Comprehensive Determination of Prostate Tumor ETS Gene Status in Clinical Samples Using the CLIA Decipher Assay
2017; Elsevier BV; Volume: 19; Issue: 3 Linguagem: Inglês
10.1016/j.jmoldx.2017.01.007
ISSN1943-7811
AutoresAlba Torres, Mohammed Alshalalfa, Scott A. Tomlins, Nicholas Erho, Ewan A. Gibb, Jijumon Chelliserry, Lony Lim, Lucia L.C. Lam, Sheila F. Faraj, Stephania Martins Bezerra, Elai Davicioni, Kasra Yousefi, Ashley E. Ross, George J. Netto, Edward M. Schaeffer, Tamara L. Lotan,
Tópico(s)Molecular Biology Techniques and Applications
ResumoETS family gene fusions are common in prostate cancer and molecularly define a tumor subset. ERG is the most commonly rearranged, leading to its overexpression, followed by ETV1, ETV4, and ETV5, and these alterations are generally mutually exclusive. We validated the Decipher prostate cancer assay to detect ETS alterations in a Clinical Laboratory Improvement Amendments–accredited laboratory. Benchmarking against ERG immunohistochemistry and ETV1/4/5 RNA in situ hybridization, we examined the accuracy, precision, and reproducibility of gene expression ETS models using formalin-fixed, paraffin-embedded samples. The m-ERG model achieved an area under curve of 95%, with 93% sensitivity and 98% specificity to predict ERG immunohistochemistry status. The m-ETV1, -ETV4, and -ETV5 models achieved areas under curve of 98%, 88%, and 99%, respectively. The models had 100% robustness for ETS status, and scores were highly correlated across sample replicates. Models predicted 41.5% of a prospective radical prostatectomy cohort (n = 4036) to be ERG+, 6.3% ETV1+, 1% ETV4+, and 0.4% ETV5+. Of prostate tumor biopsy samples (n = 509), 41.2% were ERG+, 8.6% ETV1+, 0.4% ETV4+, and none ETV5+. Higher Decipher risk status tumors were more likely to be ETS+ (ERG or ETV1/4/5) in the radical prostatectomy and the biopsy cohorts (P < 0.05). These results support the utility of microarray-based ETS status prediction models for molecular classification of prostate tumors. ETS family gene fusions are common in prostate cancer and molecularly define a tumor subset. ERG is the most commonly rearranged, leading to its overexpression, followed by ETV1, ETV4, and ETV5, and these alterations are generally mutually exclusive. We validated the Decipher prostate cancer assay to detect ETS alterations in a Clinical Laboratory Improvement Amendments–accredited laboratory. Benchmarking against ERG immunohistochemistry and ETV1/4/5 RNA in situ hybridization, we examined the accuracy, precision, and reproducibility of gene expression ETS models using formalin-fixed, paraffin-embedded samples. The m-ERG model achieved an area under curve of 95%, with 93% sensitivity and 98% specificity to predict ERG immunohistochemistry status. The m-ETV1, -ETV4, and -ETV5 models achieved areas under curve of 98%, 88%, and 99%, respectively. The models had 100% robustness for ETS status, and scores were highly correlated across sample replicates. Models predicted 41.5% of a prospective radical prostatectomy cohort (n = 4036) to be ERG+, 6.3% ETV1+, 1% ETV4+, and 0.4% ETV5+. Of prostate tumor biopsy samples (n = 509), 41.2% were ERG+, 8.6% ETV1+, 0.4% ETV4+, and none ETV5+. Higher Decipher risk status tumors were more likely to be ETS+ (ERG or ETV1/4/5) in the radical prostatectomy and the biopsy cohorts (P < 0.05). These results support the utility of microarray-based ETS status prediction models for molecular classification of prostate tumors. The landscape of somatic genomic alterations in primary prostate cancer has largely been elucidated.1Cancer Genome Atlas Research NetworkThe molecular taxonomy of primary prostate cancer.Cell. 2015; 163: 1011-1025Abstract Full Text Full Text PDF PubMed Scopus (1842) Google Scholar The most common alterations are gene rearrangements resulting in overexpression of ETS gene family transcription factors, including ERG most commonly, and ETV1, ETV4, ETV5, and FLI1.1Cancer Genome Atlas Research NetworkThe molecular taxonomy of primary prostate cancer.Cell. 2015; 163: 1011-1025Abstract Full Text Full Text PDF PubMed Scopus (1842) Google Scholar, 2Tomlins S.A. Rhodes D.R. Perner S. Dhanasekaran S.M. Mehra R. Sun X.W. Varambally S. Cao X. Tchinda J. Kuefer R. Lee C. Montie J.E. Shah R.B. Pienta K.J. Rubin M.A. Chinnaiyan A.M. 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Rubin M.A. TMPRSS2:ERG fusion-associated deletions provide insight into the heterogeneity of prostate cancer.Cancer Res. 2006; 66: 8337-8341Crossref PubMed Scopus (431) Google Scholar or by immunohistochemistry (IHC) to detect ERG protein overexpression,6Pettersson A. Graff R.E. Bauer S.R. Pitt M.J. Lis R.T. Stack E.C. Martin N.E. Kunz L. Penney K.L. Ligon A.H. Suppan C. Flavin R. Sesso H.D. Rider J.R. Sweeney C. Stampfer M.J. Fiorentino M. Kantoff P.W. Sanda M.G. Giovannucci E.L. Ding E.L. Loda M. Mucci L.A. The TMPRSS2:ERG rearrangement, ERG expression, and prostate cancer outcomes: a cohort study and meta-analysis.Cancer Epidemiol Biomarkers Prev. 2012; 21: 1497-1509Crossref PubMed Scopus (247) Google Scholar, 24Chaux A. Albadine R. Toubaji A. Hicks J. Meeker A. Platz E.A. De Marzo A.M. Netto G.J. 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The TMPRSS2:ERG rearrangement, ERG expression, and prostate cancer outcomes: a cohort study and meta-analysis.Cancer Epidemiol Biomarkers Prev. 2012; 21: 1497-1509Crossref PubMed Scopus (247) Google Scholar, 24Chaux A. Albadine R. Toubaji A. Hicks J. Meeker A. Platz E.A. De Marzo A.M. Netto G.J. Immunohistochemistry for ERG expression as a surrogate for TMPRSS2-ERG fusion detection in prostatic adenocarcinomas.Am J Surg Pathol. 2011; 35: 1014-1020Crossref PubMed Scopus (129) Google Scholar However, the lack of reliable antibodies for ETV1, ETV4, and ETV5 has meant that RNA in situ hybridization (RISH) is the only reliable method to detect fusions involving these genes in clinical specimens.26Kunju L.P. Carskadon S. Siddiqui J. Tomlins S.A. Chinnaiyan A.M. Palanisamy N. Novel RNA hybridization method for the in situ detection of ETV1, ETV4, and ETV5 gene fusions in prostate cancer.Appl Immunohistochem Mol Morphol. 2014; 22: e32-e40Crossref PubMed Scopus (21) Google Scholar Thus, multiple assays and different methods are required to fully characterize ETS status in FFPE clinical material, and this presents a challenge for many diagnostic pathology laboratories. To address this issue, we validated a method for comprehensive determination of prostate tumor ETS status in FFPE tissue using a clinical high-resolution gene expression microarray assay, the Decipher prostate cancer classifier.27Erho N. Crisan A. Vergara I.A. Mitra A.P. Ghadessi M. Buerki C. Bergstralh E.J. Kollmeyer T. Fink S. Haddad Z. Zimmermann B. Sierocinski T. Ballman K.V. Triche T.J. Black P.C. Karnes R.J. Klee G. Davicioni E. Jenkins R.B. Discovery and validation of a prostate cancer genomic classifier that predicts early metastasis following radical prostatectomy.PLoS One. 2013; 8: e66855Crossref PubMed Scopus (425) Google Scholar, 28Ross A.E. Johnson M.H. Yousefi K. Davicioni E. Netto G.J. Marchionni L. Fedor H.L. Glavaris S. Choeurng V. Buerki C. Erho N. Lam L.L. Humphreys E.B. Faraj S. Bezerra S.M. Han M. Partin A.W. Trock B.J. Schaeffer E.M. Tissue-based genomics augments post-prostatectomy risk stratification in a natural history cohort of intermediate- and high-risk men.Eur Urol. 2016; 69: 157-165Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 29Knudsen B.S. Kim H.L. Erho N. Shin H. Alshalalfa M. Lam L.L. Tenggara I. Chadwich K. Van Der Kwast T. Fleshner N. Davicioni E. Carroll P.R. Cooperberg M.R. Chan J.M. Simko J.P. Application of a clinical whole-transcriptome assay for staging and prognosis of prostate cancer diagnosed in needle core biopsy specimens.J Mol Diagn. 2016; 18: 395-406Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar Decipher is run on the Affymetrix Human Exon 1.0 ST microarray, and beyond determination and reporting of a validated metastasis signature, genome-wide gene expression data are automatically collected on all cases, enabling simultaneous comprehensive assessment of ETS status along with tumor risk score. We previously developed and validated a model to predict ERG DNA-FISH status from Decipher Genomics Resource Information Database (GRID) gene expression microarray data in a set of primary prostate tumors from the Mayo Clinic.9Tomlins S.A. Alshalalfa M. Davicioni E. Erho N. Yousefi K. Zhao S. Haddad Z. Den R.B. Dicker A.P. Trock B.J. DeMarzo A.M. Ross A.E. Schaeffer E.M. Klein E.A. Magi-Galluzzi C. Karnes R.J. Jenkins R.B. Feng F.Y. Characterization of 1577 primary prostate cancers reveals novel biological and clinicopathologic insights into molecular subtypes.Eur Urol. 2015; 68: 555-567Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar Here, we expand to include models for ETV1, ETV4, and ETV5 status and validate the four models against gold standard IHC and RISH in a total of 456 prostate tumor cases from the Johns Hopkins Hospitals. Finally, we interrogate ETS status in >4500 clinical prostate tumor samples run on the Decipher GRID transcriptome database. After institutional review board approval, a total of 456 unique patients were selected from two overlapping and previously published radical prostatectomy (RP) cohorts at Johns Hopkins.28Ross A.E. Johnson M.H. Yousefi K. Davicioni E. Netto G.J. Marchionni L. Fedor H.L. Glavaris S. Choeurng V. Buerki C. Erho N. Lam L.L. Humphreys E.B. Faraj S. Bezerra S.M. Han M. Partin A.W. Trock B.J. Schaeffer E.M. Tissue-based genomics augments post-prostatectomy risk stratification in a natural history cohort of intermediate- and high-risk men.Eur Urol. 2016; 69: 157-165Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 30Johnson M.H. Ross A.E. Alshalalfa M. Erho N. Yousefi K. Glavaris S. Fedor H. Han M. Faraj S.F. Bezerra S.M. Netto G. Partin A.W. Trock B.J. Davicioni E. Schaeffer E.M. SPINK1 defines a molecular subtype of prostate cancer in men with more rapid progression in an at risk, natural history radical prostatectomy cohort.J Urol. 2016; 196: 1436-1444Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 31Faraj S.F. Bezerra S.M. Yousefi K. Fedor H. Glavaris S. Han M. Partin A.W. Humphreys E. Tosoian J. Johnson M.H. Davicioni E. Trock B.J. Schaeffer E.M. Ross A.E. Netto G.J. Clinical validation of the 2005 ISUP Gleason grading system in a cohort of intermediate and high risk men undergoing radical prostatectomy.PLoS One. 2016; 11: e0146189Crossref Scopus (8) Google Scholar These cohorts were originally designed in Decipher validation studies to test for prognostic markers and were highly enriched for adverse oncologic outcomes. Tumor tissue from the dominant tumor nodule and benign tissue were sampled in quadruplicate on 16 individual tissue microarrays (TMAs) using 0.6-mm cores. Tissues were simultaneously punched with a 1-mm punch for Decipher assay. Expression profiles from 536 benign prostate tissues at RP from Decipher GRID and 65 LNCaP expression profiles were used as controls. LNCaP cells are negative for ERG rearrangement, but they harbor a rearrangement inserting the entire ETV1 locus into a transcriptionally active locus on chromosome 14.4Tomlins S.A. Laxman B. Dhanasekaran S.M. Helgeson B.E. Cao X. Morris D.S. Menon A. Jing X. Cao Q. Han B. Yu J. Wang L. Montie J.E. Rubin M.A. Pienta K.J. Roulston D. Shah R.B. Varambally S. Mehra R. Chinnaiyan A.M. Distinct classes of chromosomal rearrangements create oncogenic ETS gene fusions in prostate cancer.Nature. 2007; 448: 595-599Crossref PubMed Scopus (652) Google Scholar For model precision evaluation, 110 FFPE tumor samples from 11 patients (10 each) were profiled. These samples were run in different batches, with different operators and using different reagent lots. For model evaluation in prospective samples, we evaluated de-identified expression profiles from 4036 prospective RP tumor samples and 509 prospective biopsy samples available for research in the Decipher GRID. RNA extraction from FFPE tissues, amplification, labeling, and hybridization to Affymetrix Human Exon 1.0 ST microarrays was performed in a Clinical Laboratory Improvement Amendments (CLIA)-certified clinical laboratory using the Decipher prostate cancer classifier (GenomeDx Biosciences, San Diego, CA) as described previously.27Erho N. Crisan A. Vergara I.A. Mitra A.P. Ghadessi M. Buerki C. Bergstralh E.J. Kollmeyer T. Fink S. Haddad Z. Zimmermann B. Sierocinski T. Ballman K.V. Triche T.J. Black P.C. Karnes R.J. Klee G. Davicioni E. Jenkins R.B. Discovery and validation of a prostate cancer genomic classifier that predicts early metastasis following radical prostatectomy.PLoS One. 2013; 8: e66855Crossref PubMed Scopus (425) Google Scholar, 28Ross A.E. Johnson M.H. Yousefi K. Davicioni E. Netto G.J. Marchionni L. Fedor H.L. Glavaris S. Choeurng V. Buerki C. Erho N. Lam L.L. Humphreys E.B. Faraj S. Bezerra S.M. Han M. Partin A.W. Trock B.J. Schaeffer E.M. Tissue-based genomics augments post-prostatectomy risk stratification in a natural history cohort of intermediate- and high-risk men.Eur Urol. 2016; 69: 157-165Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 30Johnson M.H. Ross A.E. Alshalalfa M. Erho N. Yousefi K. Glavaris S. Fedor H. Han M. Faraj S.F. Bezerra S.M. Netto G. Partin A.W. Trock B.J. Davicioni E. Schaeffer E.M. SPINK1 defines a molecular subtype of prostate cancer in men with more rapid progression in an at risk, natural history radical prostatectomy cohort.J Urol. 2016; 196: 1436-1444Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar The SCAN algorithm was used for individual patient profile pre-processing and normalization. ERG IHC was performed on the Ventana Benchmark autostaining system using a rabbit monoclonal antibody (EPR3864, C-terminal) after antigen retrieval in CC1 buffer followed by detection with the Optiview HRP system (Roche/Ventana Medical Systems, Tucson, AZ). One important caveat to note for this antibody is that it reacts with the C-terminal of the protein and can cross-react with FLI1,32Tomlins S.A. Palanisamy N. Brenner J.C. Stall J.N. Siddiqui J. Thomas D.G. Lucas D.R. Chinnaiyan A.M. Kunju L.P. Usefulness of a monoclonal ERG/FLI1 antibody for immunohistochemical discrimination of Ewing family tumors.Am J Clin Pathol. 2013; 139: 771-779Crossref PubMed Scopus (28) Google Scholar another ETS gene documented to be rearranged in a small number of prostate cancer cases.5Paulo P. Barros-Silva J.D. Ribeiro F.R. Ramalho-Carvalho J. Jeronimo C. Henrique R. Lind G.E. Skotheim R.I. Lothe R.A. Teixeira M.R. FLI1 is a novel ETS transcription factor involved in gene fusions in prostate cancer.Genes Chromosomes Cancer. 2012; 51: 240-249Crossref PubMed Scopus (67) Google Scholar However, on comparison of ERG immunostaining with EPR3864 and a mouse monoclonal anti-ERG antibody reacting to the N-terminus (9FY; Biocare Medical, Concord CA) which does not cross-react with FLI1, we found that only 1 of 120 cases showed discordance between the two antibodies, suggesting that cross-reactivity of EPR3864 with FLI1 may rarely be a problem. Each tissue microarray spot containing tumor was visually dichotomously scored for the presence or absence of nuclear ERG signal by a urologic pathologist blinded to the gene expression data (T.L.L.). A spot was considered to be ERG+ if any tumor nuclei showed ERG positivity, using endothelial cells as an internal positive control in all cases. A tumor was considered ERG+ if all sampled spots were scored as ERG+, and as ERG− if all sampled spots were scored as ERG− (Figure 1). In the case of mixed scoring in a given tumor (some spots negative and some spots positive, 3.5% of cases, 16 of 456 cases), the case was excluded from the validation because of heterogeneity. This low percentage of cases that are heterogeneous for ERG status is consistent with what has been reported in other studies, and these cases may represent collision tumors.5Paulo P. Barros-Silva J.D. Ribeiro F.R. Ramalho-Carvalho J. Jeronimo C. Henrique R. Lind G.E. Skotheim R.I. Lothe R.A. Teixeira M.R. FLI1 is a novel ETS transcription factor involved in gene fusions in prostate cancer.Genes Chromosomes Cancer. 2012; 51: 240-249Crossref PubMed Scopus (67) Google Scholar, 12Gumuskaya B. Gurel B. Fedor H. Tan H.L. Weier C.A. Hicks J.L. Haffner M.C. Lotan T.L. De Marzo A.M. Assessing the order of critical alterations in prostate cancer development and progression by IHC: further evidence that PTEN loss occurs subsequent to ERG gene fusion.Prostate Cancer Prostatic Dis. 2013; 16: 209-215Crossref PubMed Scopus (50) Google Scholar, 13Fontugne J. Davis K. Palanisamy N. Udager A. Mehra R. McDaniel A.S. Siddiqui J. Rubin M.A. Mosquera J.M. Tomlins S.A. Clonal evaluation of prostate cancer foci in biopsies with discontinuous tumor involvement by dual ERG/SPINK1 immunohistochemistry.Mod Pathol. 2016; 29: 157-165Crossref PubMed Scopus (22) Google Scholar, 33Krohn A. Freudenthaler F. Harasimowicz S. Kluth M. Fuchs S. Burkhardt L. Stahl P. C Tsourlakis M. Bauer M. Tennstedt P. Graefen M. Steurer S. Sirma H. Sauter G. Schlomm T. Simon R. Minner S. Heterogeneity and chronology of PTEN deletion and ERG fusion in prostate cancer.Mod Pathol. 2014; 27: 1612-1620Crossref PubMed Scopus (55) Google Scholar To evaluate reproducibility of the ERG IHC, a total of 132 prostate tumors from the Hopkins cohort were independently sampled in duplicate on more than one TMA. Of these, 127 had interpretable ERG IHC results, with 95.3% (121 of 127) showing agreement in ERG status across the two TMA sets. Chromogenic in situ hybridization for ETV1/4/5 RNA was performed with the RNAscope FFPE kit 2.5 from Advanced Cell Diagnostics (Hayward, CA) according to the manufacturer's recommendations. ETV1 (NM_004956), ETV4 (NM_001986.2), and ETV5 (NM_004454.2) probes, validated in a recent study, were used.26Kunju L.P. Carskadon S. Siddiqui J. Tomlins S.A. Chinnaiyan A.M. Palanisamy N. Novel RNA hybridization method for the in situ detection of ETV1, ETV4, and ETV5 gene fusions in prostate cancer.Appl Immunohistochem Mol Morphol. 2014; 22: e32-e40Crossref PubMed Scopus (21) Google Scholar Probes for PPIB (NM_000942.4) were used as positive control. We revalidated the assay in our own laboratory, using cases known by sequencing to be positive for ETV1, ETV4, or ETV5 fusions,34Weier C. Haffner M.C. Mosbruger T. Esopi D.M. Hicks J. Zheng Q. Fedor H. Isaacs W.B. De Marzo A.M. Nelson W.G. Yegnasubramanian S. Nucleotide resolution analysis of TMPRSS2 and ERG rearrangements in prostate cancer.J Pathol. 2013;
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