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.

TP53 structure-function relationships in metastatic castrate-sensitive prostate cancer and the impact of APR-246 treatment.

PURPOSE: Despite well-informed work in several malignancies, the phenotypic effects of TP53 mutations in metastatic castration-sensitive prostate cancer (mCSPC) progression and metastasis are not clear. We characterized the structure-function and clinical impact of TP53 mutations in mCSPC. PATIENTS AND METHODS: We performed an international retrospective review of men with mCSPC who underwent next-generation sequencing and were stratified according to TP53 mutational status and metastatic burden. Clinical outcomes included radiographic progression-free survival (rPFS) and overall survival (OS) evaluated with Kaplan-Meier and multivariable Cox regression. We also utilized isogenic cancer cell lines to assess the effect of TP53 mutations and APR-246 treatment on migration, invasion, colony formation in vitro, and tumor growth in vivo. Preclinical experimental observations were compared using t-tests and ANOVA. RESULTS: Dominant-negative (DN) TP53 mutations were enriched in patients with synchronous (vs. metachronous) (20.7% vs. 6.3%, p < 0.01) and polymetastatic (vs. oligometastatic) (14.4% vs. 7.9%, p < 0.01) disease. On multivariable analysis, DN mutations were associated with worse rPFS (hazards ratio [HR] = 1.97, 95% confidence interval [CI]: 1.31-2.98) and overall survival [OS] (HR = 2.05, 95% CI: 1.14-3.68) compared to TP53 wild type (WT). In vitro, 22Rv1 TP53 R175H cells possessed stronger migration, invasion, colony formation ability, and cellular movement pathway enrichment in RNA sequencing analysis compared to 22Rv1 TP53 WT cells. Treatment with APR-246 reversed the effects of TP53 mutations in vitro and inhibited 22Rv1 TP53 R175H tumor growth in vivo in a dosage-dependent manner. CONCLUSIONS: DN TP53 mutations correlated with worse prognosis in prostate cancer patients and higher metastatic potential, which could be counteracted by APR-246 treatment suggesting a potential future therapeutic avenue.

Whole Slide Imaging-Based Prediction of TP53 Mutations Identifies an Aggressive Disease Phenotype in Prostate Cancer.

In prostate cancer, there is an urgent need for objective prognostic biomarkers that identify the metastatic potential of a tumor at an early stage. While recent analyses indicated TP53 mutations as candidate biomarkers, molecular profiling in a clinical setting is complicated by tumor heterogeneity. Deep learning models that predict the spatial presence of TP53 mutations in whole slide images (WSI) offer the potential to mitigate this issue. To assess the potential of WSIs as proxies for spatially resolved profiling and as biomarkers for aggressive disease, we developed TiDo, a deep learning model that achieves state-of-the-art performance in predicting TP53 mutations from WSIs of primary prostate tumors. In an independent multifocal cohort, the model showed successful generalization at both the patient and lesion level. Analysis of model predictions revealed that false positive (FP) predictions could at least partially be explained by TP53 deletions, suggesting that some FP carry an alteration that leads to the same histological phenotype as TP53 mutations. Comparative expression and histologic cell type analyses identified a TP53-like cellular phenotype triggered by expression of pathways affecting stromal composition. Together, these findings indicate that WSI-based models might not be able to perfectly predict the spatial presence of individual TP53 mutations but they have the potential to elucidate the prognosis of a tumor by depicting a downstream phenotype associated with aggressive disease biomarkers. SIGNIFICANCE: Deep learning models predicting TP53 mutations from whole slide images of prostate cancer capture histologic phenotypes associated with stromal composition, lymph node metastasis, and biochemical recurrence, indicating their potential as in silico prognostic biomarkers. See related commentary by Bordeleau, p. 2809.

Clinical and Genomic Differences Between Advanced Molecular Imaging-detected and Conventional Imaging-detected Metachronous Oligometastatic Castration-sensitive Prostate Cancer.

In metastatic castration-sensitive prostate cancer (mCSPC), disease volume plays an integral role in guiding treatment recommendations, including selection of docetaxel therapy, metastasis-directed therapy, and radiation to the prostate. Although there are multiple definitions of disease volume, they have commonly been studied in the context of metastases detected via conventional imaging (CIM). One such numeric definition of disease volume, termed oligometastasis, is heavily dependent on the sensitivity of the imaging modality. We performed an international multi-institutional retrospective review of men with metachronous oligometastatic CSPC (omCSPC), detected via either advanced molecular imaging alone (AMIM) or CIM. Patients were compared with respect to clinical and genomic features using the Mann-Whitney U test, Pearson's χ2 test, and Kaplan-Meier overall survival (OS) analyses with a log-rank test. A total of 295 patients were included for analysis. Patients with CIM-omCSPC had significantly higher Gleason grade group (p = 0.032), higher prostate-specific antigen at omCSPC diagnosis (8.0 vs 1.7 ng/ml; p < 0.001), more frequent pathogenic TP53 mutations (28% vs 17%; p = 0.030), and worse 10-yr OS (85% vs 100%; p < 0.001). This is the first report of clinical and biological differences between AMIM-detected and CIM-detected omCSPC. Our findings are particularly important for ongoing and planned clinical trials in omCSPC. PATIENT SUMMARY: Metastatic prostate cancer with just a few metastases only detected via newer scanning methods (called molecular imaging) is associated with fewer high-risk DNA mutations and better survival in comparison to metastatic cancer detected via conventional scan methods.

WNT Pathway Mutations in Metachronous Oligometastatic Castration-Sensitive Prostate Cancer.

PURPOSE: WNT signaling is a cellular pathway that has been implicated in the development and progression of prostate cancer. Oligometastatic castration-sensitive prostate cancer (omCSPC) represents a unique state of disease in which metastasis-directed therapy (MDT) has demonstrated improvement in progression-free survival. Herein, we investigate the clinical implications of genomic alterations in the WNT signaling cascade in men with omCSPC. METHODS AND MATERIALS: We performed an international multi-institutional retrospective study of 277 men with metachronous omCSPC who underwent targeted DNA sequencing of their primary/metastatic tumor. Patients were classified by presence or absence of pathogenic WNT pathway mutations (in the genes APC, RNF43, and CTNNB1). Pearson χ2 and Mann-Whitney U tests were used to determine differences in clinical factors between genomic strata. Kaplan-Meier survival curves were generated for radiographic progression-free survival and overall survival, stratified according to WNT pathway mutation status. RESULTS: A pathogenic WNT pathway mutation was detected in 11.2% of patients. Patients with WNT pathway mutations were more likely to have visceral metastases (22.6% vs 2.8%; P < .01) and less likely to have regional lymph node metastases (29.0% vs 50.4%; P = .02). At time of oligometastasis, these patients were treated with MDT alone (33.9%), MDT + limited course of systemic therapy (20.6%), systemic therapy alone (22.4%), or observation (defined as no treatment for ≥6 months after metastatic diagnosis). Multivariable cox regression demonstrated WNT pathway mutations associated with significantly worse overall survival (hazard ratio, 3.87; 95% confidence interval, 1.25-12.00). CONCLUSIONS: Somatic WNT pathway alterations are present in approximately 11% of patients with omCSPC and are associated with an increased likelihood of visceral metastases. Although these patients have a worse natural history, they may benefit from MDT.

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

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

Genomic biomarkers to guide precision radiotherapy in prostate cancer.

Our ability to prognosticate the clinical course of patients with cancer has historically been limited to clinical, histopathological, and radiographic features. It has long been clear however, that these data alone do not adequately capture the heterogeneity and breadth of disease trajectories experienced by patients. The advent of efficient genomic sequencing has led to a revolution in cancer care as we try to understand and personalize treatment specific to patient clinico-genomic phenotypes. Within prostate cancer, emerging evidence suggests that tumor genomics (e.g., DNA, RNA, and epigenetics) can be utilized to inform clinical decision making. In addition to providing discriminatory information about prognosis, it is likely tumor genomics also hold a key in predicting response to oncologic therapies which could be used to further tailor treatment recommendations. Herein we review select literature surrounding the use of tumor genomics within the management of prostate cancer, specifically leaning toward analytically validated and clinically tested genomic biomarkers utilized in radiotherapy and/or adjunctive therapies given with radiotherapy.

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.

Shallow whole-genome sequencing: a useful, easy to apply molecular technique for CNA detection on FFPE tumor tissue-a glioma-driven study.

Copy number alterations (CNAs) have increasingly become part of the diagnostic algorithm of glial tumors. Alterations such as homozygous deletion of CDKN2A/B, 7 +/ 10 - chromosome copy number changes or EGFR amplification are predictive of a poor prognosis. The codeletion of chromosome arms 1p and 19q, typically associated with oligodendroglioma, implies a more favorable prognosis. Detection of this codeletion by the current diagnostic standard, being fluorescence in situ hybridization (FISH), is sometimes however subject to technical and interpretation problems. In this study, we evaluated CNA detection by shallow whole-genome sequencing (sWGS) as an inexpensive, complementary molecular technique. A cohort of 36 glioma tissue samples, enriched with "difficult" and "ambiguous" cases, was analyzed by sWGS. sWGS results were compared with FISH assays of chromosomes 1p and 19q. In addition, CNAs relevant to glioblastoma diagnosis were explored. In 4/36 samples, EGFR (7p11.2) amplifications and homozygous loss of CDKN2A/B were identified by sWGS. Six out of 8 IDH-wild-type glioblastomas demonstrated a prognostic chromosome 7/chromosome 10 signature. In 11/36 samples, local interstitial and terminal 1p/19q alterations were detected by sWGS, implying that FISH's targeted nature might promote false arm-level extrapolations. In this cohort, differences in overall survival between patients with and without codeletion were better pronounced by the sequencing-based distinction (likelihood ratio of 7.48) in comparison to FISH groupings (likelihood ratio of 0.97 at diagnosis and 1.79 ± 0.62 at reobservation), suggesting sWGS is more accurate than FISH. We recognized adverse effects of tissue block age on FISH signals. In addition, we show how sWGS reveals relevant aberrations beyond the 1p/19q state, such as EGFR amplification, combined gain of chromosome 7 and loss of chromosome 10, and homozygous loss of CDKN2A/B. The findings presented by this study might stimulate implementation of sWGS as a complementary, easy to apply technique for copy number detection.

Definitions of disease burden across the spectrum of metastatic castration-sensitive prostate cancer: comparison by disease outcomes and genomics.

BACKGROUND: Several definitions have attempted to stratify metastatic castrate-sensitive prostate cancer (mCSPC) into low and high-volume states. However, at this time, comparison of these definitions is limited. Here we aim to compare definitions of metastatic volume in mCSPC with respect to clinical outcomes and mutational profiles. METHODS: We performed a retrospective review of patients with biochemically recurrent or mCSPC whose tumors underwent somatic targeted sequencing. 294 patients were included with median follow-up of 58.3 months. Patients were classified into low and high-volume disease per CHAARTED, STAMPEDE, and two numeric (≤3 and ≤5) definitions. Endpoints including radiographic progression-free survival (rPFS), time to development of castration resistance (tdCRPC), and overall survival (OS) were evaluated with Kaplan-Meier survival curves and log-rank test. The incidence of driver mutations between definitions were compared. RESULTS: Median OS and tdCRPC were shorter for high-volume than low-volume disease for all four definitions. In the majority of patients (84.7%) metastatic volume classification did not change across all four definitions. High volume disease was significantly associated with worse OS for all four definitions (CHAARTED: HR 2.89; p < 0.01, STAMPEDE: HR 3.82; p < 0.01, numeric ≤3: HR 4.67; p < 0.01, numeric ≤5: HR 3.76; p < 0.01) however, were similar for high (p = 0.95) and low volume (p = 0.79) disease across all four definitions. Those with discordant classification tended to have more aggressive clinical behavior and mutational profiles. Patients with low-volume disease and TP53 mutation experienced a more aggressive course with rPFS more closely mirroring high-volume disease. CONCLUSIONS: The spectrum of mCSPC was confirmed across four different metastatic definitions for clinical endpoints and genetics. All definitions were generally similar in classification of patients, outcomes, and genetic makeup. Given these findings, the simplicity of numerical definitions might be preferred, especially when integrating metastasis directed therapy. Incorporation of tumor genetics may allow further refinement of current metastatic definitions.

Tissue- and Blood-derived Genomic Biomarkers for Metastatic Hormone-sensitive Prostate Cancer: A Systematic Review.

CONTEXT: Multiple studies have reported on the genomic characteristics of metastatic hormone-sensitive prostate cancer (mHSPC). The impact of these findings on prognostication, treatment selection, and clinical trial design remains unclear. OBJECTIVE: To summarise genomic alteration prevalences in liquid and/or tissue biopsies, infer their clinical implications, and compare genomic alteration frequencies across different disease states and clinical phenotypes. EVIDENCE ACQUISITION: The PubMed and Web of Knowledge databases were systematically searched up to January 2021. Quality assessment was performed using the Joanna Briggs Institute Critical Appraisal tools. EVIDENCE SYNTHESIS: In total, 11 studies encompassing 1682 mHSPC patients were included. High-volume disease was associated with more frequent alterations in TP53, DNA damage repair, and Wnt pathways. Tumours from patients with de novo mHSPC were enriched for alterations in TP53 and CDK12 compared with recurrent disease. Alterations in AR, TP53, cell cycle signalling, and MYC were associated with a poorer clinical outcome. A comparative analysis of gene alteration frequencies across disease states revealed a relative increase from localised to castration-resistant tumours, with noteworthy enrichment of CTNNB1 alterations in mHSPC (5%), which warrants further investigation. This study was limited by variability in methodology and definitions used among the eligible studies, including differences in sequencing methods, analytes (being either tissue or liquid), alteration calling thresholds, and target patient populations with a relative under-representation of recurrent metastatic disease. CONCLUSIONS: Several genomic alterations are associated with differential prognosis and clinical phenotypes in mHSPC. We urge that emerging data on these potential predictive biomarkers must be validated in biomarker-driven randomised controlled trials before any clinical implementation. Alignment of the assay methodology and reporting will be critical for ensuring rapid scalability. PATIENT SUMMARY: We reviewed current data on genomic alterations of metastatic hormone-sensitive prostate cancer, and summarised key genomic subtypes that associate with specific clinical phenotypes and treatment outcomes.

Combined Longitudinal Clinical and Autopsy Phenomic Assessment in Lethal Metastatic Prostate Cancer: Recommendations for Advancing Precision Medicine.

BACKGROUND: Systematic identification of data essential for outcome prediction in metastatic prostate cancer (mPC) would accelerate development of precision oncology. OBJECTIVE: To identify novel phenotypes and features associated with mPC outcome, and to identify biomarker and data requirements to be tested in future precision oncology trials. DESIGN SETTING AND PARTICIPANTS: We analyzed deep longitudinal clinical, neuroendocrine expression, and autopsy data of 33 men who died from mPC between 1995 and 2004 (PELICAN33), and related findings to mPC biomarkers reported in the literature. INTERVENTION: Thirty-three men prospectively consented to participate in an integrated clinical-molecular rapid autopsy study of mPC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Data exploration with correction for multiple testing and survival analysis from the time of diagnosis to time to death and time to first occurrence of severe pain as outcomes were carried out. The effect of seven complications on the modeled probability of dying within 2 yr after presenting with the complication was evaluated using logistic regression. RESULTS AND LIMITATIONS: Feature exploration revealed novel phenotypes related to mPC outcome. Four complications (pleural effusion, severe anemia, severe or controlled pain, and bone fracture) predict the likelihood of death within 2 yr. Men with Gleason grade group 5 cancers developed severe pain sooner than those with lower-grade tumors. Surprisingly, neuroendocrine (NE) differentiation was frequently observed in the setting of high serum prostate-specific antigen (PSA) levels (≥30 ng/ml). In 4/33 patients, no controlled (requiring analgesics) or severe pain was detected, and strikingly, 14/15 metastatic sites studied in these men did not express NE markers, suggesting an inverse relationship between NE differentiation and pain in mPC. Intracranial subdural metastasis is common (36%) and is usually clinically undetected. Categorization of "skeletal-related events" complications used in recent studies likely obscures the understanding of spinal cord compression and fracture. Early death from prostate cancer was identified in a subgroup of men with a low longitudinal PSA bandwidth. Cachexia is common (body mass index <0.89 in 24/31 patients) but limited to the last year of life. Biomarker review identified 30 categories of mPC biomarkers in need of winnowing in future trials. All findings require validation in larger cohorts, preferably alongside data from this study. CONCLUSIONS: The study identified novel outcome subgroups for future validation and provides "vision for mPC precision oncology 2020-2050" draft recommendations for future data collection and biomarker studies. PATIENT SUMMARY: To better understand variation in metastatic prostate cancer behavior, we assembled and analyzed longitudinal clinical and autopsy records in 33 men. We identified novel outcomes, phenotypes, and aspects of disease burden to be tested and refined in future trials.

The Mutational Landscape of Metastatic Castration-sensitive Prostate Cancer: The Spectrum Theory Revisited.

BACKGROUND: Emerging data suggest that metastasis is a spectrum of disease burden rather than a binary state, and local therapies, such as radiation, might improve outcomes in oligometastasis. However, current definitions of oligometastasis are solely numerical. OBJECTIVE: To characterize the somatic mutational landscape across the disease spectrum of metastatic castration-sensitive prostate cancer (mCSPC) to elucidate a biological definition of oligometastatic CSPC. DESIGN, SETTING, AND PARTICIPANTS: This was a retrospective study of men with mCSPC who underwent clinical-grade sequencing of their tumors (269 primary tumor, 25 metastatic sites). Patients were classified as having biochemically recurrent (ie, micrometastatic), metachronous oligometastatic (≤5 lesions), metachronous polymetastatic (>5 lesions), or de novo metastatic (metastasis at diagnosis) disease. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We measured the frequency of driver mutations across metastatic classifications and the genomic associations with radiographic progression-free survival (rPFS) and time to castrate-resistant prostate cancer (CRPC). RESULTS AND LIMITATIONS: The frequency of driver mutations in TP53 (p = 0.01), WNT (p = 0.08), and cell cycle (p = 0.04) genes increased across the mCSPC spectrum. TP53 mutation was associated with shorter rPFS (26.7 vs 48.6 mo; p = 0.002), and time to CRPC (95.6 vs 155.8 mo; p = 0.02) in men with oligometastasis, and identified men with polymetastasis with better rPFS (TP53 wild-type, 42.7 mo; TP53 mutated, 18.5 mo; p = 0.01). Mutations in TP53 (incidence rate ratio [IRR] 1.45; p = 0.004) and DNA double-strand break repair (IRR 1.61; p < 0.001) were associated with a higher number of metastases. Mutations in TP53 were also independently associated with shorter rPFS (hazard ratio [HR] 1.59; p = 0.03) and the development of CRPC (HR 1.71; p = 0.01) on multivariable analysis. This study was limited by its retrospective nature, sample size, and the use of commercially available sequencing platforms, resulting in a limited predefined set of genes examined. CONCLUSIONS: Somatic mutational profiles reveal a spectrum of metastatic biology that helps in redefining oligometastasis beyond a simple binary state of lesion enumeration. PATIENT SUMMARY: Oligometastatic prostate cancer is typically defined as less than three to five metastatic lesions and evidence suggests that using radiation or surgery to treat these sites improves clinical outcomes. As of now, treatment decisions for oligometastasis are solely defined according to the number of lesions. However, this study suggests that tumor mutational profiles can provide a biological definition of oligometastasis and complement currently used numerical definitions.

Comparative analysis of somatic variant calling on matched FF and FFPE WGS samples.

BACKGROUND: Research grade Fresh Frozen (FF) DNA material is not yet routinely collected in clinical practice. Many hospitals, however, collect and store Formalin Fixed Paraffin Embedded (FFPE) tumor samples. Consequently, the sample size of whole genome cancer cohort studies could be increased tremendously by including FFPE samples, although the presence of artefacts might obfuscate the variant calling. To assess whether FFPE material can be used for cohort studies, we performed an in-depth comparison of somatic SNVs called on matching FF and FFPE Whole Genome Sequence (WGS) samples extracted from the same tumor. METHODS: Four variant callers (i.e. Strelka2, Mutect2, VarScan2 and Shimmer) were used to call somatic variants on matching FF and FFPE WGS samples from a metastatic prostate tumor. Using the variants identified by these callers, we developed a heuristic to maximize the overlap between the FF and its FFPE counterpart in terms of sensitivity and precision. The proposed variant calling approach was then validated on nine matched primary samples. Finally, we assessed what fraction of the discrepancy could be attributed to intra-tumor heterogeneity (ITH), by comparing the overlap in clonal and subclonal somatic variants. RESULTS: We first compared variants between an FF and an FFPE sample from a metastatic prostate tumor, showing that on average 50% of the calls in the FF are recovered in the FFPE sample, with notable differences between callers. Combining the variants of the different callers using a simple heuristic, increases both the precision and the sensitivity of the variant calling. Validating the heuristic on nine additional matched FF-FFPE samples, resulted in an average F1-score of 0.58 and an outperformance of any of the individual callers. In addition, we could show that part of the discrepancy between the FF and the FFPE samples can be attributed to ITH. CONCLUSION: This study illustrates that when using the correct variant calling strategy, the majority of clonal SNVs can be recovered in an FFPE sample with high precision and sensitivity. These results suggest that somatic variants derived from WGS of FFPE material can be used in cohort studies.

Shallow whole-genome sequencing of plasma cell-free DNA accurately differentiates small from non-small cell lung carcinoma.

BACKGROUND: Accurate lung cancer classification is crucial to guide therapeutic decisions. However, histological subtyping by pathologists requires tumor tissue-a necessity that is often intrinsically associated with procedural difficulties. The analysis of circulating tumor DNA present in minimal-invasive blood samples, referred to as liquid biopsies, could therefore emerge as an attractive alternative. METHODS: Concerning adenocarcinoma, squamous cell carcinoma, and small cell carcinoma, our proof of concept study investigates the potential of liquid biopsy-derived copy number alterations, derived from single-end shallow whole-genome sequencing (coverage 0.1-0.5×), across 51 advanced stage lung cancer patients. RESULTS: Genomic abnormality testing reveals anomalies in 86.3% of the liquid biopsies (16/20 for adenocarcinoma, 13/16 for squamous cell, and 15/15 for small cell carcinoma). We demonstrate that copy number profiles from formalin-fixed paraffin-embedded tumor biopsies are well represented by their liquid equivalent. This is especially valid within the small cell carcinoma group, where paired profiles have an average Pearson correlation of 0.86 (95% CI 0.79-0.93). A predictive model trained with public data, derived from 843 tissue biopsies, shows that liquid biopsies exhibit multiple deviations that reflect histological classification. Most notably, distinguishing small from non-small cell lung cancer is characterized by an area under the curve of 0.98 during receiver operating characteristic analysis. Additionally, we investigated how deeper paired-end sequencing, which will eventually become feasible for routine diagnosis, empowers tumor read enrichment by insert size filtering: for all of the 29 resequenced liquid biopsies, the tumor fraction could be increased in silico, thereby "rescuing" three out of five cases with previously undetectable alterations. CONCLUSIONS: Copy number profiling of cell-free DNA enables histological classification. Since shallow whole-genome sequencing is inexpensive and often fully operational at routine molecular laboratories, this finding has current diagnostic potential, especially for patients with lesions that are difficult to reach.

Oligometastatic prostate cancer: The game is afoot.

Oligometastatic prostate cancer represents an intermediate state between a localized tumor and widespread metastatic disease. Its specific clinical features suggest the existence of a distinct biology which still needs to be elucidated. New imaging techniques like prostate specific membrane antigen (PSMA) PET scans have shown to perform well in the staging and restaging of this category of patients, at different phases of disease evolution. Despite limited prospective evidence, metastasis-directed therapies (MDT) are emerging as valid treatment options able to postpone systemic therapies and probably improve survival outcome. The aim of this review is to shed light on the clinical scenario of prostate cancer patients with limited metastatic disease burden and highlight the role of MDT strategies in this setting.

Randomized Phase 1 Trial of Pembrolizumab with Sequential Versus Concomitant Stereotactic Body Radiotherapy in Metastatic Urothelial Carcinoma.

Preclinical data indicate that radiotherapy works synergistically with pembrolizumab, but the effect and toxicity of this combination may depend on radiotherapy timing. We conducted a randomized phase 1 trial combining pembrolizumab with either sequential (A) or concomitant (B) stereotactic body radiotherapy (SBRT) in metastatic urothelial carcinoma (mUC). No dose-limiting toxicity occurred. Treatment-related adverse events (trAEs; Common Terminology Criteria for Adverse Events v4.0) of grade 1-2 occurred in six of nine and all nine patients in arms A and B, respectively. One grade 3 trAE occurred in arm B. No grade 4-5 trAEs occurred. Overall response rates of 0% and 44.4% were noted in arms A and B, respectively, as per Response Evaluation Criteria in Solid Tumors v1.1. The trial was not powered to compare efficacy between arms. Targeted sequencing of tissue DNA and circulating tumor DNA (ctDNA) revealed high genomic concordance. Treatment response was associated with ctDNA fraction decline. We conclude that sequential and concomitant SBRT can be safely combined with pembrolizumab in mUC and that the effect of SBRT timing on efficacy is worth exploring further. PATIENT SUMMARY: This study assessed the safety of pembrolizumab combined with radiotherapy at two different time points in metastatic bladder cancer. We conclude that the combination treatment was well tolerated.

Discovery and validation of a serum microRNA signature to characterize oligo- and polymetastatic prostate cancer: not ready for prime time.

PURPOSE: Patients with oligometastatic prostate cancer (PC) may benefit from metastasis-directed therapy (MDT), delaying disease progression and the start of palliative systemic treatment. However, a significant proportion of oligometastatic PC patients progress to polymetastatic PC within a year following MDT, suggesting an underestimation of the metastatic load by current staging modalities. Molecular markers could help to identify true oligometastatic patients eligible for MDT. METHODS: Patients with asymptomatic biochemical recurrence following primary PC treatment were classified as oligo- or polymetastatic based on 18F-choline PET/CT imaging. Oligometastatic patients had up to three metastases at baseline and did not progress to more than three lesions following MDT or surveillance within 1 year of diagnosis of metastases. Polymetastatic patients had > 3 metastases at baseline or developed > 3 metastases within 1 year following imaging. A model aiming to prospectively distinguish oligo- and polymetastatic PC patients was trained using clinicopathological parameters and serum-derived microRNA expression profiles from a discovery cohort of 20 oligometastatic and 20 polymetastatic PC patients. To confirm the models predictive performance, it was applied on biomarker data obtained from an independent validation cohort of 44 patients with oligometastatic and 39 patients with polymetastatic disease. RESULTS: Oligometastatic PC patients had a more favorable prognosis compared to polymetastatic ones, as defined by a significantly longer median CRPC-free survival (not reached versus 38 months; 95% confidence interval 31-45 months with P < 0.001). Despite the good performance of a predictive model trained on the discovery cohort, with an AUC of 0.833 (0.693-0.973; 95% CI) and a sensitivity of 0.894 (0.714-1.000; 95% CI) for oligometastatic disease, none of the miRNA targets were found to be differentially expressed between oligo- and polymetastatic PC patients in the signature validation cohort. The multivariate model had an AUC of 0.393 (0.534 after cross-validation) and therefore, no predictive ability. CONCLUSIONS: Although PC patients with oligometastatic disease had a more favorable prognosis, no serum-derived biomarkers allowing for prospective discrimination of oligo- and polymetastatic prostate cancer patients could be identified.