genomics service laboratories to assess the current state of NGS pre-analytical processing of FFPE. Significant differences in pre-analytical capabilities were observed. Key aspects are highlighted and recommendations are made to improve the current practice in translational research.

Evaluation of positive Rift Valley fever virus formalin-fixed paraffin embedded samples as a source of sequence data for retrospective phylogenetic analysis.

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Mubemba, B; Thompson, P N; Odendaal, L; Coetzee, P; Venter, E H

2017-05-01

Rift Valley fever (RVF), caused by an arthropod borne Phlebovirus in the family Bunyaviridae, is a haemorrhagic disease that affects ruminants and humans. Due to the zoonotic nature of the virus, a biosafety level 3 laboratory is required for isolation of the virus. Fresh and frozen samples are the preferred sample type for isolation and acquisition of sequence data. However, these samples are scarce in addition to posing a health risk to laboratory personnel. Archived formalin-fixed, paraffin-embedded (FFPE) tissue samples are safe and readily available, however FFPE derived RNA is in most cases degraded and cross-linked in peptide bonds and it is unknown whether the sample type would be suitable as reference material for retrospective phylogenetic studies. A RT-PCR assay targeting a 490 nt portion of the structural G N glycoprotein encoding gene of the RVFV M-segment was applied to total RNA extracted from archived RVFV positive FFPE samples. Several attempts to obtain target amplicons were unsuccessful. FFPE samples were then analysed using next generation sequencing (NGS), i.e. Truseq ® (Illumina) and sequenced on the Miseq ® genome analyser (Illumina). Using reference mapping, gapped virus sequence data of varying degrees of shallow depth was aligned to a reference sequence. However, the NGS did not yield long enough contigs that consistently covered the same genome regions in all samples to allow phylogenetic analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Technical reproducibility of single-nucleotide and size-based DNA biomarker assessment using DNA extracted from formalin-fixed, paraffin-embedded tissues.

Science.gov (United States)

Zhang, Shenli; Tan, Iain B; Sapari, Nur S; Grabsch, Heike I; Okines, Alicia; Smyth, Elizabeth C; Aoyama, Toru; Hewitt, Lindsay C; Inam, Imran; Bottomley, Dan; Nankivell, Matthew; Stenning, Sally P; Cunningham, David; Wotherspoon, Andrew; Tsuburaya, Akira; Yoshikawa, Takaki; Soong, Richie; Tan, Patrick

2015-05-01

DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymorphisms using DNA extracted from FFPE material. By using the MassARRAY iPLEX system, we investigated polymorphisms in DPYD (rs1801159 and rs3918290), UMPS (rs1801019), ERCC1 (rs11615), ERCC1 (rs3212986), and ERCC2 (rs13181) in 56 FFPE DNA samples. By using PCR, followed by size-based gel electrophoresis, we also examined TYMS 5' untranslated region 2R/3R repeats and GSTT1 deletions in 50 FFPE DNA samples and 34 DNAs extracted from fresh-frozen tissues and cell lines. Each polymorphism was analyzed by two independent runs. We found that iPLEX biomarker assays measuring single-nucleotide polymorphisms provided consistent concordant results. However, by using FFPE DNA, size-based PCR biomarkers (GSTT1 and TYMS 5' untranslated region) were discrepant in 32.7% (16/49, with exact 95% CI, 19.9%-47.5%; exact binomial confidence limit test) and 4.2% (2/48, with exact 95% CI, 0.5%-14.3%) of cases, respectively, whereas no discrepancies were observed using intact genomic DNA. Our findings suggest that DNA from FFPE material can be used to reliably test single-nucleotide polymorphisms. However, results based on size-based PCR biomarkers, and particularly GSTT1 deletions, using FFPE DNA need to be interpreted with caution. Independent repeated assays should be performed on all cases to assess potential discrepancies. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Feasibility of using tissue microarray cores of paraffin-embedded breast cancer tissue for measurement of gene expression: a proof-of-concept study.

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Drury, Suzanne; Salter, Janine; Baehner, Frederick L; Shak, Steven; Dowsett, Mitch

2010-06-01

To determine whether 0.6 mm cores of formalin-fixed paraffin-embedded (FFPE) tissue, as commonly used to construct immunohistochemical tissue microarrays, may be a valid alternative to tissue sections as source material for quantitative real-time PCR-based transcriptional profiling of breast cancer. Four matched 0.6 mm cores of invasive breast tumour and two 10 microm whole sections were taken from eight FFPE blocks. RNA was extracted and reverse transcribed, and TaqMan assays were performed on the 21 genes of the Oncotype DX Breast Cancer assay. Expression of the 16 recurrence-related genes was normalised to the set of five reference genes, and the recurrence score (RS) was calculated. RNA yield was lower from 0.6 mm cores than from 10 microm whole sections, but was still more than sufficient to perform the assay. RS and single gene data from cores were highly comparable with those from whole sections (RS p=0.005). Greater variability was seen between cores than between sections. FFPE sections are preferable to 0.6 mm cores for RNA profiling in order to maximise RNA yield and to allow for standard histopathological assessment. However, 0.6 mm cores are sufficient and would be appropriate to use for large cohort studies.

Enhancement of Pathologist's Routine Practice: Reuse of DNA Extracted from Immunostained Formalin-fixed Paraffin-embedded (FFPE) Slides in Downstream Molecular Analysis of Cancer.

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Al-Attas, Asmaa; Assidi, Mourad; Al-Maghrabi, Jaudah; Dallol, Ashraf; Schulten, Hans-Juergen; Abu-Elmagd, Muhammad; Chaudhary, Adeel; Abuzenadah, Adel; Budowle, Bruce; Buhmeida, Abdelbaset; Al-Qahtani, Mohammed

To date, the conventional formalin-fixed, paraffin-embedded (FFPE) technique is the gold-standard for preserving histomorphology. Once FFPE tissues are stained, slides are routinely archived along with their blocks at biobanks/hospitals. However, the reuse of fixed and stained biospecimens as DNA source is not a common routine practice worldwide and, thus, indicates the need of studies to investigate the feasibility of extracting DNA from already immunohistochemistry (IHC) FFPE-stained slides and its possible reuse in subsequent downstream molecular analyses. FFPE IHC slides from colorectal cancer (CRC) patients were prepared and stored in the CEGMR Biobank. The workflow consists of digitalization of IHC stained slide's image, removing the slide cover-slip, crude dissection and DNA extraction. Following DNA quality assessment, mutation analysis of CTNNB1 and methylation profile of CDH1 were performed. High-quality DNA was obtained allowing 60% concordance between CDH1 methylation and membranous E-cadherin expression pattern. Clean CTNNB1 DNA chromatograms with evenly-spaced peaks were observed. This study is a proof of concept to recycle and reuse DNA from IHC stained slides with suitable concentration and integrity for further downstream molecular applications. These findings will enhance the pathologists' knowledge, attitudes and practices (KAP) towards the use of these biospecimens and support the implementation of this approach in clinical pathology practice. Therefore, the scientific community will benefit from the largest comprehensive database of human fully annotated FFPE biospecimens already available at their disposal in order to demystify the complexity and the heterogeneity of many challenging diseases and foster the transition towards precision medicine. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

Overexpression of p53 in Nigerian breast cancers and its ...

African Journals Online (AJOL)

This study sought to determine the expression of p53 protein as well as the relationship with oestrogen receptor (ER) and progesterone receptor (PR) proteins. Methodology: Formalin-fixed, paraffin-embedded tissue samples of diagnosed invasive breast cancer were obtained from the Department of Anatomic and ...

A simple and cost-effective method of DNA extraction from small formalin-fixed paraffin-embedded tissue for molecular oncologic testing.

Science.gov (United States)

Snow, Anthony N; Stence, Aaron A; Pruessner, Jonathan A; Bossler, Aaron D; Ma, Deqin

2014-01-01

Extraction of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is a critical step in molecular oncologic testing. As molecular oncology testing becomes more important for prognostic and therapeutic decision making and tissue specimens become smaller due to earlier detection of suspicious lesions and the use of fine needle aspiration methods for tissue collection, it becomes more challenging for the typical molecular pathology laboratory to obtain reliable test results. We developed a DNA extraction method to obtain sufficient quantity and high quality genomic DNA from limited FFPE tissue for molecular oncology testing using a combination of H&E stained slides, a matrix capture method and the Qiagen DNA column. THREE DNA EXTRACTION METHODS WERE COMPARED: our standard procedure of manually scraping tissue from unstained slides followed by DNA extraction using the QIAamp FFPE column (Qiagen, Valencia, CA), a glue capture method (Pinpoint Solution, Zymo Research Corp, Inc) on H&E stained slides followed by DNA extraction using either the QIAamp column or the column included with the Pinpoint kit (Zymo Research). The DNA extraction protocol was optimized. Statistical analysis was performed using the paired two-sample student's t-test. The combination of the matrix capture method with the QIAamp column gave an equivalent amount of DNA as our standard extraction method using the unstained slides and a 4.6-fold higher DNA yield than using the Zymo column included in the Pinpoint Slide Solution kit. Several molecular tests were performed and DNA purified using the new method gave the same results as for the previous methods. Using H&E stained slides allows visual confirmation of tumor cells during microdissection. The Pinpoint solution made removal of specific tissue from the slides easier and reduced the risk of contamination and tissue loss. This DNA extraction method is simple, cost-effective, and blends with our current workflow requiring no additional equipment.

Detection of hepatitis C viral RNA sequences in fresh and paraffin-embedded liver biopsy specimens of non-A, non-B hepatitis patients

NARCIS (Netherlands)

Bresters, D.; Cuypers, H. T.; Reesink, H. W.; Chamuleau, R. A.; Schipper, M. E.; Boeser-Nunnink, B. D.; Lelie, P. N.; Jansen, P. L.

1992-01-01

In this study methods of HCV-RNA detection in fresh frozen and formalin-fixed, paraffin-embedded liver biopsies are described. Of 22 untreated chronic non-A, non-B hepatitis patients and 6 control patients, a plasma sample and part of a liver biopsy were freshly frozen for hepatitis C virus (HCV)

Diagnostic sensitivity and specificity of in situ hybridization and immunohistochemistry for Eastern equine encephalitis virus and West Nile virus in formalin-fixed, paraffin-embedded brain tissue of horses.

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Pennick, Kate E; McKnight, Christy A; Patterson, Jon S; Latimer, Kenneth S; Maes, Roger K; Wise, Annabel G; Kiupel, Matti

2012-03-01

Immunohistochemistry (IHC) and in situ hybridization (ISH) can be used either to detect or to differentiate between Eastern equine encephalitis virus (EEEV) and West Nile virus (WNV) within formalin-fixed, paraffin-embedded (FFPE) brain tissue of horses. To compare the diagnostic sensitivity and specificity of ISH and IHC, FFPE brain tissue from 20 EEEV-positive horses and 16 WNV-positive horses were tested with both EEEV and WNV oligoprobes and EEEV- and WNV-specific antibodies. Reverse transcription polymerase chain reaction (RT-PCR) for detection of EEEV and WNV was used as the gold standard to confirm infection. All horses that tested positive for EEEV by RT-PCR also tested positive by IHC and ISH, except for 1 case that was false-negative by ISH. In contrast, all horses that tested positive for WNV by RT-PCR tested negative by IHC and only 2 horses tested positive by ISH. No false-positives were detected with either method for both viruses. Both IHC and ISH are highly specific and sensitive diagnostic methods to detect EEEV in equine FFPE brain tissues, although neither appear effective for the diagnosis of WNV in equine neurologic cases.

Detection of African swine fever virus from formalin fixed and non-fixed tissues by polymerase chain reaction

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P. D. Luka

2014-10-01

Full Text Available Aim: Formalin fixing and paraffin embedding of tissue samples is one of the techniques for preserving the structural integrity of cells for a very long time. However, extraction and analysis of genomic material from formalin fixed tissue (FFT remains a challenge despite numerous attempts to develop a more effective method. The success of polymerase chain reaction (PCR depends on the quality of DNA extract. Materials and Methods: Here we assessed the conventional method of DNA extraction from FFT for African swine fever virus (ASFV detection. The modified conventional method gave a higher quality DNA when compared with commercially available DNA extraction kits (QIAamp® DNA Mini Kit, DNeasy® Blood and Tissue Kit, and ZR Genomic DNA™ Tissue MiniPrep. Results: An average A260/A280 DNA purity of 0.86-1.68 and 3.22-5.32 μg DNA/mg for formalin fixed and non-fixed tissues, respectively using a conventional method. In a reproducible and three times repeat PCR, the ASFV DNA expected product size of 278 bp was obtained from the DNA extract of the conventional method but not from the DNA extract of the commercial kits. Conclusion: The present study has demonstrated that the conventional method extracts ASFV genome better than commercial kit. In summary, the commercial kit extraction appeared not suitable to purify ASFV DNA from FFT. We, therefore, recommend that the use of the conventional method be considered for African swine fever DNA extraction from FFT.

Chromosomal aberrations in bladder cancer: fresh versus formalin fixed paraffin embedded tissue and targeted FISH versus wide microarray-based CGH analysis.

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Elena Panzeri

Full Text Available Bladder carcinogenesis is believed to follow two alternative pathways driven by the loss of chromosome 9 and the gain of chromosome 7, albeit other nonrandom copy number alterations (CNAs were identified. However, confirmation studies are needed since many aspects of this model remain unclear and considerable heterogeneity among cases has emerged. One of the purposes of this study was to evaluate the performance of a targeted test (UroVysion assay widely used for the detection of Transitional Cell Carcinoma (TCC of the bladder, in two different types of material derived from the same tumor. We compared the results of UroVysion test performed on Freshly Isolated interphasic Nuclei (FIN and on Formalin Fixed Paraffin Embedded (FFPE tissues from 22 TCCs and we didn't find substantial differences. A second goal was to assess the concordance between array-CGH profiles and the targeted chromosomal profiles of UroVysion assay on an additional set of 10 TCCs, in order to evaluate whether UroVysion is an adequately sensitive method for the identification of selected aneuploidies and nonrandom CNAs in TCCs. Our results confirmed the importance of global genomic screening methods, that is array based CGH, to comprehensively determine the genomic profiles of large series of TCCs tumors. However, this technique has yet some limitations, such as not being able to detect low level mosaicism, or not detecting any change in the number of copies for a kind of compensatory effect due to the presence of high cellular heterogeneity. Thus, it is still advisable to use complementary techniques such as array-CGH and FISH, as the former is able to detect alterations at the genome level not excluding any chromosome, but the latter is able to maintain the individual data at the level of single cells, even if it focuses on few genomic regions.

Detection of SYT-SSX mutant transcripts in formalin-fixed paraffin-embedded sarcoma tissues using one-step reverse transcriptase real-time PCR.

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Norlelawati, A T; Mohd Danial, G; Nora, H; Nadia, O; Zatur Rawihah, K; Nor Zamzila, A; Naznin, M

2016-04-01

Synovial sarcoma (SS) is a rare cancer and accounts for 5-10% of adult soft tissue sarcomas. Making an accurate diagnosis is difficult due to the overlapping histological features of SS with other types of sarcomas and the non-specific immunohistochemistry profile findings. Molecular testing is thus considered necessary to confirm the diagnosis since more than 90% of SS cases carry the transcript of t(X;18)(p11.2;q11.2). The purpose of this study is to diagnose SS at molecular level by testing for t(X;18) fusion-transcript expression through One-step reverse transcriptase real-time Polymerase Chain Reaction (PCR). Formalin-fixed paraffin-embedded tissue blocks of 23 cases of soft tissue sarcomas, which included 5 and 8 cases reported as SS as the primary diagnosis and differential diagnosis respectively, were retrieved from the Department of Pathology, Tengku Ampuan Afzan Hospital, Kuantan, Pahang. RNA was purified from the tissue block sections and then subjected to One-step reverse transcriptase real-time PCR using sequence specific hydrolysis probes for simultaneous detection of either SYT-SSX1 or SYT-SSX2 fusion transcript. Of the 23 cases, 4 cases were found to be positive for SYT-SSX fusion transcript in which 2 were diagnosed as SS whereas in the 2 other cases, SS was the differential diagnosis. Three cases were excluded due to failure of both amplification assays SYT-SSX and control β-2-microglobulin. The remaining 16 cases were negative for the fusion transcript. This study has shown that the application of One-Step reverse transcriptase real time PCR for the detection SYT-SSX transcript is feasible as an aid in confirming the diagnosis of synovial sarcoma.

Combined in situ zymography, immunofluorescence, and staining of iron oxide particles in paraffin-embedded, zinc-fixed tissue sections.

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Haeckel, Akvile; Schoenzart, Lena; Appler, Franziska; Schnorr, Joerg; Taupitz, Matthias; Hamm, Bernd; Schellenberger, Eyk

2012-01-01

Superparamagnetic iron oxide particles are used as potent contrast agents in magnetic resonance imaging. In histology, these particles are frequently visualized by Prussian blue iron staining of aldehyde-fixed, paraffin-embedded tissues. Recently, zinc salt-based fixative was shown to preserve enzyme activity in paraffin-embedded tissues. In this study, we demonstrate that zinc fixation allows combining in situ zymography with fluorescence immunohistochemistry (IHC) and iron staining for advanced biologic investigation of iron oxide particle accumulation. Very small iron oxide particles, developed for magnetic resonance angiography, were applied intravenously to BALB/c nude mice. After 3 hours, spleens were explanted and subjected to zinc fixation and paraffin embedding. Cut tissue sections were further processed to in situ zymography, IHC, and Prussian blue staining procedures. The combination of in situ zymography as well as IHC with subsequent Prussian blue iron staining on zinc-fixed paraffin-embedded tissues resulted in excellent histologic images of enzyme activity, protease distribution, and iron oxide particle accumulation. The combination of all three stains on a single section allowed direct comparison with only moderate degradation of fluorescein isothiocyanate-labeled substrate. This protocol is useful for investigating the biologic environment of accumulating iron oxide particles, with excellent preservation of morphology.

Validation of putative reference genes for normalization of Q-RT-PCR data from paraffin-embedded lymphoid tissue

DEFF Research Database (Denmark)

Green, Tina Marie; de Stricker, Karin; Møller, Michael Boe

2009-01-01

Normalization of quantitative reverse transcription-PCR (Q-RT-PCR) data to appropriate tissue-specific reference genes is an essential part of interpreting the results. This study aimed to determine the most appropriate reference genes for normalizing gene expressions in lymphatic tissue...... was 0.93 (Pnormalization with the appropriate reference genes. Thus, we show that formalin-fixed, paraffin-embedded lymphoid samples are suitable for Q-RT-PCR when using thoroughly validated reference genes....

MicroRNAs are suitable for assessment as biomarkers from formalin-fixed paraffin-embedded tissue, and miR-24 represents an appropriate reference microRNA for diffuse large B-cell lymphoma studies.

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Culpin, Rachel Emily; Sieniawski, Michal; Proctor, Stephen John; Menon, Geetha; Mainou-Fowler, Tryfonia

2013-03-01

Tissue biopsy specimens in the form of formalin-fixed paraffin-embedded tissue (FFPET) represent a valuable resource for biomarker identification and validation. However, to date, they remain an underused asset due to uncertainty regarding RNA extraction and the reliability of downstream techniques, including quantitative RT-PCR. Recently, much interest has emerged in the study of microRNAs; small single-stranded RNAs with a role in transcriptional regulation, that are thought to be well preserved in FFPET. In this study, we show that microRNA expression is comparable between FFPET and matched fresh-frozen samples (miR-17-5p: p=0.01, miR-92: p=0.003), and demonstrate that no significant deterioration in expression occurs over prolonged FFPET storage (p=0.06). Furthermore, microRNA expression is equivalent dependant on RNA extraction method (p<0.001) or DNAse treatment of total RNA (p<0.001). Finally, we validate miR-24 as a suitable reference microRNA for diffuse large B-cell lymphoma (DLBCL) FFPET studies.

Investigation of Epstein-Barr virus DNA in formalin-fixed and paraffin- embedded breast cancer tissues.

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Kalkan, Ahmet; Ozdarendeli, Aykut; Bulut, Yasemin; Yekeler, Hayrettin; Cobanoglu, Bengu; Doymaz, Mehmet Z

2005-01-01

To investigate etiological role of Epstein-Barr virus (EBV) DNA in breast cancer. The presence of EBV DNA in 57 breast cancer tissues was investigated with a sensitive PCR assay. The breast cancer tissues were from invasive ductular (n=28), lobular (n=20) and other miscellaneous carcinomas (n=9). Tissues from normal breasts and patients with various benign breast diseases (n=55): fibrocystic disease (n=34), fibroadenoma (n=16), hyperplasia, and granulomatous mastitis (n=5), were used as control samples. EBV DNA was detected in 13 (23%) cancerous tissues (7 ductular, 4 lobular, 2 other carcinoma) and 19 (35%) in the control tissues. The difference between EBV presence in malignant and benign tissues was not statistically significant (p>0.05). The presence of EBV DNA was detected almost equally in both breast cancer and normal tissues, which indicates no etiological role for EBV in breast cancer. We suggest further etiological studies. Copyright (c) 2005 S. Karger AG, Basel.

Validation of tumor markers in central nervous system germ cell tumors by real-time reverse transcriptase polymerase chain reaction using formalin-fixed paraffin-embedded tissues.

Science.gov (United States)

Kim, Dowhan; Lee, Da Hye; Choi, Junjeong; Shim, Kyu Won; Kim, Se Hoon

2013-01-01

The therapeutic protocols for treatment of germinomas and non-germinomatous germ cell tumors (NGGCTs) are completely different, so it is important to distinguish pure germinomas from NGGCTs. As it can be difficult to diagnose by morphology alone, immunohisto-chemistry (IHC) has been widely used as an ancillary test to improve diagnostic accuracy. However, IHC has limitations due to the misinterpretation of results or the aberrant loss of immunoreactivity. However, real-time RT-PCR has certain advantages over IHC, including its quantitative nature. The aim of our study was to evaluate the usefulness of real-time RT-PCR on formalin-fixed paraffin-embedded (FFPE) tissue blocks for the diagnosis of germ cell tumors of the central nervous system. We selected eight markers of germ cell tumors using a literature search, and validated them using real-time RT-PCR. Among them, POU5F1, NANOG and TGFB2 were statistically significant (P=0.05) in multiple comparisons (MANOVA) of three groups (pure germinomas, mature teratomas and malignant germ cell tumors). Two-group (pure germinomas and NGGCTs) discriminant analysis achieved a 70.0% success rate in cross-validation. We concluded that real-time RT-PCR using FFPE tissue has adequate validating power comparable to IHC in the diagnosis of central nervous system germ cell tumors; therefore, when IHC is not available, not conclusive or not informative, RT-PCR is a potential alternative to a repeat biopsy.

A Method to Correlate mRNA Expression Datasets Obtained from Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Tissue Samples: A Matter of Thresholds.

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Dana A M Mustafa

Full Text Available Gene expression profiling of tumors is a successful tool for the discovery of new cancer biomarkers and potential targets for the development of new therapeutic strategies. Reliable profiling is preferably performed on fresh frozen (FF tissues in which the quality of nucleic acids is better preserved than in formalin-fixed paraffin-embedded (FFPE material. However, since snap-freezing of biopsy materials is often not part of daily routine in pathology laboratories, one may have to rely on archival FFPE material. Procedures to retrieve the RNAs from FFPE materials have been developed and therefore, datasets obtained from FFPE and FF materials need to be made compatible to ensure reliable comparisons are possible.To develop an efficient method to compare gene expression profiles obtained from FFPE and FF samples using the same platform.Twenty-six FFPE-FF sample pairs of the same tumors representing various cancer types, and two FFPE-FF sample pairs of breast cancer cell lines, were included. Total RNA was extracted and gene expression profiling was carried out using Illumina's Whole-Genome cDNA-mediated Annealing, Selection, extension and Ligation (WG-DASL V3 arrays, enabling the simultaneous detection of 24,526 mRNA transcripts. A sample exclusion criterion was created based on the expression of 11 stably expressed reference genes. Pearson correlation at the probe level was calculated for paired FFPE-FF, and three cut-off values were chosen. Spearman correlation coefficients between the matched FFPE and FF samples were calculated for three probe lists with varying levels of significance and compared to the correlation based on all measured probes. Unsupervised hierarchical cluster analysis was performed to verify performance of the included probe lists to compare matched FPPE-FF samples.Twenty-seven FFPE-FF pairs passed the sample exclusion criterion. From the profiles of 27 FFPE and FF matched samples, the best correlating probes were identified

Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients.

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Sirirat Seekhuntod

Full Text Available Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC. The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR assay to detect KRAS mutations.We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D. We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay.Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%, G12D (25.83% and G12V (12.50% in codon 12.The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues.

An ER activity profile including ER, PR, Bcl-2 and IGF-IR may have potential as selection criterion for letrozole or tamoxifen treatment of patients with advanced breast cancer

DEFF Research Database (Denmark)

Henriksen, Katrine L; Rasmussen, Birgitte B; Lykkesfeldt, Anne E

2009-01-01

microarrays from formalin fixed paraffin embedded primary tumor material from a subgroup of patients (9.4%), who have participated in the international, randomized, phase III clinical trial PO25 comparing letrozole with tamoxifen in 907 patients with advanced breast cancer. The expression levels of ER...

Measurement of Gene Expression in Archival Paraffin-Embedded Tissues

Science.gov (United States)

Cronin, Maureen; Pho, Mylan; Dutta, Debjani; Stephans, James C.; Shak, Steven; Kiefer, Michael C.; Esteban, Jose M.; Baker, Joffre B.

2004-01-01

Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10-μm FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests. PMID:14695316

Molecular diagnosis of skin infections using paraffin-embedded tissue - review and interdisciplinary consensus.

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Sunderkötter, Cord; Becker, Karsten; Kutzner, Heinz; Meyer, Thomas; Blödorn-Schlicht, Norbert; Reischl, Udo; Nenoff, Pietro; Geißdörfer, Walter; Gräser, Yvonne; Herrmann, Mathias; Kühn, Joachim; Bogdan, Christian

2018-02-01

Nucleic acid amplification techniques (NATs), such as PCR, are highly sensitive and specific methods that have become valuable supplements to culture and serology in the diagnosis of infectious disorders. However, especially when using formalin-fixed and paraffin-embedded tissue, these techniques are associated with both false-negative and false-positive results, a pitfall that is frequently misjudged. Representatives of the German Society of Hygiene and Microbiology (DGHM) and the German Society of Dermatology (DDG) therefore set out to develop a consensus - in the form of a review article - on the appropriate indications for NATs using paraffin-embedded tissue, its contraindications, and the key points to be considered in the pre- and post-analytical phase. Given that fresh, naive tissue is preferably to be used in the workup of a suspected infection, PCR analysis on paraffin sections represents an exception. The latter may be considered if an infection is suspected at a later point in time and fresh tissue has not been preserved or can no longer be obtained. Potential indications include confirmation of histologically suspected infections with Leishmania spp., Bartonella spp., Rickettsia spp., or in case of ecthyma contagiosum. Infections with, for example, mycobacteria or RNA viruses, on the other hand, are not considered useful indications for NATs using paraffin sections. In order to avoid misinterpretation of test results, it is essential that laboratory reports on NATs using paraffin-embedded tissue contain information on the indication/diagnostic circumstances, the required and chosen pre-analytical steps, the limitations of the method, and on diagnostic alternatives. © 2018 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd.

Early experience with formalin-fixed paraffin-embedded (FFPE) based commercial clinical genomic profiling of gliomas-robust and informative with caveats.

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Movassaghi, Masoud; Shabihkhani, Maryam; Hojat, Seyed A; Williams, Ryan R; Chung, Lawrance K; Im, Kyuseok; Lucey, Gregory M; Wei, Bowen; Mareninov, Sergey; Wang, Michael W; Ng, Denise W; Tashjian, Randy S; Magaki, Shino; Perez-Rosendahl, Mari; Yang, Isaac; Khanlou, Negar; Vinters, Harry V; Liau, Linda M; Nghiemphu, Phioanh L; Lai, Albert; Cloughesy, Timothy F; Yong, William H

2017-08-01

Commercial targeted genomic profiling with next generation sequencing using formalin-fixed paraffin embedded (FFPE) tissue has recently entered into clinical use for diagnosis and for the guiding of therapy. However, there is limited independent data regarding the accuracy or robustness of commercial genomic profiling in gliomas. As part of patient care, FFPE samples of gliomas from 71 patients were submitted for targeted genomic profiling to one commonly used commercial vendor, Foundation Medicine. Genomic alterations were determined for the following grades or groups of gliomas; Grade I/II, Grade III, primary glioblastomas (GBMs), recurrent primary GBMs, and secondary GBMs. In addition, FFPE samples from the same patients were independently assessed with conventional methods such as immunohistochemistry (IHC), Quantitative real-time PCR (qRT-PCR), or Fluorescence in situ hybridization (FISH) for three genetic alterations: IDH1 mutations, EGFR amplification, and EGFRvIII expression. A total of 100 altered genes were detected by the aforementioned targeted genomic profiling assay. The number of different genomic alterations was significantly different between the five groups of gliomas and consistent with the literature. CDKN2A/B, TP53, and TERT were the most common genomic alterations seen in primary GBMs, whereas IDH1, TP53, and PIK3CA were the most common in secondary GBMs. Targeted genomic profiling demonstrated 92.3%-100% concordance with conventional methods. The targeted genomic profiling report provided an average of 5.5 drugs, and listed an average of 8.4 clinical trials for the 71 glioma patients studied but only a third of the trials were appropriate for glioma patients. In this limited comparison study, this commercial next generation sequencing based-targeted genomic profiling showed a high concordance rate with conventional methods for the 3 genetic alterations and identified mutations expected for the type of glioma. While it may not be feasible to

Hormone Receptor Expression Analyses in Neoplastic and Non-Neoplastic Canine Mammary Tissue by a Bead Based Multiplex Branched DNA Assay: A Gene Expression Study in Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Samples.

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Annika Mohr

Full Text Available Immunohistochemistry (IHC is currently considered the method of choice for steroid hormone receptor status evaluation in human breast cancer and, therefore, it is commonly utilized for assessing canine mammary tumors. In case of low hormone receptor expression, IHC is limited and thus is complemented by molecular analyses. In the present study, a multiplex bDNA assay was evaluated as a method for hormone receptor gene expression detection in canine mammary tissues. Estrogen receptor (ESR1, progesterone receptor (PGR, prolactin receptor (PRLR and growth hormone receptor (GHR gene expressions were evaluated in neoplastic and non-neoplastic canine mammary tissues. A set of 119 fresh frozen and 180 formalin-fixed, paraffin-embedded (FFPE was comparatively analyzed and used for assay evaluation. Furthermore, a possible association between the hormone receptor expression in different histological subtypes of canine malignant mammary tumors and the castration status, breed and invasive growth of the tumor were analyzed. The multiplex bDNA assay proved to be more sensitive for fresh frozen specimens. Hormone receptor expression found was significantly decreased in malignant mammary tumors in comparison to non-neoplastic tissue and benign mammary tumors. Among the histological subtypes the lowest gene expression levels of ESR1, PGR and PRLR were found in solid, anaplastic and ductal carcinomas. In summary, the evaluation showed that the measurement of hormone receptors with the multiplex bDNA assay represents a practicable method for obtaining detailed quantitative information about gene expression in canine mammary tissue for future studies. Still, comparison with IHC or quantitative real-time PCR is needed for further validation of the present method.

Genome-wide comparison of paired fresh frozen and formalin-fixed paraffin-embedded gliomas by custom BAC and oligonucleotide array comparative genomic hybridization: facilitating analysis of archival gliomas.

Science.gov (United States)

Mohapatra, Gayatry; Engler, David A; Starbuck, Kristen D; Kim, James C; Bernay, Derek C; Scangas, George A; Rousseau, Audrey; Batchelor, Tracy T; Betensky, Rebecca A; Louis, David N

2011-04-01

Array comparative genomic hybridization (aCGH) is a powerful tool for detecting DNA copy number alterations (CNA). Because diffuse malignant gliomas are often sampled by small biopsies, formalin-fixed paraffin-embedded (FFPE) blocks are often the only tissue available for genetic analysis; FFPE tissues are also needed to study the intratumoral heterogeneity that characterizes these neoplasms. In this paper, we present a combination of evaluations and technical advances that provide strong support for the ready use of oligonucleotide aCGH on FFPE diffuse gliomas. We first compared aCGH using bacterial artificial chromosome (BAC) arrays in 45 paired frozen and FFPE gliomas, and demonstrate a high concordance rate between FFPE and frozen DNA in an individual clone-level analysis of sensitivity and specificity, assuring that under certain array conditions, frozen and FFPE DNA can perform nearly identically. However, because oligonucleotide arrays offer advantages to BAC arrays in genomic coverage and practical availability, we next developed a method of labeling DNA from FFPE tissue that allows efficient hybridization to oligonucleotide arrays. To demonstrate utility in FFPE tissues, we applied this approach to biphasic anaplastic oligoastrocytomas and demonstrate CNA differences between DNA obtained from the two components. Therefore, BAC and oligonucleotide aCGH can be sensitive and specific tools for detecting CNAs in FFPE DNA, and novel labeling techniques enable the routine use of oligonucleotide arrays for FFPE DNA. In combination, these advances should facilitate genome-wide analysis of rare, small and/or histologically heterogeneous gliomas from FFPE tissues.

High-risk Human Papillomavirus Determination in Formalin-fixed, Paraffin-embedded Cervical Tissue Using the Roche Cobas 4800 System: A Comparative Study With Liquid-based Cytology.

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Tardío, Juan C; Cambero, Olivia; Sánchez-Estévez, Carolina; Sánchez-García, Ana B; Angulo, Fernando; Moreno, Amalia

2017-11-14

Roche cobas 4800 human papillomavirus (HPV) test is an automated real-time polymerase chain reaction-based system that allows the simultaneous detection of 14 human papillomavirus high-risk (HR-HPV) genotypes. This test is Food and Drug Administration approved since 2011 for HPV determination in liquid-based cytologic samples, but a clinically validated technique for formalin-fixed, paraffin-embedded (FFPE) tissue specimens is presently not commercially available. In our laboratory, we have developed an HPV detection procedure in FFPE tissue by cobas 4800 HPV test. In order to validate our method, we retrospectively studied 165 FFPE cervical biopsy and conization specimens with varied diagnoses from our files. In 50 of them, we contrasted the results with those obtained from simultaneous liquid-based cytologies from the same patients. Finally, seeking the possible complementary clinical usefulness of the procedure, we compared the HPV genotypes detected in cervical intraepithelial neoplasia grade 1 (CIN1)-diagnosed biopsies from 20 patients with a subsequent high-grade CIN (CIN2+) diagnosis with those from another group of 20 patients without a posterior CIN2+ diagnosis. Eighty-seven percent of the assays provided informative results. HR-HPV was detected in 28 of 32 (88%) invasive cervical squamous carcinomas. Coincidental HR-HPV genotypes were obtained in 32 of 50 (64%) cases with simultaneous cervical biopsy and liquid-based cytologic samples. A significant higher risk of progression to CIN2+ was found when HPV16 (P=0.022) or any HR-HPV genotype (P=0.037) was detected in CIN1 biopsies. The reported procedure provides an automated, technically time-saving, easy to integrate into laboratory routine, and reliable method of HR-HPV determination in FFPE specimens.

Detection of mucormycetes and other pathogenic fungi in formalin fixed paraffin embedded and fresh tissues using the extended region of 28S rDNA.

Science.gov (United States)

Gade, Lalitha; Hurst, Steven; Balajee, S Arunmozhi; Lockhart, Shawn R; Litvintseva, Anastasia P

2017-06-01

Molecular methods of detection based on DNA-sequencing of the internal transcribed spacer 1 and 2 (ITS1 and ITS2) or 5΄ end region of 28S (D1-D2 region) of ribosomal RNA gene (rDNA) have been used extensively for molecular identification and detection of fungal infections. However, these regions are not always informative for identification of mucormycetes and other rare fungal pathogens as they often contain large introns, heterogenic regions, and/or cannot be PCR-amplified using broad range fungal PCR primers. In addition, because of the difficulties of recovering intact fungal DNA from human specimens, smaller regions of DNA are more useful for the direct detection of fungal DNA in tissues and fluids. In this study, we investigated the utility of 12F/13R PCR primers targeting a 200-230 bp region of the extended 28S region of rDNA for molecular identification of fungal DNA in formalin fixed paraffin embedded tissues and other clinical specimens. We demonstrated that this region can be successfully used for identification of all genera and some species of clinically relevant mucormycetes, as well as other medically important fungi, such as Aspergillus, Fusarium, Coccidioides, and Cryptococcus. We also demonstrated that PCR amplification and direct sequencing of the extended 28S region of rDNA was more sensitive compared to targeting the ITS2 region, as we were able to detect and identify mucormycetes and other fungal pathogens in tissues from patients with histopathological and/or culture evidence of fungal infections that were negative with PCR using ITS-specific primers. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Genome-wide comparison of paired fresh frozen and formalin-fixed paraffin-embedded gliomas by custom BAC and oligonucleotide array comparative genomic hybridization: facilitating analysis of archival gliomas

Science.gov (United States)

Mohapatra, Gayatry; Engler, David A.; Starbuck, Kristen D.; Kim, James C.; Bernay, Derek C.; Scangas, George A.; Rousseau, Audrey; Batchelor, Tracy T.; Betensky, Rebecca A.; Louis, David N.

2010-01-01

Molecular genetic analysis of cancer is rapidly evolving as a result of improvement in genomic technologies and the growing applicability of such analyses to clinical oncology. Array based comparative genomic hybridization (aCGH) is a powerful tool for detecting DNA copy number alterations (CNA), particularly in solid tumors, and has been applied to the study of malignant gliomas. In the clinical setting, however, gliomas are often sampled by small biopsies and thus formalin-fixed paraffin-embedded (FFPE) blocks are often the only tissue available for genetic analysis, especially for rare types of gliomas. Moreover, the biological basis for the marked intratumoral heterogeneity in gliomas is most readily addressed in FFPE material. Therefore, for gliomas, the ability to use DNA from FFPE tissue is essential for both clinical and research applications. In this study, we have constructed a custom bacterial artificial chromosome (BAC) array and show excellent sensitivity and specificity for detecting CNAs in a panel of paired frozen and FFPE glioma samples. Our study demonstrates a high concordance rate between CNAs detected in FFPE compared to frozen DNA. We have also developed a method of labeling DNA from FFPE tissue that allows efficient hybridization to oligonucleotide arrays. This labeling technique was applied to a panel of biphasic anaplastic oligoastrocytomas (AOA) to identify genetic changes unique to each component. Together, results from these studies suggest that BAC and oligonucleotide aCGH are sensitive tools for detecting CNAs in FFPE DNA, and can enable genome-wide analysis of rare, small and/or histologically heterogeneous gliomas. PMID:21080181

The relative test performance characteristics of two commercial assays for the detection of Mycobacterium tuberculosis complex in paraffin-fixed human biopsy specimens

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Broukhanski George

2008-09-01

Full Text Available Abstract The Seeplex™ TB Detection-2 assay (Rockville, MD is a nested endpoint PCR for the Mycobacterium tuberculosis complex (MTBC targets IS6110 and MPB64 that utilizes dual priming oligonucleotide technology. When used to detect the presence of MTBC DNA in formalin-fixed paraffin-embedded tissue specimens, the sensitivity and specificity of this assay is equivalent to a labor-intensive traditional endpoint PCR assay and is more sensitive than a commercial real-time PCR assay.

Detection of MDM2/CDK4 amplification in lipomatous soft tissue tumors from formalin-fixed, paraffin-embedded tissue: comparison of multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH).

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Creytens, David; van Gorp, Joost; Ferdinande, Liesbeth; Speel, Ernst-Jan; Libbrecht, Louis

2015-02-01

In this study, the detection of MDM2 and CDK4 amplification was evaluated in lipomatous soft tissue tumors using multiplex ligation-dependent probe amplification (MLPA), a PCR-based technique, in comparison with fluorescence in situ hybridization (FISH). These 2 techniques were evaluated in a series of 77 formalin-fixed, paraffin-embedded lipomatous tumors (27 benign adipose tumors, 28 atypical lipomatous tumors/well-differentiated liposarcomas, 18 dedifferentiated liposarcomas, and 4 pleomorphic liposarcomas). Using MLPA, with a cut-off ratio of >2, 36/71 samples (22 atypical lipomatous tumors/well-differentiated liposarcomas, and 14 dedifferentiated liposarcomas) showed MDM2 and CDK4 amplification. Using FISH as gold standard, MLPA showed a sensitivity of 90% (36/40) and a specificity of 100% (31/31) in detecting amplification of MDM2 and CDK4 in lipomatous soft tissue tumors. In case of high-level amplification (MDM2-CDK4/CEP12 ratio >5), concordance was 100%. Four cases of atypical lipomatous tumor/well-differentiated liposarcoma (4/26, 15%) with a low MDM2 and CDK4 amplification level (MDM2-CDK4/CEP12 ratio ranging between 2 and 2.5) detected by FISH showed no amplification by MLPA, although gain of MDM2 and CDK4 (ratios ranging between 1.6 and 1.9) was seen with MLPA. No amplification was detected in benign lipomatous tumors and pleomorphic liposarcomas. Furthermore, there was a very high concordance between the ratios obtained by FISH and MLPA. In conclusion, MLPA proves to be an appropriate and straightforward technique for screening MDM2/CDK4 amplification in lipomatous tumors, especially when a correct cut-off value and reference samples are chosen, and could be considered a good alternative to FISH to determine MDM2 and CDK4 amplification in liposarcomas. Moreover, because MLPA, as a multiplex technique, allows simultaneous detection of multiple chromosomal changes of interest, it could be in the future a very reliable and fast molecular analysis on

Gene expression profiles in paraffin-embedded core biopsy tissue predict response to chemotherapy in women with locally advanced breast cancer.

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Gianni, Luca; Zambetti, Milvia; Clark, Kim; Baker, Joffre; Cronin, Maureen; Wu, Jenny; Mariani, Gabriella; Rodriguez, Jaime; Carcangiu, Marialuisa; Watson, Drew; Valagussa, Pinuccia; Rouzier, Roman; Symmans, W Fraser; Ross, Jeffrey S; Hortobagyi, Gabriel N; Pusztai, Lajos; Shak, Steven

2005-10-10

We sought to identify gene expression markers that predict the likelihood of chemotherapy response. We also tested whether chemotherapy response is correlated with the 21-gene Recurrence Score assay that quantifies recurrence risk. Patients with locally advanced breast cancer received neoadjuvant paclitaxel and doxorubicin. RNA was extracted from the pretreatment formalin-fixed paraffin-embedded core biopsies. The expression of 384 genes was quantified using reverse transcriptase polymerase chain reaction and correlated with pathologic complete response (pCR). The performance of genes predicting for pCR was tested in patients from an independent neoadjuvant study where gene expression was obtained using DNA microarrays. Of 89 assessable patients (mean age, 49.9 years; mean tumor size, 6.4 cm), 11 (12%) had a pCR. Eighty-six genes correlated with pCR (unadjusted P < .05); pCR was more likely with higher expression of proliferation-related genes and immune-related genes, and with lower expression of estrogen receptor (ER) -related genes. In 82 independent patients treated with neoadjuvant paclitaxel and doxorubicin, DNA microarray data were available for 79 of the 86 genes. In univariate analysis, 24 genes correlated with pCR with P < .05 (false discovery, four genes) and 32 genes showed correlation with P < .1 (false discovery, eight genes). The Recurrence Score was positively associated with the likelihood of pCR (P = .005), suggesting that the patients who are at greatest recurrence risk are more likely to have chemotherapy benefit. Quantitative expression of ER-related genes, proliferation genes, and immune-related genes are strong predictors of pCR in women with locally advanced breast cancer receiving neoadjuvant anthracyclines and paclitaxel.

Preservation of biomolecules in breast cancer tissue by a formalin-free histology system

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Morales Azorides R

2008-01-01

Full Text Available Abstract Background The potential problems associated with the use of formalin in histology, such as health hazards, degradation of RNA and cross-linking of proteins are well recognized. We describe the utilization of a formalin-free fixation and processing system for tissue detection of two important biopredictors in breast cancer – estrogen receptor and HER2 – at the RNA and protein levels. Methods Parallel sections of 62 cases of breast cancer were fixed in an alcohol-based molecular fixative and in formalin. Molecular fixative samples were processed by a novel formalin-free microwave-assisted processing system that preserves DNA, RNA and proteins. Formalin-fixed samples were processed using the conventional method. Estrogen receptor was assessed by immunohistochemistry and real-time PCR. HER2 was assessed by immunohistochemistry, FISH, CISH and real-time PCR. Results The immunohistochemical reaction for estrogen receptor was similar in molecular- and formalin-fixed samples (Spearman Rank R = 0.83, p Conclusion The formalin-free tissue fixation and processing system is a practical platform for evaluation of biomolecular markers in breast cancer and it allows reliable DNA and RNA and protein studies.

Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples Efeitos das etapas de tratamento e processamento do tecido na PCR para detecção de Mycobacterium tuberculosis em amostras fixadas em formalina e incluídas em parafina

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Denise Barcelos

2008-12-01

Full Text Available Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10% non-buffered or 10% buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10% non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.O desenvolvimento e a padronização de métodos confiáveis para a detecção de Mycobacterium tuberculosis em amostras clínicas é um objetivo importante nos laboratórios de todo o mundo. Neste trabalho, fragmentos de pulmão e baço de paciente que morreu com o diagnóstico de tuberculose miliar foram usados para avaliar a influência do tipo de fixador e dos protocolos de fixação e inclusão em parafina na performance da PCR. Fragmentos de tecido foram fixados por quatro h a 48 h, usando formalina não tamponada a 10% ou formalina tamponada a 10% e incluídos em parafina pura ou misturada a cera de abelha. As amostras foram submetidas a PCR para amplificação do gene da beta-actina humana e

Digital dewaxing of Raman signals: discrimination between nevi and melanoma spectra obtained from paraffin-embedded skin biopsies.

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Tfayli, Ali; Gobinet, Cyril; Vrabie, Valeriu; Huez, Regis; Manfait, Michel; Piot, Olivier

2009-05-01

Malignant melanoma (MM) is the most severe tumor affecting the skin and accounts for three quarters of all skin cancer deaths. Raman spectroscopy is a promising nondestructive tool that has been increasingly used for characterization of the molecular features of cancerous tissues. Different multivariate statistical analysis techniques are used in order to extract relevant information that can be considered as functional spectroscopic descriptors of a particular pathology. Paraffin embedding (waxing) is a highly efficient process used to conserve biopsies in tumor banks for several years. However, the use of non-dewaxed formalin-fixed paraffin-embedded tissues for Raman spectroscopic investigations remains very restricted, limiting the development of the technique as a routine analytical tool for biomedical purposes. This is due to the highly intense signal of paraffin, which masks important vibrations of the biological tissues. In addition to being time consuming and chemical intensive, chemical dewaxing methods are not efficient and they leave traces of the paraffin in tissues, which affects the Raman signal. In the present study, we use independent component analysis (ICA) on Raman spectral images collected on melanoma and nevus samples. The sources obtained from these images are then used to eliminate, using non-negativity constrained least squares (NCLS), the paraffin contribution from each individual spectrum of the spectral images of nevi and melanomas. Corrected spectra of both types of lesion are then compared and classified into dendrograms using hierarchical cluster analysis (HCA).

A gene-protein assay for human epidermal growth factor receptor 2 (HER2: brightfield tricolor visualization of HER2 protein, the HER2 gene, and chromosome 17 centromere (CEN17 in formalin-fixed, paraffin-embedded breast cancer tissue sections

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Nitta Hiroaki

2012-05-01

Full Text Available Abstract Background The eligibility of breast cancer patients for human epidermal growth factor receptor 2 (HER2-directed therapies is determined by the HER2 gene amplification and/or HER2 protein overexpression status of the breast tumor as determined by in situ hybridization (ISH or immunohistochemistry (IHC, respectively. Our objective was to combine the US Food and Drug Administration (FDA-approved HER2 & chromosome 17 centromere (CEN17 brightfield ISH (BISH and HER2 IHC assays into a single automated HER2 gene-protein assay allowing simultaneous detection of all three targets in a single tissue section. Methods The HER2 gene-protein assay was optimized using formalin-fixed, paraffin-embedded (FFPE samples of the xenograft tumors MCF7 [HER2 negative (non-amplified gene, protein negative] and Calu-3 [HER2 positive (amplified gene, protein positive]. HER2 IHC was performed using a rabbit monoclonal anti-HER2 antibody (clone 4B5 and a conventional 3,3'-diaminobenzidine IHC detection. The HER2 & CEN17 BISH signals were visualized using horseradish peroxidase-based silver and alkaline phosphatase-based red detection systems, respectively with a cocktail of 2,4-dinitrophenyl-labeled HER2 and digoxigenin-labeled CEN17 probes. The performance of the gene-protein assay on tissue microarray slides containing 189 randomly selected FFPE clinical breast cancer tissue cores was compared to that of the separate HER2 IHC and HER2 & CEN17 BISH assays. Results HER2 protein detection was optimal when the HER2 IHC protocol was used before (rather than after the BISH protocol. The sequential use of HER2 IHC and HER2 & CEN17 BISH detection steps on FFPE xenograft tumor sections appropriately co-localized the HER2 protein, HER2 gene, and CEN17 signals after mitigating the silver background staining by using a naphthol phosphate-containing hybridization buffer for the hybridization step. The HER2 protein and HER2 gene status obtained using the multiplex HER2 gene

Genome-wide massively parallel sequencing of formaldehyde fixed-paraffin embedded (FFPE tumor tissues for copy-number- and mutation-analysis.

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Michal R Schweiger

Full Text Available BACKGROUND: Cancer re-sequencing programs rely on DNA isolated from fresh snap frozen tissues, the preparation of which is combined with additional preservation efforts. Tissue samples at pathology departments are routinely stored as formalin-fixed and paraffin-embedded (FFPE samples and their use would open up access to a variety of clinical trials. However, FFPE preparation is incompatible with many down-stream molecular biology techniques such as PCR based amplification methods and gene expression studies. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated the sample quality requirements of FFPE tissues for massively parallel short-read sequencing approaches. We evaluated key variables of pre-fixation, fixation related and post-fixation processes that occur in routine medical service (e.g. degree of autolysis, duration of fixation and of storage. We also investigated the influence of tissue storage time on sequencing quality by using material that was up to 18 years old. Finally, we analyzed normal and tumor breast tissues using the Sequencing by Synthesis technique (Illumina Genome Analyzer, Solexa to simultaneously localize genome-wide copy number alterations and to detect genomic variations such as substitutions and point-deletions and/or insertions in FFPE tissue samples. CONCLUSIONS/SIGNIFICANCE: The application of second generation sequencing techniques on small amounts of FFPE material opens up the possibility to analyze tissue samples which have been collected during routine clinical work as well as in the context of clinical trials. This is in particular important since FFPE samples are amply available from surgical tumor resections and histopathological diagnosis, and comprise tissue from precursor lesions, primary tumors, lymphogenic and/or hematogenic metastases. Large-scale studies using this tissue material will result in a better prediction of the prognosis of cancer patients and the early identification of patients which

The Cellient System for Paraffin Histology Can Be Combined with HPV Testing and Morphotyping the Vaginal Microbiome Thanks to BoonFixing

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Mathilde E. Boon

2013-01-01

Full Text Available The Cellient Automated Cell Block System (Hologic can be used to process cervical scrapes to paraffin sections. For the first study on this subject, cervical scrapes were fixed in the formalin-free fixative BoonFix. This pilot study was limited to cases classified as atypical squamous lesion of unknown significance (ASCUS and high-grade squamous lesion (HSIL as diagnosed in the ThinPrep slide. The Cellient paraffin sections were classified into negative, atypical, CIN 1, CIN 2, and CIN 3. Multiple HPV genotypes were encountered in 79% of the scrapes. This study showed that the Cellient system for paraffin sections can be combined with HPV testing thanks to the formalin-free BoonFix. In two additional studies it was shown that such samples can also be used for morphotyping the vaginal microbiome and preparing cytologic ThinPrep slides.

The Cellient System for Paraffin Histology Can Be Combined with HPV Testing and Morphotyping the Vaginal Microbiome Thanks to BoonFixing.

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Boon, Mathilde E

2013-01-01

The Cellient Automated Cell Block System (Hologic) can be used to process cervical scrapes to paraffin sections. For the first study on this subject, cervical scrapes were fixed in the formalin-free fixative BoonFix. This pilot study was limited to cases classified as atypical squamous lesion of unknown significance (ASCUS) and high-grade squamous lesion (HSIL) as diagnosed in the ThinPrep slide. The Cellient paraffin sections were classified into negative, atypical, CIN 1, CIN 2, and CIN 3. Multiple HPV genotypes were encountered in 79% of the scrapes. This study showed that the Cellient system for paraffin sections can be combined with HPV testing thanks to the formalin-free BoonFix. In two additional studies it was shown that such samples can also be used for morphotyping the vaginal microbiome and preparing cytologic ThinPrep slides.

Survivorship patterns of histopathological variants and molecular ...

African Journals Online (AJOL)

Objective: To study the relationship of histopathological characteristics, molecular subtypes of breast cancer and survival in a low resource setting. Design: Tumours from prospectively ascertained patients newly diagnosed with breast cancer were analyzed. Formalin-fixed and paraffin-embedded sections were constructed ...

PAM50 breast cancer intrinsic subtypes and effect of gemcitabine in advanced breast cancer patients

DEFF Research Database (Denmark)

Jørgensen, Charlotte Levin Tykjær; Nielsen, Torsten O; Bjerre, Karsten D

2014-01-01

BACKGROUND: In vitro studies suggest basal breast cancers are more sensitive to gemcitabine relative to other intrinsic subtypes. The main objective of this study was to use specimens from a randomized clinical trial to evaluate whether the basal-like subtype identifies patients with advanced...... breast cancer who benefit from gemcitabine plus docetaxel (GD) compared to single agent docetaxel (D). MATERIAL AND METHODS: From patients randomly assigned to GD or D, RNA was isolated from archival formalin-fixed, paraffin-embedded primary breast tumor tissue and used for PAM50 intrinsic subtyping...... chemotherapy were analyzed by the Kaplan-Meier method, and Cox proportional hazards regression models. Data analysis was performed independently by the Danish Breast Cancer Cooperative Group (DBCG) statistical core and all statistical tests were two-sided. RESULTS: RNA from 270 patients was evaluable; 84...

Claudin-Low Breast Cancer; Clinical & Pathological Characteristics.

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Kay Dias

Full Text Available Claudin-low breast cancer is a molecular type of breast cancer originally identified by gene expression profiling and reportedly associated with poor survival. Claudin-low tumors have been recognised to preferentially display a triple-negative phenotype, however only a minority of triple-negative breast cancers are claudin-low. We sought to identify an immunohistochemical profile for claudin-low tumors that could facilitate their identification in formalin fixed paraffin embedded tumor material. First, an in silico collection of ~1600 human breast cancer expression profiles was assembled and all claudin-low tumors identified. Second, genes differentially expressed between claudin-low tumors and all other molecular subtypes of breast cancer were identified. Third, a number of these top differentially expressed genes were tested using immunohistochemistry for expression in a diverse panel of breast cancer cell lines to determine their specificity for claudin-low tumors. Finally, the immunohistochemical panel found to be most characteristic of claudin-low tumors was examined in a cohort of 942 formalin fixed paraffin embedded human breast cancers with >10 years clinical follow-up to evaluate the clinico-pathologic and survival characteristics of this tumor subtype. Using this approach we determined that claudin-low breast cancer is typically negative for ER, PR, HER2, claudin 3, claudin 4, claudin 7 and E-cadherin. Claudin-low tumors identified with this immunohistochemical panel, were associated with young age of onset, higher tumor grade, larger tumor size, extensive lymphocytic infiltrate and a circumscribed tumor margin. Patients with claudin-low tumors had a worse overall survival when compared to patients with luminal A type breast cancer. Interestingly, claudin-low tumors were associated with a low local recurrence rate following breast conserving therapy. In conclusion, a limited panel of antibodies can facilitate the identification of

Immunohistochemical detection of SWC3, CD2, CD3, CD4 and CD8 antigens in paraformaldehyde fixed and paraffin embedded porcine lymphoid tissue

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Tingstedt, Jens Erik; Tornehave, Ditte; Lind, Peter

2003-01-01

Identification of the different cell types of the immune system is important for in situ studies on the pathogenesis of infectious diseases in various animals, including the pig. Unfortunately, many monoclonal anti-leukocyte antibodies are only useful for staining frozen tissue sections with inhe......Identification of the different cell types of the immune system is important for in situ studies on the pathogenesis of infectious diseases in various animals, including the pig. Unfortunately, many monoclonal anti-leukocyte antibodies are only useful for staining frozen tissue sections...... with inherent poor tissue morphology, and are not readily adapted to formaldehyde fixed and paraffin embedded tissue with well preserved morphology. Seven well characterised monoclonal antibodies against porcine leukocyte antigens were tested on neutral buffered paraformaldehyde fixed and paraffin embedded...

Immunohistochemical detection of VHS virus in paraffin-embedded specimens of rainbow trout (Oncorhynchus mykiss); The influence of primary antibody, fixative, and antigen unmasking on method sensitivity

DEFF Research Database (Denmark)

Evensen, O.; Olesen, Niels Jørgen

1997-01-01

performed on parallel specimens, and the virus titer (TCID50/ml) was determined. Purified nucleocapsid protein (N-protein) of the virus was incorporated in an artificial antigen substrate (polymerized bovine serum albumin), fixed as described above, and embedded in paraffin wax. Microwave unmasking...... was performed an formalin-, PLP-, and Bouin's fluid-fixed specimens. The presence of virus peptides in situ or N-protein in the artificial antigen substrates was Visualized using an immunohistochemical method based on alkaline phosphatase or peroxidase and one polyclonal and five monoclonal polypeptide......-specific antibodies. VHS virus was identified in situ in specimens with high virus titers (10(7-8) TCID50/ml) regardless of the fixative and without the need of an unmasking procedure. A pronounced masking effect was observed for the cross-linking formalin and PLP fixatives. Regardless of the primary antibodies used...

Establishment of a Pcr Technique for Determination of Htlv-1 Infection in Paraffin-Embedded Tissues

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M Rastin

2007-04-01

Full Text Available Introduction: HTLV-1 , the first known human retrovirus belongs to oncovirus subfamily of retroviruses. The major characteristic of HTLV-1 is its highly restricted geographic prevalence. Northern part of Khorasan is an endemic region of HTLV-1 infection. Epidemiological studies can help in designing preventive programs for HTLV-1 infection. The aim of this study was the establishment of a PCR technique for determination of HTLV-1 infection in paraffin-embedded tissues. Methods: In this experimental laboratory study for establishment of a technique, PCR was initially optimized using Beta-actin primers on various formalin fixed paraffin-embedded tissues from liver, spleen, skin and lymph nodes. The optimized concentration of Mgcl2 was 2mm, primer was 8 pmol. Optimized concentration of DNA was different according to the kind of tissue. HTLV-1 infection was determined by applying tax, pol, env and LTR primers on 50 paraffin-embedded lymph node tissues . The reporoducibility of this technique was shown for skin and lymph node tissues infected with HTLV-1. Resuls: In 50 lymph node tissues, one case with pathologic diagnosis of NHL was positive with all 5 sets of primers (tax, Pol, env and LTR primers and the other case was positive with only two sets of tax primers but was negative with pol, env and LTR primers. The prevalence of infection was 2% among lymph node specimens. (1 of 50 specimens and if the second case is considered, the prevalence would be 4%. Conclusion: Comparison of the results of this study with another study on blood specimens (seroprevalence2.3% was not statistically significant thus confirming the results of one another. (P=0.883

Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis.

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Vukmirovic, Milica; Herazo-Maya, Jose D; Blackmon, John; Skodric-Trifunovic, Vesna; Jovanovic, Dragana; Pavlovic, Sonja; Stojsic, Jelena; Zeljkovic, Vesna; Yan, Xiting; Homer, Robert; Stefanovic, Branko; Kaminski, Naftali

2017-01-12

Idiopathic Pulmonary Fibrosis (IPF) is a lethal lung disease of unknown etiology. A major limitation in transcriptomic profiling of lung tissue in IPF has been a dependence on snap-frozen fresh tissues (FF). In this project we sought to determine whether genome scale transcript profiling using RNA Sequencing (RNA-Seq) could be applied to archived Formalin-Fixed Paraffin-Embedded (FFPE) IPF tissues. We isolated total RNA from 7 IPF and 5 control FFPE lung tissues and performed 50 base pair paired-end sequencing on Illumina 2000 HiSeq. TopHat2 was used to map sequencing reads to the human genome. On average ~62 million reads (53.4% of ~116 million reads) were mapped per sample. 4,131 genes were differentially expressed between IPF and controls (1,920 increased and 2,211 decreased (FDR < 0.05). We compared our results to differentially expressed genes calculated from a previously published dataset generated from FF tissues analyzed on Agilent microarrays (GSE47460). The overlap of differentially expressed genes was very high (760 increased and 1,413 decreased, FDR < 0.05). Only 92 differentially expressed genes changed in opposite directions. Pathway enrichment analysis performed using MetaCore confirmed numerous IPF relevant genes and pathways including extracellular remodeling, TGF-beta, and WNT. Gene network analysis of MMP7, a highly differentially expressed gene in both datasets, revealed the same canonical pathways and gene network candidates in RNA-Seq and microarray data. For validation by NanoString nCounter® we selected 35 genes that had a fold change of 2 in at least one dataset (10 discordant, 10 significantly differentially expressed in one dataset only and 15 concordant genes). High concordance of fold change and FDR was observed for each type of the samples (FF vs FFPE) with both microarrays (r = 0.92) and RNA-Seq (r = 0.90) and the number of discordant genes was reduced to four. Our results demonstrate that RNA sequencing of RNA

Technical validation of an RT-qPCR in vitro diagnostic test system for the determination of breast cancer molecular subtypes by quantification of ERBB2, ESR1, PGR and MKI67 mRNA levels from formalin-fixed paraffin-embedded breast tumor specimens.

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Laible, Mark; Schlombs, Kornelia; Kaiser, Katharina; Veltrup, Elke; Herlein, Stefanie; Lakis, Sotiris; Stöhr, Robert; Eidt, Sebastian; Hartmann, Arndt; Wirtz, Ralph M; Sahin, Ugur

2016-07-07

MammaTyper is a novel CE-marked in vitro diagnostic RT-qPCR assay which assigns routinely processed breast cancer specimens into the molecular subtypes Luminal A-like, Luminal B-like (HER2 positive or negative), HER2 positive (non-luminal) and Triple negative (ductal) according to the mRNA expression of ERBB2, ESR1, PGR and MKI67 and the St Gallen consensus surrogate clinical definition. Until now and regarding formalin-fixed, paraffin-embedded material (FFPE), this has been a task mostly accomplished by immunohistochemistry (IHC). However the discrepancy rates of IHC for the four breast cancer biomarkers are frequently under debate, especially for Ki-67 which carries the highest degree of inter- and even intra-observer variability. Herein we describe a series of studies in FFPE specimens which aim to fully validate the analytical performance of the MammaTyper assay, including the site to site reproducibility of the individual marker measurements. Tumor RNA was extracted with the novel RNXtract RNA extraction kit. Synthetic RNA was used to assess the sensitivity of the RNXtract kit. DNA and RNA specific qPCR assays were used so as to determine analyte specificity of RNXtract. For the assessment of limit of blank, limit of detection, analytical measurement range and PCR efficiency of the MammaTyper kit serial dilutions of samples were used. Analytical precision studies of MammaTyper were built around two different real time PCR platforms and involved breast tumor samples belonging to different subtypes analyzed across multiple sites and under various stipulated conditions. The MammaTyper assay robustness was tested against RNA input variations, alternative extraction methods and tumor cell content. Individual assays were linear up to at least 32.33 and 33.56 Cqs (quantification cycles) for the two qPCR platforms tested. PCR efficiency ranged from 99 to 109 %. In qPCR platform 1, estimates for assay specific inter-site standard deviations (SD) were between 0.14 and

Technical validation of an RT-qPCR in vitro diagnostic test system for the determination of breast cancer molecular subtypes by quantification of ERBB2, ESR1, PGR and MKI67 mRNA levels from formalin-fixed paraffin-embedded breast tumor specimens

International Nuclear Information System (INIS)

Laible, Mark; Schlombs, Kornelia; Kaiser, Katharina; Veltrup, Elke; Herlein, Stefanie; Lakis, Sotiris; Stöhr, Robert; Eidt, Sebastian; Hartmann, Arndt; Wirtz, Ralph M.; Sahin, Ugur

2016-01-01

MammaTyper is a novel CE-marked in vitro diagnostic RT-qPCR assay which assigns routinely processed breast cancer specimens into the molecular subtypes Luminal A-like, Luminal B-like (HER2 positive or negative), HER2 positive (non-luminal) and Triple negative (ductal) according to the mRNA expression of ERBB2, ESR1, PGR and MKI67 and the St Gallen consensus surrogate clinical definition. Until now and regarding formalin-fixed, paraffin-embedded material (FFPE), this has been a task mostly accomplished by immunohistochemistry (IHC). However the discrepancy rates of IHC for the four breast cancer biomarkers are frequently under debate, especially for Ki-67 which carries the highest degree of inter- and even intra-observer variability. Herein we describe a series of studies in FFPE specimens which aim to fully validate the analytical performance of the MammaTyper assay, including the site to site reproducibility of the individual marker measurements. Tumor RNA was extracted with the novel RNXtract RNA extraction kit. Synthetic RNA was used to assess the sensitivity of the RNXtract kit. DNA and RNA specific qPCR assays were used so as to determine analyte specificity of RNXtract. For the assessment of limit of blank, limit of detection, analytical measurement range and PCR efficiency of the MammaTyper kit serial dilutions of samples were used. Analytical precision studies of MammaTyper were built around two different real time PCR platforms and involved breast tumor samples belonging to different subtypes analyzed across multiple sites and under various stipulated conditions. The MammaTyper assay robustness was tested against RNA input variations, alternative extraction methods and tumor cell content. Individual assays were linear up to at least 32.33 and 33.56 Cqs (quantification cycles) for the two qPCR platforms tested. PCR efficiency ranged from 99 to 109 %. In qPCR platform 1, estimates for assay specific inter-site standard deviations (SD) were between 0.14 and 0

Detection of apoptotic cells in tumour paraffin sections

International Nuclear Information System (INIS)

Pizem, J.; Coer, A.

2003-01-01

Apoptosis is a distinct form of cell death characterised by specific morphological features and regulated by complex molecular mechanisms. Its deregulation is fundamental for tumour growth and progression and, moreover, anticancer therapies suppress tumour growth mainly by induction of apoptosis. Since the extent of apoptosis in a tumour may have prognostic as well as therapeutic implications, much effort has been invested in developing specific methods that can be routinely used to detect apoptotic cells in archival formalin- fixed paraffin-embedded tissue. Complex molecular pathways are involved in the regulation of apoptosis. Pro-apoptotic signals trigger activation of caspases that specifically cleave target proteins. Cleavage of proteins (caspase substrates) is responsible for morphological changes of apoptotic cells and DNA fragmentation. In the last decade, detection of apoptotic cells in formalin-fixed tumour tissue sections has been based mainly on morphology and characteristic DNA fragmentation. Recently, specific antibodies to activated caspases and cleaved target proteins (including cytokeratin 18, actin and PARP) have been produced that enable accurate detection of apoptosis in paraffin sections. (author)

The storage period of the formalin-fixed paraffin-embedded tumor blocks does not influence the concentration and purity of the isolated DNA in a series of 83 renal and thyroid carcinomas.

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Nechifor-Boilă, Adela Corina; Loghin, Andrada; Vacariu, Victor; Halaţiu, Vasile Bogdan; Borda, Angela

2015-01-01

Optimal recovery of nucleic acids from formalin-fixed paraffin-embedded (FFPE) tissues is highly dependent on a series of pre-extraction steps, mainly related (but not limited) to fixation. The aim of our study was to investigate if the storage period of the FFPE blocks had a significant effect on the isolated DNA. We examined the quantity and purity of the isolated DNA from 83 FFPE blocks, corresponding to malignant thyroid (n=28) and renal (n=55) carcinomas that had been stored in our department for up to eight years. The DNA extraction protocol was based on a precipitation method (MasterPure™ DNA Purification Kit, Epicentre), in accordance to the manufacturer instructions, optimized in our laboratory. A spectrophotometer was used to determine the yield (A260) and purity (A260/A280 ratio) of the isolated DNA. We successfully isolated good DNA quantity and purity from all our study cases (mean concentration: 223.4 ± 104.16 ng/μL; mean A260/A280 ratio: 1.68 ± 0.09). Moreover, no statistically significant differences were observed between tumor blocks stored for 2-3 years and 7-8 years, respectively, both in terms of DNA quantity (p=0.196) and purity (p=0.663). In conclusion, we successfully validated an efficient, reproducible DNA extraction technique that provided a good range of DNA concentrations and purity, regardless the type of tissue (thyroid or kidney). Moreover, we demonstrated that the storage period of the FFPE blocks does not have a significant influence on the DNA quantity and purity.

Optical redox imaging of fixed unstained tissue slides to identify biomarkers for breast cancer diagnosis/prognosis: feasibility study

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Xu, He N.; Tchou, Julia; Li, Yusheng; Feng, Min; Zhang, Paul; Quinn, William J.; Baur, Joseph A.; Li, Lin Z.

2018-02-01

We previously showed that optical redox imaging (ORI) of snap-frozen breast biopsies by the Chance redox scanner readily discriminates cancer from normal tissue. Moreover, indices of redox heterogeneity differentiate among tumor xenografts with different metastatic potential. These observations suggest that ORI of fluorescence of NADH and oxidized flavoproteins (Fp) may provide diagnostic/prognostic value for clinical applications. In this work, we investigate whether ORI of formalin-fixed-paraffin-embedded (FFPE) unstained clinical tissue slides of breast tumors is feasible and comparable to ORI of snap-frozen tumors. If ORI of FFPE is validated, it will enhance the versatility of ORI as a novel diagnostic/prognostic assay as FFPE samples are readily available. ORI of fixed tissue slides was performed using a fluorescence microscope equipped with a precision automated stage and appropriate optical filters. We developed a vignette correction algorithm to remove the tiling effect of stitched-images. The preliminary data from imaging fixed slides of breast tumor xenografts showed intratumor redox heterogeneity patterns similar to that of the frozen tissues imaged by the Chance redox scanner. From ORI of human breast tissue slides we identified certain redox differences among normal, ductal carcinoma in situ, and invasive carcinoma. We found paraformaldehyde fixation causes no change in NADH signals but enhances Fp signals of fresh muscle fibers. We also investigated the stability of the fluorescence microscope and reproducibility of tissue slide fluorescence signals. We plan to validate the diagnostic/prognostic value of ORI using clinically annotated breast cancer sample set from patients with long-term follow-up data.

Diagnosis of Nocardia paucivorans central nervous system infection by DNA sequencing from paraffin-embedded tissue.

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Schiaroli, Elisabetta; Pasticci, Maria Bruna; De Carolis, Elena; Mello, Enrica; Pallotto, Carlo; Leli, Christian; De Socio, Giuseppe Vittorio; Baldelli, Franco; Sanguinetti, Maurizio; Mencacci, Antonella

2016-06-01

Infections by Nocardia spp. are generally regarded as opportunistic diseases in immunocompromised patients, but can also affect immunocompetent subjects. Such infections represent an important diagnostic challenge for clinicians and microbiologists, and diagnosis is frequently delayed or even conducted post mortem. A 54-year-old man was admitted to our hospital because of ventriculitis and relapsing brain abscess. Five months prior, this patient had undergone external ventricular drain and surgery for a cerebellar abscess. Histopathology demonstrated pyogenic inflammatory reaction, microbiologic investigations proved negative and empiric antimicrobial therapy was administered for a total of eight weeks. Six weeks later, the patient developed relapsing neurologic manifestations. On reviewing the patient's clinical history it emerged that the patient had suffered pneumonia two months prior to neurosurgery, treated with amoxicillin/clavulanate 3g a day and levofloxacin 500mg a day for three weeks. On the CNS relapsing manifestations, nocardiosis was suspected and DNA sequencing from the formalin-fixed paraffin-embedded cerebellar tissue collected during neurosurgery allowed diagnosis of Nocardia paucivorans infection. The patient received medical therapy for 11 months. At follow-up, eight months after treatment was discontinued, the patient was aymptomatic. Nocardia spp. infections need to be suspected not only in immunocompromised, but also in immunocompetent patients. Proper samples need to be collected for proper microbiologic investigations. Paraffin-embedded tissue genomic sequencing can be a useful tool for diagnosis of nocardiosis.

A SIMPLE PARAFFIN EMBEDDED PROTOCOL FOR FISH EGG, EMBRYO, AND LARVAE

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Gratiana Eka Wijayanti

2017-06-01

Full Text Available This paper describes a simple protocol of paraffin-embedded histological section for fish eggs, embryo and larvae of the hard-lipped barb and the giant gourami. The specimens were fixed in Bouin solution, washed in 70% ethanol, then were dehydrated in a series of ethanol solution of increasing concentration until absolute ethanol was reached. The specimens were cleared in graded xylene and were infiltrated with liquid paraffin then were embedded in pure paraffin. Upon sectioning, at 4–5 µm thick the specimens were attached to the gelatin-coated glass slide and let to dry at room temperature or 37°C overnight. The specimens were deparaffinized in xylene, rehydrated then were stained with hematoxylin and eosin. After being dehydrated in graded ethanol, the specimens were cleared in xylene and were mounted with an organic mounting agent. Any step in preparing histological section including samples collection, fixation, dehydration, infiltration and embedding might contribute to the quality of histological features. A proper knowledge of the tissues beeing processed, fixative solution and the histological techniques is essential to gain good results. Bouin fixative is preferable to fix fish larvae and produce a good histological feature. Decalcification is necessary to produce a good histological section on the specimens containing bone.

The Cellient System for Paraffin Histology Can Be Combined with HPV Testing and Morphotyping the Vaginal Microbiome Thanks to BoonFixing

OpenAIRE

Boon, Mathilde E.

2013-01-01

The Cellient Automated Cell Block System (Hologic) can be used to process cervical scrapes to paraffin sections. For the first study on this subject, cervical scrapes were fixed in the formalin-free fixative BoonFix. This pilot study was limited to cases classified as atypical squamous lesion of unknown significance (ASCUS) and high-grade squamous lesion (HSIL) as diagnosed in the ThinPrep slide. The Cellient paraffin sections were classified into negative, atypical, CIN 1, CIN 2, and CIN 3. ...

Improving the Prediction of Prostate Cancer Overall Survival by Supplementing Readily Available Clinical Data with Gene Expression Levels of IGFBP3 and F3 in Formalin-Fixed Paraffin Embedded Core Needle Biopsy Material.

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Zhuochun Peng

Full Text Available A previously reported expression signature of three genes (IGFBP3, F3 and VGLL3 was shown to have potential prognostic value in estimating overall and cancer-specific survivals at diagnosis of prostate cancer in a pilot cohort study using freshly frozen Fine Needle Aspiration (FNA samples.We carried out a new cohort study with 241 prostate cancer patients diagnosed from 2004-2007 with a follow-up exceeding 6 years in order to verify the prognostic value of gene expression signature in formalin fixed paraffin embedded (FFPE prostate core needle biopsy tissue samples. The cohort consisted of four patient groups with different survival times and death causes. A four multiplex one-step RT-qPCR test kit, designed and optimized for measuring the expression signature in FFPE core needle biopsy samples, was used. In archive FFPE biopsy samples the expression differences of two genes (IGFBP3 and F3 were measured. The survival time predictions using the current clinical parameters only, such as age at diagnosis, Gleason score, PSA value and tumor stage, and clinical parameters supplemented with the expression levels of IGFBP3 and F3, were compared.When combined with currently used clinical parameters, the gene expression levels of IGFBP3 and F3 are improving the prediction of survival time as compared to using clinical parameters alone.The assessment of IGFBP3 and F3 gene expression levels in FFPE prostate cancer tissue would provide an improved survival prediction for prostate cancer patients at the time of diagnosis.

Simultaneous phenotyping and genotyping (FICTION-methodology) on paraffin sections and cytologic specimens: a comparison of 2 different protocols

DEFF Research Database (Denmark)

Bzorek, M.Sr.; Hansen, L.; Petersen, Bodil Laub

2008-01-01

for the investigation of neoplasms)]. However, few studies have been applied to the technical problems posed by antigen retrieval and accessibility of genetic probes to target-DNA, using formalin-fixed, paraffin-embedded tissue. In this study, we compared 2 immunofluorescence detection systems, the 3-step IF (TIF......) method against the Tyramide Signal Amplification techniques (TSA). The FICTION-TSA technique significantly improved the sensitivity for detection of the immunophenotypic markers without influencing specific probe hybridization to target-DNA, compared with the results obtained with the TIF method....... The reaction product of the TSA system was robust to the following FISH procedure in contrast to the TIF technique. The TSA technique used also allowed synchronous detection of nuclear antigens and FISH signals using both fusion (IgH/CCND1) and break-apart (CCND1) probes on formalin-fixed paraffin...

Droplet digital PCR (ddPCR) vs quantitative real-time PCR (qPCR) approach for detection and quantification of Merkel cell polyomavirus (MCPyV) DNA in formalin fixed paraffin embedded (FFPE) cutaneous biopsies.

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Arvia, Rosaria; Sollai, Mauro; Pierucci, Federica; Urso, Carmelo; Massi, Daniela; Zakrzewska, Krystyna

2017-08-01

Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma and high viral load in the skin was proposed as a risk factor for the occurrence of this tumour. MCPyV DNA was detected, with lower frequency, in different skin cancers but since the viral load was usually low, the real prevalence of viral DNA could be underestimated. To evaluate the performance of two assays (qPCR and ddPCR) for MCPyV detection and quantification in formalin fixed paraffin embedded (FFPE) tissue samples. Both assays were designed to simultaneous detection and quantification of both MCPyV as well as house-keeping DNA in clinical samples. The performance of MCPyV quantification was investigated using serial dilutions of cloned target DNA. We also evaluated the applicability of both tests for the analysis of 76 FFPE cutaneous biopsies. The two approaches resulted equivalent with regard to the reproducibility and repeatability and showed a high degree of linearity in the dynamic range tested in the present study. Moreover, qPCR was able to quantify ≥10 5 copies per reaction, while the upper limit of ddPCR was 10 4 copies. There was not significant difference between viral load measured by the two methods The detection limit of both tests was 0,15 copies per reaction, however, the number of positive samples obtained by ddPCR was higher than that obtained by qPCR (45% and 37% respectively). The ddPCR represents a better method for detection of MCPyV in FFPE biopsies, mostly these containing low copies number of viral genome. Copyright © 2017 Elsevier B.V. All rights reserved.

Terahertz Imaging of Three-Dimensional Dehydrated Breast Cancer Tumors

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Bowman, Tyler; Wu, Yuhao; Gauch, John; Campbell, Lucas K.; El-Shenawee, Magda

2017-06-01

This work presents the application of terahertz imaging to three-dimensional formalin-fixed, paraffin-embedded human breast cancer tumors. The results demonstrate the capability of terahertz for in-depth scanning to produce cross section images without the need to slice the tumor. Samples of tumors excised from women diagnosed with infiltrating ductal carcinoma and lobular carcinoma are investigated using a pulsed terahertz time domain imaging system. A time of flight estimation is used to obtain vertical and horizontal cross section images of tumor tissues embedded in paraffin block. Strong agreement is shown comparing the terahertz images obtained by electronically scanning the tumor in-depth in comparison with histopathology images. The detection of cancer tissue inside the block is found to be accurate to depths over 1 mm. Image processing techniques are applied to provide improved contrast and automation of the obtained terahertz images. In particular, unsharp masking and edge detection methods are found to be most effective for three-dimensional block imaging.

Paraffin-based immunohistochemistry in the evaluation of glomerular diseases in renal biopsies

International Nuclear Information System (INIS)

Rathore, M.U.; Khadim, M.T.; Atique, M.

2012-01-01

Objective: To determine sensitivity and specificity of paraffin-based immunohistochemistry in the evaluation of glomerular diseases in renal biopsies using immunofluorescence as gold standard. Study Design: Cross-sectional analytical study. Place and Duration of Study: Department of Histopathology, Armed Forces Institute of Pathology, Rawalpindi, from August 2008 to August 2009. Methodology: Seventy renal biopsy specimens fulfilling the inclusion criteria for light microscopy and immuno-fluorescence during the study period were evaluated. Antibodies to immunoglobulins (IgG, IgA, and IgM) and components of complement system (C3) were applied on 70 formalin-fixed paraffin-embedded renal biopsy specimens previously classified by means of light microscopy and immunofluorescence (IF). Staining for these antibodies was recorded as positive and negative for immunohistochemistry (IHC) and IF in paired proportions presuming IF as gold standard test. The sensitivity, specificity, positive predictive value and negative predictive value of individual antibody were calculated. Results: Of 70 patients, mean age was 33 +- 18 years ranging from 2 to 80 years. Forty five (64%) were males and 25 (36%) were females. The sensitivity, specificity and predictive values of individual antibodies to IgG, IgA, IgM and C3 were very low and generally in the range of 40 - 60%. Conclusion: The sensitivity, specificity and predictive values of immunohistochemistry on formalin-fixed paraffin-embedded renal biopsy specimens were very low and therefore, not suitable for evaluation of renal biopsies in current circumstances. (author)

Prediction of the prognosis of breast cancer in routine histologic specimens using a simplified, low-cost gene expression signature

DEFF Research Database (Denmark)

Marcell, S.A.; Balazs, A.; Emese, A.

2013-01-01

Prediction of the prognosis of breast cancer in routine histologic specimens using a simplified, low-cost gene expression signature Background: Grade 2 breast carcinomas do not form a uniform prognostic group. Aim: To extend the number of patients and the investigated genes of a previously...... grade 2 breast carcinomas into prognostic groups. Gene expression was investigated by polymerase chain reaction in 249 formalin-fixed, paraffin-embedded breast tumors. The results were correlated with relapse-free survival. Results: Histologically grade 2 carcinomas were split into good and a poor...... identified prognostic signature described by the authors that reflect chromosomal instability in order to refine characterization of grade 2 breast cancers and identify driver genes. Methods: Using publicly available databases, the authors selected 9 target and 3 housekeeping genes that are capable to divide...

Chromogenic In Situ Hybridization and p16/Ki67 Dual Staining on Formalin-Fixed Paraffin-Embedded Cervical Specimens: Correlation with HPV-DNA Test, E6/E7 mRNA Test, and Potential Clinical Applications

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Roberta Zappacosta

2013-01-01

Full Text Available Although HPV-DNA test and E6/E7 mRNA analyses remain the current standard for the confirmation of human papillomavirus (HPV infections in cytological specimens, no universally adopted techniques exist for the detection of HPV in formalin-fixed paraffin-embedded samples. Particularly, in routine laboratories, molecular assays are still time-consuming and would require a high level of expertise. In this study, we investigated the possible use of a novel HPV tyramide-based chromogenic in situ hybridization (CISH technology to locate HPV on tissue specimens. Then, we evaluate the potential usefulness of p16INK4a/Ki-67 double stain on histological samples, to identify cervical cells expressing HPV E6/E7 oncogenes. In our series, CISH showed a clear signal in 95.2% of the specimens and reached a sensitivity of 86.5%. CISH positivity always matched with HPV-DNA positivity, while 100% of cases with punctated signal joined with cervical intraepithelial neoplasia grade 2 or worse (CIN2+. p16/Ki67 immunohistochemistry gave an interpretable result in 100% of the cases. The use of dual stain significantly increased the agreement between pathologists, which reached 100%. Concordance between dual stain and E6/E7 mRNA test was 89%. In our series, both CISH and p16INK4a/Ki67 dual stain demonstrated high grade of performances. In particular, CISH would help to distinguish episomal from integrated HPV, in order to allow conclusions regarding the prognosis of the lesion, while p16INK4a/Ki67 dual stain approach would confer a high level of standardization to the diagnostic procedure.

Chromogenic In Situ Hybridization and p16/Ki67 Dual Staining on Formalin-Fixed Paraffin-Embedded Cervical Specimens: Correlation with HPV-DNA Test, E6/E7 mRNA Test, and Potential Clinical Applications

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Zappacosta, Roberta; Colasante, Antonella; Viola, Patrizia; D'Antuono, Tommaso; Lattanzio, Giuseppe; Capanna, Serena; Gatta, Daniela Maria Pia; Rosini, Sandra

2013-01-01

Although HPV-DNA test and E6/E7 mRNA analyses remain the current standard for the confirmation of human papillomavirus (HPV) infections in cytological specimens, no universally adopted techniques exist for the detection of HPV in formalin-fixed paraffin-embedded samples. Particularly, in routine laboratories, molecular assays are still time-consuming and would require a high level of expertise. In this study, we investigated the possible use of a novel HPV tyramide-based chromogenic in situ hybridization (CISH) technology to locate HPV on tissue specimens. Then, we evaluate the potential usefulness of p16INK4a/Ki-67 double stain on histological samples, to identify cervical cells expressing HPV E6/E7 oncogenes. In our series, CISH showed a clear signal in 95.2% of the specimens and reached a sensitivity of 86.5%. CISH positivity always matched with HPV-DNA positivity, while 100% of cases with punctated signal joined with cervical intraepithelial neoplasia grade 2 or worse (CIN2+). p16/Ki67 immunohistochemistry gave an interpretable result in 100% of the cases. The use of dual stain significantly increased the agreement between pathologists, which reached 100%. Concordance between dual stain and E6/E7 mRNA test was 89%. In our series, both CISH and p16INK4a/Ki67 dual stain demonstrated high grade of performances. In particular, CISH would help to distinguish episomal from integrated HPV, in order to allow conclusions regarding the prognosis of the lesion, while p16INK4a/Ki67 dual stain approach would confer a high level of standardization to the diagnostic procedure. PMID:24369532

Quality assessment of DNA derived from up to 30 years old formalin fixed paraffin embedded (FFPE) tissue for PCR-based methylation analysis using SMART-MSP and MS-HRM.

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Kristensen, Lasse S; Wojdacz, Tomasz K; Thestrup, Britta B; Wiuf, Carsten; Hager, Henrik; Hansen, Lise Lotte

2009-12-21

The High Resolution Melting (HRM) technology has recently been introduced as a rapid and robust analysis tool for the detection of DNA methylation. The methylation status of multiple tumor suppressor genes may serve as biomarkers for early cancer diagnostics, for prediction of prognosis and for prediction of response to treatment. Therefore, it is important that methodologies for detection of DNA methylation continue to evolve. Sensitive Melting Analysis after Real Time - Methylation Specific PCR (SMART-MSP) and Methylation Sensitive - High Resolution Melting (MS-HRM) are two methods for single locus DNA methylation detection based on HRM. Here, we have assessed the quality of DNA extracted from up to 30 years old Formalin Fixed Paraffin Embedded (FFPE) tissue for DNA methylation analysis using SMART-MSP and MS-HRM. The quality assessment was performed on DNA extracted from 54 Non-Small Cell Lung Cancer (NSCLC) samples derived from FFPE tissue, collected over 30 years and grouped into five years intervals. For each sample, the methylation levels of the CDKN2A (p16) and RARB promoters were estimated using SMART-MSP and MS-HRM assays designed to assess the methylation status of the same CpG positions. This allowed for a direct comparison of the methylation levels estimated by the two methods for each sample. CDKN2A promoter methylation levels were successfully determined by SMART-MSP and MS-HRM in all 54 samples. Identical methylation estimates were obtained by the two methods in 46 of the samples. The methylation levels of the RARB promoter were successfully determined by SMART-MSP in all samples. When using MS-HRM to assess RARB methylation five samples failed to amplify and 15 samples showed a melting profile characteristic for heterogeneous methylation. Twenty-seven of the remaining 34 samples, for which the methylation level could be estimated, gave the same result as observed when using SMART-MSP. MS-HRM and SMART-MSP can be successfully used for single locus

The Resin-Embedded Cornea Prepared Via Rapid Processing Protocol : A Good Histomorphometric Target for Clinical Investigation in Ophthalmology and Optometry

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Cheah, Pike See; Mohidin, Norhani; Mohd Ali, Bariah; Maung, Myint; Latif, Azian Abdul

2008-01-01

This study illustrates and quantifies the changes on corneal tissue between the paraffin-embedded and resin-embedded blocks and thus, selects a better target in investigational ophthalmology and optometry via light microscopy. Corneas of two cynomolgus monkeys (Macaca fascicularis) were used in this study. The formalin-fixed cornea was prepared in paraffin block via the conventional tissue processing protocol (4-day protocol) and stained with haematoxylin and eosin. The glutaraldehyde-fixed cornea was prepared in resin block via the rapid and modified tissue processing procedure (1.2-day protocol) and stained with toluidine blue. The paraffin-embedded sample exhibits various undesired tissue damage and artifact such as thinner epithelium (due to the substantial volumic extraction from the tissue), thicker stroma layer (due to the separation of lamellae and the presence of voids) and the distorted endothelium. In contrast, the resin-embedded corneal tissue has demonstrated satisfactory corneal ultrastructural preservation. The rapid and modified tissue processing method for preparing the resin-embedded is particularly beneficial to accelerate the microscopic evaluation in ophthalmology and optometry. PMID:22570589

Replacing xylene with n-heptane for paraffin embedding.

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Stockert, J C; López-Arias, B; Del Castillo, P; Romero, A; Blázquez-Castro, A

2012-10-01

In standard histological technique, aromatic solvents such as xylene and toluene are used as clearing agents between ethanol dehydration and paraffin embedding. In addition, these solvents are used for de-waxing paraffin sections. Unfortunately, these solvents are harmful and therefore adequate substitutes would be useful. We suggest the use of n-heptane as a convenient substitute for xylene. Paraffin sections of rat tissues processed with n-heptane and stained with hematoxylin-eosin or Masson's trichrome showed proper embedment, well preserved morphology and excellent staining.

Cytology specimens offer an effective alternative to formalin-fixed tissue as demonstrated by novel automated detection for ALK break-apart FISH testing and immunohistochemistry in lung adenocarcinoma.

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Rosenblum, Frida; Hutchinson, Lloyd M; Garver, Joann; Woda, Bruce; Cosar, Ediz; Kurian, Elizabeth M

2014-11-01

Minimally invasive sampling by cytology or core needle biopsy often provides an initial diagnosis for treatment in patients with lung nodules. From these limited specimens, multiple molecular studies are frequently requested. Current guidelines from the US Food and Drug Administration recommend using formalin-fixed paraffin-embedded tissue sections for the detection of anaplastic lymphoma kinase (ALK) gene rearrangement by fluorescence in situ hybridization (FISH). The authors compared alcohol-fixed and formalin-fixed cytology specimens using a novel automated detection for ALK rearrangements by FISH and immunohistochemistry (IHC). ALK FISH testing was performed on 129 lung adenocarcinomas from 71 cytology cases and 58 biopsy/resection specimens using Papanicolaou staining with integrated cytomorphology. IHC with the ALK D5F3 antibody was performed on cases with residual material (88 of 129 cases). The mean age of the patients was 66 years; there were 62 women and 67 men. ALK gene rearrangement was present in 4% of cytology specimens (3 of 71 specimens) and 7% of surgical specimens (4 of 58 specimens). FISH in 13 cases was technically unsuccessful. Of the 7 FISH-positive cases, only 2 cytology cases (4%) and 2 surgical cases (6%) were found to be positive with the ALK antibody, demonstrating 80% concordance. The one case found to be negative for ALK by IHC demonstrated a variant rearrangement of the ALK 2p23 gene locus by FISH. The results of the current study validate the usefulness of alcohol-fixed and/or formalin-fixed cytology specimens for ALK rearrangement by a novel automated FISH method. IHC using the D5F3 antibody for ALK is specific in this limited cohort. The authors also demonstrated that alcohol-fixed cytology specimens can be used for ALK rearrangement by automated FISH, alone or in conjunction with IHC. © 2014 American Cancer Society.

Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay.

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Cronin, Maureen; Pho, Mylan; Dutta, Debjani; Stephans, James C; Shak, Steven; Kiefer, Michael C; Esteban, Jose M; Baker, Joffre B

2004-01-01

Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10- micro m FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.

Detection of Chlamydia in postmortal formalin-fixed tissue

DEFF Research Database (Denmark)

Lundemose, AG; Lundemose, JB; Birkelund, Svend

1989-01-01

A procedure to detect Chlamydia in postmortal formalin-fixed tissue is described. Monoclonal antibodies against a genus specific chlamydia epitope were used in immunofluorescence to detect chlamydia inclusions in formalin-fixed tissue sections. Lung sections from chlamydia-infected mice were....... Background and non-specific fluorescence were reduced by treating the tissue sections with trypsin, rabbit serum and Evans blue counterstain. Besides giving an exact diagnosis at autopsy, the method provides the possibility of determining the occurrence of chlamydia infections in various tissues, based...

Comparação de três protocolos distintos para extração de RNA de amostras fixadas em formalina e emblocadas em parafina Comparison of three different protocols for extracting RNA from formalin-fixed paraffin-embedded tissues

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Gisele Rodrigues Gouveia

2011-12-01

Full Text Available INTRODUÇÃO: Tecidos fixados em formalina e emblocados em parafina (FFEP são importantes fontes de amostras para estudos retrospectivos. Apesar de sua capacidade de preservação de proteínas e morfologia celular, a formalina interfere negativamente em testes de biologia molecular por fragmentar e modificar quimicamente os ácidos nucleicos, particularmente o ácido ribonucleico (RNA. OBJETIVO: Comparar a eficiência de três diferentes protocolos de extração de RNA para análise de expressão gênica de tecidos FFEP. MATERIAL E MÉTODOS: Amostras de linfonodo humano FEEP foram submetidas à extração de RNA utilizando-se os kits Ambion e Arcturus Bioscience e o método clássico de Trizol. Após a extração, o RNA foi quantificado e testado quanto à sua capacidade de amplificaç��o pela reação em cadeia da polimerase em tempo real (RT-PCR utilizando primers do gene endógeno gliceraldeído-3 fosfato desidrogenase (GAPDH. DISCUSSÃO/CONCLUSÃO: Todos os protocolos testados produziram quantidades adequadas e suficientes de RNA total, entretanto, somente os protocolos com uso dos kits Ambion e Arcturus produziram RNA capaz de ser amplificado pela PCR.INTRODUCTION: Formalin fixed paraffin embedded (FFPE tissues are an important sample source for retrospective studies. Despite its ability to preserve proteins and cell morphology, formalin hinders Molecular Biology tests once it fragments and chemically modifies nucleic acids, particularly RNA. OBJECTIVE: To compare the efficiency of three different RNA extraction protocols for gene expression analysis of FFEP tissues. MATERIAL AND METHODS: RNA was extracted from FFPE samples of human lymph by means of Ambion and Arcturus Bioscience kits and the classical Trizol method. After extraction, RNA was quantified and tested for amplification through real time polymerase chain reaction (RT-PCR using glyceraldehydes-3 phosphate dehydrogenase (GAPDH endogenous gene primers. DISCUSSION

Is TIMP-1 immunoreactivity alone or in combination with other markers a predictor of benefit from anthracyclines in the BR9601 adjuvant breast cancer chemotherapy trial?

DEFF Research Database (Denmark)

Munro, Alison F.; Bartels, Annette; Balslev, Eva

2013-01-01

copy number can be used to predict benefit from epirubicin (E) containing chemotherapy compared with cyclophosphamide, methotrexate and fluorouracil (CMF) treatment. METHODS: For the purpose of this study, formalin fixed paraffin embedded tumor tissue from women recruited into the BR9601 clinical trial...

Role of chromogenic in situ hybridization (CISH) in the evaluation of HER2 status in breast carcinoma: comparison with immunohistochemistry and FISH.

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Li-Ning-T, Elsa; Ronchetti, Ruben; Torres-Cabala, Carlos; Merino, Maria J

2005-10-01

We report our experience with Chromogenic in Situ Hybridization (CISH) for the evaluation of HER2 amplification on 55 cases of formalin-fixed, paraffin-embedded invasive breast carcinomas of different histology. All the results were corrected for chromosome 17 aneusomy and compared with immunohistochemistry (IHC); a subset of cases was compared to FISH. Thirty-one of 32 cases in which FISH and CISH were performed yielded the same results. CISH and IHC showed a good concordance in the 0/1+ and 3+ category, while a poor agreement with weakly protein overexpression was confirmed. Chromosome 17 analysis was necessary in cases with a low number of HER2 gene copies. CISH is a useful tool to evaluate breast cancer HER2 status that can be easily implemented in a laboratory of surgical pathology.

Thiel embalming fluid--a new way to revive formalin-fixed cadaveric specimens.

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Hunter, Amanda; Eisma, Roos; Lamb, Clare

2014-09-01

By soft fixing cadavers using the Thiel embalming method, our cadavers now exhibit a greater degree of flexibility and color retention compared to that of traditional formalin-fixed cadavers. The aim of this experiment was to discover whether Thiel embalming fluid could be used to revive and soften the muscles of formalin-fixed prosected specimens. Earlier this year, two severely dehydrated formalin-fixed forearm and hand specimens were fully submerged in a tank containing Thiel embalming fluid. After a period of six months the specimens were removed from the tank and noticeable changes were observed in flexibility, quality of the tissue, and color of the specimens. © 2014 Wiley Periodicals, Inc.

Eradication of breast cancer with bone metastasis by autologous formalin-fixed tumor vaccine (AFTV) combined with palliative radiation therapy and adjuvant chemotherapy: a case report.

Science.gov (United States)

Kuranishi, Fumito; Ohno, Tadao

2013-06-04

Skeletal metastasis of breast carcinoma is refractory to intensive chemo-radiation therapy and therefore is assumed impossible to cure. Here, we report an advanced case of breast cancer with vertebra-Th7 metastasis that showed complete response to combined treatments with formalin-fixed autologous tumor vaccine (AFTV), palliative radiation therapy with 36 Gy, and adjuvant chemotherapy with standardized CEF (cyclophosphamide, epirubicin, and 5FU), zoledronic acid, and aromatase inhibitors following mastectomy for the breast tumor. The patient has been disease-free for more than 4 years after the mammary surgery and remains well with no evidence of metastasis or local recurrence. Thus, a combination of AFTV, palliative radiation therapy, and adjuvant chemotherapy may be an effective treatment for this devastating disease.

Prognostic role of a multigene reverse transcriptase-PCR assay in patients with node-negative breast cancer not receiving adjuvant systemic therapy.

Science.gov (United States)

Esteva, Francisco J; Sahin, Aysegul A; Cristofanilli, Massimo; Coombes, Kevin; Lee, Sang-Joon; Baker, Joffre; Cronin, Maureen; Walker, Michael; Watson, Drew; Shak, Steven; Hortobagyi, Gabriel N

2005-05-01

To test the ability of a reverse transcriptase-PCR (RT-PCR) assay, based on gene expression profiles, to accurately determine the risk of recurrence in patients with node-negative breast cancer who did not receive systemic therapy using formalin-fixed, paraffin-embedded tissue. A secondary objective was to determine whether the quantitative RT-PCR data correlated with immunohistochemistry assay data regarding estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status. We obtained archival paraffin-embedded tissue from patients with invasive breast cancer but no axillary lymph node involvement who had received no adjuvant systemic therapy and been followed for at least 5 years. RNA was extracted from three 10-microm-thick sections. The expression of 16 cancer-related genes and 5 reference genes was quantified using RT-PCR. A gene expression algorithm was used to calculate a recurrence score for each patient. We then assessed the ability of the test to accurately predict distant recurrence-free survival in this population. We identified 149 eligible patients. Median age at diagnosis was 59 years; mean tumor diameter was 2 cm; and 69% of tumors were estrogen receptor positive. Median follow-up was 18 years. The 5-year disease-free survival rate for the group was 80%. The 21 gene-based recurrence score was not predictive of distant disease recurrence. However, a high concordance between RT-PCR and immunohistochemical assays for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status was noted. RT-PCR can be done on paraffin-embedded tissue to validate the large numbers of genes associated with breast cancer recurrence. However, further work needs to be done to develop an assay to identify the likelihood of recurrent disease in patients with node-negative breast cancer who do not receive adjuvant tamoxifen or chemotherapy.

Quantification of microRNA-21 and microRNA-125b in melanoma tissue

DEFF Research Database (Denmark)

Wandler, Anne; Riber-Hansen, Rikke; Hager, Henrik

2017-01-01

Although microRNAs (miRNAs) have emerged as potent mediators of melanoma development and progression, a precise understanding of their oncogenic role remains unclear. In this study, we analysed formalin-fixed and paraffin-embedded tissues from two separate melanoma cohorts and from a series...... the important involvement of different miRNAs in melanoma biology and may serve as solid basics for further miRNA investigations in melanoma formalin-fixed and paraffin-embedded tissue. In particular, there is increased expression of miR-21 in melanomas compared with benign nevi....

A high-throughput method to detect RNA profiling by integration of RT-MLPA with next generation sequencing technology.

Science.gov (United States)

Wang, Jing; Yang, Xue; Chen, Haofeng; Wang, Xuewei; Wang, Xiangyu; Fang, Yi; Jia, Zhenyu; Gao, Jidong

2017-07-11

RNA in formalin-fixed and paraffin-embedded (FFPE) tissues provides large amount of information indicating disease stages, histological tumor types and grades, as well as clinical outcomes. However, Detection of RNA expression levels in formalin-fixed and paraffin-embedded samples is extremely difficult due to poor RNA quality. Here we developed a high-throughput method, Reverse Transcription-Multiple Ligation-dependent Probe Sequencing (RT-MLPSeq), to determine expression levels of multiple transcripts in FFPE samples. By combining Reverse Transcription-Multiple Ligation-dependent Amplification method and next generation sequencing technology, RT-MLPSeq overcomes the limit of probe length in multiplex ligation-dependent probe amplification assay and thus could detect expression levels of transcripts without quantitative limitations. We proved that different RT-MLPSeq probes targeting on the same transcripts have highly consistent results and the starting RNA/cDNA input could be as little as 1 ng. RT-MLPSeq also presented consistent relative RNA levels of selected 13 genes with reverse transcription quantitative PCR. Finally, we demonstrated the application of the new RT-MLPSeq method by measuring the mRNA expression levels of 21 genes which can be used for accurate calculation of the breast cancer recurrence score - an index that has been widely used for managing breast cancer patients.

Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing.

Directory of Open Access Journals (Sweden)

Sarah M Hykin

Full Text Available For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles, attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens-particularly for use in phylogenetic analyses-has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp. We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens

Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing.

Science.gov (United States)

Hykin, Sarah M; Bi, Ke; McGuire, Jimmy A

2015-01-01

For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens-particularly for use in phylogenetic analyses-has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for

Phase-contrast X-ray computed tomography of non-formalin fixed biological objects

Energy Technology Data Exchange (ETDEWEB)

Takeda, Tohoru E-mail: ttakeda@md.tsukuba.ac.jp; Momose, Atsushi; Wu, Jin; Zeniya, Tsutomu; Yu Quanwen; Thet Thet Lwin; Itai, Yuji

2001-07-21

Using a monolithic X-ray interferometer having the view size of 25 mmx25 mm, phase-contrast X-ray CT (PCCT) was performed for non-formalin fixed livers of two normal rats and a rabbit transplanted with VX-2 cancer. PCCT images of liver and cancer lesions resembled well those obtained by formalin fixed samples.

Detection and quantitation analysis of cocaine and metabolites in fixed liver tissue and formalin solutions.

Science.gov (United States)

Cingolani, Mariano; Cippitelli, Marcello; Froldi, Rino; Gambaro, Veniero; Tassoni, Giovanna

2004-01-01

This study reports the results of the detection and quantitation of cocaine and its metabolites in liver tissues fixed in formalin and in the formalin solutions in which the same tissues were fixed. Toxicological analyses were performed on formalin-fixed liver samples from four cases of death of cocaine abusers and on formalin solutions (10% buffered, pH 7) in which the samples were preserved. Analyses carried out at the time of autopsy on body fluids and tissues allowed identification of cocaine and the metabolite benzoylecgonine. Liver tissue samples were preserved in formalin solutions for four weeks before analysis. Results only showed the presence of benzoylecgonine in the studied materials. The mean levels of recovery of benzoylecgonine in fixed tissues were 12.31% in liver and 84.47% in formalin from liver. Results indicated that benzoylecgonine has good stability, even in biological specimens subjected to chemical fixation.

Phase-contrast X-ray computed tomography of non-formalin fixed biological objects

Science.gov (United States)

Takeda, Tohoru; Momose, Atsushi; Wu, Jin; Zeniya, Tsutomu; Yu, Quanwen; Thet-Thet-Lwin; Itai, Yuji

2001-07-01

Using a monolithic X-ray interferometer having the view size of 25 mm×25 mm, phase-contrast X-ray CT (PCCT) was performed for non-formalin fixed livers of two normal rats and a rabbit transplanted with VX-2 cancer. PCCT images of liver and cancer lesions resembled well those obtained by formalin fixed samples.

EFFECT OF EMBEDDING METHODS VERSUS FIXATIVE TYPE ON KARYOMETRIC MEASURES

NARCIS (Netherlands)

BOON, ME; VANDERPOEL, HG; TAN, CJA; KOK, LP

The influence of fixation and embedding methods in seven urologic tumor samples was studied karyometrically for 12 preparatory techniques. Routine histologic formalin fixation was compared with Carbowax and Kryofix fixatives. Also, histologic material was studied embedded in paraffin and plastic

RNA-Seq-based toxicogenomic assessment of fresh frozen and formalin-fixed tissues yields similar mechanistic insights.

Science.gov (United States)

Auerbach, Scott S; Phadke, Dhiral P; Mav, Deepak; Holmgren, Stephanie; Gao, Yuan; Xie, Bin; Shin, Joo Heon; Shah, Ruchir R; Merrick, B Alex; Tice, Raymond R

2015-07-01

Formalin-fixed, paraffin-embedded (FFPE) pathology specimens represent a potentially vast resource for transcriptomic-based biomarker discovery. We present here a comparison of results from a whole transcriptome RNA-Seq analysis of RNA extracted from fresh frozen and FFPE livers. The samples were derived from rats exposed to aflatoxin B1 (AFB1 ) and a corresponding set of control animals. Principal components analysis indicated that samples were separated in the two groups representing presence or absence of chemical exposure, both in fresh frozen and FFPE sample types. Sixty-five percent of the differentially expressed transcripts (AFB1 vs. controls) in fresh frozen samples were also differentially expressed in FFPE samples (overlap significance: P < 0.0001). Genomic signature and gene set analysis of AFB1 differentially expressed transcript lists indicated highly similar results between fresh frozen and FFPE at the level of chemogenomic signatures (i.e., single chemical/dose/duration elicited transcriptomic signatures), mechanistic and pathology signatures, biological processes, canonical pathways and transcription factor networks. Overall, our results suggest that similar hypotheses about the biological mechanism of toxicity would be formulated from fresh frozen and FFPE samples. These results indicate that phenotypically anchored archival specimens represent a potentially informative resource for signature-based biomarker discovery and mechanistic characterization of toxicity. Copyright © 2014 John Wiley & Sons, Ltd.

Improved reproducibility in genome-wide DNA methylation analysis for PAXgene® fixed samples compared to restored FFPE DNA

DEFF Research Database (Denmark)

Andersen, Gitte Brinch; Hager, Henrik; Hansen, Lise Lotte

2014-01-01

Chip. Quantitative DNA methylation analysis demonstrated that the methylation profile in PAXgene-fixed tissues showed, in comparison with restored FFPE samples, a higher concordance with the profile detected in frozen samples. We demonstrate, for the first time, that DNA from PAXgene conserved tissue performs better......Formalin fixation has been the standard method for conservation of clinical specimens for decades. However, a major drawback is the high degradation of nucleic acids, which complicates its use in genome-wide analyses. Unbiased identification of biomarkers, however, requires genome-wide studies......, precluding the use of the valuable archives of specimens with long-term follow-up data. Therefore, restoration protocols for DNA from formalin-fixed and paraffin-embedded (FFPE) samples have been developed, although they are cost-intensive and time-consuming. An alternative to FFPE and snap...

Analytic performance studies and clinical reproducibility of a real-time PCR assay for the detection of epidermal growth factor receptor gene mutations in formalin-fixed paraffin-embedded tissue specimens of non-small cell lung cancer

International Nuclear Information System (INIS)

O’Donnell, Patrick; Shieh, Felice; Wei, Wen; Lawrence, H Jeffrey; Wu, Lin; Schilling, Robert; Bloom, Kenneth; Maltzman, Warren; Anderson, Steven; Soviero, Stephen; Ferguson, Jane; Shyu, Johnny; Current, Robert; Rehage, Taraneh; Tsai, Julie; Christensen, Mari; Tran, Ha Bich; Chien, Sean Shih-Chang

2013-01-01

Epidermal growth factor receptor (EGFR) gene mutations identify patients with non-small cell lung cancer (NSCLC) who have a high likelihood of benefiting from treatment with anti-EGFR tyrosine kinase inhibitors. Sanger sequencing is widely used for mutation detection but can be technically challenging, resulting in longer turn-around-time, with limited sensitivity for low levels of mutations. This manuscript details the technical performance verification studies and external clinical reproducibility studies of the cobas EGFR Mutation Test, a rapid multiplex real-time PCR assay designed to detect 41 mutations in exons 18, 19, 20 and 21. The assay’s limit of detection was determined using 25 formalin-fixed paraffin-embedded tissue (FFPET)-derived and plasmid DNA blends. Assay performance for a panel of 201 specimens was compared against Sanger sequencing with resolution of discordant specimens by quantitative massively parallel pyrosequencing (MPP). Internal and external reproducibility was assessed using specimens tested in duplicate by different operators, using different reagent lots, instruments and at different sites. The effects on the performance of the cobas EGFR test of endogenous substances and nine therapeutic drugs were evaluated in ten FFPET specimens. Other tests included an evaluation of the effects of necrosis, micro-organisms and homologous DNA sequences on assay performance, and the inclusivity of the assay for less frequent mutations. A >95% hit rate was obtained in blends with >5% mutant alleles, as determined by MPP analysis, at a total DNA input of 150 ng. The overall percent agreement between Sanger sequencing and the cobas test was 96.7% (negative percent agreement 97.5%; positive percent agreement 95.8%). Assay repeatability was 98% when tested with two operators, instruments, and reagent lots. In the external reproducibility study, the agreement was > 99% across all sites, all operators and all reagent lots for 11/12 tumors tested. Test

The prognostic value of oncogenic antigen 519 (OA-519) expression and proliferative activity detected by antibody MIB-1 in node-negative breast cancer

DEFF Research Database (Denmark)

Jensen, V; Ladekarl, M; Holm-Nielsen, P

1995-01-01

of invasion of skin or deep fascia (= T1N0M0 and T2N0M0). The median follow-up time was 104 months (range 5-143 months). Immunohistochemical analysis of OA-519 expression was performed on formalin-fixed, paraffin-embedded tissue. The proliferative activity was estimated using a Ki-67 equivalent monoclonal...

Analytical validation of the PAM50-based Prosigna Breast Cancer Prognostic Gene Signature Assay and nCounter Analysis System using formalin-fixed paraffin-embedded breast tumor specimens

International Nuclear Information System (INIS)

Nielsen, Torsten; Storhoff, James; Wallden, Brett; Schaper, Carl; Ferree, Sean; Liu, Shuzhen; Gao, Dongxia; Barry, Garrett; Dowidar, Naeem; Maysuria, Malini

2014-01-01

NanoString’s Prosigna™ Breast Cancer Prognostic Gene Signature Assay is based on the PAM50 gene expression signature. The test outputs a risk of recurrence (ROR) score, risk category, and intrinsic subtype (Luminal A/B, HER2-enriched, Basal-like). The studies described here were designed to validate the analytical performance of the test on the nCounter Analysis System across multiple laboratories. Analytical precision was measured by testing five breast tumor RNA samples across 3 sites. Reproducibility was measured by testing replicate tissue sections from 43 FFPE breast tumor blocks across 3 sites following independent pathology review at each site. The RNA input range was validated by comparing assay results at the extremes of the specified range to the nominal RNA input level. Interference was evaluated by including non-tumor tissue into the test. The measured standard deviation (SD) was less than 1 ROR unit within the analytical precision study and the measured total SD was 2.9 ROR units within the reproducibility study. The ROR scores for RNA inputs at the extremes of the range were the same as those at the nominal input level. Assay results were stable in the presence of moderate amounts of surrounding non-tumor tissue (<70% by area). The analytical performance of NanoString’s Prosigna assay has been validated using FFPE breast tumor specimens across multiple clinical testing laboratories

Evaluation of the Branched-Chain DNA Assay for Measurement of RNA in Formalin-Fixed Tissues

Science.gov (United States)

Knudsen, Beatrice S.; Allen, April N.; McLerran, Dale F.; Vessella, Robert L.; Karademos, Jonathan; Davies, Joan E.; Maqsodi, Botoul; McMaster, Gary K.; Kristal, Alan R.

2008-01-01

We evaluated the branched-chain DNA (bDNA) assay QuantiGene Reagent System to measure RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. The QuantiGene Reagent System does not require RNA isolation, avoids enzymatic preamplification, and has a simple workflow. Five selected genes were measured by bDNA assay; quantitative polymerase chain reaction (qPCR) was used as a reference method. Mixed-effect statistical models were used to partition the overall variance into components attributable to xenograft, sample, and assay. For FFPE tissues, the coefficients of reliability were significantly higher for the bDNA assay (93–100%) than for qPCR (82.4–95%). Correlations between qPCRFROZEN, the gold standard, and bDNAFFPE ranged from 0.60 to 0.94, similar to those from qPCRFROZEN and qPCRFFPE. Additionally, the sensitivity of the bDNA assay in tissue homogenates was 10-fold higher than in purified RNA. In 9- to 13-year-old blocks with poor RNA quality, the bDNA assay allowed the correct identification of the overexpression of known cancer genes. In conclusion, the QuantiGene Reagent System is considerably more reliable, reproducible, and sensitive than qPCR, providing an alternative method for the measurement of gene expression in FFPE tissues. It also appears to be well suited for the clinical analysis of FFPE tissues with diagnostic or prognostic gene expression biomarker panels for use in patient treatment and management. PMID:18276773

Recommendations for Collection and Handling of Specimens From Group Breast Cancer Clinical Trials

Science.gov (United States)

Leyland-Jones, Brian R.; Ambrosone, Christine B.; Bartlett, John; Ellis, Matthew J.C.; Enos, Rebecca A.; Raji, Adekunle; Pins, Michael R.; Zujewski, Jo Anne; Hewitt, Stephen M.; Forbes, John F.; Abramovitz, Mark; Braga, Sofia; Cardoso, Fatima; Harbeck, Nadia; Denkert, Carsten; Jewell, Scott D.

2008-01-01

Recommendations for specimen collection and handling have been developed for adoption across breast cancer clinical trials conducted by the Breast International Group (BIG)-sponsored Groups and the National Cancer Institute (NCI)-sponsored North American Cooperative Groups. These recommendations are meant to promote identifiable standards for specimen collection and handling within and across breast cancer trials, such that the variability in collection/handling practices that currently exists is minimized and specimen condition and quality are enhanced, thereby maximizing results from specimen-based diagnostic testing and research. Three working groups were formed from the Cooperative Group Banking Committee, BIG groups, and North American breast cancer cooperative groups to identify standards for collection and handling of (1) formalin-fixed, paraffin-embedded (FFPE) tissue; (2) blood and its components; and (3) fresh/frozen tissue from breast cancer trials. The working groups collected standard operating procedures from multiple group specimen banks, administered a survey on banking practices to those banks, and engaged in a series of discussions from 2005 to 2007. Their contributions were synthesized into this document, which focuses primarily on collection and handling of specimens to the point of shipment to the central bank, although also offers some guidance to central banks. Major recommendations include submission of an FFPE block, whole blood, and serial serum or plasma from breast cancer clinical trials, and use of one fixative and buffer type (10% neutral phosphate-buffered formalin, pH 7) for FFPE tissue across trials. Recommendations for proper handling and shipping were developed for blood, serum, plasma, FFPE, and fresh/frozen tissue. PMID:18955459

Evaluation of a fluorescence-labelled oligonucleotide tide probe targeting 23S rRNA for in situ detection of Salmonella serovars in paraffin-embedded tissue sections and their rapid identification in bacterial smears

DEFF Research Database (Denmark)

Nordentoft, Steen; Christensen, H.; Wegener, Henrik Caspar

1997-01-01

with the probe. The probe did not hybridize to serovars from subspecies IIIa (S. arizonae) or to S. bongori. No cross-reaction to 64 other strains of the family Enterobacteriaceae or 18 other bacterial strains outside this family was observed. The probe was tested with sections of formalin-fixed, paraffin...

Fibrinogen Demonstration in Oral Lichen Planus: An Immunofluorescence Study on Archival Tissues.

Science.gov (United States)

Shirol, Pallavi D; Naik, Veena; Kale, Alka

2015-10-01

Lichen planus is a premalignant condition with minimal diagnostic aids. This study is an attempt to use paraffin embedded sections of lichen planus with immunofluorescein stain and to evaluate the immunofluorescent sections to establish pattern of fibrinogen deposition. Thirty-five paraffin embedded sections of old and new cases of oral lichen planus (study group) and five normal oral mucosa (control group) were chosen. Two sections of each (H & E) case were taken, one was stained with hematoxylin and eosin and another with fluorescein isothiocynate conjugate (FITC) polyclonal rabbit antibody against fibrinogen. Fluorescent findings were examined with a fluorescent microscope. A high statistical significant correlation was found in respect to fluorescence positivity, intensity of fluorescence and distribution of fluorescence each with p < 0.0001 and fluorescence at blood vessel walls (p = 0.0003). This study suggested that paraffin embedded sections can be successfully used in direct immunofluorescence staining in routine set up where only formalin fixed tissues are received. Paraffin embedded sections can be successfully used in direct immunofluorescence staining when only formalin fixed tissues are received.

Detection of Mycobacterium tuberculosis Complex in Paraffin-Embedded Tissues by the New Automated Abbott RealTime MTB Assay.

Science.gov (United States)

Fu, Yung-Chieh; Liao, I-Chuang; Chen, Hung-Mo; Yan, Jing-Jou

2016-07-01

The Abbott RealTime MTB assay, launched in June 2014, has been shown to have a competitive performance in the detection of the Mycobacterium tuberculosis (MTB) complex in respiratory specimens. The present study was conducted to investigate the usefulness of the Abbott MTB Realtime assay in the detection of MTB in formalin-fixed paraffin-embedded (FFPE) tissues. A total of 96 FFPE specimens obtained from microbiologically proven MTB cases (N=60) and nontuberculous Mycobacterium cases (N=36) were analyzed. The performance of the Abbott MTB Realtime assay was compared with that of the Roche Cobas TaqMan MTB assay. The overall sensitivity and specificity of the Abbott assay were 63.3% and 97.2%, respectively, compared with 11.7% and 100% for the Cobas assay. The detection rate of the Abbott assay was much higher among 37 acid-fast-positive specimens than among 23 acid-fast-negative specimens (89.3% versus 21.7%, respectively). The detection rate of the assay was higher among 29 resection specimens than among 31 small biopsy specimens (86.2% versus 41.9%, respectively). Our results suggest that the Abbott RealTime MTB assay can be used to differentiate MTB from nontuberculous mycobacterial infections in acid-fast-positive FFPE tissues. © 2016 by the Association of Clinical Scientists, Inc.

Standardization and optimization of fluorescence in situ hybridization (FISH) for HER-2 assessment in breast cancer: A single center experience.

Science.gov (United States)

Bogdanovska-Todorovska, Magdalena; Petrushevska, Gordana; Janevska, Vesna; Spasevska, Liljana; Kostadinova-Kunovska, Slavica

2018-05-20

Accurate assessment of human epidermal growth factor receptor 2 (HER-2) is crucial in selecting patients for targeted therapy. Commonly used methods for HER-2 testing are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Here we presented the implementation, optimization and standardization of two FISH protocols using breast cancer samples and assessed the impact of pre-analytical and analytical factors on HER-2 testing. Formalin fixed paraffin embedded (FFPE) tissue samples from 70 breast cancer patients were tested for HER-2 using PathVysion™ HER-2 DNA Probe Kit and two different paraffin pretreatment kits, Vysis/Abbott Paraffin Pretreatment Reagent Kit (40 samples) and DAKO Histology FISH Accessory Kit (30 samples). The concordance between FISH and IHC results was determined. Pre-analytical and analytical factors (i.e., fixation, baking, digestion, and post-hybridization washing) affected the efficiency and quality of hybridization. The overall hybridization success in our study was 98.6% (69/70); the failure rate was 1.4%. The DAKO pretreatment kit was more time-efficient and resulted in more uniform signals that were easier to interpret, compared to the Vysis/Abbott kit. The overall concordance between IHC and FISH was 84.06%, kappa coefficient 0.5976 (p characteristics. Differences in the pre-analytical and analytical steps can affect the hybridization quality and efficiency. The use of DAKO pretreatment kit is time-saving and cost-effective.

Distinct microbiological signatures associated with triple negative breast cancer.

Science.gov (United States)

Banerjee, Sagarika; Wei, Zhi; Tan, Fei; Peck, Kristen N; Shih, Natalie; Feldman, Michael; Rebbeck, Timothy R; Alwine, James C; Robertson, Erle S

2015-10-15

Infectious agents are the third highest human cancer risk factor and may have a greater role in the origin and/or progression of cancers, and related pathogenesis. Thus, knowing the specific viruses and microbial agents associated with a cancer type may provide insights into cause, diagnosis and treatment. We utilized a pan-pathogen array technology to identify the microbial signatures associated with triple negative breast cancer (TNBC). This technology detects low copy number and fragmented genomes extracted from formalin-fixed paraffin embedded archival tissues. The results, validated by PCR and sequencing, define a microbial signature present in TNBC tissue which was underrepresented in normal tissue. Hierarchical clustering analysis displayed two broad microbial signatures, one prevalent in bacteria and parasites and one prevalent in viruses. These signatures demonstrate a new paradigm in our understanding of the link between microorganisms and cancer, as causative or commensal in the tumor microenvironment and provide new diagnostic potential.

Lack of Evidence for a Relationship between High Risk Human Papillomaviruses and Breast Cancer in Iranian Patients.

Science.gov (United States)

Doosti, Masoud; Bakhshesh, Mehran; Zahir, Shokouh Taghipour; Shayestehpour, Mohammad; Karimi-Zarchi, Mojgan

2016-01-01

Whether there is any relationship between human papilloma virus (HPV) and breast carcinoma is not clear. Some previous studies have indicated a possible role in oncogenesis in the breast. In this study, we therefore analyzed the presence of HPV infection in breast tissues of Iranian women from Yazd city. In a cross-sectional study, formalin-fixed paraffin-embedded tissues from 87 patients with breast cancer and 84 cases with breast fibrocystic lesions (control group) were selected from a tissue archive. Grade of tumors and fibrocystic tissues were determined by two pathologists. The nested-PCR method was performed for detection of HPVs in samples. HPV genotypes were determined by sequencing and the phylogenetic tree depicted by MEGA software. Of the 87 women with breast cancer, 22.9% (20 isolates) had positive results for HPV DNA. In the control group no HPV was detected. The HPV genotypes in positive samples were HPV-16 (35%) HPV-18 (15%), HPV-6 (45%) and HPV-11 (5%). The data did not approved a significant correlation between tissue pathology of breast cancer and the HPV genotype frequency. The data did not provide any evidence for a role of high risk HPV types in oncogenesis in the breast.

MicroRNA Expression in Formalin-fixed Paraffin-embedded Cancer Tissue

DEFF Research Database (Denmark)

Boisen, Mogens Karsbøl; Dehlendorff, Christian; Linnemann, Dorte

2015-01-01

of miRNA expression in FFPE tissue samples from patients with colorectal (CRC) and pancreatic (PC) cancer and to quantify the variability associated with sample age and fixation. METHODS: High-throughput miRNA profiling results from 203 CRC and 256 PC FFPE samples as well as from 37 paired frozen....../FFPE samples from nine other CRC tumors (methodological samples) were used. Candidate reference miRNAs were identified by their correlation with global mean expression. The stability of reference genes was analyzed according to published methods. The association between sample age and global mean mi...... to global mean expression in each cancer type. Nine of these miRNAs were present in both lists, and miR-103a-3p was the most stable reference miRNA for both CRC and PC FFPE tissue. The optimal number of reference miRNAs was 4 in CRC and 10 in PC. Sample age had a significant effect on global mi...

The Immunoexpression of Glucocorticoid Receptors in Breast Carcinomas, Lactational Change, and Normal Breast Epithelium and Its Possible Role in Mammary Carcinogenesis

Directory of Open Access Journals (Sweden)

Raja Alyusuf

2017-01-01

Full Text Available The role of estrogen and progesterone receptors in breast cancer biology is well established. In contrast, other steroid hormones are less well studied. Glucocorticoids (GCs are known to play a role in mammary development and differentiation; thus, it is of interest to attempt to delineate their immunoexpression across a spectrum of mammary epithelia. Aim. To delineate the distribution pattern of glucocorticoid receptors (GRs in malignant versus nonmalignant epithelium with particular emphasis on lactational epithelium. Materials and Methods. Immunohistochemistry (IHC for GRs was performed on archival formalin-fixed paraffin-embedded tissue blocks of 96 cases comprising 52 invasive carcinomas, 21 cases with lactational change, and 23 cases showing normal mammary tissue histology. Results. Results reveal an overexpression of GRs in mammary malignant epithelium as compared to both normal and lactational groups individually and combined. GR overexpression is significantly more pronounced in HER-2-negative cancers. Discussion. This is the first study to compare GR expression in human lactating epithelium versus malignant and normal epithelium. The article discusses the literature related to the pathobiology of GCs in the breast with special emphasis on breast cancer. Conclusion. The lactational epithelium did not show overexpression of GR, while GR was overexpressed in mammary NST (ductal carcinoma, particularly HER-2-negative cancers.

Rapid virtual hematoxylin and eosin histology of breast tissue specimens using a compact fluorescence nonlinear microscope.

Science.gov (United States)

Cahill, Lucas C; Giacomelli, Michael G; Yoshitake, Tadayuki; Vardeh, Hilde; Faulkner-Jones, Beverly E; Connolly, James L; Sun, Chi-Kuang; Fujimoto, James G

2018-01-01

Up to 40% of patients undergoing breast conserving surgery for breast cancer require repeat surgeries due to close to or positive margins. The lengthy processing required for evaluating surgical margins by standard paraffin-embedded histology precludes its use during surgery and therefore, technologies for rapid evaluation of surgical pathology could improve the treatment of breast cancer by reducing the number of surgeries required. We demonstrate real-time histological evaluation of breast cancer surgical specimens by staining specimens with acridine orange (AO) and sulforhodamine 101 (SR101) analogously to hematoxylin and eosin (H&E) and then imaging the specimens with fluorescence nonlinear microscopy (NLM) using a compact femtosecond fiber laser. A video-rate computational light absorption model was used to produce realistic virtual H&E images of tissue in real time and in three dimensions. NLM imaging could be performed to depths of 100 μm below the tissue surface, which is important since many surgical specimens require subsurface evaluation due to contamination artifacts on the tissue surface from electrocautery, surgical ink, or debris from specimen handling. We validate this method by expert review of NLM images compared to formalin-fixed, paraffin-embedded (FFPE) H&E histology. Diagnostically important features such as normal terminal ductal lobular units, fibrous and adipose stromal parenchyma, inflammation, invasive carcinoma, and in situ lobular and ductal carcinoma were present in NLM images associated with pathologies identified on standard FFPE H&E histology. We demonstrate that AO and SR101 were extracted to undetectable levels after FFPE processing and fluorescence in situ hybridization (FISH) HER2 amplification status was unaffected by the NLM imaging protocol. This method potentially enables cost-effective, real-time histological guidance of surgical resections.

Optimisation and validation of methods to assess single nucleotide polymorphisms (SNPs) in archival histological material

DEFF Research Database (Denmark)

Andreassen, C N; Sørensen, Flemming Brandt; Overgaard

2004-01-01

only archival specimens are available. This study was conducted to validate protocols optimised for assessment of SNPs based on paraffin embedded, formalin fixed tissue samples.PATIENTS AND METHODS: In 137 breast cancer patients, three TGFB1 SNPs were assessed based on archival histological specimens...... precipitation).RESULTS: Assessment of SNPs based on archival histological material is encumbered by a number of obstacles and pitfalls. However, these can be widely overcome by careful optimisation of the methods used for sample selection, DNA extraction and PCR. Within 130 samples that fulfil the criteria...

RNA Sequencing of Formalin-Fixed, Paraffin-Embedded Specimens for Gene Expression Quantification and Data Mining

Directory of Open Access Journals (Sweden)

Yan Guo

2016-01-01

Full Text Available Background. Proper rRNA depletion is crucial for the successful utilization of FFPE specimens when studying gene expression. We performed a study to evaluate two major rRNA depletion methods: Ribo-Zero and RNase H. RNAs extracted from 4 samples were treated with the two rRNA depletion methods in duplicate and sequenced (N=16. We evaluated their reducibility, ability to detect RNA, and ability to molecularly subtype these triple negative breast cancer specimens. Results. Both rRNA depletion methods produced consistent data between the technical replicates. We found that the RNase H method produced higher quality RNAseq data as compared to the Ribo-Zero method. In addition, we evaluated the RNAseq data generated from the FFPE tissue samples for noncoding RNA, including lncRNA, enhancer/super enhancer RNA, and single nucleotide variation (SNV. We found that the RNase H is more suitable for detecting high-quality, noncoding RNAs as compared to the Ribo-Zero and provided more consistent molecular subtype identification between replicates. Unfortunately, neither method produced reliable SNV data. Conclusions. In conclusion, for FFPE specimens, the RNase H rRNA depletion method performed better than the Ribo-Zero. Neither method generates data sufficient for SNV detection.

Chromogenic in situ hybridization (CISH): a novel alternative in screening archival breast cancer tissue samples for HER-2/neu status

OpenAIRE

Madrid, Manuelito A; Lo, Raymundo W

2004-01-01

Background Chromogenic in situ hybridization (CISH) is emerging as a practical, cost-effective, and valid alternative to fluorescent in situ hybridization in testing for gene alteration, especially in centers primarily working with immunohistochemistry (IHC). Methods We assessed Her-2/neu alteration using CISH on formalin-fixed paraffin-embedded primary invasive ductal carcinoma tumors in which IHC (CB11 antibody) had previously been performed, and we compared the results with IHC. The 160 se...

In situ hybridization for the detection of infectious laryngotracheitis virus in sections of trachea from experimentally infected chickens

DEFF Research Database (Denmark)

Nielsen, O.L.; Handberg, Kurt; Jørgensen, Poul Henrik

1998-01-01

An in situ hybridization procedure for the detection of infectious laryngotracheitis virus (ILTV) in experimentally infected chickens is described. Formalin-fixed, paraffin-embedded sections of trachea, taken from chickens on days 3-10 post-inoculation (p.i.) with ILTV were hybridized with a mixt......An in situ hybridization procedure for the detection of infectious laryngotracheitis virus (ILTV) in experimentally infected chickens is described. Formalin-fixed, paraffin-embedded sections of trachea, taken from chickens on days 3-10 post-inoculation (p.i.) with ILTV were hybridized...... on day 5 p.i. No hybridization was observed in 3 out of 3 chickens examined on day 10 p.i. ILTV nucleic acid was detected in nuclei of degenerated tracheal epithelial cells and in intranuclear inclusion bodies of syncytia....

Metadata: JPST000084 [jPOST repository metadata[Archive

Lifescience Database Archive (English)

Full Text Available alysis Formalin-fixed and paraffin-embedded (FFPE) sections mounted on microscope...s system, we could identify 1090 phosphopeptides from a single FFPE section obtained from a microscope slide

Cadmium, Zinc, and Selenium Levels in Carcinoma of the Human Prostate

National Research Council Canada - National Science Library

Sarafanov, Andrey; Centeno, Jose A

2008-01-01

.... The objectives are: 1) to establish reliability of using formalin-fixed paraffin-embedded (FFPE) prostate tissue for analysis of Zn, Se and Cd tissue by comparing their levels in the fresh specimen...

HER2 evaluation using the novel rabbit monoclonal antibody SP3 and CISH in tissue microarrays of invasive breast carcinomas.

Science.gov (United States)

Ricardo, Sara Alexandra Vinhas; Milanezi, Fernanda; Carvalho, Sílvia Teresa; Leitão, Dina Raquel Aguilera; Schmitt, Fernando Carlos Lander

2007-09-01

Laboratory methods for HER2 assessment currently include immunohistochemical (IHC) methods (measuring protein overexpression) and fluorescence in situ hybridisation (FISH) (measuring gene amplification). The measure of HER2 protein by IHC is usually assessed by the mouse monoclonal antibody CB11, and polyclonal antibodies (Herceptest) directed against the internal portion of the receptor. Recently, chromogenic in situ hybridisation (CISH), in which HER2 is detected by a peroxidase reaction and the gene amplification can be determined by regular bright-field microscopy, has emerged as an alternative to FISH. To evaluate the status of HER2 in tissue microarrays (TMAs) of invasive breast cancer using the novel rabbit monoclonal antibody SP3 directed against the external portion of HER2, and correlate the results with CB11 and CISH. IHC was performed with two antibodies (CB11 and SP3) and CISH for HER2 in 10 TMA blocks with 190 formalin-fixed paraffin-embedded cases of invasive breast carcinomas. The correlation between SP3 and CB11 was significant (pCISH (pCISH, shows that this novel antibody is a reliable candidate to evaluate the expression of HER2 in breast cancer.

Fluorescence in situ hybridization on formalin-fixed and paraffin-embedded tissue

DEFF Research Database (Denmark)

Laub Petersen, Bodil; Zeuthen, Mette Christa; Pedersen, Sanni

2004-01-01

Fluorescence in situ hybridization (FISH) is widely used to study numerical and structural genetic abnormalities in both metaphase and interphase cells. The technique is based on the hybridization of labeled probes to complementary sequences in the DNA or RNA of the cells. Interphase FISH is most...... in time lapse between removal of tissue and fixation, duration of fixation, enzymatic pretreatment, hybridization conditions, and posthybridization washing conditions are important factors in the hybridization. In this study, we have listed the results of a systematic approach to improve FISH on isolated...

MiR-34b is associated with clinical outcome in triple-negative breast cancer patients

Directory of Open Access Journals (Sweden)

Svoboda Marek

2012-03-01

Full Text Available Abstract Background Breast cancer is the most common malignancy with the highest incidence rates among women worldwide. Triple-negative breast cancer (TNBC represents the major phenotype of basal-like molecular subtype of breast cancer, characterized by higher incidence in young women and a very poor prognosis. MicroRNAs (miRNAs are small non-coding RNAs playing significant role in the pathogenesis of many cancers including breast cancer. Therefore, miRNAs are also potential prognostic and/or predictive biomarkers in triple-negative breast cancer patients. Methods Thirty-nine TNBC patients with available formalin-fixed paraffin-embedded (FFPE tissues were enrolled in the study. MiR-34a, miR-34b, and miR-34c were analyzed using qRT-PCR and correlated to clinico-pathological features of TNBC patients. Results Expression levels of miR-34b significantly correlate with disease free survival (DFS (p = 0.0020, log-rank test and overall survival (OS (p = 0.0008, log-rank test of TNBC patients. No other significant associations between miR-34a, miR-34b, and miR-34c with available clinical pathological data were observed. Conclusions MiR-34b expression negatively correlates with disease free survival and overall survival in TNBC patients. Thus, miR-34b may present a new promising prognostic biomarker in TNBC patients, but independent validations are necessary.

CRTP-13: ABC-GCB Expression Signatures in Human B-cell Lymphoma on the NanoString Platform | FNLCR

Science.gov (United States)

The CLIA Molecular Diagnostics Laboratory within the Cancer Research Technology Program will perform messenger RNA isolation and expression analysis specific to a 20-gene panel on formalin-fixed paraffin-embedded (FFPE) patient samples using the Nano

Prevalence of human papillomavirus genotypes associated with cervical and breast cancers in iran.

Science.gov (United States)

Hossein, Rassi; Behzad, Salehi; Tahar, Mohammadian; Azadeh, Nahavandi Araghi

2013-12-01

Cancer is a multi-step disease, and infection with a DNA virus could play a role in one or more of the steps in this pathogenic process. High-risk human papillomaviruses (HPV) are the causative agent of several cancers. In this study, we determined the prevalence and genotype distribution of HPV infection among Iranian patients with cervix lesions (CL) and breast cancer (BC). The study group consisted of postoperative tissues from patients diagnosed with cervix lesions and breast cancer. We analyzed 250 formalin-fixed, paraffin-embedded tissue blocks from 100 cervix lesions and 150 breast cancer samples. Verification of each cancer reported in a relative was sought through the pathology reports of the hospital records. Cervix lesions were collected from 100 patients with squamous metaplasia (SM, n=50), cervical intraepithelial neoplasia (CINI, n=18, CINII or III, n=8), and cervical carcinoma (CC, n=24). In this study we evaluated the prevalence of HPV by multiplex PCR in cervix lesions and breast cancer. For paraffin-embedded tissues, DNA extracted by the simple boiling method yielded higher proportions of successful gene amplification (99%) for b-actin gene. Overall prevalence of HPV infection was 6% in the SM group, 34.61% in the CIN group, 75% in the CC group, and 34.66% in the BC group. Furthermore, MY09/11 consensus PCR failed to detect 44 (55.69%) of all HPV infections and interestingly, the predominant genotype detected in all cancers was the oncogenic variant HPV16/18; about 34% of women aged 24 to 54 were infected with at least one type of HPV. Our results demonstrate that DNA derived from archival tissues that archived for less than 8 years could be used successfully for HPV genotyping by multiplex PCR. Infection with HPV was prevalent among Iranian women with CC and BC. The results indicate a likely causal role for high-risk HPV in CC and BC, and also offer the possibility of primary prevention of these cancers by vaccination against HPV in Iran.

Antigen retrieval, blocking, detection and visualisation systems in immunohistochemistry: a review and practical evaluation of tyramide and rolling circle amplification systems.

Science.gov (United States)

Warford, Anthony; Akbar, Hameed; Riberio, Deise

2014-11-01

To achieve specificity and sensitivity using immunohistochemistry it is necessary to combine the application of validated primary antibodies with optimised pre-treatment, detection and visualisation steps. The influence of these surrounding procedures is reviewed. A practical evaluation of tyramide signal amplification and rolling circle amplification detection methods is provided in which formalin fixed paraffin embedded sections of adenocarcinomas of breast, colon and lung together with squamous metaplasia of lung were immunostained with CD20 and CK19 primary antibodies. The results indicate that the detection systems are of comparable sensitivity and specificity. Copyright © 2014 Elsevier Inc. All rights reserved.

Detection of viral infection by immunofluorescence in formalin-fixed tissues, pretreated with trypsin

Directory of Open Access Journals (Sweden)

O. M. Barth

1988-06-01

Full Text Available The presence of viral antigen in sections from formalin-fixed and paraffin-embedded human tissues was demonstrated by trypsin digestion followed by direct or indirect immunofluorescence. The specimens may be used for retrospective diagnosis. The immunofluorescence technique has to be adapted to the suspected virus infection on the basis of previous histopathology study. Variations of trypsin concentration time and temperature of incubation, expose different viral antigens and have to be previously tested for each unknown system. For measles virus detection in lung a stronger digestion has to be applied as compared to adenovirus or respiratory disease viruses in the same tisue. Flavivirus in liver tissue needs a weaker digestion. The reproducibility of the method makes it useful as a routine technique in diagnosis of virus infection.A presença de antígeno viral em cortes de tecidos humanos fixados em formol e emblocados em parafina foi demonstrada pela digestão com tripsina foi demonstrada pela ingestão com tripsina seguida de imunofluorescência direta ou indireta. Os espécimens podem ser utilizados para diagnoses retrospectivas. A técnica da imunofluorescência deve ser adaptada à infecção viral suspeita segundo diagnosie histopatológica prévia. Os parâmetros para a digestão do tecido pela tripsina, relacionados à concentração, duração de atuação e temperatura, expõem diferentes antígenos virais e devem ser previamente testados para cada sistema a ser estabelecido. Uma digestão mais intensa deve ser aplicada para a detecção do vírus do sarampo em tecido pulmonar do que para adenovírus ou vírus respiratório sincicial no mesmo tecido. Por outro lado, o vírus da febre amarela em tecido de fígado necessita de uma digestão mais fraca.

Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization.

Science.gov (United States)

Pauletti, G; Godolphin, W; Press, M F; Slamon, D J

1996-07-04

Amplification and overexpression of the HER-2/neu gene occurs in 25-30% of human breast cancers. This genetic alteration is associated with a poor clinical prognosis in women with either node negative or node positive breast cancers. The initial studies testing this association were somewhat controversial and this controversy was due in large part to significant heterogeneity in both the methods and/or reagents used in testing archival material for the presence of the alteration. These methods included a number of solid matrix blotting techniques for DNA, RNA and protein as well as immunohistochemistry. Fluorescence in situ hybridization (FISH) represents the newest methodologic approach for testing for this genetic alteration. In this study, FISH is compared to Southern, Northern and Western blot analyses as well as immunohistochemistry in a large cohort of archival human breast cancer specimens. FISH was found to be superior to all other methodologies tested in assessing formalin fixed, paraffin embedded material for HER-2/neu amplification. The results from this study also confirm that overexpression of HER-2/neu rarely occurs in the absence of gene amplification in breast cancer (approximately 3% of cases). This method of analysis is rapid, reproducible and extremely reliable in detecting presence of HER-2/neu gene amplification and should have clinical utility.

The relationship of platinum resistance and ERCC1 protein expression in epithelial ovarian cancer

DEFF Research Database (Denmark)

Steffensen, Karina Dahl; Waldstrøm, Marianne; Jakobsen, Anders

2009-01-01

: Formalin-fixed, paraffin-embedded tissue sections from 101 patients with newly diagnosed ovarian cancer were used for immunohistochemical staining for the ERCC1 protein. All patients received carboplatin-paclitaxel combination chemotherapy. RESULTS: Excision repair cross-complementation group 1 enzyme...

Highly sensitive KRAS mutation detection from formalin-fixed paraffin-embedded biopsies and circulating tumour cells using wild-type blocking polymerase chain reaction and Sanger sequencing.

Science.gov (United States)

Huang, Meggie Mo Chao; Leong, Sai Mun; Chua, Hui Wen; Tucker, Steven; Cheong, Wai Chye; Chiu, Lily; Li, Mo-Huang; Koay, Evelyn Siew-Chuan

2014-08-01

Among patients with colorectal cancer (CRC), KRAS mutations were reported to occur in 30-51 % of all cases. CRC patients with KRAS mutations were reported to be non-responsive to anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MoAb) treatment in many clinical trials. Hence, accurate detection of KRAS mutations would be critical in guiding the use of anti-EGFR MoAb therapies in CRC. In this study, we carried out a detailed investigation of the efficacy of a wild-type (WT) blocking real-time polymerase chain reaction (PCR), employing WT KRAS locked nucleic acid blockers, and Sanger sequencing, for KRAS mutation detection in rare cells. Analyses were first conducted on cell lines to optimize the assay protocol which was subsequently applied to peripheral blood and tissue samples from patients with CRC. The optimized assay provided a superior sensitivity enabling detection of as little as two cells with mutated KRAS in the background of 10(4) WT cells (0.02 %). The feasibility of this assay was further investigated to assess the KRAS status of 45 colorectal tissue samples, which had been tested previously, using a conventional PCR sequencing approach. The analysis showed a mutational discordance between these two methods in 4 of 18 WT cases. Our results present a simple, effective, and robust method for KRAS mutation detection in both paraffin embedded tissues and circulating tumour cells, at single-cell level. The method greatly enhances the detection sensitivity and alleviates the need of exhaustively removing co-enriched contaminating lymphocytes.

Antikeratin antibodies in routine diagnostic pathology. A comparison of 10 different commercial antikeratins

DEFF Research Database (Denmark)

Mygind, H; Nielsen, B; Moe, D

1988-01-01

Ten commercially available antikeratin antisera were tested immunohistochemically on fresh frozen and formalin-fixed paraffin-embedded tissue. Eight of the antisera were in addition tested on protein-immunoblottings. For six of the antisera a good correspondence was found between our immunoblots ...

Association of ERCC1 protein expression to platinum resistance in epithelial ovarian cancer

DEFF Research Database (Denmark)

Dahl Steffensen, Karina; Waldstrøm, Marianne; Jakobsen, Anders

was to investigate if immunohistochemical expression of ERCC1 protein was associated with resistance to standard combination carboplatin and paclitaxel chemotherapy in newly diagnosed ovarian cancer patients. Methods: Formalin-fixed, paraffin-embedded tissue sections from 101 patients with newly diagnosed ovarian...

Increased angiotensin II type 1 receptor expression in temporal arteries from patients with giant cell arteritis

DEFF Research Database (Denmark)

Dimitrijevic, Ivan; Malmsjö, Malin; Andersson, Christina

2009-01-01

-AT(2) antibodies, was performed on formalin-fixed and paraffin-embedded temporal arteries. MAIN OUTCOME MEASURES: AT(1) and AT(2) receptor immunostaining intensity was quantified. RESULTS: Hematoxylin-eosin-stained sections of temporal arteries from patients with GCA showed intimal hyperplasia...

A generally applicable sequential alkaline phosphatase immunohistochemical double staining

NARCIS (Netherlands)

van der Loos, Chris M.; Teeling, Peter

2008-01-01

A universal type of sequential double alkaline phosphatase immunohistochemical staining is described that can be used for formalin-fixed, paraffin-embedded and cryostat tissue sections from human and mouse origin. It consists of two alkaline phosphatase detection systems including enzymatic

Liver fibrosis and regeneration in dogs and cats: An immunohistochemical approach

NARCIS (Netherlands)

IJzer, J.

2008-01-01

In this thesis we focus on liver tissue repair processes in canine and feline hepatitis, on formalin fixed paraffin embedded archival liver specimens. Hepatitis was diagnosed using histological standard criteria, and always includes hepatocellular cell death and an inflammatory infiltrate.

Gene expression patterns in formalin-fixed, paraffin-embedded core biopsies predict docetaxel chemosensitivity in breast cancer patients.

Science.gov (United States)

Chang, Jenny C; Makris, Andreas; Gutierrez, M Carolina; Hilsenbeck, Susan G; Hackett, James R; Jeong, Jennie; Liu, Mei-Lan; Baker, Joffre; Clark-Langone, Kim; Baehner, Frederick L; Sexton, Krsytal; Mohsin, Syed; Gray, Tara; Alvarez, Laura; Chamness, Gary C; Osborne, C Kent; Shak, Steven

2008-03-01

Previously, we had identified gene expression patterns that predicted response to neoadjuvant docetaxel. Other studies have validated that a high Recurrence Score (RS) by the 21-gene RT-PCR assay is predictive of worse prognosis but better response to chemotherapy. We investigated whether tumor expression of these 21 genes and other candidate genes can predict response to docetaxel. Core biopsies from 97 patients were obtained before treatment with neoadjuvant docetaxel (4 cycles, 100 mg/m2 q3 weeks). Three 10-microm FFPE sections were submitted for quantitative RT-PCR assays of 192 genes that were selected from our previous work and the literature. Of the 97 patients, 81 (84%) had sufficient invasive cancer, 80 (82%) had sufficient RNA for QRTPCR assay, and 72 (74%) had clinical response data. Mean age was 48.5 years, and the median tumor size was 6 cm. Clinical complete responses (CR) were observed in 12 (17%), partial responses in 41 (57%), stable disease in 17 (24%), and progressive disease in 2 patients (3%). A significant relationship (P<0.05) between gene expression and CR was observed for 14 genes, including CYBA. CR was associated with lower expression of the ER gene group and higher expression of the proliferation gene group from the 21 gene assay. Of note, CR was more likely with a high RS (P=0.008). We have established molecular profiles of sensitivity to docetaxel. RT-PCR technology provides a potential platform for a predictive test of docetaxel chemosensitivity using small amounts of routinely processed material.

COMPARISON OF THE QUALITY OF RNA ISOLATED FROM THE RAINBOW TROUT (Oncorhynchus mykiss TISSUE IN FOUR DIFFERENT WAYS

Directory of Open Access Journals (Sweden)

Irena Vardić

2004-03-01

Full Text Available Rapid and accurate diagnostic procedures for identification of reared fish diseases are important in order to reduce serious losses in relation of diseases outbrakes. Therefore, molecular biology methods are required for such types of investigations. First level in these experiments are DNA or RNA isolation. Tissue preparation for isolation of RNA, which is used in further RT-PCR (reverse transcription-polymerise chain reaction analysis is the key step on which is the whole process of analysis dependent. Our goal was to compare quality and integrity of RNA isolated from the rainbow trout tissue, which was prepared in four different ways: fresh tissue, frozen tissue, in formalin-fixed, paraffin embedded tissue as well as in methacarn-fixed, paraffin embedded tissue. Isolated RNA was analyzed in gel electrophoresis on non-denaturated, 1% agarose gel. Quality and integrity of RNA was proved by RT-PCR reaction with primers for ß-actin gene. Additional, prepared tissue was tested on presence of two fish viruses: viral haemorrhagic septicaemia (VHS virus and infectious haematopoietic necrosis (IHN virus in RT-PCR reaction with primers specific for these viruses. RNA isolated from fresh and frozen tissue was of high quality, integrity and quantity. RNA isolated from in methacarn-fixed, paraffin embedded tissue was quite disintegrated, but in RT-PCR with primers for ß-actin gave expected products. These products were absent after RT-PCR reaction with in formallin-fixed, paraffin-embedded tissue. That agrees with the facts from the literature about very agressive affect of formalin as a fixative on RNA in tissue. Inspected fish were not infected with VHS and IHN viruses and that was in agreement with results of clinical examination and pathological analysis. According to our knowledge, this is the first successful RNA isolation from in methacarn-fixed, paraffin embedded fish tissue. Isolated RNA can be used for further analysis in RT-PCR reaction. This

Application of fluorescent in situ hybridisation for demonstration of Coxiella burnetti in placentas from ruminant abortions

DEFF Research Database (Denmark)

Jensen, Tim Kåre; Montgomery, Donald L.; Jaeger, Paula T.

2007-01-01

A fluorescent in situ hybridisation (FISH) assay targeting 16S ribosomal RNA was developed for detection of the zoonotic bacterium Coxiella burnetii in formalin-fixed, paraffin-embedded tissue, and applied on placentas from ruminant abortions. The applicability of the FISH assay was compared...

Methylation-associated Silencing of microRNA-126 and its Host Gene EGFL7 in Malignant Pleural Mesothelioma

DEFF Research Database (Denmark)

Andersen, Morten; Trapani, Davide; Ravn, Jesper

2015-01-01

gene EGF-like domain, multiple 7 (EGFL7). MATERIALS AND METHODS: Resected formalin-fixed paraffin-embedded MPM tissues from 29 patients, 14 patient-matched non-neoplastic pleura (NNP) specimens, 5 MPM diagnostic biopsies (DB), and 5 samples of pneumothorax-induced benign reactive mesothelial...

Expression of p53 in oligodendrogliomas

NARCIS (Netherlands)

J.M. Kros (Johan); J.J.C.J. Godschalk (J. J C J); K.K. Krishnadath (Kausilia); C.G. van Eden (C.)

1993-01-01

textabstractThe expression of the nuclear protein p53 in oligodendrogliomas was investigated by immunohistochemistry, using a monoclonal anti-p53 antibody (DO-7) on formalin-fixed, paraffin-embedded material in 84 histologically verified cases, and compared with the histopathological grade and

Expression of p53 in oligodendrogliomas

NARCIS (Netherlands)

Kros, J. M.; Godschalk, J. J.; Krishnadath, K. K.; van Eden, C. G.

1993-01-01

The expression of the nuclear protein p53 in oligodendrogliomas was investigated by immunohistochemistry, using a monoclonal anti-p53 antibody (DO-7) on formalin-fixed, paraffin-embedded material in 84 histologically verified cases, and compared with the histopathological grade and survival.

Comparison of microRNA expression using different preservation methods of matched psoriatic skin samples

DEFF Research Database (Denmark)

Løvendorf, Marianne B; Zibert, John R; Hagedorn, Peter H

2012-01-01

MicroRNAs are non-coding RNA molecules modulating gene expression post-transcriptionally. Formalin-fixed, paraffin-embedding (FFPE) is a standard preservation method often used in clinical practices, but induces RNA degradation. Extracting high-quality RNA from human skin can be challenging as skin...

Temporal lobe pathology in amyotrophic lateral sclerosis. Do amyotrophic lateral sclerosis and Alzheimer's disease share a common etiological factor?

NARCIS (Netherlands)

Smitt, P. A.; Troost, D.; Louwerse, E. S.; de Jong, J. M.; van Kessel, D. T.; de Leeuw, M. A.

1993-01-01

An autopsy study was performed on temporal lobe samples from 20 non-demented patients with amyotrophic lateral sclerosis (ALS), 17 age-matched non-demented controls and 4 Alzheimer's disease (AD) patients. Formalin fixed, paraffin embedded sections from the hippocampus with adjacent parahippocampal

Concordance of genotype for polymorphisms in DNA isolated from peripheral blood and colorectal cancer tumor samples

NARCIS (Netherlands)

van Huis-Tanja, Lieke; Kweekel, Dinemarie; Gelderblom, Hans; Koopman, Miriam; Punt, Kees; Guchelaar, Henk-Jan; van der Straaten, Tahar

2013-01-01

Background & aim: Results from different pharmacogenetic association studies in colorectal cancer are often conflicting. Both peripheral blood and formalin-fixed, paraffin-embedded (FFPE) tissue are routinely used as DNA source. This could cause bias due to somatic alterations in tumor tissue, such

Phylogeny and distribution of an unknown Treponema sp. associated with porcine colitis by using in situ hybridization and laser capture microdissection (LCM)

DEFF Research Database (Denmark)

Mølbak, Lars; Schou, Kirstine Klitgaard; Jensen, Tim Kåre

Helical-shaped bacteria resembling Spirochaetes commonly are present in the gastrointestinal tract of animals and humans. Culturing of Spirochaetes is in general fastidious and not always successful. Here, a new DNA isolation approach for prokaryotic cells in formalin-fixed paraffin-embedded tissue...

Mucin expression patterns in histological grades of colonic cancers ...

African Journals Online (AJOL)

Pathological expression of mucins has been noted in cancer development and progression. This study sought to identify and quantify the types of mucins produced during various histological grades of colon cancer and to assess the diagnostic significance. Methods: Formalin fixed, paraffin-embedded tissue blocks, ...

Resistance to first line platinum paclitaxel chemotherapy in serous epithelial ovarian cancer

DEFF Research Database (Denmark)

Steffensen, Karina Dahl; Smoter, Marta; Waldstrøm, Marianne

2014-01-01

of sensitivity to platinum/paclitaxel treatment. The primary aim of the study was to investigate whether ERCC1 and Tau protein expression correlates with patient outcome in newly diagnosed epithelial ovarian cancer (EOC) patients. Formalin-fixed, paraffin-embedded tissue sections from 227 newly diagnosed EOC...

Fluorescence-based codetection with protein markers reveals distinct cellular compartments for altered MicroRNA expression in solid tumors

DEFF Research Database (Denmark)

Sempere, Lorenzo F; Preis, Meir; Yezefski, Todd

2010-01-01

of altered miRNA expression in solid tumors, we developed a sensitive fluorescence-based in situ hybridization (ISH) method to visualize miRNA accumulation within individual cells in formalin-fixed, paraffin-embedded tissue specimens. This ISH method was implemented to be compatible with routine clinical...

Long noncoding RNA metastasis-associated lung adenocarcinoma ...

African Journals Online (AJOL)

Subjects and methods: The relative expression of MALAT1 was determined in 37 human glioblastoma formalin-fixed paraffin embedded (FFPE) tissue samples and 10 FFPE non-neoplastic brain tissues using quantitative reverse transcription polymerase chain reaction (qRT-PCR) technology. Results: The current results ...

Standardization and optimization of fluorescence in situ hybridization (FISH for HER-2 assessment in breast cancer: A single center experience

Directory of Open Access Journals (Sweden)

Magdalena Bogdanovska-Todorovska

2018-05-01

Full Text Available Accurate assessment of human epidermal growth factor receptor 2 (HER-2 is crucial in selecting patients for targeted therapy. Commonly used methods for HER-2 testing are immunohistochemistry (IHC and fluorescence in situ hybridization (FISH. Here we presented the implementation, optimization and standardization of two FISH protocols using breast cancer samples and assessed the impact of pre-analytical and analytical factors on HER-2 testing. Formalin fixed paraffin embedded (FFPE tissue samples from 70 breast cancer patients were tested for HER-2 using PathVysion™ HER-2 DNA Probe Kit and two different paraffin pretreatment kits, Vysis/Abbott Paraffin Pretreatment Reagent Kit (40 samples and DAKO Histology FISH Accessory Kit (30 samples. The concordance between FISH and IHC results was determined. Pre-analytical and analytical factors (i.e., fixation, baking, digestion, and post-hybridization washing affected the efficiency and quality of hybridization. The overall hybridization success in our study was 98.6% (69/70; the failure rate was 1.4%. The DAKO pretreatment kit was more time-efficient and resulted in more uniform signals that were easier to interpret, compared to the Vysis/Abbott kit. The overall concordance between IHC and FISH was 84.06%, kappa coefficient 0.5976 (p < 0.0001. The greatest discordance (82% between IHC and FISH was observed in IHC 2+ group. A standardized FISH protocol for HER-2 assessment, with high hybridization efficiency, is necessary due to variability in tissue processing and individual tissue characteristics. Differences in the pre-analytical and analytical steps can affect the hybridization quality and efficiency. The use of DAKO pretreatment kit is time-saving and cost-effective.

Formalin fixation increases deamination mutation signature but should not lead to false positive mutations in clinical practice.

Directory of Open Access Journals (Sweden)

Leah M Prentice

Full Text Available Genomic analysis of cancer tissues is an essential aspect of personalized oncology treatment. Though it has been suggested that formalin fixation of patient tissues may be suboptimal for molecular studies, this tissue processing approach remains the industry standard. Therefore clinical molecular laboratories must be able to work with formalin fixed, paraffin embedded (FFPE material. This study examines the effects of pre-analytic variables introduced by routine pathology processing on specimens used for clinical reports produced by next-generation sequencing technology. Tissue resected from three colorectal cancer patients was subjected to 2, 15, 24, and 48 hour fixation times in neutral buffered formalin. DNA was extracted from all tissues twice, once with uracil-N-glycosylase (UNG treatment to counter deamination effects, and once without. Of note, deamination events at methylated cytosine, as found at CpG sites, remains unaffected by UNG. After extraction a two-step PCR targeted sequencing method was performed using the Illumina MiSeq and the data was analyzed via a custom-built bioinformatics pipeline, including filtration of reads with mapping quality T/A mutations that is not represented in DNA treated with UNG. This suggests these errors may be due to deamination events triggered by a longer fixation time. However the allelic frequency of these events remained below the limit of detection for reportable mutations in this assay (<2%. We do however recommend that suspected intratumoral heterogeneity events be verified by re-sequencing the same FFPE block.

Gene expression in triple-negative breast cancer in relation to survival.

Science.gov (United States)

Wang, Shuyang; Beeghly-Fadiel, Alicia; Cai, Qiuyin; Cai, Hui; Guo, Xingyi; Shi, Liang; Wu, Jie; Ye, Fei; Qiu, Qingchao; Zheng, Ying; Zheng, Wei; Bao, Ping-Ping; Shu, Xiao-Ou

2018-05-10

The identification of biomarkers related to the prognosis of triple-negative breast cancer (TNBC) is critically important for improved understanding of the biology that drives TNBC progression. We evaluated gene expression in total RNA isolated from formalin-fixed paraffin-embedded tumor samples using the NanoString nCounter assay for 469 TNBC cases from the Shanghai Breast Cancer Survival Study. We used Cox regression to quantify Hazard Ratios (HR) and corresponding confidence intervals (CI) for overall survival (OS) and disease-free survival (DFS) in models that included adjustment for breast cancer intrinsic subtype. Of 302 genes in our discovery analysis, 22 were further evaluated in relation to OS among 134 TNBC cases from the Nashville Breast Health Study and the Southern Community Cohort Study; 16 genes were further evaluated in relation to DFS in 335 TNBC cases from four gene expression omnibus datasets. Fixed-effect meta-analysis was used to combine results across data sources. Twofold higher expression of EOMES (HR 0.90, 95% CI 0.83-0.97), RASGRP1 (HR 0.89, 95% CI 0.82-0.97), and SOD2 (HR 0.80, 95% CI 0.66-0.96) was associated with better OS. Twofold higher expression of EOMES (HR 0.89, 95% CI 0.81-0.97) and RASGRP1 (HR 0.87, 95% CI 0.81-0.95) was also associated with better DFS. On the contrary, a doubling of FA2H (HR 1.14, 95% CI 1.06-1.22) and GSPT1 (HR 1.33, 95% CI 1.14-1.55) expression was associated with shorter DFS. We identified five genes (EOMES, FA2H, GSPT1, RASGRP1, and SOD2) that may serve as potential prognostic biomarkers and/or therapeutic targets for TNBC.

Evaluation of a panel of antibodies for the immunohistochemical identification of immune cells in paraffin-embedded lymphoid tissues of new- and old-world camelids.

Science.gov (United States)

Uhde, Ann-Kathrin; Lehmbecker, Annika; Baumgärtner, Wolfgang; Spitzbarth, Ingo

2017-02-01

Different species of camelids play an important role in the epidemiology of various emerging infectious diseases such as Middle East respiratory syndrome. For precise investigations of the immunopathogenesis in these host species, appropriate immunohistochemical markers are highly needed in order to phenotype distinct immune cells populations in camelids. So far, specific immunohistochemical markers for camelid immune cells are rarely commercially available, and cross-reactivity studies are restricted to the use of frozen dromedary tissues. To bridge this gap, 14 commercially available primary antibodies were tested for their suitability to demonstrate immune cell populations on formalin fixed paraffin-embedded (FFPE) tissue sections of dromedaries, Bactrian camels, llamas, and alpacas in the present study. Out of these, 9 antibodies directed against CD3, CD20, CD79α, HLA-DR, Iba-1, myeloid/histiocyte antigen, CD204, CD208, and CD68 antigen exhibited distinct immunoreaction patterns to certain camelid immune cell subsets. The distribution of these antigens was comparatively evaluated in different anatomical compartments of thymus, spleen, mesenteric, and tracheobronchial lymph nodes. The presented results will provide a basis for further investigations in camelids, especially with respect to the role of the immune response in certain infectious diseases, which harbor a considerable risk to spill over to other species. Copyright © 2017 Elsevier B.V. All rights reserved.

Divisional role of quantitative HER2 testing in breast cancer.

Science.gov (United States)

Yamamoto-Ibusuki, Mutsuko; Yamamoto, Yutaka; Fu, Peifen; Yamamoto, Satoko; Fujiwara, Saori; Honda, Yumi; Iyama, Ken-ichi; Iwase, Hirotaka

2015-03-01

Human epidermal growth factor receptor 2 (HER2) is amplified in human breast cancers in which therapy targeted to HER2 significantly improves patient outcome. We re-visited the use of real-time quantitative polymerase chain reaction (qPCR)-based assays using formalin-fixed paraffin-embedded (FFPE) tissues as alternative methods and investigated their particular clinical relevance. DNA and RNA were isolated from FFPE specimens and HER2 status was assessed by qPCR in 249 consecutive patients with primary breast cancer. Concordance with results forg immunohistochemistry (IHC) and in situ hybridization (ISH), clinical characteristics and survival was assessed. HER2 gene copy number had a stronger correlation with clinicopathological characteristics and excellent concordance with IHC/ISH results (Sensitivity: 96.7 %; concordance: 99.2 %). HER2 gene expression showed inadequate sensitivity, rendering it unsuitable to determine HER2 status (Sensitivity: 46.7 %; concordance: 92.1 %), but lower HER2 gene expression, leading to the classification of many cases as "false negative", contributed to a prediction of better prognosis within the HER2-amplified subpopulation. Quantitative HER2 assessments are suggested to have evolved their accuracy in this decade, which can be a potential alternative for HER2 diagnosis in line with the in situ method, while HER2 gene expression levels could provide additional information regarding prognosis or therapeutic strategy within a HER2-amplified subpopulation.

[Molecular classification of breast cancer patients obtained through the technique of chromogenic in situ hybridization (CISH)].

Science.gov (United States)

Fernández, Angel; Reigosa, Aldo

2013-12-01

Breast cancer is a heterogeneous disease composed of a growing number of biological subtypes, with substantial variability of the disease progression within each category. The aim of this research was to classify the samples object of study according to the molecular classes of breast cancer: luminal A, luminal B, HER2 and triple negative, as a result of the state of HER2 amplification obtained by the technique of chromogenic in situ hybridization (CISH). The sample consisted of 200 biopsies fixed in 10% formalin, processed by standard techniques up to paraffin embedding, corresponding to patients diagnosed with invasive ductal carcinoma of the breast. These biopsies were obtained from patients from private practice and the Institute of Oncology "Dr. Miguel Pérez Carreño", for immunohistochemistry (IHC) of hormone receptors and HER2 made in the Hospital Metropolitano del Norte, Valencia, Venezuela. The molecular classification of the patient's tumors considering the expression of estrogen and progesterone receptors by IHC and HER2 amplification by CISH, allowed those cases originally classified as unknown, since they had an indeterminate (2+) outcome for HER2 expression by IHC, to be grouped into the different molecular classes. Also, this classification permitted that some cases, initially considered as belonging to a molecular class, were assigned to another class, after the revaluation of the HER2 status by CISH.

Does the correlation between EBNA-1 and p63 expression in breast carcinomas provide a clue to tumorigenesis in Epstein-Barr virus-related breast malignancies?

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Ribeiro-Silva A.

2004-01-01

Full Text Available Several investigators have identified Epstein-Barr virus (EBV particles in breast carcinomas, a fact that supports a role for EBV in mammary tumorigenesis. The possible mechanism involved in this process is not clear. The present study was carried out in an attempt to determine whether there is a relationship between latent infection with EBV and p53 and p63 expression in breast carcinomas. Immunohistochemistry developed with 3.3-diaminobenzidine tetrahydrochloride was performed in 85 formalin-fixed paraffin-embedded breast carcinomas using anti-EBV EBNA-1, anti-p63, anti-p53, anti-estrogen receptor (ER and anti-progesterone receptor (PR antibodies. The cases were selected to represent each of the various histologic types: intraductal carcinoma (N = 12, grade I invasive ductal carcinoma (N = 15, grade II invasive ductal carcinoma (N = 15, grade III invasive ductal carcinoma (N = 15, tubular carcinoma (N = 8, lobular carcinoma (N = 10, and medullary carcinoma (N = 10. The ductal breast carcinomas were graded I, II and III based on the Scarff-Bloom and Richardson grading system modified by Elston and Ellis. One slide containing at least 1000 neoplastic cells was examined in each case. ER, PR, p63, p53 and EBNA-1 were positive in 60, 40, 11.8, 21.2 and 37.6% of carcinomas, respectively. There was a correlation between EBNA-1 and p63 expression (P < 0.001, but not between EBNA-1 and p53 (P = 0.10. These data suggest a possible role for p63 in the mammary tumorigenesis associated with Epstein-Barr virus infection.

Evaluation of Human Epidermal Growth Factor Receptor 2 (HER2) Gene Status in Human Breast Cancer Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Specimens by Fluorescence In Situ Hybridization (FISH).

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Hwang, Harry C; Gown, Allen M

2016-01-01

Current standard of care requires that HER2 gene testing be performed on all newly diagnosed invasive breast cancers in order to determine eligibility for anti-HER2 antibody therapy and should be performed in accordance with current ASCO-CAP guidelines (Hammond et al., J Clin Oncol 29(15):e458, 2011; Wolff et al., J Clin Oncol 31(31):3997-4013, 2013). Here we describe a HER2 FISH methodology to evaluate HER2 gene status in FFPE breast tumor specimens.

MethylMeter(®): bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples.

Science.gov (United States)

McCarthy, David; Pulverer, Walter; Weinhaeusel, Andreas; Diago, Oscar R; Hogan, Daniel J; Ostertag, Derek; Hanna, Michelle M

2016-06-01

Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter(®). Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas.

c-erbB2 and topoisomerase IIα protein expression independently predict poor survival in primary human breast cancer: a retrospective study

International Nuclear Information System (INIS)

Fritz, Peter; Cabrera, Cristina M; Dippon, Jürgen; Gerteis, Andreas; Simon, Wolfgang; Aulitzky, Walter E; Kuip, Heiko van der

2005-01-01

c-erbB2 (also known as HER-2/neu) and topoisomerase IIα are frequently overexpressed in breast cancer. The aim of the study was to analyze retrospectively whether the expression of c-erbB2 and topoisomerase IIα protein influences the long-term outcome of patients with primary breast cancer. In this study c-erbB2 and topoisomerase IIα protein were evaluated by immunohistochemistry in formalin-fixed paraffin-embedded tissue from 225 samples of primary breast cancer, obtained between 1986 and 1998. The prognostic value of these markers was analyzed. Of 225 primary breast tumor samples, 78 (34.7%) showed overexpression of either c-erbB2 (9.8%) or topoisomerase IIα protein (24.9%), whereas in 21 tumors (9.3%) both proteins were found to be overexpressed. Patients lacking both c-erbB2 and topoisomerase IIα overexpression had the best long-term survival. Overexpression of either c-erbB2 or topoisomerase IIα was associated with shortened survival, whereas patients overexpressing both c-erbB2 and topoisomerase IIα showed the worst disease outcome (P < 0.0001). Treatment with anthracyclines was not capable of reversing the negative prognostic impact of topoisomerase IIα or c-erbB2 overexpression. The results of this exploratory study suggest that protein expression of c-erbB2 and topoisomerase IIα in primary breast cancer tissues are independent prognostic factors and are not exclusively predictive factors for anthracycline response in patients with primary breast cancer

The human placenta from heavy smokers: evaluation of vasoactive peptides by immunohistochemistry

DEFF Research Database (Denmark)

Clausen, H V; Larsen, L Grupe; Jørgensen, A

2007-01-01

The study aimed to demonstrate the expression of nitric oxide converting enzyme, nitric oxide synthase (e-NOS), and endothelin-1 (Et-1) in formalin-fixed paraffin-embedded placental tissue, and to demonstrate a difference in staining intensity between heavy smokers and non-smokers. Term placentas...... from pregnancies from otherwise healthy women smoking 15 or more cigarettes per day (heavy smokers) and term placentas from a matching group of non-smokers were included. The antibodies for Et-1 and e-NOS are recommended for cryostat sections. We evaluated the antibodies on paraffin-embedded tissue...

Relative shrinkage of adipocytes by paraffin in proportion to plastic embedding in human adipose tissue before and after weight loss.

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Verhoef, Sanne P M; van Dijk, Paul; Westerterp, Klaas R

2013-01-01

Adipocyte size is a major modulator of endocrine functioning of adipose tissue and methods allowing accurate determination of adipocyte size are important to study energy metabolism. The aim of this study was to assess the relative shrinkage of adipocytes before and after weight loss by comparing adipose tissue from the same subjects embedded in paraffin and plastic. 18 healthy subjects (5 males and 13 females) aged 20-50 y with a BMI of 28-38 kg/m² followed a very low energy diet for 8 weeks. Adipose tissue biopsies were taken prior to and after weight loss and were processed for paraffin and plastic sections. Parameters of adipocyte size were determined with computer image analysis. Mean adipocyte size was smaller in paraffin compared to plastic embedded tissue both before (66 ± 4 vs. 103 ± 5 μm, P paraffin embedded tissue in proportion to plastic embedded tissue was not significantly different before and after weight loss (73 and 69%, respectively). Shrinkage due to the type of embedding of the adipose tissue can be ignored when comparing before and after weight loss. Plastic embedding of adipose tissue provides more accurate and sensitive results. © 2013 Asian Oceanian Association for the Study of Obesity . Published by Elsevier Ltd. All rights reserved.

Specific detection of Pasteurella multocida in chickens with fowl cholera and in pig lung tissues using fluorescent rRNA in situ hybridization

DEFF Research Database (Denmark)

Mbuthia, P.G.; Christensen, H.; Boye, Mette

2001-01-01

in formalin-fixed paraffin-embedded lung tissues from experimental fowl cholera in chickens and infections in pigs. In chicken lung tissues P. multocida cells were detected singly, in pairs, as microcolonies, and as massive colonies within air capillaries (septa and lumen), parabronchial septa, and blood...... and fast method for specific detection of P. multocida in histological formalin-fixed tissues. The test was replicable and reproducible and is recommended as a supplementary test for diagnosis and as a tool in pathogenesis studies of fowl cholera and respiratory tract infections in pigs due to P. multocida....

A simple osmium post-fixation paraffin-embedment technique to identify lipid accumulation in fish liver using medaka (Oryziaslatipes) eggs and eleutheroembryos as lipid rich models

International Nuclear Information System (INIS)

Mondon, J.A.; Howitt, J.; Tosiano, M.; Kwok, K.W.H.; Hinton, D.E.

2011-01-01

Highlights: → Hepatic lipidosis in fish liver is often misdiagnosed or overlooked. → Specific histological fat stains and cryostat sections are not commonly used. → Standard paraffin processing removes lipid leaving vacuoles of unknown origin. → Osmium post-fixed paraffin-embedment is a cost effective alternative. → Medaka trials show suitability for lipid visualization in tissues from egg to adult. - Abstract: Hepatic lipidosis is a non-specific biomarker of effect from pollution exposure in fish. Fatty liver is often misdiagnosed or overlooked in histological assessments due to the decreasing application of specific fat procedures and stains. For example, ethanol dehydration in standard paraffin processing removes lipids, leaving vacuoles of which the precise nature is unknown. Lipids can be identified using osmium post-fixation in semi-thin resin sections or transmission electron microscopy. However, both are expensive and technically demanding procedures, often not available for routine environmental risk assessment and monitoring programs. The current emphasis to reduce and refine animal toxicity testing, requires refinement of the suite of histopathological techniques currently available to maximize information gained from using fish for toxicity testing and as bio-indicators of environmental quality. This investigation has successfully modified an osmium post-fixation technique to conserve lipids in paraffin-embedded tissues using medaka (Oryzias latipes) eleutheroembryos and eggs (embryos) as lipid rich models.

One-Step Preservation of Phosphoproteins and Tissue Morphology at Room Temperature for Diagnostic and Research Specimens

Science.gov (United States)

Mueller, Claudius; Edmiston, Kirsten H.; Carpenter, Calvin; Gaffney, Eoin; Ryan, Ciara; Ward, Ronan; White, Susan; Memeo, Lorenzo; Colarossi, Cristina; Petricoin, Emanuel F.; Liotta, Lance A.; Espina, Virginia

2011-01-01

Background There is an urgent need to measure phosphorylated cell signaling proteins in cancer tissue for the individualization of molecular targeted kinase inhibitor therapy. However, phosphoproteins fluctuate rapidly following tissue procurement. Snap-freezing preserves phosphoproteins, but is unavailable in most clinics and compromises diagnostic morphology. Formalin fixation preserves tissue histomorphology, but penetrates tissue slowly, and is unsuitable for stabilizing phosphoproteins. We originated and evaluated a novel one-step biomarker and histology preservative (BHP) chemistry that stabilizes signaling protein phosphorylation and retains formalin-like tissue histomorphology with equivalent immunohistochemistry in a single paraffin block. Results Total protein yield extracted from BHP-fixed, routine paraffin-embedded mouse liver was 100% compared to snap-frozen tissue. The abundance of 14 phosphorylated proteins was found to be stable over extended fixation times in BHP fixed paraffin embedded human colon mucosa. Compared to matched snap-frozen tissue, 8 phosphoproteins were equally preserved in mouse liver, while AMPKβ1 Ser108 was slightly elevated after BHP fixation. More than 25 tissues from mouse, cat and human specimens were evaluated for preservation of histomorphology. Selected tissues were evaluated in a multi-site, independent pathology review. Tissue fixed with BHP showed equivalent preservation of cytoplasmic and membrane cytomorphology, with significantly better nuclear chromatin preservation by BHP compared to formalin. Immunohistochemical staining of 13 non-phosphorylated proteins, including estrogen receptor alpha, progesterone receptor, Ki-67 and Her2, was equal to or stronger in BHP compared to formalin. BHP demonstrated significantly improved immunohistochemical detection of phosphorylated proteins ERK Thr202/Tyr204, GSK3-α/β Ser21/Ser9, p38-MAPK Thr180/Tyr182, eIF4G Ser1108 and Acetyl-CoA Carboxylase Ser79. Conclusion In a single

One-step preservation of phosphoproteins and tissue morphology at room temperature for diagnostic and research specimens.

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Claudius Mueller

Full Text Available BACKGROUND: There is an urgent need to measure phosphorylated cell signaling proteins in cancer tissue for the individualization of molecular targeted kinase inhibitor therapy. However, phosphoproteins fluctuate rapidly following tissue procurement. Snap-freezing preserves phosphoproteins, but is unavailable in most clinics and compromises diagnostic morphology. Formalin fixation preserves tissue histomorphology, but penetrates tissue slowly, and is unsuitable for stabilizing phosphoproteins. We originated and evaluated a novel one-step biomarker and histology preservative (BHP chemistry that stabilizes signaling protein phosphorylation and retains formalin-like tissue histomorphology with equivalent immunohistochemistry in a single paraffin block. RESULTS: Total protein yield extracted from BHP-fixed, routine paraffin-embedded mouse liver was 100% compared to snap-frozen tissue. The abundance of 14 phosphorylated proteins was found to be stable over extended fixation times in BHP fixed paraffin embedded human colon mucosa. Compared to matched snap-frozen tissue, 8 phosphoproteins were equally preserved in mouse liver, while AMPKβ1 Ser108 was slightly elevated after BHP fixation. More than 25 tissues from mouse, cat and human specimens were evaluated for preservation of histomorphology. Selected tissues were evaluated in a multi-site, independent pathology review. Tissue fixed with BHP showed equivalent preservation of cytoplasmic and membrane cytomorphology, with significantly better nuclear chromatin preservation by BHP compared to formalin. Immunohistochemical staining of 13 non-phosphorylated proteins, including estrogen receptor alpha, progesterone receptor, Ki-67 and Her2, was equal to or stronger in BHP compared to formalin. BHP demonstrated significantly improved immunohistochemical detection of phosphorylated proteins ERK Thr202/Tyr204, GSK3-α/β Ser21/Ser9, p38-MAPK Thr180/Tyr182, eIF4G Ser1108 and Acetyl-CoA Carboxylase Ser79

Molecular Diagnosis of Polycystic Echinococcosis Due to Echinococcus vogeli in a Paraguayan Immigrant in Argentina

Science.gov (United States)

Frider, B.; Alvarez Rodriguez, J.; Amante, M.; Pestalardo, M. L.; Cazorla, A.; Bresson-Hadni, S.; Millon, L.

2013-01-01

Polycystic echinococcosis due to Echinococcus vogeli is a rare parasitic infection that occurs in rural areas of Central and South America. Only molecular identification performed on formalin-fixed paraffin-embedded liver tissue samples gave an unequivocal diagnosis of this disease in a Paraguayan immigrant in Argentina. PMID:23824768

Comprehensive gene expression profiling and immunohistochemical studies support application of immunophenotypic algorithm for molecular subtype classification in diffuse large B-cell lymphoma

DEFF Research Database (Denmark)

Visco, C; Li, Y; Xu-Monette, Z Y

2012-01-01

on formalin-fixed, paraffin-embedded tissue samples. Sections were stained with antibodies reactive with CD10, GCET1, FOXP1, MUM1 and BCL6 and cases were classified following a rationale of sequential steps of differentiation of B cells. Cutoffs for each marker were obtained using receiver...

The use of special stains in liver biopsy interpretation: Implications ...

African Journals Online (AJOL)

Materials and Methods: The formalin fixed paraffin embedded blocks of liver biopsies reported in two histopathology laboratories between 2008 and 2013 were retrieved. These were stained with H and E and the following standard special stains for liver tissue histology – Perl's Prussian blue, reticulin, Sirius red, Shikata ...

Immunophenotyping of Gastrointestinal Mesenchymal Tumours in ...

African Journals Online (AJOL)

METHODS: Materials were formalin fixed paraffin embedded blocks of GMT diagnosed in Lagos Nigeria between January 1995 and February 2007. Sections were stained with CD117, CD34, SMA, S100 and Desmin antibodies at the research Laboratory of The Leeds General Infirmary, United Kingdom following standard ...

Lack of evidence for KRAS oncogenic mutations in triple-negative breast cancer

International Nuclear Information System (INIS)

Sánchez-Muñoz, Alfonso; Gallego, Elena; Luque, Vanessa de; Pérez-Rivas, Luís G; Vicioso, Luís; Ribelles, Nuria; Lozano, José; Alba, Emilio

2010-01-01

Mutational analysis of the KRAS gene has recently been established as a complementary in vitro diagnostic tool for the identification of patients with colorectal cancer who will not benefit from anti-epidermal growth factor receptor (EGFR) therapies. Assessment of the mutation status of KRAS might also be of potential relevance in other EGFR-overexpressing tumors, such as those occurring in breast cancer. Although KRAS is mutated in only a minor fraction of breast tumors (5%), about 60% of the basal-like subtype express EGFR and, therefore could be targeted by EGFR inhibitors. We aimed to study the mutation frequency of KRAS in that subtype of breast tumors to provide a molecular basis for the evaluation of anti-EGFR therapies. Total, genomic DNA was obtained from a group of 35 formalin-fixed paraffin-embedded, triple-negative breast tumor samples. Among these, 77.1% (27/35) were defined as basal-like by immunostaining specific for the established surrogate markers cytokeratin (CK) 5/6 and/or EGFR. KRAS mutational status was determined in the purified DNA samples by Real Time (RT)-PCR using primers specific for the detection of wild-type KRAS or the following seven oncogenic somatic mutations: Gly12Ala, Gly12Asp, Gly12Arg, Gly12Cys, Gly12Ser, Gly12Val and Gly13Asp. We found no evidence of KRAS oncogenic mutations in all analyzed tumors. This study indicates that KRAS mutations are very infrequent in triple-negative breast tumors and that EGFR inhibitors may be of potential benefit in the treatment of basal-like breast tumors, which overexpress EGFR in about 60% of all cases

Identification of 5-hydroxytryptamine-producing cells by detection of fluorescence in paraffin-embedded tissue sections

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Y. Kaneko

2016-09-01

Full Text Available 5-Hydroxytryptamine (5-HT produced by enterochromaffin (EC cells is an important enteric mucosal signaling ligand and has been implicated in several gastrointestinal diseases, including inflammatory bowel disease and functional disorders such as irritable bowel syndrome. The present study reports a new, simple and rapid visualization method of 5-HT-producing EC cells utilizing detection of autofluorescence in paraffin-embedded tissue sections after formalin fixation. In human samples, there was a high incidence of autofluorescence+ cells in the 5-HT+ cells in the pyloric, small intestinal and colonic glands, while co-localization was lacking between autofluorescence+ and gastrin+ cells in the pyloric and small intestinal glands. Autofluorescence+ EC cells were detected in the colon of mice and rats. Autofluorescence+ cells were also observed in 5-HT+ β cells in the pancreatic islets of Langerhans in pregnant mice, while non-pregnant mouse pancreatic islet cells showed no 5-HT immunoreactivity or autofluorescence. These results suggest that autofluorescence+ cells are identical to 5-HT+ cells, and the source of autofluorescence may be 5-HT itself or molecules related to its synthesis or degradation. This autofluorescence signal detection method may be applicable for monitoring of inflammatory status of inflammatory bowel diseases in both the experimental and clinical settings.

The expression of prostate-specific antigen in invasive breast carcinoma and its relationship with routine clinicopathologic parameters

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Fereshteh Mohammadizadeh

2012-01-01

Full Text Available Background: Invasive breast carcinoma is one of the most common cancers of women. Parameters such as lymph node status, tumor grade, and the status of hormone receptors are among the main prognostic determinants of this cancer. Immunohistochemical detection of prostate-specific antigen (PSA is widely used to identify metastatic prostatic adenocarcinoma. However, its immunoreactivity has been found in some non-prostatic tissues. This study was conducted to assess PSA expression in invasive breast carcinoma and its relationship with routine clinicopathologic parameters. Materials and Methods: 100 formalin-fixed and paraffin-embedded invasive breast carcinoma tissue specimens from the pathology archive of Alzahra hospital (Isfahan, Iran were studied for the expression of estrogen receptor (ER, progesterone receptor (PR, HER2/neu, and PSA by immunohistochemistry. Stained sections were classified according to the intensity of staining and the percentage of cells showing PSA staining. The relationship between PSA expression and other markers, age, lymph node status, tumor subtype, and tumor grade was then studied. Results: No association was found between PSA expression on one hand and PR, Her2/neu, age, lymph node status, tumor grade, and tumor subtype on the other. PSA score was reversely correlated with ER expression (P = 0.015. Conclusion: Despite the reverse relationship between PSA expression and the immunoreactivity of ER, PSA expression was not correlated with other prognostic factors. Therefore, the detection of PSA by immunohistochemistry does not seem to be a significant prognostic parameter in patients with invasive breast carcinoma.

Single-Nucleotide Polymorphism-Microarray Ploidy Analysis of Paraffin-Embedded Products of Conception in Recurrent Pregnancy Loss Evaluations.

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Maslow, Bat-Sheva L; Budinetz, Tara; Sueldo, Carolina; Anspach, Erica; Engmann, Lawrence; Benadiva, Claudio; Nulsen, John C

2015-07-01

To compare the analysis of chromosome number from paraffin-embedded products of conception using single-nucleotide polymorphism (SNP) microarray with the recommended screening for the evaluation of couples presenting with recurrent pregnancy loss who do not have previous fetal cytogenetic data. We performed a retrospective cohort study including all women who presented for a new evaluation of recurrent pregnancy loss over a 2-year period (January 1, 2012, to December 31, 2013). All participants had at least two documented first-trimester losses and both the recommended screening tests and SNP microarray performed on at least one paraffin-embedded products of conception sample. Single-nucleotide polymorphism microarray identifies all 24 chromosomes (22 autosomes, X, and Y). Forty-two women with a total of 178 losses were included in the study. Paraffin-embedded products of conception from 62 losses were sent for SNP microarray. Single-nucleotide polymorphism microarray successfully diagnosed fetal chromosome number in 71% (44/62) of samples, of which 43% (19/44) were euploid and 57% (25/44) were noneuploid. Seven of 42 (17%) participants had abnormalities on recurrent pregnancy loss screening. The per-person detection rate for a cause of pregnancy loss was significantly higher in the SNP microarray (0.50; 95% confidence interval [CI] 0.36-0.64) compared with recurrent pregnancy loss evaluation (0.17; 95% CI 0.08-0.31) (P=.002). Participants with one or more euploid loss identified on paraffin-embedded products of conception were significantly more likely to have an abnormality on recurrent pregnancy loss screening than those with only noneuploid results (P=.028). The significance remained when controlling for age, number of losses, number of samples, and total pregnancies. These results suggest that SNP microarray testing of paraffin-embedded products of conception is a valuable tool for the evaluation of recurrent pregnancy loss in patients without prior fetal

Healing effect of Sanguisorba officinalis L extract on second-degree ...

African Journals Online (AJOL)

The skin tissues were fixed with 10 % formalin. After fixation, samples were embedded in paraffin, cut into 3 mm frozen sections with a cryostat microtome, then stained with hematoxylin eosin reagent. Collagen fiber, inflammatory cell, blood vessel and granulation tissue of the skin tissues were examined under a microscope ...

The negative predictive value of p16INK4a to assess the outcome of cervical intraepithelial neoplasia 1 in the uterine cervix

DEFF Research Database (Denmark)

Hariri, Jalil; Øster, Anne

2007-01-01

The immunohistochemical expression of p16 in formalin-fixed and paraffin-embedded histological sections was evaluated in a retrospective study comprising a low-grade group of 100 cases of cervical intraepithelial neoplasia (CIN) 1, a high-grade group of 50 cases of CIN 2 to 3, and a benign group...

Isolation and Characterization of Prostate Cancer Stem Cells

Science.gov (United States)

2013-10-01

et al. Targeting cancer stem cells by inhibiting Wnt, Notch, and Hedgehog pathways. Nat Rev Clin Oncol 2011;8:97–106. 26] Guise T. Examining the...Nitrogen or fixed in formalin and paraffin-embedded to evaluate anatomy and glandular architecture. The remainder of the tissue was mechan- ically and

Immunohistochemical expression of latent membrane protein 1 ...

African Journals Online (AJOL)

Methods: Archival formalin-fixed, paraffin-embedded NPC biopsies were evaluated in 23 Moroccan patients for the presence of LMP1 and p53 using immunohistochemistry (IHC). Results: No LMP1 expression was observed whereas 8 of 23 cases (34. 7%) had detectable p53 protein in the nuclei of tumor cells.

Multimodal Raman-fluorescence spectroscopy of formalin fixed samples is able to discriminate brain tumors from dysplastic tissue

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Anand, Suresh; Cicchi, Riccardo; Giordano, Flavio; Buccoliero, Anna Maria; Pavone, Francesco Saverio

2014-05-01

In the recent years, there has been a considerable surge in the application of spectroscopy for disease diagnosis. Raman and fluorescence spectra provide characteristic spectral profile related to biochemical and morphological changes when tissues progress from normal state towards malignancy. Spectroscopic techniques offer the advantage of being minimally invasive compared to traditional histopathology, real time and quantitative. In biomedical optical diagnostics, freshly excised specimens are preferred for making ex-vivo spectroscopic measurements. With regard to fresh tissues, if the lab is located far away from the clinic it could pose a problem as spectral measurements have to be performed immediately after dissection. Tissue samples are usually placed in a fixative agent such as 4% formaldehyde to preserve the samples before processing them for routine histopathological studies. Fixation prevents the tissues from decomposition by arresting autolysis. In the present study, we intend to investigate the possibility of using formalin fixed samples for discrimination of brain tumours from dysplastic tissue using Raman and fluorescence spectroscopy. Formalin fixed samples were washed with phosphate buffered saline for about 5 minutes in order to remove the effects of formalin during spectroscopic measurements. In case of fluorescence spectroscopy, changes in spectral profile have been observed in the region between 550-670 nm between dysplastic and tumor samples. For Raman measurements, we found significant differences in the spectral profiles between dysplasia and tumor. In conclusion, formalin fixed samples can be potentially used for the spectroscopic discrimination of tumor against dysplastic tissue in brain samples.

Targeted biomarker profiling of matched primary and metastatic estrogen receptor positive breast cancers.

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Erica B Schleifman

Full Text Available Patients with newly diagnosed, early stage estrogen receptor positive (ER+ breast cancer often show disease free survival in excess of five years following surgery and systemic adjuvant therapy. An important question is whether diagnostic tumor tissue from the primary lesion offers an accurate molecular portrait of the cancer post recurrence and thus may be used for predictive diagnostic purposes for patients with relapsed, metastatic disease. As the class I phosphatidylinositol 3' kinase (PI3K pathway is frequently activated in ER+ breast cancer and has been linked to acquired resistance to hormonal therapy, we hypothesized pathway status could evolve over time and treatment. Biomarker analyses were conducted on matched, asynchronous primary and metastatic tumors from 77 patients with ER+ breast cancer. We examined whether PIK3CA and AKT1 alterations or PTEN and Ki67 levels showed differences between primary and metastatic samples. We also sought to look more broadly at gene expression markers reflective of proliferation, molecular subtype, and key receptors and signaling pathways using an mRNA analysis platform developed on the Fluidigm BioMark™ microfluidics system to measure the relative expression of 90 breast cancer related genes in formalin-fixed paraffin-embedded (FFPE tissue. Application of this panel of biomarker assays to matched tumor pairs showed a high concordance between primary and metastatic tissue, with generally few changes in mutation status, proliferative markers, or gene expression between matched samples. The collection of assays described here has been optimized for FFPE tissue and may have utility in exploratory analyses to identify patient subsets responsive to targeted therapies.

A case study of rabies diagnosis from formalin-fixed brain material : short communication

Directory of Open Access Journals (Sweden)

J. Coertse

2011-05-01

Full Text Available Rabies is caused by several Lyssavirus species, a group of negative sense RNA viruses. Although rabies is preventable, it is often neglected particularly in developing countries in the face of many competing public and veterinary health priorities. Epidemiological information based on laboratory-based surveillance data is critical to adequately strategise control and prevention plans. In this regard the fluorescent antibody test for rabies virus antigen in brain tissues is still considered the basic requirement for laboratory confirmation of animal cases. Occasionally brain tissues from suspected rabid animals are still submitted in formalin, although this has been discouraged for a number of years. Immunohistochemical testing or a modified fluorescent antibody technique can be performed on such samples. However, this method is cumbersome and cannot distinguish between different Lyssavirus species. Owing to RNA degradation in formalin-fixed tissues, conventional RT-PCR methodologies have also been proven to be unreliable. This report is concerned with a rabies case in a domestic dog from an area in South Africa where rabies is not common. Typing of the virus involved was therefore important, but the only available sample was submitted as a formalin-fixed specimen. A real-time RT-PCR method was therefore applied and it was possible to confirmrabies and obtain phylogenetic information that indicated a close relationship between this virus and the canid rabies virus variants from another province (KwaZulu-Natal in South Africa.

A Molecular Framework for Understanding DCIS

Science.gov (United States)

2016-10-01

formalin fixation and paraffin embedding. Utilizing Duke’s Breast Data Mart We have identified the Duke Breast Data Mart as a valuable resource ...IRB (eIRB), CTMS (eResearch), Finance ( SAP and PDC), HR (Faculty and Staff) and other base operational systems to present a complete view of research...Hannon and Duke groups, given separate physical locations. Changes in use or care of human subjects Nothing to report. Products Nothing to report. 5

Nigerian Veterinary Journal - Vol 38, No 3 (2017)

African Journals Online (AJOL)

Formalin-fixed and paraffin-embedded tissues of chickens are useful for retrospective studies on pathology of H5N1 Highly Pathogenic Avian Influenza Virus (HPAI) outbreaks in Nigeria · EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT. B.O. Akanbi, S Fereidouni, V.O. ...

Molecular detection of Coxiella burnetii from the formalin-fixed tissues of Q fever patients with acute hepatitis.

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Young-Rock Jang

Full Text Available Serologic diagnosis is one of the most widely used diagnostic methods for Q fever, but the window period in antibody response of 2 to 3 weeks after symptom onset results in significant diagnostic delay. We investigated the diagnostic utility of Q fever PCR from formalin-fixed liver tissues in Q fever patients with acute hepatitis.We reviewed the clinical and laboratory data in patients with Q fever hepatitis who underwent liver biopsy during a 17-year period, and whose biopsied tissues were available. We also selected patients who revealed granuloma in liver biopsy and with no Q fever diagnosis within the last 3 years as control. Acute Q fever hepatitis was diagnosed if two or more of the following clinical, serologic, or histopathologic criteria were met: (1 an infectious hepatitis-like clinical feature such as fever (≥ 38°C with elevated hepatic transaminase levels; (2 exhibition of a phase II immunoglobulin G (IgG antibodies titer by IFA of ≥ 1:128 in single determination, or a four-fold or greater rise between two separate samples obtained two or more weeks apart; (3 histologic finding of biopsy tissue showing characteristic fibrin ring granuloma.A total of 11 patients with acute Q fever hepatitis were selected and analyzed. Of the 11 patients, 3 (27% had exposure to zoonotic risk factors and 7 (63% met the serologic criteria. Granulomas with either circumferential or radiating fibrin deposition were observed in 10 cases on liver biopsy and in 1 case on bone marrow biopsy. 8 (73% revealed positive Coxiella burnetii PCR from their formalin-fixed liver tissues. In contrast, none of 10 patients with alternative diagnosis who had hepatic granuloma revealed positive C. burnetii PCR from their formalin-fixed liver tissues.Q fever PCR from formalin-fixed liver tissues appears to be a useful adjunct for diagnosing Q fever hepatitis.

Measurements of T1 and T2 over time in formalin-fixed human whole-brain specimens

International Nuclear Information System (INIS)

Tovi, M.; Ericsson, A.

1992-01-01

T1 and T2 were measured in 5 formalin-fixed human whole-brain specimens as a function of time. Gray matter/white matter contrast reversal was observed around the 4th day and was considered to be due to the greater decrease in T1 in gray than in white matter. A possible explanation for this is that the decomposition of the myelin phospholipid structure by formalin somewhat counteracts the general reductive effect of the fixation procedure on relaxation times. (orig.)

Predicting response to primary chemotherapy: gene expression profiling of paraffin-embedded core biopsy tissue.

Science.gov (United States)

Mina, Lida; Soule, Sharon E; Badve, Sunil; Baehner, Fredrick L; Baker, Joffre; Cronin, Maureen; Watson, Drew; Liu, Mei-Lan; Sledge, George W; Shak, Steve; Miller, Kathy D

2007-06-01

Primary chemotherapy provides an ideal opportunity to correlate gene expression with response to treatment. We used paraffin-embedded core biopsies from a completed phase II trial to identify genes that correlate with response to primary chemotherapy. Patients with newly diagnosed stage II or III breast cancer were treated with sequential doxorubicin 75 mg/M2 q2 wks x 3 and docetaxel 40 mg/M2 weekly x 6; treatment order was randomly assigned. Pretreatment core biopsy samples were interrogated for genes that might correlate with pathologic complete response (pCR). In addition to the individual genes, the correlation of the Oncotype DX Recurrence Score with pCR was examined. Of 70 patients enrolled in the parent trial, core biopsies samples with sufficient RNA for gene analyses were available from 45 patients; 9 (20%) had inflammatory breast cancer (IBC). Six (14%) patients achieved a pCR. Twenty-two of the 274 candidate genes assessed correlated with pCR (p < 0.05). Genes correlating with pCR could be grouped into three large clusters: angiogenesis-related genes, proliferation related genes, and invasion-related genes. Expression of estrogen receptor (ER)-related genes and Recurrence Score did not correlate with pCR. In an exploratory analysis we compared gene expression in IBC to non-inflammatory breast cancer; twenty-four (9%) of the genes were differentially expressed (p < 0.05), 5 were upregulated and 19 were downregulated in IBC. Gene expression analysis on core biopsy samples is feasible and identifies candidate genes that correlate with pCR to primary chemotherapy. Gene expression in IBC differs significantly from noninflammatory breast cancer.

CDX2 downregulation is associated with poor differentiation and MMR deficiency in colon cancer

DEFF Research Database (Denmark)

Olsen, J.; Eiholm, Susanne; Kirkeby, L T

2016-01-01

adjacent tissue were fixed in liquid nitrogen for RNA extraction or in formalin and paraffin embedded (FFPE) for immunohistochemical staining. CDX2 mRNA expression was evaluated by RT-qPCR. FFPE sections were stained for MLH1, MSH2, MSH6, PMS2, and CDX2. RESULTS: A total of 191 patient samples were...

Advantages of infrared transflection micro spectroscopy and paraffin-embedded sample preparation for biological studies

Science.gov (United States)

Yao, Jie; Li, Qian; Zhou, Bo; Wang, Dan; Wu, Rie

2018-04-01

Fourier-Transform Infrared micro-spectroscopy is an excellent method for biological analyses. In this paper, series metal coating films on ITO glass were prepared by the electrochemical method and the different thicknesses of paraffin embedding rat's brain tissue on the substrates were studied by IR micro-spetroscopy in attenuated total reflection (ATR) mode and transflection mode respectively. The Co-Ni-Cu alloy coating film with low cost is good reflection substrates for the IR analysis. The infrared microscopic transflection mode needs not to touch the sample at all and can get the IR spectra with higher signal to noise ratios. The Paraffin-embedding method allows tissues to be stored for a long time for re-analysis to ensure the traceability of the sample. Also it isolates the sample from the metal and avoids the interaction of biological tissue with the metals. The best thickness of the tissues is 4 μm.

Avaliação de dois métodos de extração de DNA de material parafinado para amplificação em PCR Evaluation of two methods of DNA extraction from paraffin-embedded material for PCR amplification

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Luciana Estevam Simonato

2007-04-01

Full Text Available INTRODUÇÃO: Diversos métodos para extração de DNA a partir de tecidos biológicos inclusos em parafina encontram-se descritos na literatura, sendo o sucesso desse procedimento de grande importância para a realização de métodos moleculares de diagnóstico empregando a reação em cadeia da polimerase (PCR. OBJETIVO: Este estudo avaliou dois métodos de extração de DNA de material parafinado, visando à amplificação do DNA genômico pela técnica da PCR. MATERIAL E MÉTODO: Foram utilizadas 35 amostras de casos de carcinoma epidermóide de assoalho bucal diagnosticados e tratados no Centro de Oncologia Bucal da Universidade Estadual Paulista (Unesp. Os métodos de extração de DNA avaliados incluíram: 1. digestão com proteinase K seguida por purificação com Chelex 100® (BioRad; e 2. sistema QIAamp DNA minikit® (Qiagen. O DNA obtido foi quantificado por espectrofotometria e amplificado pela técnica da PCR, utilizando-se oligonucleotídeos iniciadores para betaglobina. RESULTADOS: A concentração de DNA obtido do material extraído com o primeiro método apresentou média de 120,62 ng/µl com razão entre as leituras das absorbâncias 260/280 variando de 0,8 a 1,41. Para as amostras extraídas com o segundo procedimento, o rendimento médio foi de 67,38 ng/µl, no entanto a razão 260/280 variou entre 1,11 e 2,53. O material foi submetido à PCR e, das 35 amostras extraídas com cada método, respectivamente, 29 e 30 apresentaram sinal positivo. CONCLUSÃO: Os dois métodos utilizados para obtenção de DNA de material parafinado apresentaram desempenho semelhante, revelando que ambos têm potencial para auxiliar na prática da biologia molecular diagnóstica, assim como no estudo diagnóstico retrospectivo em material parafinizado.BACKGROUND: There are several methods for DNA extraction from formalin-fixed paraffin-embedded tissues reported in the literature. High success rate on this procedure is important for the use of

[Comparison of two different real-time PCR systems in postmortem diagnosis of tuberculosis in paraffin-embedded tissues].

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Yağmur, Gülhan; Albayrak, Nurhan; Daş, Taner; Yıldırım, Muzaffer; Ozgün, Ayşe; Büyük, Yalçın

2014-10-01

Tuberculosis (TB) is one of those infections with high morbidity and mortality in all around the world. Hundreds of people died from this disease without diagnosed or due to resistant strains in Turkey. Therefore, it is important to identify postmortem cases who have died from tuberculosis. Molecular methods have been widely used as well as conventional methods in the diagnosis of tuberculosis. The aim of this study was to compare the two different real-time polymerase chain reaction (Rt-PCR) system in the postmortem diagnosis of Mycobacterium tuberculosis infections in paraffin-embedded tissues. A total of 40 paraffin-embedded tissue samples [lung (n= 35), brain (n= 2), heart (n= 2), lymph node (n= 1)] in which histopathologic findings consistent with TB (necrotizing granulomatous inflammation, gelatinous caseous pneumonia, necrotic fibrous nodul) obtained from 37 autopsy cases (31 male, 6 female; age range: 25-85 yrs) were included in the study. Paraffin-embedded tissues were deparafinized with xylene and ethyl alcohol and then DNA isolation was done with QIAsymphony DSP Virus/Pathogen Midi kit in the QIAsymphony device. DNA amplification process was performed by Rt-PCR using the kit Artus® M. tuberculosis RG-PCR in the Rotor-Gene® Q device (Qiagen, Germany). Likewise, after deparafinization process, samples placed in the cartridge and isolation and Rt-PCR was performed by Xpert® MTB/RIF (Cepheid, USA) system, simultaneosly. Seventeen and 20 out of the 40 paraffin-embedded tissues yielded positive results with Qiagen and Xpert system, respectively. M.tuberculosis DNA was found positive in 13 (32.5%) and negative in 16 (40%) of the samples by both of the systems, exhibiting 72.5% (29/40) of concordance. On the other hand, seven (17.5%) samples that were positive with Xpert system yielded negative result with the Qiagen, while four (10%) samples that were positive with Qiagen yielded negative result with the Xpert system. Of the 20 positive cases detected with

Papillary neoplasia of the breast: immunohistochemically defined myoepithelial cells in the diagnosis of benign and malignant papillary breast neoplasms.

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Raju, U B; Lee, M W; Zarbo, R J; Crissman, J D

1989-11-01

The presence or absence of myoepithelial cells (ME) has been considered as an important feature in the differential diagnosis of benign and malignant papillary lesions of the breast. We evaluated the distribution of myoepithelial cells in formalin-fixed paraffin-embedded tissue sections of 25 papillomas and 18 papillary carcinomas by ABC immunoperoxidase technique with antibodies to muscle actin (HHF-35) and high molecular weight (HMW) keratin (clone 34BE12, cytokeratins 1, 5, 10, and 14; reacting preferentially with ME cells) and an antiserum to S-100 protein. Also included in the study were eight cases of micropapillary ductal carcinoma in situ (DCIS) having a few fibrovascular cores and five peripheral papillomas with accompanying ductal carcinoma in situ or atypical hyperplasia. The antibodies to muscle actin were sensitive and relatively specific for ME cells of the breast and uniformly labeled ME cells in all 25 papillomas. ME cells were absent or extremely sparse in papillary carcinomas. They were present focally in some of the fibrovascular cores of the micropapillary DCIS, and a mixed pattern was observed in peripheral papillomas with areas of carcinoma. HMW keratin was variably expressed in ME cells in most cases with positive internal controls and was present in several normal ductal and papilloma epithelial cells but not in epithelial cells of papillary carcinomas. HMW keratin, although less specific for ME cells, was a useful adjunct because of its reactivity with ME cells as well as hyperplastic epithelial cells in papillomas, which resulted in a combined positive reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

In situ detection of estrogen receptor dimers in breast carcinoma cells in archival materials using proximity ligation assay (PLA).

Science.gov (United States)

Iwabuchi, Erina; Miki, Yasuhiro; Ono, Katsuhiko; Onodera, Yoshiaki; Suzuki, Takashi; Hirakawa, Hisashi; Ishida, Takanori; Ohuchi, Noriaki; Sasano, Hironobu

2017-01-01

Estrogen receptor (ER) is required for carcinoma cell proliferation in the great majority of breast cancer and also functions as a dimer. ER dimeric proteins have been largely identified by BRET/FRET analyses but their in situ visualization have not yet been reported. Recently, in situ Proximity Ligation Assay (PLA) has been developed as the methods detecting protein interactions in situ. Therefore, in this study we firstly demonstrated the dimerization of ERα in breast carcinoma cell lines and tissues using PLA. The human breast carcinoma cell lines MCF-7, T-47D and MDA-MB-231 were used in this study. Cells were treated with ER agonist or antagonist and fixed in 4% PFA, and ER dimers were subsequently detected using PLA. The evaluation of ER dimers in breast carcinoma cell lines were quantified by measuring the area of dots localized in the nuclei using image analysis. We also firstly demonstrated the visualization of ER dimer patterns in 10% formalin-fixed paraffin-embedded tissues of breast cancer using PLA technique. Estradiol (E2) administration induced ERα homodimers in the nuclei of MCF-7 and T-47D but not in ER-negative MDA-MB-231. 4-OH tamoxifen also induced ERα homodimers but the subcellular localization of these ERα homodimers was predominant in cytoplasm instead of the nuclei induced by E2 treatment. ICI182,780 treatment did decrease the number of formation of ERα homodimers in MCF-7. In breast cancer patients, ERα PLA score was significantly correlated positively with ERα- or PgR (progesterone receptor) immunohistochemical scores and inversely with Ki-67-labeling index, respectively. We also demonstrated the ERα/β heterodimer as well as ERα homodimers in both breast carcinoma cell lines and surgical pathology specimens. In summary, we did firstly succeed in the visualization of ER dimeric proteins using PLA method. The evaluation of ER dimer patterns could provide pivotal information as to the prediction of response to endocrine therapy of

Modeling formalin fixation and histological processing with ribonuclease A: effects of ethanol dehydration on reversal of formaldehyde cross-links.

Science.gov (United States)

Fowler, Carol B; O'Leary, Timothy J; Mason, Jeffrey T

2008-07-01

Understanding the chemistry of protein modification by formaldehyde fixation and subsequent tissue processing is central to developing improved methods for antigen retrieval in immunohistochemistry and for recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissues for proteomic analysis. Our initial studies of single proteins, such as bovine pancreatic ribonuclease A (RNase A), in 10% buffered formalin solution revealed that upon removal of excess formaldehyde, monomeric RNase A exhibiting normal immunoreactivity could be recovered by heating at 60 degrees C for 30 min at pH 4. We next studied tissue surrogates, which are gelatin-like plugs of fixed proteins that have sufficient physical integrity to be processed using normal tissue histology. Following histological processing, proteins could be extracted from the tissue surrogates by combining heat, detergent, and a protein denaturant. However, gel electrophoresis revealed that the surrogate extracts contained a mixture of monomeric and multimeric proteins. This suggested that during the subsequent steps of tissue processing protein-formaldehyde adducts undergo further modifications that are not observed in aqueous proteins. As a first step toward understanding these additional modifications we have performed a comparative evaluation of RNase A following fixation in buffered formaldehyde alone and after subsequent dehydration in 100% ethanol by combining gel electrophoresis, chemical modification, and circular dichroism spectroscopic studies. Our results reveal that ethanol-induced rearrangement of the conformation of fixed RNase A leads to protein aggregation through the formation of large geometrically compatible hydrophobic beta-sheets that are likely stabilized by formaldehyde cross-links, hydrogen bonds, and van der Waals interactions. It requires substantial energy to reverse the formaldehyde cross-links within these sheets and regenerate protein monomers free of formaldehyde modifications

Copy number and loss of heterozygosity detected by SNP array of formalin-fixed tissues using whole-genome amplification.

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Angela Stokes

Full Text Available The requirement for large amounts of good quality DNA for whole-genome applications prohibits their use for small, laser capture micro-dissected (LCM, and/or rare clinical samples, which are also often formalin-fixed and paraffin-embedded (FFPE. Whole-genome amplification of DNA from these samples could, potentially, overcome these limitations. However, little is known about the artefacts introduced by amplification of FFPE-derived DNA with regard to genotyping, and subsequent copy number and loss of heterozygosity (LOH analyses. Using a ligation adaptor amplification method, we present data from a total of 22 Affymetrix SNP 6.0 experiments, using matched paired amplified and non-amplified DNA from 10 LCM FFPE normal and dysplastic oral epithelial tissues, and an internal method control. An average of 76.5% of SNPs were called in both matched amplified and non-amplified DNA samples, and concordance was a promising 82.4%. Paired analysis for copy number, LOH, and both combined, showed that copy number changes were reduced in amplified DNA, but were 99.5% concordant when detected, amplifications were the changes most likely to be 'missed', only 30% of non-amplified LOH changes were identified in amplified pairs, and when copy number and LOH are combined ∼50% of gene changes detected in the unamplified DNA were also detected in the amplified DNA and within these changes, 86.5% were concordant for both copy number and LOH status. However, there are also changes introduced as ∼20% of changes in the amplified DNA are not detected in the non-amplified DNA. An integrative network biology approach revealed that changes in amplified DNA of dysplastic oral epithelium localize to topologically critical regions of the human protein-protein interaction network, suggesting their functional implication in the pathobiology of this disease. Taken together, our results support the use of amplification of FFPE-derived DNA, provided sufficient samples are used

miR-671-5p inhibits epithelial-to-mesenchymal transition by downregulating FOXM1 expression in breast cancer

Science.gov (United States)

Tan, Xiaohui; Fu, Yebo; Chen, Liang; Lee, Woojin; Lai, Yinglei; Rezaei, Katayoon; Tabbara, Sana; Latham, Patricia; Teal, Christine B.; Man, Yan-Gao; Siegel, Robert S.; Brem, Rachel F.; Fu, Sidney W.

2016-01-01

MicroRNA (miRNA) dysfunction is associated with a variety of human diseases, including cancer. Our previous study showed that miR-671-5p was deregulated throughout breast cancer progression. Here, we report for the first time that miR-671-5p is a tumor-suppressor miRNA in breast tumorigenesis. We found that expression of miR-671-5p was decreased significantly in invasive ductal carcinoma (IDC) compared to normal in microdissected formalin-fixed, paraffin-embedded (FFPE) tissues. Forkhead Box M1 (FOXM1), an oncogenic transcription factor, was predicted as one of the direct targets of miR-671-5p, which was subsequently confirmed by luciferase assays. Forced expression of miR-671-5p in breast cancer cell lines downregulated FOXM1 expression, and attenuated the proliferation and invasion in breast cancer cell lines. Notably, overexpression of miR-671-5p resulted in a shift from epithelial-to-mesenchymal transition (EMT) to mesenchymal-to-epithelial transition (MET) phenotypes in MDA-MB-231 breast cancer cells and induced S-phase arrest. Moreover, miR-671-5p sensitized breast cancer cells to cisplatin, 5-fluorouracil (5-FU) and epirubicin exposure. Host cell reactivation (HCR) assays showed that miR-671-5p reduces DNA repair capability in post-drug exposed breast cancer cells. cDNA microarray data revealed that differentially expressed genes when miR-671-5p was transfected are associated with cell proliferation, invasion, cell cycle, and EMT. These data indicate that miR-671-5p functions as a tumor suppressor miRNA in breast cancer by directly targeting FOXM1. Hence, miR-671-5p may serve as a novel therapeutic target for breast cancer management. PMID:26588055

Detection of apoptosis in paraffin embedded tissues: the influence of tissue type and fixation

Czech Academy of Sciences Publication Activity Database

Matalová, Eva; Dubská, L.; Míšek, Ivan

2002-01-01

Roč. 71, č. 4 (2002), s. 529-533 ISSN 0001-7213 R&D Projects: GA ČR GP204/02/P112; GA AV ČR KSK6005114 Keywords : apoptosis * TUNEL test * paraffin embedded tissues Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.370, year: 2002

Analytical Validation of a Highly Quantitative, Sensitive, Accurate, and Reproducible Assay (HERmark® for the Measurement of HER2 Total Protein and HER2 Homodimers in FFPE Breast Cancer Tumor Specimens

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Jeffrey S. Larson

2010-01-01

Full Text Available We report here the results of the analytical validation of assays that measure HER2 total protein (H2T and HER2 homodimer (H2D expression in Formalin Fixed Paraffin Embedded (FFPE breast cancer tumors as well as cell line controls. The assays are based on the VeraTag technology platform and are commercially available through a central CAP-accredited clinical reference laboratory. The accuracy of H2T measurements spans a broad dynamic range (2-3 logs as evaluated by comparison with cross-validating technologies. The measurement of H2T expression demonstrates a sensitivity that is approximately 7–10 times greater than conventional immunohistochemistry (IHC (HercepTest. The HERmark assay is a quantitative assay that sensitively and reproducibly measures continuous H2T and H2D protein expression levels and therefore may have the potential to stratify patients more accurately with respect to response to HER2-targeted therapies than current methods which rely on semiquantitative protein measurements (IHC or on indirect assessments of gene amplification (FISH.

Diagnosis of breast cancer at dynamic MRI in patients with breast augmentation by paraffin or silicone injection

International Nuclear Information System (INIS)

Youk, J.H.; Son, E.J.; Kim, E.-K.; Kim, J.-A.; Kim, M.J.; Kwak, J.Y.; Lee, S.M.

2009-01-01

Aim: To determine the diagnostic performance of dynamic magnetic resonance imaging (MRI) for breast cancer in breasts augmented with liquid paraffin or silicone injection. Materials and methods: Among 62 patients with breast augmentation by liquid paraffin or silicone injection who had undergone dynamic breast MRI at our institution, 27 women, who had pathological diagnosis or at least 1-year MRI follow-up, were included in this retrospective study and their MRI images were reviewed. For enhancing lesions on MRI, the morphological features, enhancement kinetics, and BI-RADS assessment category were analysed. The lesion characteristics at MRI were correlated with the final diagnosis based on the histopathological result or at least 1-year MRI follow-up. Results: Of the 27 patients, 17 enhancing lesions in 13 patients were found on MRI. All six lesions that were confirmed as malignancy showed suspicious morphological findings and type 2 or 3 enhancement kinetics, assigned to BI-RADS category 4 or 5. Of the remaining 11 benign lesions, 10 showed benign-favouring morphological findings, and all showed type 1 enhancement kinetics, assigned to BI-RADS category 2 or 4. Conclusion: In patients with breasts injected with foreign material, MRI was used to successfully diagnose malignant breast lesions and could be the diagnostic method of choice. Analysis of the morphological and kinetic features at MRI in conjunction with clinical findings is essential.

Staining Methods for Normal and Regenerative Myelin in the Nervous System.

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Carriel, Víctor; Campos, Antonio; Alaminos, Miguel; Raimondo, Stefania; Geuna, Stefano

2017-01-01

Histochemical techniques enable the specific identification of myelin by light microscopy. Here we describe three histochemical methods for the staining of myelin suitable for formalin-fixed and paraffin-embedded materials. The first method is conventional luxol fast blue (LFB) method which stains myelin in blue and Nissl bodies and mast cells in purple. The second method is a LBF-based method called MCOLL, which specifically stains the myelin as well the collagen fibers and cells, giving an integrated overview of the histology and myelin content of the tissue. Finally, we describe the osmium tetroxide method, which consist in the osmication of previously fixed tissues. Osmication is performed prior the embedding of tissues in paraffin giving a permanent positive reaction for myelin as well as other lipids present in the tissue.

Comparison of 5 Ki-67 antibodies regarding reproducibility and capacity to predict prognosis in breast cancer: does the antibody matter?

Science.gov (United States)

Ács, Balázs; Kulka, Janina; Kovács, Kristóf Attila; Teleki, Ivett; Tőkés, Anna-Mária; Meczker, Ágnes; Győrffy, Balázs; Madaras, Lilla; Krenács, Tibor; Szász, Attila Marcell

2017-07-01

Although several antibodies are available for immunohistochemical detection of Ki-67, even the most commonly used MIB-1 has not been validated yet. Our aim was to compare 5 commercially available antibodies for detection of Ki-67 in terms of agreement and their ability in predicting prognosis of breast cancer. Tissue microarrays were constructed from 378 breast cancer patients' representative formalin-fixed, paraffin-embedded tumor blocks. Five antibodies were used to detect Ki-67 expression: MIB-1 using chromogenic detection and immunofluorescent-labeled MIB-1, SP-6, 30-9, poly, and B56. Semiquantitative assessment was performed by 2 pathologists independently on digitized slides. To compare the 5 antibodies, intraclass correlation and concordance correlation coefficient were used. All the antibodies but immunofluorescent-labeled MIB-1 (at 20% and 30% thresholds, P=.993 and P=.342, respectively) and B56 (at 30% threshold, P=.288) separated high- and low-risk patient groups. However, there were a significant difference (P values for all comparisons≤.005) and a moderate concordance (intraclass correlation, 0.645) between their Ki-67 labeling index scores. The highest concordance was found between MIB-1 and poly (concordance correlation coefficient=0.785) antibodies. None of the antibodies except Ki-67 labeling index as detected by poly (P=.031) at 20% threshold and lymph node status (Pantibodies in their capacity to detect proliferating tumor cells and to separate low- and high-risk breast cancer patient groups. Copyright © 2017 Elsevier Inc. All rights reserved.

Dual color chromogenic in situ hybridization for determination of HER2 status in breast cancer: a large comparative study to current state of the art fluorescence in situ hybridization

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Mollerup Jens

2012-02-01

Full Text Available Abstract Background Chromogenic in situ hybridization (CISH is fast becoming a well established technique for easy and sensitive determination of HER2 gene status in breast cancer. However, for the chromogenic method to achieve status as a safe and reliable technique, the method needs to be validated against already known and validated FISH techniques. Methods Here it is reported from a comparative study where HER2 gene status obtained by HER2 CISH pharmDx™ Kit was compared to HER2 gene status obtained by the FDA approved HER2 FISH pharmDx™ Kit and the PathVysion HER-2 DNA probe Kit. The study included 365 formalin fixed and paraffin-embedded invasive breast cancer tissue specimens collected consecutively at a US reference laboratory. Results The data obtained revealed an overall HER2 status concordance of approximately 98% for comparisons of HER2 CISH pharmDx™ Kit to both HER2 FISH pharmDx™ Kit and PathVysion HER-2 DNA Probe Kit. Conclusions The concordance between results obtained using the recently FDA approved HER2 CISH pharmDx™ Kit with previously FDA approved FISH techniques for HER2 gene status determination indicate that the HER2 CISH pharmDx™ Kit is a reliable chromogenic alternative to fluorescence-based methods.

Dual color chromogenic in situ hybridization for determination of HER2 status in breast cancer: a large comparative study to current state of the art fluorescence in situ hybridization

Science.gov (United States)

2012-01-01

Background Chromogenic in situ hybridization (CISH) is fast becoming a well established technique for easy and sensitive determination of HER2 gene status in breast cancer. However, for the chromogenic method to achieve status as a safe and reliable technique, the method needs to be validated against already known and validated FISH techniques. Methods Here it is reported from a comparative study where HER2 gene status obtained by HER2 CISH pharmDx™ Kit was compared to HER2 gene status obtained by the FDA approved HER2 FISH pharmDx™ Kit and the PathVysion HER-2 DNA probe Kit. The study included 365 formalin fixed and paraffin-embedded invasive breast cancer tissue specimens collected consecutively at a US reference laboratory. Results The data obtained revealed an overall HER2 status concordance of approximately 98% for comparisons of HER2 CISH pharmDx™ Kit to both HER2 FISH pharmDx™ Kit and PathVysion HER-2 DNA Probe Kit. Conclusions The concordance between results obtained using the recently FDA approved HER2 CISH pharmDx™ Kit with previously FDA approved FISH techniques for HER2 gene status determination indicate that the HER2 CISH pharmDx™ Kit is a reliable chromogenic alternative to fluorescence-based methods. PMID:22333181

Identifying Corneal Infections in Formalin-Fixed Specimens Using Next Generation Sequencing.

Science.gov (United States)

Li, Zhigang; Breitwieser, Florian P; Lu, Jennifer; Jun, Albert S; Asnaghi, Laura; Salzberg, Steven L; Eberhart, Charles G

2018-01-01

We test the ability of next-generation sequencing, combined with computational analysis, to identify a range of organisms causing infectious keratitis. This retrospective study evaluated 16 cases of infectious keratitis and four control corneas in formalin-fixed tissues from the pathology laboratory. Infectious cases also were analyzed in the microbiology laboratory using culture, polymerase chain reaction, and direct staining. Classified sequence reads were analyzed with two different metagenomics classification engines, Kraken and Centrifuge, and visualized using the Pavian software tool. Sequencing generated 20 to 46 million reads per sample. On average, 96% of the reads were classified as human, 0.3% corresponded to known vectors or contaminant sequences, 1.7% represented microbial sequences, and 2.4% could not be classified. The two computational strategies successfully identified the fungal, bacterial, and amoebal pathogens in most patients, including all four bacterial and mycobacterial cases, five of six fungal cases, three of three Acanthamoeba cases, and one of three herpetic keratitis cases. In several cases, additional potential pathogens also were identified. In one case with cytomegalovirus identified by Kraken and Centrifuge, the virus was confirmed by direct testing, while two where Staphylococcus aureus or cytomegalovirus were identified by Centrifuge but not Kraken could not be confirmed. Confirmation was not attempted for an additional three potential pathogens identified by Kraken and 11 identified by Centrifuge. Next generation sequencing combined with computational analysis can identify a wide range of pathogens in formalin-fixed corneal specimens, with potential applications in clinical diagnostics and research.

CD8+ T cell infiltration in breast and colon cancer: A histologic and statistical analysis.

Directory of Open Access Journals (Sweden)

James Ziai

Full Text Available The prevalence of cytotoxic tumor infiltrating lymphocytes (TILs has demonstrated prognostic value in multiple tumor types. In particular, CD8 counts (in combination with CD3 and CD45RO have been shown to be superior to traditional UICC staging in colon cancer patients and higher total CD8 counts have been associated with better survival in breast cancer patients. However, immune infiltrate heterogeneity can lead to potentially significant misrepresentations of marker prevalence in routine histologic sections. We examined step sections of breast and colorectal cancer samples for CD8+ T cell prevalence by standard chromogenic immunohistochemistry to determine marker variability and inform practice of T cell biomarker assessment in formalin-fixed, paraffin-embedded (FFPE tissue samples. Stained sections were digitally imaged and CD8+ lymphocytes within defined regions of interest (ROI including the tumor and surrounding stroma were enumerated. Statistical analyses of CD8+ cell count variability using a linear model/ANOVA framework between patients as well as between levels within a patient sample were performed. Our results show that CD8+ T-cell distribution is highly homogeneous within a standard tissue sample in both colorectal and breast carcinomas. As such, cytotoxic T cell prevalence by immunohistochemistry on a single level or even from a subsample of biopsy fragments taken from that level can be considered representative of cytotoxic T cell infiltration for the entire tumor section within the block. These findings support the technical validity of biomarker strategies relying on CD8 immunohistochemistry.

Bone Metastasis in Advanced Breast Cancer: Analysis of Gene Expression Microarray.

Science.gov (United States)

Cosphiadi, Irawan; Atmakusumah, Tubagus D; Siregar, Nurjati C; Muthalib, Abdul; Harahap, Alida; Mansyur, Muchtarruddin

2018-03-08

Approximately 30% to 40% of breast cancer recurrences involve bone metastasis (BM). Certain genes have been linked to BM; however, none have been able to predict bone involvement. In this study, we analyzed gene expression profiles in advanced breast cancer patients to elucidate genes that can be used to predict BM. A total of 92 advanced breast cancer patients, including 46 patients with BM and 46 patients without BM, were identified for this study. Immunohistochemistry and gene expression analysis was performed on 81 formalin-fixed paraffin-embedded samples. Data were collected through medical records, and gene expression of 200 selected genes compiled from 6 previous studies was performed using NanoString nCounter. Genetic expression profiles showed that 22 genes were significantly differentially expressed between breast cancer patients with metastasis in bone and other organs (BM+) and non-BM, whereas subjects with only BM showed 17 significantly differentially expressed genes. The following genes were associated with an increasing incidence of BM in the BM+ group: estrogen receptor 1 (ESR1), GATA binding protein 3 (GATA3), and melanophilin with an area under the curve (AUC) of 0.804. In the BM group, the following genes were associated with an increasing incidence of BM: ESR1, progesterone receptor, B-cell lymphoma 2, Rab escort protein, N-acetyltransferase 1, GATA3, annexin A9, and chromosome 9 open reading frame 116. ESR1 and GATA3 showed an increased strength of association with an AUC of 0.928. A combination of the identified 3 genes in BM+ and 8 genes in BM showed better prediction than did each individual gene, and this combination can be used as a training set. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Comparison between single PCR and nested PCR in detection of human papilloma viruses in paraffin-embedded OSCC and fresh oral mucosa.

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Jalouli, Miranda; Jalouli, Jamshid; Ibrahim, Salah O; Hirsch, Jan-Michaél; Sand, Lars

2015-01-01

Infection with human papilloma virus (HPV) has been implicated as one of the risk factors for the development of oropharyngeal cancer. Many different HPV tests exist, and information regarding their specific technical, analytical, and clinical properties is increasing. This study aimed to compare the level of detection of HPV using two reliable polymerase chain reaction (PCR) methods, nested PCR (NPCR) and single PCR (SPCR), in archival paraffin-embedded oral squamous cell carcinoma (OSCC) samples and fresh oral mucosa specimens. The presence of HPV genome in two groups of tissue samples was analyzed: (i) 57 paraffin-embedded OSCC samples from Sudan and (ii) eight healthy fresh oral mucosal samples from Swedish volunteers. The specimens were tested by SPCR with primer pair MY9/MY11 and NPCR using GP5+/GP6+ primer sets. Eighteen (32%) out of the 57 paraffin-embedded OSCC samples, and five (62%) out of the eight fresh clinically healthy samples were found to be HPV-positive with NPCR. With SPCR, four (7%) out of the paraffin-embedded OSCC samples were HPV-positive. A statistically significant difference between HPV-positive and -negative samples was found when comparing NPCR and SPCR in OSCC and fresh oral mucosa (pnested PCR increased the positivity rate, efficiency rate and sensitivity of HPV detection in oral samples significantly and should be considered as the method of choice. Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Real-time and label free determination of ligand binding-kinetics to primary cancer tissue specimens; a novel tool for the assessment of biomarker targeting

DEFF Research Database (Denmark)

Clausen, Thomas Mandel; Ayres Pereira, Marina; Oo, Htoo Zarni

2016-01-01

crystal microbalance (QCM) enabled biosensor technology. We analysed the interaction between the rVAR2 protein and its placental-like chondroitin sulfate (pl-CS) receptor in primary human placenta tissue and in breast and prostate tumour specimens in situ. rVAR2 interacted with FFPE human placenta...... and cancer tissue with an affinity in the nanomolar range, and showed no detectable interaction with pl-CS negative normal tissue. We further validated the method by including analysis with the androgen receptor N-20 antibody (anti-AR). As the KD value produced by this method is independent of the number......In clinical oncology, diagnosis and evaluation of optimal treatment strategies are mostly based on histopathological examination combined with immunohistochemical (IHC) expression analysis of cancer-associated antigens in formalin fixed paraffin-embedded (FFPE) tissue biopsies. However, informative...

New comprehensive denaturing-gradient-gel-electrophoresis assay for KRAS mutation detection applied to paraffin-embedded tumours

NARCIS (Netherlands)

Hayes, VM; Westra, JL; Verlind, E; Bleeker, W; Plukker, JT; Hofstra, RMW; Buys, CHCM

2000-01-01

A comprehensive mutation detection assay is presented for the entire coding region and all splice site junctions of the KRAS oncogene. The assay is based on denaturing gradient gel electrophoresis and applicable to archival paraffin-embedded tumour material. All KRAS amplicons are analysed within

Prognostic and predictive value of p-Akt, EGFR, and p-mTOR in early breast cancer

International Nuclear Information System (INIS)

Lazaridis, Georgios; Lambaki, Sofia; Karayannopoulou, Georgia; Eleftheraki, Anastasia G.; Papaspirou, Irene; Bobos, Mattheos; Efstratiou, Ioannis; Pentheroudakis, George; Zamboglou, Nikolaos; Fountzilas, George; Aristotle Univ. of Thessaloniki School of Medicine, Thessaloniki

2014-01-01

There are scarce data available on the prognostic/predictive value of p-Akt and p-mTOR protein expression in patients with high-risk early breast cancer. Formalin-fixed paraffin-embedded (FFPE) tumor tissue samples from 997 patients participating in two adjuvant phase III trials were assessed for EGFR, PTEN, p-Akt, p-mTOR protein expression, and PIK3CA mutational status. These markers were evaluated for associations with each other and with selected patient and tumor characteristics, immunohistochemical subtypes, disease-free survival (DFS), and overall survival (OS). p-mTOR protein expression was negatively associated with EGFR and positively associated with PTEN, with p-Akt473, and with the presence of PIK3CA mutations. EGFR expression was positively associated with p-Akt473, p-Akt308, and PIK3CA wild-type tumors. Finally, p-Akt308 was positively associated with p-Akt473 expression. In univariate analysis, EGFR (p = 0.016) and the coexpression of EGFR and p-mTOR (p = 0.015) were associated with poor OS. Among patients with p-Akt308-negative or low-expressing tumors, those treated with hormonal therapy were associated with decreased risk for both relapse and death (p = 0.013 and p [de

Comparative analysis of two immunohistochemical methods for antigen retrieval in the optical lobe of the honeybee Apis mellifera: Myosin-v assay

Directory of Open Access Journals (Sweden)

LUCIANA KAREN CALÁBRIA

2010-01-01

Full Text Available The present study compared two heating methods currently used for antigen retrieval (AR immunostaining: the microwave oven and the steam cooker. Myosin-V, a molecular motor involved in vesicle transport, was used as a neuronal marker in honeybee Apis mellifera brains fixed in formalin. Overall, the steam cooker showed the most satisfactory AR results. At 100 ºC, tissue morphology was maintained and revealed epitope recovery, while evaporation of the AR solution was markedly reduced; this is important for stabilizing the sodium citrate molarity of the AR buffer and reducing background effects. Standardization of heat-mediated AR of formalin-fixed and paraffin-embedded tissue sections results in more reliable immunostaining of the honeybee brain.

Rapid, sensitive, type specific PCR detection of the E7 region of human papillomavirus type 16 and 18 from paraffin embedded sections of cervical carcinoma

DEFF Research Database (Denmark)

Lesnikova, Iana; Lidang, Marianne; Hamilton-Dutoit, Stephen Jacques

2010-01-01

ABSTRACT: Human papillomavirus (HPV) infection, and in particularly infection with HPVs 16 and 18 is a central carcinogenic factor in the uterine cervix. We established and optimized a PCR assay for the detection and discrimination of HPV types 16 and 18 in archival formaldehyde fixed and paraffin...... embedded (FFPE) sections of cervical cancer. Tissue blocks from 35 cases of in situ or invasive cervical squamouscell carcinoma and surrogate FFPE sections containing the cell lines HeLa and SiHa were tested for HPV 16 and HPV18 and for the housekeeping gene beta-actin by conventional PCR using type...

Rapid, sensitive, type specific PCR detection of the E7 region of human papillomavirus type 16 and 18 from paraffin embedded sections of cervical carcinoma

DEFF Research Database (Denmark)

Lesnikova, Iana; Lidang, Marianne; Hamilton-Dutoit, Steven

2010-01-01

ABSTRACT: Human papillomavirus (HPV) infection, and in particularly infection with HPVs 16 and 18, is a central carcinogenic factor in the uterine cervix. We established and optimized a PCR assay for the detection and discrimination of HPV types 16 and 18 in archival formaldehyde fixed and paraffin...... embedded (FFPE) sections of cervical cancer.Tissue blocks from 35 cases of in situ or invasive cervical squamous cell carcinoma and surrogate FFPE sections containing the cell lines HeLa and SiHa were tested for HPV 16 and HPV18 by conventional PCR using type specific primers, and for the housekeeping gene...

Polymerase chain reaction analysis for viruses in paraffin-embedded myocardium from dogs with dilated cardiomyopathy or myocarditis.

Science.gov (United States)

Maxson, T R; Meurs, K M; Lehmkuhl, L B; Magnon, A L; Weisbrode, S E; Atkins, C E

2001-01-01

To perform polymerase chain reaction (PCR) analysis on paraffin-embedded myocardium from dogs with dilated cardiomyopathy (DCM) and dogs with myocarditis to screen for canine parvovirus, adenovirus types 1 and 2, and herpesvirus. Myocardial specimens from 18 dogs with an antemortem diagnosis of DCM and 9 dogs with a histopathologic diagnosis of myocarditis were evaluated. Paraffin-embedded myocardial specimens were screened for viral genome by PCR analysis. Positive-control specimens were developed from cell cultures as well as paraffin-embedded tissue specimens from dogs with clinical and histopathologic diagnoses of viral infection with canine parvovirus, adenovirus types 1 and 2, and herpesvirus. The histologic characteristics of all myocardial specimens were classified regarding extent, location, and type of inflammation and fibrosis. Canine adenovirus type 1 was amplified from 1 specimen from a dog with DCM. Canine parvovirus, adenovirus type 2, and herpesvirus were not amplified from any myocardial specimens. Histologic analysis of specimens from dogs with DCM revealed variable amounts of fibrosis; myocardial inflammation was observed in 1 affected dog. Histopathologic analysis of specimens from dogs with myocarditis disclosed variable degrees of inflammation and fibrosis. Viral agents canine parvovirus, adenovirus types 1 and 2, and herpesvirus are not commonly associated with DCM or active myocarditis in dogs. Additional studies evaluating for nucleic acid from viruses that less commonly affect dogs or different types of infectious agents may be warranted to gain insight into the cause of DCM and myocarditis in dogs.

Annual Progress Report FY-91. Volume 1 and 2.

Science.gov (United States)

1992-03-12

Yancy LTC MC. The Effect of Face Mask CPAP on 258 Radionuclide Ventilation Perfusion Scanning of the Lung in the Setting of Postoperative...infarction, 109, 111, 464 myocardial ischemia, 464 nail bands, 125 narcolepsy, 274 narcotics, 458, 461 nasal CPAP , 257, 258 natural history, 46, 53, 101...formalin-fixed, paraffin-embedded) or flash frozen for immune cell phenotyping. Routine histochemistry, special stains, and immune cell phenotyping

Effect of sample preparation techniques on the concentrations and distributions of elements in biological tissues using µSRXRF: a comparative study

International Nuclear Information System (INIS)

Al-Ebraheem, A; Dao, E; Desouza, E; McNeill, F E; Farquharson, M J; Li, C; Wainman, B C

2015-01-01

Routine tissue sample preparation using chemical fixatives is known to preserve the morphology of the tissue being studied. A competitive method, cryofixation followed by freeze drying, involves no chemical agents and maintains the biological function of the tissue. The possible effects of both sample preparation techniques in terms of the distribution of bio-metals (calcium (Ca), copper (Cu) zinc (Zn), and iron (Fe) specifically) in human skin tissue samples was investigated. Micro synchrotron radiation x-ray fluorescence (μSRXRF) was used to map bio-metal distribution in epidermal and dermal layers of human skin samples from various locations of the body that have been prepared using both techniques. For Ca, Cu and Zn, there were statistically significant differences between the epidermis and dermis using the freeze drying technique (p = 0.02, p < 0.01, and p < 0.01, respectively). Also using the formalin fixed, paraffin embedded technique the levels of Ca, Cu and Zn, were significantly different between the epidermis and dermis layers (p = 0.03, p < 0.01, and p < 0.01, respectively). However, the difference in levels of Fe between the epidermis and dermis was unclear and further analysis was required. The epidermis was further divided into two sub-layers, one mainly composed of the stratum corneum and the other deeper layer, the stratum basale. It was found that the difference between the distribution of Fe in the two epidermal layers using the freeze drying technique resulted in a statistically significant difference (p = 0.012). This same region also showed a difference in Fe using the formalin fixed, paraffin embedded technique (p < 0.01). The formalin fixed, paraffin embedded technique also showed a difference between the deeper epidermal layer and the dermis (p < 0.01). It can be concluded that studies involving Ca, Cu and Zn might show similar results using both sample preparation techniques, however studies involving Fe would need more

Intra-tumour IgA1 is common in cancer and is correlated with poor prognosis in bladder cancer.

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Charlotte Welinder

2016-08-01

Full Text Available A high frequency of IgA1-positive tumour cells was found in tissue micro-arrays of oesophagus, colon, testis, lung, breast, bladder and ovarian cancer. IgA1 was observed in the cytoplasm and the plasma membrane. A correlation was found between intra-tumour IgA1 and poor overall survival in a large cohort of bladder cancer patients (n = 99, p = 0.011, log-rank test. The number of IgA1-positive tumour cells was also found to be higher in female than male bladder cancer patients. The presence of IgA1 was confirmed in formalin-fixed paraffin-embedded ovarian carcinoma samples using LC-MS/MS analysis. Uptake of IgA1 was also observed in breast cancer and melanoma cell lines when cultivated in the presence of serum from healthy individuals, indicating a possible origin of the IgA1 antibodies in cancer cells.

The role of microRNA-200 in progression of human colorectal and breast cancer.

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Linda Bojmar

Full Text Available The role of the epithelial-mesenchymal transition (EMT in cancer has been studied extensively in vitro, but involvement of the EMT in tumorigenesis in vivo is largely unknown. We investigated the potential of microRNAs as clinical markers and analyzed participation of the EMT-associated microRNA-200-ZEB-E-cadherin pathway in cancer progression. Expression of the microRNA-200 family was quantified by real-time RT-PCR analysis of fresh-frozen and microdissected formalin-fixed paraffin-embedded primary colorectal tumors, normal colon mucosa, and matched liver metastases. MicroRNA expression was validated by in situ hybridization and after in vitro culture of the malignant cells. To assess EMT as a predictive marker, factors considered relevant in colorectal cancer were investigated in 98 primary breast tumors from a treatment-randomized study. Associations between the studied EMT-markers were found in primary breast tumors and in colorectal liver metastases. MicroRNA-200 expression in epithelial cells was lower in malignant mucosa than in normal mucosa, and was also decreased in metastatic compared to non-metastatic colorectal cancer. Low microRNA-200 expression in colorectal liver metastases was associated with bad prognosis. In breast cancer, low levels of microRNA-200 were related to reduced survival and high expression of microRNA-200 was predictive of benefit from radiotheraphy. MicroRNA-200 was associated with ER positive status, and inversely correlated to HER2 and overactivation of the PI3K/AKT pathway, that was associated with high ZEB1 mRNA expression. Our findings suggest that the stability of microRNAs makes them suitable as clinical markers and that the EMT-related microRNA-200-ZEB-E-cadherin signaling pathway is connected to established clinical characteristics and can give useful prognostic and treatment-predictive information in progressive breast and colorectal cancers.

miR-4443 Participates in the Malignancy of Breast Cancer.

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Xiu Chen

Full Text Available Chemo-resistance is the leading cause of failure in cancer therapy, however, much remains to be understood about the intrinsic mechanisms. In the present study, we discovered the novel miR-4443 that regulated malignancy of breast cancer both in vitro and in vivo.We examined the expression of miR-4443 in MDA-MB-231/S and MDA-MB-231 Epirubicin-resistant cell lines with 76 breast cancer formalin-fixed paraffin-embedded tissues by real-time PCR. Also, we investigated the loss- and gain-functions of miR-4443 by MTT assay and flow cytometry. Furthermore, we detected miR-4443 mediated tissue inhibitor of metalloproteinase 2 expression in cells by TargetScan, RT-qPCR and western blot.We identified the up-regulated expression of miR-4443 in Epi-resistant cell lines versus MDA-MB-231/S cell(Epi versus S and in post-chemotherapy FFPE tissues, along with statistically differential expressions in PR(partial response versus SD(stable disease/PD(progressive disease patients. Overexpression of miR-4443 increased the IC50 value of Epi for the target cells transfected, while inhibition of miR-4443 could restored sensitivity of the target cells to Epi. Besides, down-regulation of endogenous miR-4443 by miRNA-inhibitors significantly enhanced Epi-induced apoptosis while up-regulation of miR-4443 by miRNA-mimics lead to less Epi-induced apoptotic cells. Consequently, changes in TIMP2 mRNA and protein expression revealed that miR-4443 mimics suppressed expression of TIMP2 and induced migration in breast cancer cells. Furthermore, TIMP2 expression associated with better prognosis(HR = 0.721, 95%CI: 0.529-0.983.We revealed that miR-4443 induced malignancy of breast cancer mainly in chemo-resistance aspect for the very first time, providing a novel biomarker in breast cancer diagnosis and therapy.

Light microscopic identification and semiquantification of polyethylene particles in methylmethacrylate and paraffin-embedded experimental bone implant specimens

DEFF Research Database (Denmark)

Rahbek, O; Kold, S; Overgaard, S

2005-01-01

The aim of this study was to evaluate the identification of polyethylene (PE) particles in relatively thick methylmethacrylate (MMA) sections widely used in bone implant research. The sensitivity and specificity were compared between decalcified paraffin-embedded oil red O (ORO) stained and MMA-e...

Prognostic value of human apurinic/apyrimidinic endonuclease 1 (APE1 expression in breast cancer.

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Joohyun Woo

Full Text Available Human apurinic/apyrimidinic endonuclease 1 (APE1 is an essential protein for DNA base excision repair (BER and redox regulation. The ability of cancer cells to recognize DNA damage and initiate DNA repair is an important mechanism for therapeutic resistance. Several recent studies have suggested that APE1 expression levels and/or subcellular dysregulation may be used to indicate the sensitivity of tumors to radiotherapy or chemotherapy. In this study, we assessed the prognostic significance of APE1 and differences in APE1 expression levels according to breast cancer molecular subtypes. We analyzed formalin-fixed, paraffin-embedded tumor tissue sections from 243 cases diagnosed as invasive breast cancer at Ewha Womans University Medical Center between January 2003 and December 2008. Immunohistochemistry was performed and the nuclear level of APE1 was scored by taking into account the percentage of positive cells. Medical records were reviewed to investigate clinicopathologic characteristics. We found that nuclear APE1 high-level expression (proportion ≥50% in breast cancer showed a tendency towards unfavorable prognosis regarding disease-free survival (p = 0.093. However, there was no significant difference in overall survival between low and high-level expression groups (p = 0.294. Interestingly, within the Ki-67 low-level expression group, APE1 low-level expression was significantly associated with poor overall survival (p = 0.007. A significant positive correlation was observed between APE1 nuclear expression and estrogen receptor status (75.7% vs. 59.7%, p = 0.022. Also, the luminal A subtype was the most commonly observed breast cancer subtype in the APE1 high-level expression group (61.6% vs. 45.2%, p = 0.000. This study suggests that APE1 expression may be associated with breast cancer prognosis. In particular, its role as a prognostic factor would be significant for breast cancers with a low Ki-67 proliferation index

Molecular discrimination of Echinococcus granulosus and Echinococcus multilocularis by sequencing and a new PCR-RFLP method with the potential use for other Echinococcus species

OpenAIRE

ŞAKALAR, Çağrı; KUK, Salih; ERENSOY, Ahmet; DAĞLI, Adile Ferda; ÖZERCAN, İbrahim Hanifi

2015-01-01

To develop a novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol using a new genomic marker sequence and a novel set of restriction enzymes in order to detect and discriminate 2 Echinococcus species, E. granulosus and E. multilocularis, found in formalin-fixed paraffin-embedded (FFPE) human tissues. Materials and methods: DNA was isolated from 11 FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis. A mitochondrial...

TruSeq Stranded mRNA and Total RNA Sample Preparation Kits

Science.gov (United States)

Total RNA-Seq enabled by ribosomal RNA (rRNA) reduction is compatible with formalin-fixed paraffin embedded (FFPE) samples, which contain potentially critical biological information. The family of TruSeq Stranded Total RNA sample preparation kits provides a unique combination of unmatched data quality for both mRNA and whole-transcriptome analyses, robust interrogation of both standard and low-quality samples and workflows compatible with a wide range of study designs.

Stroma Based Prognosticators Incorporating Differences between African and European Americans

Science.gov (United States)

2017-10-01

markers, and uses standard Formalin-fixed Paraffin-embedded (FFPE) tissue, which is a challenge for molecular biology . In the first year, we profiled...1_Supplement/1053.7 Lernhardt, W., Fiedler, F. Lasitschka, H. Kremling, F. Zinnhammer, F. Autschbach, D. Mercola, K. Schütze, J. Rassweiler. Raman micro ...07/31/22 Mechanisms of temporal gene regulation in Chlamydia Role: Co-investigator. 2% effort. Major Goal: Systems biology of the infection cycle

Metaplasia of the parietal layer of Bowman's capsule. A histopathological survey of the human kidney

OpenAIRE

Haensly, William E.; Lee, J.C.

1986-01-01

Human kidney sections taken at autopsy were examined to determine the incidence of metaplasia of the Bowman's parietal epithelium. Autopsy records were consulted to determine if there was any correlation between clinical disease, histopathological changes in organ systems and metaplasia of Bowman's capsule. The sections represented both sexes in 9 age groups from 2 to 87 years. The sections were fixed in neutral formalin, embedded in paraffin, sectioned at 6 pm...

Elastic scattering spectroscopy findings in formalin-fixed oral squamous cell carcinoma specimens

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Swinson, B.; Elmaaytah, M.; Jerjes, W.; Hopper, C.

2005-11-01

Oral squamous cell carcinoma (OSCC) has been shown to spread locally and infiltrate adjacent bone or via the lymphatic system to the cervical lymph nodes. This usually necessitates a surgical neck dissection and either a local or segmental resection for bone clearance. While histopathology remains the gold standard for tissue diagnosis, several new diagnostic techniques are being developed that rely on physical and biochemical changes that mirror or precede malignant changes within tissue. The aim of this study was to compare findings of Elastic Scattering Spectroscopy (ESS) with histopathology on formalin-fixed specimens of both neck lymph node dissections and de-calcified archival bone from patients with OSCC. We wished to see if this technique could be used as an adjunct or alternative to histopathology in defining cervical nodal involvement and if it could be used to identify bone resection margins positive for tumour. 130 lymph nodes were examined from 13 patients. The nodes were formalin-fixed, bivalved and examined by ESS. The intensity of the spectrum at 4 points was considered for comparison; at 360nm, 450nm, 630nm and 690nm. 341 spectra were taken from the mandibular specimens of 21 patients, of which 231 spectra were taken from histologically positive sites and the rest were normal. The nodes and bone specimens were then routinely processed with haematoxylin and eosin-stained sections, examined histopathologically, and the results compared. Using Linear Discriminant Analysis (LDA) as a statistical method, a sensitivity of 98% and a specificity of 68% was obtained for the neck nodes and a sensitivity of 87% and a specificity of 80% for the bone margins.

Limited numbers of apoptotic cells in fresh paraffin embedded bone marrow samples of patients with myelodysplastic syndrome

NARCIS (Netherlands)

Brada, SJL; van de Loosdrecht, AA; Koudstaal, J; de Wolf, JTM; Vellenga, E

In myelodysplasia (MDS) the precise mechanism of ineffective erythropoiesis is not fully elucidated, but it is suggested that apoptosis may contribute to this process. We performed TdT-mediated dUTP-nick end labelling (TUNEL) staining of paraffin embedded bone marrow specimens to assess the amount

Development and validation of a gene profile predicting benefit of postmastectomy radiotherapy in patients with high-risk breast cancer: a study of gene expression in the DBCG82bc cohort.

Science.gov (United States)

Tramm, Trine; Mohammed, Hayat; Myhre, Simen; Kyndi, Marianne; Alsner, Jan; Børresen-Dale, Anne-Lise; Sørlie, Therese; Frigessi, Arnoldo; Overgaard, Jens

2014-10-15

To identify genes predicting benefit of radiotherapy in patients with high-risk breast cancer treated with systemic therapy and randomized to receive or not receive postmastectomy radiotherapy (PMRT). The study was based on the Danish Breast Cancer Cooperative Group (DBCG82bc) cohort. Gene-expression analysis was performed in a training set of frozen tumor tissue from 191 patients. Genes were identified through the Lasso method with the endpoint being locoregional recurrence (LRR). A weighted gene-expression index (DBCG-RT profile) was calculated and transferred to quantitative real-time PCR (qRT-PCR) in corresponding formalin-fixed, paraffin-embedded (FFPE) samples, before validation in FFPE from 112 additional patients. Seven genes were identified, and the derived DBCG-RT profile divided the 191 patients into "high LRR risk" and "low LRR risk" groups. PMRT significantly reduced risk of LRR in "high LRR risk" patients, whereas "low LRR risk" patients showed no additional reduction in LRR rate. Technical transfer of the DBCG-RT profile to FFPE/qRT-PCR was successful, and the predictive impact was successfully validated in another 112 patients. A DBCG-RT gene profile was identified and validated, identifying patients with very low risk of LRR and no benefit from PMRT. The profile may provide a method to individualize treatment with PMRT. ©2014 American Association for Cancer Research.

A Machine Learned Classifier That Uses Gene Expression Data to Accurately Predict Estrogen Receptor Status

Science.gov (United States)

Bastani, Meysam; Vos, Larissa; Asgarian, Nasimeh; Deschenes, Jean; Graham, Kathryn; Mackey, John; Greiner, Russell

2013-01-01

Background Selecting the appropriate treatment for breast cancer requires accurately determining the estrogen receptor (ER) status of the tumor. However, the standard for determining this status, immunohistochemical analysis of formalin-fixed paraffin embedded samples, suffers from numerous technical and reproducibility issues. Assessment of ER-status based on RNA expression can provide more objective, quantitative and reproducible test results. Methods To learn a parsimonious RNA-based classifier of hormone receptor status, we applied a machine learning tool to a training dataset of gene expression microarray data obtained from 176 frozen breast tumors, whose ER-status was determined by applying ASCO-CAP guidelines to standardized immunohistochemical testing of formalin fixed tumor. Results This produced a three-gene classifier that can predict the ER-status of a novel tumor, with a cross-validation accuracy of 93.17±2.44%. When applied to an independent validation set and to four other public databases, some on different platforms, this classifier obtained over 90% accuracy in each. In addition, we found that this prediction rule separated the patients' recurrence-free survival curves with a hazard ratio lower than the one based on the IHC analysis of ER-status. Conclusions Our efficient and parsimonious classifier lends itself to high throughput, highly accurate and low-cost RNA-based assessments of ER-status, suitable for routine high-throughput clinical use. This analytic method provides a proof-of-principle that may be applicable to developing effective RNA-based tests for other biomarkers and conditions. PMID:24312637

From High-Throughput Microarray-Based Screening to Clinical Application: The Development of a Second Generation Multigene Test for Breast Cancer Prognosis

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Carsten Denkert

2013-08-01

Full Text Available Several multigene tests have been developed for breast cancer patients to predict the individual risk of recurrence. Most of the first generation tests rely on proliferation-associated genes and are commonly carried out in central reference laboratories. Here, we describe the development of a second generation multigene assay, the EndoPredict test, a prognostic multigene expression test for estrogen receptor (ER positive, human epidermal growth factor receptor (HER2 negative (ER+/HER2− breast cancer patients. The EndoPredict gene signature was initially established in a large high-throughput microarray-based screening study. The key steps for biomarker identification are discussed in detail, in comparison to the establishment of other multigene signatures. After biomarker selection, genes and algorithms were transferred to a diagnostic platform (reverse transcription quantitative PCR (RT-qPCR to allow for assaying formalin-fixed, paraffin-embedded (FFPE samples. A comprehensive analytical validation was performed and a prospective proficiency testing study with seven pathological laboratories finally proved that EndoPredict can be reliably used in the decentralized setting. Three independent large clinical validation studies (n = 2,257 demonstrated that EndoPredict offers independent prognostic information beyond current clinicopathological parameters and clinical guidelines. The review article summarizes several important steps that should be considered for the development process of a second generation multigene test and offers a means for transferring a microarray signature from the research laboratory to clinical practice.

Putting the colours into chromogenic in situ hybridization (CISH).

Science.gov (United States)

Shipley, J

2006-09-01

Recurrent genomic alterations are the hallmarks of particular cancers. Application of molecular cytogenetic technologies to tumour material in order to detect these alterations has become important for molecular diagnostics and research. A dual-colour chromogenic in situ hybridization (dc-CISH) method described recently in the Journal of Pathology has the advantage of visualizing two probes simultaneously with the ability to discern morphological features. In addition, the bright field microscopy required is readily accessible to many laboratories. The approach has been validated by comparison of results with standard analyses for HER-2 amplification status in formalin-fixed, paraffin-embedded breast cancers and is applicable to the analysis of other clinically relevant genomic aberrations as well as of use in research investigations. Copyright (c) 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Validation of 31 of the most commonly used immunohistochemical antibodies in cytology prepared using the Cellient(®) automated cell block system.

Science.gov (United States)

Montgomery, Eric; Gao, Chen; de Luca, Julie; Bower, Jessie; Attwood, Kristropher; Ylagan, Lourdes

2014-12-01

The Cellient(®) cell block system has become available as an alternative, partially automated method to create cell blocks in cytology. We sought to show a validation method for immunohistochemical (IHC) staining on the Cellient cell block system (CCB) in comparison with the formalin fixed paraffin embedded traditional cell block (TCB). Immunohistochemical staining was performed using 31 antibodies on 38 patient samples for a total of 326 slides. Split samples were processed using both methods by following the Cellient(®) manufacturer's recommendations for the Cellient cell block (CCB) and the Histogel method for preparing the traditional cell block (TCB). Interpretation was performed by three pathologists and two cytotechnologists. Immunohistochemical stains were scored as: 0/1+ (negative) and 2/3+ (positive). Inter-rater agreement for each antibody was evaluated for CCB and TCB, as well as the intra-rater agreement between TCB and CCB between observers. Interobserver staining concordance for the TCB was obtained with statistical significance (P Cellient system are reliable and concordant with stains performed on the same split samples processed via a formalin fixed-paraffin embedded (FFPE) block. The Cellient system is a welcome adjunct to cytology work-flow by producing cell block material of sufficient quality to allow the use of routine IHC. © 2014 Wiley Periodicals, Inc.

Head and Neck Squamous Cell Carcinomas Do Not Express EGFRvIII

Energy Technology Data Exchange (ETDEWEB)

Melchers, Lieuwe J., E-mail: l.j.melchers@umcg.nl [Department of Oral and Maxillofacial Surgery, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Clausen, Martijn J.A.M. [Department of Oral and Maxillofacial Surgery, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Mastik, Mirjam F. [Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Slagter-Menkema, Lorian [Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Department of Otorhinolaryngology/Head and Neck Surgery, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Langendijk, Johannes A. [Department of Radiation Oncology, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Laan, Bernard F.A.M. van der [Department of Otorhinolaryngology/Head and Neck Surgery, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Wal, Jacqueline E. van der; Vegt, Bert van der [Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Roodenburg, Jan L.N. [Department of Oral and Maxillofacial Surgery, University Medical Center Groningen, University of Groningen, Groningen (Netherlands); Schuuring, Ed [Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen (Netherlands)

2014-10-01

Purpose: To assess the prevalence of EGFRvIII, a specific variant of EGFR (epidermal growth factor receptor), in 3 well-defined cohorts of head and neck squamous cell carcinoma (HNSCC). Methods and Materials: Immunohistochemistry for the specific detection of EGFRvIII using the L8A4 antibody was optimized on formalin-fixed, paraffin-embedded tissue using glioblastoma tissue. It was compared with EGFR and EGFRvIII RNA expression using a specific reverse transcription–polymerase chain reaction also optimized for formalin-fixed, paraffin-embedded tissue. Tissue microarrays including 531 HNSCCs of various stages with complete clinicopathologic and follow-up data were tested for the presence of EGFRvIII. Results: None of the 531 cases showed EGFRvIII protein expression. Using an immunohistochemistry protocol reported by others revealed cytoplasmic staining in 8% of cases. Reverse transcription–polymerase chain reaction for the EGFRvIII transcript of the 28 highest cytoplasmic staining cases, as well as 69 negative cases, did not show expression in any of the tested cases, suggesting aspecific staining by a nonoptimal protocol. Conclusions: The EGFRvIII mutation is not present in HNSCC. Therefore, EGFRvIII does not influence treatment response in HNSCC and is not a usable clinical prognostic marker.

Head and Neck Squamous Cell Carcinomas Do Not Express EGFRvIII

International Nuclear Information System (INIS)

Melchers, Lieuwe J.; Clausen, Martijn J.A.M.; Mastik, Mirjam F.; Slagter-Menkema, Lorian; Langendijk, Johannes A.; Laan, Bernard F.A.M. van der; Wal, Jacqueline E. van der; Vegt, Bert van der; Roodenburg, Jan L.N.; Schuuring, Ed

2014-01-01

Purpose: To assess the prevalence of EGFRvIII, a specific variant of EGFR (epidermal growth factor receptor), in 3 well-defined cohorts of head and neck squamous cell carcinoma (HNSCC). Methods and Materials: Immunohistochemistry for the specific detection of EGFRvIII using the L8A4 antibody was optimized on formalin-fixed, paraffin-embedded tissue using glioblastoma tissue. It was compared with EGFR and EGFRvIII RNA expression using a specific reverse transcription–polymerase chain reaction also optimized for formalin-fixed, paraffin-embedded tissue. Tissue microarrays including 531 HNSCCs of various stages with complete clinicopathologic and follow-up data were tested for the presence of EGFRvIII. Results: None of the 531 cases showed EGFRvIII protein expression. Using an immunohistochemistry protocol reported by others revealed cytoplasmic staining in 8% of cases. Reverse transcription–polymerase chain reaction for the EGFRvIII transcript of the 28 highest cytoplasmic staining cases, as well as 69 negative cases, did not show expression in any of the tested cases, suggesting aspecific staining by a nonoptimal protocol. Conclusions: The EGFRvIII mutation is not present in HNSCC. Therefore, EGFRvIII does not influence treatment response in HNSCC and is not a usable clinical prognostic marker

Amebic lung abscess with coexisting lung adenocarcinoma: a unusual case of amebiasis

OpenAIRE

Zhu, Hailong; Min, Xiangyang; Li, Shuai; Feng, Meng; Zhang, Guofeng; Yi, Xianghua

2014-01-01

Amebic lung abscess with concurrent lung cancer, but without either a liver abscess or amebic colitis, is extremely uncommon. Here, we report a 70-year-old man presenting with pulmonary amebiasis and coexisting lung adenocarcinoma. During his first-time hospitalization, the diagnosis of lung amebiasis was confirmed by morphological observation and PCR in formalin-fixed and paraffin-embedded sediments of pleural effusion. Almost four months later, the patient was readmitted to hospital for sim...

PREVALENCE OF HUMAN PAPILLOMAVIRUS GENOTYPES IN LOW AND HIGH GRADE SQUAMOUS INTRAEPITHELIAL LESIONS AT CERVICAL TISSUE

OpenAIRE

Prasetyo, Rizki Eko; Mastutik, Gondo; Mustokoweni, Sjahjenny

2017-01-01

HPV infection is known to cause cervical cancer. This study aimed to identify the variant of HPV genotypes of cervical precancerous lesions from low grade squamous intraepithelial lesion  (LSIL) and high grade squamous intraepithelial lesion (HSIL). This was an explorative study using formalin fix paraffin embedded (FFPE) from cervical precancerous lesions at Dr. Soetomo Hospital, Surabaya. DNA was extracted from FFPE and hybridized for HPV genotyping using Ampliquality HPV Type Express kit (...

Development of Mouse Models of Ovarian Cancer for Studying Tumor Biology and Testing Novel Molecularly Targeted Therapeutic Strategies

Science.gov (United States)

2011-09-01

classifying ovarian carcinomas (OvCas) based largely on their degree of resemblance to nonneoplastic epithelia in the female genital tract . However...In: Kurman RJ, Ellenson LH, Ronnett BM, editors. Blaustein’s pathology of the female genital tract . 6th ed. New York: Springer; 2011. 6. Ahmed AA...abnormalities. The genital tract and other major organs were collected, fixed in 10% (v/v) buffered formalin, embedded in paraffin, and pro- cessed for staining

Histology without formalin?

Science.gov (United States)

Buesa, René J

2008-12-01

Because formalin is toxic, carcinogenic, and a poor preserver of nucleic acids, for more than 20 years, there have been numerous attempts to find a substitute, with as many different alternative fixatives, none totally successful. With a fast penetration, formaldehyde is a slow and reversible fixative that requires 24 to 48 hours to completely bind to tissue; thus, any surgical specimen arriving to the laboratory between 8 AM and 4 PM and processed conventionally for the slides to be ready the following day will be only between 30% and 66% bound and even less fixed when the dehydration starts, resulting in an additional and also incomplete alcoholic fixation. This causes infiltration problems and can affect subsequent tests, especially immunohistochemistry. Formaldehyde fixation is tissue thickness independent between 16 microm and 4 mm but is faster at above room temperature, so the fixation of specimens with less than 24 hours in formalin can be improved if the fixing stations in the conventional tissue processors are set at 40 degrees C. If the safety measures are improved to offer a work environment with a time weighted average level of 0.4 ppm, and the contact with formalin is reduced to a minimum by discouraging its neutralization and limiting the recycling practice to filtering methods, formalin could remain as the routine fixative, with modified methacarn for those specimens requiring nucleic acids studies. This is a preferred solution than having to validate all the standard and special procedures, including those US Food and Drug Administration approved, if formalin is replaced by another fixative without its advantages. To the question posed in the title of this article, the answer is "Yes, it can be done, but that is neither likely nor worth it!"

High-risk human papillomavirus (HPV) DNA sequences in metaplastic breast carcinomas of Mexican women

International Nuclear Information System (INIS)

Herrera-Goepfert, Roberto; Vela-Chávez, Teresa; Carrillo-García, Adela; Lizano-Soberón, Marcela; Amador-Molina, Alfredo; Oñate-Ocaña, Luis F; Hallmann, Rita Sotelo-Regil

2013-01-01

Metaplastic carcinoma, an uncommon subtype of breast cancer, is part of the spectrum of basal-like, triple receptor-negative breast carcinomas. The present study examined 20 surgical specimens of metaplastic breast carcinomas, for the presence of high-risk Human papillomavirus (HPV), which is suspected to be a potential carcinogenic agent for breast carcinoma. Mastectomy specimens from patients harboring metaplastic breast carcinoma, as defined by the World Health Organization (WHO), and who attended the Instituto Nacional de Cancerologia in Mexico City, were retrieved from the files of the Department of Pathology accumulated during a 16-year period (1995–2008). Demographic and clinical information was obtained from patients’ medical records. DNA was extracted from formalin-fixed, paraffin-embedded tumors and HPV type-specific amplification was performed by means of Polymerase chain reaction (PCR). Quantitative Real-time (RT) PCR was conducted in HPV positive cases. Statistically, the association of continuous or categorical variables with HPV status was tested by the Student t, the Chi square, or Fisher’s exact tests, as appropriate. High-risk HPV DNA was detected in eight (40%) of 20 metaplastic breast carcinomas: seven (87.5%) HPV-16 and one (12.5%) HPV-18. Mean age of patients with HPV-positive cases was 49 years (range 24–72 years), the same as for HPV-negative cases (range, 30–73 years). There were not striking differences between HPV + and HPV– metaplastic carcinomas regarding clinical findings. Nearly all cases were negative for estrogen, progesterone and Human epidermal growth factor receptor 2 (HER2), but positive for Epidermal growth factor receptor (EGFR). High-risk HPV has been strongly associated with conventional breast carcinomas, although the subtle mechanism of neoplastic transformation is poorly understood. In Mexican patients, the prevalence of HPV infection among metaplastic breast carcinomas is higher than in non-metaplastic ones

In vitro and in vivo MMP gene expression localisation by In Situ-RT-PCR in cell culture and paraffin embedded human breast cancer cell line xenografts

International Nuclear Information System (INIS)

Haupt, Larisa M; Thompson, Erik W; Trezise, Ann EO; Irving, Rachel E; Irving, Michael G; Griffiths, Lyn R

2006-01-01

Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origin is difficult. We utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was examined in the MDA-MB-231 and MCF-7 HBC cell lines following exposure to Concanavalin A (Con A). In vivo, we examined their expression in archival paraffin embedded xenografts derived from a range of HBC cell lines of varied invasive and metastatic potential. Mouse xenografts are heterogenous, containing neoplastic human parenchyma with mouse stroma and vasculature and provide a reproducible in vivo model system correlated to the human disease state. In vitro, exposure to Con A increased MT1-MMP gene expression in MDA-MB-231 cells and decreased MT1-MMP gene expression in MCF-7 cells. MMP-1 and MMP-3 gene expression remained unchanged in both cell lines. In vivo, stromal cells recruited into each xenograft demonstrated differences in localised levels of MMP gene expression. Specifically, MDA-MB-231, MDA-MB-435 and Hs578T HBC cell lines are able to influence MMP gene expression in the surrounding stroma. We have demonstrated the applicability and sensitivity of IS-RT-PCR for the examination of MMP gene expression both in vitro and in

Modified paraffin wax for improvement of histological analysis efficiency.

Science.gov (United States)

Lim, Jin Ik; Lim, Kook-Jin; Choi, Jin-Young; Lee, Yong-Keun

2010-08-01

Paraffin wax is usually used as an embedding medium for histological analysis of natural tissue. However, it is not easy to obtain enough numbers of satisfactory sectioned slices because of the difference in mechanical properties between the paraffin and embedded tissue. We describe a modified paraffin wax that can improve the histological analysis efficiency of natural tissue, composed of paraffin and ethylene vinyl acetate (EVA) resin (0, 3, 5, and 10 wt %). Softening temperature of the paraffin/EVA media was similar to that of paraffin (50-60 degrees C). The paraffin/EVA media dissolved completely in xylene after 30 min at 50 degrees C. Physical properties such as the amount of load under the same compressive displacement, elastic recovery, and crystal intensity increased with increased EVA content. EVA medium (5 wt %) was regarded as an optimal composition, based on the sectioning efficiency measured by the numbers of unimpaired sectioned slices, amount of load under the same compressive displacement, and elastic recovery test. Based on the staining test of sectioned slices embedded in a 5 wt % EVA medium by hematoxylin and eosin (H&E), Masson trichrome (MT), and other staining tests, it was concluded that the modified paraffin wax can improve the histological analysis efficiency with various natural tissues. (c) 2010 Wiley-Liss, Inc.

Staining of E-selectin ligands on paraffin-embedded sections of tumor tissue.

Science.gov (United States)

Carrascal, Mylène A; Talina, Catarina; Borralho, Paula; Gonçalo Mineiro, A; Henriques, Ana Raquel; Pen, Cláudia; Martins, Manuela; Braga, Sofia; Sackstein, Robert; Videira, Paula A

2018-05-02

The E-selectin ligands expressed by cancer cells mediate adhesion of circulating cancer cells to endothelial cells, as well as within tissue microenvironments important for tumor progression and metastasis. The identification of E-selectin ligands within cancer tissue could yield new biomarkers for patient stratification and aid in identifying novel therapeutic targets. The determinants of selectin ligands consist of sialylated tetrasaccharides, the sialyl Lewis X and A (sLe X and sLe A ), displayed on protein or lipid scaffolds. Standardized procedures for immunohistochemistry make use of the antibodies against sLe X and/or sLe A . However, antibody binding does not define E-selectin binding activity. In this study, we developed an immunohistochemical staining technique, using E-selectin-human Ig Fc chimera (E-Ig) to characterize the expression and localization of E-selectin binding sites on paraffin-embedded sections of different cancer tissue. E-Ig successfully stained cancer cells with high specificity. The E-Ig staining show high reactivity scores in colon and lung adenocarcinoma and moderate reactivity in triple negative breast cancer. Compared with reactivity of antibody against sLe X/A , the E-Ig staining presented higher specificity to cancer tissue with better defined borders and less background. The E-Ig staining technique allows the qualitative and semi-quantitative analysis of E-selectin binding activity on cancer cells. The development of accurate techniques for detection of selectin ligands may contribute to better diagnostic and better understanding of the molecular basis of tumor progression and metastasis.

Microdissecção e captura a laser na investigação do gene TP53 em tecidos incluídos em parafina Laser-capture microdissection for TP53 gene analysis in paraffin-embedded tissues

Directory of Open Access Journals (Sweden)

Shadia Muhammad Ihlaseh

2007-02-01

characterize its enormous potential for diagnosis and research. OBJECTIVE: This study describes the standardization of LCM and DNA extraction from formalin fixed and paraffin-embedded tissues. MATERIAL AND METHOD: The gene TP53 exon 8 and the cyclophilin gene were studied in normal and neoplastic liver and kidney samples from a chemical carcinogenesis model in rat. DNA extraction was confirmed by nested-PCR. RESULTS: Histological sections preparation for LCM and the nested-PCR procedures were standardized; 48.3% amplifications of the gene TP53 exon 8 and 51.7% of the cyclophilin gene samples were obtained. When at least one of the gene segments was considered, 79.3% samples presented amplification. DISCUSSION AND CONCLUSION: Procedures for DNA extraction from formalin fixed and paraffin-embedded tissues collected by LCM were standardized. They can be useful for DNA collection for molecular studies.

Zoonotic onchocerciasis in Hiroshima, Japan, and molecular analysis of a paraffin section of the agent for a reliable identification

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Fukuda M.

2011-05-01

Full Text Available Japan is a country of high specific diversity of Onchocerca with eight species, the adults of two not yet known. Onchocerca dewittei japonica, a common filarial parasite of wild boar, had been proved to be the agent of five zoonotic onchocerciasis in Kyushu island with morphological and molecular studies. The sixth case, at Hiroshima in the main island, was identified to the same Onchocerca species, based on adult characters observed on histological sections. To consolidate the identification, mitochondrial cytochrome c oxidase subunit 1 (CO1 gene analysis was attempted with the formalin-fixed, paraffin-embedded parasite specimen. The sequence (196 bp of a CO1 gene fragment of the parasite successfully PCR-amplified agreed well with those of O. dewittei japonica registered in GenBank, confirming the morphological identification. Moreover a comparison with the CO1 gene sequences of six other Onchocerca species in GenBank excluded the possibility that Onchocerca sp. from wild boar and Onchocerca sp. type A from cattle in Japan, were the causative agents in this case. Mitochondrial DNA analysis proved to be a valuable tool to support the morphological method for the discrimination of zoonotic Onchocerca species in a histological specimen.

Chromogenic in situ hybridization (CISH) to detect HER2 gene amplification in breast and gastric cancer: comparison with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).

Science.gov (United States)

Kiyose, Shinichiro; Igarashi, Hisaki; Nagura, Kiyoko; Kamo, Takaharu; Kawane, Kazunori; Mori, Hiroki; Ozawa, Takachika; Maeda, Matsuyoshi; Konno, Keisuke; Hoshino, Hideaki; Konno, Hiroyuki; Ogura, Hiroyuki; Shinmura, Kazuya; Hattori, Naohiko; Sugimura, Haruhiko

2012-11-01

The chromogenic in situ hybridization (CISH) assay, designed to detect the amplification of the HER2 gene in formalin-fixed, paraffin-embedded (FFPE) breast cancer (BC) and gastric cancer (GC) tissue specimens, was evaluated in 125 FFPE BC cases and 198 FFPE GC cases for which the HER2 status had been predetermined using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the 125 BC cases and the 198 gastric cases, we found a very good concordance (98.4% and 99.0%, respectively) between CISH and FISH. In particular, we evaluated the polysomy cases, as these cases often have ambiguous treatment options in clinical practice. The polysomy of chromosome 17 was defined as the presence of three or more CEP17 signals in at least 10% of the tumor cells. In the 50 BC cases and 54 GC cases displaying chromosome 17 polysomy, the concordance between FISH and CISH was 98.0% and 98.1%, respectively. These results indicate that CISH could provide an accurate and practical alternative to FISH for the clinical diagnosis of HER2 gene amplification in FFPE BC and FFPE GC samples. © 2012 The Authors. Pathology International © 2012 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.

Terahertz spectroscopy for the study of paraffin-embedded gastric cancer samples

Science.gov (United States)

Wahaia, Faustino; Kasalynas, Irmantas; Seliuta, Dalius; Molis, Gediminas; Urbanowicz, Andrzej; Carvalho Silva, Catia D.; Carneiro, Fatima; Valusis, Gintaras; Granja, Pedro L.

2015-01-01

Terahertz (THz) spectroscopy constitute promising technique for biomedical applications as a complementary and powerful tool for diseases screening specially for early cancer diagnostic. The THz radiation is not harmful to biological tissues. As increased blood supply in cancer-affected tissues and consequent local increase in tissue water content makes THz technology a potentially attractive. In the present work, samples of healthy and adenocarcinoma-affected gastric tissue were analyzed using transmission time-domain THz spectroscopy (THz-TDS). The work shows the capability of the technique to distinguish between normal and cancerous regions in dried and paraffin-embedded samples. Plots of absorption coefficient α and refractive index n of normal and cancer affected tissues, are presented and the conditions for discrimination between normal and affected tissues are discussed.

Cellular localization of 2-[3H]deoxy-D-glucose from paraffin-embedded brains

International Nuclear Information System (INIS)

Durham, D.; Woolsey, T.A.; Kruger, L.

1981-01-01

Results of experiments in which regional neuronal activity is revealed by a 2-[ 3 H]deoxy-D-glucose ( 3 H-2-DG)-paraffin section-emulsion autoradiography method are described. The trigeminal pathway of freely behaving mice was activated differentially by selective patterns of whisker removal. One hour after injection of concentrated 3 H-2-DG, the animals were perfused systemically with a periodate/lysine/paraformaldehyde mixture the brains were embedded in paraffin, and serial sections were taken and coated with emulsion for autoradiography. Diffusion of the isotope out of the tissue was assessed visually and by liquid scintillation counting. While substantial loss of 3 H isotope into the embedding fluids (about 95%) was found, the scintillation counts and the autoradiograms showed good fixation of the isotope in situ, no evidence of isotope movement into the emulsion, and no gradients of diffusion in the sectioned material. Patterns of regional labeling were similar to those reported from brains prepared by conventional 2-[ 14 C]deoxy-D-glucose ( 14 C-2-DG) autoradiography; Trigeminal structures associated with the intact (stimulated) whiskers were labeled relatively heavily, indicating that label uptake is specific with respect to neuronal activity. In the cortex, the patterns of label corresponded directly and precisely to those barrels known to receive inputs from the intact whiskers. Distribution of silver grains in the cortex and in the brainstem was correlated directly with neuronal profiles. Clearly, this approach offers considerable technical advantages, in particular, the ease with which the histological material is prepared. The resolution of the autoradiograms and the quality of the histology are excellent

Implementation of immunohistochemistry on frozen ear notch tissue samples in diagnosis of bovine viral diarrhea virus in persistently infected cattle

Directory of Open Access Journals (Sweden)

Bedeković Tomislav

2011-12-01

Full Text Available Abstract Background Bovine viral diarrhea is a contagious disease of domestic and wild ruminants and one of the most economically important diseases in cattle. Bovine viral diarrhea virus belongs to the genus Pestivirus, within the family Flaviviridae. The identification and elimination of the persistently infected animals from herds is the initial step in the control and eradication programs. It is therefore necessary to have reliable methods for diagnosis of bovine viral diarrhea virus. One of those methods is immunohistochemistry. Immunohistochemistry on formalin fixed, paraffin embedded tissue is a routine technique in diagnosis of persistently infected cattle from ear notch tissue samples. However, such technique is inappropriate due to complicated tissue fixation process and it requires more days for preparation. On the contrary, immunohistochemistry on frozen tissue was usually applied on organs from dead animals. In this paper, for the first time, the imunohistochemistry on frozen ear notch tissue samples was described. Findings Seventeen ear notch tissue samples were obtained during the period 2008-2009 from persistently infected cattle. Samples were fixed in liquid nitrogen and stored on -20°C until testing. Ear notch tissue samples from all persistently infected cattle showed positive results with good section quality and possibility to determinate type of infected cells. Conclusions Although the number of samples was limited, this study indicated that immunohistochemistry on formalin fixed paraffin embedded tissue can be successfully replaced with immunohistochemistry on frozen ear notch tissue samples in diagnosis of persistently infected cattle.

Comparison of central HER2 testing with quantitative total HER2 expression and HER2 homodimer measurements using a novel proximity-based assay.

Science.gov (United States)

Huang, Weidong; Reinholz, Monica; Weidler, Jodi; Yolanda, Lie; Paquet, Agnes; Whitcomb, Jeannette; Lingle, Wilma; Jenkins, Robert B; Chen, Beiyun; Larson, Jeffrey S; Tan, Yuping; Sherwood, Thomas; Bates, Michael; Perez, Edith A

2010-08-01

The accuracy and reliability of immunohistochemical analysis and in situ hybridization for the assessment of HER2 status remains a subject of debate. We developed a novel assay (HERmark Breast Cancer Assay, Monogram Biosciences, South San Francisco, CA) that provides precise quantification of total HER2 protein expression (H2T) and HER2 homodimers (H2D) in formalin-fixed, paraffin-embedded tissue specimens. H2T and H2D results of 237 breast cancers were compared with those of immunohistochemical studies and fluorescence in situ hybridization (FISH) centrally performed at the Mayo Clinic, Rochester, MN. H2T described a continuum across a wide dynamic range ( approximately 2.5 log). Excluding the equivocal cases, HERmark showed 98% concordance with immunohistochemical studies for positive and negative assay values. For the 94 immunohistochemically equivocal cases, 67% and 39% concordance values were observed between HERmark and FISH for positive and negative assay values, respectively. Polysomy 17 in the absence of HER2 gene amplification did not result in HER2 overexpression as evaluated quantitatively using the HERmark assay.

Novel Growth Factor as Prognostic Marker for Estrogen-Independence in Breast Cancer

National Research Council Canada - National Science Library

Serrero, Ginette

2002-01-01

...-independence and tamoxifen resistance. Here, wave conducted a study with 206 paraffin embedded human breast cancer biopsies and measured PCDGF expression by immunohistochemistry using an anti-PCDGF developed in our laboratory...

Efeito dos fixadores formalina e Bouin na preservação de biópsias do endométrio de éguas após inclusão em resina plástica Effect of formalin and Bouin fixation upon the mare's endometrial biopsies embedded in plastic resin

Directory of Open Access Journals (Sweden)

D. Amaral

2004-02-01

Full Text Available Biópsias do endométrio de 16 éguas sexualmente maduras, em estro e diestro, foram processadas para microscopia de luz utilizando-se fixação em formalina ou Bouin e inclusão em resina plástica à base de glicol metacrilato. Análises morfológicas de 46 biópsias demonstraram que o epitélio de revestimento do endométrio, o epitélio glandular, as fibras do tecido conjuntivo e os diferentes tipos celulares presentes na lâmina própria, tais como fibroblastos, plasmócitos, mastócitos e macrófagos, apresentaram-se melhor preservados quando os fragmentos de tecidos foram fixados em formalina. O epitélio de revestimento mostrou grau mais acentuado de retração tecidual nas biópsias fixadas em Bouin, independente da fase do ciclo estral. A fixação em formalina aliada à inclusão em resina plástica resultou em melhor resolução das células ao microscópio de luz, permitindo um estudo citológico mais acurado do endométrio eqüino.Endometrial biopsies were performed in 16 mares at estrus and diestrus and tissues were processed for light microscopy using formalin or Bouin fixatives and plastic resin glycol methacrylate for embedding. Results of the tissue processing demonstrated that the luminal and glandular epithelium, connective tissue fibers and many cell types present in the lamina propria such as fibroblasts, plasmocytes, mast cells and macrophages were best preserved in formalin fixed samples. The luminal epithelium showed increased shrinkage in Bouin fixed specimens when compared to formalin fixed ones. Those morphological findings were present throughout the estral cycle. The formalin fixation procedure associated with plastic resin embedding yielded increased tissue resolution as seen by light microscopy, and allowed a more accurate cytological study of the endometrium of the mare.

Underpinning the repurposing of anthracyclines towards colorectal cancer

DEFF Research Database (Denmark)

Nygård, Sune Boris; Christensen, Ib Jarle; Smith, David Hersi

2013-01-01

Abstract Objective. We propose a repurposing strategy where anthracyclines are reintroduced to a subgroup of patients with metastatic colorectal cancer with the highest likelihood of response. In breast cancer, DNA topoisomerase II alpha gene (TOP2A) alterations predict incremental benefit...... of anthracyclines, but this association has not been investigated in colorectal cancer. Frequency analysis of TOP2A gene alterations in colorectal cancer and the association with prognosis are evaluated and the challenges of using a TOP2A/CEN-17 FISH probe combination are addressed. Material and methods. Formalin......-fixed, paraffin-embedded material from 154 stage III colorectal cancer patients included in the RANX05 clinical trial was retrospectively assessed for TOP2A gene alterations using FISH. The TOP2A/CEN-17 ratio as well as the TOP2A gene copy number alone was used to define gene alterations and associations between...

Exposure to depleted uranium does not alter the co-expression of HER-2/neu and p53 in breast cancer patients

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Al-Toriahi Kaswer M

2011-03-01

Full Text Available Abstract Background Amongst the extensive literature on immunohistochemical profile of breast cancer, very little is found on populations exposed to a potential risk factor such as depleted uranium. This study looked at the immunohistochemical expression of HER-2/neu (c-erbB2 and p53 in different histological types of breast cancer found in the middle Euphrates region of Iraq, where the population has been exposed to high levels of depleted uranium. Findings The present investigation was performed over a period starting from September 2008 to April 2009. Formalin-fixed, paraffin-embedded blocks from 70 patients with breast cancer (62 ductal and 8 lobular carcinoma were included in this study. A group of 25 patients with fibroadenoma was included as a comparative group, and 20 samples of normal breast tissue sections were used as controls. Labeled streptavidin-biotin (LSAB+ complex method was employed for immunohistochemical detection of HER-2/neu and p53. The detection rate of HER-2/neu and p53 immunohistochemical expression were 47.14% and 35.71% respectively in malignant tumors; expression was negative in the comparative and control groups (p HER-2/neu immunostaining was significantly associated with histological type, tumor size, nodal involvement, and recurrence of breast carcinoma (p p Both biomarkers were positively correlated with each other. Furthermore, all the cases that co-expressed both HER-2/neu and p53 showed the most unfavorable biopathological profile. Conclusion P53 and HER-2/neu over-expression play an important role in pathogenesis of breast carcinoma. The findings indicate that in regions exposed to high levels of depleted uranium, although p53 and HER-2/neu overexpression are both high, correlation of their expression with age, grade, tumor size, recurrence and lymph node involvement is similar to studies that have been conducted on populations not exposed to depleted uranium. HER-2/neu expression in breast cancer was higher

Mass-spectrometry analysis of histone post-translational modifications in pathology tissue using the PAT-H-MS approach

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Roberta Noberini

2016-06-01

Full Text Available Aberrant histone post-translational modifications (hPTMs have been implicated with various pathologies, including cancer, and may represent useful epigenetic biomarkers. The data described here provide a mass spectrometry-based quantitative analysis of hPTMs from formalin-fixed paraffin-embedded (FFPE tissues, from which histones were extracted through the recently developed PAT-H-MS method. First, we analyzed FFPE samples from mouse spleen and liver or human breast cancer up to six years old, together with their corresponding fresh frozen tissue. We then combined the PAT-H-MS approach with a histone-focused version of the super-SILAC strategy-using a mix of histones from four breast cancer cell lines as a spike-in standard- to accurately quantify hPTMs from breast cancer specimens belonging to different subtypes. The data, which are associated with a recent publication (Pathology tissue-quantitative mass spectrometry analysis to profile histone post-translational modification patterns in patient samples (Noberini, 2015 [1], are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier http://www.ebi.ac.uk/pride/archive/projects/PXD002669.

A machine learned classifier that uses gene expression data to accurately predict estrogen receptor status.

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Meysam Bastani

Full Text Available BACKGROUND: Selecting the appropriate treatment for breast cancer requires accurately determining the estrogen receptor (ER status of the tumor. However, the standard for determining this status, immunohistochemical analysis of formalin-fixed paraffin embedded samples, suffers from numerous technical and reproducibility issues. Assessment of ER-status based on RNA expression can provide more objective, quantitative and reproducible test results. METHODS: To learn a parsimonious RNA-based classifier of hormone receptor status, we applied a machine learning tool to a training dataset of gene expression microarray data obtained from 176 frozen breast tumors, whose ER-status was determined by applying ASCO-CAP guidelines to standardized immunohistochemical testing of formalin fixed tumor. RESULTS: This produced a three-gene classifier that can predict the ER-status of a novel tumor, with a cross-validation accuracy of 93.17±2.44%. When applied to an independent validation set and to four other public databases, some on different platforms, this classifier obtained over 90% accuracy in each. In addition, we found that this prediction rule separated the patients' recurrence-free survival curves with a hazard ratio lower than the one based on the IHC analysis of ER-status. CONCLUSIONS: Our efficient and parsimonious classifier lends itself to high throughput, highly accurate and low-cost RNA-based assessments of ER-status, suitable for routine high-throughput clinical use. This analytic method provides a proof-of-principle that may be applicable to developing effective RNA-based tests for other biomarkers and conditions.

Histopathologic Effects of Dirofilaria Immitis Microfilaria on Internal Organs of Dog Confirming by PCR Technique

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S Simsek

2012-06-01

Full Text Available Background: The heartworm disease is an infectious disease of dogs with Dirofilaria immitis combined with cardiovascular and circulatory abnormalities. The heartworm disease can become a serious health risk when associated with a severe infection. In this study, a male, 8 year-old dog that died suddenly was necropsied and all tissues were examined grossly.Methods: Major organs including heart, lungs, liver, spleen, kidneys, brain, eyes, and testis were fixed in 10% neutral formalin, embedded in paraffin, sectioned at 5-µm thickness, stained with hematoxylin and eosin, and examined with a light microscope. For each examined organ, paraffin-embedded tissues were cut and placed in eppendorf tubes for genomic DNA extraction. PCR was performed using two sets of primers for amplification of a 302 bp ITS-2 gene fragment and a 203 bp cytochrome oxidase subunit 1 (CO1 gene fragment of D. immitis.Results: During the necropsy examination, 46 adult D. immitis were found in the portal vein, right ventricle, and atrium of the heart and pulmonary trunk. Microscopically, microfilarias were found throughout the vessels of different organs including lungs, kidneys, liver, heart, brain, and spleen. All tissues examined by PCR were positive for D. immitis ITS-2 and CO1.Conclusion: PCR technique now represents an effective method for identification of D. immitis from formalin-fixed samples.

The PAXgene(® tissue system preserves phosphoproteins in human tissue specimens and enables comprehensive protein biomarker research.

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Sibylle Gündisch

Full Text Available Precise quantitation of protein biomarkers in clinical tissue specimens is a prerequisite for accurate and effective diagnosis, prognosis, and personalized medicine. Although progress is being made, protein analysis from formalin-fixed and paraffin-embedded tissues is still challenging. In previous reports, we showed that the novel formalin-free tissue preservation technology, the PAXgene Tissue System, allows the extraction of intact and immunoreactive proteins from PAXgene-fixed and paraffin-embedded (PFPE tissues. In the current study, we focused on the analysis of phosphoproteins and the applicability of two-dimensional gel electrophoresis (2D-PAGE and enzyme-linked immunosorbent assay (ELISA to the analysis of a variety of malignant and non-malignant human tissues. Using western blot analysis, we found that phosphoproteins are quantitatively preserved in PFPE tissues, and signal intensities are comparable to that in paired, frozen tissues. Furthermore, proteins extracted from PFPE samples are suitable for 2D-PAGE and can be quantified by ELISA specific for denatured proteins. In summary, the PAXgene Tissue System reliably preserves phosphoproteins in human tissue samples, even after prolonged fixation or stabilization times, and is compatible with methods for protein analysis such as 2D-PAGE and ELISA. We conclude that the PAXgene Tissue System has the potential to serve as a versatile tissue fixative for modern pathology.

BRCA Mutation-Related and Claudin-Low Breast Cancer: Blood Relatives or Stepsisters.

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Madaras, Lilla; Balint, Nora; Gyorffy, Balazs; Tokes, Anna-Maria; Barshack, Iris; Yosepovich, Ady; Friedman, Eitan; Paluch-Shimon, Shani; Zippel, Dov; Baghy, Kornelia; Timar, Jozsef; Kovalszky, Ilona; Kulka, Janina; Szasz, Attila Marcell

2016-01-01

BRCA mutation-associated (BRCAmut) breast cancer represents a heterogeneous group displaying certain molecular features. Claudin-low breast cancers (CLBC) overlap with characteristics of BRCAmut tumors; therefore, we have investigated whether these are identical subtypes. Using public gene expression data, CLDN, CDH1, 9-cell line claudin-low predictor (9CLCLP) and PAM50 expression was evaluated in BRCAmut and BRCA wild-type (BRCAwt) breast cancer cases focusing on their possible overlap with the CLBC subtype. A separate formalin-fixed, paraffin-embedded (FFPE) cohort of 22 BRCAmut and 19 BRCAwt tumor tissues was used for immunohistochemical examination of AR, CD24, CD44, CK5/6, claudin-1, -3, -4 and -7, E-cadherin, EGFR, estrogen receptor (ER), EZH2, HER2, Ki67, p53, progesterone receptor (PgR) and vimentin expression. In the data sets, CLDN1 (ROC = 0.785, p < 0.001), CDH1 (ROC = 0.785, p < 0.001), CLDN7 (ROC = 0.723, p < 0.001), CLDN3 (ROC = 0.696, p = 0.020) and CLDN4 (ROC = 0.685, p = 0.027) were expressed at higher level in BRCAmut than BRCAwt tumor tissue. The PAM50 subtype differed from the assigned immunohistochemistry (IHC)-based subtype in 30%. Based on accessible 9CLCLP predictor genes, BRCAmut breast cancer does not display the claudin-low phenotype. Utilizing FFPE samples, claudins were evidently expressed in both BRCAmut and BRCAwt cases. However, at the protein level, only claudin-3 expression was higher in BRCAmut tumors, while claudin-1, -4 and -7 and E-cadherin expression was lower compared to BRCAwt cases. A CD24low/CD44high phenotype was found in BRCAmut tumors upon comparison with BRCAwt cases (p < 0.001 and p = 0.001, respectively). There is a prominent correlation between the genes under focus herein and BRCA mutation status. BRCAmut tumors bear stem cell characteristics displaying a distinct cell adhesion molecule profile characterized by high expression of CDH1 and CLDN4 according to public gene expression data set analysis, and higher

The accessory magnocellular neurosecretory system of the rostral human hypothalamus

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Møller, Morten; Busch, Johannes R.; Jacobsen, Christina

2018-01-01

magnocellular neurons were often located along the blood vessels and projections of some of these neurons penetrated the vascular endothelium. The accessory magnocellular cell bodies expressed either neurophysin I or neurophysin II immunoreactivity. Summarizing, the accessory magnocellular system in the human......The morphology and neurophysin expression of the magnocellular accessory neuroendocrine system located in the rostral human hypothalamus is investigated in a series of brains obtained at autopsy. The hypothalami were fixed in formalin and embedded in paraffin, or after cryoprotection, frozen...

Post-mortem testing; germline BRCA1/2 variant detection using archival FFPE non-tumor tissue

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Petersen, Annabeth Høgh; Jørgensen, Mads Malik Aagaard; Nielsen, Henriette Roed

2016-01-01

Accurate estimation of cancer risk in HBOC families often requires BRCA1/2 testing, but this may be impossible in deceased family members. Previous, testing archival formalin-fixed, paraffin-embedded (FFPE) tissue for germline BRCA1/2 variants was unsuccessful, except for the Jewish founder mutat...... samples from non-tumor tissue. Accurate genetic counseling is achievable in families where variant testing would otherwise be impossible.European Journal of Human Genetics advance online publication, 6 January 2016; doi:10.1038/ejhg.2015.268....

Semi-Nested Real-Time Reverse Transcription Polymerase Chain Reaction Methods for the Successful Quantitation of Cytokeratin mRNA Expression Levels for the Subtyping of Non-Small-Cell Lung Carcinoma Using Paraffin-Embedded and Microdissected Lung Biopsy Specimens

International Nuclear Information System (INIS)

Nakanishi, Yoko; Shimizu, Tetsuo; Tsujino, Ichiro; Obana, Yukari; Seki, Toshimi; Fuchinoue, Fumi; Ohni, Sumie; Oinuma, Toshinori; Kusumi, Yoshiaki; Yamada, Tsutomu; Takahashi, Noriaki; Hashimoto, Shu; Nemoto, Norimichi

2013-01-01

In patients with inoperable advanced non-small cell lung carcinomas (NSCLCs), histological subtyping using small-mount biopsy specimens was often required to decide the indications for drug treatment. The aim of this study was to assess the utility of highly sensitive mRNA quantitation for the subtyping of advanced NSCLC using small formalin fixing and paraffin embedding (FFPE) biopsy samples. Cytokeratin (CK) 6, CK7, CK14, CK18, and thyroid transcription factor (TTF)-1 mRNA expression levels were measured using semi-nested real-time quantitative (snq) reverse-transcribed polymerase chain reaction (RT-PCR) in microdissected tumor cells collected from 52 lung biopsies. Our results using the present snqRT-PCR method showed an improvement in mRNA quantitation from small FFPE samples, and the mRNA expression level using snqRT-PCR was correlated with the immunohistochemical protein expression level. CK7, CK18, and TTF-1 mRNA were expressed at significantly higher levels (P<0.05) in adenocarcinoma (AD) than in squamous cell carcinoma (SQ), while CK6 and CK14 mRNA expression was significantly higher (P<0.05) in SQ than in AD. Each histology-specific CK, particularly CK18 in AD and CK6 in SQ, were shown to be correlated with a poor prognosis (P=0.02, 0.02, respectively). Our results demonstrated that a quantitative CK subtype mRNA analysis from lung biopsy samples can be useful for predicting the histology subtype and prognosis of advanced NSCLC

Determining Optimal Microwave Antigen Retrieval Conditions for Microtubule-Associated Protein 2 Immunohistochemistry in the Guinea Pig Brain

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2002-12-01

sections of formalin-fixed guinea pig brains using different MAP-2 monoclonal antibodies. Brain sections were boiled in sodium citrate, citric acid...citric acid solution at pH 6.0 is the optimal microwave-assisted AR method for immunolabeling MAP-2 in formalin-fixed, paraffin-processed guinea pig brain...studies on archival guinea pig brain paraffin blocks, ultimately relaxing the use of additional animals to evaluate changes in MAP-2 expression between chemical warfare nerve agent-treated and control samples.

Patients with primary breast and primary female genital tract diffuse large B cell lymphoma have a high frequency of MYD88 and CD79B mutations.

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Cao, Xin-Xin; Li, Jian; Cai, Hao; Zhang, Wei; Duan, Ming-Hui; Zhou, Dao-Bin

2017-11-01

This study is to retrospectively evaluate the prevalence of MYD88 and CD79B mutations and the clinicopathologic characteristics of patients with primary diffuse large B cell lymphoma (DLBCL) of the female genital tract and breast. The characteristics, treatments, and outcomes of 19 patients diagnosed with primary DLBCL of the female genital tract and breast, who had formalin-fixed and paraffin-embedded tissues obtained from diagnostic samples diagnosed between January 2004 and June 2016, were analyzed retrospectively. Nineteen female patients (7 with primary breast and 12 with primary female genital tract DLBCL) were included in this retrospective study. Eleven patients (57.9%) carried a MYD88 mutation, including 10 with MYD8 L265P and 1 with the MYD88 L265S mutation. Seven patients (36.8%) harbored a CD79B mutation, which included two cases with CD79B Y196H, two cases with CD79B Y196N, one case with CD79B Y196D, one case with CD79B Y196F, and one case with CD79B Y196X. Four cases had both MYD88 and CD79B mutations. The clinicopathologic parameters, progression-free survival (PFS), and overall survival (OS) of the MYD88 mutation-carrying group were not significantly different from those of the MYD88 wild-type group except for higher LDH levels. Six patients received cyclophosphamide, doxorubicin, vincristine, and prednisolone (CHOP), while 13 patients received rituximab plus CHOP, and 13 patients received central nervous system prophylaxis. The median OS and PFS were 73 and 56 months, respectively. Patients with primary breast and primary female genital tract DLBCL have a high frequency of MYD88 and CD79B mutations. The presence of these mutations does not affect survival but may offer additional therapeutic options.

Characterization of Melanin Radicals in Paraffin-embedded Malignant Melanoma and Nevus Pigmentosus Using X-band EPR and EPR Imaging.

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Nakagawa, Kouichi; Minakawa, Satoko; Sawamura, Daisuke; Hara, Hideyuki

2017-01-01

Continuous wave electron paramagnetic resonance (CW EPR) and X-band (9 GHz) EPR imaging (EPRI) were used to nondestructively investigate the possible differentiation between malignant melanoma (MM) and nevus pigmentosus (NP) melanin radicals in paraffin-embedded specimens. The EPR spectra of both samples were analyzed using linewidth, spectral pattern, and X-band EPRI. The CW-EPR spectra of the MM showed an additional signal overlap. Eumelanin- and pheomelanin-related radicals were observed in the MM specimens. The EPR results revealed that the peak-to-peak linewidths (ΔH pp ) of paraffin-embedded MM and NP samples were 0.65 ± 0.01 and 0.69 ± 0.01 mT, respectively. The g-value was 2.005 for both samples. Moreover, the two-dimensional (2D) EPRI of the MM showed different signal intensities at the different tumor stages, unlike the NP, which displayed fewer variations in signal intensity. Thus, the present results suggest that EPR and 2D EPRI can be useful for characterization of the two melanin radicals in the MM and for determination of their size and concentration.

Improvised double-embedding technique of minute biopsies: a mega boon to histopathology laboratory.

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Yadav, Lokendra; Thomas, Sarega; Kini, Usha

2015-01-01

Optimal orientation of minute mucosal biopsies is essential for a definite diagnosis in gastrointestinal pathology or to visualize neural plexuses in Hirschsprung disease. The problem of minute size of the biopsy and its orientation gets compounded when they are from neonates and mandates exhaustive strip cuts, thus delaying reporting. A modified agar-paraffin technique is aimed to make tissue embedding efficient and user-friendly by inking mapping biopsies (one or more) either fresh or fixed with surgical coloring inks followed by embedding first in agar after orientation and followed thereafter by processing, re-embedding in paraffin wax, sectioning and staining. The tissues in agar paraffin block were found to be well processed, firm, held secure and well preserved. The blocks were easy to cut, with serial sections of thickness 2-3 μ and easy to spread. The colored inks remained permanently on the tissues both in the block as well as on the sections which helped in easy identification of tissues. Agar did not interfere with any stain such as Hematoxylin and Eosin or with histochemical stains, enzyme histochemistry or immunohistochemistry. Inking biopsies and pooling them in a block when obtained from the same patient reduced the number of tissue blocks. The modified agar-paraffin embedding technique is a simple reliable user friendly method that can greatly improve the quality of diagnostic information from minute biopsies by optimal orientation, better quality of sections, faster turnaround time and cost-effectiveness by economizing on the number of paraffin blocks, manpower, chemical reagents and laboratory infrastructure.

Improvised double-embedding technique of minute biopsies: A mega boon to histopathology laboratory

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Lokendra Yadav

2015-01-01

Full Text Available Introduction: Optimal orientation of minute mucosal biopsies is essential for a definite diagnosis in gastrointestinal pathology or to visualize neural plexuses in Hirschsprung disease. The problem of minute size of the biopsy and its orientation gets compounded when they are from neonates and mandates exhaustive strip cuts, thus delaying reporting. Aim: A modified agar-paraffin technique is aimed to make tissue embedding efficient and user-friendly by inking mapping biopsies (one or more either fresh or fixed with surgical coloring inks followed by embedding first in agar after orientation and followed thereafter by processing, re-embedding in paraffin wax, sectioning and staining. Results: The tissues in agar paraffin block were found to be well processed, firm, held secure and well preserved. The blocks were easy to cut, with serial sections of thickness 2-3 μ and easy to spread. The colored inks remained permanently on the tissues both in the block as well as on the sections which helped in easy identification of tissues. Agar did not interfere with any stain such as Hematoxylin and Eosin or with histochemical stains, enzyme histochemistry or immunohistochemistry. Inking biopsies and pooling them in a block when obtained from the same patient reduced the number of tissue blocks. Conclusion: The modified agar-paraffin embedding technique is a simple reliable user friendly method that can greatly improve the quality of diagnostic information from minute biopsies by optimal orientation, better quality of sections, faster turnaround time and cost-effectiveness by economizing on the number of paraffin blocks, manpower, chemical reagents and laboratory infrastructure.

Cerebral phaeohyphomycosis in a green iguana (Iguana iguana).

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Olias, P; Hammer, M; Klopfleisch, R

2010-07-01

Cerebral phaeohyphomycosis was diagnosed in a 4-year-old green iguana (Iguana iguana) with paroxysmal spastic paralysis of all limbs and circling motion. Formalin-fixed tissues were collected at necropsy examination and submitted for evaluation. The left cerebrum and the left ventricle were replaced by a solid brown coloured mass. Microscopical examination revealed the presence of necrotizing and granulomatous encephalitis affecting the cerebrum, cerebellum and brainstem, with severe vasculitis and intralesional dematiaceous fungal hyphae. No other lesions or fungi were found in other organs. Fungi were identified as Oidiodendron spp. by sequence analysis of the internal transcribed spacer (ITS) region 1 extracted from formalin-fixed and paraffin wax-embedded brain tissue. This case represents the first report of phaeohyphomycosis with tropism for the central nervous system in a reptile. In the absence of fresh tissue, the diagnosis in such cases may be assisted by molecular analysis of fixed tissue specimens. (c) 2009 Elsevier Ltd. All rights reserved.

Automated Analysis of Protein Expression and Gene Amplification within the Same Cells of Paraffin-Embedded Tumour Tissue

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Timo Gaiser

2010-01-01

Full Text Available Background: The simultaneous detection of protein expression and gene copy number changes in patient samples, like paraffin-embedded tissue sections, is challenging since the procedures of immunohistochemistry (IHC and Fluorescence in situ Hybridization (FISH negatively influence each other which often results in suboptimal staining. Therefore, we developed a novel automated algorithm based on relocation which allows subsequent detection of protein content and gene copy number changes within the same cell.

Immunohistochemical localisation of keratin and luminal epithelial antigen in myoepithelial and luminal epithelial cells of human mammary and salivary gland tumours.

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Nathrath, W B; Wilson, P D; Trejdosiewicz, L K

1982-01-01

Rabbit antisera to human 40-63 000 MW epidermal keratin, one batch with restricted distribution of reactivity from an initial (aK1) and one with "broad spectrum" distribution of reactivity from a late bleeding (aK), and to "luminal epithelial antigen" (aLEA) were applied to formalin fixed paraffin embedded sections of human normal and neoplastic mammary and salivary glands using an indirect immunoperoxidase method. aK1 reacted with myoepithelial cells, aLEA with luminal epithelial cells and aK with both cell types in normal mammary and salivary gland. In breast carcinomas the majority of intraluminal and infiltrating carcinoma cells reacted with aLEA but not with aK1 which reacted only with surrounding myoepithelial cells. aK reacted with both myoepithelial cells and with intraluminal and infiltrating tumour cells. In the salivary gland adenomas the majority of cells reacted with aK, and those cells arranged in a tubular fashion reacted with aLEA.

Analysis of hepcidin expression: in situ hybridization and quantitative polymerase chain reaction from paraffin sections.

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Sakuraoka, Yuhki; Sawada, Tokihiko; Shiraki, Takayuki; Park, Kyunghwa; Sakurai, Yuhichiro; Tomosugi, Naohisa; Kubota, Keiichi

2012-07-28

To establish methods for quantitative polymerase chain reaction (PCR) for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of hepatocellular carcinoma (HCC). Total RNA from paraffin-embedded sections was isolated from 68 paraffin-embedded samples of HCC. Samples came from 54 male and 14 female patients with a mean age of 66.8 ± 7.8 years. Quantitative PCR was performed. Immunohistochemistry and in situ hybridization for hepcidin were also performed. Quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections of HCC was performed successfully. The expression level of hepcidin mRNA in cancer tissues was significantly higher than that in non-cancer tissues. A method of in situ hybridization for hepcidin was established successfully, and this demonstrated that hepcidin mRNA was expressed in non-cancerous tissue but absent in cancerous tissue. We have established novel methods for quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of HCC.

FTA Cards for Preservation of Nucleic Acids for Molecular Assays: A Review on the Use of Cytologic/Tissue Samples.

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da Cunha Santos, Gilda

2018-03-01

- Traditional methods for storing histologic and cytologic specimens for future use in molecular assays have consisted of either snap-freezing with cryopreservation or formalin-fixing, paraffin-embedding the samples. Although snap-freezing with cryopreservation is recommended for better preservation of nucleic acids, the infrastructure and space required for archiving impose challenges for high-volume pathology laboratories. Cost-effective, long-term storage at room temperature; relatively easy shipment; and standardized handling can be achieved with formalin-fixed, paraffin-embedded samples, but formalin fixation induces fragmentation and chemical modification of nucleic acids. Advances in next-generation sequencing platforms, coupled with an increase in diagnostic, prognostic, and predictive molecular biomarkers have created a demand for high-quality nucleic acids. To address issues of the quality of nucleic acid and logistics in sample acquisition, alternatives for specimen preservation and long-term storage have been described and include novel universal tissue fixatives, stabilizers, and technologies. - To collect, retrieve, and review information from studies describing the use of nucleic acids recovered from cytologic/tissue specimens stored on Flinders Technology Associates (FTA, GE Whatman, Maidstone, Kent, United Kingdom) cards for downstream molecular applications. - An electronic literature search in the PubMed (National Center for Biotechnology Information, Bethesda, Maryland) database allowed the selection of manuscripts addressing the use of FTA cards for storage of cytologic samples for molecular analysis. Only articles published in English were retrieved. - The use of FTA cards is a versatile method for fostering multicenter, international collaborations and clinical trials that require centralized testing, long-distance shipment, and high-quality nucleic acids for molecular techniques. Studies with controlled temperature are required to test the

Progesterone receptor isoform A may regulate the effects of neoadjuvant aglepristone in canine mammary carcinoma

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Guil-Luna, Silvia; Stenvang, Jan; Brünner, Nils

2014-01-01

RNA expression of progesterone receptor isoforms A and B in mammary carcinomas in dogs treated with 20 mg/Kg of aglepristone (n¿=¿22) or vehicle (n¿=¿5) twice before surgery.ResultsFormalin-fixed, paraffin-embedded tissue samples taken before and after treatment were used to analyse total progesterone receptor......-receptor positive and isoform-A positive tumours in aglepristone-treated dogs.ConclusionsThese findings suggest that the antiproliferative effects of aglepristone in canine mammary carcinomas are mediated by progesterone receptor isoform A....

Utility of cytopathological specimens and an automated image analysis for the evaluation of HER2 status and intratumor heterogeneity in breast carcinoma.

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Arihiro, Koji; Oda, Miyo; Ogawa, Katsunari; Kaneko, Yoshie; Shimizu, Tomomi; Tanaka, Yuna; Marubashi, Yukari; Ishida, Katsunari; Takai, Chikako; Taoka, Chie; Kimura, Shuji; Shiroma, Noriyuki

2016-12-01

Although updated HER2 testing guidelines have been improved by a collaboration between the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) in 2013, HER2 evaluation is still problematic because of issues involving CEP17 polysomy, heterogeneity, and HER2 score 2+ cases. The aim of this retrospective study was to evaluate the relationship between HER2 gene heterogeneity, or so called CEP17 polysomy, using breast carcinoma cells sampled by scraping and the IHC score graded by automated image analysis using whole slide image. We randomly selected 23 breast carcinoma cases with a HER2 score 0, 24 cases with a HER2 score 1+, 24 cases with HER2 score 2+, and 23 cases with HER2 score 3+ from the records of patients with breast cancer at Hiroshima University Hospital. We compared the results of fluorescent in situ hybridization (FISH) using formalin-fixed, paraffin-embedded (FFPE) tissues and cytological samples and compared the HER2 score calculated using an automated image analysis using wholly scanned slide images and visual counting. We successfully performed the FISH assay in 78 of 94 cases (83%) using FFPE tissues and in all 94 (100%) cases using cytological samples. Frequency of both HER2 amplification and CEP17 polysomy was higher when cytological samples were used than when FFPE tissue was used. Frequency of HER2 heterogeneity using cytological samples was higher that than using FFPE tissue, except for the IHC score 3+ cases. When assessment of HER2 status based on FISH using FFPE tissue cannot be accomplished, FISH using cytological samples should be considered. When intensity of HER2 is heterogeneous in the tumor tissue, particularly in cases regarded as score 2+, they should be evaluated by automated image analysis using the whole slide image. Copyright © 2016 Elsevier GmbH. All rights reserved.

Profiling the HER3/PI3K Pathway in Breast Tumors Using Proximity-Directed Assays Identifies Correlations between Protein Complexes and Phosphoproteins

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Mukherjee, Ali; Badal, Youssouf; Nguyen, Xuan-Thao; Miller, Johanna; Chenna, Ahmed; Tahir, Hasan; Newton, Alicia; Parry, Gordon; Williams, Stephen

2011-01-01

Background The identification of patients for targeted antineoplastic therapies requires accurate measurement of therapeutic targets and associated signaling complexes. HER3 signaling through heterodimerization is an important growth-promoting mechanism in several tumor types and may be a principal resistance mechanism by which EGFR and HER2 expressing tumors elude targeted therapies. Current methods that can study these interactions are inadequate for formalin-fixed, paraffin-embedded (FFPE) tumor samples. Methodology and Principal Findings Herein, we describe a panel of proximity-directed assays capable of measuring protein-interactions and phosphorylation in FFPE samples in the HER3/PI3K/Akt pathway and examine the capability of these assays to inform on the functional state of the pathway. We used FFPE breast cancer cell line and tumor models for this study. In breast cancer cell lines we observe both ligand-dependent and independent activation of the pathway and strong correlations between measured activation of key analytes. When selected cell lines are treated with HER2 inhibitors, we not only observe the expected molecular effects based on mechanism of action knowledge, but also novel effects of HER2 inhibition on key targets in the HER receptor pathway. Significantly, in a xenograft model of delayed tumor fixation, HER3 phosphorylation is unstable, while alternate measures of pathway activation, such as formation of the HER3PI3K complex is preserved. Measurements in breast tumor samples showed correlations between HER3 phosphorylation and receptor interactions, obviating the need to use phosphorylation as a surrogate for HER3 activation. Significance This assay system is capable of quantitatively measuring therapeutically relevant responses and enables molecular profiling of receptor networks in both preclinical and tumor models. PMID:21297994

Anatomic Dissection of Arachnoid Membranes Encircling the Pituitary Stalk on Fresh, Non-Formalin-Fixed Specimens: Anatomoradiologic Correlations and Clinical Applications in Craniopharyngioma Surgery.

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Ciappetta, Pasqualino; Pescatori, Lorenzo

2017-12-01

The anatomy of the arachnoid membranes and cisternal spaces around the pituitary stalk has not been yet exhaustively described and understood. In this study, we performed a detailed anatomic study on fresh, non-formalin-fixed cadavers of the arachnoid membranes encircling the pituitary stalk and correlate our anatomic findings with magnetic resonance imaging (MRI). Ten fresh, non-formalin-fixed, non-silicon-injected adult cadaveric heads were analyzed in this study. The membrane and cisterns that were studied for our study were as follows: 1) the diaphragma sellae and its dural components; 2) the basal arachnoid membrane; 3) the Liliequist membrane with its diencephalic and mesencephalic portion; 4) the medial carotid membrane; 5) the chiasmatic cistern; and 6) the pituitary stalk. MRI examinations of the sellar region were performed in 15 healthy volunteers (9 men, mean age 40 years; and 6 women mean age, 37 years) to visualize the arachnoid membrane encircling the pituitary stalk. MRI examinations were performed with a 3-T unit. A 3-dimensional constructive interference in steady state pulse magnetic resonance sequence was used. All the membranes examined were visualized clearly in all the dissections performed. Their 3-dimensional organization around the pituitary stalk was clarified and confirmed by MRI. Our study gives a detailed description of the pituitary stalk arachnoid sheets on fresh, non-formalin-fixed cadavers. This technique allowed us to clearly identify a funnel-shaped arachnoid collar encircling the pituitary stalk and delimiting a distinct cisternal space belonging to the stalk itself. Copyright © 2017 Elsevier Inc. All rights reserved.

Correlation between quantitative HER-2 protein expression and risk for brain metastases in HER-2+ advanced breast cancer patients receiving trastuzumab-containing therapy.

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Duchnowska, Renata; Biernat, Wojciech; Szostakiewicz, Barbara; Sperinde, Jeff; Piette, Fanny; Haddad, Mojgan; Paquet, Agnes; Lie, Yolanda; Czartoryska-Arłukowicz, Bogumiła; Wysocki, Piotr; Jankowski, Tomasz; Radecka, Barbara; Foszczynska-Kłoda, Małgorzata; Litwiniuk, Maria; Debska, Sylwia; Weidler, Jodi; Huang, Weidong; Buyse, Marc; Bates, Michael; Jassem, Jacek

2012-01-01

Patients with human epidermal growth factor receptor (HER)-2+ breast cancer are at particularly high risk for brain metastases; however, the biological basis is not fully understood. Using a novel HER-2 assay, we investigated the correlation between quantitative HER-2 expression in primary breast cancers and the time to brain metastasis (TTBM) in HER-2+ advanced breast cancer patients treated with trastuzumab. The study group included 142 consecutive patients who were administered trastuzumab-based therapy for HER-2+ metastatic breast cancer. HER-2/neu gene copy number was quantified as the HER-2/centromeric probe for chromosome 17 (CEP17) ratio by central laboratory fluorescence in situ hybridization (FISH). HER-2 protein was quantified as total HER-2 protein expression (H2T) by the HERmark® assay (Monogram Biosciences, Inc., South San Francisco, CA) in formalin-fixed, paraffin-embedded tumor samples. HER-2 variables were correlated with clinical features and TTBM was measured from the initiation of trastuzumab-containing therapy. A higher H2T level (continuous variable) was correlated with shorter TTBM, whereas HER-2 amplification by FISH and a continuous HER-2/CEP17 ratio were not predictive (p = .013, .28, and .25, respectively). In the subset of patients that was centrally determined by FISH to be HER-2+, an above-the-median H2T level was significantly associated with a shorter TTBM (hazard ratio, [HR], 2.4; p = .005), whereas this was not true for the median HER-2/CEP17 ratio by FISH (p = .4). Correlation between a continuous H2T level and TTBM was confirmed on multivariate analysis (HR, 3.3; p = .024). These data reveal a strong relationship between the quantitative HER-2 protein expression level and the risk for brain relapse in HER-2+ advanced breast cancer patients. Consequently, quantitative assessment of HER-2 protein expression may inform and facilitate refinements in therapeutic treatment strategies for selected subpopulations of patients in this

In situ zymography and immunolabeling in fixed and decalcified craniofacial tissues.

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Porto, Isabel M; Rocha, Lenaldo B; Rossi, Marcos A; Gerlach, Raquel F

2009-07-01

In situ zymography is a very important technique that shows the proteolytic activity in sections and allows researchers to observe the specific sites of proteolysis in tissues or cells. It is normally performed in non-fixed frozen sections and is not routinely performed in calcified tissues. In this study, we describe a technique that maintains proteolytic activity in fixed and decalcified sections obtained after routine paraffin sectioning in conventional microtome and cryostat sections. We used adult rat hemimandibles, which presented bone, enamel, and dentine matrices; the substrate used was dye-quenched-gelatin. Gelatinolytic activity was colocalized with MMP-2 using fluorescent antibodies. Specific proteolytic activity was observed in all sections, compatible with metalloproteinase activity, particularly in dentine and bone. Furthermore, matrix metalloproteinase-2 was colocalized to the sites of green fluorescence in dentine. In conclusion, the technique presented here will allow in situ zymography reactions in fixed, decalcified, and paraffin-embedded tissues, and we showed that paraformaldehyde-lysine-periodate-fixed cryostat sections are suitable for colocalization of gelatinolytic activity and protein labeling with antibodies.

Comparação de três protocolos de extração de DNA a partir de tecido fixado em formol e incluído em parafina Comparison of three DNA extraction protocols from formaldehyde-fixed and paraffin-embedded tissues

Directory of Open Access Journals (Sweden)

José Veríssimo Fernandes

2004-06-01

Full Text Available OBJETIVO: Padronizar um método alternativo para extração de DNA a partir de tecido fixado em formol e conservado em arquivos de blocos de parafina, visando à realização de estudos retrospectivos. MÉTODOS: Comparou-se a eficiência de protocolos de extração de DNA a partir de tecido parafinado, para análise por reação em cadeia de polimerase (PCR, tomando-se como parâmetro um protocolo baseado em um kit comercial. Foram feitas extrações do DNA de 60 espécimes por três métodos: o protocolo A, baseado no kit GlassMAX; o B, utilizando-se o kit GFX TM; e o C, tendo como base o método de Banerjee et al.(2. A integridade e a suficiência do DNA presente na amostra foram avaliadas pela amplificação por PCR de um segmento de 110pb do gene da beta-globina humana, com visualização por meio de eletroforese em gel de poliacrilamida, corado pela prata. Resultados: Das 60 amostras analisadas, 45 apresentaram resultado positivo na PCR quando o DNA foi extraído por qualquer um dos três protocolos. Em seis amostras, a amplificação foi positiva apenas para o DNA extraído pelos protocolos A e C. Em três amostras, o resultado foi positivo apenas para o DNA extraído pelo protocolo A, e em duas, apenas para o DNA extraído pelo protocolo C. CONCLUSÕES: O protocolo C apresentou desempenho semelhante ao do protocolo A, com as vantagens de apresentar menor custo, dispensar o uso de kit comercial, além de não utilizar solventes orgânicos, revelando-se uma alternativa viável para a obtenção de DNA a partir de tecido fixado em formol e incluído em parafina.OBJECTIVE: To set up a method for DNA extraction from paraffin embedded cervical cancer specimens, previously formalin-fixed, aiming to accomplish retrospective analysis. METHODS: Sixty specimens were submitted to DNA extraction by three different methods. All of them involved digestion of the tissues by proteinase K, followed by DNA purification, based in three different approaches

Identification of BRCA1-deficient ovarian cancers

DEFF Research Database (Denmark)

Skytte, Anne-Bine; Waldstrøm, Marianne; Rasmussen, Anders Aamann

2011-01-01

of offering genetic counseling and due to beneficial effects of PARP inhibitor treatment in this group. Since DNA sequencing is expensive and time-consuming efforts have been devoted to develop more indirect methods for BRCA screening that can improve the selection of patients for sequence-based BRCA testing....... Design. BRCA1-immunohistochemistry (IHC), fluorescence in-situ hybridization (FISH) and methylation analyses were performed on formalin-fixed, paraffin-embedded ovarian cancer tissue. Sample: 54 ovarian cancers; 15 BRCA1 cancers, 4 BRCA2 cancers, 10 cancers from patients with a family history...

A prognostic profile of hypoxia-induced genes for localised high-grade soft tissue sarcoma

DEFF Research Database (Denmark)

Aggerholm-Pedersen, Ninna; Sørensen, Brita Singers; Overgaard, Jens

2016-01-01

sarcoma (STS). METHODS: The hypoxia-induced gene quantification was performed by real-time quantitative PCR (RT-qPCR) of formalin-fixed, paraffin-embedded tissue samples. The gene expression cut-points were determined in a test cohort of 55 STS patients and used to allocate each patient into a more......BACKGROUND: For decades, tumour hypoxia has been pursued as a cancer treatment target. However, prognostic and predictive biomarkers are essential for the use of this target in the clinic. This study investigates the prognostic value of a hypoxia-induced gene profile in localised soft tissue...

Immunocytochemistry of formalin-fixed human brain tissues: microwave irradiation of free-floating sections.

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Shiurba, R A; Spooner, E T; Ishiguro, K; Takahashi, M; Yoshida, R; Wheelock, T R; Imahori, K; Cataldo, A M; Nixon, R A

1998-01-01

Formalin fixation, the chemical process in which formaldehyde binds to cells and tissues, is widely used to preserve human brain specimens from autolytic decomposition. Ultrastructure of cellular and mitochondrial membranes is markedly altered by vesiculation, but this does not interfere with diagnostic evaluation of neurohistology by light microscopy. Serious difficulties are encountered, however, when immunocytochemical staining is attempted. Antigens that are immunoreactive in unfixed frozen sections and protein extracts appear to be concealed or destroyed in formalin-fixed tissues. In dilute aqueous solution, formaldehyde is in equilibrium with methylene glycol and its polymeric hydrates, the balance by far in favor of methylene glyco. Carbonylic formaldehyde is a reactive electrophilic species well known for crosslinking functional groups in tissue proteins, nucleic acids, and polysaccharides. Some of its methylene crosslinks are readily hydrolyzed. Others are stable and irreversible. During immunostaining reactions, intra- and inter-molecular links between macromolecules limit antibody permeation of tissue sections, alter protein secondary structure, and reduce accessibility of antigenic determinants . Accordingly, immunoreactivity is diminished for many antigens. Tissues are rapidly penetrated by methylene glycol, but formaldehyde binding to cellular constituents is relatively slow, increasing progressively until equilibrium is reached. In addition, prolonged storage in formalin may result in acidification of human brain specimens. Low pH favors dissociation of methylene glycol into formaldehyde, further reducing both classical staining and antigen detectability. Various procedures have been devised to counter the antigen masking effects of formaldehyde. Examples include pretreatment of tissue sections with proteases, formic acid, or ultrasound. Recently, heating of mounted sections in ionic salt solution by microwave energy was found to restore many

[Immunoexpression of c-erbB-2 in intraductal proliferative lesions of the female breast].

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Oliveira, Agliberto Barbosa de; De Luca, Laurival Antônio; Carvalho, Grigna Teixeira; Arias, Victor Eduardo Arua; Carvalho, Lídia Raquel de; Assunção, Maria do Carmo

2004-01-01

Genetic modifications are related to genesis and development of cancer. Neoplasias in various organs express the c-erbB-2 oncogene. In intraductal proliferations of the breast it has been assessed as a risk factor for subsequent development of carcinoma. The c-erbB-2 immunoexpression in intraductal epithelial proliferations and the relationship with histopathological characteristics of ductal carcinoma in situ (DCIS) were evaluated. File material from 88 women, which were tissue samples formalin-fixed, paraffin-embedded blocks, was used. Of these 51 presented with DCIS and 37 with ductal hyperplasia without atypia. Ages of the women ranged from 35 to 76 years. All cases were reviewed and nuclear grade, presence of necrosis, preponderance of histological subtype and its extension were verified. Specimens were obtained for the c-erB-2 immunohistochemical study of 84 of the women in question. No expression of the oncogene was verified in the hyperplasias without atypias and in tissues adjacent to all tissue samples. Expression of c-erbB-2 was verified in 9 (19.1%) of the DCIS (p = 0.0001). Immunoexpression was not related to the extension of the lesions. The c-erbB-2 immunoexpression in DCIS was correlated to the histological subtype (p = 0.019), necrosis (p = 0.0066), nuclear grade (p = 0.0084) and Van Nuys Classification (p = 0.039). Expression of c-erbB-2 was significant in proliferative lesions with risk (DCIS) and was correlated to histopathological characteristics: high nuclear grade, presence of necrosis and comedy subtype. There was no expression in the hyperplasias without atypias and adjacent tissues.

Matched rabbit monoclonal antibodies against αv-series integrins reveal a novel αvβ3-LIBS epitope, and permit routine staining of archival paraffin samples of human tumors

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Goodman, Simon L.; Grote, Hans Juergen; Wilm, Claudia

2012-01-01

Summary The relationship between integrin expression and function in pathologies is often contentious as comparisons between human pathological expression and expression in cell lines is difficult. In addition, the expression of even integrins αvβ6 and αvβ8 in tumor cell lines is not comprehensively documented. Here, we describe rabbit monoclonal antibodies (RabMabs) against the extracellular domains of αv integrins that react with both native integrins and formalin fixed, paraffin embedded (FFPE) human tissues. These RabMabs, against αvβ3 (EM22703), αvβ5 (EM09902), αvβ6 (EM05201), αvβ8 (EM13309), and pan-αv (EM01309), recognize individual integrin chains in Western blots and in flow cytometry. EM22703 detected a ligand-induced binding site (LIBS), reporting an epitope enhanced by the binding of an RGD-peptide to αvβ3. αvβ8 was rarely expressed in human tumor specimens, and weakly expressed in non-small-cell lung carcinoma (NSCLC). However, ovarian carcinoma cell lines expressed αvβ8, as did some melanoma cells, whereas U87MG glioma lacked αvβ8 expression. We observed an unexpected strong expression of αvβ6 in tumor samples of invasive ductal breast adenoma, colorectal carcinoma (CRC), and NSCLC. αvβ3 was strongly expressed in some invasive NSCLC cohorts. Interestingly, PC3 prostate cell and human prostate tumors did not express αvβ3. The RabMabs stained plasma membranes in FFPE-immunohistochemistry (IHC) samples of tumor cell lines from lung, ovary, colon, prostate, squamous cell carcinoma of head and neck (SCCHN), breast, and pancreas carcinomas. The RabMabs are unique tools for probing αv integrin biology, and suggest that especially αvβ6 and αvβ8 biologies still have much to reveal. PMID:23213423

Polysomy of chromosome 17 in breast cancer tumors showing an overexpression of ERBB2: a study of 175 cases using fluorescence in situ hybridization and immunohistochemistry

International Nuclear Information System (INIS)

Salido, Marta; Solé, Francesc; Tusquets, Ignasi; Corominas, Josep M; Suarez, Marta; Espinet, Blanca; Corzo, Cristina; Bellet, Meritxell; Fabregat, Xavier; Serrano, Sergi

2005-01-01

One of the most common genetic aberrations associated with breast cancer is the amplification and overexpression of the ERBB2 proto-oncogene located at chromosome 17, bands q12-21. The amplification/overexpression occurs in 25 to 30% of all breast cancers. In breast cancer, aneusomy of chromosome 17, either monosomy or polysomy, is frequently observed by conventional cytogenetics and fluorescence in situ hybridization (FISH). The aim of this study was to discover whether or not numerical aberrations on chromosome 17 have a correlation to the amplification or overexpression of the ERBB2 gene and to analyze their clinical implications in subgroups showing 2+ or 3+ positive scores by immunohistochemistry (IHC). We used FISH on a series of 175 formalin-fixed paraffin-embedded breast carcinomas to detect ERBB2 amplification, using a dual-probe system for the simultaneous enumeration of the ERBB2 gene and the centromeric region of chromosome 17, as well as using IHC to detect overexpression. We analyzed clinical and pathological variables in a subgroup of patients with 2+ and 3+ IHC scores (147 patients), to describe any differences in clinicopathological characteristics between polysomic and non-polysomic cases with the use of the χ 2 test. We found 13% of cases presenting polysomy, and three cases presented monosomy 17 (2%). According to the status of the ERBB2 gene, instances of polysomy 17 were more frequently observed in non-amplified cases than in FISH-amplified cases, suggesting that the mechanism for ERBB2 amplification is independent of polysomy 17. Polysomy 17 was detected in patients with 2+ and 3+ IHC scores. We found that nodal involvement was more frequent in polysomic than in non-polysomic cases (P = 0.046). The determination of the copy number of chromosome 17 should be incorporated into the assesment of ERBB2 status. It might also be helpful to differentiate a subgroup of breast cancer patients with polysomy of chromosome 17 and overexpression of ERBB2

Determination of HER2 gene amplification by chromogenic in situ hybridization (CISH) in archival breast carcinoma.

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Zhao, Jianxin; Wu, Rina; Au, Alfred; Marquez, Abbey; Yu, Yibing; Shi, Zuorong

2002-06-01

To compare the efficacy of chromogenic in situ hybridization (CISH(TM)) with fluorescence in situ (FISH) hybridization and immunohistochemistry (IHC) in determination of the HER2 status in human breast cancer. HER2 gene amplification was determined on formalin-fixed paraffin-embedded (FFPE) sections of 62 invasive breast cancers by FISH and followed by CISH using a digoxigenin (DIG)-labeled HER2 DNA probe generated by Subtraction Probe Technology (SPT(TM)), and a biotin-labeled chromosome 17 centromeric (chr.17cen) probe. The sections were heat treated and enzyme digested. After in situ hybridization, the HER2 probe was detected with fluorescein (FITC)-anti-DIG for FISH, followed by peroxidase-anti-FITC and diaminobenzidine (DAB) for CISH. The chr.17cen probe was detected with peroxidase-streptavidin and DAB. For CISH application, HER2 gene copies or chromosome 17 centromeres and morphology of cells were easily visualized simultaneously with a 40x objective under bright-field microscope in hematoxylin-counterstained sections. IHC study of HER2 overexpression was performed on adjacent sections using a panel of three HER2 antibodies (TAB 250, CB11, A0485), and staining was scored according to the criteria specified in the HercepTest. HER2 gene amplification detected by CISH was visualized typically as large DAB-stained clusters or by many dots in the nucleus. FISH and CISH identified HER2 gene amplification in 19% of the tumors. Chromosome 17 polysomy was detected in 31% of the tumors. HER2 overexpression was demonstrated in 19% (TAB 250), 23% (CB11), and 36% (A0485) of the tumors. Complete concordance between the results of CISH with FISH, TAB 250, CB11, and A0485 was seen in 100%, 97%, 94%, and 84% of the cases, respectively. By permitting observation of morphology using a bright-field microscope, CISH is an accurate, practical, and economical approach to screen HER2 status in breast cancers. It is a useful methodology for confirming ambiguous IHC results.

Epstein-Barr virus, human papillomavirus and mouse mammary tumour virus as multiple viruses in breast cancer.

Science.gov (United States)

Glenn, Wendy K; Heng, Benjamin; Delprado, Warick; Iacopetta, Barry; Whitaker, Noel J; Lawson, James S

2012-01-01

The purpose of this investigation is to determine if Epstein Barr virus (EBV), high risk human papillomavirus (HPV), and mouse mammary tumour viruses (MMTV) co-exist in some breast cancers. All the specimens were from women residing in Australia. For investigations based on standard PCR, we used fresh frozen DNA extracts from 50 unselected invasive breast cancers. For normal breast specimens, we used DNA extracts from epithelial cells from milk donated by 40 lactating women. For investigations based on in situ PCR we used 27 unselected archival formalin fixed breast cancer specimens and 18 unselected archival formalin fixed normal breast specimens from women who had breast reduction surgery. Thirteen of these fixed breast cancer specimens were ductal carcinoma in situ (dcis) and 14 were predominantly invasive ductal carcinomas (idc). EBV sequences were identified in 68%, high risk HPV sequences in 50%, and MMTV sequences in 78% of DNA extracted from 50 invasive breast cancer specimens. These same viruses were identified in selected normal and breast cancer specimens by in situ PCR. Sequences from more than one viral type were identified in 72% of the same breast cancer specimens. Normal controls showed these viruses were also present in epithelial cells in human milk - EBV (35%), HPV, 20%) and MMTV (32%) of 40 milk samples from normal lactating women, with multiple viruses being identified in 13% of the same milk samples. We conclude that (i) EBV, HPV and MMTV gene sequences are present and co-exist in many human breast cancers, (ii) the presence of these viruses in breast cancer is associated with young age of diagnosis and possibly an increased grade of breast cancer.

Technical note on the validation of a semi-automated image analysis software application for estrogen and progesterone receptor detection in breast cancer

Science.gov (United States)

2011-01-01

Background The immunohistochemical detection of estrogen (ER) and progesterone (PR) receptors in breast cancer is routinely used for prognostic and predictive testing. Whole slide digitalization supported by dedicated software tools allows quantization of the image objects (e.g. cell membrane, nuclei) and an unbiased analysis of immunostaining results. Validation studies of image analysis applications for the detection of ER and PR in breast cancer specimens provided strong concordance between the pathologist's manual assessment of slides and scoring performed using different software applications. Methods The effectiveness of two connected semi-automated image analysis software (NuclearQuant v. 1.13 application for Pannoramic™ Viewer v. 1.14) for determination of ER and PR status in formalin-fixed paraffin embedded breast cancer specimens immunostained with the automated Leica Bond Max system was studied. First the detection algorithm was calibrated to the scores provided an independent assessors (pathologist), using selected areas from 38 small digital slides (created from 16 cases) containing a mean number of 195 cells. Each cell was manually marked and scored according to the Allred-system combining frequency and intensity scores. The performance of the calibrated algorithm was tested on 16 cases (14 invasive ductal carcinoma, 2 invasive lobular carcinoma) against the pathologist's manual scoring of digital slides. Results The detection was calibrated to 87 percent object detection agreement and almost perfect Total Score agreement (Cohen's kappa 0.859, quadratic weighted kappa 0.986) from slight or moderate agreement at the start of the study, using the un-calibrated algorithm. The performance of the application was tested against the pathologist's manual scoring of digital slides on 53 regions of interest of 16 ER and PR slides covering all positivity ranges, and the quadratic weighted kappa provided almost perfect agreement (κ = 0.981) among the two

Significance of genomic instability in breast cancer in atomic bomb survivors: analysis of microarray-comparative genomic hybridization

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Oikawa Masahiro

2011-12-01

Full Text Available Abstract Background It has been postulated that ionizing radiation induces breast cancers among atomic bomb (A-bomb survivors. We have reported a higher incidence of HER2 and C-MYC oncogene amplification in breast cancers from A-bomb survivors. The purpose of this study was to clarify the effect of A-bomb radiation exposure on genomic instability (GIN, which is an important hallmark of carcinogenesis, in archival formalin-fixed paraffin-embedded (FFPE tissues of breast cancer by using microarray-comparative genomic hybridization (aCGH. Methods Tumor DNA was extracted from FFPE tissues of invasive ductal cancers from 15 survivors who were exposed at 1.5 km or less from the hypocenter and 13 calendar year-matched non-exposed patients followed by aCGH analysis using a high-density oligonucleotide microarray. The total length of copy number aberrations (CNA was used as an indicator of GIN, and correlation with clinicopathological factors were statistically tested. Results The mean of the derivative log ratio spread (DLRSpread, which estimates the noise by calculating the spread of log ratio differences between consecutive probes for all chromosomes, was 0.54 (range, 0.26 to 1.05. The concordance of results between aCGH and fluorescence in situ hybridization (FISH for HER2 gene amplification was 88%. The incidence of HER2 amplification and histological grade was significantly higher in the A-bomb survivors than control group (P = 0.04, respectively. The total length of CNA tended to be larger in the A-bomb survivors (P = 0.15. Correlation analysis of CNA and clinicopathological factors revealed that DLRSpread was negatively correlated with that significantly (P = 0.034, r = -0.40. Multivariate analysis with covariance revealed that the exposure to A-bomb was a significant (P = 0.005 independent factor which was associated with larger total length of CNA of breast cancers. Conclusions Thus, archival FFPE tissues from A-bomb survivors are useful for

Significance of genomic instability in breast cancer in atomic bomb survivors: analysis of microarray-comparative genomic hybridization

International Nuclear Information System (INIS)

Oikawa, Masahiro; Yoshiura, Koh-ichiro; Kondo, Hisayoshi; Miura, Shiro; Nagayasu, Takeshi; Nakashima, Masahiro

2011-01-01

It has been postulated that ionizing radiation induces breast cancers among atomic bomb (A-bomb) survivors. We have reported a higher incidence of HER2 and C-MYC oncogene amplification in breast cancers from A-bomb survivors. The purpose of this study was to clarify the effect of A-bomb radiation exposure on genomic instability (GIN), which is an important hallmark of carcinogenesis, in archival formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer by using microarray-comparative genomic hybridization (aCGH). Tumor DNA was extracted from FFPE tissues of invasive ductal cancers from 15 survivors who were exposed at 1.5 km or less from the hypocenter and 13 calendar year-matched non-exposed patients followed by aCGH analysis using a high-density oligonucleotide microarray. The total length of copy number aberrations (CNA) was used as an indicator of GIN, and correlation with clinicopathological factors were statistically tested. The mean of the derivative log ratio spread (DLRSpread), which estimates the noise by calculating the spread of log ratio differences between consecutive probes for all chromosomes, was 0.54 (range, 0.26 to 1.05). The concordance of results between aCGH and fluorescence in situ hybridization (FISH) for HER2 gene amplification was 88%. The incidence of HER2 amplification and histological grade was significantly higher in the A-bomb survivors than control group (P = 0.04, respectively). The total length of CNA tended to be larger in the A-bomb survivors (P = 0.15). Correlation analysis of CNA and clinicopathological factors revealed that DLRSpread was negatively correlated with that significantly (P = 0.034, r = -0.40). Multivariate analysis with covariance revealed that the exposure to A-bomb was a significant (P = 0.005) independent factor which was associated with larger total length of CNA of breast cancers. Thus, archival FFPE tissues from A-bomb survivors are useful for genome-wide aCGH analysis. Our results suggested that A

Significance of genomic instability in breast cancer in atomic bomb survivors: analysis of microarray-comparative genomic hybridization.

Science.gov (United States)

Oikawa, Masahiro; Yoshiura, Koh-ichiro; Kondo, Hisayoshi; Miura, Shiro; Nagayasu, Takeshi; Nakashima, Masahiro

2011-12-07

It has been postulated that ionizing radiation induces breast cancers among atomic bomb (A-bomb) survivors. We have reported a higher incidence of HER2 and C-MYC oncogene amplification in breast cancers from A-bomb survivors. The purpose of this study was to clarify the effect of A-bomb radiation exposure on genomic instability (GIN), which is an important hallmark of carcinogenesis, in archival formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer by using microarray-comparative genomic hybridization (aCGH). Tumor DNA was extracted from FFPE tissues of invasive ductal cancers from 15 survivors who were exposed at 1.5 km or less from the hypocenter and 13 calendar year-matched non-exposed patients followed by aCGH analysis using a high-density oligonucleotide microarray. The total length of copy number aberrations (CNA) was used as an indicator of GIN, and correlation with clinicopathological factors were statistically tested. The mean of the derivative log ratio spread (DLRSpread), which estimates the noise by calculating the spread of log ratio differences between consecutive probes for all chromosomes, was 0.54 (range, 0.26 to 1.05). The concordance of results between aCGH and fluorescence in situ hybridization (FISH) for HER2 gene amplification was 88%. The incidence of HER2 amplification and histological grade was significantly higher in the A-bomb survivors than control group (P = 0.04, respectively). The total length of CNA tended to be larger in the A-bomb survivors (P = 0.15). Correlation analysis of CNA and clinicopathological factors revealed that DLRSpread was negatively correlated with that significantly (P = 0.034, r = -0.40). Multivariate analysis with covariance revealed that the exposure to A-bomb was a significant (P = 0.005) independent factor which was associated with larger total length of CNA of breast cancers. Thus, archival FFPE tissues from A-bomb survivors are useful for genome-wide aCGH analysis. Our results suggested that A

Characterization of paraffin based breast tissue equivalent phantom using a CdTe detector pulse height analysis.

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Cubukcu, Solen; Yücel, Haluk

2016-12-01

In this study, paraffin was selected as a base material and mixed with different amounts of CaSO 4 ·2H 2 O and H 3 BO 3 compounds in order to mimic breast tissue. Slab phantoms were produced with suitable mixture ratios of the additives in the melted paraffin. Subsequently, these were characterized in terms of first half-value layer (HVL) in the mammographic X-ray range using a pulse-height spectroscopic analysis with a CdTe detector. Irradiations were performed in the energy range of 23-35 kV p under broad beam conditions from Mo/Mo and Mo/Rh target/filter combinations. X-ray spectra were acquired with a CdTe detector without and with phantom material interposition in increments of 1 cm thickness and then evaluated to obtain the transmission data. The net integral areas of the spectra for the slabs were used to plot the transmission curves and these curves were fitted to the Archer model function. The results obtained for the slabs were compared with those of standard mammographic phantoms such as CIRS BR series phantoms and polymethylmethacrylate plates (PMMA). From the evaluated transmission curves, the mass attenuation coefficients and HVLs of some mixtures are close to those of the commercially available standard mammography phantoms. Results indicated that when a suitable proportion of H 3 BO 3 and CaSO 4 ·2H 2 O is added to the paraffin, the resulting material may be a good candidate for a breast tissue equivalent phantom.

Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues.

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Chat-Uthai, Nunthawut; Vejvisithsakul, Pichpisith; Udommethaporn, Sutthirat; Meesiri, Puttarakun; Danthanawanit, Chetiya; Wongchai, Yannawan; Teerapakpinyo, Chinachote; Shuangshoti, Shanop; Poungvarin, Naravat

2018-01-01

The protein kinase BRAF is one of the key players in regulating cellular responses to extracellular signals. Somatic mutations of the BRAF gene, causing constitutive activation of BRAF, have been found in various types of human cancers such as malignant melanoma, and colorectal cancer. BRAF V600E and V600K, most commonly observed mutations in these cancers, may predict response to targeted therapies. Many techniques suffer from a lack of diagnostic sensitivity in mutation analysis in clinical samples with a low cancer cell percentage or poor-quality fragmented DNA. Here we present allele-specific real-time PCR assay for amplifying 35- to 45-base target sequences in BRAF gene. Forward primer designed for BRAF V600E detection is capable of recognizing both types of BRAF V600E mutation, i.e. V600E1 (c.1799T>A) and V600E2 (c.1799_1800delTGinsAA), as well as complex tandem mutation caused by nucleotide changes in codons 600 and 601. We utilized this assay to analyze Thai formalin-fixed paraffin-embedded tissues. Forty-eight percent of 178 Thai colorectal cancer tissues has KRAS mutation detected by highly sensitive commercial assays. Although these DNA samples contain low overall yield of amplifiable DNA, our newly-developed assay successfully revealed BRAF V600 mutations in 6 of 93 formalin-fixed paraffin-embedded colorectal cancer tissues which KRAS mutation was not detected. Ultra-short PCR assay with forward mutation-specific primers is potentially useful to detect BRAF V600 mutations in highly fragmented DNA specimens from cancer patients.

Quantitative description of the morphology and ossification center in the axial skeleton of 20-week gestation formalin-fixed human fetuses using magnetic resonance images.

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Chabert, Steren; Villalobos, Manuel; Ulloa, Patricia; Salas, Rodrigo; Tejos, Cristian; San Martin, Sebastian; Pereda, Jaime

2012-03-01

Human tissues are usually studied using a series of two-dimensional visualizations of in vivo or cutout specimens. However, there is no precise anatomical description of some of the processes of human fetal development. The purpose of our study is to develop a quantitative description of the normal axial skeleton by means of high-resolution three-dimensional magnetic resonance (MR) images, collected from six normal 20-week-old human fetuses fixed in formaldehyde. Fetuses were collected after spontaneous abortion and subsequently fixed with formalin. They were imaged using a 1.5 T MR scanner with an isotropic spatial resolution of 200 µm. The correct tissue discrimination between ossified and cartilaginous bones was confirmed by comparing the images achieved by MR scans and computerized axial tomographies. The vertebral column was segmented out from each image using a specially developed semi-automatic algorithm. Vertebral body dimensions and inter-vertebral distances were larger in the lumbar region, in agreement with the beginning of the ossification process from the thoracolumbar region toward the sacral and cephalic ends. In this article, we demonstrate the feasibility of using MR images to study the ossification process in formalin-fixed fetal tissues. A quantitative description of the ossification centers of vertebral bodies and arches is presented. © 2012 John Wiley & Sons, Ltd.

Lack of prognostic and predictive value of CA IX in radiotherapy of squamous cell carcinoma of the head and neck with known modifiable hypoxia: An evaluation of the DAHANCA 5 study

DEFF Research Database (Denmark)

Eriksen, Jesper Grau; Overgaard, Jens

2007-01-01

BACKGROUND AND PURPOSE: CA IX is suggested to be an endogenous marker of hypoxia in tumours like squamous cell carcinomas of the head and neck (HNSCC). The aim of the present study was to investigate whether CA IX served as a prognostic factor for outcome in a large population of HNSCC and if CA IX...... was able to discriminate the tumours that did benefit from hypoxic modification with nimorazole. MATERIALS AND METHODS: Paraffin-embedded formalin-fixed pre-treatment tumour tissue was available from 320 of the 414 patients treated in the randomized DAHANCA 5 protocol with primary radiotherapy...

Cell-based quantification of biomarkers from an ultra-fast microfluidic immunofluorescent staining: application to human breast cancer cell lines

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Migliozzi, D.; Nguyen, H. T.; Gijs, M. A. M.

2018-02-01

Immunohistochemistry (IHC) is one of the main techniques currently used in the clinics for biomarker characterization. It consists in colorimetric labeling with specific antibodies followed by microscopy analysis. The results are then used for diagnosis and therapeutic targeting. Well-known drawbacks of such protocols are their limited accuracy and precision, which prevent the clinicians from having quantitative and robust IHC results. With our work, we combined rapid microfluidic immunofluorescent staining with efficient image-based cell segmentation and signal quantification to increase the robustness of both experimental and analytical protocols. The experimental protocol is very simple and based on fast-fluidic-exchange in a microfluidic chamber created on top of the formalin-fixed-paraffin-embedded (FFPE) slide by clamping it a silicon chip with a polydimethyl siloxane (PDMS) sealing ring. The image-processing protocol is based on enhancement and subsequent thresholding of the local contrast of the obtained fluorescence image. As a case study, given that the human epidermal growth factor receptor 2 (HER2) protein is often used as a biomarker for breast cancer, we applied our method to HER2+ and HER2- cell lines. We report very fast (5 minutes) immunofluorescence staining of both HER2 and cytokeratin (a marker used to define the tumor region) on FFPE slides. The image-processing program can segment cells correctly and give a cell-based quantitative immunofluorescent signal. With this method, we found a reproducible well-defined separation for the HER2-to-cytokeratin ratio for positive and negative control samples.

Confirmation of immunoglobulin heavy chain rearrangement by polymerase chain reaction using surgically obtained, paraffin-embedded samples to diagnose primary palate mucosa-associated lymphoid tissue lymphoma: A case study

Directory of Open Access Journals (Sweden)

Shigehiro Abe

2015-01-01

Conclusion: We suggest that, if histological examination is ambiguous or fresh material is insufficient, PCR can be performed using paraffin-embedded materials to definitively diagnose low-grade lymphomas, such as MALT lymphoma.

An immunohistochemical and fluorescence in situ hybridization-based comparison between the Oracle HER2 Bond Immunohistochemical System, Dako HercepTest, and Vysis PathVysion HER2 FISH using both commercially validated and modified ASCO/CAP and United Kingdom HER2 IHC scoring guidelines.

LENUS (Irish Health Repository)

O'Grady, Anthony

2010-12-01

Immunohistochemistry (IHC) is used as the frontline assay to determine HER2 status in invasive breast cancer patients. The aim of the study was to compare the performance of the Leica Oracle HER2 Bond IHC System (Oracle) with the current most readily accepted Dako HercepTest (HercepTest), using both commercially validated and modified ASCO\\/CAP and UK HER2 IHC scoring guidelines. A total of 445 breast cancer samples from 3 international clinical HER2 referral centers were stained with the 2 test systems and scored in a blinded fashion by experienced pathologists. The overall agreement between the 2 tests in a 3×3 (negative, equivocal and positive) analysis shows a concordance of 86.7% and 86.3%, respectively when analyzed using commercially validated and modified ASCO\\/CAP and UK HER2 IHC scoring guidelines. There is a good concordance between the Oracle and the HercepTest. The advantages of a complete fully automated test such as the Oracle include standardization of key analytical factors and improved turn around time. The implementation of the modified ASCO\\/CAP and UK HER2 IHC scoring guidelines has minimal effect on either assay interpretation, showing that Oracle can be used as a methodology for accurately determining HER2 IHC status in formalin fixed, paraffin-embedded breast cancer tissue.

An immunohistochemical and fluorescence in situ hybridization-based comparison between the Oracle HER2 Bond Immunohistochemical System, Dako HercepTest, and Vysis PathVysion HER2 FISH using both commercially validated and modified ASCO/CAP and United Kingdom HER2 IHC scoring guidelines.

Science.gov (United States)

O'Grady, Anthony; Allen, David; Happerfield, Lisa; Johnson, Nicola; Provenzano, Elena; Pinder, Sarah E; Tee, Lilian; Gu, Mai; Kay, Elaine W

2010-12-01

Immunohistochemistry (IHC) is used as the frontline assay to determine HER2 status in invasive breast cancer patients. The aim of the study was to compare the performance of the Leica Oracle HER2 Bond IHC System (Oracle) with the current most readily accepted Dako HercepTest (HercepTest), using both commercially validated and modified ASCO/CAP and UK HER2 IHC scoring guidelines. A total of 445 breast cancer samples from 3 international clinical HER2 referral centers were stained with the 2 test systems and scored in a blinded fashion by experienced pathologists. The overall agreement between the 2 tests in a 3×3 (negative, equivocal and positive) analysis shows a concordance of 86.7% and 86.3%, respectively when analyzed using commercially validated and modified ASCO/CAP and UK HER2 IHC scoring guidelines. There is a good concordance between the Oracle and the HercepTest. The advantages of a complete fully automated test such as the Oracle include standardization of key analytical factors and improved turn around time. The implementation of the modified ASCO/CAP and UK HER2 IHC scoring guidelines has minimal effect on either assay interpretation, showing that Oracle can be used as a methodology for accurately determining HER2 IHC status in formalin fixed, paraffin-embedded breast cancer tissue.

Multi-elemental imaging of paraffin-embedded human samples by laser-induced breakdown spectroscopy

Science.gov (United States)

Moncayo, S.; Trichard, F.; Busser, B.; Sabatier-Vincent, M.; Pelascini, F.; Pinel, N.; Templier, I.; Charles, J.; Sancey, L.; Motto-Ros, V.

2017-07-01

Chemical elements play central roles for physiological homeostasis in human cells, and their dysregulation might lead to a certain number of pathologies. Novel imaging techniques that improve the work of pathologists for tissue analysis and diagnostics are continuously sought. We report the use of Laser-Induced Breakdown Spectroscopy (LIBS) to perform multi-elemental images of human paraffin-embedded skin samples on the entire biopsy scale in a complementary and compatible way with microscope histopathological examination. A specific instrumental configuration is proposed in order to detect most of the elements of medical interest (i.e. P, Al, Mg, Na, Zn, Si, Fe, and Cu). As an example of medical application, we selected and analysed skin biopsies, including healthy skin tissue, cutaneous metastasis of melanoma, Merkel-cell carcinoma and squamous cell carcinoma. Clear distinctions in the distribution of chemical elements are observed from the different samples investigated. This study demonstrates the high complementarity of LIBS elemental imaging with conventional histopathology, opening new opportunities for any medical application involving metals.

The effect of tissue decalcification on mRNA retention within bone for in-situ hybridization studies.

Science.gov (United States)

Walsh, L; Freemont, A J; Hoyland, J A

1993-06-01

Tissue decalcification is a routine part of the preparation of bone tissue for histological studies. Although in-situ hybridization has been employed to localize mRNA of collagenous and non-collagenous bone related proteins in skeletal tissue, little is known regarding the effects of decalcifying agents on mRNA retention within tissue. In this study in-situ hybridization using an oligonucleotide probe (i.e. a poly d(T) probe) to detect total messenger RNA has been employed to investigate the effects of the decalcifying agents nitric acid, formic acid and EDTA on mRNA retention compared to undeacalcified tissue. The results show that formalin fixation and EDTA decalcification preserve substantial amounts of mRNA within the tissue. In particular, this study illustrates that it is possible to perform in-situ hybridization on formalin fixed decalcified paraffin embedded tissue.

Ewing's Sarcoma: An Analysis of miRNA Expression Profiles and Target Genes in Paraffin-Embedded Primary Tumor Tissue.

Science.gov (United States)

Parafioriti, Antonina; Bason, Caterina; Armiraglio, Elisabetta; Calciano, Lucia; Daolio, Primo Andrea; Berardocco, Martina; Di Bernardo, Andrea; Colosimo, Alessia; Luksch, Roberto; Berardi, Anna C

2016-04-30

The molecular mechanism responsible for Ewing's Sarcoma (ES) remains largely unknown. MicroRNAs (miRNAs), a class of small non-coding RNAs able to regulate gene expression, are deregulated in tumors and may serve as a tool for diagnosis and prediction. However, the status of miRNAs in ES has not yet been thoroughly investigated. This study compared global miRNAs expression in paraffin-embedded tumor tissue samples from 20 ES patients, affected by primary untreated tumors, with miRNAs expressed in normal human mesenchymal stromal cells (MSCs) by microarray analysis. A miRTarBase database was used to identify the predicted target genes for differentially expressed miRNAs. The miRNAs microarray analysis revealed distinct patterns of miRNAs expression between ES samples and normal MSCs. 58 of the 954 analyzed miRNAs were significantly differentially expressed in ES samples compared to MSCs. Moreover, the qRT-PCR analysis carried out on three selected miRNAs showed that miR-181b, miR-1915 and miR-1275 were significantly aberrantly regulated, confirming the microarray results. Bio-database analysis identified BCL-2 as a bona fide target gene of the miR-21, miR-181a, miR-181b, miR-29a, miR-29b, miR-497, miR-195, miR-let-7a, miR-34a and miR-1915. Using paraffin-embedded tissues from ES patients, this study has identified several potential target miRNAs and one gene that might be considered a novel critical biomarker for ES pathogenesis.

Antibody validation and scoring guidelines for ABCG2 immunohistochemical staining in formalin-fixed paraffin-embedded colon cancer tissue

DEFF Research Database (Denmark)

Cederbye, Camilla Natasha; Palshof, Jesper Andreas; Hansen, Tine Plato

2016-01-01

cancer (CRC), probably because of the use of different antibodies and scoring approaches. In this study, we systematically studied six commercially available anti-ABCG2 antibodies, using cell lines with up-regulation of ABCG2, and selected one antibody for validation in CRC tissue. Furthermore, we...... sections, especially when more than one core was used. In conclusion, here, we provide validated results to guide future studies on the associations between ABCG2 immunoreactivity in tumor cells and the benefits of chemotherapeutic treatment in patients with CRC...

Estrogen receptors in human thyroid gland. An immunohistochemical study

International Nuclear Information System (INIS)

Arain, Shaukat A.; Shah, Munawar H.; Jamal, Qamar; Meo, Sultan A.

2003-01-01

The objective of this study is to determine the estrogen receptors (ER) status (present in the nucleous of cell) in the thyroid gland tissues. For this purpose 50 previously diagnosed cases of various thyroid lesions were selected from the Surgical Pathology Records of Pathology Department, Basic Medical Sciences Institute,Jinnah Postgraduate. Medical Center,Karachi,Pakistan between March and August 2000.The staining was performed on formalin fixed paraffin embeded tissues using monoclonal anti-ER anti-body (clone1D5).Out of 50 cases,8 were noduler goiter,9 cases of adenoma 19 papillary carcinoma, 10 follicular and 4 cases were of medullary carcinoma. Surrounding normal tissue was available in 25 (50%) cases, 4 non-neoplastic and 21 neoplastic lesions.Out of 50 cases ,10(20%) and 40(80%) were females, the youngest patient was a 15-year-old female and the eldest patient was a 56-years-old male. Despite the availability of normal thyroid tissue and a wide range of lesions, none of our cases showed the positive staining. In contrary to many earlier reports by immunohistochemical method using monoclonal antibody (clone1D5) on formalin- fixed praffin-embedded thyroid tissues, the ER is not detectable. The effect of Estrogen on thyroid gland may be indirect one. (author)

The frequency of p53, Ki67, CD99 and Fli-1 protein expression in paraffin-embedded tissue blocks in Ewing’s sarcoma

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Bagheri Hossein-Abadi Z

2011-06-01

Full Text Available "n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Ewing sarcoma family tumors (ESFTs are among the most malignant tumors in children and young adults. ESFTs include Ewing sarcoma (ES and peripheral primitive neuroectodermal tumors (pPNETs. As there seemed to be few studies on the molecular biology of ESFTs, we investigated the frequency of CD99, Ki67, p53 and Fli-1 protein expression in 15 Iranian patients with ESFTs. In addition, the correlation between expression rate of these proteins and various clinical factors, including age, sex and survival was computed."n"nMethods: The expression of the aforesaid proteins was studied by immunohisto-chemistry in formalin-fixed and paraffin-embedded blocks of 15 ESFTs specimens. Stained sections were classified according to the percentage of stained tumor cells."n"nResults: The results showed the membrane expression of CD99 protein in all of the specimens. The nuclear expression of Fli-1 protein was observed in 86.7% and the over-expression of p53 nuclear protein was seen in 53.3% of the specimens. The expression rate of Ki67 protein was 60%. Although a significant correlation was not shown between the expression levels of Ki67, p53 or Fli-1 proteins with age, sex or survival of the patients, there was a significant

Evaluation and Adaptation of a Laboratory-Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens.

Science.gov (United States)

Loudig, Olivier; Wang, Tao; Ye, Kenny; Lin, Juan; Wang, Yihong; Ramnauth, Andrew; Liu, Christina; Stark, Azadeh; Chitale, Dhananjay; Greenlee, Robert; Multerer, Deborah; Honda, Stacey; Daida, Yihe; Spencer Feigelson, Heather; Glass, Andrew; Couch, Fergus J; Rohan, Thomas; Ben-Dov, Iddo Z

2017-03-14

Formalin-fixed paraffin-embedded (FFPE) specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS) specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p) differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases) and women who did not develop invasive breast cancer within the same time interval (control). Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens.

Use of a solid-phase radioimmunoassay and formalin-fixed whole bacterial antigen in the detection of antigen-specific immunoglobulin in prostatic fluid.

OpenAIRE

Shortliffe, L M; Wehner, N; Stamey, T A

1981-01-01

The prostatic fluid of two patients with Escherichia coli bacterial prostatitis was analyzed for evidence of a local immune response to bacterial infection. A solid-phase radioimmunoassay was modified to measure the immunoglobulin (Ig)A and IgG antigen-specific antibody responses to infecting bacteria in serum and prostatic fluid from patient. Formalin-fixed whole E. coli were used as antigen. In one patient with acute E. coli prostatic infection, measurements of antigen-specific antibody con...

Transparency-enhancing technology allows three-dimensional assessment of gastrointestinal mucosa: A porcine model.

Science.gov (United States)

Mizutani, Hiroya; Ono, Satoshi; Ushiku, Tetsuo; Kudo, Yotaro; Ikemura, Masako; Kageyama, Natsuko; Yamamichi, Nobutake; Fujishiro, Mitsuhiro; Someya, Takao; Fukayama, Masashi; Koike, Kazuhiko; Onodera, Hiroshi

2018-02-01

Although high-resolution three-dimensional imaging of endoscopically resected gastrointestinal specimens can help elucidating morphological features of gastrointestinal mucosa or tumor, there are no established methods to achieve this without breaking specimens apart. We evaluated the utility of transparency-enhancing technology for three-dimensional assessment of gastrointestinal mucosa in porcine models. Esophagus, stomach, and colon mucosa samples obtained from a sacrificed swine were formalin-fixed and paraffin-embedded, and subsequently deparaffinized for analysis. The samples were fluorescently stained, optically cleared using transparency-enhancing technology: ilLUmination of Cleared organs to IDentify target molecules method (LUCID), and visualized using laser scanning microscopy. After observation, all specimens were paraffin-embedded again and evaluated by conventional histopathological assessment to measure the impact of transparency-enhancing procedures. As a result, microscopic observation revealed horizontal section views of mucosa at deeper levels and enabled the three-dimensional image reconstruction of glandular and vascular structures. Besides, paraffin-embedded specimens after transparency-enhancing procedures were all assessed appropriately by conventional histopathological staining. These results suggest that transparency-enhancing technology may be feasible for clinical application and enable the three-dimensional structural analysis of endoscopic resected specimen non-destructively. Although there remain many limitations or problems to be solved, this promising technology might represent a novel histopathological method for evaluating gastrointestinal cancers. © 2018 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

Preparation of tissue samples for X-ray fluorescence microscopy

International Nuclear Information System (INIS)

Chwiej, Joanna; Szczerbowska-Boruchowska, Magdalena; Lankosz, Marek; Wojcik, Slawomir; Falkenberg, Gerald; Stegowski, Zdzislaw; Setkowicz, Zuzanna

2005-01-01

As is well-known, trace elements, especially metals, play an important role in the pathogenesis of many disorders. The topographic and quantitative elemental analysis of pathologically changed tissues may shed some new light on processes leading to the degeneration of cells in the case of selected diseases. An ideal and powerful tool for such purpose is the Synchrotron Microbeam X-ray Fluorescence technique. It enables the carrying out of investigations of the elemental composition of tissues even at the single cell level. The tissue samples for histopathological investigations are routinely fixed and embedded in paraffin. The authors try to verify the usefulness of such prepared tissue sections for elemental analysis with the use of X-ray fluorescence microscopy. Studies were performed on rat brain samples. Changes in elemental composition caused by fixation in formalin or paraformaldehyde and embedding in paraffin were examined. Measurements were carried out at the bending magnet beamline L of the Hamburger Synchrotronstrahlungslabor HASYLAB in Hamburg. The decrease in mass per unit area of K, Br and the increase in P, S, Fe, Cu and Zn in the tissue were observed as a result of the fixation. For the samples embedded in paraffin, a lower level of most elements was observed. Additionally, for these samples, changes in the composition of some elements were not uniform for different analyzed areas of rat brain

High-risk human papilloma virus in archival tissues of oral pathosis and normal oral mucosa

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Raghu Dhanapal

2015-01-01

Full Text Available Objectives: Oral cancer ranks third among all cancers in the Indian population. Human papilloma virus (HPV plays a significant role in oral carcinogenesis. Population-based subtype variations are present in the HPV prevalence. This study gives an emphasis on the parameters to be considered in formalin fixed paraffin embedded tissues for polymerase chain reaction (PCR-based research work. Materials and Methods: Cross-sectional study on archival paraffin-embedded tissue samples of oral squamous cell carcinoma (OSCC, epithelial dysplasia, and normal oral mucosa surrounding impacted tooth was amplified by PCR for the E6 gene of HPV type 16 and E1 gene of HPV type 18. Results: HPV 18 was positive in three OSCC cases. There was no statistically significant association of the positivity of HPV with the age, gender or habit. The HPV positive patients had a tobacco habit and were of a younger age group. Conclusion: The presence of HPV in carcinomatous tissue highlights the possible role of HPV in carcinogenesis and archival paraffin embedded tissue specimen can be used for this analysis. Recent studies on genomic analyses have highlighted that the HPV positive tumors are a separate subgroup based on genomic sequencing. The results of a larger retrospective study will help further in our understanding of the role of HPV in carcinogenesis, this study could form the baseline for such follow-up studies.

High-risk human papilloma virus in archival tissues of oral pathosis and normal oral mucosa.

Science.gov (United States)

Dhanapal, Raghu; Ranganathan, K; Kondaiah, Paturu; Devi, R Uma; Joshua, Elizabeth; Saraswathi, T R

2015-01-01

Oral cancer ranks third among all cancers in the Indian population. Human papilloma virus (HPV) plays a significant role in oral carcinogenesis. Population-based subtype variations are present in the HPV prevalence. This study gives an emphasis on the parameters to be considered in formalin fixed paraffin embedded tissues for polymerase chain reaction (PCR)-based research work. Cross-sectional study on archival paraffin-embedded tissue samples of oral squamous cell carcinoma (OSCC), epithelial dysplasia, and normal oral mucosa surrounding impacted tooth was amplified by PCR for the E6 gene of HPV type 16 and E1 gene of HPV type 18. HPV 18 was positive in three OSCC cases. There was no statistically significant association of the positivity of HPV with the age, gender or habit. The HPV positive patients had a tobacco habit and were of a younger age group. The presence of HPV in carcinomatous tissue highlights the possible role of HPV in carcinogenesis and archival paraffin embedded tissue specimen can be used for this analysis. Recent studies on genomic analyses have highlighted that the HPV positive tumors are a separate subgroup based on genomic sequencing. The results of a larger retrospective study will help further in our understanding of the role of HPV in carcinogenesis, this study could form the baseline for such follow-up studies.

Preparation of tissue samples for X-ray fluorescence microscopy

Energy Technology Data Exchange (ETDEWEB)

Chwiej, Joanna [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, 30-059 Cracow (Poland)]. E-mail: jchwiej@novell.ftj.agh.edu.pl; Szczerbowska-Boruchowska, Magdalena [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, 30-059 Cracow (Poland); Lankosz, Marek [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, 30-059 Cracow (Poland); Wojcik, Slawomir [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, 30-059 Cracow (Poland); Falkenberg, Gerald [Hamburger Synchrotronstrahlungslabor at Deutsches Elektronen-Synchrotron, Notkestr. 85, Hamburg (Germany); Stegowski, Zdzislaw [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, 30-059 Cracow (Poland); Setkowicz, Zuzanna [Department of Neuroanatomy, Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060 Cracow (Poland)

2005-12-15

As is well-known, trace elements, especially metals, play an important role in the pathogenesis of many disorders. The topographic and quantitative elemental analysis of pathologically changed tissues may shed some new light on processes leading to the degeneration of cells in the case of selected diseases. An ideal and powerful tool for such purpose is the Synchrotron Microbeam X-ray Fluorescence technique. It enables the carrying out of investigations of the elemental composition of tissues even at the single cell level. The tissue samples for histopathological investigations are routinely fixed and embedded in paraffin. The authors try to verify the usefulness of such prepared tissue sections for elemental analysis with the use of X-ray fluorescence microscopy. Studies were performed on rat brain samples. Changes in elemental composition caused by fixation in formalin or paraformaldehyde and embedding in paraffin were examined. Measurements were carried out at the bending magnet beamline L of the Hamburger Synchrotronstrahlungslabor HASYLAB in Hamburg. The decrease in mass per unit area of K, Br and the increase in P, S, Fe, Cu and Zn in the tissue were observed as a result of the fixation. For the samples embedded in paraffin, a lower level of most elements was observed. Additionally, for these samples, changes in the composition of some elements were not uniform for different analyzed areas of rat brain.

An immunohistochemical approach to differentiate hepatic lipidosis from hepatic phospholipidosis in rats.

Science.gov (United States)

Obert, Leslie A; Sobocinski, Gregg P; Bobrowski, Walter F; Metz, Alan L; Rolsma, Mark D; Altrogge, Douglas M; Dunstan, Robert W

2007-08-01

Hepatocellular vacuolation can be a diagnostic challenge since cytoplasmic accumulations of various substances (lipid, water, phospholipids, glycogen, and plasma) can have a similar morphology. Cytoplasmic accumulation of phospholipids following administration of cationic amphiphilic drugs (CAD) can be particularly difficult to differentiate from nonphosphorylated lipid accumulations at the light microscopic level. Histochemical methods (Sudan Black, Oil Red-O, Nile Blue, etc.) can be used to identify both nonphosphorylated and/or phosphorylated lipid accumulations, but these techniques require non-paraffin-embedded tissue and are only moderately sensitive. Thus, electron microscopy is often utilized to achieve a definitive diagnosis based upon the characteristic morphologic features of phospholipid accumulations; however, this is a low throughput and labor intense procedure. In this report, we describe the use of immunohistochemical staining for LAMP-2 (a lysosome-associated protein) and adipophilin (a protein that forms the membrane around non-lysosomal lipid droplets) to differentiate phospholipidosis and lipidosis, respectively in the livers of rats. This staining procedure can be performed on formalin-fixed paraffin embedded tissues, is more sensitive than histochemistry, and easier to perform than ultrastructural evaluation.

Pathology and diagnosis of avian bornavirus infection in wild Canada geese (Branta canadensis), trumpeter swans (Cygnus buccinator) and mute swans (Cygnus olor) in Canada: a retrospective study.

Science.gov (United States)

Delnatte, Pauline; Ojkic, Davor; Delay, Josepha; Campbell, Doug; Crawshaw, Graham; Smith, Dale A

2013-04-01

Nine hundred and fifty-five pathology cases collected in Ontario between 1992 and 2011 from wild free-ranging Canada geese, trumpeter swans and mute swans were retrospectively evaluated for the pathology associated with avian bornavirus (ABV) infection. Cases were selected based on the presence of upper gastrointestinal impaction, central nervous system histopathology or clinical history suggestive of ABV infection. The proportion of birds meeting at least one of these criteria was significantly higher at the Toronto Zoo (30/132) than elsewhere in Ontario (21/823). Central, peripheral and autonomic nervous tissues were examined for the presence of lymphocytes and plasma cells on histopathology. The presence of virus was assessed by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) on frozen brains and on formalin-fixed paraffin-embedded tissues. Among selected cases, 86.3% (44/51) were considered positive on histopathology, 56.8% (29/51) were positive by immunohistochemistry, and RT-PCR was positive on 88.2% (15/17) of the frozen brains and 78.4% (40/51) of the formalin-fixed paraffin-embedded samples. Histopathological lesions included gliosis and lymphoplasmacytic perivascular cuffing in brain (97.7%), spinal cord (50%), peripheral nerves (55.5%) and myenteric ganglia or nerves (62.8%), resembling lesions described in parrots affected with proventricular dilatation disease. Partial amino acid sequences of the nucleocapsid gene from seven geese were 100% identical amongst themselves and 98.1 to 100% identical to the waterfowl sequences recently described in the USA. Although ABV has been identified in apparently healthy geese, our study confirmed that ABV can also be associated with significant disease in wild waterfowl species.

MVP immunohistochemistry is a useful adjunct in distinguishing leiomyosarcoma from leiomyoma and leiomyoma with bizarre nuclei.

Science.gov (United States)

Lintel, Nicholas J; Luebker, Stephen A; Lele, Subodh M; Koepsell, Scott A

2018-03-01

Morphologically, distinguishing between leiomyoma (LM) and leiomyosarcoma (LMS) is not always straightforward, especially with benign variants such as bizarre leiomyoma (BLM). To identify potential markers of malignancy in uterine smooth muscle tumors, proteomic studies were performed followed by assessment of protein expression by immunohistochemistry. Archival formalin-fixed, paraffin-embedded tissues from tumors (n = 23) diagnosed as LM, BLM, and LMS (using published criteria) were selected for the study. Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry was applied to pooled samples of formalin-fixed, paraffin-embedded LM and LMS tumor tissue to assay the relative protein quantities and look for expression patterns differentiating the 2 tumor types. A total of 592 proteins were quantified, and 10 proteins were differentially expressed between LM and LMS. Select proteins were chosen for evaluation by immunohistochemistry (IHC) based on antibody availability and biologic relevance in the literature. IHC was performed on a tissue microarray, and intensity was evaluated using imaging software. Major vault protein (MVP) and catechol O-methyltransferase had 3.05 and 13.94 times higher expression in LMS relative to LM by sequential window acquisition of all theoretical fragment ion spectra mass spectrometry, respectively. By IHC, MVP (clone 1014; Santa Cruz Biotechnology, Dallas, TX) was found to be 50% sensitive and 100% specific when comparing LMS to LM. Catechol O-methyltransferase (clone FL-271; Santa Cruz Biotechnology) had a sensitivity of 38% and a specificity of 88%. Six of 7 BLM had expression of MVP similar to LM. Immunohistochemical staining for MVP is a useful adjunct in distinguishing LMS from LM and BLM in difficult cases. Copyright © 2018. Published by Elsevier Inc.

A Unique Immunofluorescence Protocol to Detect Protein Expression in Vascular Tissues: Tacking a Long Standing Pathological Hitch

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Puneet GANDHI

2018-01-01

Full Text Available Objective: Autofluorescence induced interference is one of the major drawbacks in immunofluorescence analysis of formalin-fixed paraffin-embedded tissues, as it decreases the signal-to-noise ratio of specific labeling. Apart from aldehyde-fixation induced artifacts; collagen and elastin, red blood cells and endogenous fluorescent pigment lipofuscin are prime sources of autofluorescence in vascular and aging tissues. We describe herein, an optimized indirect-immunofluorescence method for archival formalin-fixed paraffin-embedded tissues tissues and cryo sections, using a combination of 3-reagents in a specific order, to achieve optimal fluorescence signals and imaging. Material and Method: Human telomerase reverse transcriptase, a protein implicated as a proliferation marker, was chosen relevant to its expression in solid tumors along with 3 other intracellular proteins exhibiting nuclear and/or cytoplasmic expression. Staining was performed on 10 glioma tissue sections along with 5 of their cryo sections, 5 sections each of hepatocellular, lung, papillary-thyroid and renal cell carcinoma, with 10 non-malignant brain tissue samples serving as control. Specimens were imaged using epifluorescence microscopy, followed by software-based quantification of fluorescence signals for statistical analysis and validation. Results: We observed that the combined application of sodium-borohydride followed by crystal violet before antigen retrieval and a Sudan black B treatment after secondary antibody application proved to be most efficacious for masking autofluorescence/non-specific background in vascular tissues. Conclusion: This unique trio-methodology provides quantifiable observations with maximized fluorescence signal intensity of the target protein for longer retention time of the signal even after prolonged storage. The results can be extrapolated to other human tissues for different protein targets.

HER2 testing on core needle biopsy specimens from primary breast cancers: interobserver reproducibility and concordance with surgically resected specimens

International Nuclear Information System (INIS)

Tsuda, Hitoshi; Kurosumi, Masafumi; Umemura, Shinobu; Yamamoto, Sohei; Kobayashi, Takayuki; Osamura, Robert Yoshiyuki

2010-01-01

Accurate evaluation of human epidermal growth factor receptor type-2 (HER2) status based on core needle biopsy (CNB) specimens is mandatory for identification of patients with primary breast cancer who will benefit from primary systemic therapy with trastuzumab. The aim of the present study was to validate the application of HER2 testing with CNB specimens from primary breast cancers in terms of interobserver reproducibility and comparison with surgically resected specimens. A total of 100 pairs of archival formalin-fixed paraffin-embedded CNB and surgically resected specimens of invasive breast carcinomas were cut into sections. All 100 paired sections were subjected to HER2 testing by immunohistochemistry (IHC) and 27 paired sections were subjected to that by fluorescence in situ hybridization (FISH), the results being evaluated by three and two observers, respectively. Interobserver agreement levels in terms of judgment and the concordance of consensus scores between CNB samples and the corresponding surgically resected specimens were estimated as the percentage agreement and κ statistic. In CNB specimens, the percentage interobserver agreement of HER2 scoring by IHC was 76% (κ = 0.71) for 3 × 3 categories (0-1+ versus 2+ versus 3+) and 90% (κ = 0.80) for 2 × 2 categories (0-2+ versus 3+). These levels were close to the corresponding ones for the surgically resected specimens: 80% (κ = 0.77) for 3 × 3 categories and 92% (κ = 0.88) for 2 × 2 categories. Concordance of consensus for HER2 scores determined by IHC between CNB and the corresponding surgical specimens was 87% (κ = 0.77) for 3 × 3 categories, and 94% (κ = 0.83) for 2 × 2 categories. Among the 13 tumors showing discordance in the mean IHC scores between the CNB and surgical specimens, the results of consensus for FISH results were concordant in 11. The rate of successful FISH analysis and the FISH positivity rate in cases with a HER2 IHC score of 2+ differed among specimens processed at

HER2 testing on core needle biopsy specimens from primary breast cancers: interobserver reproducibility and concordance with surgically resected specimens

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Yamamoto Sohei

2010-10-01

Full Text Available Abstract Background Accurate evaluation of human epidermal growth factor receptor type-2 (HER2 status based on core needle biopsy (CNB specimens is mandatory for identification of patients with primary breast cancer who will benefit from primary systemic therapy with trastuzumab. The aim of the present study was to validate the application of HER2 testing with CNB specimens from primary breast cancers in terms of interobserver reproducibility and comparison with surgically resected specimens. Methods A total of 100 pairs of archival formalin-fixed paraffin-embedded CNB and surgically resected specimens of invasive breast carcinomas were cut into sections. All 100 paired sections were subjected to HER2 testing by immunohistochemistry (IHC and 27 paired sections were subjected to that by fluorescence in situ hybridization (FISH, the results being evaluated by three and two observers, respectively. Interobserver agreement levels in terms of judgment and the concordance of consensus scores between CNB samples and the corresponding surgically resected specimens were estimated as the percentage agreement and κ statistic. Results In CNB specimens, the percentage interobserver agreement of HER2 scoring by IHC was 76% (κ = 0.71 for 3 × 3 categories (0-1+ versus 2+ versus 3+ and 90% (κ = 0.80 for 2 × 2 categories (0-2+ versus 3+. These levels were close to the corresponding ones for the surgically resected specimens: 80% (κ = 0.77 for 3 × 3 categories and 92% (κ = 0.88 for 2 × 2 categories. Concordance of consensus for HER2 scores determined by IHC between CNB and the corresponding surgical specimens was 87% (κ = 0.77 for 3 × 3 categories, and 94% (κ = 0.83 for 2 × 2 categories. Among the 13 tumors showing discordance in the mean IHC scores between the CNB and surgical specimens, the results of consensus for FISH results were concordant in 11. The rate of successful FISH analysis and the FISH positivity rate in cases with a HER2 IHC score of

Comparison of Different Double Immunostaining Protocols for Paraffin Embedded Liver Tissue

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Alexander Schütz

1999-01-01

Full Text Available Most of the double immunostaining protocols that have been introduced so far have been developed for application on fresh frozen material or based on different species antibodies. In liver tissue, general problems of double immunostaining techniques are further complicated by tissue‐specific difficulties, such as necrosis or high intracellular protein content. To assess a reliable double immunostaining protocol for archived, paraffin embedded liver tissue, different protocols based on the use of same species primary antibodies were evaluated in terms of sensitivity, specificity and non‐specific background staining in pathological liver specimens. We compared peroxidase–anti‐peroxidase, alkaline phosphatase–anti‐alkaline phosphatase (PAP/APAP, labelled‐avidin–biotin (LAB/LAB and digoxigenin–anti‐digoxigenin (dig–a‐dig/PAP techniques using different cytokeratin antibodies and an antibody against PCNA. Comparison of the double immunostaining techniques revealed a high sensitivity and specificity in all procedures. Sections, which were stained employing PAP/APAP‐technique, displayed a higher background staining compared to sections which were treated with the LAB/LAB or dig–a‐dig/PAP protocol. In contrast to the dig–a‐dig/PAP protocol, the LAB/LAB technique provides a better time/cost relationship. Therefore, we would like to recommend a modified LAB/LAB protocol for simultaneous detection of different antigens in archived liver tissue.

Molecular screening by polymerase chain reaction detects panleukopenia virus DNA in formalin-fixed hearts from cats with idiopathic cardiomyopathy and myocarditis.

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Meurs, K M; Fox, P R; Magnon, A L; Liu, S; Towbin, J A

2000-01-01

Viral myocarditis has been suggested as an etiology for cardiomyopathy in several mammalian species. Myocarditis and idiopathic cardiomyopathy have been reported in the domestic cat, although a viral etiology has not been demonstrated. Because of the continuing interest in the potential relationship between viral myocarditis and cardiomyopathy, we evaluated hearts from cats with spontaneous, idiopathic cardiomyopathy for viral genomic material within myocytes by polymerase chain reaction, and for the presence of myocarditis by light microscopy. Thirty-one (31) formalin-fixed hearts from domestic cats who died of idiopathic cardiomyopathy were randomly selected from pathology archives. Seventeen (17) formalin-fixed hearts from healthy cats were similarly selected as normal controls. The polymerase chain reaction (PCR) was used to evaluate myocardial tissue for the presence of viral genome from feline panleukopenia virus, herpes virus, calici virus, and corona virus. Hearts were examined using light microscopy for histologic evidence of myocarditis according to the Dallas criteria. Panleukopenia virus was identified by PCR in 10 of 31 cats with cardiomyopathy but in none of the controls. Neither cardiomyopathic or control cats tested positive by PCR for herpes virus, calici virus, and corona virus. Myocarditis was detected by histologic examination in 18 of 31 cardiomyopathic cats and in none of 17 control cats. Myocarditis and or feline panleukopenia virus genome was detected in felines with idiopathic hypertrophic, dilated, and restrictive cardiomyopathy, suggesting a possible role of viral infection and inflammation in the pathogenesis of cardiomyopathy in this species.

Zinc presence in invasive ductal carcinoma of the breast and its correlation with oestrogen receptor status

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Farquharson, M J [Department of Medical Physics and Applied Radiation Sciences, McMaster University, 1280 Main St W Hamilton, Ontario, L8S 4L8 (Canada); Al-Ebraheem, A [Department of Radiography, City Community and Health Sciences, City University, London, EC1V 0HB (United Kingdom); Geraki, K [Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxon, OX11 0DE (United Kingdom); Leek, R; Jubb, A; Harris, A L [Cancer Research UK, Oxford Cancer Centre, Molecular Oncology Laboratories, University of Oxford, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, 0X3 9DS (United Kingdom)], E-mail: farquhm@mcmaster.ca

2009-07-07

Zinc is known to play an important role in many cellular processes, and the levels of zinc are controlled by specific transporters from the ZIP (SLC39A) influx transporter group and the ZnT (SLC30A) efflux transporter group. The distribution of zinc was measured in 59 samples of invasive ductal carcinoma of breast using synchrotron radiation micro probe x-ray fluorescence facilities. The samples were formalin fixed paraffin embedded tissue micro arrays (TMAs) enabling a high throughput of samples and allowing us to correlate the distribution of trace metals with tumour cell distribution and, for the first time, important biological variables. The samples were divided into two classes, 34 oestrogen receptor positive (ER+ve) and 25 oestrogen receptor negative (ER-ve) based on quantitative immunohistochemistry assessment. The overall levels of zinc (i.e. in tumour and surrounding tissue) in the ER+ve samples were on average 60% higher than those in the ER-ve samples. The zinc levels were higher in the ER+ve tumour areas compared to the ER-ve tumour areas with the mean levels in the ER+ve samples being approximately 80% higher than the mean ER-ve levels. However, the non-tumour tissue regions of the samples contained on average the same levels of zinc in both types of breast cancers. The relative levels of zinc in tumour areas of the tissue were compared with levels in areas of non-tumour surrounding tissue. There was a significant increase in zinc in the tumour regions of the ER+ve samples compared to the surrounding regions (P < 0.001) and a non-significant increase in the ER-ve samples. When comparing the increase in zinc in the tumour regions expressed as a percentage of the surrounding non-tumour tissue zinc level in the same sample, a significant difference between the ER+ve and ER-ve samples was found (P < 0.01)

Zinc presence in invasive ductal carcinoma of the breast and its correlation with oestrogen receptor status

International Nuclear Information System (INIS)

Farquharson, M J; Al-Ebraheem, A; Geraki, K; Leek, R; Jubb, A; Harris, A L

2009-01-01

Zinc is known to play an important role in many cellular processes, and the levels of zinc are controlled by specific transporters from the ZIP (SLC39A) influx transporter group and the ZnT (SLC30A) efflux transporter group. The distribution of zinc was measured in 59 samples of invasive ductal carcinoma of breast using synchrotron radiation micro probe x-ray fluorescence facilities. The samples were formalin fixed paraffin embedded tissue micro arrays (TMAs) enabling a high throughput of samples and allowing us to correlate the distribution of trace metals with tumour cell distribution and, for the first time, important biological variables. The samples were divided into two classes, 34 oestrogen receptor positive (ER+ve) and 25 oestrogen receptor negative (ER-ve) based on quantitative immunohistochemistry assessment. The overall levels of zinc (i.e. in tumour and surrounding tissue) in the ER+ve samples were on average 60% higher than those in the ER-ve samples. The zinc levels were higher in the ER+ve tumour areas compared to the ER-ve tumour areas with the mean levels in the ER+ve samples being approximately 80% higher than the mean ER-ve levels. However, the non-tumour tissue regions of the samples contained on average the same levels of zinc in both types of breast cancers. The relative levels of zinc in tumour areas of the tissue were compared with levels in areas of non-tumour surrounding tissue. There was a significant increase in zinc in the tumour regions of the ER+ve samples compared to the surrounding regions (P < 0.001) and a non-significant increase in the ER-ve samples. When comparing the increase in zinc in the tumour regions expressed as a percentage of the surrounding non-tumour tissue zinc level in the same sample, a significant difference between the ER+ve and ER-ve samples was found (P < 0.01).

Her-2/neu expression in node-negative breast cancer: direct tissue quantitation by computerized image analysis and association of overexpression with increased risk of recurrent disease.

Science.gov (United States)

Press, M F; Pike, M C; Chazin, V R; Hung, G; Udove, J A; Markowicz, M; Danyluk, J; Godolphin, W; Sliwkowski, M; Akita, R

1993-10-15

The HER-2/neu proto-oncogene (also known as c-erb B-2) is homologous with, but distinct from, the epidermal growth factor receptor. Amplification of this gene in node-positive breast cancers has been shown to correlate with both earlier relapse and shorter overall survival. In node-negative breast cancer patients, the subgroup for which accurate prognostic data could make a significant contribution to treatment decisions, the prognostic utility of HER-2/neu amplification and/or overexpression has been controversial. The purpose of this report is to address the issues surrounding this controversy and to evaluate the prognostic utility of overexpression in a carefully followed group of patients using appropriately characterized reagents and methods. In this report we present data from a study of HER-2/neu expression designed specifically to test whether or not overexpression is associated with an increased risk of recurrence in node-negative breast cancers. From a cohort of 704 women with node-negative breast cancer who experienced recurrent disease (relapsed cases) 105 were matched with 105 women with no recurrence (disease-free controls) after the equivalent follow-up period. Immunohistochemistry was used to assess HER-2/neu expression in archival tissue blocks from both relapsed cases and their matched disease-free controls. Importantly, a series of molecularly characterized breast cancer specimens were used to confirm that the antibody used was of sufficient sensitivity and specificity to identify those cancers overexpressing the HER-2/neu protein in this formalin-fixed, paraffin-embedded tissue cohort. In addition, a quantitative approach was developed to more accurately assess the amount of HER-2/neu protein identified by immunostaining tumor tissue. This was done using a purified HER-2/neu protein synthesized in a bacterial expression vector and protein lysates derived from a series of cell lines, engineered to express a defined range of HER-2/neu oncoprotein

Design and Testing of Digitally Manufactured Paraffin Acrylonitrile-Butadiene-Styrene Hybrid Rocket Motors

OpenAIRE

McCulley, Jonathan M.

2013-01-01

This research investigates the application of additive manufacturing techniques for fabricating hybrid rocket fuel grains composed of porous Acrylonitrile-butadiene-styrene impregnated with paraffin wax. The digitally manufactured ABS substrate provides mechanical support for the paraffin fuel material and serves as an additional fuel component. The embedded paraffin provides an enhanced fuel regression rate while having no detrimental effect on the thermodynamic burn properties of the fuel g...

Sensitivity of HER-2/neu antibodies in archival tissue samples: potential source of error in immunohistochemical studies of oncogene expression.

Science.gov (United States)

Press, M F; Hung, G; Godolphin, W; Slamon, D J

1994-05-15

HER-2/neu oncogene amplification and overexpression of breast cancer tissue has been correlated with poor prognosis in women with both node-positive and node-negative disease. However, several studies have not confirmed this association. Review of these studies reveals the presence of considerable methodological variability including differences in study size, follow-up time, techniques and reagents. The majority of papers with clinical follow-up information are immunohistochemical studies using archival, paraffin-embedded breast cancers, and a variety of HER-2/neu antibodies have been used in these studies. Very little information, however, is available about the ability of the antibodies to detect overexpression following tissue processing for paraffin-embedding. Therefore, a series of antibodies, reported in the literature or commercially available, were evaluated to assess their sensitivity and specificity as immunohistochemical reagents. Paraffin-embedded samples of 187 breast cancers, previously characterized as frozen specimens for HER-2/neu amplification by Southern blot and for overexpression by Northern blot, Western blot, and immunohistochemistry, were used. Two multitumor paraffin-embedded tissue blocks were prepared from the previously analyzed breast cancers as a panel of cases to test a series of previously studied and/or commercially available anti-HER-2/neu antibodies. Immunohistochemical staining results obtained with 7 polyclonal and 21 monoclonal antibodies in sections from paraffin-embedded blocks of these breast cancers were compared. The ability of these antibodies to detect overexpression was extremely variable, providing an important explantation for the variable overexpression rate reported in the literature.

A rapid method of reprocessing for electronic microscopy of cut histological in paraffin

International Nuclear Information System (INIS)

Hernandez Chavarri, F.; Vargas Montero, M.; Rivera, P.; Carranza, A.

2000-01-01

A simple and rapid method is described for re-processing of light microscopy paraffin sections to observe they under transmission electron microscopy (TEM) and scanning electron microscopy (SEM) The paraffin-embedded tissue is sectioned and deparaffinized in toluene; then exposed to osmium vapor under microwave irradiation using a domestic microwave oven. The tissues were embedded in epoxy resin, polymerized and ultrathin sectioned. The method requires a relatively short time (about 30 minutes for TEM and 15 for SEM), and produces a reasonable quality of the ultrastructure for diagnostic purposes. (Author) [es

Ewing’s Sarcoma: An Analysis of miRNA Expression Profiles and Target Genes in Paraffin-Embedded Primary Tumor Tissue

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Antonina Parafioriti

2016-04-01

Full Text Available The molecular mechanism responsible for Ewing’s Sarcoma (ES remains largely unknown. MicroRNAs (miRNAs, a class of small non-coding RNAs able to regulate gene expression, are deregulated in tumors and may serve as a tool for diagnosis and prediction. However, the status of miRNAs in ES has not yet been thoroughly investigated. This study compared global miRNAs expression in paraffin-embedded tumor tissue samples from 20 ES patients, affected by primary untreated tumors, with miRNAs expressed in normal human mesenchymal stromal cells (MSCs by microarray analysis. A miRTarBase database was used to identify the predicted target genes for differentially expressed miRNAs. The miRNAs microarray analysis revealed distinct patterns of miRNAs expression between ES samples and normal MSCs. 58 of the 954 analyzed miRNAs were significantly differentially expressed in ES samples compared to MSCs. Moreover, the qRT-PCR analysis carried out on three selected miRNAs showed that miR-181b, miR-1915 and miR-1275 were significantly aberrantly regulated, confirming the microarray results. Bio-database analysis identified BCL-2 as a bona fide target gene of the miR-21, miR-181a, miR-181b, miR-29a, miR-29b, miR-497, miR-195, miR-let-7a, miR-34a and miR-1915. Using paraffin-embedded tissues from ES patients, this study has identified several potential target miRNAs and one gene that might be considered a novel critical biomarker for ES pathogenesis.

Phenotypic characterisation of cell populations in the brains of horses experimentally infected with West Nile virus.

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Delcambre, G H; Liu, J; Streit, W J; Shaw, G P J; Vallario, K; Herrington, J; Wenzlow, N; Barr, K L; Long, M T

2017-11-01

West Nile virus (WNV), a mosquito borne member of the Flaviviridae, is one of the most commonly diagnosed agents of viral encephalitis in horses and people worldwide. A cassette of markers for formalin-fixed paraffin-embedded tissue and an archive of tissues from experimental infections in the horse were used to investigate the equine neuroimmune response to WNV meningoencephalomyelitis to phenotype the early response to WNV infection in the horse. Quantitative analysis using archived tissue from experimentally infected horses. The thalamus and hindbrain from 2 groups of 6 horses were compared and consisted of a culture positive tissues from WNV experimentally horses, in the other, normal horses. Formalin-fixed paraffin-embedded tissue from the thalamus and hindbrain were immunolabeled for microglia, astrocytes, B cells, macrophages/neutrophils, CD3 + T cells. Fresh frozen tissues were immunolabeled for CD4 + and CD8 + T lymphocyte cell markers. Cell counts were obtained using a computer software program. Differences, after meeting assumptions of abnormality, were computed using a general linear model with a Tukey test (Phorses, Iba-1 + microglia, CD3 + T lymphocyte and MAC387 + macrophage staining were significantly increased. The T cell response for the WNV-challenged horses was mixed, composed of CD4 + and CD8 + T lymphocytes. A limited astrocyte response was also observed in WNV-challenged horses, and MAC387 + and B cells were the least abundant cell populations. The results of this study were limited by a single collection time post-infection. Furthermore, a comprehensive analysis of cellular phenotypes is needed for naturally infected horses. Unfortunately, in clinical horses, there is high variability of sampling in terms of days post-infection and tissue handling. The data show that WNV-challenged horses recruit a mixed T cell population at the onset of neurologic disease. © 2017 EVJ Ltd.

Real-time PCR improves Helicobacter pylori detection in patients with peptic ulcer bleeding.

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María José Ramírez-Lázaro

Full Text Available BACKGROUND AND AIMS: Histological and rapid urease tests to detect H. pylori in biopsy specimens obtained during peptic ulcer bleeding episodes (PUB often produce false-negative results. We aimed to examine whether immunohistochemistry and real-time PCR can improve the sensitivity of these biopsies. PATIENTS AND METHODS: We selected 52 histology-negative formalin-fixed paraffin-embedded biopsy specimens obtained during PUB episodes. Additional tests showed 10 were true negatives and 42 were false negatives. We also selected 17 histology-positive biopsy specimens obtained during PUB to use as controls. We performed immunohistochemistry staining and real-time PCR for 16S rRNA, ureA, and 23S rRNA for H. pylori genes on all specimens. RESULTS: All controls were positive for H. pylori on all PCR assays and immunohistochemical staining. Regarding the 52 initially negative biopsies, all PCR tests were significantly more sensitive than immunohistochemical staining (p<0.01. Sensitivity and specificity were 55% and 80% for 16S rRNA PCR, 43% and 90% for ureA PCR, 41% and 80% for 23S rRNA PCR, and 7% and 100% for immunohistochemical staining, respectively. Combined analysis of PCR assays for two genes were significantly more sensitive than ureA or 23S rRNA PCR tests alone (p<0.05 and marginally better than 16S rRNA PCR alone. The best combination was 16S rRNA+ureA, with a sensitivity of 64% and a specificity of 80%. CONCLUSIONS: Real-time PCR improves the detection of H. pylori infection in histology-negative formalin-fixed paraffin-embedded biopsy samples obtained during PUB episodes. The low reported prevalence of H. pylori in PUB may be due to the failure of conventional tests to detect infection.

Concordance of DNA methylation profiles between breast core biopsy and surgical excision specimens containing ductal carcinoma in situ (DCIS).

Science.gov (United States)

Chen, Youdinghuan; Marotti, Jonathan D; Jenson, Erik G; Onega, Tracy L; Johnson, Kevin C; Christensen, Brock C

2017-08-01

The utility and reliability of assessing molecular biomarkers for translational applications on pre-operative core biopsy specimens assume consistency of molecular profiles with larger surgical specimens. Whether DNA methylation in ductal carcinoma in situ (DCIS), measured in core biopsy and surgical specimens are similar, remains unclear. Here, we compared genome-scale DNA methylation measured in matched core biopsy and surgical specimens from DCIS, including specific DNA methylation biomarkers of subsequent invasive cancer. DNA was extracted from guided 2mm cores of formalin fixed paraffin embedded (FFPE) specimens, bisulfite-modified, and measured on the Illumina HumanMethylation450 BeadChip. DNA methylation profiles of core biopsies exhibited high concordance with matched surgical specimens. Within-subject variability in DNA methylation was significantly lower than between-subject variability (all Pcore biopsy and surgical specimens, 15%, and a pathway analysis of these CpGs indicated enrichment for genes related with wound healing. Our results indicate that DNA methylation measured in core biopsies are representative of the matched surgical specimens and suggest that DCIS biomarkers measured in core biopsies can inform clinical decision-making. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Polymerase chain reaction detection of retinoblastoma gene deletions in paraffin-embedded mouse lung adenocarcinomas

International Nuclear Information System (INIS)

Churchill, M.E.; Gemmell, M.A.; Woloschak, G.E.

1991-01-01

A Polymerase chain reaction (PCR) technique was used to detect deletions in the mouse retinoblastoma (mRb) gene using microtomed sections from paraffin-embedded radiation-induced and spontaneous tumors as the DNA source. Six mRb gene exon fragments were amplified in a 40-cycle, 3-temperature PCR protocol. Absence of any of these fragments relative to control PCR products on a Southern blot indicated a deletion of that portion of the mRb gene. Tumors chosen for analysis were lung adenocarcinomas that were judged to be the cause of death. Spontaneous tumors as well as those from irradiated mice (569 cGy of 60 Co γ rays or 60 cGy of JANUS neutrons) were analyzed. Tumors in six neutron-irradiated mice also had no mRb deletions. However, one of six tumors from γ-irradiated mice and 6 of 18 spontaneous tumors from unirradiated mice showed a deletion in one or both mRb alleles. All deletions detected were in the 5' region of the mRb gene

A new substitute for formalin: Application to embalming cadavers.

Science.gov (United States)

Haizuka, Yoshinori; Nagase, Miki; Takashino, Satoshi; Kobayashi, Yasushi; Fujikura, Yoshihisa; Matsumura, George

2018-01-01

The development of formalin-free fixatives is an urgent issue in gross anatomy because of the health hazard and the tissue-hardening actions of formalin. We recently identified the fixative, antimicrobial, and preservative effects of N-vinyl-2-pyrrolidone (NVP), a precursor of the water-soluble macromolecular polymer polyvinylpyrrolidone, in animal experiments. The aim of the present study is to investigate whether NVP solution can be used as an alternative to formalin in human cadaveric dissection. Twelve donated cadavers were infused with NVP via the femoral and common carotid arteries using a peristaltic pump. Experienced teaching staff members in our department dissected the cadavers and examined their macroanatomical properties. The NVP-embalmed corpses showed no sign of decomposition or fungal growth. The bodies remained soft and flexible. Notably, the shoulder, elbow, wrist, phalangeal, hip, knee, cervical spine, and temporomandibular joints were highly mobile, almost equivalent to those of living individuals. The range of motion of most joints was greater in the NVP-fixed than formalin-fixed cadavers. Under the dermis, the subcutaneous fat was markedly reduced and the connective tissues were transparent, so the ligaments, cutaneous nerves, and veins were easily discernible. The abdominal wall and the visceral organs remained pliable and elastic, resembling those of fresh cadavers. The lungs, liver, and gastrointestinal tract were moveable in the thoracic and abdominal cavities and were readily isolated. NVP can be used successfully as a fixative and preservative solution for human cadavers; furthermore, NVP-embalmed bodies could be valuable for learning clinical skills and for training, and for developing innovative medical devices. Clin. Anat. 31:90-98, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Phase-contrast X-ray CT imaging of the kidney. Differences between ethanol fixation and formalin fixation

International Nuclear Information System (INIS)

Shirai, Ryota; Kunii, Takuya; Maruyama, Hiroko; Takeda, Tohoru; Yoneyama, Akio; Lwin, Thet Thet

2012-01-01

A phase-contrast X-ray imaging technique using an X-ray interferometer that provides approximately 1000 times higher sensitivity than the conventional X-ray imaging method for low-atomic number elements based on the difference in the mass attenuation coefficient has recently been developed. In the present study, we compared rat kidneys fixed in 100% ethanol and in 10% formalin to evaluate the effects of ethanol in enhancing image contrast in phase-contrast imaging because ethanol causes significant dehydration of tissues and enhances density differences between tissue components. The experiments were conducted at the Photon Factory in Tsukuba, and the X-ray energy was set at 35 keV. Fine anatomical structures in the kidney such as the glomeruli, tubules, and vessels were observed. Particularly clear renal images were obtained with ethanol fixation. The pixel value ratio between the cortex and medulla was about 43% in ethanol-fixed kidneys and 21% in formalin-fixed kidneys. In other words, the contrast in ethanol-fixed kidneys was about two times higher than that in formalin-fixed kidneys. Histological examination showed significantly condensed features in the cortex. The results of this study suggest that the ethanol fixation technique may be useful for enhancing the image contrast of renal structures in the phase-contrast X-ray imaging technique. (author)

Comparación de métodos de extracción de ADN en tejidos parafinados y utilidad para amplificación por PCR

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Javier Alonso Baena Del Valle

2013-01-01

Formalin-fixed and paraffin-embedded tissues are a source of important molecular findings in clinical and scientific, demonstrating that the DNA extracted from these is suitable for amplification by polymerase chain reaction (PCR. In this study, we tested three methods of DNA extraction, in order to compare the efficiency of these DNA for RCP amplification. Three samples were used, corresponding to a lung biopsy, endometrial curettage and lymph node, all fixed in 10% formaldehyde and embedded in paraffin. Three different methods were used for DNA extraction (extraction by salting out, modified Sambrook method and commercial kit The DNA obtained was analyzed by spectrophotometry, and gel electrophoresis was performed in 1% agarose to check if the DNA was amplifiable. PCR was performed for exon 3 of caveolin-1 gene. All methods resulted in a good product of genomic DNA, obtaining more quality and purity in the salting out and commercial kit methods. Also, we obtained amplification of the product by these two methods, without favorable results with the DNA extracted by the modified "Preparation of Genomic DNA from Mouse Tails and Other Small samples, according to Sambrook et al." The DNA obtained from FFPE can be amplified by several methods, among them, salting out extraction is an easy, effective and low toxicity for obtaining good quality DNA for PCR amplification.

Measuring ERCC1 protein expression in cancer specimens

DEFF Research Database (Denmark)

Smith, David Hersi; Fiehn, Anne-Marie Kanstrup; Fogh, Louise

2014-01-01

Platinum chemotherapy remains part of standard therapies in the management of a variety of cancers. Severe side effects and a high degree of resistance to platinum drugs have led numerous researchers to search for predictive biomarkers, which could aid in identifying patients that are the most......, the specificity of antibody 4F9 was tested by immunoblotting, immunohistochemistry and immunofluorescence. Scoring guidelines to aid in the evaluation of ERCC1 tumor expression were developed and evaluated in archival formalin-fixed paraffin embedded colorectal cancer specimens. Antibody 4F9 was found...... to be specific by all methods applied and it was possible to evaluate the ERCC1 expression in the majority (85%) of colorectal cancer tumor specimens....

Left-sided breast cancer irradiation using rotational and fixed-field radiotherapy

International Nuclear Information System (INIS)

Qi, X. Sharon; Liu, Tian X.; Liu, Arthur K.; Newman, Francis; Rabinovitch, Rachel; Kavanagh, Brian; Hu, Y. Angie

2014-01-01

The 3-dimensional conformal radiotherapy (3DCRT) technique is the standard for breast cancer radiotherapy. During treatment planning, not only the coverage of the planning target volume (PTV) but also the minimization of the dose to critical structures, such as the lung, heart, and contralateral breast tissue, need to be considered. Because of the complexity and variations of patient anatomy, more advanced radiotherapy techniques are sometimes desired to better meet the planning goals. In this study, we evaluated external-beam radiation treatment techniques for left breast cancer using various delivery platforms: fixed-field including TomoDirect (TD), static intensity-modulated radiotherapy (sIMRT), and rotational radiotherapy including Elekta volumetric-modulated arc therapy (VMAT) and tomotherapy helical (TH). A total of 10 patients with left-sided breast cancer who did or did not have positive lymph nodes and were previously treated with 3DCRT/sIMRT to the entire breast were selected, their treatment was planned with Monaco VMAT, TD, and TH. Dosimetric parameters including PTV coverage, organ-at-risk (OAR) sparing, dose-volume histograms, and target minimum/maximum/mean doses were evaluated. It is found that for plans providing comparable PTV coverage, the Elekta VMAT plans were generally more inhomogeneous than the TH and TD plans. For the cases with regional node involvement, the average mean doses administered to the heart were 9.2 (± 5.2) and 8.8 (± 3.0) Gy in the VMAT and TH plans compared with 11.9 (± 6.4) and 11.8 (± 9.2) Gy for the 3DCRT and TD plans, respectively, with slightly higher doses given to the contralateral lung or breast or both. On average, the total monitor units for VMAT plans are 11.6% of those TH plans. Our studies have shown that VMAT and TH plans offer certain dosimetric advantages over fixed-field IMRT plans for advanced breast cancer requiring regional nodal treatment. However, for early-stage breast cancer fixed

Translating tumor biology into personalized treatment planning: analytical performance characteristics of the Oncotype DX? Colon Cancer Assay

OpenAIRE

Clark-Langone, Kim M; Sangli, Chithra; Krishnakumar, Jayadevi; Watson, Drew

2010-01-01

Abstract Background The Oncotype DX® Colon Cancer Assay is a new diagnostic test for determining the likelihood of recurrence in stage II colon cancer patients after surgical resection using fixed paraffin embedded (FPE) primary colon tumor tissue. Like the Oncotype DX Breast Cancer Assay, this is a high complexity, multi-analyte, reverse transcription (RT) polymerase chain reaction (PCR) assay that measures the expression levels of specific cancer-related genes. By capturing the biology unde...

Confocal Microscopy of Unfixed Breast Needle Core Biopsies: A Comparison to Fixed and Stained Sections

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Zavislan James M

2009-08-01

Full Text Available Abstract Background Needle core biopsy, often in conjunction with ultrasonic or stereotactic guided techniques, is frequently used to diagnose breast carcinoma in women. Confocal scanning laser microscopy (CSLM is a technology that provides real-time digital images of tissues with cellular resolution. This paper reports the progress in developing techniques to rapidly screen needle core breast biopsy and surgical specimens at the point of care. CSLM requires minimal tissue processing and has the potential to reduce the time from excision to diagnosis. Following imaging, specimens can still be submitted for standard histopathological preparation. Methods Needle core breast specimens from 49 patients were imaged at the time of biopsy. These lesions had been characterized under the Breast Imaging Reporting And Data System (BI-RADS as category 3, 4 or 5. The core biopsies were imaged with the CSLM before fixation. Samples were treated with 5% citric acid and glycerin USP to enhance nuclear visibility in the reflectance confocal images. Immediately following imaging, the specimens were fixed in buffered formalin and submitted for histological processing and pathological diagnosis. CSLM images were then compared to the standard histology. Results The pathologic diagnoses by standard histology were 7 invasive ductal carcinomas, 2 invasive lobular carcinomas, 3 ductal carcinomas in-situ (CIS, 21 fibrocystic changes/proliferative conditions, 9 fibroadenomas, and 5 other/benign; two were excluded due to imaging difficulties. Morphologic and cellular features of benign and cancerous lesions were identified in the confocal images and were comparable to standard histologic sections of the same tissue. Conclusion CSLM is a technique with the potential to screen needle core biopsy specimens in real-time. The confocal images contained sufficient information to identify stromal reactions such as fibrosis and cellular proliferations such as intra-ductal and

Confocal Microscopy of Unfixed Breast Needle Core Biopsies: A Comparison to Fixed and Stained Sections

Science.gov (United States)

2009-01-01

Background Needle core biopsy, often in conjunction with ultrasonic or stereotactic guided techniques, is frequently used to diagnose breast carcinoma in women. Confocal scanning laser microscopy (CSLM) is a technology that provides real-time digital images of tissues with cellular resolution. This paper reports the progress in developing techniques to rapidly screen needle core breast biopsy and surgical specimens at the point of care. CSLM requires minimal tissue processing and has the potential to reduce the time from excision to diagnosis. Following imaging, specimens can still be submitted for standard histopathological preparation. Methods Needle core breast specimens from 49 patients were imaged at the time of biopsy. These lesions had been characterized under the Breast Imaging Reporting And Data System (BI-RADS) as category 3, 4 or 5. The core biopsies were imaged with the CSLM before fixation. Samples were treated with 5% citric acid and glycerin USP to enhance nuclear visibility in the reflectance confocal images. Immediately following imaging, the specimens were fixed in buffered formalin and submitted for histological processing and pathological diagnosis. CSLM images were then compared to the standard histology. Results The pathologic diagnoses by standard histology were 7 invasive ductal carcinomas, 2 invasive lobular carcinomas, 3 ductal carcinomas in-situ (CIS), 21 fibrocystic changes/proliferative conditions, 9 fibroadenomas, and 5 other/benign; two were excluded due to imaging difficulties. Morphologic and cellular features of benign and cancerous lesions were identified in the confocal images and were comparable to standard histologic sections of the same tissue. Conclusion CSLM is a technique with the potential to screen needle core biopsy specimens in real-time. The confocal images contained sufficient information to identify stromal reactions such as fibrosis and cellular proliferations such as intra-ductal and infiltrating carcinoma, and

SISH/CISH or qPCR as alternative techniques to FISH for determination of HER2 amplification status on breast tumors core needle biopsies: a multicenter experience based on 840 cases.

Science.gov (United States)

Jacquemier, Jocelyne; Spyratos, Frédérique; Esterni, Benjamin; Mozziconacci, Marie-Joëlle; Antoine, Martine; Arnould, Laurent; Lizard, Sarab; Bertheau, Philippe; Lehmann-Che, Jacqueline; Fournier, Cécile Blanc; Krieger, Sophie; Bibeau, Frédéric; Lamy, Pierre-Jean; Chenard, Marie Pierre; Legrain, Michèle; Guinebretière, Jean-Marc; Loussouarn, Delphine; Macgrogan, Gaëtan; Hostein, Isabelle; Mathieu, Marie Christine; Lacroix, Ludovic; Valent, Alexander; Robin, Yves Marie; Revillion, Françoise; Triki, Magali Lacroix; Seaume, Aline; Salomon, Anne Vincent; de Cremoux, Patricia; Portefaix, Geneviève; Xerri, Luc; Vacher, Sophie; Bièche, Ivan; Penault-Llorca, Frédérique

2013-07-22

Until now, FISH has been the gold standard technique to identify HER2 amplification status in ambiguous cases of breast cancer. Alternative techniques have been developed to increase the capacities of investigating HER2 amplification status. The aims of this multicenter study in a large series of breast cancer patients were to prospectively compare the level of performance of CISH, SISH, and qPCR alternative techniques on paraffin-embedded core biopsies with "gold standard FISH" for evaluation of HER2 amplification status. This study was performed on 840 cases scored by immunohistochemistry (IHC): 0=317 (38%), 1+=183 (22%), 2+=109 (13%), 3+=231 (27%). Each of the 15 French centers participating in the study analyzed 56 breast carcinoma cases diagnosed on fixed paraffin-embedded core biopsies. HER2 amplification status was determined by commercially available FISH used as the reference technique with determination of the HER2/CEN17 ratio or HER2 copy number status. The alternative techniques performed on the same cases were commercially available SISH or CISH and a common qPCR method especially designed for the study including a set of 10 primer pairs: 2 for HER2 (exons 8 and 26), 5 to evaluate chromosome 17 polysomy TAOK1, UTP6, MRM1, MKS1, SSTR2 and 3 for diploidy control TSN, LAP3 and ADAMTS16. The concordance between IHC and FISH was 96% to 95% based on the HER2/CEN17 ratio (n=766) or HER2 copy number (n=840), respectively. The concordance of the alternative techniques with FISH was excellent: 97% and 98% for SISH (498 and 587 cases), 98% and 75% for CISH (108 and 204 cases) and 95% and 93% (699 and 773 cases) for qPCR based on the HER2/CEN17 ratio or HER2 copy number, respectively. Similarly, sensitivity ranged from 99% to 95% for SISH, 100% to 99% for CISH and 89% to 80% for qPCR. The concordance with FISH (ratio) in the 2+ cases was 89% for SISH, 100% for CISH and 93% for qPCR. These alternative techniques showed an excellent concordance with FISH in core

Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers.

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Iwamoto, Ryo; Takagi, Mika; Akatsuka, Jun-Ichi; Ono, Ken-Ichiro; Kishi, Yoshiro; Mekada, Eisuke

2016-04-01

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.

Pathology and biofilms in a porcine model of heamatogenous osteomyelitis

DEFF Research Database (Denmark)

Johansen, Louise Kruse

with saline. Following euthanasia, 11 days after inoculation the animals were necropsied and macroscopic bone lesions were recorded. Tissue was sampled from the lesions and following fixation in formalin and embedding in paraffin used for immunohistochemistry and peptide nuclei acid in situ hybridisation (PNA...

Background staining of visualization systems in immunohistochemistry: comparison of the Avidin-Biotin Complex system and the EnVision+ system.

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Vosse, Bettine A H; Seelentag, Walter; Bachmann, Astrid; Bosman, Fred T; Yan, Pu

2007-03-01

The aim of this study was to evaluate specific immunostaining and background staining in formalin-fixed, paraffin-embedded human tissues with the 2 most frequently used immunohistochemical detection systems, Avidin-Biotin-Peroxidase (ABC) and EnVision+. A series of fixed tissues, including breast, colon, kidney, larynx, liver, lung, ovary, pancreas, prostate, stomach, and tonsil, was used in the study. Three monoclonal antibodies, 1 against a nuclear antigen (Ki-67), 1 against a cytoplasmic antigen (cytokeratin), and 1 against a cytoplasmic and membrane-associated antigen and a polyclonal antibody against a nuclear and cytoplasmic antigen (S-100) were selected for these studies. When the ABC system was applied, immunostaining was performed with and without blocking of endogenous avidin-binding activity. The intensity of specific immunostaining and the percentage of stained cells were comparable for the 2 detection systems. The use of ABC caused widespread cytoplasmic and rare nuclear background staining in a variety of normal and tumor cells. A very strong background staining was observed in colon, gastric mucosa, liver, and kidney. Blocking avidin-binding capacity reduced background staining, but complete blocking was difficult to attain. With the EnVision+ system no background staining occurred. Given the efficiency of the detection, equal for both systems or higher with EnVision+, and the significant background problem with ABC, we advocate the routine use of the EnVision+ system.

Detection of PAX8/PPARG and RET/PTC Rearrangements Is Feasible in Routine Air-Dried Fine Needle Aspiration Smears

DEFF Research Database (Denmark)

Ferraz, Carolina; Rehfeld, Christian; Krogdahl, Annelise

2012-01-01

Background: The diagnostic limitations of fine needle aspiration (FNA), like the indeterminate category, can be partially overcome by molecular analysis. As PAX8/PPARG and RET/PTC rearrangements have been detected in follicular thyroid carcinomas (FTCs) and papillary thyroid carcinomas (PTCs......), their detection in FNA smears could improve the FNA diagnosis. To date, these rearrangements have never been analyzed in routine air-dried FNA smears, but only in frozen tissue, formalin-fixed paraffin-embedded (FFPE) tissue, and in fresh FNA material. Fixed routine air-dried FNA samples have hitherto been judged...... as generally not suitable for testing these rearrangements in a clinical setting. Therefore, the objective of the present study was to investigate the feasibility of extracting RNA from routine air-dried FNA smears for the detection of these rearrangements with real-time polymerase chain reaction (RT...

Cutaneous lesions in pet rabbits following subcutaneous administration of a novel bivalent vaccine against myxomatosis and rabbit haemorrhagic disease.

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Selleri, Paolo; Di Girolamo, Nicola; Vögtlin, Andrea; Fileccia, Ivan; Hoop, Richard; Bongiovanni, Laura

2014-12-01

A novel bivalent vaccine to protect against myxomatosis and rabbit haemorrhagic disease is commercially available for pet rabbits. To describe the appearance of cutaneous lesions arising in pet rabbits positive for myxoma virus (MV) by RT-PCR evaluation shortly after vaccination. Four pet rabbits presenting with papular, crusting skin lesions ~10 days after vaccination. Histological evaluation of formalin-fixed skin biopsies obtained from lesional skin (case 1). Real-time polymerase chain reaction (RT-PCR) evaluation of paraffin-embedded tissue from skin biopsies (case 1) and crusts obtained from the lesion surface (cases 2-4) for myxoma virus are reported as cycle threshold (Ct ) values. Lesions affecting the ear pinna, dorsal aspect of the nose, vulva and/or conjunctiva are reported. Histopathological findings included severe ulcerative, necrotizing dermatitis and intralesional cytoplasmic inclusion bodies in myxoma cells. DNA was amplified from all the paraffin-embedded skin biopsies (Ct  = 34-35) and crusts (Ct  = 20-24). Although a wild virus challenge cannot be definitively excluded, veterinarians and pet-owners should be aware that cutaneous lesions have been observed after vaccination with this novel vaccine in low numbers of rabbits. © 2014 ESVD and ACVD.

Biomarker discovery for colon cancer using a 761 gene RT-PCR assay

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Hackett James R

2007-08-01

Full Text Available Abstract Background Reverse transcription PCR (RT-PCR is widely recognized to be the gold standard method for quantifying gene expression. Studies using RT-PCR technology as a discovery tool have historically been limited to relatively small gene sets compared to other gene expression platforms such as microarrays. We have recently shown that TaqMan® RT-PCR can be scaled up to profile expression for 192 genes in fixed paraffin-embedded (FPE clinical study tumor specimens. This technology has also been used to develop and commercialize a widely used clinical test for breast cancer prognosis and prediction, the Onco typeDX™ assay. A similar need exists in colon cancer for a test that provides information on the likelihood of disease recurrence in colon cancer (prognosis and the likelihood of tumor response to standard chemotherapy regimens (prediction. We have now scaled our RT-PCR assay to efficiently screen 761 biomarkers across hundreds of patient samples and applied this process to biomarker discovery in colon cancer. This screening strategy remains attractive due to the inherent advantages of maintaining platform consistency from discovery through clinical application. Results RNA was extracted from formalin fixed paraffin embedded (FPE tissue, as old as 28 years, from 354 patients enrolled in NSABP C-01 and C-02 colon cancer studies. Multiplexed reverse transcription reactions were performed using a gene specific primer pool containing 761 unique primers. PCR was performed as independent TaqMan® reactions for each candidate gene. Hierarchal clustering demonstrates that genes expected to co-express form obvious, distinct and in certain cases very tightly correlated clusters, validating the reliability of this technical approach to biomarker discovery. Conclusion We have developed a high throughput, quantitatively precise multi-analyte gene expression platform for biomarker discovery that approaches low density DNA arrays in numbers of

Evaluation of different tissue de-paraffinization procedures for infrared spectral imaging.

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Nallala, Jayakrupakar; Lloyd, Gavin Rhys; Stone, Nicholas

2015-04-07

In infrared spectral histopathology, paraffin embedded tissues are often de-paraffinized using chemical agents such as xylene and hexane. These chemicals are known to be toxic and the routine de-waxing procedure is time consuming. A comparative study was carried out to identify alternate de-paraffinization methods by using paraffin oil and electronic de-paraffinization (using a mathematical computer algorithm) and their effectiveness was compared to xylene and hexane. Sixteen adjacent tissue sections obtained from a single block of a normal colon tissue were de-paraffinized using xylene, hexane and paraffin oil (+ hexane wash) at five different time points each for comparison. One section was reserved unprocessed for electronic de-paraffinization based on a modified extended multiplicative signal correction (EMSC). IR imaging was carried out on these tissue sections. Coefficients based on the fit of a pure paraffin model to the IR images were then calculated to estimate the amount of paraffin remaining after processing. Results indicate that on average xylene removes more paraffin in comparison to hexane and paraffin oil although the differences were small. This makes paraffin oil, followed by a hexane wash, an interesting and less toxic alternative method of de-paraffinization. However, none of the chemical methods removed paraffin completely from the tissues at any given time point. Moreover, paraffin was removed more easily from the glandular regions than the connective tissue regions indicating a form of differential paraffin retention based on the histology. In such cases, the use of electronic de-paraffinization to neutralize such variances across different tissue regions might be considered. Moreover it is faster, reduces scatter artefacts by index matching and enables samples to be easily stored for further analysis if required.

Quantifying Short-Chain Chlorinated Paraffin Congener Groups

NARCIS (Netherlands)

Yuan, Bo; Bogdal, Christian; Berger, Urs; MacLeod, Matthew; Gebbink, Wouter A.; Alsberg, Tomas; Wit, de Cynthia A.

2017-01-01

Accurate quantification of short-chain chlorinated paraffins (SCCPs) poses an exceptional challenge to analytical chemists. SCCPs are complex mixtures of chlorinated alkanes with variable chain length and chlorination level; congeners with a fixed chain length (n) and number of chlorines (m) are

Improved clonality detection in Hodgkin lymphoma using a semi-nested modification of the BIOMED-2 PCR assay for IGH and IGK rearrangements: A paraffin-embedded tissue study.

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Han, Shusen; Masaki, Ayako; Sakamoto, Yuma; Takino, Hisashi; Murase, Takayuki; Iida, Shinsuke; Inagaki, Hiroshi

2018-05-01

The BIOMED-2 PCR protocols targeting IGH and IGK genes may be useful for detecting clonality in Hodgkin lymphoma (HL). The clonality detection rates, however, have not been very high with these methods using paraffin-embedded tumor sections. We previously described the usefulness of the semi-nested BIOMED-2 IGH assay in B-cell malignancies. In this study, we devised a novel semi-nested BIOMED-2 IGK assay. Employing 58 cases of classical HL, we carried out the standard BIOMED-2, BIOMED-2 followed by BIOMED-2 re-amplification, and BIOMED-2 followed by semi-nested BIOMED-2, all targeting IGH and IGK, using paraffin-embedded tissues. In both IGH and IGK assays, semi-nested assays yielded significantly higher clonality detection rates than the standard assays and re-amplification assays. Clonality was detected in 13/58 (22.4%) classical HL cases using the standard IGH/IGK assays while it was detected in 38/58 (65.5%) cases using semi-nested IGH/IGK assays. The detection rates were not associated with the HL subtypes, CD30-positive cell density, CD20-positive cell density, or Epstein-Barr virus (EBV) positivity. In conclusion, tumor clonality was detected in nearly two-thirds of classical HL cases using semi-nested BIOMED-2 IGH/IGK assays using paraffin tumor sections. These semi-nested assays may be useful when the standard IGH/IGK assays fail to detect clonality in histopathologically suspected HLs. © 2018 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

Utilization of Cell-Transfer Technique for Molecular Testing on Hematoxylin-Eosin-Stained Sections: A Viable Option for Small Biopsies That Lack Tumor Tissues in Paraffin Block.

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Wu, Howard H; Jovonovich, Stephen M; Randolph, Melissa; Post, Kristin M; Sen, Joyashree D; Curless, Kendra; Cheng, Liang

2016-12-01

- In some instances the standard method of doing molecular testing from formalin-fixed, paraffin-embedded block is not possible because of limited tissue. Tumor cell-enriched cell-transfer technique has been proven useful for performing immunocytochemistry and molecular testing on cytologic smears. - To establish the cell-transfer technique as a viable option for isolating tumor cells from hematoxylin-eosin (H&E)-stained slides. - Molecular testing was performed by using the cell-transfer technique on 97 archived H&E-stained slides from a variety of different tumors. Results were compared to the conventional method of molecular testing. - Polymerase chain reaction-based molecular testing via the cell-transfer technique was successfully performed on 82 of 97 samples (85%). This included 39 of 47 cases for EGFR, 10 of 11 cases for BRAF, and 33 of 39 cases for KRAS mutations. Eighty-one of 82 cell-transfer technique samples (99%) showed agreement with previous standard method results, including 4 mutations and 35 wild-type alleles for EGFR, 4 mutations and 6 wild-type alleles for BRAF, and 11 mutations and 21 wild-type alleles for KRAS. There was only 1 discrepancy: a cell-transfer technique with a false-negative >KRAS result (wild type versus G12C). - Molecular testing performed on H&E-stained sections via cell-transfer technique is useful when tissue from cell blocks and small surgical biopsy samples is exhausted and the only available material for testing is on H&E-stained slides.

Macrodissection prior to closed system RT-qPCR is not necessary for estrogen receptor and HER2 concordance with IHC/FISH in breast cancer.

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Gupta, Swati; Mani, Navin R; Carvajal-Hausdorf, Daniel E; Bossuyt, Veerle; Ho, Kenneth; Weidler, Jodi; Wong, Wendy; Rhees, Brian; Bates, Michael; Rimm, David L

2018-06-01

An on-demand, closed RT-qPCR, the GeneXpert (GX) system, has the potential to provide biomarker information in low-resourced settings and elsewhere. We used this system with a research use only version of the Breast Cancer STRAT4 cartridge that measures the mRNA expression levels of ERBB2, ESR1, PGR, and MKi67. Here we evaluated the impact of non-macrodissected (non m-d) versus macrodissected (m-d) samples using STRAT4 on formalin-fixed, paraffin-embedded (FFPE) core needle biopsies. Two cohorts were assessed: (1) 60 FFPE infiltrating ductal carcinoma (IDCA) cases and (2) 20 FFPE IDCA cases with ductal carcinoma in situ (DCIS) with a range of HER2 expression as determined by clinical immunohistochemistry and fluorescence in situ hybridization (IHC/FISH). We observed about half of the core needle biopsy area as invasive tumor in both IDCA (mean = 51.5%) and IDCA with DCIS (mean = 53.5%) cohorts, but also found the mRNA levels were independent of tumor area. We found excellent agreement of the mRNA transcript level between the paired samples, m-d versus non m-d, for ERBB2, ESR1, PGR, and MKi67 for both the IDCA and IDCA with DCIS cohorts. No significant difference (P > 0.99) was observed when we compared the mRNA transcript level between the paired samples m-d versus non m-d. In addition, we noted a significant concordance (P < 0.001) between RT-qPCR and IHC/FISH for HER2-positivity, ER-positivity, and PR-positivity, independent of specimen dissection. These data suggest that mRNA expression for ERBB2, ESR, and PGR is sufficiently low in surrounding tissue cells such that macrodissection is not required for assessment of key breast cancer mRNA markers and is independent of the amount of input tumor. This approach may be valuable in settings lacking pathology expertise or using specimen types, such as fine-needle aspirates, where it may be challenging to separate non-tumor from tumor tissue.

Chromogenic in situ hybridization (CISH): a novel alternative in screening archival breast cancer tissue samples for HER-2/neu status.

Science.gov (United States)

Madrid, Manuelito A; Lo, Raymundo W

2004-01-01

Chromogenic in situ hybridization (CISH) is emerging as a practical, cost-effective, and valid alternative to fluorescent in situ hybridization in testing for gene alteration, especially in centers primarily working with immunohistochemistry (IHC). We assessed Her-2/neu alteration using CISH on formalin-fixed paraffin-embedded primary invasive ductal carcinoma tumors in which IHC (CB11 antibody) had previously been performed, and we compared the results with IHC. The 160 selected cases were equally stratified randomly into the four IHC categories (scores of 0, 1+, 2+, and 3+). We also compared age at diagnosis and tumor histologic grade with IHC and CISH Her-2/neu. We were able to perform and evaluate CISH successfully on all cases. The agreement between 3+ IHC and CISH-amplified cases as well as between all IHC and CISH Her-2/neu negative cases was 100%, and the concordance on all positive cases was 72.50%, with an overall agreement of 86.25%. All the discordant cases had 2+ IHC scores. Although we noted Her-2/neu positivity more in premenopausal women, the age at diagnosis was not significantly associated with IHC or CISH results. Similarly, although the small group of well-differentiated tumors was apparently Her-2/neu negative in both tests, no significant association was noted between any tumor histologic grade and either IHC or CISH results. CISH is easily integrated into routine testing in our laboratory. It is a necessary adjunct in determining the subset of non-amplified IHC-positive invasive tumors that will not benefit from trastuzumab therapy. Those cases with 2+ IHC results will be triaged and subjected to CISH. Her-2/neu testing should be done on all breast cancer cases regardless of age at presentation and tumor histologic grade.

Chromogenic in situ hybridization (CISH): a novel alternative in screening archival breast cancer tissue samples for HER-2/neu status

International Nuclear Information System (INIS)

Madrid, Manuelito A; Lo, Raymundo W

2004-01-01

Chromogenic in situ hybridization (CISH) is emerging as a practical, cost-effective, and valid alternative to fluorescent in situ hybridization in testing for gene alteration, especially in centers primarily working with immunohistochemistry (IHC). We assessed Her-2/neu alteration using CISH on formalin-fixed paraffin-embedded primary invasive ductal carcinoma tumors in which IHC (CB11 antibody) had previously been performed, and we compared the results with IHC. The 160 selected cases were equally stratified randomly into the four IHC categories (scores of 0, 1+, 2+, and 3+). We also compared age at diagnosis and tumor histologic grade with IHC and CISH Her-2/neu. We were able to perform and evaluate CISH successfully on all cases. The agreement between 3+ IHC and CISH-amplified cases as well as between all IHC and CISH Her-2/neu negative cases was 100%, and the concordance on all positive cases was 72.50%, with an overall agreement of 86.25%. All the discordant cases had 2+ IHC scores. Although we noted Her-2/neu positivity more in premenopausal women, the age at diagnosis was not significantly associated with IHC or CISH results. Similarly, although the small group of well-differentiated tumors was apparently Her-2/neu negative in both tests, no significant association was noted between any tumor histologic grade and either IHC or CISH results. CISH is easily integrated into routine testing in our laboratory. It is a necessary adjunct in determining the subset of non-amplified IHC-positive invasive tumors that will not benefit from trastuzumab therapy. Those cases with 2+ IHC results will be triaged and subjected to CISH. Her-2/neu testing should be done on all breast cancer cases regardless of age at presentation and tumor histologic grade

Probing focal cortical dysplasia in formalin fixed samples using tissue optical spectroscopy

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Anand, Suresh; Cicchi, Riccardo; Giordano, Flavio; Buccoliero, Anna Maria; Conti, Valerio; Guerrini, Renzo; Pavone, Francesco Saverio

2016-03-01

Focal cortical dysplasia (FCD) is one of most common causes of intractable epilepsy in pediatric population and these are often insensitive to anti-epileptic drugs. FCD is characterized by a disarray in localized regions of the cerebral cortex and abnormal neurons which results them to misfire with incorrect signals. Resective neurosurgery to remove or disconnect the affected parts from the rest of the brain seems to be a viable option to treat FCD. Before neurosurgery the subject could undergo imaging studies including magnetic resonance imaging (MRI) or computed tomography (CT) scans. On the downside FCD could be elusive in MRI images and may be practically invisible in CT scans. Furthermore, unnecessary removal of normal tissues is to be taken into consideration as this could lead to neurological defects. In this context, optical spectroscopy have been widely investigated as an alternative technique for the detection of abnormal tissues in different organ sites. Disease progression is accompanied by a number of architectural, biochemical and morphological changes. These variations are reflected in the spectral intensity and line shape. Here, in this proof of concept study we propose to investigate the application of tissue optical spectroscopy based on fluorescence excitation at two wavelength 378 and 445 nm coupled along with Raman spectroscopy for the detection of FCD on formalin fixed tissue specimens from pediatric subjects. For fluorescence at both the excitation wavelengths FCD showed a decreased intensity at longer wavelength when compared to normal tissues. Also, differences exist in the Raman spectral profiles of normal and FCD.

Decentral gene expression analysis: analytical validation of the Endopredict genomic multianalyte breast cancer prognosis test

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Kronenwett Ralf

2012-10-01

Full Text Available Abstract Background EndoPredict (EP is a clinically validated multianalyte gene expression test to predict distant metastasis in ER-positive, HER2-negative breast cancer treated with endocrine therapy alone. The test is based on the combined analysis of 12 genes in formalin-fixed, paraffin-embedded (FFPE tissue by reverse transcription-quantitative real-time PCR (RT-qPCR. Recently, it was shown that EP is feasible for reliable decentralized assessment of gene expression. The aim of this study was the analytical validation of the performance characteristics of the assay and its verification in a molecular-pathological routine laboratory. Methods Gene expression values to calculate the EP score were assayed by one-step RT-qPCR using RNA from FFPE tumor tissue. Limit of blank, limit of detection, linear range, and PCR efficiency were assessed for each of the 12 PCR assays using serial samples dilutions. Different breast cancer samples were used to evaluate RNA input range, precision and inter-laboratory variability. Results PCR assays were linear up to Cq values between 35.1 and 37.2. Amplification efficiencies ranged from 75% to 101%. The RNA input range without considerable change of the EP score was between 0.16 and 18.5 ng/μl. Analysis of precision (variation of day, day time, instrument, operator, reagent lots resulted in a total noise (standard deviation of 0.16 EP score units on a scale from 0 to 15. The major part of the total noise (SD 0.14 was caused by the replicate-to-replicate noise of the PCR assays (repeatability and was not associated with different operating conditions (reproducibility. Performance characteristics established in the manufacturer’s laboratory were verified in a routine molecular pathology laboratory. Comparison of 10 tumor samples analyzed in two different laboratories showed a Pearson coefficient of 0.995 and a mean deviation of 0.15 score units. Conclusions The EP test showed reproducible performance

Decentral gene expression analysis: analytical validation of the Endopredict genomic multianalyte breast cancer prognosis test

International Nuclear Information System (INIS)

Kronenwett, Ralf; Brase, Jan C; Weber, Karsten E; Fisch, Karin; Müller, Berit M; Schmidt, Marcus; Filipits, Martin; Dubsky, Peter; Petry, Christoph; Dietel, Manfred; Denkert, Carsten; Bohmann, Kerstin; Prinzler, Judith; Sinn, Bruno V; Haufe, Franziska; Roth, Claudia; Averdick, Manuela; Ropers, Tanja; Windbergs, Claudia

2012-01-01

EndoPredict (EP) is a clinically validated multianalyte gene expression test to predict distant metastasis in ER-positive, HER2-negative breast cancer treated with endocrine therapy alone. The test is based on the combined analysis of 12 genes in formalin-fixed, paraffin-embedded (FFPE) tissue by reverse transcription-quantitative real-time PCR (RT-qPCR). Recently, it was shown that EP is feasible for reliable decentralized assessment of gene expression. The aim of this study was the analytical validation of the performance characteristics of the assay and its verification in a molecular-pathological routine laboratory. Gene expression values to calculate the EP score were assayed by one-step RT-qPCR using RNA from FFPE tumor tissue. Limit of blank, limit of detection, linear range, and PCR efficiency were assessed for each of the 12 PCR assays using serial samples dilutions. Different breast cancer samples were used to evaluate RNA input range, precision and inter-laboratory variability. PCR assays were linear up to C q values between 35.1 and 37.2. Amplification efficiencies ranged from 75% to 101%. The RNA input range without considerable change of the EP score was between 0.16 and 18.5 ng/μl. Analysis of precision (variation of day, day time, instrument, operator, reagent lots) resulted in a total noise (standard deviation) of 0.16 EP score units on a scale from 0 to 15. The major part of the total noise (SD 0.14) was caused by the replicate-to-replicate noise of the PCR assays (repeatability) and was not associated with different operating conditions (reproducibility). Performance characteristics established in the manufacturer’s laboratory were verified in a routine molecular pathology laboratory. Comparison of 10 tumor samples analyzed in two different laboratories showed a Pearson coefficient of 0.995 and a mean deviation of 0.15 score units. The EP test showed reproducible performance characteristics with good precision and negligible laboratory

Detection of Mycobacterium tuberculosis complex in formalin-fixed, paraffin-embedded tissue specimens with necrotizing granulomatous inflammation by strand displacement amplification

DEFF Research Database (Denmark)

Johansen, Isik Somuncu; Thomsen, Vibeke Østergaard; Forsgren, Arne

2004-01-01

prospectively and 19 retrospectively collected FFPE samples from various sources with granulomatous inflammation and results were compared to tuberculosis notification. Of the prospective samples, 20 were from patients who were notified as having tuberculosis and the assay was positive in 18 (90%). Specificity...... culture and negative in the remaining. The sensitivity and specificity in 19 archival samples was 40% and 100%, respectively, compared to notification data. The assay provided rapid, correct diagnosis on different sources of FFPE samples collected prospectively and therefore offers an important...

Detection of HPV-DNA by a PCR-based method in formalin-fixed, paraffin-embedded tissue from rare endocervical carcinoma types.

Science.gov (United States)

Nofech-Mozes, Sharon; Khalifa, Mahmoud M; Ismiil, Nadia; Dubé, Valerie; Saad, Reda S; Sun, Peizhu; Seth, Arun; Ghorab, Zeina

2010-01-01

High-risk human papilloma virus (HPV) seems to play a role in the pathogenesis of cervical squamous neoplasia and adenocarcinomas of the mucinous and endometrioid cell types. Cervical serous, clear cell, and small cell carcinomas differ from the conventional endocervical adenocarcinoma in their clinical characteristics. The data on the role of HPV in their pathogenesis are limited. In this study, we examined the presence of high-risk HPV-DNA in rare types of cervical carcinoma using polymerase chain reaction-based test. In-house cervical serous, clear cell, and small cell carcinoma cases accessioned between 2000 and 2008 were tested for HPV by polymerase chain reaction amplification of DNA extracted from deparaffinized sections using Roche AMPLICOR HPV Amplification Detection and Control Kits. The kit detects all 13 high-risk HPV-DNA genotypes. The positive cut-off point for AMPLICOR HPV Test was A450 = 0.2. We identified 4 serous, 3 clear cell, 1 mixed clear cell and serous, and 5 small cell carcinomas. High-risk HPV-DNA tested positive in 3 out of 4 serous carcinomas, 2 out of 3 cervical clear cell carcinomas, and all 5 cases of small cell carcinoma and the mixed cell type. Our report documents HPV status in a series of archival unusual types of adenocarcinoma of the uterine cervix. It suggests a robust association between high-risk HPV and these rare subtypes. Despite their unique clinical setting and morphologic appearance, the majority of these tumors likely share a common HPV-mediated carcinogenic pathway. Our observation is particularly significant in cervical cancer prevention as we enter the HPV vaccination era.

Identification of cell proliferation, immune response and cell migration as critical pathways in a prognostic signature for HER2+:ERα- breast cancer.

Directory of Open Access Journals (Sweden)

Jeffrey C Liu

Full Text Available Multi-gene prognostic signatures derived from primary tumor biopsies can guide clinicians in designing an appropriate course of treatment. Identifying genes and pathways most essential to a signature performance may facilitate clinical application, provide insights into cancer progression, and uncover potentially new therapeutic targets. We previously developed a 17-gene prognostic signature (HTICS for HER2+:ERα- breast cancer patients, using genes that are differentially expressed in tumor initiating cells (TICs versus non-TICs from MMTV-Her2/neu mammary tumors. Here we probed the pathways and genes that underlie the prognostic power of HTICS.We used Leave-One Out, Data Combination Test, Gene Set Enrichment Analysis (GSEA, Correlation and Substitution analyses together with Receiver Operating Characteristic (ROC and Kaplan-Meier survival analysis to identify critical biological pathways within HTICS. Publically available cohorts with gene expression and clinical outcome were used to assess prognosis. NanoString technology was used to detect gene expression in formalin-fixed paraffin embedded (FFPE tissues.We show that three major biological pathways: cell proliferation, immune response, and cell migration, drive the prognostic power of HTICS, which is further tuned by Homeostatic and Glycan metabolic signalling. A 6-gene minimal Core that retained a significant prognostic power, albeit less than HTICS, also comprised the proliferation/immune/migration pathways. Finally, we developed NanoString probes that could detect expression of HTICS genes and their substitutions in FFPE samples.Our results demonstrate that the prognostic power of a signature is driven by the biological processes it monitors, identify cell proliferation, immune response and cell migration as critical pathways for HER2+:ERα- cancer progression, and defines substitutes and Core genes that should facilitate clinical application of HTICS.

Optimized Clinical Use of RNALater and FFPE Samples for Quantitative Proteomics

DEFF Research Database (Denmark)

Bennike, Tue Bjerg; Kastaniegaard, Kenneth; Padurariu, Simona

2015-01-01

Introduction and Objectives The availability of patient samples is essential for clinical proteomic research. Biobanks worldwide store mainly samples stabilized in RNAlater as well as formalin-fixed and paraffin embedded (FFPE) biopsies. Biobank material is a potential source for clinical...... we compare to FFPE and frozen samples being the control. Methods From the sigmoideum of two healthy participants’ twenty-four biopsies were extracted using endoscopy. The biopsies was stabilized either by being directly frozen, RNAlater, FFPE or incubated for 30 min at room temperature prior to FFPE...... information. Conclusion We have demonstrated that quantitative proteome analysis and pathway mapping of samples stabilized in RNAlater as well as by FFPE is feasible with minimal impact on the quality of protein quantification and post-translational modifications....

Keratin, luminal epithelial antigen and carcinoembryonic antigen in human urinary bladder carcinomas. An immunohistochemical study.

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Nathrath, W B; Arnholdt, H; Wilson, P D

1982-01-01

14 urinary bladder carcinomas of all main types were investigated with antisera to "broad spectrum keratin" (aK), "luminal epithelial antigen" (aLEA) and carcinoembryonic antigen (aCEA), using an indirect immunoperoxidase method on formalin fixed paraffin embedded sections. Keratin and LEA were both present in normal transitional epithelium, papilloma and carcinoma in situ whereas CEA was absent. Transitional cell carcinomas reacted with both aK and aLEA whereas CEA was seen only in a few foci. In squamous metaplasia and squamous carcinoma reaction with aK was particularly strong, while LEA was almost lacking and CEA was present in necrotic centres. In adenocarcinomas aK and aLEA reacted equally while aCEA reacted only on the surface.

Acurácia do exame citológico no diagnóstico de processos inflamatórios e proliferativos dos animais domésticos Accuracy of cytological exam for diagnosis of inflammatory and proliferative process in domestic animals

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R.M.C. Guedes

2000-10-01

Full Text Available The purpose of this study was to demonstrate the importance and accuracy of cytology as a diagnostic method for proliferative and inflammatory processes in domestic animals. The cytology samples were collected by biopsy or during necropsy, using fine needle aspiration or imprint techniques in 80 dogs, 4 cats, 3 goats, 2 cows and 1 horse. Histopathology of formalin fixed tissues processed by the routine embedding paraffin technique was used as a confirmatory diagnostic test. Cytology results agreed with histopathological findings in 75 (83.3% cases. They held higher accord in round cell tumor cases. Therefore, this study shows that cytology is a valuable diagnostic method for diagnosis of proliferative process in domestic animals, especially for round cells tumors.

The Proteome of Primary Prostate Cancer

DEFF Research Database (Denmark)

Iglesias-Gato, Diego; Wikström, Pernilla; Tyanova, Stefka

2016-01-01

for disease aggressiveness. DESIGN, SETTING, AND PARTICIPANTS: Mass spectrometry was used for genome-scale quantitative proteomic profiling of 28 prostate tumors (Gleason score 6-9) and neighboring nonmalignant tissue in eight cases, obtained from formalin-fixed paraffin-embedded prostatectomy samples. Two...... changes occurring during prostate cancer (PCa) initiation and progression can result in clinically relevant discoveries. OBJECTIVES: To study cellular processes altered in PCa using system-wide quantitative analysis of changes in protein expression in clinical samples and to identify prognostic biomarkers......BACKGROUND: Clinical management of the prostate needs improved prognostic tests and treatment strategies. Because proteins are the ultimate effectors of most cellular reactions, are targets for drug actions and constitute potential biomarkers; a quantitative systemic overview of the proteome...

A new technique for Gram staining paraffin-embedded tissue.

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Engbaek, K; Johansen, K S; Jensen, M E

1979-01-01

Five techniques for Gram staining bacteria in paraffin sections were compared on serial sections of pulmonary tissues from eight bacteriological necropsies. Brown and Hopp's method was the most satisfactory for distinguishing Gram-positive and Gram-negative bacteria. However, this method cannot be recommended as the preparations were frequently overstained, and the Gram-negative bacteria were stained indistinctly. A modification of Brown and Hopps' method was developed which stains larger numbers of Gram-negative bacteria and differentiates well between different cell types and connective tissue, and there is no risk of overstaining. PMID:86548

Typing and grading of cytological category C5 breast lesions

International Nuclear Information System (INIS)

Zafar, N.; Jamal, S.; Mamoon, N.; Luqman, M.; Anwar, M.

2005-01-01

Objective: To determine the tumour type, cytological grade and nuclear grade on fine-needle aspiration smears in cytological category C5 breast lesions and compare them with histopathological findings. Subjects and Methods: Out of all patients referred to AFIP, Rawalpindi for fine-needle aspiration of breast masses, those adjudged C5 (malignant) were chosen for this study. History, clinical details and mammographic findings were noted. Aspirated smears were examined and an attempt was made to ascertain tumour type, cytological grade and nuclear grade. On excision of these lesions, the cytological findings were compared with those on paraffin-embedded histological sections. Results: A total of 71 patients were included in this study. Of these, 64 (90.14%) were cytologically diagnosed as ductal carcinoma, 4 (5.63%) lobular carcinoma, 2 (2.82%) mucinous carcinoma and 1 (1.41%) as medullary carcinoma. Seventy (98.60%) tumours were correctly typed on aspiration smears. Sixty-eight (95.77%) cases were cytologically graded with accuracy. Nuclear grading was even better on cytology and, excluding one malignant lymphoma, all 70 (100%) smears were assessed correctly. Conclusion: Overall efficiency of the tumour typing, cytological grading and nuclear grading on aspirated material turned out to be quite accurate. In expert hands, cytological examination can be of great help in pre-operative planning and in cases where tumour morphology in paraffin-embedded material has been distorted by neo-adjuvant therapy prior to excision. (author)

Long-lasting complete response status of advanced stage IV gall bladder cancer and colon cancer after combined treatment including autologous formalin-fixed tumor vaccine: two case reports.

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Imaoka, Yuki; Kuranishi, Fumito; Miyazaki, Tsubasa; Yasuda, Hiroko; Ohno, Tadao

2017-09-11

The prognosis of advanced (stage IV) cancer of the digestive organs is very poor. We have previously reported a case of advanced breast cancer with bone metastasis that was successfully treated with combined treatments including autologous formalin-fixed tumor vaccine (AFTV). Herein, we report the success of this approach in advanced stage IV (heavily metastasized) cases of gall bladder cancer and colon cancer. Case 1: A 61-year-old woman with stage IV gall bladder cancer (liver metastasis and lymph node metastasis) underwent surgery in May 2011, including partial resection of the liver. She was treated with AFTV as the first-line adjuvant therapy, followed by conventional chemotherapy. This patient is still alive without any recurrence, as confirmed with computed tomography, for more than 5 years. Case 2: A 64-year-old man with stage IV colon cancer (multiple para-aortic lymph node metastases and direct abdominal wall invasion) underwent non-curative surgery in May 2006. Following conventional chemotherapy, two courses of AFTV and radiation therapy were administered sequentially. This patient has had no recurrence for more than 5 years. We report the success of combination therapy including AFTV in cases of liver-metastasized gall bladder cancer and abdominal wall-metastasized colon cancer. Both patients experienced long-lasting, complete remission. Therefore, combination therapies including AFTV should be considered in patients with advanced cancer of the digestive organs.

First detection and genotyping of Giardia intestinalis in stool samples collected from children in Ghazni Province, eastern Afghanistan and evaluation of the PCR assay in formalin-fixed specimens.

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Lass, Anna; Karanis, Panagiotis; Korzeniewski, Krzysztof

2017-08-01

It is estimated that faecal-orally transmitted diseases are common in Afghanistan, as a consequence of poor hygienic standards of life and widespread contamination of water and food with both human and animal faeces. However, there is little information in the literature concerning infections caused by intestinal parasites in the Afghan population. In this study, we report the occurrence of Giardia intestinalis assemblages (A and B) in formalin-fixed stool samples collected from 245 Afghan schoolchildren living in Ghazni Province in eastern Afghanistan. Detection of the parasite's DNA and genotyping was performed using real-time PCR, specific to the β-giardin gene of G. intestinalis. Positive results were recorded in 52 (21.2%) samples. Genotyping was successful in 39 faecal samples and showed the predominance of assemblage B of G. intestinalis in this population (15 assemblage A and 24 assemblage B). Co-infection with both genotypes A and B was detected in four samples. Additionally, we evaluated the effect of 10% buffered formalin fixative on the detection of G. intestinalis DNA using real-time PCR and nested PCR characterised by different lengths of PCR products (74 and 479 bp, respectively). The human faeces containing the Giardia cysts were tested for 16 weeks. Amplification of G. intestinalis DNA with real-time PCR was possible up to 6 weeks of preservation of stool sample in formalin, compared to only 2 weeks with nested PCR. This suggests that real-time PCR is a more suitable tool in cases where stool samples have to be kept in formalin for longer periods of time.

Expression of glucocorticoid and progesterone nuclear receptor genes in archival breast cancer tissue

International Nuclear Information System (INIS)

Smith, Robert A; Lea, Rod A; Curran, Joanne E; Weinstein, Stephen R; Griffiths, Lyn R

2003-01-01

Previous studies in our laboratory have shown associations of specific nuclear receptor gene variants with sporadic breast cancer. In order to investigate these findings further, we conducted the present study to determine whether expression levels of the progesterone and glucocorticoid nuclear receptor genes vary in different breast cancer grades. RNA was extracted from paraffin-embedded archival breast tumour tissue and converted into cDNA. Sample cDNA underwent PCR using labelled primers to enable quantitation of mRNA expression. Expression data were normalized against the 18S ribosomal gene multiplex and analyzed using analysis of variance. Analysis of variance indicated a variable level of expression of both genes with regard to breast cancer grade (P = 0.00033 for glucocorticoid receptor and P = 0.023 for progesterone receptor). Statistical analysis indicated that expression of the progesterone nuclear receptor is elevated in late grade breast cancer tissue

Imaging of metastatic lymph nodes by X-ray phase-contrast micro-tomography.

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Torben Haugaard Jensen

Full Text Available Invasive cancer causes a change in density in the affected tissue, which can be visualized by x-ray phase-contrast tomography. However, the diagnostic value of this method has so far not been investigated in detail. Therefore, the purpose of this study was, in a blinded manner, to investigate whether malignancy could be revealed by non-invasive x-ray phase-contrast tomography in lymph nodes from breast cancer patients. Seventeen formalin-fixed paraffin-embedded lymph nodes from 10 female patients (age range 37-83 years diagnosed with invasive ductal carcinomas were analyzed by X-ray phase-contrast tomography. Ten lymph nodes had metastatic deposits and 7 were benign. The phase-contrast images were analyzed according to standards for conventional CT images looking for characteristics usually only visible by pathological examinations. Histopathology was used as reference. The result of this study was that the diagnostic sensitivity of the image analysis for detecting malignancy was 100% and the specificity was 87%. The positive predictive value was 91% for detecting malignancy and the negative predictive value was 100%. We conclude that x-ray phase-contrast imaging can accurately detect density variations to obtain information regarding lymph node involvement previously inaccessible with standard absorption x-ray imaging.

Molecular Pathology: A Requirement for Precision Medicine in Cancer.

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Dietel, Manfred

2016-01-01

The increasing importance of targeting drugs and check-point inhibitors in the treatment of several tumor entities (breast, colon, lung, malignant melanoma, lymphoma, etc.) and the necessity of a companion diagnostic (HER2, (pan)RAS, EGFR, ALK, BRAF, ROS1, MET, PD-L1, etc.) is leading to new challenges for surgical pathology. Since almost all the biomarkers to be specifically detected are tissue based, a precise and reliable diagnostic is absolutely crucial. To meet this challenge surgical pathology has adapted a number of molecular methods (semi-quantitative immunohistochemistry, fluorescence in situ hybridization, PCR and its multiple variants, (pyro/Sanger) sequencing, next generation sequencing (amplicon, whole exome, whole genome), DNA arrays, methylation analyses, etc.) to be applicable for formalin-fixed paraffin-embedded tissue. Reading a patient's tissue as 'deeply' as possible and obtaining information on the morphological, genetic, proteomic and epigenetic background are the tasks of pathologists and molecular biologists and provide the clinicians with information relevant for precision medicine. Intensified cooperation between clinicians and pathologists will provide the basis of improved clinical drug selection and guide development of new cancer gene therapies and molecularly targeted drugs by research units and the pharmaceutical industry. © 2016 S. Karger GmbH, Freiburg.

Kiss-1/GPR54 protein expression in breast cancer.

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Papaoiconomou, Eleni; Lymperi, Maria; Petraki, Constantina; Philippou, Anastassios; Msaouel, Pavlos; Michalopoulou, Fani; Kafiri, Georgia; Vassilakos, George; Zografos, Georgios; Koutsilieris, Michael

2014-03-01

Numerous studies have shown that the Kiss-1 gene countervails the metastatic aptitude of several cancer cell lines and solid-tumor neoplasias. However, there still remains ambiguity regarding its role in breast cancer and literature has arisen asserting that Kiss-1 expression may be linked to an aggressive phenotype and malignant progression. Herein, we investigated the protein expression of Kiss-1 and its receptor GPR54 in breast cancer tissues compared to non-cancerous mammary tissues. Paraffin-fixed cancer tissues from 43 women with resected breast adenocarcinomas and 11 specimens derived from women suffering from fibrocystic disease, serving as controls, were immunostained with Kiss-1 and GPR54 antibodies. Kiss-1 and GPR54 protein expression levels were significantly higher in breast cancer compared to fibrocystic tissues (pbreast cancer and fibrocystic disease specimens. Kiss-1/GPR54 expression was found to be significantly higher in breast cancer compared to non-malignant mammary tissues.

Differing lectin-binding patterns of malignant melanoma and nevocellular and Spitz nevi.

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Kohchiyama, A; Oka, D; Ueki, H

1987-01-01

The lectin-binding patterns of primary malignant melanoma, nevocellular nevus, and Spitz nevus were studied on formalin-fixed, paraffin-embedded sections using a series of biotinylated lectins--concanavalin A (ConA), Ricinus communis agglutinin-1 (RCA1), dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA), maclura pomifera agglutinin (MPA), peanut agglutinin (PNA), wheat germ agglutinin (WGA), and Ulex europeus agglutinin-1(UEA1)--and employing the avidin-biotin-peroxidase complex method. In nevocellular and Spitz nevi, all of the nevus cells were positively stained with ConA and RCA1. No positive staining was observed, however, with the other lectins and no change in binding patterns occurred following neuraminidase pretreatment. In malignant melanoma, all of the melanoma cells were positively stained with ConA and RCA1, and some were also stained with MPA, PNA, and WGA. In addition, DBA, SBA, MPA, PNA, and WGA labeled all of the melanoma cells after neuraminidase pretreatment. No positive staining was observed with UEA1 despite neuraminidase pretreatment. The present results showed that malignant melanoma and nevocellular and Spitz nevi have different lectin-binding patterns and different responses to neuraminidase pretreatment. We, therefore, believe that the lectin staining on paraffin-embedded sections can be a useful probe for the differentiation of these diseases.

Exploring DNA methylation changes in promoter, intragenic, and intergenic regions as early and late events in breast cancer formation

International Nuclear Information System (INIS)

Rauscher, Garth H.; Kresovich, Jacob K.; Poulin, Matthew; Yan, Liying; Macias, Virgilia; Mahmoud, Abeer M.; Al-Alem, Umaima; Kajdacsy-Balla, Andre; Wiley, Elizabeth L.; Tonetti, Debra; Ehrlich, Melanie

2015-01-01

Breast cancer formation is associated with frequent changes in DNA methylation but the extent of very early alterations in DNA methylation and the biological significance of cancer-associated epigenetic changes need further elucidation. Pyrosequencing was done on bisulfite-treated DNA from formalin-fixed, paraffin-embedded sections containing invasive tumor and paired samples of histologically normal tissue adjacent to the cancers as well as control reduction mammoplasty samples from unaffected women. The DNA regions studied were promoters (BRCA1, CD44, ESR1, GSTM2, GSTP1, MAGEA1, MSI1, NFE2L3, RASSF1A, RUNX3, SIX3 and TFF1), far-upstream regions (EN1, PAX3, PITX2, and SGK1), introns (APC, EGFR, LHX2, RFX1 and SOX9) and the LINE-1 and satellite 2 DNA repeats. These choices were based upon previous literature or publicly available DNA methylome profiles. The percent methylation was averaged across neighboring CpG sites. Most of the assayed gene regions displayed hypermethylation in cancer vs. adjacent tissue but the TFF1 and MAGEA1 regions were significantly hypomethylated (p ≤0.001). Importantly, six of the 16 regions examined in a large collection of patients (105 – 129) and in 15-18 reduction mammoplasty samples were already aberrantly methylated in adjacent, histologically normal tissue vs. non-cancerous mammoplasty samples (p ≤0.01). In addition, examination of transcriptome and DNA methylation databases indicated that methylation at three non-promoter regions (far-upstream EN1 and PITX2 and intronic LHX2) was associated with higher gene expression, unlike the inverse associations between cancer DNA hypermethylation and cancer-altered gene expression usually reported. These three non-promoter regions also exhibited normal tissue-specific hypermethylation positively associated with differentiation-related gene expression (in muscle progenitor cells vs. many other types of normal cells). The importance of considering the exact DNA region analyzed and the

Incorporating pathology in the practice of infectious disease: myths and reality.

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Guarner, Jeannette

2014-10-15

The role pathology plays in establishing or excluding infectious diseases has been established. However, as the practice of pathology has become subspecialized, there is not enough infectious disease specimen volume to have a pathologist dedicated full time to this crosscutting subspecialty. So, what are the myths and realities of a practicing infectious disease pathologist in the hospital setting? Infectious disease clinicians tend to consult pathologists when there are questions regarding terminology used in pathology reports; when there is the need to perform additional studies on formalin-fixed, paraffin-embedded tissues; and when there is an interest in seeing biopsies or resections obtained from patients and in obtaining photographs for presentations. Pathologists consult infectious disease pathologists when there is a need to review diverse inflammatory reactions; for identification of fungi, parasites, or unknown structures; to define the need to use special stains and other techniques in order to identify organisms in tissues that have been formalin fixed; and to help with terminology to be used in reports. This review explores in more detail why and how these consultations occur. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Validation and utilization of a TFE3 break-apart FISH assay for Xp11.2 translocation renal cell carcinoma and alveolar soft part sarcoma.

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Pradhan, Dinesh; Roy, Somak; Quiroga-Garza, Gabriela; Cieply, Kathleen; Mahaffey, Alyssa L; Bastacky, Sheldon; Dhir, Rajiv; Parwani, Anil V

2015-09-29

Xp11.2 or TFE3 translocation renal cell carcinomas (RCC) and alveolar soft part sarcoma (ASPS) are characterized by chromosome translocations involving the Xp11.2 breakpoint resulting in transcription factor TFE3 gene fusions. The most common translocations documented in TFE3 RCCs are t(X;1) (p11.2;q21) and t(X;17) (p11.2;q25) which leads to fusion of TFE3 gene on Xp11.2 with PRCC or ASPL respectively. TFE3 immunohistochemistry (IHC) has been inconsistent over time due to background staining problems in part related to fixation issues. Karyotyping to detect TFE3 gene rearrangement requires typically unavailable fresh tissue. Reverse transcriptase-polymerase chain reaction (RT-PCR) is generally very challenging due to degradation of RNA in archival material. The study objective was to develop and validate a TFE3 break-apart fluorescence in situ hybridization (FISH) assay to confirm Xp11 translocation RCCs and ASPS. Representative sections of formalin-fixed paraffin-embedded tissue blocks were selected in 40 possible cases. Approximately 60 tumor cells were analyzed in the targeted region. The validation of TFE3 FISH was done with 11 negative and two positive cases. Cut off for a positive result was validated as >7.15 % positive nuclei with any pattern of break-apart signals. FISH evaluation was done blinded of the immunohistochemical or karyotype data. Three out of forty cases were positive for the TFE3 break-apart signals by FISH. The negative cases were reported as clear cell RCC with papillary features (10), clear cell RCC with sarcomatoid areas (2), Papillary RCC with clear cell areas (9), Chromophobe RCC (2), RCC, unclassified type (3) and renal medullary carcinoma (1). 3 of the negative cases were consultation cases for renal tumor with unknown histology. Seven negative cases were soft tissue tumor suspicious for ASPS. Our study validates the utility of TFE3 break-apart FISH on formalin-fixed paraffin-embedded tissue sections for diagnosis and confirmation of

The distribution pattern of ERα expression, ESR1 genetic variation and expression of growth factor receptors: association with breast cancer prognosis in Russian patients treated with adjuvant tamoxifen.

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Babyshkina, Nataliya; Vtorushin, Sergey; Zavyalova, Marina; Patalyak, Stanislav; Dronova, Tatyana; Litviakov, Nikolay; Slonimskaya, Elena; Kzhyshkowska, Julia; Cherdyntseva, Nadejda; Choynzonov, Evgeny

2017-08-01

Identification of additional biomarkers associated with ER genomic and nongenomic pathways could be very useful to distinguish patients who will benefit from tamoxifen treatment. The aim of this study was to analyze the prognostic significance of the distribution pattern of ERα expression, ESR1 gene single-nucleotide polymorphisms and expression levels of growth factor receptors in Russian hormone receptor-positive breast cancer patients treated with adjuvant tamoxifen. Formalin-fixed paraffin-embedded tumor tissue samples from 97 patients were examined for the distribution pattern of ERα expression, as well as for EGFR and TGF-βR1 expression by immunohistochemistry. Genotypes for ESR1 +30T>C (rs2077647) and ESR1 2014G>A (rs2228480) were analyzed using a TaqMan assay. Progression-free survival (PFS) was used as an endpoint for the survival analyses. We found that patients with the heterogeneous distribution of ERα expression had poor prognosis on tamoxifen treatment (P = 0.021). We identified a high EGFR expression in patients who developed distant metastasis or recurrence during tamoxifen treatment (a tamoxifen-resistant group-TR) in contrast to the distant metastasis-free patients (a tamoxifen-sensitive group-TS) (80.0 vs. 41.9 %, respectively, P = 0.009). Carriers of the ESR12014A mutant allele were more prevalent among the TR patients compared to the TS patients (26.3 vs. 8.0 %, respectively, P = 0.009). EGFR expression and the distribution pattern of ERα expression were associated with the response to tamoxifen by both univariate and multivariate logistic regression analyses. The presence of these markers either alone or in combination was correlated with the worse PFS for all patients. Analysis of the distribution pattern of ERα expression and the EGFR status in tumor tissue may be valuable for patient selection for tamoxifen adjuvant therapy.

Improved method for extraction and detection of Helicobacter pylori DNA in formalin-fixed paraffin embedded gastric biopsies using laser micro-dissection

Directory of Open Access Journals (Sweden)

María Fernanda Loayza

2015-01-01

• The use of thin purification columns with 35 μL of elution buffer. The mean of DNA concentration obtained from 25 LM cut sections was 1.94± 0 .16 ng/μL, and it was efficiently amplified with qPCR in a Bio Rad iCycler instrument. The LM can improve the sample selection and DNA extraction for molecular analysis of H. pylori associated with human gastric epithelium.

Clinical array-based karyotyping of breast cancer with equivocal HER2 status resolves gene copy number and reveals chromosome 17 complexity

International Nuclear Information System (INIS)

Gunn, Shelly; Gorre, Mercedes; Mohammed, Mansoor; Yeh, I-Tien; Lytvak, Irina; Tirtorahardjo, Budi; Dzidic, Natasha; Zadeh, Soheila; Kim, Jaeweon; McCaskill, Chris; Lim, Lony

2010-01-01

HER2 gene copy status, and concomitant administration of trastuzumab (Herceptin), remains one of the best examples of targeted cancer therapy based on understanding the genomic etiology of disease. However, newly diagnosed breast cancer cases with equivocal HER2 results present a challenge for the oncologist who must make treatment decisions despite the patient's unresolved HER2 status. In some cases both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are reported as equivocal, whereas in other cases IHC results and FISH are discordant for positive versus negative results. The recent validation of array-based, molecular karyotyping for clinical oncology testing provides an alternative method for determination of HER2 gene copy number status in cases remaining unresolved by traditional methods. In the current study, DNA extracted from 20 formalin fixed paraffin embedded (FFPE) tissue samples from newly diagnosed cases of invasive ductal carcinoma referred to our laboratory with unresolved HER2 status, were analyzed using a clinically validated genomic array containing 127 probes covering the HER2 amplicon, the pericentromeric regions, and both chromosome 17 arms. Array-based comparative genomic hybridization (array CGH) analysis of chromosome 17 resolved HER2 gene status in [20/20] (100%) of cases and revealed additional chromosome 17 copy number changes in [18/20] (90%) of cases. Array CGH analysis also revealed two false positives and one false negative by FISH due to 'ratio skewing' caused by chromosomal gains and losses in the centromeric region. All cases with complex rearrangements of chromosome 17 showed genome-wide chromosomal instability. These results illustrate the analytical power of array-based genomic analysis as a clinical laboratory technique for resolution of HER2 status in breast cancer cases with equivocal results. The frequency of complex chromosome 17 abnormalities in these cases suggests that the two

Commercial formalin substitutes for histopathology

DEFF Research Database (Denmark)

Prentø, P; Lyon, H

1997-01-01

We compared the performance of six commercial fixatives proposed to be formalin substitutes with the performance of buffered formalin, Clarke's ethanol-acetic acid, and ethanol, using rat liver, small intestine, and kidney. We investigated the rate of penetration, mode of fixation, extent of prot...... was obtained by combining formalin fixation with antigen retrieval. We conclude that none of the proposed commercial substitutes for buffered formalin are adequate for critical histology or histopathology....

Cell Line Derived 5-FU and Irinotecan Drug-Sensitivity Profiles Evaluated in Adjuvant Colon Cancer Trial Data

DEFF Research Database (Denmark)

Buhl, Ida Kappel; Gerster, Sarah; Delorenzi, Mauro

2016-01-01

patients who benefitted from the addition of irinotecan to 5-FU, we used gene expression profiles based on cell lines and clinical tumor material. These profiles were applied to expression data obtained from pretreatment formalin fixed paraffin embedded (FFPE) tumor tissue from 636 stage III colon cancer...... patients enrolled in the PETACC-3 prospective randomized clinical trial. A 5-FU profile developed similarly was assessed by comparing the PETACC-3 cohort with a cohort of 359 stage II colon cancer patients who underwent surgery but received no adjuvant therapy. RESULTS: There was no statistically...... to identify colon cancer patients who may benefit from 5-FU, however, any biomarker predicting benefit for adjuvant 5-FU must be rigorously evaluated in independent cohorts. Given differences between the two study cohorts, the present results should be further validated....

Use of MicroRNA biomarkers to distinguish enchondroma from low-grade chondrosarcoma.

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Zhang, Liang; Yang, Maozhou; Mayer, Theodore; Johnstone, Brian; Les, Clifford; Frisch, Nicholas; Parsons, Theodore; Mi, Qing-Sheng; Gibson, Gary

2017-03-01

Establishing a definitive diagnosis between benign enchondroma versus low-grade chondrosarcoma presents a potential challenge to both clinicians and pathologists. microRNAs (small non-coding RNAs) have proven to be effective biomarkers for the identification of tumors and tumor progression. We present analysis, both array and quantitative PCR, that shows consistently and substantially increased expression of two microRNAs, miRs-181a and -138, in low-grade chondrosarcomas compared with enchondromas. The data suggest these microRNAs would provide an analytical distinction between the chondrosarcoma and benign neoplasms that can be performed in formalin-fixed paraffin-embedded specimens. Together with recent publications, these data indicate that miRs-181a and -138 also play a role in tumor development and homeostasis and may provide new targets for the development of much needed therapeutic intervention.

Quantitative Proteomics Identifies Activation of Hallmark Pathways of Cancer in Patient Melanoma.

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Byrum, Stephanie D; Larson, Signe K; Avaritt, Nathan L; Moreland, Linley E; Mackintosh, Samuel G; Cheung, Wang L; Tackett, Alan J

2013-03-01

Molecular pathways regulating melanoma initiation and progression are potential targets of therapeutic development for this aggressive cancer. Identification and molecular analysis of these pathways in patients has been primarily restricted to targeted studies on individual proteins. Here, we report the most comprehensive analysis of formalin-fixed paraffin-embedded human melanoma tissues using quantitative proteomics. From 61 patient samples, we identified 171 proteins varying in abundance among benign nevi, primary melanoma, and metastatic melanoma. Seventy-three percent of these proteins were validated by immunohistochemistry staining of malignant melanoma tissues from the Human Protein Atlas database. Our results reveal that molecular pathways involved with tumor cell proliferation, motility, and apoptosis are mis-regulated in melanoma. These data provide the most comprehensive proteome resource on patient melanoma and reveal insight into the molecular mechanisms driving melanoma progression.

Urokinase-type plasminogen activator receptor (uPAR) expression is associated with T-stage and survival in urothelial carcinoma of the bladder

DEFF Research Database (Denmark)

Dohn, Line Hammer; Illemann, Martin; Høyer-Hansen, Gunilla

2015-01-01

OBJECTIVES: To evaluate the expression-and localization pattern of the urokinase-type plasminogen activator receptor (uPAR), focusing on its clinical implications in patients with urothelial neoplasia of the bladder treated with radical cystectomy. uPAR is a central molecule in tissue remodeling...... during cancer invasion and metastasis and is an established prognostic marker in cancer. The expression and localization of uPAR and its prognostic significance is only limitedly investigated in urothelial bladder neoplasia. MATERIALS AND METHODS: The expression-and localization pattern of u......PAR was investigated in formalin-fixed paraffin-embedded tumor tissue from 149 patients treated with radical cystectomy between 1988 and 2005. uPAR expression was determined by immunohistochemistry and scored as either negative or positive. Separate values were obtained for cancer cells, macrophages...

Saffold virus infection associated with human myocarditis

DEFF Research Database (Denmark)

Nielsen, Trine Skov; Nielsen, Alex Yde; Banner, Jytte

2016-01-01

BACKGROUND: Saffold virus was described in 2007 as one of the first human viruses within the genus cardioviruses. Cardioviruses may cause severe infections of the myocardium in animals, and several studies have associated saffold virus with human disease. As a result, saffold virus has been...... isolated from different anatomical compartments, including the myocardium, but, until now, it has not been possible to demonstrate the accompanying histopathological signs of inflammation. OBJECTIVES: The aim of the study was to examine if saffold virus is capable of causing invasive infection in the human...... myocardium. STUDY DESIGN: Using real-time PCR, we retrospectively examined formalin-fixed paraffin embedded cardiac tissue specimens from 150 deceased individuals diagnosed with myocarditis at autopsy. The results were compared with histological findings. RESULTS AND CONCLUSIONS: Saffold virus was detected...

Human parvovirus B19 infection and hydrops fetalis in Rio de Janeiro, Brazil

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Rita CN Cubel

1996-04-01

Full Text Available Formalin-fixed paraffin embedded lung and liver tissue from 23 cases of non immune hydrops fetalis and five control cases, in which hydrops were due to syphilis (3 and genetic causes (2, were examined for the presence of human parvovirus B19 by DNA hybridisation. Using in situ hybridisation with a biotynilated probe one positive case was detected. Using 32P-labelled probes in a dot blot assay format, five further positives were obtained. These were all confirmed as positive by a nested polymerase chain reaction assay. Electron microscopy revealed virus in all these five positive cases. The six B19 DNA positive cases of hydrops fetalis were from 1974, 1980, 1982, 1987 and 1988, four of which occurred during the second half of the year, confirming the seasonality of the disease.

Transcriptional changes induced by bevacizumab combination therapy in responding and non-responding recurrent glioblastoma patients

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Urup, Thomas; Staunstrup, Line Maersk; Michaelsen, Signe Regner

2017-01-01

Background: Bevacizumab combined with chemotherapy produces clinical durable response in 25-30% of recurrent glioblastoma patients. This group of patients has shown improved survival and quality of life. The aim of this study was to investigate changes in gene expression associated with response...... and resistance to bevacizumab combination therapy.Methods: Recurrent glioblastoma patients who had biomarker-accessible tumor tissue surgically removed both before bevacizumab treatment and at time of progression were included. Patients were grouped into responders (n = 7) and non-responders (n = 14). Gene...... expression profiling of formalin-fixed paraffin-embedded tumor tissue was performed using RNA-sequencing.Results: By comparing pretreatment samples of responders with those of non-responders no significant difference was observed. In a paired comparison analysis of pre- and posttreatment samples of non...

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil.

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Silva, Marcio Roberto; Rocha, Adalgiza da Silva; da Costa, Ronaldo Rodrigues; de Alencar, Andrea Padilha; de Oliveira, Vania Maria; Fonseca Júnior, Antônio Augusto; Sales, Mariana Lázaro; Issa, Marina de Azevedo; Filho, Paulo Martins Soares; Pereira, Omara Tereza Vianello; dos Santos, Eduardo Calazans; Mendes, Rejane Silva; Ferreira, Angela Maria de Jesus; Mota, Pedro Moacyr Pinto Coelho; Suffys, Philip Noel; Guimarães, Mark Drew Crosland

2013-05-01

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

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Marcio Roberto Silva

2013-05-01

Full Text Available In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis. Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ and Stonebrink (SB-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks. One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6% of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.

In situ hybridization for the detection of rust fungi in paraffin embedded plant tissue sections.

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Ellison, Mitchell A; McMahon, Michael B; Bonde, Morris R; Palmer, Cristi L; Luster, Douglas G

2016-01-01

Rust fungi are obligate pathogens with multiple life stages often including different spore types and multiple plant hosts. While individual rust pathogens are often associated with specific plants, a wide range of plant species are infected with rust fungi. To study the interactions between these important pathogenic fungi and their host plants, one must be able to differentiate fungal tissue from plant tissue. This can be accomplished using the In situ hybridization (ISH) protocol described here. To validate reproducibility using the ISH protocol, samples of Chrysanthemum × morifolium infected with Puccinia horiana, Gladiolus × hortulanus infected with Uromyces transversalis and Glycine max infected with Phakopsora pachyrhizi were tested alongside uninfected leaf tissue samples. The results of these tests show that this technique clearly distinguishes between rust pathogens and their respective host plant tissues. This ISH protocol is applicable to rust fungi and potentially other plant pathogenic fungi as well. It has been shown here that this protocol can be applied to pathogens from different genera of rust fungi with no background staining of plant tissue. We encourage the use of this protocol for the study of plant pathogenic fungi in paraffin embedded sections of host plant tissue.

Cytologic assessment of estrogen receptor, progesterone receptor, and HER2 status in metastatic breast carcinoma.

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Pareja, Fresia; Murray, Melissa P; Jean, Ryan Des; Konno, Fumiko; Friedlander, Maria; Lin, Oscar; Edelweiss, Marcia

2017-01-01

Discordance in the receptor status between primary breast carcinomas (PBC) and corresponding metastasis is well documented. Interrogation of the receptor status of metastatic breast carcinoma (MBC) in cytology material is common practice; however, its utility has not been thoroughly validated. We studied patients with MBC, and evaluated the concordance rates of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) between PBC surgical specimens and corresponding MBC cell blocks (CBs). We correlated the findings with clinicopathologic variables and with the fixation methods used. We searched for patients with MBC diagnosed on cytology from 2007 to 2009 and selected those with ER, PR and HER2 tested in both the PBC surgical specimens and the MBC CBs. We included CBs fixed in formalin and methanol based solution (CytoLyt®). All slides were reevaluated by cytopathologists. Clinical information was retrieved from the medical records. We studied 65 patients with PBC and MBC paired specimens. The concordance rates between PBC and MBC were 78.5%, 58.5% and 96.9%, for ER, PR and HER2, respectively. When discordant, PR status switched from positive (PBC) to negative (MBC) in most cases (23/27). The PR concordance rate was 45.2% for CBs fixed in formalin and 70.6% for those fixed with CytoLyt® (p=0.047). The ER, PR and HER2 concordance rates between the PBC and MBC CBs are similar to those reported in paired surgical specimens. PR status was the most prevalent discordance and was not accompanied by a switch in ER.

DNA methylation–based immune response signature improves patient diagnosis in multiple cancers

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Jeschke, Jana; Bizet, Martin; Calonne, Emilie; Dedeurwaerder, Sarah; Garaud, Soizic; Koch, Alexander; Larsimont, Denis; Salgado, Roberto; Van den Eynden, Gert; Willard Gallo, Karen; Defrance, Matthieu; Sotiriou, Christos

2017-01-01

BACKGROUND. The tumor immune response is increasingly associated with better clinical outcomes in breast and other cancers. However, the evaluation of tumor-infiltrating lymphocytes (TILs) relies on histopathological measurements with limited accuracy and reproducibility. Here, we profiled DNA methylation markers to identify a methylation of TIL (MeTIL) signature that recapitulates TIL evaluations and their prognostic value for long-term outcomes in breast cancer (BC). METHODS. MeTIL signature scores were correlated with clinical endpoints reflecting overall or disease-free survival and a pathologic complete response to preoperative anthracycline therapy in 3 BC cohorts from the Jules Bordet Institute in Brussels and in other cancer types from The Cancer Genome Atlas. RESULTS. The MeTIL signature measured TIL distributions in a sensitive manner and predicted survival and response to chemotherapy in BC better than did histopathological assessment of TILs or gene expression–based immune markers, respectively. The MeTIL signature also improved the prediction of survival in other malignancies, including melanoma and lung cancer. Furthermore, the MeTIL signature predicted differences in survival for malignancies in which TILs were not known to have a prognostic value. Finally, we showed that MeTIL markers can be determined by bisulfite pyrosequencing of small amounts of DNA from formalin-fixed, paraffin-embedded tumor tissue, supporting clinical applications for this methodology. CONCLUSIONS. This study highlights the power of DNA methylation to evaluate tumor immune responses and the potential of this approach to improve the diagnosis and treatment of breast and other cancers. FUNDING. This work was funded by the Fonds National de la Recherche Scientifique (FNRS) and Télévie, the INNOVIRIS Brussels Region BRUBREAST Project, the IUAP P7/03 program, the Belgian “Foundation against Cancer,” the Breast Cancer Research Foundation (BCRF), and the Fonds Gaston Ithier

Spectral embedding based active contour (SEAC): application to breast lesion segmentation on DCE-MRI

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Agner, Shannon C.; Xu, Jun; Rosen, Mark; Karthigeyan, Sudha; Englander, Sarah; Madabhushi, Anant

2011-03-01

Spectral embedding (SE), a graph-based manifold learning method, has previously been shown to be useful in high dimensional data classification. In this work, we present a novel SE based active contour (SEAC) segmentation scheme and demonstrate its applications in lesion segmentation on breast dynamic contrast enhance magnetic resonance imaging (DCE-MRI). In this work, we employ SE on DCE-MRI on a per voxel basis to embed the high dimensional time series intensity vector into a reduced dimensional space, where the reduced embedding space is characterized by the principal eigenvectors. The orthogonal eigenvector-based data representation allows for computation of strong tensor gradients in the spectrally embedded space and also yields improved region statistics that serve as optimal stopping criteria for SEAC. We demonstrate both analytically and empirically that the tensor gradients in the spectrally embedded space are stronger than the corresponding gradients in the original grayscale intensity space. On a total of 50 breast DCE-MRI studies, SEAC yielded a mean absolute difference (MAD) of 3.2+/-2.1 pixels and mean Dice similarity coefficient (DSC) of 0.74+/-0.13 compared to manual ground truth segmentation. An active contour in conjunction with fuzzy c-means (FCM+AC), a commonly used segmentation method for breast DCE-MRI, produced a corresponding MAD of 7.2+/-7.4 pixels and mean DSC of 0.58+/-0.32. In conjunction with a set of 6 quantitative morphological features automatically extracted from the SEAC derived lesion boundary, a support vector machine (SVM) classifier yielded an area under the curve (AUC) of 0.73, for discriminating between 10 benign and 30 malignant lesions; the corresponding SVM classifier with the FCM+AC derived morphological features yielded an AUC of 0.65.

Anisotropy of the apparent frequency dependence of backscatter in formalin fixed human myocardium.

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Hall, C S; Verdonk, E D; Wickline, S A; Perez, J E; Miller, J G

1997-01-01

Measurements of the frequency dependence of ultrasonic backscatter are presented for specific angles of insonification for regions of infarcted and noninfarcted human myocardium. A 5-MHz transducer was used to insonify cylindrical cores taken from 7 noninfarcted regions and 12 infarcted regions of the left ventricular free wall of 6 formalin-fixed human hearts explanted because of ischemic cardiomyopathy. The dependence of apparent (uncompensated for diffraction effects and attenuation) backscatter on frequency was approximated by a power-law dependence, magnitude of B(f)2 = afn. Under ideal conditions in a lossless medium, the effect of not compensating for the effects of diffraction and attenuation leads to the value of n to be 2.0 for Rayleigh scatterers while the frequency dependence of the fully compensated backscatter coefficient would be f4. The value of n was determined over the frequency range, 3-7 MHz. Both nonifarcted and infarcted myocardium exhibited anisotropy of the frequency dependence of backscatter, with maxima occurring at angles that were perpendicular to the predominant myofiber direction and minima when parallel to the fibers. Perpendicular insonification yielded results for n of 1.8 +/- 0.1 for noninfarcted myocardium and 1.2 +/- 0.1 for infarcted myocardium while parallel insonification yielded results of 0.4 +/- 0.1 for noninfarcted and 0.0 +/- 0.1 for infarcted myocardium. The functional form of the angle-dependent backscatter is similar for both noninfarcted and infarcted myocardium, although the frequency dependence is clearly different for both tissue states for all angles of insonification. The results of this study indicate that the anisotropy of the frequency dependence of backscatter may play a significant role in ultrasonic imaging and is an important consideration for ultrasonic tissue characterization in myocardium.

Molecular and immunohistochemical profiling of invasive micropapillary carcinoma of the breast

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Thomas A

2014-10-01

Full Text Available Alexandra Thomas,1 Ryan W Askeland,2 Natalya V Guseva,2 Ramakrishna Sompallae,2,3 Deqin Ma2 1Department of Internal Medicine, 2Department of Pathology, 3Bioinformatics Division, Iowa Institute of Human Genetics, University of Iowa Hospitals and Clinics, Iowa City, IA, USABackground: In this study, molecular and immunohistochemical profiling of invasive micropapillary carcinoma of the breast was used to identify potentially useful markers for targeted therapies with a focus on BRAF V600E mutation.Methods: Formalin-fixed, paraffin-embedded tumor blocks from seven patients were identified from the archives at our institution and tumor registry from 1997 to 2012. Massively parallel (Next-generation sequencing was performed using the Ion AmpliSeq™ Cancer Hotspot Panel version 2 (Life Technologies, Carlsbad, CA, USA. Mutation analysis for BRAF V600E was performed using a single nucleotide primer extension assay. Immunohistochemistry studies for estrogen receptor (ER, progesterone receptor (PR, Her2/Neu, phosphatase and tensin homolog (PTEN, and non-metastatic protein 23 homologue 1 (NM23H1 were performed using the same tumor blocks. Staining for ER, PR, and Her2/Neu was scored according to American Society of Clinical Oncology/College of American Pathologists guidelines, and a four-tier system, ie, strong homogenous, heterogeneous, positive with negative foci, reduced in more than 50%, and lost in all or majority was used for PTEN and NM23H1 staining.Results: No pathogenic mutations were identified in the tumors by next-generation sequencing. The lack of BRAF V600E mutation was confirmed by single nucleotide primer extension assay. All tumors were positive for ER and PR, and showed no overexpression of Her2/Neu. Loss of or reduced PTEN expression was observed in six of seven cases and was associated with lymph node metastasis. Reduced NM23H1 expression was observed in three of seven cases, all of which had concurrent PTEN loss.Conclusion: No somatic

Touch imprint cytology with massively parallel sequencing (TIC-seq): a simple and rapid method to snapshot genetic alterations in tumors.

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Amemiya, Kenji; Hirotsu, Yosuke; Goto, Taichiro; Nakagomi, Hiroshi; Mochizuki, Hitoshi; Oyama, Toshio; Omata, Masao

2016-12-01

Identifying genetic alterations in tumors is critical for molecular targeting of therapy. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissue is usually employed for genetic analysis. However, DNA extracted from FFPE tissue is often not suitable for analysis because of its low levels and poor quality. Additionally, FFPE sample preparation is time-consuming. To provide early treatment for cancer patients, a more rapid and robust method is required for precision medicine. We present a simple method for genetic analysis, called touch imprint cytology combined with massively paralleled sequencing (touch imprint cytology [TIC]-seq), to detect somatic mutations in tumors. We prepared FFPE tissues and TIC specimens from tumors in nine lung cancer patients and one patient with breast cancer. We found that the quality and quantity of TIC DNA was higher than that of FFPE DNA, which requires microdissection to enrich DNA from target tissues. Targeted sequencing using a next-generation sequencer obtained sufficient sequence data using TIC DNA. Most (92%) somatic mutations in lung primary tumors were found to be consistent between TIC and FFPE DNA. We also applied TIC DNA to primary and metastatic tumor tissues to analyze tumor heterogeneity in a breast cancer patient, and showed that common and distinct mutations among primary and metastatic sites could be classified into two distinct histological subtypes. TIC-seq is an alternative and feasible method to analyze genomic alterations in tumors by simply touching the cut surface of specimens to slides. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Preventing Paraffin-Related Injury

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Dehran Swart

2009-07-01

Full Text Available Paraffin (called kerosene in North America and other parts of the world is the most commonly used fuel in ‎non-electrified dwellings worldwide. It is especially popular in Africa and South Asia. Although paraffin ‎offers many advantages – especially its comparatively low cost to produce – it poses two major risks of ‎injury. First, paraffin poisoning is common, either through ingestion or through inhalation of smoke and ‎fumes. Second, paraffin is highly flammable, and poses fire risk through multiple causes. This commentary ‎discusses strategies to prevent paraffin-related injury. Prevention of paraffin-related injury must be through ‎multiple strategies, and should include policy-oriented change, changes to the safety of home environments, ‎and behavioral changes targeting how individuals store and use paraffin and paraffin appliances. We review ‎successful prevention strategies in each of these domains and discuss appropriate research and community ‎initiatives that should be implemented to improve paraffin safety among at-risk populations.‎

Small RNA sequencing reveals metastasis-related microRNAs in lung adenocarcinoma

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Daugaard, Iben; Venø, Morten T.; Yan, Yan

2017-01-01

The majority of lung cancer deaths are caused by metastatic disease. MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression and miRNA dysregulation can contribute to metastatic progression. Here, small RNA sequencing was used to profile the miRNA and piwi-interacting RNA (piRNA......) transcriptomes in relation to lung cancer metastasis. RNA-seq was performed using RNA extracted from formalin-fixed paraffin embedded (FFPE) lung adenocarcinomas (LAC) and brain metastases from 8 patients, and LACs from 8 patients without detectable metastatic disease. Impact on miRNA and piRNA transcriptomes...... was subtle with 9 miRNAs and 8 piRNAs demonstrating differential expression between metastasizing and non-metastasizing LACs. For piRNAs, decreased expression of piR-57125 was the most significantly associated with distant metastasis. Validation by RT-qPCR in a LAC cohort comprising 52 patients confirmed...

Efficient Identification of miRNAs for Classification of Tumor Origin

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Søkilde, Rolf; Vincent, Martin; Møller, Anne K

2014-01-01

Carcinomas of unknown primary origin constitute 3% to 5% of all newly diagnosed metastatic cancers, with the primary source difficult to classify with current histological methods. Effective cancer treatment depends on early and accurate identification of the tumor; patients with metastases...... of unknown origin have poor prognosis and short survival. Because miRNA expression is highly tissue specific, the miRNA profile of a metastasis may be used to identify its origin. We therefore evaluated the potential of miRNA profiling to identify the primary tumor of known metastases. Two hundred eight...... formalin-fixed, paraffin-embedded samples, representing 15 different histologies, were profiled on a locked nucleic acid-enhanced microarray platform, which allows for highly sensitive and specific detection of miRNA. On the basis of these data, we developed and cross-validated a novel classification...

Investigating intra-tumor heterogeneity and expression gradients of miR-21, miR-92a and miR-200c and their potential of predicting lymph node metastases in early colorectal cancer

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Jepsen, Rikke Karlin; Novotny, Guy Wayne; Klarskov, Louise Laurberg

2016-01-01

Introduction miR-21, miR-92a and miR-200c are regulators of pathways involved in migration, intravasation and metastasis, and their tumor expression levels have been proposed as potential prognostic markers in colorectal cancer (CRC). In two parallel cohorts we examine intra-tumor expression levels...... in early stage CRC tissue in order to determine intra-tumor heterogeneity, potential systematic intra-tumor expression gradients of the miRNAs and to investigate the association to metastatic disease in early stage CRC. Material and methods Two parallel studies on archived formalin-fixed paraffin......-embedded (FFPE) CRC tissue. Intra-tumor and inter-patient variances were analyzed in 9 early metastatic CRCs by measuring expression levels by qRT-PCR on isolated tissue samples from luminal, central and invasive border zones. Associations between miRNA expression levels and early metastasizing tumors...

p16 as a diagnostic marker of cervical neoplasia: a tissue microarray study of 796 archival specimens

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Lesnikova, Iana; Lidang, Marianne; Hamilton-Dutoit, Stephen

2009-01-01

from archival formalin fixed, paraffin-embedded donor tissues from 796 patients, and included cases of cervical intraepithelial neoplasia (CIN)1 (n = 249), CIN2 (n = 233), CIN3 (n = 181), and invasive cervical carcinoma (n = 133). p16INK4a expression was scored using two different protocols: 1......BACKGROUND: To evaluate the usefulness of this biomarker in the diagnosis of cases of cervical neoplasia we studied the immunohistochemical expression of p16INK4a in a large series of archival cervical biopsies arranged into tissue microarray format. METHODS: TMAs were constructed with tissue cores...... dysplasia or the presence of invasive carcinoma. CONCLUSION: Immunohistochemical analysis of p16INK4a expression is a useful diagnostic tool. Expression is related to the degree of histological dysplasia, suggesting that it may have prognostic and predicative value in the management of cervical neoplasia....

Prostate-specific antigen-positive extramammary Paget's disease--association with prostate cancer

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Hammer, Anne; Hager, Henrik; Steiniche, Torben

2008-01-01

Extramammary Paget's disease (EMPD) is a rare intraepidermal adenocarcinoma that primarily affects the anogenital region. Cases of EMPD reacting with PSA (prostate-specific antigen) have previously been associated with underlying prostate cancer. However, a recent case of EMPD in our department has...... led us to question the value of PSA as an indicator of underlying prostate cancer. Clinical and pathological data were obtained for 16 cases of EMPD. Formalin-fixed, paraffin-embedded tissue blocks from the primary skin lesions were investigated using PSA and other immunohistochemical markers. 5...... of the 16 cases of EMPD stained positive for PSA (2 women and 3 men). However, no reactivity was seen for the prostatic marker P501S. Three of the five patients had been diagnosed with internal malignant disease-two with prostate cancer, stage 1. Immunohistochemical investigations of the tumour specimens...

Distinction between papillary thyroid hyperplasia and papillary thyroid carcinoma by immunohistochemical staining for cytokeratin 19, galectin-3, and HBME-1.

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Casey, Mary B; Lohse, Christine M; Lloyd, Ricardo V

2003-01-01

The histopathology of papillary thyroid hyperplasia and papillary thyroid carcinoma is similar enough to cause a diagnostic dilemma in a few cases. Both lesions may have papillary fronds with fibrovascular cores, nuclear crowding, and nuclear anisocytosis. Formalin- fixed paraffin-embedded tissues from 30 randomly selected patients with papillary thyroid hyperplasia and an equal number from patients with papillary thyroid carcinoma were analyzed for expression of cytokeratin 19 (CK19), galectin-3, and HBME-1. Cases of papillary thyroid carcinoma had moderate to strong CK19, galectin-3, and HBME-1 reactivity although both CK19 and galectin-3 showed positive staining in a significant number of nonneoplastic thyroid cases. HBME-1 was uncommon in the nonneoplastic cases. These results indicate that HBME-1 may be useful in helping to distinguish papillary thyroid carcinoma from hyperplasia in diagnostically difficult cases.

Expression of the lysosomal-associated membrane protein-1 (LAMP-1) in astrocytomas

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Jensen, Stine S; Aaberg-Jessen, Charlotte; Christensen, Karina G

2013-01-01

Targeting of lysosomes is a novel therapeutic anti-cancer strategy for killing the otherwise apoptosis-resistant cancer cells. Such strategies are urgently needed for treatment of brain tumors, especially the glioblastoma, which is the most frequent and most malignant type. The aim of the present...... study was to investigate the presence of lysosomes in astrocytic brain tumors focussing also on the therapy resistant tumor stem cells. Expression of the lysosomal marker LAMP-1 (lysosomal-associated membrane protein-1) was investigated by immunohistochemistry in 112 formalin fixed paraffin embedded...... in the individual tumor grades. LAMP-1/GFAP showed pronounced co-expression and LAMP-1/CD133 was co-expressed as well suggesting that tumor cells including the proposed tumor stem cells contain lysosomes. The results suggest that high amounts of lysosomes are present in glioblastomas and in the proposed tumor stem...