Publications by authors named "Kevin Beja"

20 Publications

  • Page 1 of 1

Evolution of Castration-Resistant Prostate Cancer in ctDNA during Sequential Androgen Receptor Pathway Inhibition.

Clin Cancer Res 2021 Aug 3;27(16):4610-4623. Epub 2021 Jun 3.

Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, British Columbia, Canada.

Purpose: Cross-resistance renders multiple lines of androgen receptor (AR) signaling inhibitors increasingly futile in metastatic castration-resistant prostate cancer (mCRPC). We sought to determine acquired genomic contributors to cross-resistance.

Experimental Design: We collected 458 serial plasma cell-free DNA samples at baseline and progression timepoints from 202 patients with mCRPC receiving sequential AR signaling inhibitors (abiraterone and enzalutamide) in a randomized phase II clinical trial (NCT02125357). We utilized deep targeted and whole-exome sequencing to compare baseline and posttreatment somatic genomic profiles in circulating tumor DNA (ctDNA).

Results: Patient ctDNA abundance was correlated across plasma collections and independently prognostic for sequential therapy response and overall survival. Most driver alterations in established prostate cancer genes were consistently detected in ctDNA over time. However, shifts in somatic populations after treatment were identified in 53% of patients, particularly after strong treatment responses. Treatment-associated changes converged upon the gene, with an average 50% increase in copy number, changes in mutation frequencies, and a 2.5-fold increase in the proportion of patients carrying AR ligand binding domain truncating rearrangements.

Conclusions: Our data show that the dominant genotype continues to evolve during sequential lines of AR inhibition and drives acquired resistance in patients with mCRPC.
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http://dx.doi.org/10.1158/1078-0432.CCR-21-1625DOI Listing
August 2021

, , and Defects Differentially Shape Prostate Tumor Driver Genomics and Clinical Aggression.

Clin Cancer Res 2021 Mar 7;27(6):1650-1662. Epub 2021 Jan 7.

Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

Purpose: DNA damage repair (DDR) defects are common across cancer types and can indicate therapeutic vulnerability. Optimal exploitation of DDR defects in prostate cancer requires new diagnostic strategies and a better understanding of associated clinical genomic features.

Experimental Design: We performed targeted sequencing of 1,615 plasma cell-free DNA samples from 879 patients with metastatic prostate cancer. Depth-based copy-number calls and heterozygous SNP imbalance were leveraged to expose DDR-mutant allelic configuration and categorize mechanisms of biallelic loss. We used split-read structural variation analysis to characterize tumor suppressor rearrangements. Patient-matched archival primary tissue was analyzed identically.

Results: , and were the most frequently disrupted DDR genes in circulating tumor DNA (ctDNA), collectively mutated in 15% of evaluable cases. Biallelic gene disruption via second somatic alteration or mutant allele-specific imbalance was identified in 79% of patients. A further 2% exhibited homozygous deletions. Tumor suppressors , and were controlled via disruptive chromosomal rearrangements in defective samples, but via oncogene amplification in context of defects. mutations were rare in cases with defects. DDR mutations were re-detected across 94% of serial ctDNA samples and in all available archival primary tissues, indicating they arose prior to metastatic progression. Loss of and , but not , was associated with poor clinical outcomes.

Conclusions: , and defects are each linked to distinct prostate cancer driver genomics and aggression. The consistency of DDR status in longitudinal samples and resolution of allelic status underscores the potential for ctDNA as a diagnostic tool.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-3708DOI Listing
March 2021

Activating AKT1 and PIK3CA Mutations in Metastatic Castration-Resistant Prostate Cancer.

Eur Urol 2020 12 22;78(6):834-844. Epub 2020 May 22.

Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada. Electronic address:

Background: Activating mutations in AKT1 and PIK3CA are undercharacterised in metastatic castration-resistant prostate cancer (mCRPC), but are linked to activation of phosphatidylinositol 3-kinase (PI3K) signalling and sensitivity to pathway inhibitors in other cancers.

Objective: To determine the prevalence, genomic context, and clinical associations of AKT1/PIK3CA activating mutations in mCRPC.

Design, Setting, And Participants: We analysed targeted cell-free DNA (cfDNA) sequencing data from 599 metastatic prostate cancer patients with circulating tumour DNA (ctDNA) content above 2%.

Outcome Measurements And Statistical Analysis: In patients with AKT1/PIK3CA mutations, cfDNA was subjected to PTEN intron sequencing and matched diagnostic tumour tissue was analysed when possible.

Results And Limitations: Of the patients, 6.0% (36/599) harboured somatic clonal activating mutation(s) in AKT1 or PIK3CA. Mutant allele-specific imbalance was common. Clonal mutations in mCRPC ctDNA were typically detected in pretreatment primary tissue and were consistent across serial ctDNA collections. AKT1/PIK3CA-mutant mCRPC had fewer androgen receptor (AR) gene copies than AKT1/PIK3CA wild-type mCRPC (median 4.7 vs 10.3, p =  0.003). AKT1 mutations were mutually exclusive with PTEN alterations. Patients with and without AKT1/PIK3CA mutations showed similar clinical outcomes with standard of care treatments. A heavily pretreated mCRPC patient with an AKT1 mutation experienced a 50% decline in prostate-specific antigen with Akt inhibitor (ipatasertib) monotherapy. Ipatasertib also had a marked antitumour effect in a patient-derived xenograft harbouring an AKT1 mutation. Limitations include the inability to assess AKT1/PIK3CA correlatives in ctDNA-negative patients.

Conclusions: AKT1/PIK3CA activating mutations are relatively common and delineate a distinct mCRPC molecular subtype with low-level AR copy gain. Clonal prevalence and evidence of mutant allele selection propose PI3K pathway dependency in selected patients. The use of cfDNA screening enables prospective clinical trials to test PI3K pathway inhibitors in this population.

Patient Summary: Of advanced prostate cancer cases, 6% have activating mutations in the genes AKT1 or PIK3CA. These mutations can be identified using a blood test and may help select patients suitable for clinical trials of phosphatidylinositol 3-kinase inhibitors.
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http://dx.doi.org/10.1016/j.eururo.2020.04.058DOI Listing
December 2020

Identification of Hypermutation and Defective Mismatch Repair in ctDNA from Metastatic Prostate Cancer.

Clin Cancer Res 2020 03 19;26(5):1114-1125. Epub 2019 Nov 19.

Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, British Columbia, Canada.

Purpose: DNA mismatch repair defects (MMRd) and tumor hypermutation are rare and under-characterized in metastatic prostate cancer (mPC). Furthermore, because hypermutated MMRd prostate cancers can respond to immune checkpoint inhibitors, there is an urgent need for practical detection tools.

Experimental Design: We analyzed plasma cell-free DNA-targeted sequencing data from 433 patients with mPC with circulating tumor DNA (ctDNA) purity ≥2%. Samples with somatic hypermutation were subjected to 185 × whole-exome sequencing and capture of mismatch repair gene introns. Archival tissue was analyzed with targeted sequencing and IHC.

Results: Sixteen patients (3.7%) had somatic hypermutation with MMRd etiology, evidenced by deleterious alterations in , or , microsatellite instability, and characteristic trinucleotide signatures. ctDNA was concordant with mismatch repair protein IHC and DNA sequencing of tumor tissue. Tumor suppressors such as , and were inactivated by mutation rather than copy-number loss. Hotspot mutations in oncogenes such as , and were common, and the androgen receptor ()-ligand binding domain was mutated in 9 of 16 patients. We observed high intrapatient clonal diversity, evidenced by subclonal driver mutations and shifts in mutation allele frequency over time. Patients with hypermutation and MMRd etiology in ctDNA had a poor response to AR inhibition and inferior survival compared with a control cohort.

Conclusions: Hypermutated MMRd mPC is associated with oncogene activation and subclonal diversity, which may contribute to a clinically aggressive disposition in selected patients. In patients with detectable ctDNA, cell-free DNA sequencing is a practical tool to prioritize this subtype for immunotherapy..
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http://dx.doi.org/10.1158/1078-0432.CCR-19-1623DOI Listing
March 2020

Evaluation of Commercial Circulating Tumor DNA Test in Metastatic Prostate Cancer.

JCO Precis Oncol 2019 12;3. Epub 2019 Jun 12.

University of British Columbia, Vancouver, British Columbia, Canada.

Purpose: Circulating tumor DNA (ctDNA) sequencing provides a minimally invasive method for tumor molecular stratification. Commercial ctDNA sequencing is increasingly used in the clinic, but its accuracy in metastatic prostate cancer is untested. We compared the commercial Guardant360 ctDNA test against an academic sequencing approach for profiling metastatic prostate cancer.

Patients And Methods: Plasma cell-free DNA was collected between September 2016 and April 2018 from 24 patients with clinically progressive metastatic prostate cancer representing a range of clinical scenarios. Each sample was analyzed using Guardant360 and a research panel encompassing 73 prostate cancer genes. Concordance of somatic mutation and copy number calls was evaluated between the two approaches.

Results: Targeted sequencing independently confirmed 94% of somatic mutations identified by Guardant360 at an allele fraction greater than 1%. amplifications and mutations were detected with high concordance in 14 patients, with only three discordant subclonal mutations at an allele fraction lower than 0.5%. Many somatic mutations identified by Guardant360 at an allele fraction lower than 1% seemed to represent subclonal passenger events or non-prostate-derived clones. Most of the non- gene amplifications reported by Guardant360 represented single copy gains. The research approach detected several clinically relevant DNA repair gene alterations not reported by Guardant360, including four germline truncating / mutations, two somatic stop gain mutations, one biallelic deletion, 11 stop gain reversal mutations in a patient treated with olaparib, and a hypermutator phenotype in a patient sample with 42 mutations per megabase.

Conclusion: Guardant360 accurately identifies somatic ctDNA mutations in patients with metastatic prostate cancer, but low allele frequency mutations should be interpreted with caution. Test utility in metastatic prostate cancer is currently limited by the lack of reporting on actionable deletions, rearrangements, and germline mutations.
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http://dx.doi.org/10.1200/PO.19.00014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446428PMC
June 2019

A Phase II Study of PX-866 in Patients With Recurrent or Metastatic Castration-resistant Prostate Cancer: Canadian Cancer Trials Group Study IND205.

Clin Genitourin Cancer 2019 06 15;17(3):201-208.e1. Epub 2019 Mar 15.

Canadian Cancer Trials Group, Kingston, ON, Canada.

Background: In PTEN-loss models, the phosphatidylinositol 3-kinase (PI3K)/AKT and androgen receptor signaling pathways cross-regulate by reciprocal feedback whereby inhibition of one activates the other, creating a rationale for co-targeting. We studied the irreversible, pan-isoform inhibitor of Class I PI-3K PX-866 singly (part A) and with abiraterone acetate (AA) in patients on AA with rising prostate-specific antigen (PSA) (part B).

Patients And Methods: The primary endpoint was lack of progression at 12 weeks. Exploratory endpoints included changes in circulating tumor cells (CTC), pharmacodynamic studies on platelets (part A), and archival tumor exploration of PTEN as predictor of response (part B).

Results: A total of 43 and 25 patients accrued to parts A and B, respectively. In part A, 14 (33%) patients were progression-free at 12 weeks, with 2 partial objective responses and 1 confirmed PSA response. Favorable CTC conversion (< 5 CTC/7.5 mL) occurred in 6 (24%) of 25 evaluable patients. In part B, 11 of 25 patients had measurable disease. Six (24%) patients were progression-free at 12 weeks. No objective or PSA responses were observed. For all 68 patients, the most common toxicities were diarrhea (53 patients), nausea (36), anorexia (24), fatigue (22), and vomiting (20). Among 17 patients for whom PTEN testing was possible, 3 had PTEN homozygous deletion and 14 had no change. No correlation between PTEN status and response was seen.

Conclusions: PX-866 had modest single agent activity. Adding AA to PX-866 showed no evidence of resistance reversal. Strategies to combine PI3K inhibition with androgen receptor-targeted therapies could consider initiation earlier, combination with other agents, and/or recruiting a selected population.
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http://dx.doi.org/10.1016/j.clgc.2019.03.005DOI Listing
June 2019

Genetic and Molecular Analysis of Essential Genes in Centromeric Heterochromatin of the Left Arm of Chromosome 3 in .

G3 (Bethesda) 2019 05 7;9(5):1581-1595. Epub 2019 May 7.

Department of Molecular Biology and Biochemistry (MBB), Simon Fraser University, 8888 University Dr, Burnaby BC V5A 1S6 and

A large portion of the genome is contained within heterochromatic regions of chromosomes, predominantly at centromeres and telomeres. The remaining euchromatic portions of the genome have been extensively characterized with respect to gene organization, function and regulation. However, it has been difficult to derive similar data for sequences within centromeric (centric) heterochromatin because these regions have not been as amenable to analysis by standard genetic and molecular tools. Here we present an updated genetic and molecular analysis of chromosome 3L centric heterochromatin (3L Het). We have generated and characterized a number of new, overlapping deficiencies (Dfs) which remove regions of 3L Het. These Dfs were critically important reagents in our subsequent genetic analysis for the isolation and characterization of lethal point mutations in the region. The assignment of these mutations to genetically-defined essential loci was followed by matching them to gene models derived from genome sequence data: this was done by using molecular mapping plus sequence analysis of mutant alleles, thereby aligning genetic and physical maps of the region. We also identified putative essential gene sequences in 3L Het by using RNA interference to target candidate gene sequences. We report that at least 25, or just under 2/3 of loci in 3L Het, are essential for viability and/or fertility. This work contributes to the functional annotation of centric heterochromatin in , and the genetic and molecular tools generated should help to provide important insights into the organization and functions of gene sequences in 3L Het.
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http://dx.doi.org/10.1534/g3.119.0003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6505167PMC
May 2019

Circulating Tumor DNA Abundance and Potential Utility in De Novo Metastatic Prostate Cancer.

Eur Urol 2019 04 10;75(4):667-675. Epub 2019 Jan 10.

Department of Urologic Sciences, Vancouver Prostate Centre, University of British Columbia, Vancouver, Canada. Electronic address:

Background: Several systemic therapeutic options exist for metastatic castrate-sensitive prostate cancer (mCSPC). Circulating tumor DNA (ctDNA) can molecularly profile metastatic castration-resistant prostate cancer and can influence decision-making, but remains untested in mCSPC.

Objective: To determine ctDNA abundance at de novo mCSPC diagnosis and whether ctDNA provides complementary clinically relevant information to a prostate biopsy.

Design, Setting, And Participants: We collected plasma cell-free DNA (cfDNA) from 53 patients newly diagnosed with mCSPC and, where possible, during treatment. Targeted sequencing was performed on cfDNA and DNA from diagnostic prostate tissue.

Results And Limitations: The median ctDNA fraction was 11% (range 0-84%) among untreated patients but was lower (1.0%, range 0-51%) among patients after short-term (median 22d) androgen deprivation therapy (ADT). TP53 mutations and DNA repair defects were identified in 47% and 21% of the cohort, respectively. The concordance for mutation detection in matched samples was 80%. Combined ctDNA and tissue analysis identified potential driver alterations in 94% of patients, whereas ctDNA or prostate biopsy alone was insufficient in 19 cases (36%). Limitations include the use of a narrow gene panel and undersampling of primary disease by prostate biopsy.

Conclusions: ctDNA provides additional information to a prostate biopsy in men with de novo mCSPC, but ADT rapidly reduces ctDNA availability. Primary tissue and ctDNA share relevant somatic alterations, suggesting that either is suitable for molecular subtyping in de novo mCSPC. The optimal approach for biomarker development should utilize both a tissue and liquid biopsy at diagnosis, as neither captures clinically relevant somatic alterations in all patients.

Patient Summary: In men with advanced prostate cancer, tumor DNA shed into the bloodstream can be measured via a blood test. The information from this test provides complementary information to a prostate needle biopsy and could be used to guide management strategies. Sequencing data were deposited in the European Genome-phenome Archive (EGA) under study identifier EGAS00001003351.
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http://dx.doi.org/10.1016/j.eururo.2018.12.042DOI Listing
April 2019

Frequent mutation of the untranslated region in prostate cancer.

Commun Biol 2018 24;1:122. Epub 2018 Aug 24.

Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, BC, V6H 3Z6, Canada.

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 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. lies in head-on orientation with the androgen-regulated non-coding gene , resulting in strong prostate lineage-specific bidirectional transcription across the 3'-UTR. This suggests transcriptional activity as a cause for the localized hypermutation. The indel-dominant pattern of somatic mutation extends into the coding region, where it is shaped by clonal selection to yield a cluster of non-frameshift indels inside the forkhead domain. Somatic 3'-UTR mutations may prove useful for diagnostic and screening approaches, given their high frequency and lineage specificity.
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http://dx.doi.org/10.1038/s42003-018-0128-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123809PMC
August 2018

Isolation and genome sequencing of individual circulating tumor cells using hydrogel encapsulation and laser capture microdissection.

Lab Chip 2018 06;18(12):1736-1749

Department of Mechanical Engineering, University of British Columbia, Canada.

Circulating tumor cells (CTCs) are malignant cells released into the bloodstream with the potential to form metastases in secondary sites. These cells, acquired non-invasively, represent a sample of highly relevant tumor tissue that is an alternative to difficult and low-yield tumor biopsies. In recent years, there has been growing interest in genomic profiling of CTCs to enable longitudinal monitoring of the tumor's adaptive response to therapy. However, due to their extreme rarity, genotyping CTCs has proved challenging. Relevant mutations can be masked by leukocyte contamination in isolates. Heterogeneity between subpopulations of tumor cells poses an additional obstacle. Recent advances in single-cell sequencing can overcome these limitations but isolation of single CTCs is prone to cell loss and is prohibitively difficult and time consuming. To address these limitations, we developed a single cell sample preparation and genome sequencing pipeline that combines biophysical enrichment and single cell isolation using laser capture microdissection (LCM). A key component of this process is the encapsulation of enriched CTC sample in a hydrogel matrix, which enhances the efficiency of single-cell isolation by LCM, and is compatible with downstream sequencing. We validated this process by sequencing of single CTCs and cell free DNA (cfDNA) from a single patient with castration resistant prostate cancer. Identical mutations were observed in prostate cancer driver genes (TP53, PTEN, FOXA1) in both single CTCs and cfDNA. However, two independently isolated CTCs also had identical missense mutations in the genes for ATR serine/threonine kinase, KMT2C histone methyltransferase, and FANCC DNA damage repair gene. These mutations may be missed by bulk sequencing libraries, whereas single cell sequencing could potentially enable the characterization of key CTC subpopulations that arise during metastasis.
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http://dx.doi.org/10.1039/c8lc00184gDOI Listing
June 2018

Circulating Tumor DNA Genomics Correlate with Resistance to Abiraterone and Enzalutamide in Prostate Cancer.

Cancer Discov 2018 04 24;8(4):444-457. Epub 2018 Jan 24.

Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

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 and were strongly associated with poor clinical outcomes independently of clinical prognostic factors and circulating tumor DNA abundance. Somatic alterations in , previously linked to reduced tumor dependency on AR signaling, were also independently associated with rapid resistance. Although detection of amplifications did not outperform standard prognostic biomarkers, 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. 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. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-0937DOI Listing
April 2018

Concordance of Circulating Tumor DNA and Matched Metastatic Tissue Biopsy in Prostate Cancer.

J Natl Cancer Inst 2017 Dec;109(12)

Department of Urologic Sciences, Vancouver Prostate Centre, University of British Columbia, Vancouver, Canada; Institute of Biosciences and Medical Technology, University of Tampere, Tampere, Finland; Department of Medicine and Department of Radiation Oncology, UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA; Oregon Health and Science University (OHSU) Knight Cancer Institute, Portland, OR; Department of Urology, University of California, Davis, School of Medicine, Sacremento, CA; Department of Medical Oncology, British Columbia Cancer Agency, Vancouver, Canada.

Background: Real-time knowledge of the somatic genome can influence management of patients with metastatic castration-resistant prostate cancer (mCRPC). While routine metastatic tissue biopsy is challenging in mCRPC, plasma circulating tumor DNA (ctDNA) has emerged as a minimally invasive tool to sample the tumor genome. However, no systematic comparisons of matched "liquid" and "solid" biopsies have been performed that would enable ctDNA profiling to replace the need for direct tissue sampling.

Methods: We performed targeted sequencing across 72 clinically relevant genes in 45 plasma cell-free DNA (cfDNA) samples collected at time of metastatic tissue biopsy. We compared ctDNA alterations with exome sequencing data generated from matched tissue and quantified the concordance of mutations and copy number alterations using the Fisher exact test and Pearson correlations.

Results: Seventy-five point six percent of cfDNA samples had a ctDNA proportion greater than 2% of total cfDNA. In these patients, all somatic mutations identified in matched metastatic tissue biopsies were concurrently present in ctDNA. Furthermore, the hierarchy of variant allele fractions for shared mutations was remarkably similar between ctDNA and tissue. Copy number profiles between matched liquid and solid biopsy were highly correlated, and individual copy number calls in clinically actionable genes were 88.9% concordant. Detected alterations included AR amplifications in 22 (64.7%) samples, SPOP mutations in three (8.8%) samples, and inactivating alterations in tumor suppressors TP53 , PTEN , RB1 , APC , CDKN1B , BRCA2 , and PIK3R1 . In several patients, ctDNA sequencing revealed robust changes not present in paired solid biopsy, including clinically relevant alterations in the AR, WNT, and PI3K pathways.

Conclusions: Our study shows that, in the majority of patients, a ctDNA assay is sufficient to identify all driver DNA alterations present in matched metastatic tissue and supports development of DNA biomarkers to guide mCRPC patient management based on ctDNA alone.
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http://dx.doi.org/10.1093/jnci/djx118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6440274PMC
December 2017

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

Clin Cancer Res 2017 Nov 25;23(22):6802-6811. Epub 2017 Aug 25.

Vancouver Prostate Centre and Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada.

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. 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 ( = 72 genes), and expression profiling using NanoString platform ( = 163 genes) were performed to explore changes in critical prostate cancer pathways linked to aggression and resistance. Three of 52 patients had only microfocal residual cancer at prostatectomy. The most common alterations included fusion ( = 32), mutation or deletion ( = 11), deletion ( = 6), ( = 6), and ( = 4) mutation, with no significant enrichment in posttreated specimens. We did not observe 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. 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. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-1034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5690882PMC
November 2017

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

Clin Cancer Res 2017 Nov 31;23(21):6487-6497. Epub 2017 Jul 31.

Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, British Columbia, Canada.

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. 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. 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 , or , and 70% harboring a mutation or disrupting rearrangement affecting chromatin modifiers such as Targetable alterations in MAPK/ERK or PI3K/AKT/mTOR pathways were robustly detected, including amplification of (20% of patients) and activating hotspot mutations in A (20%), with the latter mutually exclusive to truncating mutations in A novel gene fusion was identified in consecutive samples from one patient. 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. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-1140DOI Listing
November 2017

Analysis of Circulating Cell-Free DNA Identifies Multiclonal Heterogeneity of Reversion Mutations Associated with Resistance to PARP Inhibitors.

Cancer Discov 2017 09 27;7(9):999-1005. Epub 2017 Apr 27.

Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco (UCSF), San Francisco, California.

Approximately 20% of metastatic prostate cancers harbor mutations in genes required for DNA repair by homologous recombination repair (HRR) such as 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 mutation reversion. Whether similar mechanisms operate in prostate cancer, and could be detected in liquid biopsies, is unclear. Here, we identify 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. The mechanisms of clinical resistance to PARPi in DNA repair-deficient prostate cancer have not been described. Here, we show 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. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-0146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5581695PMC
September 2017

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

Eur Urol 2017 07 1;72(1):34-42. Epub 2017 Mar 1.

Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, British Columbia, Canada. Electronic address:

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.
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http://dx.doi.org/10.1016/j.eururo.2017.02.023DOI Listing
July 2017

Genome-wide chemical mapping of O-GlcNAcylated proteins in Drosophila melanogaster.

Nat Chem Biol 2017 02 5;13(2):161-167. Epub 2016 Dec 5.

Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada.

N-Acetylglucosamine β-O-linked to nucleocytoplasmic proteins (O-GlcNAc) is implicated in the regulation of gene expression in organisms, from humans to Drosophila melanogaster. Within Drosophila, O-GlcNAc transferase (OGT) is one of the Polycomb group proteins (PcGs) that act through Polycomb group response elements (PREs) to silence homeotic (HOX) and other PcG target genes. Using Drosophila, we identify new O-GlcNAcylated PcG proteins and develop an antibody-free metabolic feeding approach to chemoselectively map genomic loci enriched in O-GlcNAc using next-generation sequencing. We find that O-GlcNAc is distributed to specific genomic loci both in cells and in vivo. Many of these loci overlap with PREs, but O-GlcNAc is also present at other loci lacking PREs. Loss of OGT leads to altered gene expression not only at loci containing PREs but also at loci lacking PREs, including several heterochromatic genes. These data suggest that O-GlcNAc acts through multiple mechanisms to regulate gene expression in Drosophila.
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http://dx.doi.org/10.1038/nchembio.2247DOI Listing
February 2017

SiNVICT: ultra-sensitive detection of single nucleotide variants and indels in circulating tumour DNA.

Bioinformatics 2017 01 16;33(1):26-34. Epub 2016 Aug 16.

School of Computing Science.

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/sinvictSupplementary information: Supplementary data are available at Bioinformatics online.

Contact: [email protected]
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http://dx.doi.org/10.1093/bioinformatics/btw536DOI Listing
January 2017

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

JAMA Oncol 2016 Dec;2(12):1598-1606

Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada2Department of Medical Oncology, British Columbia Cancer Agency, Vancouver, British Columbia, Canada.

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.
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http://dx.doi.org/10.1001/jamaoncol.2016.0494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5097690PMC
December 2016

Moving Toward Personalized Care: Liquid Biopsy Predicts Response to Cisplatin in an Unusual Case of BRCA2-Null Neuroendocrine Prostate Cancer.

Clin Genitourin Cancer 2016 Apr 24;14(2):e233-6. Epub 2015 Dec 24.

Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada. Electronic address:

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http://dx.doi.org/10.1016/j.clgc.2015.12.023DOI Listing
April 2016
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