Integrated analyses highlight interactions between the three-dimensional genome and DNA, RNA and epigenomic alterations in metastatic prostate cancer.

The impact of variations in the three-dimensional structure of the genome has been recognized, but solid cancer tissue studies are limited. Here, we performed integrated deep Hi-C sequencing with matched whole-genome sequencing, whole-genome bisulfite sequencing, 5-hydroxymethylcytosine (5hmC) sequencing and RNA sequencing across a cohort of 80 biopsy samples from patients with metastatic castration-resistant prostate cancer. Dramatic differences were present in gene expression, 5-methylcytosine/5hmC methylation and in structural variation versus mutation rate between A and B (open and closed) chromatin compartments. A subset of tumors exhibited depleted regional chromatin contacts at the AR locus, linked to extrachromosomal circular DNA (ecDNA) and worse response to AR signaling inhibitors. We also identified topological subtypes associated with stark differences in methylation structure, gene expression and prognosis. Our data suggested that DNA interactions may predispose to structural variant formation, exemplified by the recurrent TMPRSS2-ERG fusion. This comprehensive integrated sequencing effort represents a unique clinical tumor resource.

Toward Informed Selection and Interpretation of Clinical Genomic Tests in Prostate Cancer.

Clinical genomic testing of patient germline, tumor tissue, or plasma cell-free DNA can enable a personalized approach to cancer management and treatment. In prostate cancer (PCa), broad genotyping tests are now widely used to identify germline and/or somatic alterations in BRCA2 and other DNA damage repair genes. Alterations in these genes can confer cancer sensitivity to poly (ADP-ribose) polymerase inhibitors, are linked with poor prognosis, and can have potential hereditary cancer implications for family members. However, there is huge variability in genomic tests and reporting standards, meaning that for successful implementation of testing in clinical practice, end users must carefully select the most appropriate test for a given patient and critically interpret the results. In this white paper, we outline key pre- and post-test considerations for choosing a genomic test and evaluating reported variants, specifically for patients with advanced PCa. Test choice must be based on clinical context and disease state, availability and suitability of tumor tissue, and the genes and regions that are covered by the test. We describe strategies to recognize false positives or negatives in test results, including frameworks to assess low tumor fraction, subclonal alterations, clonal hematopoiesis, and pathogenic versus nonpathogenic variants. We assume that improved understanding among health care professionals and researchers of the nuances associated with genomic testing will ultimately lead to optimal patient care and clinical decision making.

Prediction of plasma ctDNA fraction and prognostic implications of liquid biopsy in advanced prostate cancer.

No consensus strategies exist for prognosticating metastatic castration-resistant prostate cancer (mCRPC). Circulating tumor DNA fraction (ctDNA%) is increasingly reported by commercial and laboratory tests but its utility for risk stratification is unclear. Here, we intersect ctDNA%, treatment outcomes, and clinical characteristics across 738 plasma samples from 491 male mCRPC patients from two randomized multicentre phase II trials and a prospective province-wide blood biobanking program. ctDNA% correlates with serum and radiographic metrics of disease burden and is highest in patients with liver metastases. ctDNA% strongly predicts overall survival, progression-free survival, and treatment response independent of therapeutic context and outperformed established prognostic clinical factors. Recognizing that ctDNA-based biomarker genotyping is limited by low ctDNA% in some patients, we leverage the relationship between clinical prognostic factors and ctDNA% to develop a clinically-interpretable machine-learning tool that predicts whether a patient has sufficient ctDNA% for informative ctDNA genotyping (available online: https://www.ctDNA.org ). Our results affirm ctDNA% as an actionable tool for patient risk stratification and provide a practical framework for optimized biomarker testing.

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.

Molecular features of prostate cancer after neoadjuvant therapy in the phase 3 CALGB 90203 trial.

BACKGROUND: The phase 3 CALGB 90203 (Alliance) trial evaluated neoadjuvant chemohormonal therapy for high-risk localized prostate cancer before radical prostatectomy. We dissected the molecular features of post-treated tumors with long-term clinical outcomes to explore mechanisms of response and resistance to chemohormonal therapy. METHODS: We evaluated 471 radical prostatectomy tumors, including 294 samples from 166 patients treated with 6 cycles of docetaxel plus androgen deprivation therapy before radical prostatectomy and 177 samples from 97 patients in the control arm (radical prostatectomy alone). Targeted DNA sequencing and RNA expression of tumor foci and adjacent noncancer regions were analyzed in conjunction with pathologic changes and clinical outcomes. RESULTS: Tumor fraction estimated from DNA sequencing was significantly lower in post-treated tumor tissues after chemohormonal therapy compared with controls. Higher tumor fraction after chemohormonal therapy was associated with aggressive pathologic features and poor outcomes, including prostate-specific antigen-progression-free survival. SPOP alterations were infrequently detected after chemohormonal therapy, while TP53 alterations were enriched and associated with shorter overall survival. Residual tumor fraction after chemohormonal therapy was linked to higher expression of androgen receptor-regulated genes, cell cycle genes, and neuroendocrine genes, suggesting persistent populations of active prostate cancer cells. Supervised clustering of post-treated high-tumor-fraction tissues identified a group of patients with elevated cell cycle-related gene expression and poor clinical outcomes. CONCLUSIONS: Distinct recurrent prostate cancer genomic and transcriptomic features are observed after exposure to docetaxel and androgen deprivation therapy. Tumor fraction assessed by DNA sequencing quantifies pathologic response and could be a useful trial endpoint or prognostic biomarker. TP53 alterations and high cell cycle transcriptomic activity are linked to aggressive residual disease, despite potent chemohormonal therapy.

Phase 1b study of enzalutamide plus CC-115, a dual mTORC1/2 and DNA-PK inhibitor, in men with metastatic castration-resistant prostate cancer (mCRPC).

BACKGROUND: CC-115, a dual mTORC1/2 and DNA-PK inhibitor, has promising antitumour activity when combined with androgen receptor (AR) inhibition in pre-clinical models. METHODS: Phase 1b multicentre trial evaluating enzalutamide with escalating doses of CC-115 in AR inhibitor-naive mCRPC patients (n = 41). Primary endpoints were safety and RP2D. Secondary endpoints included PSA response, time-to-PSA progression, and radiographic progression. RESULTS: Common adverse effects included rash (31.7% Grades 1-2 (Gr); 31.7% Gr 3), pruritis (43.9% Gr 1-2), diarrhoea (37% Gr 1-2), and hypertension (17% Gr 1-2; 9.8% Gr 3). CC-115 RP2D was 5 mg twice a day. In 40 evaluable patients, 80% achieved ≥50% reduction in PSA (PSA50), and 58% achieved ≥90% reduction in PSA (PSA90) by 12 weeks. Median time-to-PSA progression was 14.7 months and median rPFS was 22.1 months. Stratification by PI3K alterations demonstrated a non-statistically significant trend towards improved PSA50 response (PSA50 of 94% vs. 67%, p = 0.08). Exploratory pre-clinical analysis suggested CC-115 inhibited mTOR pathway strongly, but may be insufficient to inhibit DNA-PK at RP2D. CONCLUSIONS: The combination of enzalutamide and CC-115 was well tolerated. A non-statistically significant trend towards improved PSA response was observed in patients harbouring PI3K pathway alterations, suggesting potential predictive biomarkers of response to a PI3K/AKT/mTOR pathway inhibitor. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02833883.

Practical Guidance on Establishing a Molecular Testing Pathway for Alterations in Homologous Recombination Repair Genes in Clinical Practice for Patients with Metastatic Prostate Cancer.

CONTEXT: Prostate cancer is a molecularly heterogeneous disease that is amenable to diagnostic testing to identify patients potentially eligible for personalised treatments inform familial risk and provide relevant information about potential prognosis. Several guidelines support the integration of genomic testing in a shared decision-making framework so that both health care professionals (HCPs) and patients are involved in determining the best treatment approach. OBJECTIVE: To review current guidelines on molecular diagnostic testing for homologous recombination repair (HRR) gene alterations in patients with metastatic prostate cancer, with the aim of providing practical considerations for effective guideline implementation and establishment of an appropriate pathway for molecular diagnostic testing. EVIDENCE ACQUISITION: We undertook a nonsystematic narrative review of the literature using PubMed to identify current guidelines and recommendations on molecular diagnostic testing for BRCA and/or homologous recombination repair gene alterations (HRRm) in patients with prostate cancer. In addition, selected articles that included BRCA/HRRm testing in clinical trials in metastatic castration-resistant prostate cancer and real-world evidence were also evaluated. Websites for relevant societies were reviewed for molecular diagnostic guidelines not published on PubMed. EVIDENCE SYNTHESIS: Our review of guidelines published by several international societies that include molecular testing in prostate cancer identified variations in molecular testing approaches. The review of testing approaches used in clinical trials and real-world settings also highlighted several aspects that require improvement. Therefore, we compiled practical guidance for establishing an appropriate BRCA/HRR mutation testing pathway. CONCLUSIONS: While there are several challenges to molecular testing and interpretation of test results that require enhancement, a multidisciplinary team approach will empower HCPs and their institutions to improve on or initiate their own molecular testing pathways. This in turn will lead to improvements in management strategies for patients with metastatic prostate cancer, for whom better treatment outcomes is a significant unmet need. PATIENT SUMMARY: Establishing a molecular testing pathway in clinical practice for patients with metastatic castration-resistant prostate cancer will lead to fairer and more equal access to personalised treatments. This should lead to better outcomes, particularly for patients whose disease has spread to other areas of the body.

The Genomic and Epigenomic Landscape of Double-Negative Metastatic Prostate Cancer.

Systemic targeted therapy in prostate cancer is primarily focused on ablating androgen signaling. Androgen deprivation therapy and second-generation androgen receptor (AR)-targeted therapy selectively favor the development of treatment-resistant subtypes of metastatic castration-resistant prostate cancer (mCRPC), defined by AR and neuroendocrine (NE) markers. Molecular drivers of double-negative (AR-/NE-) mCRPC are poorly defined. In this study, we comprehensively characterized treatment-emergent mCRPC by integrating matched RNA sequencing, whole-genome sequencing, and whole-genome bisulfite sequencing from 210 tumors. AR-/NE- tumors were clinically and molecularly distinct from other mCRPC subtypes, with the shortest survival, amplification of the chromatin remodeler CHD7, and PTEN loss. Methylation changes in CHD7 candidate enhancers were linked to elevated CHD7 expression in AR-/NE+ tumors. Genome-wide methylation analysis nominated Krüppel-like factor 5 (KLF5) as a driver of the AR-/NE- phenotype, and KLF5 activity was linked to RB1 loss. These observations reveal the aggressiveness of AR-/NE- mCRPC and could facilitate the identification of therapeutic targets in this highly aggressive disease. SIGNIFICANCE: Comprehensive characterization of the five subtypes of metastatic castration-resistant prostate cancer identified transcription factors that drive each subtype and showed that the double-negative subtype has the worst prognosis.

Genetic and Genomic Testing for Prostate Cancer: Beyond DNA Repair.

Significant progress has been made in genetic and genomic testing for prostate cancer across the disease spectrum. Molecular profiling is increasingly relevant for routine clinical management, fueled in part by advancements in testing technology and integration of biomarkers into clinical trials. In metastatic prostate cancer, defects in DNA damage response genes are now established predictors of benefit to US Food and Drug Administration-approved poly (ADP-ribose) polymerase inhibitors and immune checkpoint inhibitors, and trials are actively investigating these and other targeted treatment strategies in earlier disease states. Excitingly, opportunities for molecularly informed management beyond DNA damage response genes are also maturing. Germline genetic variants (eg, BRCA2 or MSH2/6) and polygenic germline risk scores are being investigated to inform cancer screening and active surveillance in at-risk carriers. RNA expression tests have recently gained traction in localized prostate cancer, enabling patient risk stratification and tailored treatment intensification via radiotherapy and/or androgen deprivation therapy for localized or salvage treatment. Finally, emerging minimally invasive circulating tumor DNA technology promises to enhance biomarker testing in advanced disease pending additional methodological and clinical validation. Collectively, genetic and genomic tests are rapidly becoming indispensable tools for informing the optimal clinical management of prostate cancer.

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.

Early On-treatment Changes in Circulating Tumor DNA Fraction and Response to Enzalutamide or Abiraterone in Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: Androgen receptor pathway inhibitors (ARPI) are standard of care for treatment-naïve metastatic castration-resistant prostate cancer (mCRPC), but rapid resistance is common. Early identification of resistance will improve management strategies. We investigated whether changes in circulating tumor DNA (ctDNA) fraction during ARPI treatment are linked with mCRPC clinical outcomes. EXPERIMENTAL DESIGN: Plasma cell-free DNA was collected from 81 patients with mCRPC at baseline and after 4 weeks of first-line ARPI treatment during two prospective multicenter observational studies (NCT02426333; NCT02471469). ctDNA fraction was calculated from somatic mutations in targeted sequencing and genome copy-number profiles. Samples were classified into detected versus undetected ctDNA. Outcome measurements were progression-free survival (PFS) and overall survival (OS). Nondurable treatment response was defined as PFS ≤6 months. RESULTS: ctDNA was detected in 48/81 (59%) baseline and 29/81 (36%) 4-week samples. ctDNA fraction for samples with detected ctDNA was lower at 4 weeks versus baseline (median 5.0% versus 14.5%, P = 0.017). PFS and OS were shortest for patients with persistent ctDNA at 4 weeks (univariate HR, 4.79; 95% CI, 2.62-8.77 and univariate HR, 5.49; 95% CI, 2.76-10.91, respectively), independent of clinical prognostic factors. For patients exhibiting change from detected to undetected ctDNA by 4 weeks, there was no significant PFS difference versus patients with baseline undetected ctDNA. ctDNA change had a positive predictive value of 88% and negative predictive value of 92% for identifying nondurable responses. CONCLUSIONS: Early changes in ctDNA fraction are strongly linked to duration of first-line ARPI treatment benefit and survival in mCRPC and may inform early therapy switches or treatment intensification. See related commentary by Sartor, p. 2745.

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.

Towards clinical implementation of circulating tumor DNA in metastatic prostate cancer: Opportunities for integration and pitfalls to interpretation.

Plasma circulating tumor DNA (ctDNA) represents short fragments of tumor-derived DNA released into the bloodstream primarily from cancer cells undergoing apoptosis. In metastatic castration-resistant prostate cancer (mCRPC), characterizing genomic alterations in ctDNA identifies mutations, copy number alterations, and structural rearrangements with predictive and prognostic biomarker utility. These associations with clinical outcomes have resulted in ctDNA increasingly incorporated into routine clinical care. In this review, we summarize current and emerging applications for ctDNA analysis in metastatic prostate cancer, including outcome prediction, treatment selection, and characterization of treatment resistance. We also discuss potential pitfalls with interpreting ctDNA findings, namely false negatives arising from low tumor content and optimal assay design, including correction for clonal hematopoiesis of indeterminate potential and germline variants. Understanding the influence of these limitations on interpretation of ctDNA results is necessary to overcome barriers to clinical implementation. Nevertheless, as assay availability and technology continue to improve, recognizing both opportunities and shortcomings of ctDNA analysis will retain relevance with informing the implementation of precision-oncology initiatives for metastatic prostate cancer.

The cell-free DNA methylome captures distinctions between localized and metastatic prostate tumors.

Metastatic prostate cancer remains a major clinical challenge and metastatic lesions are highly heterogeneous and difficult to biopsy. Liquid biopsy provides opportunities to gain insights into the underlying biology. Here, using the highly sensitive enrichment-based sequencing technology, we provide analysis of 60 and 175 plasma DNA methylomes from patients with localized and metastatic prostate cancer, respectively. We show that the cell-free DNA methylome can capture variations beyond the tumor. A global hypermethylation in metastatic samples is observed, coupled with hypomethylation in the pericentromeric regions. Hypermethylation at the promoter of a glucocorticoid receptor gene NR3C1 is associated with a decreased immune signature. The cell-free DNA methylome is reflective of clinical outcomes and can distinguish different disease types with 0.989 prediction accuracy. Finally, we show the ability of predicting copy number alterations from the data, providing opportunities for joint genetic and epigenetic analysis on limited biological samples.

TP53 Alterations Are Associated With Poor Survival in Patients With Primary Mediastinal Nonseminoma Germ Cell Tumors.

BACKGROUND: Primary mediastinal nonseminoma germ cell tumors (PMNSGCT) are a subgroup of nonseminoma germ cell tumors (GCT) with poor prognosis. In this study, PMNSGCT-specific genomic landscape was analyzed and correlated with clinical outcomes. METHODS: DNA was extracted and sequenced from 28 archival tumor tissue of patients with mediastinal GCT (3 seminoma and 25 nonseminoma). Overall survival (OS) and association with gene alterations were estimated using the Kaplan-Meier and univariate Cox regression methods. RESULTS: Three patients (11%) had a karyotype XXY, 17/28 (61%) tumor samples presented chromosome 12p amplification. Somatic mutations were detected in 19/28 (68%) samples. The most frequently mutated genes were: TP53 (13/28; 46%), KIT (5/28; 18%), and KRAS (5/28; 18%). Deleterious TP53 alterations were associated with significantly reduced overall survival (HR: 7.16; P = .012). CONCLUSIONS: TP53 alterations are common in PMNSGCT and are associated with reduced overall survival, potentially underlying the poor sensitivity to chemotherapy observed in these patients.

Long-Term Outcomes and Genetic Predictors of Response to Metastasis-Directed Therapy Versus Observation in Oligometastatic Prostate Cancer: Analysis of STOMP and ORIOLE Trials.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.The initial STOMP and ORIOLE trial reports suggested that metastasis-directed therapy (MDT) in oligometastatic castration-sensitive prostate cancer (omCSPC) was associated with improved treatment outcomes. Here, we present long-term outcomes of MDT in omCSPC by pooling STOMP and ORIOLE and assess the ability of a high-risk mutational signature to risk stratify outcomes after MDT. The primary end point was progression-free survival (PFS) calculated using the Kaplan-Meier method. High-risk mutations were defined as pathogenic somatic mutations within ATM, BRCA1/2, Rb1, or TP53. The median follow-up for the whole group was 52.5 months. Median PFS was prolonged with MDT compared with observation (pooled hazard ratio [HR], 0.44; 95% CI, 0.29 to 0.66; P value < .001), with the largest benefit of MDT in patients with a high-risk mutation (HR high-risk, 0.05; HR no high-risk, 0.42; P value for interaction: .12). Within the MDT cohort, the PFS was 13.4 months in those without a high-risk mutation, compared with 7.5 months in those with a high-risk mutation (HR, 0.53; 95% CI, 0.25 to 1.11; P = .09). Long-term outcomes from the only two randomized trials in omCSPC suggest a sustained clinical benefit to MDT over observation. A high-risk mutational signature may help risk stratify treatment outcomes after MDT.