Juan Miguel Mosquera

Integrative clinical genomics of advanced prostate cancer

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 clinically actionable information that could impact treatment decisions for these affected individuals.

Exome sequencing identifies recurrent SPOP, FOXA1 and MED12 mutations in prostate cancer

Prostate cancer is the second most common cancer in men worldwide and causes over 250,000 deaths each year. Overtreatment of indolent disease also results in significant morbidity. Common genetic alterations in prostate cancer include losses of NKX3.1 (8p21) and PTEN (10q23), gains of AR (the androgen receptor gene) and fusion of ETS family transcription factor genes with androgen-responsive promoters. Recurrent somatic base-pair substitutions are believed to be less contributory in prostate tumorigenesis but have not been systematically analyzed in large cohorts. Here, we sequenced the exomes of 112 prostate tumor and normal tissue pairs. New recurrent mutations were identified in multiple genes, including MED12 and FOXA1. SPOP was the most frequently mutated gene, with mutations involving the SPOP substrate-binding cleft in 6–15% of tumors across multiple independent cohorts. Prostate cancers with mutant SPOP lacked ETS family gene rearrangements and showed a distinct pattern of genomic alterations. Thus, SPOP mutations may define a new molecular subtype of prostate cancer.

Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing

As more clinically relevant cancer genes are identified, comprehensive diagnostic approaches are needed to match patients to therapies, raising the challenge of optimization and analytical validation of assays that interrogate millions of bases of cancer genomes altered by multiple mechanisms. Here we describe a test based on massively parallel DNA sequencing to characterize base substitutions, short insertions and deletions (indels), copy number alterations and selected fusions across 287 cancer-related genes from routine formalin-fixed and paraffin-embedded (FFPE) clinical specimens. We implemented a practical validation strategy with reference samples of pooled cell lines that model key determinants of accuracy, including mutant allele frequency, indel length and amplitude of copy change. Test sensitivity achieved was 95–99% across alteration types, with high specificity (positive predictive value >99%). We confirmed accuracy using 249 FFPE cancer specimens characterized by established assays. Application of the test to 2,221 clinical cases revealed clinically actionable alterations in 76% of tumors, three times the number of actionable alterations detected by current diagnostic tests.

TMPRSS2:ERG gene fusion associated with lethal prostate cancer in a watchful waiting cohort.

The identification of the TMPRSS2:ERG fusion in prostate cancer suggests that distinct molecular subtypes may define risk for disease progression. In surgical series, TMPRSS2:ERG fusion was identified in 50% of the tumors. Here, we report on a population-based cohort of men with localized prostate cancers followed by expectant (watchful waiting) therapy with 15% (17/111) TMPRSS2:ERG fusion. We identified a statistically significant association between TMPRSS2:ERG fusion and prostate cancer specific death (cumulative incidence ratio=2.7, P<0.01, 95% confidence interval=1.3–5.8). Quantitative reverse-transcription–polymerase chain reaction demonstrated high estrogen-regulated gene (ERG) expression to be associated with TMPRSS2:ERG fusion (P<0.005). These data suggest that TMPRSS2:ERG fusion prostate cancers may have a more aggressive phenotype, possibly mediated through increased ERG expression.

TMPRSS2-ERG Fusion Prostate Cancer: An Early Molecular Event Associated With Invasion

Prostate cancer (PCA) is one of the most prevalent cancers and a major leading cause of morbidity and mortality in the Western world. The TMPRSS2-ERG fusion was recently identified as a common recurrent chromosomal aberration in this malignancy. In our study, we interrogated a broad spectrum of benign, precursor, and malignant prostatic lesions to assess the TMPRSS2-ERG fusion status using a multicolor interphase fluorescence in situ hybridization assay. Samples from hospital-based cohorts consisted of 237 clinically localized PCA, 34 hormone naive metastases, 9 hormone refractory metastases, 26 high grade prostatic intraepithelial neoplasia lesions, 15 samples of benign prostatic hyperplasia, 38 of proliferative inflammatory atrophy, and 47 of benign prostatic tissue. The TMPRSS2-ERG fusion was present in 48.5% of clinically localized PCA, 30% of hormone naive metastases, 33% of hormone refractory metastases, and in 19% of high grade prostatic intraepithelial neoplasia lesions in intermingling to cancer foci. Almost all these fusion positive cases show a homogenous distribution of the fusion pattern. In contrast, none of the other samples harbored this genetic aberration. If we consider the high incidence of PCA and the high frequency of this gene fusion, TMPRSS2-ERG is the most common genetic aberration so far described in human malignancies. Furthermore, its clinical application as a biomarker and ancillary diagnostic test is promising given its high specificity.

Identification of recurrent NAB2-STAT6 gene fusions in solitary fibrous tumor by integrative sequencing

A 44-year old woman with recurrent solitary fibrous tumor (SFT)/hemangiopericytoma was enrolled in a clinical sequencing program including whole-exome and transcriptome sequencing. A gene fusion of the transcriptional repressor NAB2 with the transcriptional activator STAT6 was detected. Transcriptome sequencing of 27 additional SFTs identified the presence of a NAB2-STAT6 gene fusion in all tumors. Using RT-PCR and sequencing, we detected this fusion in all 51 SFTs, indicating high levels of recurrence. Expression of NAB2-STAT6 fusion proteins was confirmed in SFT, and the predicted fusion products harbor the early growth response (EGR)-binding domain of NAB2 fused to the activation domain of STAT6. Overexpression of the NAB2-STAT6 gene fusion induced proliferation in cultured cells and activated the expression of EGR-responsive genes. These studies establish NAB2-STAT6 as the defining driver mutation of SFT and provide an example of how neoplasia can be initiated by converting a transcriptional repressor of mitogenic pathways into a transcriptional activator.

Targeted Next-generation Sequencing of Advanced Prostate Cancer Identifies Potential Therapeutic Targets and Disease Heterogeneity

BACKGROUND Most personalized cancer care strategies involving DNA sequencing are highly reliant on acquiring sufficient fresh or frozen tissue. It has been challenging to comprehensively evaluate the genome of advanced prostate cancer (PCa) because of limited access to metastatic tissue. OBJECTIVE To demonstrate the feasibility of a novel next-generation sequencing (NGS)-based platform that can be used with archival formalin-fixed paraffin-embedded (FFPE) biopsy tissue to evaluate the spectrum of DNA alterations seen in advanced PCa. DESIGN, SETTING, AND PARTICIPANTS FFPE samples (including archival prostatectomies and prostate needle biopsies) were obtained from 45 patients representing the spectrum of disease: localized PCa, metastatic hormone-naive PCa, and metastatic castration-resistant PCa (CRPC). We also assessed paired primaries and metastases to understand disease heterogeneity and disease progression. INTERVENTION At least 50 ng of tumor DNA was extracted from FFPE samples and used for hybridization capture and NGS using the Illumina HiSeq 2000 platform. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS A total of 3320 exons of 182 cancer-associated genes and 37 introns of 14 commonly rearranged genes were evaluated for genomic alterations. RESULTS AND LIMITATIONS We obtained an average sequencing depth of >900X. Overall, 44% of CRPCs harbored genomic alterations involving the androgen receptor gene (AR), including AR copy number gain (24% of CRPCs) or AR point mutation (20% of CRPCs). Other recurrent mutations included transmembrane protease, serine 2 gene (TMPRSS2):v-ets erythroblastosis virus E26 oncogene homolog (avian) gene (ERG) fusion (44%); phosphatase and tensin homolog gene (PTEN) loss (44%); tumor protein p53 gene (TP53) mutation (40%); retinoblastoma gene (RB) loss (28%); v-myc myelocytomatosis viral oncogene homolog (avian) gene (MYC) gain (12%); and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α gene (PIK3CA) mutation (4%). There was a high incidence of genomic alterations involving key genes important for DNA repair, including breast cancer 2, early onset gene (BRCA2) loss (12%) and ataxia telangiectasia mutated gene (ATM) mutations (8%); these alterations are potentially targetable with poly(adenosine diphosphate-ribose)polymerase inhibitors. A novel and actionable rearrangement involving the v-raf murine sarcoma viral oncogene homolog B1 gene (BRAF) was also detected. CONCLUSIONS This first-in-principle study demonstrates the feasibility of performing in-depth DNA analyses using FFPE tissue and brings new insight toward understanding the genomic landscape within advanced PCa.

The oestrogen receptor alpha-regulated lncRNA NEAT1 is a critical modulator of prostate cancer

The androgen receptor (AR) plays a central role in establishing an oncogenic cascade that drives prostate cancer progression. Some prostate cancers escape androgen dependence and are often associated with an aggressive phenotype. The oestrogen receptor alpha (ERα) is expressed in prostate cancers, independent of AR status. However, the role of ERα remains elusive. Using a combination of chromatin immunoprecipitation (ChIP) and RNA-sequencing data, we identified an ERα-specific non-coding transcriptome signature. Among putatively ERα-regulated intergenic long non-coding RNAs (lncRNAs), we identified nuclear enriched abundant transcript 1 (NEAT1) as the most significantly overexpressed lncRNA in prostate cancer. Analysis of two large clinical cohorts also revealed that NEAT1 expression is associated with prostate cancer progression. Prostate cancer cells expressing high levels of NEAT1 were recalcitrant to androgen or AR antagonists. Finally, we provide evidence that NEAT1 drives oncogenic growth by altering the epigenetic landscape of target gene promoters to favour transcription.

Characterization of TMPRSS2-ERG fusion high-grade prostatic intraepithelial neoplasia and potential clinical implications.

Purpose: More than 1,300,000 prostate needle biopsies are done annually in the United States with up to 16% incidence of isolated high-grade prostatic intraepithelial neoplasia (HGPIN). HGPIN has low predictive value for identifying prostate cancer on subsequent needle biopsies in prostate-specific antigen–screened populations. In contemporary series, prostate cancer is detected in ∼20% of repeat biopsies following a diagnosis of HGPIN. Further, discrete histologic subtypes of HGPIN with clinical implication in management have not been characterized. The TMPRSS2-ERG gene fusion that has recently been described in prostate cancer has also been shown to occur in a subset of HGPIN. This may have significant clinical implications given that TMPRSS2-ERG fusion prostate cancer is associated with a more aggressive clinical course. Experimental Design: In this study, we assessed a series of HGPIN lesions and paired prostate cancer for the presence of TMPRSS2-ERG gene fusion. Results: Fusion-positive HGPIN was observed in 16% of the 143 number of lesions, and in all instances, the matching cancer shared the same fusion pattern. Sixty percent of TMPRSS2-ERG fusion prostate cancer had fusion-negative HGPIN. Conclusions: Given the more aggressive nature of TMPRSS2-ERG prostate cancer, the findings of this study raise the possibility that gene fusion-positive HGPIN lesions are harbingers of more aggressive disease. To date, pathologic, molecular, and clinical variables do not help stratify which men with HGPIN are at increased risk for a cancer diagnosis. Our results suggest that the detection of isolated TMPRSS2-ERG fusion HGPIN would improve the positive predictive value of finding TMPRSS2-ERG fusion prostate cancer in subsequent biopsies.

Prevalence of TMPRSS2-ERG Fusion Prostate Cancer among Men Undergoing Prostate Biopsy in the United States

Purpose: Fusion of the TMPRSS2 prostate-specific gene with the ERG transcription factor is a putatively oncogenic gene rearrangement that is commonly found in prostate cancer tissue from men undergoing prostatectomy. However, the prevalence of the fusion was less common in samples of transurethral resection of the prostate from a Swedish cohort of patients with incidental prostate cancer followed by watchful waiting, raising the question as to whether the high prevalence in prostatectomy specimens reflects selection bias. We sought to determine the prevalence of TMPRSS2-ERG gene fusion among prostate-specific antigen–screened men undergoing prostate biopsy in the United States. Experimental Design: We studied 140 prostate biopsies from the same number of patients for TMPRSS2-ERG fusion status with a fluorescent in situ hybridization assay. One hundred and thirty-four samples (100 cancer and 34 benign) were assessable. Results:ERG gene rearrangement was detected in 46% of prostate biopsies that were found to have prostate cancer and in 0% of benign prostate biopsies (P < 0.0001). Evaluation of morphologic features showed that cribriform growth, blue-tinged mucin, macronucleoli, and collagenous micronodules were significantly more frequent in TMPRSS2-ERG fusion–positive prostate cancer biopsies than gene fusion–negative prostate cancer biopsies (P ≤ 0.04). No significant association with Gleason score was detected. In addition, non-Caucasian patients were less likely to have positive fusion status (P = 0.02). Conclusions: This is the first prospective North American multicenter study to characterize TMPRSS2-ERG prostate cancer prevalence in a cohort of patients undergoing needle biopsy irrespective of whether or not they subsequently undergo prostatectomy. Our results show that this gene rearrangement is common among North American men who have prostate cancer on biopsy, is absent in benign prostate biopsy, and is associated with specific morphologic features. These findings indicate a need for prospective studies to evaluate the relationship of TMPRSS2-ERG rearrangement with clinical course of screening-detected prostate cancer in North American men, and a need for the development of noninvasive screening tests to detect TMPRSS2-ERG rearrangement.