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.

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 DNAtargeted sequencing data from 433 patients with mPC with circulating tumor DNA (ctDNA) purity !2%. Samples with somatic hypermutation were subjected to 185 Â 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

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.

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Background: 10% of newly diagnosed prostate cancer presents with metastases. Known as de novo metastatic castrate-sensitive prostate cancer (mCSPC), it is disproportionally responsible for >50% of prostate cancer deaths. Cancer genotyping can identify vulnerabilities exploitable by targeted therapies, and promises to help prognosticate. However, tissue from de novo mCSPC is scarce; neither prostatectomy nor metastatic biopsy is standard, and it is unknown if diagnostic biopsies are representative of synchronous metastases. The potential for plasma circulating tumor DNA (ctDNA) to inform on tumor genotype is also unknown. Methods: We performed comprehensive pathological and genomic assessment of all spatially or phenotypically-distinct tumor foci (n=523) in 43 patients with de novo mCSPC who underwent prostatectomy, pelvic lymph node dissection, and plasma collection. Results: 91% (478/523) of tissue foci had evidence of prostate cancer by targeted DNA sequencing, with a median tumor fraction of 48%. When modeling random selection of a single primary foci (mirroring biopsy tissue availability in clinic), tumor fraction was <20% in 19% of patients. Only 46% of plasma cell-free DNA samples prior to systemic therapy had a ctDNA fraction above 0.3% (limit of detection); median tumor fraction of 5% in samples with confirmed ctDNA. We observed recurrent alterations in major driver genes, including TP53, FOXA1, PTEN, and RB1, and the genomic landscape was very similar to published cohorts of castration-resistant prostate cancer (excluding AR). Primary site genomic heterogeneity was pervasive, including secondary (clonally distinct) prostate cancer populations in 14% of patients. Polyclonal seeding of metastases was detected in 26% of patients. Biallelic inactivation of TP53, PTEN, and/or RB1 was observed in 63% of tumors, and was frequently found in synchronous metastases and ctDNA. The two patients with compound disruption of TP53, PTEN, and RB1 experienced rapid progression to castration-resistance and death within two years of diagnosis, despite initial low-risk clinical features. Across the cohort, biallelic disruption of TP53 together with high-risk clinical features at diagnosis was associated with rapid progression (HR 4.64 (95% CI: 1.70-12.69); P = 0.003). Conclusions: One fifth of patients with de novo mCSPC have pervasive low tumor fraction in their primary tumor and blood plasma. Many tumors exhibit spatial heterogeneity within the primary site, with evidence of multiple clones seeding metastases. This data raises concerns about accurate tumor genotyping in routine clinical practice where needle biopsy specimens are the only available tissue for profiling. Nevertheless, some de novo mCSPC are marked by aggressive genomics and experience rapid progression to lethal disease, suggesting that tailored multi-focal genomic profiling can further segment the disease. Citation Format: Evan Warner, Kim Van der Eecken, Andrew J. Murtha, Edmond M. Kwan, Sarah W. Ng, Xinyi E. Chen, Cecily Q. Bernales, Grainne Donnellan, Elena Schonlau, Sofie Verbeke, Nicolaas Lumen, Jo Van Dorpe, Gillian Vandekerkhove, Elie Ritch, Matti Annala, Bram De Laere, Piet Ost, Alexander W. Wyatt. Multi-focal genomic dissection of synchronous primary and metastatic tissue from de novo metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 41.