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.

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 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.

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.

The 5-Hydroxymethylcytosine Landscape of Prostate Cancer.

Analysis of DNA methylation is a valuable tool to understand disease progression and is increasingly being used to create diagnostic and prognostic clinical biomarkers. While conversion of cytosine to 5-methylcytosine (5mC) commonly results in transcriptional repression, further conversion to 5-hydroxymethylcytosine (5hmC) is associated with transcriptional activation. Here we perform the first study integrating whole-genome 5hmC with DNA, 5mC, and transcriptome sequencing in clinical samples of benign, localized, and advanced prostate cancer. 5hmC is shown to mark activation of cancer drivers and downstream targets. Furthermore, 5hmC sequencing revealed profoundly altered cell states throughout the disease course, characterized by increased proliferation, oncogenic signaling, dedifferentiation, and lineage plasticity to neuroendocrine and gastrointestinal lineages. Finally, 5hmC sequencing of cell-free DNA from patients with metastatic disease proved useful as a prognostic biomarker able to identify an aggressive subtype of prostate cancer using the genes TOP2A and EZH2, previously only detectable by transcriptomic analysis of solid tumor biopsies. Overall, these findings reveal that 5hmC marks epigenomic activation in prostate cancer and identify hallmarks of prostate cancer progression with potential as biomarkers of aggressive disease. SIGNIFICANCE: In prostate cancer, 5-hydroxymethylcytosine delineates oncogene activation and stage-specific cell states and can be analyzed in liquid biopsies to detect cancer phenotypes. See related article by Wu and Attard, p. 3880.

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.

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.

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.

Technical and biological constraints on ctDNA-based genotyping.

Circulating tumor DNA (ctDNA) enables real-time genomic profiling of cancer without the need for tissue biopsy. ctDNA-based technology is seeing rapid uptake in clinical practice due to the potential to inform patient management from diagnosis to advanced disease. In metastatic disease, ctDNA can identify somatic mutations, copy-number variants (CNVs), and structural rearrangements that are predictive of therapy response. However, the ctDNA fraction (ctDNA%) is unpredictable and confounds variant detection strategies, undermining confidence in liquid biopsy results. Assay design also influences which types of genomic alterations are identifiable. Here, we describe the relationships between ctDNA%, methodology, and sensitivity-specificity for major classes of genomic alterations in prostate cancer. We provide recommendations to navigate the technical complexities that constrain the detection of clinically relevant genomic alterations in ctDNA.

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

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 AR gene, with an average 50% increase in AR copy number, changes in AR 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 AR genotype continues to evolve during sequential lines of AR inhibition and drives acquired resistance in patients with mCRPC.

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.

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.

Morphologic and genomic characterization of urothelial to sarcomatoid transition in muscle-invasive bladder cancer.

INTRODUCTION: The sarcomatoid morphology of muscle-invasive bladder cancer (MIBC) is associated with unfavorable prognosis. However, the genomic, transcriptomic, and proteomic relationship between conventional urothelial and synchronous sarcomatoid morphology is poorly defined. METHODS: We compiled a cohort of 21 MIBC patients with components of conventional urothelial and adjacent sarcomatoid morphology within the same tumor focus. We performed comprehensive pathologic and immunohistochemical characterization and in 4 selected cases, subjected both morphologic components to targeted DNA sequencing and whole transcriptome analysis. RESULTS: Synchronous sarcomatoid and urothelial morphology from the same MIBC foci shared truncal somatic mutations, indicating a common ancestral clone. However, additional mutations or copy number alterations restricted to the either component suggested divergent evolution at the genomic level. This was confirmed at the transcriptome level since while the urothelial component exhibited a basal-like subtype (TCGA2014: cluster III, LundTax: basal/squamous-like), the sarcomatoid morphology was predominantly cluster IV (claudin-low). Protein expression was consistent with a basal-like phenotype in both morphologies in 18/21 of cases. However, most cases had evidence of active epithelial-to-mesenchymal transition (E-Cad ↓ and Zeb1 or TWIST1 ↑) from urothelial toward the sarcomatoid morphology. Drug response signatures nominated different targets for each morphology and proposed agents under clinical investigation in liposarcoma or other sarcoma. PD-L1 expression was higher in the sarcomatoid than the urothelial component. CONCLUSIONS: Conventional urothelial and adjacent sarcomatoid morphologies of MIBC arise from the same common ancestor and share a basal-like phenotype. However, divergence between the morphologies at the genome, transcriptome, and proteome level suggests differential sensitivity to therapy.

Morphologic and genomic characterization of urothelial to sarcomatoid transition in muscle-invasive bladder cancer.

INTRODUCTION: The sarcomatoid morphology of muscle-invasive bladder cancer (MIBC) is associated with unfavorable prognosis. However, the genomic, transcriptomic, and proteomic relationship between conventional urothelial and synchronous sarcomatoid morphology is poorly defined. METHODS: We compiled a cohort of 21 MIBC patients with components of conventional urothelial and adjacent sarcomatoid morphology within the same tumor focus. We performed comprehensive pathologic and immunohistochemical characterization and in 4 selected cases, subjected both morphologic components to targeted DNA sequencing and whole transcriptome analysis. RESULTS: Synchronous sarcomatoid and urothelial morphology from the same MIBC foci shared truncal somatic mutations, indicating a common ancestral clone. However, additional mutations or copy number alterations restricted to the either component suggested divergent evolution at the genomic level. This was confirmed at the transcriptome level since while the urothelial component exhibited a basal-like subtype (TCGA2014: cluster III, LundTax: basal/squamous-like), the sarcomatoid morphology was predominantly cluster IV (claudin-low). Protein expression was consistent with a basal-like phenotype in both morphologies in 18/21 of cases. However, most cases had evidence of active epithelial-to-mesenchymal transition (E-Cad ↓ and Zeb1 or TWIST1 ↑) from urothelial toward the sarcomatoid morphology. Drug response signatures nominated different targets for each morphology and proposed agents under clinical investigation in liposarcoma or other sarcoma. PD-L1 expression was higher in the sarcomatoid than the urothelial component. CONCLUSIONS: Conventional urothelial and adjacent sarcomatoid morphologies of MIBC arise from the same common ancestor and share a basal-like phenotype. However, divergence between the morphologies at the genome, transcriptome, and proteome level suggests differential sensitivity to therapy.

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.

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.