Sensitive circulating tumor DNA-based residual disease detection in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is one of the leading causes of cancer-related death in women worldwide, and is characterized by a high rate of recurrence after surgery and chemotherapy. We sought to implement a circulating tumor DNA (ctDNA)-based blood test for more accurate post-operative surveillance of this disease. We analyzed 264 plasma samples collected between June 2016 and September 2021 from 63 EOC patients using tumor-guided plasma cell-free DNA analysis to detect residual disease after treatment. Assay specificity was verified using cross-patient analysis of 1,195 control samples. ctDNA was detected in 51 of 55 (93%) samples at diagnosis, and 18 of 18 (100%) samples at progression. Positive ctDNA in the last on-treatment sample was associated with rapid progression (median 1.02 versus 3.38 yr, HR = 5.63, P < 0.001) and reduced overall survival (median 2.31 versus NR yr, HR = 8.22, P < 0.001) in patients with high-grade serous cancer. In the case of 12 patients, ctDNA assays detected progression earlier than standard surveillance, with a median lead time of 5.9 mo. To approach the physical limits of ctDNA detection, five patients were analyzed using ultra-sensitive assays interrogating 479-1,856 tumor mutations, capable of tracking ctDNA fractions down to 0.0004%. Our results demonstrate that ctDNA assays achieve high sensitivity and specificity in detecting post-operative residual disease in EOC.

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.

Genomic characterization of IDH-mutant astrocytoma progression to grade 4 in the treatment setting.

As the progression of low-grade diffuse astrocytomas into grade 4 tumors significantly impacts patient prognosis, a better understanding of this process is of paramount importance for improved patient care. In this project, we analyzed matched IDH-mutant astrocytomas before and after progression to grade 4 from six patients (discovery cohort) with genome-wide sequencing, 21 additional patients with targeted sequencing, and 33 patients from Glioma Longitudinal AnalySiS cohort for validation. The Cancer Genome Atlas data from 595 diffuse gliomas provided supportive information. All patients in our discovery cohort received radiation, all but one underwent chemotherapy, and no patient received temozolomide (TMZ) before progression to grade 4 disease. One case in the discovery cohort exhibited a hypermutation signature associated with the inactivation of the MSH2 and DNMT3A genes. In other patients, the number of chromosomal rearrangements and deletions increased in grade 4 tumors. The cell cycle checkpoint gene CDKN2A, or less frequently RB1, was most commonly inactivated after receiving both chemo- and radiotherapy when compared to other treatment groups. Concomitant activating PDGFRA/MET alterations were detected in tumors that acquired a homozygous CDKN2A deletion. NRG3 gene was significantly downregulated and recurrently altered in progressed tumors. Its decreased expression was associated with poorer overall survival in both univariate and multivariate analysis. We also detected progression-related alterations in RAD51B and other DNA repair pathway genes associated with the promotion of error-prone DNA repair, potentially facilitating tumor progression. In our retrospective analysis of patient treatment and survival timelines (n = 75), the combination of postoperative radiation and chemotherapy (mainly TMZ) outperformed radiation, especially in the grade 3 tumor cohort, in which it was typically given after primary surgery. Our results provide further insight into the contribution of treatment and genetic alterations in cell cycle, growth factor signaling, and DNA repair-related genes to tumor evolution and progression.

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.

PTPRD and CNTNAP2 as markers of tumor aggressiveness in oligodendrogliomas.

Oligodendrogliomas are typically associated with the most favorable prognosis among diffuse gliomas. However, many of the tumors progress, eventually leading to patient death. To characterize the changes associated with oligodendroglioma recurrence and progression, we analyzed two recurrent oligodendroglioma tumors upon diagnosis and after tumor relapse based on whole-genome and RNA sequencing. Relapsed tumors were diagnosed as glioblastomas with an oligodendroglioma component before the World Health Organization classification update in 2016. Both patients died within 12 months after relapse. One patient carried an inactivating POLE mutation leading to a clearly hypermutated progressed tumor. Strikingly, both relapsed tumors carried focal chromosomal rearrangements in PTPRD and CNTNAP2 genes with associated decreased gene expression. TP53 mutation was also detected in both patients after tumor relapse. In The Cancer Genome Atlas (TCGA) diffuse glioma cohort, PTPRD and CNTNAP2 expression decreased by tumor grade in oligodendrogliomas and PTPRD expression also in IDH-mutant astrocytomas. Low expression of the genes was associated with poor overall survival. Our analysis provides information about aggressive oligodendrogliomas with worse prognosis and suggests that PTPRD and CNTNAP2 expression could represent an informative marker for their stratification.

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.

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.

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.

Author Correction: The evolutionary history of lethal metastatic prostate cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

AR and ERG drive the expression of prostate cancer specific long noncoding RNAs.

Long noncoding RNAs (lncRNAs) play pivotal roles in cancer development and progression, and some function in a highly cancer-specific manner. However, whether the cause of their expression is an outcome of a specific regulatory mechanism or nonspecific transcription induced by genome reorganization in cancer remains largely unknown. Here, we investigated a group of lncRNAs that we previously identified to be aberrantly expressed in prostate cancer (PC), called TPCATs. Our high-throughput real-time PCR experiments were integrated with publicly available RNA-seq and ChIP-seq data and revealed that the expression of a subset of TPCATs is driven by PC-specific transcription factors (TFs), especially androgen receptor (AR) and ETS-related gene (ERG). Our in vitro validations confirmed that AR and ERG regulated a subset of TPCATs, most notably for EPCART. Knockout of EPCART was found to reduce migration and proliferation of the PC cells in vitro. The high expression of EPCART and two other TPCATs (TPCAT-3-174133 and TPCAT-18-31849) were also associated with the biochemical recurrence of PC in prostatectomy patients and were independent prognostic markers. Our findings suggest that the expression of numerous PC-associated lncRNAs is driven by PC-specific mechanisms and not by random cellular events that occur during cancer development. Furthermore, we report three prospective prognostic markers for the early detection of advanced PC and show EPCART to be a functionally relevant lncRNA in PC.

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.

Plasma Circulating Tumor DNA and Clonal Hematopoiesis in Metastatic Renal Cell Carcinoma.

BACKGROUND: There is a lack of molecularly-informed biomarkers for patients with metastatic renal cell carcinoma (RCC). Plasma cell-free DNA (cfDNA) sequencing is a minimally-invasive alternative to tissue for profiling the genome in other cancers but relevance in metastatic RCC remains unclear. MATERIALS AND METHODS: Whole blood was collected from 55 patients with metastatic RCC. Plasma cfDNA and leukocyte DNA were subjected to targeted sequencing across 981 cancer genes. Matched tumor tissue from 14 patients was analyzed. RESULTS: Thirty-three percent of patients had evidence for RCC-derived circulating tumor DNA (ctDNA), significantly lower than patients with metastatic prostate or bladder cancer analyzed using the same approach. Among ctDNA-positive patients, ctDNA fraction averaged only 3.9% and showed no strong association with clinical variables. In these patients, the most commonly mutated genes were VHL, BAP1, and PBRM1, and matched tissue concordance was 77%. Evidence of somatic expansions unrelated to RCC, such as clonal hematopoiesis of indeterminate potential, were detected in 43% of patients. Pathogenic germline mutations in DNA repair genes were detected in 11% of patients. CtDNA-positive patients had shorter overall survival and progression-free survival on first-line therapy. Patients with evidence of clonal hematopoiesis of indeterminate potential had an intermediate prognosis compared with ctDNA-positive and -negative patients. CONCLUSIONS: CfDNA sequencing enables straightforward characterization of the somatic RCC genome in a minority of patients with metastatic RCC. Owing to low ctDNA abundance, and the presence of non-RCC derived somatic clones in circulation, cfDNA sequencing may not be a simple pan-patient alternative to tissue biopsy in metastatic RCC.

Moderate-to-strong expression of FGFR3 and TP53 alterations in a subpopulation of choroid plexus tumors.

Deregulation of fibroblast growth factor receptor (FGFR) signaling is tightly associated with numerous human malignancies, including cancer. Indeed, FGFR inhibitors are being tested as anti-tumor drugs in clinical trials. Among gliomas, FGFR3 fusions occur in IDH wild-type diffuse gliomas leading to high FGFR3 protein expression and both, FGFR3 and FGFR1, show elevated expression in aggressive ependymomas. The aim of this study was to uncover the expression of FGFR1 and FGFR3 proteins in choroid plexus tumors and to further characterize FGFR-related as well as other genetic alterations in FGFR3 expressing tumors. Expression levels of FGFR1 and FGFR3 were detected in 15 choroid plexus tumor tissues using immunohistochemistry of tissue microarrays and 6 samples were subjected to whole mount FGFR3 staining. Targeted sequencing was used for deeper molecular analysis of two FGFR3 positive cases. Moderate expression of FGFR1 or FGFR3 was evidenced in one third of the studied choroid plexus tumors. Targeted sequencing of a choroid plexus carcinoma and an atypical choroid plexus papilloma, both with moderate-to-strong FGFR3 expression, revealed lack of protein-altering mutations or fusions in FGFR1 or FGFR3, but TP53 was altered in both tumors. FGFR3 and FGFR1 proteins are expressed in a subpopulation of choroid plexus tumors. Further studies using larger cohorts of patients will allow identification of the clinicopathological implications of FGFR1 and FGFR3 expression in choroid plexus tumors.

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.