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.
Targeted agents and immunotherapies promise to transform the treatment of metastatic bladder cancer, but therapy selection will depend on practical tumor molecular stratification. Circulating tumor ...DNA (ctDNA) is established in several solid malignancies as a minimally invasive tool to profile the tumor genome in real-time, but is critically underexplored in bladder cancer.
We applied a combination of whole-exome sequencing and targeted sequencing across 50 bladder cancer driver genes to plasma cell-free DNA (cfDNA) from 51 patients with aggressive bladder cancer, including 37 with metastatic disease.
The majority of patients with metastasis, but only 14% of patients with localized disease, had ctDNA proportions above 2% of total cfDNA (median 16.5%, range 3.9%-72.6%). Twelve percent of estimable samples had evidence of genome hypermutation. We reveal an aggressive mutational landscape in metastatic bladder cancer with 95% of patients harboring deleterious alterations to
, or
, and 70% harboring a mutation or disrupting rearrangement affecting chromatin modifiers such as
Targetable alterations in MAPK/ERK or PI3K/AKT/mTOR pathways were robustly detected, including amplification of
(20% of patients) and activating hotspot mutations in
A (20%), with the latter mutually exclusive to truncating mutations in
A novel
gene fusion was identified in consecutive samples from one patient.
Our study demonstrates that ctDNA provides a practical and cost-effective snapshot of driver gene status in metastatic bladder cancer. The identification of a wide spectrum of clinically informative somatic alterations nominates ctDNA as a tool to dissect disease pathogenesis and guide therapy selection in patients with metastatic bladder cancer.
.
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.
To determine ctDNA abundance at de novo mCSPC diagnosis and whether ctDNA provides complementary clinically relevant information to a prostate biopsy.
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.
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.
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.
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.
Circulating tumor DNA (ctDNA) is highly abundant in de novo metastatic prostate cancer, but short-term androgen deprivation therapy rapidly reduces ctDNA. The best approach for tumor molecular subtyping and precision oncology should utilize both primary tissue biopsy and ctDNA.
Abstract Background Germline mutations in DNA repair genes were recently reported in 8–12% of patients with metastatic castration-resistant prostate cancer (mCRPC). It is unknown whether these ...mutations associate with differential response to androgen receptor (AR)-directed therapy. Objective To determine the clinical response of mCRPC patients with germline DNA repair defects to AR-directed therapies and to establish whether biallelic DNA repair gene loss is detectable in matched circulating tumor DNA (ctDNA). Design, setting, and participants We recruited 319 mCRPC patients and performed targeted germline sequencing of 22 DNA repair genes. In patients with deleterious germline mutations, plasma cell-free DNA was also sequenced. Outcome measurements and statistical analysis Prostate-specific antigen response and progression were assessed in relation to initial androgen deprivation therapy (ADT) and subsequent therapy for mCRPC using Kaplan–Meier analysis. Results and limitations Of the 319 patients, 24 (7.5%) had deleterious germline mutations, with BRCA2 ( n = 16) being the most frequent. Patients ( n = 22) with mutations in genes linked to homologous recombination were heterogeneous at initial presentation but, after starting ADT, progressed to mCRPC with a median time of 11.8 mo (95% confidence interval CI 5.1–18.4). The median time to prostate-specific antigen progression on first-line AR-targeted therapy in the mCRPC setting was 3.3 mo (95% CI 2.7-3.9). Ten out of 11 evaluable patients with germline BRCA2 mutations had somatic deletion of the intact allele in ctDNA. A limitation of this study is absence of a formal control cohort for comparison of clinical outcomes. Conclusions Patients with mCRPC who have germline DNA repair defects exhibit attenuated responses to AR-targeted therapy. Biallelic gene loss was robustly detected in ctDNA, suggesting that this patient subset could be prioritized for therapies exploiting defective DNA repair using a liquid biopsy. Patient summary Patients with metastatic prostate cancer and germline DNA repair defects exhibit a poor response to standard hormonal therapies, but may be prioritized for potentially more effective therapies using a blood test.
To understand functional consequences of genetic and transcriptional aberrations in prostate cancer, the proteomic changes during disease formation and progression need to be revealed. Here we report ...high-throughput mass spectrometry on clinical tissue samples of benign prostatic hyperplasia (BPH), untreated primary prostate cancer (PC) and castration resistant prostate cancer (CRPC). Each sample group shows a distinct protein profile. By integrative analysis we show that, especially in CRPC, gene copy number, DNA methylation, and RNA expression levels do not reliably predict proteomic changes. Instead, we uncover previously unrecognized molecular and pathway events, for example, several miRNA target correlations present at protein but not at mRNA level. Notably, we identify two metabolic shifts in the citric acid cycle (TCA cycle) during prostate cancer development and progression. Our proteogenomic analysis uncovers robustness against genomic and transcriptomic aberrations during prostate cancer progression, and significantly extends understanding of prostate cancer disease mechanisms.
Cancers emerge from an ongoing Darwinian evolutionary process, often leading to multiple competing subclones within a single primary tumour. This evolutionary process culminates in the formation of ...metastases, which is the cause of 90% of cancer-related deaths. However, despite its clinical importance, little is known about the principles governing the dissemination of cancer cells to distant organs. Although the hypothesis that each metastasis originates from a single tumour cell is generally supported, recent studies using mouse models of cancer demonstrated the existence of polyclonal seeding from and interclonal cooperation between multiple subclones. Here we sought definitive evidence for the existence of polyclonal seeding in human malignancy and to establish the clonal relationship among different metastases in the context of androgen-deprived metastatic prostate cancer. Using whole-genome sequencing, we characterized multiple metastases arising from prostate tumours in ten patients. Integrated analyses of subclonal architecture revealed the patterns of metastatic spread in unprecedented detail. Metastasis-to-metastasis spread was found to be common, either through de novo monoclonal seeding of daughter metastases or, in five cases, through the transfer of multiple tumour clones between metastatic sites. Lesions affecting tumour suppressor genes usually occur as single events, whereas mutations in genes involved in androgen receptor signalling commonly involve multiple, convergent events in different metastases. Our results elucidate in detail the complex patterns of metastatic spread and further our understanding of the development of resistance to androgen-deprivation therapy in prostate cancer.
Fusion genes are chromosomal aberrations that are found in many cancers and can be used as prognostic markers and drug targets in clinical practice. Fusions can lead to production of oncogenic fusion ...proteins or to enhanced expression of oncogenes. Several recent studies have reported that some fusion genes can escape microRNA regulation via 3'-untranslated region (3'-UTR) deletion. We performed whole transcriptome sequencing to identify fusion genes in glioma and discovered FGFR3-TACC3 fusions in 4 of 48 glioblastoma samples from patients both of mixed European and of Asian descent, but not in any of 43 low-grade glioma samples tested. The fusion, caused by tandem duplication on 4p16.3, led to the loss of the 3'-UTR of FGFR3, blocking gene regulation of miR-99a and enhancing expression of the fusion gene. The fusion gene was mutually exclusive with EGFR, PDGFR, or MET amplification. Using cultured glioblastoma cells and a mouse xenograft model, we found that fusion protein expression promoted cell proliferation and tumor progression, while WT FGFR3 protein was not tumorigenic, even under forced overexpression. These results demonstrated that the FGFR3-TACC3 gene fusion is expressed in human cancer and generates an oncogenic protein that promotes tumorigenesis in glioblastoma.
A significant subset of prostate cancer (PC) patients with a castration-resistant form of the disease (CRPC) show primary resistance to androgen receptor (AR)-targeting drugs developed against CRPC. ...As one explanation could be the expression of constitutively active androgen receptor splice variants (AR-Vs), our current objectives were to study AR-Vs and other AR aberrations to better understand the emergence of CRPC.
We analysed specimens from different stages of prostate cancer by next-generation sequencing and immunohistochemistry.
AR mutations and copy number variations were detected only in CRPC specimens. Genomic structural rearrangements of AR were observed in 5/30 metastatic CRPC patients, but they were not associated with expression of previously known AR-Vs. The predominant AR-Vs detected were AR-V3, AR-V7 and AR-V9, with the expression levels being significantly higher in CRPC cases compared to prostatectomy samples. Out of 25 CRPC metastases that expressed any AR variant, 17 cases harboured expression of all three of these AR-Vs. AR-V7 protein expression was highly heterogeneous and higher in CRPC compared to hormone-naïve tumours.
AR-V3, AR-V7 and AR-V9 are co-expressed in CRPC metastases highlighting the fact that inhibiting AR function via regions common to all AR-Vs is likely to provide additional benefit to patients with CRPC.
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.
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.
, and
were the most frequently disrupted DDR genes in circulating tumor DNA (ctDNA), collectively mutated in 15% of evaluable cases. Biallelic gene disruption via second somatic alteration or mutant allele-specific imbalance was identified in 79% of patients. A further 2% exhibited homozygous
deletions. Tumor suppressors
, and
were controlled via disruptive chromosomal rearrangements in
defective samples, but via oncogene amplification in context of
defects.
mutations were rare in cases with
defects. DDR mutations were re-detected across 94% of serial ctDNA samples and in all available archival primary tissues, indicating they arose prior to metastatic progression. Loss of
and
, but not
, was associated with poor clinical outcomes.
, and
defects are each linked to distinct prostate cancer driver genomics and aggression. The consistency of DDR status in longitudinal samples and resolution of allelic status underscores the potential for ctDNA as a diagnostic tool.
The molecular landscape underpinning response to the androgen receptor (AR) antagonist enzalutamide in patients with metastatic castration-resistant prostate cancer (mCRPC) is undefined. ...Consequently, there is an urgent need for practical biomarkers to guide therapy selection and elucidate resistance. Although tissue biopsies are impractical to perform routinely in the majority of patients with mCRPC, the analysis of plasma cell-free DNA (cfDNA) has recently emerged as a minimally invasive method to explore tumor characteristics.
To reveal genomic characteristics from cfDNA associated with clinical outcomes during enzalutamide treatment.
Plasma samples were obtained from August 4, 2013, to July 31, 2015, at a single academic institution (British Columbia Cancer Agency) from 65 patients with mCRPC. We collected temporal plasma samples (at baseline, 12 weeks, end of treatment) for circulating cfDNA and performed array comparative genomic hybridization copy number profiling and deep AR gene sequencing. Samples collected at end of treatment were also subjected to targeted sequencing of 19 prostate cancer-associated genes.
Enzalutamide, 160 mg, daily orally.
Prostate-specific antigen response rate (decline ≥50% from baseline confirmed ≥3 weeks later). Radiographic (as per Prostate Cancer Working Group 2 Criteria) and/or clinical progression (defined as worsening disease-related symptoms necessitating a change in anticancer therapy and/or deterioration in Eastern Cooperative Group performance status ≥2 levels).
The 65 patients had a median (interquartile range) age of 74 (68-79) years. Prostate-specific antigen response rate to enzalutamide treatment was 38% (25 of 65), while median clinical/radiographic progression-free survival was 3.5 (95% CI, 2.1-5.0) months. Cell-free DNA was isolated from 122 of 125 plasma samples, and targeted sequencing was successful in 119 of 122. AR mutations and/or copy number alterations were robustly detected in 48% (31 of 65) and 60% (18 of 30) of baseline and progression samples, respectively. Detection of AR amplification, heavily mutated AR (≥2 mutations), and RB1 loss were associated with worse progression-free survival, with hazard ratios of 2.92 (95% CI, 1.59-5.37), 3.94 (95% CI, 1.46-10.64), and 4.46 (95% CI, 2.28-8.74), respectively. AR mutations exhibited clonal selection during treatment, including an increase in glucocorticoid-sensitive AR L702H and promiscuous AR T878A in patients with prior abiraterone treatment. At the time of progression, cfDNA sequencing revealed mutations or copy number changes in all patients tested, including clinically actionable alterations in DNA damage repair genes and PI3K pathway genes, and a high frequency (4 of 14) of activating CTNNB1 mutations.
Clinically informative genomic profiling of cfDNA was feasible in nearly all patients with mCRPC and can provide important insights into enzalutamide response and resistance.
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