Deep whole-genome ctDNA chronology of treatment-resistant prostate cancer.

Circulating tumour DNA (ctDNA) in blood plasma is an emerging tool for clinical cancer genotyping and longitudinal disease monitoring1. However, owing to past emphasis on targeted and low-resolution profiling approaches, our understanding of the distinct populations that comprise bulk ctDNA is incomplete2-12. Here we perform deep whole-genome sequencing of serial plasma and synchronous metastases in patients with aggressive prostate cancer. We comprehensively assess all classes of genomic alterations and show that ctDNA contains multiple dominant populations, the evolutionary histories of which frequently indicate whole-genome doubling and shifts in mutational processes. Although tissue and ctDNA showed concordant clonally expanded cancer driver alterations, most individual metastases contributed only a minor share of total ctDNA. By comparing serial ctDNA before and after clinical progression on potent inhibitors of the androgen receptor (AR) pathway, we reveal population restructuring converging solely on AR augmentation as the dominant genomic driver of acquired treatment resistance. Finally, we leverage nucleosome footprints in ctDNA to infer mRNA expression in synchronously biopsied metastases, including treatment-induced changes in AR transcription factor signalling activity. Our results provide insights into cancer biology and show that liquid biopsy can be used as a tool for comprehensive multi-omic discovery.

Biallelic tumour suppressor loss and DNA repair defects in de novo small-cell prostate carcinoma.

Small-cell prostate carcinoma (SCPC) is an aggressive malignancy that is managed similarly to small-cell lung cancer. SCPC can evolve from prostate adenocarcinoma in response to androgen deprivation therapy, but, in rare cases, is present at initial cancer diagnosis. The molecular aetiology of de novo SCPC is incompletely understood, owing to the scarcity of tumour tissue and the short life-expectancy of patients. Through a retrospective search of our regional oncology pharmacy database, we identified 18 patients diagnosed with de novo SCPC between 2004 and 2017. Ten patients had pure SCPC pathology, and the remainder had some admixed adenocarcinoma foci, but all were treated with first-line platinum-based chemotherapy. The median overall survival was 28 months. We performed targeted DNA sequencing, whole exome sequencing and mRNA profiling on formalin-fixed paraffin-embedded archival tumour tissue. We observed frequent biallelic deletion and/or mutation of the tumour suppressor genes TP53, RB1, and PTEN, similarly to what was found in treatment-related SCPC. Indeed, at the RNA level, pure de novo SCPC closely resembled treatment-related SCPC. However, five patients had biallelic loss of DNA repair genes, including BRCA1, BRCA2, ATM, and MSH2/6, potentially underlying the high genomic instability of this rare disease variant. Two patients with pure de novo SCPC harboured ETS gene rearrangements involving androgen-driven promoters, consistent with the evolution of de novo SCPC from an androgen-driven ancestor. Overall, our results reveal a highly aggressive molecular landscape that underlies this unusual pathological variant, and suggest opportunities for targeted therapy strategies in a disease with few treatment options. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Multiregion sampling of de novo metastatic prostate cancer reveals complex polyclonality and augments clinical genotyping.

De novo metastatic prostate cancer is highly aggressive, but the paucity of routinely collected tissue has hindered genomic stratification and precision oncology. Here, we leveraged a rare study of surgical intervention in 43 de novo metastatic prostate cancers to assess somatic genotypes across 607 synchronous primary and metastatic tissue regions plus circulating tumor DNA. Intra-prostate heterogeneity was pervasive and impacted clinically relevant genes, resulting in discordant genotypes between select primary restricted regions and synchronous metastases. Additional complexity was driven by polyclonal metastatic seeding from phylogenetically related primary populations. When simulating clinical practice relying on a single tissue region, genomic heterogeneity plus variable tumor fraction across samples caused inaccurate genotyping of dominant disease; however, pooling extracted DNA from multiple biopsy cores before sequencing can rescue misassigned somatic genotypes. Our results define the relationship between synchronous treatment-sensitive primary and metastatic lesions in men with de novo metastatic prostate cancer and provide a framework for implementing genomics-guided patient management.

A genome-wide CRISPR screen maps endogenous regulators of PPARG gene expression in bladder cancer.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor central in the regulation of key cellular processes including cell metabolism, tissue differentiation, and regulation of the immune system. PPARγ is required for normal differentiation of the urothelium and is thought to be an essential driver of the luminal subtype of bladder cancer. However, the molecular components that regulate PPARG gene expression in bladder cancer remain unclear. Here, we developed an endogenous PPARG reporter system in luminal bladder cancer cells and performed genome-wide CRISPR knockout screening to identify bona fide regulators of PPARG gene expression. Functional validation of the dataset confirmed GATA3, SPT6, and the cohesin complex components SMC1A, and RAD21, as permissive upstream positive regulators of PPARG gene expression in luminal bladder cancer. In summary, this work provides a resource and biological insights to aid our understanding of PPARG regulation in bladder cancer.

A generalizable machine learning framework for classifying DNA repair defects using ctDNA exomes.

Specific classes of DNA damage repair (DDR) defect can drive sensitivity to emerging therapies for metastatic prostate cancer. However, biomarker approaches based on DDR gene sequencing do not accurately predict DDR deficiency or treatment benefit. Somatic alteration signatures may identify DDR deficiency but historically require whole-genome sequencing of tumour tissue. We assembled whole-exome sequencing data for 155 high ctDNA fraction plasma cell-free DNA and matched leukocyte DNA samples from patients with metastatic prostate or bladder cancer. Labels for DDR gene alterations were established using deep targeted sequencing. Per sample mutation and copy number features were used to train XGBoost ensemble models. Naive somatic features and trinucleotide signatures were associated with specific DDR gene alterations but insufficient to resolve each class. Conversely, XGBoost-derived models showed strong performance including an area under the curve of 0.99, 0.99 and 1.00 for identifying BRCA2, CDK12, and mismatch repair deficiency in metastatic prostate cancer. Our machine learning approach re-classified several samples exhibiting genomic features inconsistent with original labels, identified a metastatic bladder cancer sample with a homozygous BRCA2 copy loss, and outperformed an existing exome-based classifier for BRCA2 deficiency. We present DARC Sign (DnA Repair Classification SIGNatures); a public machine learning tool leveraging clinically-practical liquid biopsy specimens for simultaneously identifying multiple types of metastatic prostate cancer DDR deficiencies. We posit that it will be useful for understanding differential responses to DDR-directed therapies in ongoing clinical trials and may ultimately enable prospective identification of prostate cancers with phenotypic evidence of DDR deficiency.

Somatic Features of Response and Relapse in Non-muscle-invasive Bladder Cancer Treated with Bacillus Calmette-Guérin Immunotherapy.

BACKGROUND: High-risk non-muscle-invasive bladder cancer (NMIBC) is treated with bacillus Calmette-Guérin (BCG), but relapse is common. Improvement of patient outcomes requires better understanding of links between BCG resistance and genomic driver alterations. OBJECTIVE: To validate the prognostic impact of common genomic alterations in NMIBC pretreatment and define somatic changes present in post-BCG relapses. DESIGN, SETTING, AND PARTICIPANTS: We retrieved tumour tissues and outcomes for 90 patients with BCG-naive NMIBC initiating BCG monotherapy. Post-BCG tissue was available from 34 patients. All tissues underwent targeted sequencing of tumour and matched normal. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Associations between clinical outcomes and genomics were determined using Cox proportional hazard models. RESULTS AND LIMITATIONS: Of the patients, 58% were relapse free at data cut-off, 24% had NMIBC recurrence, and 18% experienced muscle-invasive progression. The risk of relapse was associated with ARID1A mutation (hazard ratio [HR] = 2.00; p = 0.04) and CCNE1 amplification (HR = 2.61; p = 0.02). Pre- and post-BCG tumours shared truncal driver alterations, with mutations in TERT and chromatin remodelling genes particularly conserved. However, shifts in somatic profiles were common and clinically relevant alterations in FGFR3, PIK3CA, TSC1, and TP53 were temporally variable, despite apparent clonal prevalence at one time point. Limitations include the difficulty of resolving the relative impact of BCG therapy versus surgery on genomics at relapse and biopsy bias. CONCLUSIONS: Somatic hypermutation and alterations in CCNE1 and ARID1A should be incorporated into future models predicting NMIBC BCG outcomes. Changes in tumour genomics over time highlight the importance of recent biopsy when considering targeted therapies, and suggest that relapse after BCG is due to persisting and evolving precursor populations. PATIENT SUMMARY: Changes in key cancer genes can predict bladder cancer relapse after treatment with bacillus Calmette-Guérin. Relapses after treatment can be driven by large-scale genetic changes within the cancer. These genetic changes help us understand how superficial bladder cancer can progress to be treatment resistant.

Genomic Features of Lung-Recurrent Hormone-Sensitive Prostate Cancer.

PURPOSE: Pulmonary involvement is rare in metastatic hormone-sensitive prostate cancer (mHSPC) that recurs after treatment for localized disease. Guidelines recommend intensive systemic therapy, similar to patients with liver metastases, but some lung-recurrent mHSPC may have good outcomes. Genomic features of lung metastases may clarify disease aggression, but are poorly understood since lung biopsy is rarely performed. We present a comparative assessment of genomic drivers and heterogeneity in metachronous prostate tumors and lung metastases. METHODS: We leveraged a prospective functional imaging study of 208 biochemically recurrent prostate cancers to identify 10 patients with lung-recurrent mHSPC. Histologic diagnosis was attained via thoracic surgery or fine-needle lung biopsy. We retrieved clinical data and performed multiregion sampling of primary tumors and metastases. Targeted and/or whole-exome sequencing was applied to 46 primary and 32 metastatic foci. RESULTS: Unusually for mHSPC, all patients remained alive despite a median follow-up of 11.5 years. Several patients experienced long-term freedom from systemic treatment. The genomic landscape of lung-recurrent mHSPC was typical of curable prostate cancer with frequent PTEN, SPOP, and chromosome 8p alterations, and there were no deleterious TP53 and DNA damage repair gene mutations that characterize aggressive prostate cancer. Despite a long median time to recurrence (76.8 months), copy number alterations and clonal mutations were highly conserved between metastatic and primary foci, consistent with intrapatient homogeneity and limited genomic evolution. CONCLUSION: In this retrospective hypothesis-generating study, we observed indolent genomic etiology in selected lung-recurrent mHSPC, cautioning against grouping these patients together with liver or bone-predominant mHSPC. Although our data do not generalize to all patients with lung metastases, the results encourage prospective efforts to stratify lung-recurrent mHSPC by genomic features.

BRCA2, ATM, and CDK12 Defects Differentially Shape Prostate Tumor Driver Genomics and Clinical Aggression.

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: BRCA2, ATM, and CDK12 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 BRCA2 deletions. Tumor suppressors TP53, RB1, and PTEN were controlled via disruptive chromosomal rearrangements in BRCA2-defective samples, but via oncogene amplification in context of CDK12 defects. TP53 mutations were rare in cases with ATM 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 BRCA2 and CDK12, but not ATM, was associated with poor clinical outcomes. CONCLUSIONS: BRCA2, ATM, and CDK12 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.

Plasma ctDNA is a tumor tissue surrogate and enables clinical-genomic stratification of metastatic bladder cancer.

Molecular stratification can improve the management of advanced cancers, but requires relevant tumor samples. Metastatic urothelial carcinoma (mUC) is poised to benefit given a recent expansion of treatment options and its high genomic heterogeneity. We profile minimally-invasive plasma circulating tumor DNA (ctDNA) samples from 104 mUC patients, and compare to same-patient tumor tissue obtained during invasive surgery. Patient ctDNA abundance is independently prognostic for overall survival in patients initiating first-line systemic therapy. Importantly, ctDNA analysis reproduces the somatic driver genome as described from tissue-based cohorts. Furthermore, mutation concordance between ctDNA and matched tumor tissue is 83.4%, enabling benchmarking of proposed clinical biomarkers. While 90% of mutations are identified across serial ctDNA samples, concordance for serial tumor tissue is significantly lower. Overall, our exploratory analysis demonstrates that genomic profiling of ctDNA in mUC is reliable and practical, and mitigates against disease undersampling inherent to studying archival primary tumor foci. We urge the incorporation of cell-free DNA profiling into molecularly-guided clinical trials for mUC.

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

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.

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

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 MSH2, MSH6, or MLH1, microsatellite instability, and characteristic trinucleotide signatures. ctDNA was concordant with mismatch repair protein IHC and DNA sequencing of tumor tissue. Tumor suppressors such as PTEN, RB1, and TP53 were inactivated by mutation rather than copy-number loss. Hotspot mutations in oncogenes such as AKT1, PIK3CA, and CTNNB1 were common, and the androgen receptor (AR)-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.See related commentary by Schweizer and Yu, p. 981.

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

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.

Predicting therapy response and resistance in metastatic prostate cancer with circulating tumor DNA.

The treatment of metastatic castration-resistant prostate cancer (mCRPC) is empirical, with progress to a more precision medicine approach hampered by lack of predictive biomarkers. This is due in large part to the historical difficulty of molecularly profiling a bone-predominant metastatic disease. Focus has turned to minimally invasive sources of tumor material to better understand the molecular drivers of therapy resistance. Circulating cell-free tumor DNA (ctDNA) is highly abundant in the bloodstream of mCRPC patients and appears to provide an accurate snapshot of real-time tumor genomics. Already, the analysis of androgen receptor gene alterations in the ctDNA of mCRPC patient cohorts has suggested significant potential for guiding the use of androgen receptor-directed therapy. Furthermore, the monitoring of patient ctDNA burden in the wake of systemic therapy may offer a powerful surrogate for tracking tumor responses and emerging resistant subclones. This seminar covers recent advances in mCRPC patient ctDNA profiling, emerging associations of distinct molecular subtypes with clinical outcomes, and the potential for ctDNA to augment precision oncology.

Enhancing knowledge discovery from cancer genomics data with Galaxy.

The field of cancer genomics has demonstrated the power of massively parallel sequencing techniques to inform on the genes and specific alterations that drive tumor onset and progression. Although large comprehensive sequence data sets continue to be made increasingly available, data analysis remains an ongoing challenge, particularly for laboratories lacking dedicated resources and bioinformatics expertise. To address this, we have produced a collection of Galaxy tools that represent many popular algorithms for detecting somatic genetic alterations from cancer genome and exome data. We developed new methods for parallelization of these tools within Galaxy to accelerate runtime and have demonstrated their usability and summarized their runtimes on multiple cloud service providers. Some tools represent extensions or refinement of existing toolkits to yield visualizations suited to cohort-wide cancer genomic analysis. For example, we present Oncocircos and Oncoprintplus, which generate data-rich summaries of exome-derived somatic mutation. Workflows that integrate these to achieve data integration and visualizations are demonstrated on a cohort of 96 diffuse large B-cell lymphomas and enabled the discovery of multiple candidate lymphoma-related genes. Our toolkit is available from our GitHub repository as Galaxy tool and dependency definitions and has been deployed using virtualization on multiple platforms including Docker.

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.