Genomic characterization of metastatic patterns from prospective clinical sequencing of 25,000 patients.

Metastatic progression is the main cause of death in cancer patients, whereas the underlying genomic mechanisms driving metastasis remain largely unknown. Here, we assembled MSK-MET, a pan-cancer cohort of over 25,000 patients with metastatic diseases. By analyzing genomic and clinical data from this cohort, we identified associations between genomic alterations and patterns of metastatic dissemination across 50 tumor types. We found that chromosomal instability is strongly correlated with metastatic burden in some tumor types, including prostate adenocarcinoma, lung adenocarcinoma, and HR+/HER2+ breast ductal carcinoma, but not in others, including colorectal cancer and high-grade serous ovarian cancer, where copy-number alteration patterns may be established early in tumor development. We also identified somatic alterations associated with metastatic burden and specific target organs. Our data offer a valuable resource for the investigation of the biological basis for metastatic spread and highlight the complex role of chromosomal instability in cancer progression.

Natural history, response to systemic therapy, and genomic landscape of plasmacytoid urothelial carcinoma.

BACKGROUND: Plasmacytoid urothelial carcinoma (PUC) is a rare, aggressive histologic variant of urothelial cancer characterised by a diffuse growth pattern and CDH1 mutation. We studied the efficacy of preoperative platinum-based chemotherapy in nonmetastatic PUC and immune checkpoint inhibitors (ICIs) in advanced PUC. METHODS: Cases of nonmetastatic PUC and advanced PUC treated with ICIs at our institution were identified. Outcomes were compared to those of a published cohort of patients with urothelial carcinoma not otherwise specified. RESULTS: We identified 81 patients with nonmetastatic PUC. Of the patients with localised disease who underwent neoadjuvant chemotherapy, pathologic complete response and downstaging rates were 12 and 21%, respectively. Pathologic downstaging was not associated with significant improvement in clinical outcomes. Up to 18% of localised disease and 28% of locally advanced cases had unresectable disease at the time of surgery. ICI-treated advanced PUC (N = 21) had progression-free and overall survival of 4.5 and 10.5 months, respectively, and a 38% response rate. FGFR3 and DNA damage response gene alterations were observed in 3 and 15% of cases, respectively. CONCLUSIONS: PUC is associated with high disease burden and poor chemosensitivity. Increased awareness and recognition of this disease variant will allow for new treatment strategies.

First-in-human study of naporafenib (LXH254) with or without spartalizumab in adult patients with advanced solid tumors harboring MAPK signaling pathway alterations.

BACKGROUND: We investigated naporafenib (LXH254), a pan-RAF kinase inhibitor, with or without spartalizumab, in patients with advanced solid tumors harboring MAPK pathway alterations. METHODS: This first-in-human phase 1 study had two dose-escalation arms: single-agent naporafenib (starting at 100 mg once-daily [QD]) and naporafenib (starting at the recommended dose/regimen)/spartalizumab (400 mg every 4 weeks). The naporafenib/spartalizumab dose-expansion part enrolled patients with KRAS-mutated non-small cell lung cancer (NSCLC) and NRAS-mutated melanoma. The primary objectives were to establish the maximum tolerated doses (MTD)/recommended doses for expansion (RDE) and evaluate tolerability and safety. RESULTS: A total of 142 patients were included in the naporafenib dose-escalation (n = 87), naporafenib/spartalizumab dose-escalation (n = 12) and naporafenib/spartalizumab dose-expansion (n = 43) arms. The MTD/RDE of naporafenib was 600 mg twice-daily (BID). In naporafenib escalation, five patients experienced 7 dose-limiting toxicities: decreased platelet count (1200 mg QD); neuralgia, maculopapular rash, pruritus (600 mg BID); increased blood bilirubin, hyponatremia, peripheral sensory neuropathy (800 mg BID). No DLTs occurred in the naporafenib/spartalizumab arm: the RDE was established at 400 mg BID. The most common treatment-related adverse events were rash and dermatitis acneiform (each 24.1%; naporafenib), nausea and pruritus (each 33.3%; naporafenib/spartalizumab; escalation) and rash (39.5%; naporafenib/spartalizumab; expansion). Naporafenib reduced DUSP6 expression in tumors. Two partial responses (PRs) occurred in naporafenib escalation, and 1 complete response and 3 PRs in the naporafenib/spartalizumab NRAS-mutated melanoma and KRAS-mutated NSCLC arms, respectively. CONCLUSIONS: Naporafenib, with or without spartalizumab, showed an acceptable safety profile, pharmacodynamic activity and limited antitumor activity. Additional naporafenib combination therapies are currently under investigation.

KDM6A Loss Triggers an Epigenetic Switch That Disrupts Urothelial Differentiation and Drives Cell Proliferation in Bladder Cancer.

Disruption of KDM6A, a histone lysine demethylase, is one of the most common somatic alternations in bladder cancer. Insights into how KDM6A mutations affect the epigenetic landscape to promote carcinogenesis could help reveal potential new treatment approaches. Here, we demonstrated that KDM6A loss triggers an epigenetic switch that disrupts urothelial differentiation and induces a neoplastic state characterized by increased cell proliferation. In bladder cancer cells with intact KDM6A, FOXA1 interacted with KDM6A to activate genes instructing urothelial differentiation. KDM6A-deficient cells displayed simultaneous loss of FOXA1 target binding and genome-wide redistribution of the bZIP transcription factor ATF3, which in turn repressed FOXA1-target genes and activated cell-cycle progression genes. Importantly, ATF3 depletion reversed the cell proliferation phenotype induced by KDM6A deficiency. These data establish that KDM6A loss engenders an epigenetic state that drives tumor growth in an ATF3-dependent manner, creating a potentially targetable molecular vulnerability. SIGNIFICANCE: A gain-of-function epigenetic switch that disrupts differentiation is triggered by inactivating KDM6A mutations in bladder cancer and can serve as a potential target for novel therapies.

Inherited Germline Cancer Susceptibility Gene Variants in Individuals with Non-Muscle-Invasive Bladder Cancer.

PURPOSE: Identification of inherited germline variants can guide personalized cancer screening, prevention, and treatment. Pathogenic and likely pathogenic (P/LP) germline variants in cancer predisposition genes are frequent among patients with locally advanced or metastatic urothelial carcinoma, but their prevalence and significance in patients with non-muscle-invasive bladder cancer (NMIBC), the most common form of urothelial carcinoma, is understudied. EXPERIMENTAL DESIGN: Germline analysis was conducted on paired tumor/normal sequencing results from two distinct cohorts of patients initially diagnosed with NMIBC. Associations between clinicopathologic features and clinical outcomes with the presence of P/LP germline variants in ≥76 hereditary cancer predisposition genes were analyzed. RESULTS: A similar frequency of P/LP germline variants were seen in our two NMIBC cohorts [12% (12/99) vs. 8.7% (10/115), P = 0.4]. In the combined analysis, P/LP germline variants were found only in patients with high-grade NMIBC (22/163), but none of the 46 patients with low-grade NMIBC (13.5% vs. 0%, P = 0.005). Fifteen (9.2%) patients with high-grade NMIBC had P/LP variants in DNA damage response genes, most within the nucleotide excision repair (ERCC2/3) and homologous recombination repair (BRCA1, NBN, RAD50) pathways. Contrary to prior reports in patients with NMIBC not receiving Bacillus Calmette-Guerin (BCG), P/LP germline variants were not associated with worse recurrence-free or progression-free survival in patients treated with BCG or with risk of developing upper tract urothelial carcinoma. CONCLUSIONS: Our results support offering germline counseling and testing for all patients with high-grade bladder cancer, regardless of initial tumor stage. Therapeutic strategies that target impaired DNA repair may benefit patients with high-grade NMIBC.

Noninvasive Detection of Polyclonal Acquired Resistance to FGFR Inhibition in Patients With Cholangiocarcinoma Harboring FGFR2 Alterations.

PURPOSE: Fibroblast growth factor receptor (FGFR) 2 alterations, present in 5%-15% of intrahepatic cholangiocarcinomas (IHC), are targets of FGFR-directed therapies. Acquired resistance is common among patients who respond. Biopsies at the time of acquired resistance to targeted agents may not always be feasible and may not capture the genetic heterogeneity that could exist within a patient. We studied circulating tumor DNA (ctDNA) as a less invasive means of potentially identifying genomic mechanisms of resistance to FGFR-targeted therapies. MATERIALS AND METHODS: Serial blood samples were collected from eight patients with FGFR-altered cholangiocarcinoma for ctDNA isolation and next-generation sequencing (NGS) throughout treatment and at resistance to anti-FGFR-targeted therapy. ctDNA was sequenced using a custom ultra-deep coverage NGS panel, incorporating dual index primers and unique molecular barcodes to enable high-sensitivity mutation detection. RESULTS: Thirty-one acquired mutations in FGFR2, 30/31 located in the kinase domain, were identified at resistance in six of eight patients with detectable ctDNA. Up to 13 independent FGFR2 mutations were detected per patient, indicative of striking genomic concordance among resistant subclones. CONCLUSION: ctDNA could be an effective means to longitudinally monitor for acquired resistance in FGFR2-altered IHC. The numerous acquired genetic alterations in FGFR2 suggest frequent polyclonal mechanisms of resistance that cannot be detected from single-site tissue biopsies.

Identification of a Novel Inflamed Tumor Microenvironment Signature as a Predictive Biomarker of Bacillus Calmette-Guérin Immunotherapy in Non-Muscle-Invasive Bladder Cancer.

PURPOSE: Improved risk stratification and predictive biomarkers of treatment response are needed for non-muscle-invasive bladder cancer (NMIBC). Here we assessed the clinical utility of targeted RNA and DNA molecular profiling in NMIBC. EXPERIMENTAL DESIGN: Gene expression in NMIBC samples was profiled by NanoString nCounter, an RNA quantification platform, from two independent cohorts (n = 28, n = 50); targeted panel sequencing was performed in a subgroup (n = 50). Gene signatures were externally validated using two RNA sequencing datasets of NMIBC tumors (n = 438, n = 73). Established molecular subtype classifiers and novel gene expression signatures were assessed for associations with clinicopathologic characteristics, somatic tumor mutations, and treatment outcomes. RESULTS: Molecular subtypes distinguished between low-grade Ta tumors with FGFR3 mutations and overexpression (UROMOL-class 1) and tumors with more aggressive clinicopathologic characteristics (UROMOL-classes 2 and 3), which were significantly enriched with TERT promoter mutations. However, UROMOL subclasses were not associated with recurrence after bacillus Calmette-Guérin (BCG) immunotherapy in two independent cohorts. In contrast, a novel expression signature of an inflamed tumor microenvironment (TME) was associated with improved recurrence-free survival after BCG. Expression of immune checkpoint genes (PD-L1/PD-1/CTLA-4) was associated with an inflamed TME, but not with higher recurrence rates after BCG. FGFR3 mutations and overexpression were both associated with low immune signatures. CONCLUSIONS: Assessment of the immune TME, rather than molecular subtypes, is a promising predictive biomarker of BCG response. Modulating the TME in an immunologically "cold" tumor warrants further investigation. Integrated transcriptomic and exome sequencing should improve treatment selection in NMIBC.

OncoTree: A Cancer Classification System for Precision Oncology.

PURPOSE: Cancer classification is foundational for patient care and oncology research. Systems such as International Classification of Diseases for Oncology (ICD-O), Systematized Nomenclature of Medicine Clinical Terms (SNOMED-CT), and National Cancer Institute Thesaurus (NCIt) provide large sets of cancer classification terminologies but they lack a dynamic modernized cancer classification platform that addresses the fast-evolving needs in clinical reporting of genomic sequencing results and associated oncology research. METHODS: To meet these needs, we have developed OncoTree, an open-source cancer classification system. It is maintained by a cross-institutional committee of oncologists, pathologists, scientists, and engineers, accessible via an open-source Web user interface and an application programming interface. RESULTS: OncoTree currently includes 868 tumor types across 32 organ sites. OncoTree has been adopted as the tumor classification system for American Association for Cancer Research (AACR) Project Genomics Evidence Neoplasia Information Exchange (GENIE), a large genomic and clinical data-sharing consortium, and for clinical molecular testing efforts at Memorial Sloan Kettering Cancer Center and Dana-Farber Cancer Institute. It is also used by precision oncology tools such as OncoKB and cBioPortal for Cancer Genomics. CONCLUSION: OncoTree is a dynamic and flexible community-driven cancer classification platform encompassing rare and common cancers that provides clinically relevant and appropriately granular cancer classification for clinical decision support systems and oncology research.

DNA damage repair pathway alterations in metastatic clear cell renal cell carcinoma and implications on systemic therapy.

BACKGROUND: Loss-of-function alterations in DNA damage repair (DDR) genes are associated with human tumorigenesis and may determine benefit from immune-oncology (I/O) agents as shown in colon cancer. However, biologic significance and relevance to I/O in metastatic clear cell RCC (ccRCC) are unknown. METHODS: Genomic data and treatment outcomes were retrospectively collected for patients with metastatic ccRCC. Tumor and germline DNA were subject to targeted next generation sequencing across >400 genes of interest, including 34 DDR genes. Patients were dichotomized according to underlying DDR gene alteration into (1) deleterious DDR gene alterations present (Del DDR); (2) wild-type (WT) and variants of unknown significance (VUS) DDR gene alterations present (WT/VUS DDR). Association between DDR status and therapeutic benefit was investigated separately for I/O and vascular endothelial growth factor (VEGF) tyrosine kinase inhibitor (TKI) therapy. RESULTS: Del DDR were detected in 43/229 patients (19%). The most frequently altered genes were CHEK2 and ATM. Clonality analysis was performed in 27 somatic DDR mutations and 17 were clonal (63%). For patients with I/O treatment, Del DDR status was associated with superior overall survival (log-rank p=0.049); after adjusting for International Metastatic Renal Cell Carcinoma Database Consortium risks and extent of prior therapy, the HR for Del DDR was 0.41 (95% CI: 0.14-1.14; p=0.09). No association was seen with VEGF-TKI treatment (log-rank p=0.903). CONCLUSION: Del DDR alterations are recurrent genomic events in patients with advanced RCC and were mostly clonal in this cohort. Loss-of-function events in these genes may affect outcome with I/O therapy in metastatic RCC, and these hypothesis-generating results deserve further study.

Activity of M3814, an Oral DNA-PK Inhibitor, In Combination with Topoisomerase II Inhibitors in Ovarian Cancer Models.

DNA-dependent protein kinase (DNA-PK) has been shown to play a crucial role in repair of DNA double-strand breaks, facilitating nonhomologous end-joining. DNA-PK inhibitors have the potential to block DNA repair and therefore enhance DNA-damaging agents. M3814 is a DNA-PK inhibitor that has shown preclinical activity in combination with DNA-damaging agents, including radiotherapy and topoisomerase II inhibitors. Here we evaluated the activity of M3814 in combination with multiple topoisomerase II inhibitors, doxorubicin, etoposide, and pegylated liposomal doxorubicin (PLD) in vivo, utilizing ovarian cancer xenografts. Using cell lines representative of P53 wild-type ovarian cancer (A2780), and P53 mutant ovarian cancer (SKOV3), cells were implanted in the flank of athymic nude female mice. Mice were treated with vehicle, M3814 alone, topoisomerase II inhibitor alone, and M3814 in combination with topoisomerase II inhibitor, and change in tumor volume over time was documented. The addition of M3814 was well tolerated. We demonstrated that M3814 shows limited efficacy as a single agent in ovarian cancer models. The combination of M3814 with PLD showed enhanced activity over PLD as a single agent. Further study of this combination is warranted.

Next-generation sequencing of urine specimens: A novel platform for genomic analysis in patients with non-muscle-invasive urothelial carcinoma treated with bacille Calmette-Guérin.

BACKGROUND: Biopsies from patients with high-risk (HR) non-muscle-invasive urothelial carcinoma (NMIUC), especially flat urothelial carcinoma in situ, frequently contain scant diagnostic material or denuded mucosa only, and this precludes further extensive genomic analysis. This study evaluated the use of next-generation sequencing (NGS) analysis of urine cytology material from patients with HR NMIUC in an attempt to identify genetic alterations that might correlate with clinical features and responses to bacille Calmette-Guérin (BCG) treatment. METHODS: Forty-one cytology slides from patients with HR NMIUC treated with intravesical BCG were selected for this study. Histological confirmation was available for all cases. The specimens were subjected to NGS analysis with a customized targeted exome capture assay composed of 341 genes. RESULTS: In this cohort, genomic alterations were successfully identified in all cytology samples. Mutations were detected down to a 2% allele frequency and chromosomal rearrangements including copy number alterations and gene fusions were identified. The most frequently altered genes included telomerase reverse transcriptase (TERT), tumor protein 53 (TP53), Erb-B2 receptor tyrosine kinase 2 (ERBB2), and chromatin remodeling genes such as lysine demethylase 6A (KDM6A) and AT-rich interaction domain 1A (ARID1A). For patients with matched tumor tissue, cytology specimens revealed all mutations detected in tissue as well as additional mutations, and this suggested that urine might more effectively capture the full genetic heterogeneity of disease than an individual cystectomy. Alterations in multiple genes correlated with clinical and histopathological features, including responses to BCG treatment, flat architecture versus papillary architecture, and smoking history. CONCLUSIONS: Urine specimens can replace tissue as a substrate for NGS analysis of HR NMIUC. Several genomic alterations identified in urine specimens might be associated with histological features and clinical characteristics. Cancer Cytopathol 2017;125:416-26. © 2017 American Cancer Society.

DNA Damage Response and Repair Gene Alterations Are Associated with Improved Survival in Patients with Platinum-Treated Advanced Urothelial Carcinoma.

Purpose: Platinum-based chemotherapy remains the standard treatment for advanced urothelial carcinoma by inducing DNA damage. We hypothesize that somatic alterations in DNA damage response and repair (DDR) genes are associated with improved sensitivity to platinum-based chemotherapy.Experimental Design: Patients with diagnosis of locally advanced and metastatic urothelial carcinoma treated with platinum-based chemotherapy who had exon sequencing with the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) assay were identified. Patients were dichotomized based on the presence/absence of alterations in a panel of 34 DDR genes. DDR alteration status was correlated with clinical outcomes and disease features.Results: One hundred patients were identified, of which 47 harbored alterations in DDR genes. Patients with DDR alterations had improved progression-free survival (9.3 vs. 6.0 months, log-rank P = 0.007) and overall survival (23.7 vs. 13.0 months, log-rank P = 0.006). DDR alterations were also associated with higher number mutations and copy-number alterations. A trend toward positive correlation between DDR status and nodal metastases and inverse correlation with visceral metastases were observed. Different DDR pathways also suggested variable impact on clinical outcomes.Conclusions: Somatic DDR alteration is associated with improved clinical outcomes in platinum-treated patients with advanced urothelial carcinoma. Once validated, it can improve patient selection for clinical practice and future study enrollment. Clin Cancer Res; 23(14); 3610-8. ©2017 AACR.