Kevin Beja

Genomic Alterations in Cell-Free DNA and Enzalutamide Resistance in Castration-Resistant Prostate Cancer

Importance The molecular landscape underpinning response to the androgen receptor (AR) antagonist enzalutamide in patients with metastatic castration-resistant prostate cancer (mCRPC) is undefined. Consequently, there is an urgent need for practical biomarkers to guide therapy selection and elucidate resistance. Although tissue biopsies are impractical to perform routinely in the majority of patients with mCRPC, the analysis of plasma cell-free DNA (cfDNA) has recently emerged as a minimally invasive method to explore tumor characteristics. Objective To reveal genomic characteristics from cfDNA associated with clinical outcomes during enzalutamide treatment. Design, Setting, and Participants Plasma samples were obtained from August 4, 2013, to July 31, 2015, at a single academic institution (British Columbia Cancer Agency) from 65 patients with mCRPC. We collected temporal plasma samples (at baseline, 12 weeks, end of treatment) for circulating cfDNA and performed array comparative genomic hybridization copy number profiling and deep AR gene sequencing. Samples collected at end of treatment were also subjected to targeted sequencing of 19 prostate cancer-associated genes. Exposure Enzalutamide, 160 mg, daily orally. Main Outcomes and Measures Prostate-specific antigen response rate (decline ≥50% from baseline confirmed ≥3 weeks later). Radiographic (as per Prostate Cancer Working Group 2 Criteria) and/or clinical progression (defined as worsening disease-related symptoms necessitating a change in anticancer therapy and/or deterioration in Eastern Cooperative Group performance status ≥2 levels). Results The 65 patients had a median (interquartile range) age of 74 (68-79) years. Prostate-specific antigen response rate to enzalutamide treatment was 38% (25 of 65), while median clinical/radiographic progression-free survival was 3.5 (95% CI, 2.1-5.0) months. Cell-free DNA was isolated from 122 of 125 plasma samples, and targeted sequencing was successful in 119 of 122. AR mutations and/or copy number alterations were robustly detected in 48% (31 of 65) and 60% (18 of 30) of baseline and progression samples, respectively. Detection of AR amplification, heavily mutated AR (≥2 mutations), and RB1 loss were associated with worse progression-free survival, with hazard ratios of 2.92 (95% CI, 1.59-5.37), 3.94 (95% CI, 1.46-10.64), and 4.46 (95% CI, 2.28-8.74), respectively. AR mutations exhibited clonal selection during treatment, including an increase in glucocorticoid-sensitive AR L702H and promiscuous AR T878A in patients with prior abiraterone treatment. At the time of progression, cfDNA sequencing revealed mutations or copy number changes in all patients tested, including clinically actionable alterations in DNA damage repair genes and PI3K pathway genes, and a high frequency (4 of 14) of activating CTNNB1 mutations. Conclusions and Relevance Clinically informative genomic profiling of cfDNA was feasible in nearly all patients with mCRPC and can provide important insights into enzalutamide response and resistance.

Treatment Outcomes and Tumor Loss of Heterozygosity in Germline DNA Repair–deficient Prostate Cancer

BACKGROUND Germline mutations in DNA repair genes were recently reported in 8-12% of patients with metastatic castration-resistant prostate cancer (mCRPC). It is unknown whether these mutations associate with differential response to androgen receptor (AR)-directed therapy. OBJECTIVE To determine the clinical response of mCRPC patients with germline DNA repair defects to AR-directed therapies and to establish whether biallelic DNA repair gene loss is detectable in matched circulating tumor DNA (ctDNA). DESIGN, SETTING, AND PARTICIPANTS We recruited 319 mCRPC patients and performed targeted germline sequencing of 22 DNA repair genes. In patients with deleterious germline mutations, plasma cell-free DNA was also sequenced. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Prostate-specific antigen response and progression were assessed in relation to initial androgen deprivation therapy (ADT) and subsequent therapy for mCRPC using Kaplan-Meier analysis. RESULTS AND LIMITATIONS Of the 319 patients, 24 (7.5%) had deleterious germline mutations, with BRCA2 (n=16) being the most frequent. Patients (n=22) with mutations in genes linked to homologous recombination were heterogeneous at initial presentation but, after starting ADT, progressed to mCRPC with a median time of 11.8 mo (95% confidence interval [CI] 5.1-18.4). The median time to prostate-specific antigen progression on first-line AR-targeted therapy in the mCRPC setting was 3.3 mo (95% CI 2.7-3.9). Ten out of 11 evaluable patients with germline BRCA2 mutations had somatic deletion of the intact allele in ctDNA. A limitation of this study is absence of a formal control cohort for comparison of clinical outcomes. CONCLUSIONS Patients with mCRPC who have germline DNA repair defects exhibit attenuated responses to AR-targeted therapy. Biallelic gene loss was robustly detected in ctDNA, suggesting that this patient subset could be prioritized for therapies exploiting defective DNA repair using a liquid biopsy. PATIENT SUMMARY Patients with metastatic prostate cancer and germline DNA repair defects exhibit a poor response to standard hormonal therapies, but may be prioritized for potentially more effective therapies using a blood test.

Analysis of circulating cell-free DnA identifies multiclonal heterogeneity of BRCA2 reversion mutations associated with resistance to PARP inhibitors

Approximately 20% of metastatic prostate cancers harbor mutations in genes required for DNA repair by homologous recombination repair (HRR) such as BRCA2 HRR defects confer synthetic lethality to PARP inhibitors (PARPi) such as olaparib and talazoparib. In ovarian or breast cancers, olaparib resistance has been associated with HRR restoration, including by BRCA2 mutation reversion. Whether similar mechanisms operate in prostate cancer, and could be detected in liquid biopsies, is unclear. Here, we identify BRCA2 reversion mutations associated with olaparib and talazoparib resistance in patients with prostate cancer. Analysis of circulating cell-free DNA (cfDNA) reveals reversion mutation heterogeneity not discernable from a single solid-tumor biopsy and potentially allows monitoring for the emergence of PARPi resistance.Significance: The mechanisms of clinical resistance to PARPi in DNA repair-deficient prostate cancer have not been described. Here, we show BRCA2 reversion mutations in patients with prostate cancer with metastatic disease who developed resistance to talazoparib and olaparib. Furthermore, we show that PARPi resistance is highly multiclonal and that cfDNA allows monitoring for PARPi resistance. Cancer Discov; 7(9); 999-1005. ©2017 AACR.See related commentary by Domchek, p. 937See related article by Kondrashova et al., p. 984See related article by Goodall et al., p. 1006This article is highlighted in the In This Issue feature, p. 920.

Circulating Tumor DNA Reveals Clinically Actionable Somatic Genome of Metastatic Bladder Cancer

Purpose: Targeted agents and immunotherapies promise to transform the treatment of metastatic bladder cancer, but therapy selection will depend on practical tumor molecular stratification. Circulating tumor DNA (ctDNA) is established in several solid malignancies as a minimally invasive tool to profile the tumor genome in real-time, but is critically underexplored in bladder cancer. Experimental Design: We applied a combination of whole-exome sequencing and targeted sequencing across 50 bladder cancer driver genes to plasma cell-free DNA (cfDNA) from 51 patients with aggressive bladder cancer, including 37 with metastatic disease. Results: The majority of patients with metastasis, but only 14% of patients with localized disease, had ctDNA proportions above 2% of total cfDNA (median 16.5%, range 3.9%–72.6%). Twelve percent of estimable samples had evidence of genome hypermutation. We reveal an aggressive mutational landscape in metastatic bladder cancer with 95% of patients harboring deleterious alterations to TP53, RB1, or MDM2, and 70% harboring a mutation or disrupting rearrangement affecting chromatin modifiers such as ARID1A. Targetable alterations in MAPK/ERK or PI3K/AKT/mTOR pathways were robustly detected, including amplification of ERBB2 (20% of patients) and activating hotspot mutations in PIK3CA (20%), with the latter mutually exclusive to truncating mutations in TSC1. A novel FGFR3 gene fusion was identified in consecutive samples from one patient. Conclusions: Our study demonstrates that ctDNA provides a practical and cost-effective snapshot of driver gene status in metastatic bladder cancer. The identification of a wide spectrum of clinically informative somatic alterations nominates ctDNA as a tool to dissect disease pathogenesis and guide therapy selection in patients with metastatic bladder cancer. Clin Cancer Res; 23(21); 6487–97. ©2017 AACR.

SiNVICT: Ultra-Sensitive Detection of Single Nucleotide Variants and Indels in Circulating Tumour DNA.

Motivation: Successful development and application of precision oncology approaches require robust elucidation of the genomic landscape of a patient’s cancer and, ideally, the ability to monitor therapy-induced genomic changes in the tumour in an inexpensive and minimally invasive manner. Thanks to recent advances in sequencing technologies, ‘liquid biopsy’, the sampling of patient’s bodily fluids such as blood and urine, is considered as one of the most promising approaches to achieve this goal. In many cancer patients, and especially those with advanced metastatic disease, deep sequencing of circulating cell free DNA (cfDNA) obtained from patient’s blood yields a mixture of reads originating from the normal DNA and from multiple tumour subclones—called circulating tumour DNA or ctDNA. The ctDNA/cfDNA ratio as well as the proportion of ctDNA originating from specific tumour subclones depend on multiple factors, making comprehensive detection of mutations difficult, especially at early stages of cancer. Furthermore, sensitive and accurate detection of single nucleotide variants (SNVs) and indels from cfDNA is constrained by several factors such as the sequencing errors and PCR artifacts, and mapping errors related to repeat regions within the genome. In this article, we introduce SiNVICT, a computational method that increases the sensitivity and specificity of SNV and indel detection at very low variant allele frequencies. SiNVICT has the capability to handle multiple sequencing platforms with different error properties; it minimizes false positives resulting from mapping errors and other technology specific artifacts including strand bias and low base quality at read ends. SiNVICT also has the capability to perform time-series analysis, where samples from a patient sequenced at multiple time points are jointly examined to report locations of interest where there is a possibility that certain clones were wiped out by some treatment while some subclones gained selective advantage. Results: We tested SiNVICT on simulated data as well as prostate cancer cell lines and cfDNA obtained from castration-resistant prostate cancer patients. On both simulated and biological data, SiNVICT was able to detect SNVs and indels with variant allele percentages as low as 0.5%. The lowest amounts of total DNA used for the biological data where SNVs and indels could be detected with very high sensitivity were 2.5 ng on the Ion Torrent platform and 10 ng on Illumina. With increased sequencing and mapping accuracy, SiNVICT might be utilized in clinical settings, making it possible to track the progress of point mutations and indels that are associated with resistance to cancer therapies and provide patients personalized treatment. We also compared SiNVICT with other popular SNV callers such as MuTect, VarScan2 and Freebayes. Our results show that SiNVICT performs better than these tools in most cases and allows further data exploration such as time-series analysis on cfDNA sequencing data. Availability and Implementation: SiNVICT is available at: https://sfu-compbio.github.io/sinvict Supplementary information: Supplementary data are available at Bioinformatics online. Contact: cenk@sfu.ca

Circulating tumor DNA genomics correlate with resistance to abiraterone and enzalutamide in prostate cancer

Primary resistance to androgen receptor (AR)-directed therapies in metastatic castration-resistant prostate cancer (mCRPC) is poorly understood. We randomized 202 patients with treatment-naïve mCRPC to abiraterone or enzalutamide and performed whole-exome and deep targeted 72-gene sequencing of plasma cell-free DNA prior to therapy. For these agents, which have never been directly compared, time to progression was similar. Defects in BRCA2 and ATM were strongly associated with poor clinical outcomes independently of clinical prognostic factors and circulating tumor DNA abundance. Somatic alterations in TP53, previously linked to reduced tumor dependency on AR signaling, were also independently associated with rapid resistance. Although detection of AR amplifications did not outperform standard prognostic biomarkers, AR gene structural rearrangements truncating the ligand binding domain were identified in several patients with primary resistance. These findings establish genomic drivers of resistance to first-line AR-directed therapy in mCRPC and identify potential minimally invasive biomarkers.Significance: Leveraging plasma specimens collected in a large randomized phase II trial, we report the relative impact of common circulating tumor DNA alterations on patient response to the most widely used therapies for advanced prostate cancer. Our findings suggest that liquid biopsy analysis can guide the use of AR-targeted therapy in general practice. Cancer Discov; 8(4); 444-57. ©2018 AACR.See related commentary by Jayaram et al., p. 392This article is highlighted in the In This Issue feature, p. 371.

Impact of Therapy on Genomics and Transcriptomics in High-Risk Prostate Cancer Treated with Neoadjuvant Docetaxel and Androgen Deprivation Therapy

Purpose: The combination of docetaxel chemotherapy and androgen deprivation therapy (ADT) has become a standard treatment for patients with metastatic prostate cancer. The recently accrued phase III CALGB 90203 trial was designed to investigate the clinical effectiveness of this treatment approach earlier in the disease. Specimens from this trial offer a unique opportunity to interrogate the acute molecular response to docetaxel and ADT and identify potential biomarkers. Experimental Design: We evaluated baseline clinical data, needle biopsies, and radical prostatectomy (RP) specimens from 52 (of 788) patients enrolled on CALGB 90203 at one high volume center. Pathology review, tumor and germline-targeted DNA sequencing (n = 72 genes), and expression profiling using NanoString platform (n = 163 genes) were performed to explore changes in critical prostate cancer pathways linked to aggression and resistance. Results: Three of 52 patients had only microfocal residual cancer at prostatectomy. The most common alterations included TMPRSS2-ERG fusion (n = 32), TP53 mutation or deletion (n = 11), PTEN deletion (n = 6), FOXA1 (n = 6), and SPOP (n = 4) mutation, with no significant enrichment in posttreated specimens. We did not observe AR amplification or mutations. The degree of AR signaling suppression varied among treated tumors and there was upregulation of both AR and AR-V7 expression as well as a subset of neuroendocrine and plasticity genes. Conclusions: These data support the feasibility of targeted and temporal genomic and transcriptome profiling of neoadjuvant-treated prostate cancer with limited formalin-fixed paraffin embedded tissue requirement. Characterization of the heterogeneity of treatment response and molecular outliers that arise posttreatment provides new insight into potential early markers of resistance. Clin Cancer Res; 23(22); 6802–11. ©2017 AACR.

Frequent mutation of the FOXA1 untranslated region in prostate cancer

Prostate cancer has a low somatic mutation rate but non-coding regions remain underexplored. We sequenced the untranslated regions (UTRs) of 72 established driver genes in 428 patients with metastatic prostate cancer and identified FOXA1 3′-UTR mutations in 12% of patients. The mutations were predominantly insertions or deletions, covered the entire UTR without motif enrichment, and were not detected in other cancers. FOXA1 lies in head-on orientation with the androgen-regulated non-coding gene AL121790.1, resulting in strong prostate lineage-specific bidirectional transcription across the FOXA1 3′-UTR. This suggests transcriptional activity as a cause for the localized hypermutation. The indel-dominant pattern of somatic mutation extends into the FOXA1 coding region, where it is shaped by clonal selection to yield a cluster of non-frameshift indels inside the forkhead domain. Somatic FOXA1 3′-UTR mutations may prove useful for diagnostic and screening approaches, given their high frequency and lineage specificity.Matti Annala et al. report the recurrence of new FOXA1 mutations in prostate cancer. These FOXA1 mutations in 3’ untranslated region may prove useful as diagnostic markers for prostate cancer.