PurposeTissue-based comprehensive genomic profiling (CGP) is increasingly utilized for treatment ... more PurposeTissue-based comprehensive genomic profiling (CGP) is increasingly utilized for treatment selection in patients with advanced solid tumors, however real-world tissue availability may limit widespread implementation. Here we established real-world CGP tissue availability and assessed CGP performance on consecutively received samples.Patients and MethodPost-hoc, non-prespecified analysis of 32,048 consecutive tumor tissue samples received for StrataNGS, a multiplex PCR based-CGP (PCR-CGP) test, as part of an ongoing observational trial (NCT03061305). Tumor tissue sample characteristics and PCR-CGP performance were assessed across all tested tumor samples, including exception samples not meeting minimum input requirements (<20% tumor content [TC], <2mm2 tumor surface area [TSA], DNA or RNA yield <1ng/ul, or specimen age >5yrs). Tests reporting at least one prioritized alteration or meeting all sequencing QC metrics (and ≥20% TC) were considered successful. For prosta...
Aim: Primary cutaneous neuroendocrine carcinoma, or Merkel cell carcinoma (MCC), cannot be distin... more Aim: Primary cutaneous neuroendocrine carcinoma, or Merkel cell carcinoma (MCC), cannot be distinguished morphologically from small-cell neuroendocrine carcinomas (SmCC) from other sites. Immunohistochemistry is required to confirm cutaneous origin, and is also used for detection of sentinel lymph node (SLN) metastases of MCC. Cytokeratin 20 (CK20) expression is commonly used for these purposes, but is negative in some MCC cases, and has unclear specificity. We evaluated immunohistochemistry for neurofilament and CK20 in MCC compared with SmCC from other sites. Methods and results: We evaluated neurofilament expression in 55 MCC specimens from 39 unique patients, including nine CK20-negative MCC tumours. Neurofilament expression was observed in 42 of 55 (76.4%) MCC cases, including seven of nine (77.8%) CK20-negative MCC cases. Neurofilament was expressed in nine of 12 (75%) Merkel cell polyomavirus-positive tumours and five of 10 (50%) virus-negative tumours. Compared to a standard immunohistochemical panel (cytokeratin cocktail and CK20), neurofilament was 87.5% sensitive for detecting SLN metastases. Neurofilament and CK20 expression was also assessed in 61 extracutaneous SmCC from 60 unique patients, with primary sites including lung (27), bladder (18), cervix (3), gastrointestinal tract (3), sinonasal tract (2) and other sites (7). The specificity of neurofilament and CK20 for MCC versus non-cutaneous SmCC was 96.7% and 59.0%, respectively. Conclusions: Neurofilament has superior specificity to CK20 in distinguishing MCC from non-cutaneous SmCC. Neurofilament is frequently expressed in CK20-and virus-negative MCC tumours. Limitations of neurofilament immunohistochemistry include lower sensitivity than CK20 and subtle staining in some tumours. However, our findings indicate that neurofilament is useful for excluding non-cutaneous SmCC.
Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc, Jan 25, 2017
Renal cell carcinomas with MITF aberrations demonstrate a wide morphologic spectrum, highlighting... more Renal cell carcinomas with MITF aberrations demonstrate a wide morphologic spectrum, highlighting the need to consider these entities within the differential diagnosis of renal tumors encountered in clinical practice. Herein, we describe our experience with application of clinical fluorescence in situ hybridization (FISH) assays for detection of TFE3 and TFEB gene aberrations from 85 consecutive renal cell carcinoma cases submitted to our genitourinary FISH service. Results from 170 FISH assays performed on these tumors were correlated with available clinicopathologic findings. Ninety-eight percent of renal tumors submitted for FISH evaluation were from adult patients. Thirty-one (37%) tumors were confirmed to demonstrate MITF aberrations (21 TFE3 translocation, 4 TFEB translocation, and 6 TFEB amplification cases). Overall, renal cell carcinomas with MITF aberrations demonstrated morphologic features overlapping with clear cell, papillary, or clear cell papillary renal cell carcino...
Metastasis is the primary cause of cancer-related deaths. Although The Cancer Genome Atlas has se... more Metastasis is the primary cause of cancer-related deaths. Although The Cancer Genome Atlas has sequenced primary tumour types obtained from surgical resections, much less comprehensive molecular analysis is available from clinically acquired metastatic cancers. Here we perform whole-exome and -transcriptome sequencing of 500 adult patients with metastatic solid tumours of diverse lineage and biopsy site. The most prevalent genes somatically altered in metastatic cancer included TP53, CDKN2A, PTEN, PIK3CA, and RB1. Putative pathogenic germline variants were present in 12.2% of cases of which 75% were related to defects in DNA repair. RNA sequencing complemented DNA sequencing to identify gene fusions, pathway activation, and immune profiling. Our results show that integrative sequence analysis provides a clinically relevant, multi-dimensional view of the complex molecular landscape and microenvironment of metastatic cancers.
Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 12, 2017
Purpose: Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine tumor of the skin. Mer... more Purpose: Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine tumor of the skin. Merkel cell polyomavirus (MCPyV) plays an oncogenic role in the majority of MCCs. Detection of MCPyV in MCCs has diagnostic utility and prognostic potential. We investigated whether RNAscope, an RNA in situ hybridization (ISH) assay for detection of RNA transcripts in tissues, is useful for MCPyV detection. <p>Experimental Design: We applied an RNAscope probe targeting MCPyV T antigen transcripts on tissue microarrays (TMAs) and whole tissue sections encompassing 87 MCCs from 75 patients, 14 carcinomas of other types, and benign tissues. For comparison, quantitative PCR (qPCR) was performed on 57 cases of MCC from 52 patients. </p> <p>Results: RNA-ISH demonstrated the presence of MCPyV in 37/75 (49.3%) cases. Notably, tumors from younger patients (< 73 years) had a significantly higher virus positivity than those from elderly patients (≥ 73 years) (64.9% vs. 34.2%, P ...
Potential survival benefits from treating aggressive (Gleason score, ≥7) early-stage prostate can... more Potential survival benefits from treating aggressive (Gleason score, ≥7) early-stage prostate cancer are undermined by harms from unnecessary prostate biopsy and overdiagnosis of indolent disease. To evaluate the a priori primary hypothesis that combined measurement of PCA3 and TMPRSS2:ERG (T2:ERG) RNA in the urine after digital rectal examination would improve specificity over measurement of prostate-specific antigen alone for detecting cancer with Gleason score of 7 or higher. As a secondary objective, to evaluate the potential effect of such urine RNA testing on health care costs. Prospective, multicenter diagnostic evaluation and validation in academic and community-based ambulatory urology clinics. Participants were a referred sample of men presenting for first-time prostate biopsy without preexisting prostate cancer: 516 eligible participants from among 748 prospective cohort participants in the developmental cohort and 561 eligible participants from 928 in the validation coho...
Recurrent ERG gene fusions, the most common genetic alterations in prostate cancer, drive overexp... more Recurrent ERG gene fusions, the most common genetic alterations in prostate cancer, drive overexpression of the nuclear transcription factor ERG, and are early clonal events in prostate cancer progression. The nuclear transcription factor MYC is also frequently overexpressed in prostate cancer and may play a role in tumor initiation and/or progression. The relationship between nuclear ERG and MYC protein overexpression in prostate cancer, as well as the clinicopathologic characteristics and prognosis of ERG-positive/MYC high tumors, is not well understood. Immunohistochemistry (IHC) for ERG and MYC was performed on formalin-fixed, paraffin-embedded tissue from prostate cancer tissue microarrays (TMAs), and nuclear staining was scored semi-quantitatively (IHC product score range = 0-300). Correlation between nuclear ERG and MYC protein expression and association with clinicopathologic parameters and biochemical recurrence after radical prostatectomy was assessed. 29.1% of all tumor n...
Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc, 2016
The presence of two or more prostate cancer foci separated by intervening benign tissue in a sing... more The presence of two or more prostate cancer foci separated by intervening benign tissue in a single core is a well-recognized finding on prostate biopsy. Cancer involvement can be measured by including intervening benign tissue or only including the actual cancer involved area. Importantly, this parameter is a common enrollment criterion for active surveillance protocols. We hypothesized that spatially distinct prostate cancer foci in biopsies may arise from separate clones, impacting cancer involvement assessment. Hence, we used dual ERG/SPINK1 immunohistochemistry to determine the frequency of separate clones-when separate tumor foci showed discordant ERG and/or SPINK1 status-in discontinuously involved prostate biopsy cores from two academic institutions. In our cohort of 97 prostate biopsy cores with spatially discrete tumor foci (from 80 patients), discontinuous cancer involvement including intervening tissue ranged from 20 to 100% and Gleason scores ranged from 6 to 9. Twenty-...
Penile squamous cell carcinoma (PeSCCA) is a rare malignancy for which there are limited treatmen... more Penile squamous cell carcinoma (PeSCCA) is a rare malignancy for which there are limited treatment options due to a poor understanding of the molecular alterations underlying disease development and progression. Therefore, we performed comprehensive, targeted next-generation sequencing to identify relevant somatic genomic alterations in a retrospective cohort of 60 fixed tumor samples from 43 PeSCCA cases (including 14 matched primary/metastasis pairs). We identified a median of two relevant somatic mutations and one high-level copy-number alteration per sample (range, 0-5 and 0-6, respectively). Expression of HPV and p16 was detectable in 12% and 28% of patients, respectively. Furthermore, advanced clinical stage, lack of p16 expression, and MYC and CCND1 amplifications were significantly associated with shorter time to progression or PeSCCA-specific survival. Notably, four cases harbored EGFR amplifications and one demonstrated CDK4 amplification, genes for which approved and inve...
RNA-seq by poly(A) selection is currently the most common protocol for whole transcriptome sequen... more RNA-seq by poly(A) selection is currently the most common protocol for whole transcriptome sequencing as it provides a broad, detailed, and accurate view of the RNA landscape. Unfortunately, the utility of poly(A) libraries is greatly limited when the input RNA is degraded, which is the norm for research tissues and clinical samples, especially when specimens are formalin-fixed. To facilitate the use of RNA sequencing beyond cell lines and in the clinical setting, we developed an exome-capture transcriptome protocol with greatly improved performance on degraded RNA. Capture transcriptome libraries enable measuring absolute and differential gene expression, calling genetic variants, and detecting gene fusions. Through validation against gold-standard poly(A) and Ribo-Zero libraries from intact RNA, we show that capture RNA-seq provides accurate and unbiased estimates of RNA abundance, uniform transcript coverage, and broad dynamic range. Unlike poly(A) selection and Ribo-Zero depleti...
had full access to all the data in the study and takes responsibility for the integrity of the da... more had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Tomlins, Groskopf, Wei, Chinnaiyan.
Toward development of a precision medicine framework for metastatic, castration-resistant prostat... more Toward development of a precision medicine framework for metastatic, castration-resistant prostate cancer (mCRPC), we established a multi-institutional clinical sequencing infrastructure to conduct prospective whole-exome and transcriptome sequencing of bone or soft tissue tumor biopsies from a cohort of 150 mCRPC affected individuals. Aberrations of AR, ETS genes, TP53, and PTEN were frequent (40%-60% of cases), with TP53 and AR alterations enriched in mCRPC compared to primary prostate cancer. We identified new genomic alterations in PIK3CA/B, R-spondin, BRAF/RAF1, APC, β-catenin, and ZBTB16/PLZF. Moreover, aberrations of BRCA2, BRCA1, and ATM were observed at substantially higher frequencies (19.3% overall) compared to those in primary prostate cancers. 89% of affected individuals harbored a clinically actionable aberration, including 62.7% with aberrations in AR, 65% in other cancer-related genes, and 8% with actionable pathogenic germline alterations. This cohort study provides...
Next-generation sequencing (NGS) has enabled genome-wide personalized oncology efforts at centers... more Next-generation sequencing (NGS) has enabled genome-wide personalized oncology efforts at centers and companies with the specialty expertise and infrastructure required to identify and prioritize actionable variants. Such approaches are not scalable, preventing widespread adoption. Likewise, most targeted NGS approaches fail to assess key relevant genomic alteration classes. To address these challenges, we predefined the catalog of relevant solid tumor somatic genome variants (gain-of-function or loss-of-function mutations, high-level copy number alterations, and gene fusions) through comprehensive bioinformatics analysis of >700,000 samples. To detect these variants, we developed the Oncomine Comprehensive Panel (OCP), an integrative NGS-based assay [compatible with…
ERG rearrangements in localized prostate cancer can be detected with high sensitivity and specifi... more ERG rearrangements in localized prostate cancer can be detected with high sensitivity and specificity by immunohistochemistry (IHC). However, recent data suggest that ERG IHC may be less sensitive for ERG rearrangements in castration-resistant prostate cancer (CRPC). Thus, we sought to examine ERG protein expression in a cohort of rapid autopsy patients with lethal metastatic CRPC (mCRPC). A tissue microarray (TMA) of tumor sites from these patients was evaluated for ERG, prostate-specific antigen (PSA), and androgen receptor (AR) expression by IHC and correlated with ERG rearrangement status by fluorescent in situ hybridization (FISH). IHC was scored as the product of tumor cell staining intensity (0-3) and percentage of cells positive (0-100) (overall product score range = 0-300). All 16 (100%) ERG rearrangement negative (ERG(neg) ) patients were also negative for ERG tumor cell expression (i.e., IHC product score = 0). Of the 10 ERG rearrangement positive (ERG(pos) ) patients, tw...
Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that promotes tumorigenesis by ... more Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that promotes tumorigenesis by epigenetic repression of tumor suppressor genes. Although EZH2 is overexpressed in many malignancies, activating EZH2 mutations have been described predominantly in follicular lymphoma and diffuse large B-cell lymphoma. EZH2 activating mutations in melanoma are not well described. Here, we report a case of cutaneous melanoma that displayed an EZH2 (Y641S) activating mutation detected by transcriptome sequencing as well as BRAF (V600K). The patient had concurrent follicular lymphoma that lacked both the EZH2 (Y641) and BRAF (V600K) mutations present in the patient's melanoma. Further, we screened publicly available sequence data and identified four additional melanoma cases harboring activating EZH2 mutation. Our findings provide additional evidence that activating recurrent EZH2 mutations occur in cutaneous melanoma, to our knowledge the first solid tumor shown to
Transcriptional repressors and corepressors play a critical role in cellular homeostasis and are ... more Transcriptional repressors and corepressors play a critical role in cellular homeostasis and are frequently altered in cancer. C-terminal binding protein 1 (CtBP1), a transcriptional corepressor that regulates the expression of tumor suppressors and genes involved in cell death, is known to play a role in multiple cancers. In this study, we observed the overexpression and mislocalization of CtBP1 in metastatic prostate cancer and demonstrated the functional significance of CtBP1 in prostate cancer progression. Transient and stable knockdown of CtBP1 in prostate cancer cells inhibited their proliferation and invasion. Expression profiling studies of prostate cancer cell lines revealed that multiple
BACKGROUND. The incidence and prevalence of both benign prostatic hypertrophy (BPH) and prostate ... more BACKGROUND. The incidence and prevalence of both benign prostatic hypertrophy (BPH) and prostate cancer (PCa) increase with the aging process. Our laboratory recently showed that the chemokines CXCL5 and CXCL12, which normally function as inflammatory mediators, are secreted at higher levels by aging prostate stromal fibroblasts and elicit proliferative responses from both prostate stromal fibroblast and epithelial cells. Because both CXCL5 and CXCL12 are secreted molecules, we hypothesized that their levels in patient serum might serve as biomarkers to distinguish between BPH and PCa. METHODS. Serum CXCL5 and CXCL12 levels were determined using sandwich ELISAs for 51 men demonstrating low serum PSA values of 10 ng/ml who underwent diagnostic needle biopsy for the detection of PCa. The bivariate relationship of circulating chemokine levels, age, and disease status in the prostate was tested using the Wilcoxon rank-sum test. RESULTS. Total serum CXCL12 levels were significantly higher for men who were biopsy positive compared to those who were biopsy negative for cancer and histological prostatitis (P ¼ 0.050). Among men who were biopsy negative for PCa, total serum CXCL5 levels were inversely associated with prostate volume and were significantly higher in men with concomitant BPH and histological prostatitis compared to those without evidence of prostatic disease (P < 0.003). CONCLUSIONS. The results of this pilot and feasibility study suggest that serum or plasma CXCL5 and CXCL12 levels may potentially distinguish between BPH and PCa among patients presenting with low serum PSA, and may be useful toward facilitating decisions to perform diagnostic needle biopsy in this patient population.
The mutations present in advanced cancers can be identified by integrative high-throughput sequen... more The mutations present in advanced cancers can be identified by integrative high-throughput sequencing to enable biomarker-driven clinical trials and, ultimately, treatment.
Proceedings of the National Academy of Sciences, 2009
How cancer cells bind to vascular surfaces and extravasate into target organs is an underapprecia... more How cancer cells bind to vascular surfaces and extravasate into target organs is an underappreciated, yet essential step in metastasis. We postulate that the metastatic process involves discrete adhesive interactions between circulating cancer cells and microvascular endothelial cells. Sialyl Lewis X (sLe X ) on prostate cancer (PCa) cells is thought to promote metastasis by mediating PCa cell binding to microvascular endothelial (E)-selectin. Yet, regulation of sLe X and related E-selectin ligand expression in PCa cells is a poorly understood factor in PCa metastasis. Here, we describe a glycobiological mechanism regulating E-selectin-mediated adhesion and metastatic potential of PCa cells. We demonstrate that α1,3 fucosyltransferases (FT) 3, 6, and 7 are markedly elevated in bone- and liver-metastatic PCa and dictate synthesis of sLe X and E-selectin ligands on metastatic PCa cells. Upregulated FT3, FT6, or FT7 expression induced robust PCa PC-3 cell adhesion to bone marrow (BM) e...
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Papers by Javed Siddiqui