Formalin-fixed And Paraffin-embedded Specimens Research Articles

Predominance of T-bet-positive Th1 cells in infiltrating T-lymphocytes in most of active arteritis lesions of giant cell arteritis.

Immunological mechanisms through the activation of CD4-positive T-cells have been assumed to be involved in the pathogenesis of giant cell arteritis (GCA). Many studies employing frozen tissues of temporal artery biopsy, peripheral blood lymphocytes, and plasma of GCA patients have revealed the contribution of interferon-γ and interleukin-17 in both protein and mRNA levels. However, the analyses using formalin-fixed and paraffin-embedded (FFPE) tissue specimens, in which the correlation between histopathologic pictures and immunological circumstances would be elucidated, have been limited. Here, we performed the immunohistochemical analyses of infiltrating small lymphocytes in GCA lesions using FFPE specimens, especially of the subsets of CD4-positive T-cells by immunohistochemistry with antibodies against T-bet, GATA-3, RORγT, and Foxp3, which is the differentiation-specific transcription factor for Th1, Th2, Th17, and Treg cells, respectively. In these slides, the nuclear-positive staining is much more clearly and easily identifiable than the cytoplasmic staining for cytokines. The results indicate the predominance of T-bet-positive Th1 cells in infiltrating T-cells in most of active arteritis lesions of GCA. Furthermore, our data suggest the possible immunosuppressive microenvironment induced by T-reg cells and M2-type macrophages in the arteritis lesions throughout the course of GCA inflammation.

Significance of micro-EGFR T790M mutations on EGFR-tyrosine kinase inhibitor efficacy in non-small cell lung cancer.

Small amounts of epidermal growth factor receptor (EGFR) T790M mutation (micro-T790M), which is detected using droplet digital PCR (ddPCR) but not conventional PCR, in formalin-fixed and paraffin-embedded (FFPE) samples have been investigated as a predictive factor for the efficacy of EGFR-tyrosine kinase inhibitors (TKIs). However, the predictive value of micro-T790M remains controversial, possibly owing to the failure to examine artificial T790M in FFPE specimens. Therefore, we examined the predictive value of micro-T790M in first-generation (1G), second-generation (2G), and third-generation (3G) EGFR-TKI efficacy using a new method to exclude FFPE-derived artificial mutations in our retrospective cohort. The primary objective was time to treatment failure (TTF) of 1G, 2G, and 3G EGFR-TKIs according to micro-T790M status. In total, 315 patients with EGFR-positive non-small cell lung cancer treated with 1G, 2G, and 3G EGFR-TKIs were included in this study. The proportion of patients positive formicro-T790M in the 1G, 2G, and 3G EGFR-TKI groups was 48.2%, 47.1%, and 47.6%, respectively. In the micro-T790M-positive group, the TTF was significantly longer in the 2G and 3G EGFR-TKI groups than in the 1G TKI group. No differences in the micro-T790M-negative group were observed. Micro-T790M status detected using ddPCR, eliminating false positives, may be a valuable predictor of EGFR-TKI efficacy.

Identification of a predictive biomarker and two pharmacodynamic biomarkers to ADG106 treatment, a novel anti-CD137 agonist antibody, in phase I clinical trials.

e14505 Background: ADG106 is a fully human agonistic anti-CD137 monoclonal IgG4 antibody that mediates anti-tumor activities via unique mechanisms of action. Here we provide safety and efficacy updates from our phase I trials and report the findings of a predictive biomarker and two pharmacodynamic biomarkers which correlate with patients’ clinical responses to ADG106 treatment and demonstrate target engagement, respectively. Methods: Formalin fixed and paraffin embedded (FFPE), blood and plasma specimens were collected from 92 patients enrolled in our phase I trials. We measured expression across a panel of protein biomarkers in FFPE specimens using three highly sensitive detection technologies: multiple immunohistochemical (IHC) staining of protein expression, the BD Multitest 6-color TBNK reagent for profiling immune cell subpopulations, and the MSD-ECL electrochemiluminescence assay for detection of soluble CD137. Objective tumor responses were determined using RECIST v1.1 for solid tumor patients and Lugano classification for lymphoma patients. Results: As of November 30, 2020, ADG106 has demonstrated a favorable safety profile and efficacy in the phase I clinical trials with a disease control rate of 56%. From a retrospective analysis of 28 pretreatment FFPE specimens, we identified a predictive biomarker that correlated with tumor shrinkage upon ADG106 treatment. We identified four biomarker positive specimens from two patients with lymphoma and two with solid tumors. Three out of four biomarker positive patients achieved greater than 30% tumor shrinkage after 3mg/kg or 5mg/kg ADG106 treatment. One biomarker positive patient with stable disease received a low dose ADG106 treatment at 0.5mg/kg during dose escalation. None of the 24 biomarker negative patients showed significant clinical response. A tissue microarray study confirmed expression of this predictive biomarker in a variety of tumor types suggesting a broad indication for ADG106 therapy. Our biomarker studies also demonstrated target engagement with increased NK cell proliferation and soluble CD137 upon ADG106 treatment. Analysis of safety, efficacy, PK and PD data allowed us to select a recommended dose for the upcoming phase II study. Conclusions: We identified a biomarker predictive of response to antitumor CD137 blockade by ADG106, as well as demonstrated the involvement of NK cells in ADG106 mediated anti-tumor activities. In upcoming phase II trials, we plan to enrich for populations expressing this predictive biomarker to demonstrate a clinical benefit to ADG106 therapy further validating early biomarker-based patient stratification. We will also explore the potential of selecting patients for combination treatment with anti-PD-1 therapies. Clinical trial information: NCT03802955.

Highly Sensitive Droplet Digital PCR Method for Detection of de novo EGFR T790M Mutation in Patients with Non-Small Cell Lung Cancer.

ObjectiveThe aim of this study was to investigate the allelic relation between de novo T790M and concomitant sensitizing EGFR mutations in EGFR-TKIs naïve NSCLCs and to explore whether the formalin-fixed and paraffin-embedded (FFPE) materials affect the detection of de novo EGFR T790M mutation.MethodsSpecimens of 300 consecutive EGFR-TKI naïve NSCLCs who received surgical resection between January 2016 and June 2018 were retrospectively investigated. All the snap-frozen tumor tissues from 300 NSCLCs were screened by droplet digital PCR (ddPCR) for the detection of de novo T790M mutation. The allelic relation between de novo T790M mutation and concomitant sensitizing EGFR mutations was also investigated. Furthermore, we assessed de novo T790M mutation in paired FFPE specimens of 50 patients which included tumor tissues and paired normal lung tissues of the pretreatment NSCLCs to investigate whether FFPE materials affect the detection of de novo T790M mutation.ResultsThe de novo T790M mutation was observed in four patients which included one patient of single de novo T790M mutation and three patients of de novo T790M mutation coexisting with L858R mutation. The incidence of de novo T790M in pretreatment NSCLCs who harboring EGFR mutations was 2.9% (4/139). All the de novo T790M mutations were detected in cis with the concomitant L858R mutations for the three NSCLCs. Our ddPCR method demonstrated that the frequency of de novo T790M mutation was ranging from 0.1% to 0.5% among 90% (45/50) of the FFPE tumor samples and 92% (46/50) of the paired FFPE adjacent normal lung samples. The frequency of de novo T790M mutation in the paired snap-frozen samples was all below 0.1%.ConclusionOur study demonstrated that most de novo T790M mutations were detected in cis with concomitant sensitizing mutations for pretreatment NSCLCs. Analytical cut-off of ddPCR assay for FFPE specimens should be validated carefully considering the possibility of FFPE-derived artificial gene mutations.

Quantitative Proteomics for Monitoring Renal Transplant Injury.

This study is aimed at developing a molecular diagnostics platform to enhance the interpretation of renal allograft biopsies using quantitative proteomic profiling of formalin-fixed and paraffin-embedded (FFPE) specimens. A quantitative proteomics platform composed of 1) an optimized FFPE protein sample preparation method, 2) a tandem mass tag TMT10-plex-based proteomic workflow, and 3) a systematic statistical analysis pipeline to reveal differentially expressed proteins has been developed. This platform is then tested on a small sample set (five samples per phenotype) to reveal proteomic signatures that can differentiate T-cell mediated rejection (TCMR) and polyomavirus BK nephropathy (BKPyVN) from healthy functionally stable kidney tissue (STA). Among 2798 quantified proteins, the expression levels of 740 BKPyVN and 638 TCMR associated proteins are significantly changed compared to STA specimens. Principal component analysis demonstrated good segregation of all three phenotypes investigated. Protein detection and quantitation are highly reproducible: replicate comparative analyses demonstrated 71-84% overlap of detected proteins, and the coefficient of variation for protein measurements is <15% in triplicate liquid chromatography-tandem mass spectrometry runs. Quantitative proteomics can be applied to archived FFPE specimens to differentiate different causes of renal allograft injury.

Development and validation of a DNA methylation panel for the prediction of outcome in node-positive penile squamous cell carcinoma after lymphadenectomy.

513 Background: Node-positive penile squamous cell carcinoma is lethal disease with heterogenous outcome after lymphadenectomy. Molecular prognostic factor are poorly tested in this population to individualize treatment. The aim of this study was to develop and validate the prognostic value of a DNA methylation panel in node-positive penile cancer after lymphadenectomy. Methods: Patients with node-positive penile squamous cell carcinoma, and received inguinal or ilioinguinal lymphadenectomy were eligible. The prognostic marker study had 3-stage design: first, discovery set of 23 frozen tissues was used to generate candidate CpG sites associated with overall survival; second, development set of 68 formalin-fixed and paraffin-embedded (FFPE) samples was used to construct a multivariable methylation panel for outcome stratification; third, validation set of 69 FFPE specimens from multi-center database was used to independently validate the performance of the prognostic marker. The study was approved by local ethnic committee. The primary endpoint was overall survival. The prognostic value of the DNA methylation panel value was evaluated by hazard ratio (HR), concordance index (C-index) and clinical benefit. Results: At discovery stage, 11 CpG sites were significantly associated with overall survival. In development set, we constructed a 3-CpGs-based prognostic panel for survival prediction. The HR of the panel (dichotomized using median cutoff) was 6.5 in multivariate analyses (p &lt; 0.001). Addition of methylation panel significantly improved the C-index of N stage from 0.64 to 0.75 (incremental C = 0.11, p &lt; 0.001). In the validation set, the methylation panel showed a HR of 7.8 in multivariate analyses. Addition of the molecular marker significantly improved the C-index of N stage from 0.69 to 0.78 (incremental C = 0.09, p &lt; 0.001). The main limitation was retrospective design. Conclusions: We developed and validated a methylation prognostic panel for node-positive penile cancer. The prognostic maker showed remarkable improvement in risk stratification, and need further evaluation in prospective setting.

Three-dimensional NMR microscopy of zebrafish specimens.

While zebrafish embryos in the first five days after fertilization are clear and amenable to optical analysis, older juveniles and adults are not, due to pigmentation development and tissue growth. Thus other imaging methods are needed to image adult specimens. NMR is a versatile tool for studies of biological systems and has been successfully used for in vivo zebrafish microscopy. In this work we use NMR microscopy (MRM) for assessment of zebrafish specimens, which includes imaging of formalin fixed (FF), formalin fixed and paraffin embedded (FFPE), fresh (unfixed), and FF gadolinium doped specimens. To delineate the size and shape of various organs we concentrated on 3D MRM. We have shown that at 7T a 3D NMR image can be obtained with isotropic resolution of 50μm/pxl within 10min and 25μm/pxl within 4h. Also, we have analyzed sources of contrast and have found that in FF specimens the best contrast is obtained by T1 weighting (3D FLASH, 3D FISP), whereas in FFPE specimens T2 weighting (3D RARE) is the best. We highlight an approach to perform segmentation of the organs in order to study morphological changes associated with mutations. The broader implication of this work is development of NMR methodology for high contrast and high resolution serial imaging and automated analysis of morphology of various zebrafish mutants.

Small RNA sequencing for profiling microRNAs in long-term preserved formalin-fixed and paraffin-embedded non-small cell lung cancer tumor specimens.

BackgroundThe preservation of microRNAs in formalin-fixed and paraffin-embedded (FFPE) tissue makes them particularly useful for biomarker studies. The utility of small RNA sequencing for microRNA expression profiling of FFPE samples has yet to be determined.MethodsTotal RNA was extracted from de-paraffinized and proteinase K-treated FFPE specimens (15–20 years old) of 8 human lung adenocarcinoma tumors by affinity chromatography on silica columns. MicroRNAs in the RNA preparations were quantified by the Illumina HiSeq 2000 sequencing platform with sequencing libraries prepared with the TruSeq Small RNA Sample Preparation Kit (version 2.0) to obtain unpaired reads of 50 b for small RNA fragments. MicroRNAs were also quantified using Agilent Human miRNA (release 16.0) microarrays that can detect 1,205 mature microRNAs and by quantitative reverse transcription (RT)-PCR assays.ResultsBetween 9.1–16.9 million reads were obtained by small RNA sequencing of extracted RNA samples. Of these, only 0.6–2.3% (mean = 1.5%) represented microRNAs. The sequencing method detected 454–625 microRNAs/sample (mean = 550) compared with 200–349 (mean = 286) microRNAs detected by microarray. In Spearman correlation analyses, the average correlation coefficient for the 126 microRNAs detected in all samples by both methods was 0.37, and >0.5 for 63 microRNAs. In correlation analyses of the sequencing- and RT-PCR-based measurements, the coefficients were 0.19–0.95 (mean = 0.73) and >0.7, respectively, for 7 of 9 examined microRNAs. The average inter-replicate Spearman correlation coefficient for the sequencing method was 0.81.ConclusionsSmall RNA sequencing can be used to obtain microRNA profiles of FFPE tissue specimens with performance characteristics similar to those of microarrays, in spite of the fragmentation of ribosomal and messenger RNAs that reduces the method's informative capacity. The accuracy of the method can conceivably be improved by increasing sequencing depth and/or depleting FFPE tissue RNAs of ribosomal RNA fragments.

Overview Of The Genomic Landscape Of High Risk Diffuse Large B-Cell Lymphoma Using Targeted DNA and RNA Sequencing

▪ BackgroundThe implementation of chemoimmunotherapy (R-CHOP) has dramatically improved the outcome of B-cell NHL particularly DLBCL (> 50% OS at 5 years with very few late relapses). Most failures to R-CHOP occur early (80% in the 1st 18 months), are not easily salvageable even with high dose therapy-ASCT and are not reliably predicted with current available prognostic models. There is a growing awareness of the molecular heterogeneity of DLBCL beyond well-established germinal center (GC) and non-GC subtypes. Additional genomic alterations include structural alterations (such as in the “double-hit” lymphoma (DHL) (harboring rearrangements or additional copies of MYC and BCL2 or less commonly BCL6); predominantly deregulated or activated driving pathways (ex NF-κB particularly associated with mutations in genes such as CARD11, CD79B or MYD88 in ABC subtype). The recent use of high-resolution technologies has revealed a much higher complexity of the mutational landscape in NHL. MethodsHere, we studied 35 DLBCL primary tumors from very poor outcome DLBCL patients with a median OS of 8.4 months (range .3-44.5 months). Targeted DNA and RNA sequencing using a NGS based genomic profiling assay was performed in a CLIA certified laboratory (Foundation Medicine) and genomic alterations were compared to those previously published for DLBCL: Genomic DNA and total RNA was isolated from 40µm of tissue from archived clinical formalin fixed and paraffin-embedded (FFPE) blocks. Illumina adaptor-ligated sequencing libraries were created and captured by solution hybridization using two custom DNA oligonucleotide baitsets targeting all coding exons of 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 272 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >810X unique coverage for DNA and >20,000,000 total pairs for RNA, to enable accurate detection of genomic alterations. ResultsWe identified 214 genomic alterations, an average of 6.1 per sample, in 71 genes: 87 substitutions/indels, 85 nonsense or frameshifting mutations resulting in truncation of known tumor suppressor genes, 4 gene amplifications, 15 homozygous gene deletions, and 25 translocations. In the 35 samples, 12 harbored immunoglobulin (Ig) translocations with MYC, BCL2 or BCL6 (2 MYC/IgH, 7 BCL2/IgH, and 6 BCL6/IgH or IgL) and RNA-seq allowed detection of 4 non-Ig partner BCL6 fusions, including RHOH-BCL6, ST6GAL1-BCL6, and GAPDH-BCL6. Alterations aggregated in pathways previously implicated in DLBCL pathogenesis, including chromatin modification (18%) and NF-κB signaling (16%). More than 50% of ABC-DLBCL and 27% of GCB-DLBCL carry somatic mutations in multiple genes, regulators of NF-κB signaling. Intriguingly, alterations in TP53 and MLL2 occurred at approximately double the frequency previously reported for DLBCL overall (32% vs. 17% and 47% vs 24%, respectively, Pasqualucci et al, Nature Genetics, 2011), reflecting likely the poor outcome population of our cohort. ConclusionsWe demonstrate technical feasibility of comprehensive genomic profiling of DLBCL from clinical FFPE specimens, with a low overall failure rate (1/36 samples for DNA, 2/36 for RNA), deep unique coverage and simultaneous detection of multiple rearrangements (BCL2, BCL6, Myc) using a combination of DNA and RNA sequencing. We additionally observe a potentially interesting enrichment of alterations in P53 and MLL2 in aggressive disease, with cohort expansion and more detailed genotype/phenotype correlative analysis ongoing. Disclosures:Wang:Foundation Medicine: Employment. Nahas:Foundation Medicine: Employment. Donahue:Foundation Medicine: Employment. He:Foundation Medicine: Employment. Otto:Foundation Medicine, Inc: Employment. Lipson:Foundation Medincine, Inc: Employment. Yelensky:Foundation Medicine, Inc: Employment. Ross:Foundation Medicine: Employment. Stephens:Foundation Medicine, Inc: Employment. Miller:Foundation Medicine: Employment.

Multiple Protein Analysis of Formalin-fixed and Paraffin-embedded Tissue Samples with Reverse phase Protein Arrays

Reverse-phase protein arrays (RPPAs) have become an important tool for the sensitive and high-throughput detection of proteins from minute amounts of lysates from cell lines and cryopreserved tissue. The current standard method for tissue preservation in almost all hospitals worldwide is formalin fixation and paraffin embedding, and it would be highly desirable if RPPA could also be applied to formalin-fixed and paraffin embedded (FFPE) tissue. We investigated whether the analysis of FFPE tissue lysates with RPPA would result in biologically meaningful data in two independent studies. In the first study on breast cancer samples, we assessed whether a human epidermal growth factor receptor (HER) 2 score based on immunohistochemistry (IHC) could be reproduced with RPPA. The results showed very good concordance between the IHC and RPPA classifications of HER2 expression. In the second study, we profiled FFPE tumor specimens from patients with adenocarcinoma and squamous cell carcinoma in order to find new markers for differentiating these two subtypes of non-small cell lung cancer. p21-activated kinase 2 could be identified as a new differentiation marker for squamous cell carcinoma. Overall, the results demonstrate the technical feasibility and the merits of RPPA for protein expression profiling in FFPE tissue lysates.

Potentially Prognostic miRNAs in HPV-Associated Oropharyngeal Carcinoma

Deregulation of miRNAs is associated with almost all human malignancies. Human papillomavirus (HPV)-associated oropharyngeal carcinoma (OPC) has a significantly more favorable outcome compared with HPV-negative OPCs; however, the underlying mechanisms are not well understood. Hence, the objectives of this study were to determine whether miRNA expression differed as a function of HPV status and to assess whether such miRNAs provide prognostic value beyond HPV status. Global miRNA profilings were conducted on 88 formalin-fixed and paraffin-embedded (FFPE) OPC biopsies (p16-positive: 56; p16-negative: 32), wherein the expression levels of 365 miRNAs plus 3 endogenous controls were simultaneously measured using quantitative real-time (qRT)-PCR. Seven FFPE specimens of histologically normal tonsils were used as controls. Overall, 224 miRNAs were expressed in more than 80% of the investigated samples, with 128 (57%) being significantly differentially expressed between tumor versus normal tissues (P < 0.05). Upregulated miR-20b, miR-9, and miR-9* were significantly associated with HPV/p16-status. Three miRNA sets were significantly associated with overall survival (miR-107, miR-151, miR-492; P = 0.0002), disease-free survival (miR-20b, miR-107, miR-151, miR-182, miR-361; P = 0.0001), and distant metastasis (miR-151, miR-152, miR-324-5p, miR-361, miR492; P = 0.0087), which retained significance even after adjusting for p16 status. The associated biologic functions of these miRNAs include immune surveillance, treatment resistance, invasion, and metastasis. We have identified several miRNAs, which associate with HPV status in OPC; furthermore, three candidate prognostic sets of miRNAs seem to correlate with clinical outcome, independent of p16 status. Furthermore, evaluations will offer biologic insights into the mechanisms underlying the differences between HPV-positive versus HPV-negative OPC.

Abstract 3161: Identification of HPV/p16-associated microRNAs in primary oropharyngeal carcinoma

Abstract Introduction: Deregulation of micro-RNAs (miR) is associated with oncogenesis of various malignancies, suggesting that miR expression profiling have the potential to unravel the complex biology of human tumours. It is now established that human papillomavirus (HPV)-associated oropharyngeal carcinoma (OPC) is a completely distinct entity, with a significantly more favourable outcome, compared to HPV-negative OPCs. The mechanisms underlying these differences currently remain incompletely understood; we therefore postulated that miR profilings might offer additional biological insights. Hence, the objectives of this study were to determine whether: a) there were miRs which associate with HPV (or p16) status; and b) there might be any association with clinical outcome, independent of HPV (or p16) status. Experimental Procedures: Archival formalin fixed and paraffin embedded (FFPE) diagnostic biopsy specimens were collected from 88 non-metastatic OPC samples (p16+ve:58; p16-ve:34), from patients who were treated for cure, as per institutional guidelines. Macrodissection was performed to ensure that each specimen contained &amp;gt;70% tumor cells. Seven FFPE specimens derived from histologically normal tonsils were used as controls. RNA was isolated using a commercially available extraction kit designed specifically for FFPE samples (Ambion). Expression levels of 365 miRs plus 3 endogenous controls were simultaneously measured by quantitative RT-PCR using the Human TaqMan Low Density Array (Applied Biosystem). Results: Four patient samples were excluded due to poor RNA quality. Overall, 224 miRs were expressed in &amp;gt;80% of both tumors and normal samples. Slightly more than half (128/224; 57%) of miRs were significantly differentially expressed between tumor and normal tissues (p&amp;lt;0.05); the majority of which (120/128; 94%) were up-regulated in tumor samples. The top 5 most significantly up-regulated miRs were miR-21, let-7g, miR-25, let-7f and miR-130b. It was noted that many of the deregulated miRs were located at known fragile sites of the chromosome. The top 5 most significantly p16-associated miRs included up-regulated miR-20b, miR-9, miR-9*, as well as down-regulated miR-193b and miR-23a. Further analyses identified three potential miR sets that associated with overall survival (miR-107, miR-151, miR-492; p=0.0002), disease free survival (miR-107, miR-151, miR-182, miR-20b, miR-361; p=0.0001), and distant metastasis (miR-324-5p, miR-151, miR-152, miR-361, miR492; p=0.0087), which retained significance even after adjusting for p16 status. Conclusions: We have identified a panel of p16/HPV-associated miRs in OPC, which appear to associate with clinical outcome, independent of p16 status. These data require further validation, but further evaluations of these miRs might offer biological insights into the mechanisms underlying the differences between HPV-positive vs. HPV-negative OPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3161. doi:1538-7445.AM2012-3161

1317 PROTEOMIC ANALYSIS OF LASER-MICRODISSECTED FORMALIN-FIXED AND PARAFFIN-EMBEDDED TISSUES OF LOW AND HIGH GLEASON SCORE AND METASTATIC PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research II1 Apr 20121317 PROTEOMIC ANALYSIS OF LASER-MICRODISSECTED FORMALIN-FIXED AND PARAFFIN-EMBEDDED TISSUES OF LOW AND HIGH GLEASON SCORE AND METASTATIC PROSTATE CANCER Takahiro Kimura, Toshihiro Yamamoto, Makoto Kihara, Yuko Kamata, Naoki Tanaka, Makiko Otsuji, Yasuhiko Bando, Sayaka Mikami, Bungo Furusato, Hiroyuki Takahashi, Toshihide Nishimura, and Shin Egawa Takahiro KimuraTakahiro Kimura Tokyo, Japan More articles by this author , Toshihiro YamamotoToshihiro Yamamoto Tokyo, Japan More articles by this author , Makoto KiharaMakoto Kihara Kanagawa, Japan More articles by this author , Yuko KamataYuko Kamata Tokyo, Japan More articles by this author , Naoki TanakaNaoki Tanaka Kanagawa, Japan More articles by this author , Makiko OtsujiMakiko Otsuji Kanagawa, Japan More articles by this author , Yasuhiko BandoYasuhiko Bando Tokyo, Japan More articles by this author , Sayaka MikamiSayaka Mikami Tokyo, Japan More articles by this author , Bungo FurusatoBungo Furusato Tokyo, Japan More articles by this author , Hiroyuki TakahashiHiroyuki Takahashi Tokyo, Japan More articles by this author , Toshihide NishimuraToshihide Nishimura Kanagawa, Japan More articles by this author , and Shin EgawaShin Egawa Tokyo, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1664AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES New biomarkers of prostate cancer (PCa) which distinguish between aggressive cancers with metastatic potential and cancers with low risk for progression are required. Although proteomic analysis is useful for discovery of cancer biomarkers, PCa has some difficulties. Collecting fresh and homogeneous cancer tissue from prostatectomy (RP) specimens is difficult because PCa is multifocal and heterogeneous and accuracy of pathological diagnosis of frozen section is limited. In present study, we performed shotgun LC/MS-based global proteomics using recently developed technique, laser-microdissection (LMD) of formalin-fixed and paraffin-embedded (FFPE) tissues to identify grade/stage related biomarkers from low and high Gleason score (GS) and metastatic PCa. METHODS The nanoLC-MS/MS proteomics was performed using FFPE tissues obtained from RP specimens with low GS, high GS and biopsy specimens with metastatic cancer (n=5). Normal epithelium from RP specimens were used as control. Targeted cells were microdissected and proteins were extracted, digested and subjected to LC/MS. Identified proteins were semi-quantified by spectral counting and subjected further to statistical analysis. Candidate proteins were extracted from 3D-scatter plot analysis based on G-test. Extracted candidates were validated using immunohistochemistry (IHC) in 92 RP specimens. RESULTS Total 371 proteins were identified. According to 3D-scatter plot analysis, we have extracted 11 proteins as low GS specific marker candidates and 23 proteins as high GS specific marker candidates. Validation analysis using IHC revealed that methylcrotonoyl-CoA carboxylase b (MCCC2) and fatty acid synthase (FASN) were significantly higher expressed in cancer cells comparing to normal cells (p>0.0001 and p>0.001, respectively). CONCLUSIONS We established the global clinical shotgun proteomics that could work excellently within the small patients number feasibility study in PCa. To achieve such performance, combination of LMD of FFPE specimens, high-resolution mass spectrometry, spectral counting method and 3D-scatter plot analysis are indispensable. MCCC2 and FASN were suggested as biomarkers with different expression profiles associated with grade/stage of PCa. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e534 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takahiro Kimura Tokyo, Japan More articles by this author Toshihiro Yamamoto Tokyo, Japan More articles by this author Makoto Kihara Kanagawa, Japan More articles by this author Yuko Kamata Tokyo, Japan More articles by this author Naoki Tanaka Kanagawa, Japan More articles by this author Makiko Otsuji Kanagawa, Japan More articles by this author Yasuhiko Bando Tokyo, Japan More articles by this author Sayaka Mikami Tokyo, Japan More articles by this author Bungo Furusato Tokyo, Japan More articles by this author Hiroyuki Takahashi Tokyo, Japan More articles by this author Toshihide Nishimura Kanagawa, Japan More articles by this author Shin Egawa Tokyo, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Tackling Formalin-Fixed, Paraffin-Embedded Tumor Tissue with Next-Generation Sequencing

Most tumor samples available for clinical genotyping are formalin-fixed and paraffin-embedded (FFPE), but there has been relatively little published on the suitability of such samples for next-generation sequencing approaches. A new study by Wagle and colleagues shows that a combination of hybridization-capture and deep sequencing yields high-quality data from FFPE specimens.

Poly (ADP-ribose) polymerase-1 (PARP) expression in triple-negative breast cancer.

1114 Background: Triple-negative breast cancer (TNBC) can affect germline BRCA 1 mutation carriers or BRCA1 wild-type patients; in both cases they lack expression of the biological markers used to select tailored therapy. According to BRCA1 status, tumour regression, relapse and resistance to therapy have different rates and are related to the ability of tumour cell to repair therapy induced DNA damage. In carriers of BRCA-1 mutations, homologous recombination mechanisms are usually inhibited; moreover base excision repair, a single strand repair mechanism in which PARP plays a pivotal role, can be down-regulated by PARP inhibitors. These drugs are actually considered potentially useful in carriers of BRCA-1 mutations. The goal of this study was the evaluation of quantitative expression of PARP1 in patients with BRCA-1 mutation compared to patients with BRCA1 wild type developing TNBC. Methods: We studied 25 TNBC –BRCA wild-type and 20 TNBC-BRCA1 mutated patients. RNA was extracted from unfixed and formalin-fixed and paraffin embedded (FFPE) tumor specimens in RNase-free laboratory conditions. PARP1 relative quantity (RQ) was evaluated by a quantitative RT-PCR assay. Results: The levels of PARP1 expression from FFPE specimens were confirmed on unfixed specimens. In TNBC, RQ ranged from 3,29 to 34,39; in BRCA1-mutated RQ ranged from 0 to 140. Although different RQ range, the two groups show similar distribution and RQ values overlap on log10 scale graph. Student’s t test (BRCA1 mut /BRCA1 wt): Number of samples 20/25; Media 27,4/36,5; Standard DEV 12,51/29,7; degrees of freedom 43; p Value 0,206 t: 1,28. Conclusions: The expression of PARP1 was similar and heterogeneous in the two groups. These preliminary observations could favour the hypothesis of offering PARP inhibitors to TNBC as it is proposed in BRCA1 mutated patients. This assay could be used prospectively as a predictive parameter for response to PARP1 inhibitor therapy.

Fixation of Brain Tumor Biopsy Specimens with RCL2 Results in Well‐Preserved Histomorphology, Immunohistochemistry and Nucleic Acids

RCL2 is an alcohol-based fixative reported to preserve histomorphology and nucleic acids in non-CNS neoplasms. We compared histomorphology, immunohistochemistry, DNA and RNA in brain tumor specimens preserved frozen at -80°C, and after formalin or RCL2 fixation. RCL2-fixed and paraffin-embedded (RCLPE) samples showed well-preserved histomorphology and specific immunoreactivity comparable to formalin-fixed and paraffin-embedded (FFPE) specimens testing a broad panel of antibodies. In all the analyzed cases, high-molecular weight DNA (up to a fragment length of 600 bp) was amplifyable from RCLPE samples, even after prolonged fixation times. Beta-actin (ACTB) and O6-methylguanine-methyltransferase (MGMT) gene concentrations were significantly higher in DNA isolated from RCLPE specimens as compared with FFPE specimens. Testing of MGMT promoter methylation status using methylation-specific polymerase-chain reaction (MSP) yielded conclusive results in 8/8 analyses in RCLPE and 6/8 analyses in FFPE material, respectively. Amplification of three reference genes (ABL, RAR-alpha, BCR-1) from cDNA showed good RNA preservation in frozen and RCLPE tissue specimens and significant RNA degradation in all FFPE samples. In conclusion, RCL2 fixation of brain tumor biopsies does not seem to significantly compromise histological tumor typing or immunohistochemistry and preserves nucleic acids (DNA and RNA) at a better quality than formalin fixation.

A Gynecologic Oncology Group study of frequent copy number aberrations in DNA repair genes and other genomic regions in stage I serous ovarian cancers

e16504 Background: A proof of principle array-based comparative genomic hybridization (aCGH) analysis was performed in archival formalin-fixed and paraffin-embedded (FFPE) stage I ovarian cancers (EOC) to determine if frequent (&gt;40%) copy number aberrations (CNAs) can be detected in DNA repair genes including the Fanconi anemia complementation group (FANC) and RAD51 families compared with the rest of the genome. Methods: Tumor DNA was isolated from 22 serous cancers from the GOG-175 virtual tissue bank. RPCI 19K BAC arrays were hybridized (GeneTAC HybStation) and scanned (Gene Pix 4200AL Laser Scanner). Spot fluorescence values were quantified using ImaGene, vetted for quality and loess corrected with adjustments for chip-specific spatial effects. The genome was segmented to identify regions with common copy number means (DNAcopy software). Posterior aberration probabilities for the regions were obtained using CGHcall and data was visualized and annotated using iGenomicViewer in R. Results: Several genes associated with the Fanconi DNA-damage response pathway were frequently altered in stage I serous ovarian carcinomas. A RAD51 homology DMC1 was amplified in 55% of the specimens. Genomic losses were observed in FANC-D1 (BRCA2), and RAD51L3 in 41%, and 27% of specimens, respectively. In contrast, frequent genomic losses or gains involved 13q33.1; 13 q21.31; 17p12; 17q22; 18q12.3; 9p11.2; 9p22.2–22.3; 9q33.1; 8p23.2; 21q21.3; and 5q14.2; or 8q24.3; 3q29; 12p11.1; 17q25.1; 17q25.3; 20q13.33; 20q11.21; 20q11.23; 19p13.13; and 19p13.2, respectively. Conclusions: The GOG-0175 virtual tissue bank yielded high quality DNA for detecting and mapping CNAs in archival FFPE specimens with high resolution. Frequent genomic losses and gains were observed in DNA repair genes and other genomic regions in stage I serous ovarian cancer which may promote genomic instability, resistance, metastasis and aggressiveness of this disease. No significant financial relationships to disclose.

MicroRNA Expression in Melanocytic Nevi: The Usefulness of Formalin-Fixed, Paraffin-Embedded Material for miRNA Microarray Profiling

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.

Desmoplastic Small Round Cell Tumor: A Clinicopathologic, Immunohistochemical and Molecular Study of Four Patients

Desmoplastic small round cell tumor (DSRCT) is a rare but highly aggressive tumor that usually occurs in the abdominal cavity of young males. Immunohistochemical staining plays an important role in the differentiation of DSRCT from other small round cell neoplasms. Recently, a novel translocation of chromosome 11 and 22 [t(11;22)(p13+q12)] was identified in DSRCTs, which could result in the formation of the chimeric EWS-WT1 fusion gene. Reverse transcriptasepolymerase chain reaction (RT-PCR) has been applied to detect the fusion gene in fresh tissue and confirm the diagnosis. Only a few studies using RT-PCR with formalin-fixed and paraffin-embedded (FFPE) tissue sections to detect the specific transcript have been reported. We collected four patients who had been diagnosed with DSRCT from the archives of the Department of Pathology, National Taiwan University Hospital, between 1996 and 2006. The clinical information and histologic sections of the patients were reviewed. Panel of primary antibodies including cytokeratin, vimentin, desmin and WT-1 were used for immunohistochemical staining. RNA extracted from the FFPE tissue sections was used for RT-PCR to identify the transcript of EWS-WT1. All four patients were male, with a mean age of 24 years. All of the tumors originated in the abdominal cavity and liver metastases were found in two patients. Two patients died of disease within 2 years, and the other two remained alive with disease. Histologically, the tumors were composed of nests of primitive small round cells within a desmoplastic stroma. Immunohistochemically, all the tumor cells were reactive to cytokeratin, vimentin, desmin and WT-1. The characteristic EWS-WT1 fusion gene could be identified in the FFPE specimens of all three patients who had tissue sections available for RT-PCR analysis. DSRCT is an aggressive tumor that usually occurs in the peritoneum of young males. The tumor is characterized by the expression of cytokeratin, vimentin, desmin and WT-1. RT-PCR, using paraffin-embedded tissue sections, can effectively detect the characteristic EWS-WT1 fusion gene transcript.

Detection of Mycobacterium tuberculosis Complex in Formalin-Fixed, Paraffin-Embedded Tissue Specimens with Necrotizing Granulomatous Inflammation by Strand Displacement Amplification

Rapid, reliable diagnosis of tuberculosis is essential to initiate correct treatment, avoid severe complications, and prevent transmission. Conventional microbiological methods may not be an option if samples are formalin-fixed and paraffin-embedded (FFPE) for histopathological examination. With the demonstration of necrotizing granulomatous inflammation, tuberculosis becomes an important differential diagnosis, although it was not initially suspected. Following paraffin extraction, BDProbeTec ET strand displacement amplification for detection of Mycobacterium tuberculosis complex (MTC) was applied to 47 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 was 100%. For 27 of the patients with prospectively collected FFPE specimens, culture was performed on a specimen collected at a later date from the same location. Culture revealed MTC in 14 and nontuberculous mycobacteria in four. BDProbeTec ET was positive in 13 (92.8%) of the patients with positive MTC 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 supplementary method for patients where tuberculosis was not initially suspected.