SUMMARY 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 novel genomic alterations in PIK3CA/B, R-spondin, BRAF/RAF1, APC, β-catenin and ZBTB16/PLZF. Aberrations of BRCA2, BRCA1 and ATM were observed at substantially higher frequencies (19.3% overall) than seen 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 evidence that clinical sequencing in mCRPC is feasible and could impact treatment decisions in significant numbers of affected individuals.

Characterization of the prostate cancer transcriptome and genome has identified chromosomal rearrangements and copy number gains/losses, including ETS gene fusions, PTEN loss and androgen receptor (AR) amplification, that drive prostate cancer development and progression to lethal, metastatic castrate resistant prostate cancer (CRPC)1. As less is known about the role of mutations2–4, here we sequenced the exomes of 50 lethal, heavily-pretreated metastatic CRPCs obtained at rapid autopsy (including three different foci from the same patient) and 11 treatment naïve, high-grade localized prostate cancers. We identified low overall mutation rates even in heavily treated CRPC (2.00/Mb) and confirmed the monoclonal origin of lethal CRPC. Integrating exome copy number analysis identified disruptions of CHD1, which define a subtype of ETS fusionnegative prostate cancer. Similarly, we demonstrate that ETS2, which is deleted in ~1/3 of CRPCs (commonly through TMPRSS2:ERG fusions), is also deregulated through mutation. Further, we identified recurrent mutations in multiple chromatin/histone modifying genes, including MLL2 (mutated in 8.6% of prostate cancers), and demonstrate interaction of the MLL complex with AR, which is required for AR-mediated signaling. We also identified novel recurrent mutations in the AR collaborating factor FOXA1, which is mutated in 5 of 147 (3.4%) prostate cancers (both untreated localized prostate cancer and CRPC), and showed that mutated FOXA1 represses androgen signaling and increases tumour growth. Proteins that physically interact with AR, such as the ERG gene fusion product, FOXA1, MLL2, UTX, and ASXL1 were found to be mutated in CRPC. In summary, we describe the mutational landscape of a heavily treated metastatic cancer, identify novel mechanisms of AR signaling deregulated in prostate cancer, and prioritize candidates for future study.

High-throughput sequencing of polyA+ RNA (RNA-Seq) in human cancer shows remarkable potential to identify both novel markers of disease and uncharacterized aspects of tumor biology, particularly non-coding RNA (ncRNA) species. We employed RNA-Seq on a cohort of 102 prostate tissues and cells lines and performed ab initio transcriptome assembly to discover unannotated ncRNAs. We nominated 121 such Prostate Cancer Associated Transcripts (PCATs) with cancer-specific expression patterns. Among these, we characterized PCAT-1 as a novel prostate-specific regulator of cell proliferation and target of the Polycomb Repressive Complex 2 (PRC2). We further found that high PCAT-1 and PRC2 expression stratified patient tissues into molecular subtypes distinguished by expression signatures of PCAT-1-repressed target genes. Taken together, the findings presented herein identify PCAT-1 as a novel transcriptional repressor implicated in subset of prostate cancer patients. These findings establish the utility of RNA-Seq to identify disease-associated ncRNAs that may improve the stratification of cancer subtypes.

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Through a prospective clinical sequencing program for advanced cancers, four index cases were identified which harbor gene rearrangements of FGFR2 including patients with cholangiocarcinoma, breast cancer, and prostate cancer. After extending our assessment of FGFR rearrangements across multiple tumor cohorts, we identified additional FGFR gene fusions with intact kinase domains in lung squamous cell cancer, bladder cancer, thyroid cancer, oral cancer, glioblastoma, and head and neck squamous cell cancer. All FGFR fusion partners tested exhibit oligomerization capability, suggesting a shared mode of kinase activation. Overexpression of FGFR fusion proteins induced cell proliferation. Two bladder cancer cell lines that harbor FGFR3 fusion proteins exhibited enhanced susceptibility to pharmacologic inhibition in vitro and in vivo. Due to the combinatorial possibilities of FGFR family fusion to a variety of oligomerization partners, clinical sequencing efforts which incorporate transcriptome analysis for gene fusions are poised to identify rare, targetable FGFR fusions across diverse cancer types.

SUMMARYMetastasis is the primary cause of cancer-related deaths. While The Cancer Genome Atlas (TCGA) has sequenced primary tumor 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 tumors 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 for the identification of gene fusions, pathway activation, and immune profiling. Integrative sequence analysis provides a clinically relevant, multi-dimensional view of the complex molecular landscape and microenvironment of metastatic cancers.

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 all revealed the presence of a NAB2-STAT6 gene fusion. Using RT-PCR and sequencing, we detected this fusion in 51 of 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 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.

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