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Abstract Background Both somatic copy number alterations (CNAs) and germline copy number variants (CNVs) that are prevalent in healthy individuals can appear as recurrent changes in comparative genomic hybridization (CGH) analyses of tumors. In order to identify important cancer genes CNAs and CNVs must be distinguished. Although the Database of Genomic Variants (DGV) contains a list of all known CNVs, there is no standard methodology to use the database effectively. Results We develop a prediction model that distinguishes CNVs from CNAs based on the information contained in the DGV and several other variables, including segment's length, height, closeness to a telomere or centromere and occurrence in other patients. The models are fitted on data from glioblastoma and their corresponding normal samples that were collected as part of The Cancer Genome Atlas project and hybridized to Agilent 244 K arrays. Conclusions Using the DGV alone CNVs in the test set can be correctly identified with about 85% accuracy if the outliers are removed before segmentation and with 72% accuracy if the outliers are included, and additional variables improve the prediction by about 2-3% and 12%, respectively. Final models applied to data from ovarian tumors have about 90% accuracy with all the variables and 86% accuracy with the DGV alone.

Obesity is a risk factor for cancer, but whether obesity is linked to specific genomic subtypes of cancer is unknown. We examined the relationship between obesity and tumor genotype in two clinicogenomic corpora. Obesity was associated with specific driver mutations in lung adenocarcinoma, endometrial carcinoma and cancers of unknown primaries, independent of clinical covariates, demographic factors and genetic ancestry. Obesity is therefore a driver of etiological heterogeneity in some cancers., (© 2024. The Author(s).)

Immunotherapy (ICIs) remains a mainstay for treatment of advanced clear-cell renal cell carcinoma (ccRCC). Biomarker analyses have demonstrated that gene expression profiles are associated with regimen-specific outcomes. These transcriptomic analyses used mixed sample cohorts (primary and metastatic tumor specimens) and it is unknown whether the clinical relevance of transcriptomic signatures is impacted by tissue site. We evaluated data for 1132 patients with metastatic ccRCC treated with ICI in prior studies (IMmotion151 and CheckMate-009, -010, and -025). We identified significant and reproducible differences in gene expression by tissue site. We tested the association between previously described molecular tissue clusters (MTCs) by tissue site (MTC1-primary and MTC1-metastasis) and progression-free survival (PFS) and objective response to systemic therapy. In IMmotion151, MTC2-metastasis was significantly associated with better PFS on sunitinib (hazard ratio [HR] 3.39, 95% confidence interval [CI] 1.32-8.69; p = 0.01) in comparison to MTC2-primary (HR 0.95, 95% CI 0.65-1.38; p = 0.80; p interaction  = 0.02). Evaluation of known RNA signatures in the CheckMate trials revealed that JAVELIN-metastasis was associated with better PFS on ICI (HR 0.77, 95% CI 0.62-0.97; p = 0.03) in comparison to JAVELIN-primary (HR 1.04, 95% CI 0.91-1.19; p = 0.56; p interaction  = 0.02). These results indicate that tissue site may be a relevant confounder in biomarker analyses., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: There is a significant need for effective therapies to treat recurrent/metastatic (R/M) adenoid cystic carcinoma (ACC). This study evaluated the multi-targeted, vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor (TKI) regorafenib in patients with R/M ACC., Methods: Patients with progressive R/M ACC were treated with regorafenib until disease progression, consent withdrawal, or excessive toxicity. The primary endpoints were best overall response (BOR) and 6-month progression-free survival (PFS). Genomic and transcriptomic biomarker analyses were performed in tumors from trial participants., Results: Thirty-eight patients were enrolled, including 7 (18%) patients with prior VEGFR-TKIs. No objective responses were observed. Six-month PFS was 45%, and median PFS was 7.2 months (95%CI 5.2-11.9 months). Presence of either activating NOTCH1 (22%) or KDM6A alterations (24%) was associated with decreased PFS (HR 2.6, 95%CI 1.1-6.1, p=0.03). Bulk RNA sequencing of pre-treatment tumors revealed that regorafenib clinical benefit (CB; PFS≥6 months; n=11) was associated with native enrichment of immune-related signatures. Immune deconvolution revealed a greater degree of macrophage and T-cell infiltration in CB tumors. Tumors from patients with no clinical benefit (NCB; PFS<6 months; n=9) had greater expression of signatures related to cell cycle progression (E2F targets, G2/M checkpoint)., Conclusion: The trial failed to meet the pre-specified 6-month PFS and BOR targets. We hypothesize that TKI efficacy may be reliant upon an interplay between kinase inhibition and the ACC immune microenvironment, while programs promoting cell cycle progression may contribute to TKI resistance. These observations suggest that trials evaluating CDK4/6 inhibition plus a VEGFR-TKI should be considered.

Small cell carcinomas (SMC) of the lung are now molecularly classified based on the expression of transcriptional regulators (NEUROD1, ASCL1, POU2F3, and YAP1) and DLL3, which has emerged as an investigational therapeutic target. PLCG2 has been shown to identify a distinct subpopulation of lung SMC with stem cell-like and prometastasis features and poor prognosis. We analyzed the expression of these novel neuroendocrine markers and their association with traditional neuroendocrine markers and patient outcomes in a cohort of bladder neuroendocrine carcinoma (NEC) consisting of 103 SMC and 19 large cell NEC (LCNEC) assembled in tissue microarrays. Coexpression patterns were assessed and integrated with detailed clinical annotation including overall (OS) and recurrence-free survival (RFS) and response to neoadjuvant/adjuvant chemotherapy. We identified 5 distinct molecular subtypes in bladder SMC based on the expression of ASCL1, NEUROD1, and POU2F3: ASCL1+/NEUROD1- (n = 33; 34%), ASCL1- /NEUROD1+ (n = 21; 21%), ASCL1+/NEUROD1+ (n = 17; 17%), POU2F3+ (n = 22, 22%), and ASCL1- /NEUROD1- /POU2F3- (n = 5, 5%). POU2F3+ tumors were mutually exclusive with those expressing ASCL1 and NEUROD1 and exhibited lower expression of traditional neuroendocrine markers. PLCG2 expression was noted in 33 tumors (32%) and was highly correlated with POU2F3 expression (P < .001). DLL3 expression was high in both SMC (n = 72, 82%) and LCNEC (n = 11, 85%). YAP1 expression was enriched in nonneuroendocrine components and negatively correlated with all neuroendocrine markers. In patients without metastatic disease who underwent radical cystectomy, PLCG2+ or POU2F3+ tumors had shorter RFS and OS (P < .05), but their expression was not associated with metastasis status or response to neoadjuvant/adjuvant chemotherapy. In conclusion, the NEC of the bladder can be divided into distinct molecular subtypes based on the expression of ASCL1, NEUROD1, and POU2F3. POU2F3-expressing tumors represent an ASCL1/NEUROD1-negative subset of bladder NEC characterized by lower expression of traditional neuroendocrine markers. Marker expression patterns were similar in SMC and LCNEC. Expression of PLCG2 and POU2F3 was associated with shorter RFS and OS. DLL3 was expressed at high levels in both SMC and LCNEC of the bladder, nominating it as a potential therapeutic target., (Copyright © 2024 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose: Patients with microsatellite instability-high/mismatch repair-deficient (MSI-H/dMMR) and high tumor mutational burden (TMB-H) prostate cancers are candidates for pembrolizumab. We define the genomic features, clinical course, and response to immune checkpoint blockade (ICB) in patients with MSI-H/dMMR and TMB-H prostate cancers without MSI [TMB-H/microsatellite stable (MSS)]., Experimental Design: We sequenced 3,244 tumors from 2,257 patients with prostate cancer. MSI-H/dMMR prostate cancer was defined as an MSIsensor score ≥10 or MSIsensor score ≥3 and <10 with a deleterious MMR alteration. TMB-H was defined as ≥10 mutations/megabase. PSA50 and RECIST responses were assigned. Overall survival and radiographic progression-free survival (rPFS) were compared using log-rank test., Results: Sixty-three (2.8%) men had MSI-H/dMMR, and 33 (1.5%) had TMB-H/MSS prostate cancers. Patients with MSI-H/dMMR and TMB-H/MSS tumors more commonly presented with grade group 5 and metastatic disease at diagnosis. MSI-H/dMMR tumors had higher TMB, indel, and neoantigen burden compared with TMB-H/MSS. Twenty-seven patients with MSI-H/dMMR and 8 patients with TMB-H/MSS tumors received ICB, none of whom harbored polymerase epsilon (polE) catalytic subunit mutations. About 45% of patients with MSI-H/dMMR had a RECIST response, and 65% had a PSA50 response. No patient with TMB-H/MSS had a RECIST response, and 50% had a PSA50 response. rPFS tended to be longer in patients with MSI-H/dMMR than in patients with TMB-H/MSS who received immunotherapy. Pronounced differences in genomics, TMB, or MSIsensor score were not detected between MSI-H/dMMR responders and nonresponders., Conclusions: MSI-H/dMMR prostate cancers have greater TMB, indel, and neoantigen burden than TMB-H/MSS prostate cancers, and these differences may contribute to profound and durable responses to ICB., (©2024 American Association for Cancer Research.)

Purpose: Patients with residual invasive bladder cancer after neoadjuvant chemotherapy (NAC) and radical cystectomy have a poor prognosis. Data on adjuvant therapy for these patients are conflicting. We sought to evaluate the natural history and genomic landscape of chemotherapy-resistant bladder cancer to inform patient management and clinical trials., Methods: Data were collected on patients with clinically localized muscle-invasive urothelial bladder cancer treated with NAC and cystectomy at our institution between May 15, 2001, and August 15, 2019, and completed four cycles of gemcitabine and cisplatin NAC, excluding those treated with adjuvant therapies. Survival was estimated using the Kaplan-Meier method, and multivariable Cox proportional hazards models were used to identify predictors of recurrence-free survival (RFS). Genomic alterations were identified in targeted exome sequencing (Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets) data from post-NAC specimens from a subset of patients., Results: Lymphovascular invasion (LVI) was the strongest predictor of RFS (hazard ratio, 2.15 [95% CI, 1.37 to 3.39]) on multivariable analysis. Patients with ypT2N0 disease without LVI had a significantly prolonged RFS compared with those with LVI (70% RFS at 5 years). Lymph node yield did not affect RFS. Among patients with sequencing data (n = 101), chemotherapy-resistant tumors had fewer alterations in DNA damage response genes compared with tumors from a publicly available chemotherapy-naïve cohort (15% v 29%; P = .021). Alterations in CDKN2A/B were associated with shorter RFS. PIK3CA alterations were associated with LVI. Potentially actionable alterations were identified in more than 75% of tumors., Conclusion: Although chemotherapy-resistant bladder cancer generally portends a poor prognosis, patients with organ-confined disease without LVI may be candidates for close observation without adjuvant therapy. The genomic landscape of chemotherapy-resistant tumors is similar to chemotherapy-naïve tumors. Therapeutic opportunities exist for targeted therapies as adjuvant treatment in chemotherapy-resistant disease.

Purpose: The use of systemic immune checkpoint blockade before surgery is increasing in patients with metastatic renal cell carcinoma, however, the safety and feasibility of performing consolidative cytoreductive nephrectomy after the administration of systemic therapy are not well described., Patients and Methods: A retrospective review of patients undergoing nephrectomy was performed using our prospectively maintained institutional database. Patients who received preoperative systemic immunotherapy were identified, and the risk of postoperative complications were compared to those who underwent surgery without upfront systemic treatment. Perioperative characteristics and surgical complications within 90 days following surgery were recorded., Results: Overall, we identified 220 patients who underwent cytoreductive nephrectomy from April 2015 to December 2022, of which 46 patients (21%) received systemic therapy before undergoing surgery. Unadjusted rates of surgical complications included 20% (n = 35) in patients who did not receive upfront systemic therapy and 20% (n = 9) in those who received upfront systemic immunotherapy. In our propensity score analysis, there was no statistically significant association between receipt of upfront immunotherapy and 90-day surgical complications [odds ratio (OR): 1.82, 95% confidence interval (CI): 0.59-5.14; P = 0.3]. This model, however, demonstrated an association between receipt of upfront immunotherapy and an increased odds of requiring a blood transfusion [OR: 4.53, 95% CI: 1.83-11.7; P = 0.001]., Conclusion: In our cohort, there was no significant difference in surgical complications among patients who received systemic therapy before surgery compared to those who did not receive upfront systemic therapy. Cytoreductive nephrectomy is safe and with low rates of complications following the use of systemic therapy., Competing Interests: Declaration of Competing Interest Maria Carlo has no conflicts of interest to disclose. Jonathan Coleman has no conflicts of interest to disclose. Lennert Eismann has no conflicts of interest to disclose. Darren Feldman reports research funding from Telix, BioNTech, Astellas, and Decibel Therapeutics; is a consultant for Telix and BioNTech; and reports royalties from UpToDate. Alvin Goh is a consultant for Medtronic. A. Ari Hakimi is on the Merck Advisory board. Sari Khaleel (KhaleelS@mskcc.org) has no conflicts of interest to disclose. Ritesh Kotecha reports advisory board consultation for Eisai; receives institutional research funding from Pfizer, Novartis, Takeda, and Allogene Therapeutics; and is supported in part by the Academy of Kidney Cancer Investigators of the CDMRP/DOD (KC200127). Chung-Han Lee reports honoraria from Ideology Health, Intellisphere, Research to Practice, American Institute of Continuing Medical Education, and Medscape; has a consulting or advisory role for Aveo, Cardinal Health, Eisai, Bristol Myers Squibb, Merck, Pfizer/EMD Serono, and Exelixis; and reports research funding from Eisai, Bristol Myers Squibb, Calithera Biosciences, Exelixis, Merck, and AstraZeneca. Richard Matulewicz has no conflicts of interest to disclose. Robert Motzer reports grants from Bristol Myers Squibb and Pfizer; grants and personal fees from Eisai, Exelixis, Merck, and Genentech/Roche; and personal fees from EMD Serono Research. Irina Ostrovnaya has no conflicts of interest to disclose. Stephen Reese has no conflicts of interest to disclose. Katiana Vazquez-Rivera has no conflicts of interest to disclose. Paul Russo has no conflicts of interest to disclose. Juan Arroyave Villada has no conflicts of interest to disclose. Martin Voss reports receiving commercial research grants from Bristol Myers Squibb, Pfizer, and Genentech/Roche; honoraria from Novartis and Bristol Myers Squibb; and travel/accommodation from AstraZeneca, Eisai, Novartis, and Takeda; and serving as a consultant/advisory board member for Alexion Pharmaceuticals, Aveo, Bayer, Calithera Biosciences, Corvus Pharmaceuticals, Exelixis, Eisai, Genentech, GlaxoSmithKline, Merck, Natera, Onquality Pharmaceuticals, Novartis, and Pfizer. Charlie White has no conflicts of interest to disclose., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Salivary gland cancers (SGCs) are rare, aggressive cancers without effective treatments when metastasized. We conducted a phase 2 trial evaluating nivolumab (nivo, anti-PD-1) and ipilimumab (ipi, anti-CTLA-4) in 64 patients with metastatic SGC enrolled in two histology-based cohorts (32 patients each): adenoid cystic carcinoma (ACC; cohort 1) and other SGCs (cohort 2). The primary efficacy endpoint (≥4 objective responses) was met in cohort 2 (5/32, 16%) but not in cohort 1 (2/32, 6%). Treatment safety/tolerability and progression-free survival (PFS) were secondary endpoints. Treatment-related adverse events grade ≥3 occurred in 24 of 64 (38%) patients across both cohorts, and median PFS was 4.4 months (95% confidence interval (CI): 2.4, 8.3) and 2.2 months (95% CI: 1.8, 5.3) for cohorts 1 and 2, respectively. We present whole-exome, RNA and T cell receptor (TCR) sequencing data from pre-treatment and on-treatment tumors and immune cell flow cytometry and TCR sequencing from peripheral blood at serial timepoints. Responding tumors universally demonstrated clonal expansion of pre-existing T cells and mutational contraction. Responding ACCs harbored neoantigens, including fusion-derived neoepitopes, that induced T cell responses ex vivo. This study shows that nivo+ipi has limited efficacy in ACC, albeit with infrequent, exceptional responses, and that it could be promising for non-ACC SGCs, particularly salivary duct carcinomas. ClinicalTrials.gov identifier: NCT03172624 ., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Purpose: While most small renal masses (SRM) < 4 cm have an excellent prognosis following resection, the impact of adverse T3a pathologic features on oncologic outcomes of SRMs remains unclear. We sought to compare clinical outcomes for surgically resected pT3a versus pT1a SRMs at our institution., Materials and Methods: We retrospectively reviewed records of patients who underwent radical or partial nephrectomy (RN, PN) for renal tumors <4 cm at our institution between 2010 and 2020. We compared features and outcomes of pT3a vs pT1a SRMs. Continuous and categorical variables were compared using Student's t and Pearson's chi-squared tests, respectively. Postoperative outcomes of interest including overall, cancer-specific, and recurrence-free survival (OS, CSS, and RFS) were analyzed using Kaplan-Meier method, Cox proportional hazard regression, and competing risk analysis. Analyses were performed using R statistical package (R Foundation, v4.0)., Results: We identified 1,837 patients with malignant SRMs. Predictors of postoperative pT3a upstaging included higher renal score, larger tumor size, and presence of radiologic features concerning for T3a disease (odds ratio [OR] = 5.45, 95% confidence interval [CI] 3.92-7.59, P < 0.001). On univariable modeling, pT3a SRMs had higher positive margin rates (9.6% vs 4.1%, P < 0.001), worse OS (hazard ratio [HR] = 2.9, 95% CI 1.6-5.3, P = 0.002), RFS (HR 9.32, 95% CI 2-40.1, P = 0.003), and CSS (HR = 3.6, 95% CI 1.5-8.2, P = 0.003). On multivariable modeling, pT3a status remained associated with worse RFS (HR = 2.7, 95% CI 1.04-7, P = 0.04), but not OS (HR 1.6, 95% CI = 0.83-3.1, P = 0.2); multivariable modeling was deferred for CSS due to low event rates., Conclusions: Adverse T3a pathologic features portend worse outcomes for SRMs, highlighting the crucial role of pre-operative planning and case selection. These patients have relatively poor prognosis, and should be monitored more closely and counseled for consideration of adjuvant therapy or clinical trials., Competing Interests: Declaration of Competing Interest The authors do not have any direct conflicts of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Purpose: Immune checkpoint blockade (ICB) therapy has significantly improved clinical outcomes in bladder cancer. Identification of correlates of benefit is critical to select appropriate therapy for individual patients., Methods: To reveal genetic variables associated with benefit from ICB, we performed whole-exome sequencing on tumor specimens from 88 patients with advanced bladder cancer treated with ICB., Results: We identified several genetic factors that correlated with progression-free and overall survival after ICB therapy including ARID1A mutation, tumor mutational burden, intratumoral heterogeneity, the ratio of nonsynonymous to synonymous mutations in the immunopeptidome (immune dN/dS), and tumor cell purity. In addition, we noted that neutrophil-to-lymphocyte ratio and smoking history were negatively associated with overall survival. These genetic characteristics define four molecular subtypes demonstrating differential sensitivity to ICB. We validated the association of these four subtypes with clinical benefit from ICB in an independent cohort (IMvigor210). Finally, we showed that these molecular subtypes also correlate with outcome, although with distinct relationships, among patients not treated with ICB using The Cancer Genome Atlas (TCGA) bladder cancer cohort. Using parallel RNA sequencing data, the subtypes were also shown to correlate with immune infiltration and inflammation, respectively, in the IMvigor210 and TCGA cohorts., Conclusion: Together, our study defines molecular subgroups of bladder cancer that influence benefit from ICB.

Precision oncology relies on the accurate molecular characterization of individual patients with cancer at the time of treatment initiation. However, tumor molecular profiles are not static, and cancers continually evolve because of ongoing mutagenesis and clonal selection. Here, we performed genomic analyses of primary tumors, metastases, and plasma collected from individual patients to define the concordance of actionable genomic alterations and to identify drivers of metastatic disease progression. We observed a high degree of discordance of actionable genomic alterations, with 23% discordant between primary and metastatic disease sites. Among chromatin-modifying genes, ARID1A mutations, when discordant, were exclusive to the metastatic tumor samples. Our findings indicate that the high degree of lesion-to-lesion genomic heterogeneity may be a barrier to precision oncology approaches for bladder cancer and that circulating tumor DNA profiling may be preferred to tumor sequencing for a subset of patients., Competing Interests: Author contributions Conceptualization, T.N.C., Z.C., H.W., S.C., H.A.A.-A., and D.B.S; methodology, T.N.C., Z.C., H.W., A.T.L., S.C., M.T.A.D., N.A., S.D., J.E.R., N.V., E.J.P., S.P.G., W.H., H.A.A.-A., and D.B.S.; investigation, T.N.C., Z.C., H.W., A.T.L., S.C., M.T.A.D., N.A., C.C., S.D., J.R., N.V., S.P.G., W.H., and H.A.A.-A.; resources, P.R., J.R., D.F.B., M.Y.T., B.H.B., M.B., E.J.P., G.I., H.A.A.-A., and D.B.S.; formal analysis, T.N.C., Z.C., A.T.L., S.C., M.T.A.D., I.O., and W.H.; writing, T.N.C., Z.C., A.T.L., and D.B.S.; review & editing, T.N.C., Z.C., H.A.A.-A., and D.B.S.; supervision, D.B.S. Declaration of interests D.B.S. has served as a consultant for/received honorarium from Pfizer, Loxo/Lilly Oncology, Vividion Therapeutics, Scorpion Therapeutics, Fore Therapeutics, FOG Pharma, Rain Therapeutics, and BridgeBio. H.A.A.-A. provided consultation to AstraZeneca, Janssen Biotech, Bristol-Myers-Squibb, and Paige.ai. J.E.R. has served as a consultant for Astellas, Seagen, Merck, Roche, Genentech, AstraZeneca, Janssen Biotech, Gilead, Pfizer, EMD-Serono, Mirati, Boehringer Ingelheim, Pharmacyclis, GSK, Infinity, Tyra BioSciences, Bayer, and QED Therapeutics and received honoraria from EMD-Serono. M.B. has served as a consultant for Eli Lilly and PetDx and has a patent pending on “Systems and Methods for Detecting Cancer Via cfDNA Screening.” S.D. has served as a consultant for Roche., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Accurate ancestry inference is critical for identifying genetic contributors of cancer disparities among populations. Although methods to infer genetic ancestry have historically relied upon genome-wide markers, the adaptation to targeted clinical sequencing panels presents an opportunity to incorporate ancestry inference into routine diagnostic workflows. We show that global ancestral contributions and admixture of continental populations can be quantitatively inferred using markers captured by the MSK-IMPACT clinical panel. In a pan-cancer cohort of 45,157 patients, we observed differences by ancestry in the frequency of somatic alterations, recapitulating known associations and revealing novel associations. Despite the comparable overall prevalence of driver alterations by ancestry group, the proportion of patients with clinically actionable alterations was lower for African (30%) compared with European (33%) ancestry. Although this result is largely explained by population-specific cancer subtype differences, it reveals an inequity in the degree to which different populations are served by existing precision oncology interventions., Significance: We performed a comprehensive analysis of ancestral associations with somatic mutations in a real-world pan-cancer cohort, including >5,000 non-European individuals. Using an FDA-authorized tumor sequencing panel and an FDA-recognized oncology knowledge base, we detected differences in the prevalence of clinically actionable alterations, potentially contributing to health care disparities affecting underrepresented populations. This article is highlighted in the In This Issue feature, p. 2483., (©2022 American Association for Cancer Research.)

Cancers arising from the bladder urothelium often exhibit lineage plasticity with regions of urothelial carcinoma adjacent to or admixed with regions of divergent histomorphology, most commonly squamous differentiation. To define the biologic basis for and clinical significance of this morphologic heterogeneity, here we perform integrated genomic analyses of mixed histology bladder cancers with separable regions of urothelial and squamous differentiation. We find that squamous differentiation is a marker of intratumoral genomic and immunologic heterogeneity in patients with bladder cancer and a biomarker of intrinsic immunotherapy resistance. Phylogenetic analysis confirms that in all cases the urothelial and squamous regions are derived from a common shared precursor. Despite the presence of marked genomic heterogeneity between co-existent urothelial and squamous differentiated regions, no recurrent genomic alteration exclusive to the urothelial or squamous morphologies is identified. Rather, lineage plasticity in bladder cancers with squamous differentiation is associated with loss of expression of FOXA1, GATA3, and PPARG, transcription factors critical for maintenance of urothelial cell identity. Of clinical significance, lineage plasticity and PD-L1 expression is coordinately dysregulated via FOXA1, with patients exhibiting morphologic heterogeneity pre-treatment significantly less likely to respond to immune checkpoint inhibitors., (© 2022. The Author(s).)

It is poorly understood how the tumor immune microenvironment influences disease recurrence in localized clear-cell renal cell carcinoma (ccRCC). Here we performed whole-transcriptomic profiling of 236 tumors from patients assigned to the placebo-only arm of a randomized, adjuvant clinical trial for high-risk localized ccRCC. Unbiased pathway analysis identified myeloid-derived IL6 as a key mediator. Furthermore, a novel myeloid gene signature strongly correlated with disease recurrence and overall survival on uni- and multivariate analyses and is linked to TP53 inactivation across multiple data sets. Strikingly, effector T-cell gene signatures, infiltration patterns, and exhaustion markers were not associated with disease recurrence. Targeting immunosuppressive myeloid inflammation with an adenosine A2A receptor antagonist in a novel, immunocompetent, Tp53-inactivated mouse model significantly reduced metastatic development. Our findings suggest that myeloid inflammation promotes disease recurrence in ccRCC and is targetable as well as provide a potential biomarker-based framework for the design of future immuno-oncology trials in ccRCC., Significance: Improved understanding of factors that influence metastatic development in localized ccRCC is greatly needed to aid accurate prediction of disease recurrence, clinical decision-making, and future adjuvant clinical trial design. Our analysis implicates intratumoral myeloid inflammation as a key driver of metastasis in patients and a novel immunocompetent mouse model. This article is highlighted in the In This Issue feature, p. 2221., (©2022 American Association for Cancer Research.)

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., (©2022 The Authors; Published by the American Association for Cancer Research.)

Introduction: Small cell carcinoma of the bladder (SCCB) is a rare variant of bladder cancer with poor outcomes. We evaluated long-term outcomes of nonmetastatic (M0) and metastatic (M1) SCCB and correlated pathologic response with genomic alterations of patients treated with neoadjuvant chemotherapy (NAC)., Patients and Methods: Clinical history and pathology samples from SCCB patients diagnosed at our institution were reviewed., Results: One hundred and ninety-nine SCCB patients were identified. (M0: 147 [74%]; M1: 52 [26%]). Among M0 patients, 108 underwent radical cystectomy (RC) (NAC: 71; RC only: 23; adjuvant chemotherapy: 14); 14 received chemoradiotherapy; the rest received chemotherapy alone or no cancer-directed therapy. RC-only patients had a median follow-up of 9.1 years, and median disease-free survival (DFS) and overall survival (OS) were 1.1 and 1.2 years, respectively. NAC patients had pathologic response (

Purpose: To compare oncologic outcomes and genomic alteration profiles in patients with bladder and urachal adenocarcinoma, urothelial carcinoma (UC) with glandular differentiation, and UC, not otherwise specified (NOS) undergoing surgical resection, with emphasis on response to systemic therapy., Methods: We identified patients with bladder cancer with glandular variants who underwent surgical resection at Memorial Sloan Kettering from 1995 to 2018 (surgical cohort) and/or patients who had tumor sequencing using a targeted next-generation sequencing platform (genomics cohort). Pathologic complete and partial response rates to neoadjuvant chemotherapy (NAC) and recurrence-free and cancer-specific survival were measured. Alteration frequencies between histologic subtypes were compared., Results: Thirty-seven patients with bladder adenocarcinoma, 46 with urachal adenocarcinoma, 84 with UC with glandular differentiation, and 1,049 with UC, NOS comprised the surgical cohort. Despite more advanced disease in patients with bladder and urachal adenocarcinoma, no significant differences in recurrence or cancer-specific survival by histology were observed after adjusting for stage. In patients with UC with glandular differentiation, NAC resulted in partial (≤ pT1N0) and complete (pT0N0) responses in 28% and 17%, respectively. Bladder and urachal adenocarcinoma genomic profiles resembled colorectal adenocarcinoma with frequent TP53 , KRAS , and PIK3CA alterations while the genomic profile of UC with glandular differentiation more closely resembled UC, NOS. Limitations include retrospective nature of analysis and small numbers of nonurothelial histology specimens., Conclusion: The genomic profile of bladder adenocarcinomas resembled colorectal adenocarcinomas, whereas UC with glandular differentiation more closely resembled UC, NOS. Differences in outcomes among patients with glandular bladder cancer variants undergoing surgical resection were largely driven by differences in stage. Cisplatin-based NAC demonstrated activity in UC with glandular differentiation, suggesting NAC should be considered for this histologic variant.

Purpose: Neoadjuvant gemcitabine and cisplatin (GC) followed by radical cystectomy (RC) is standard for patients with muscle-invasive bladder cancer (MIBC). On the basis of the activity of atezolizumab (A) in metastatic BC, we tested neoadjuvant GC plus A for MIBC., Methods: Eligible patients with MIBC (cT2-T4aN0M0) received a dose of A, followed 2 weeks later by GC plus A every 21 days for four cycles followed 3 weeks later by a dose of A before RC. The primary end point was non-muscle-invasive downstaging to < pT2N0., Results: Of 44 enrolled patients, 39 were evaluable. The primary end point was met, with 27 of 39 patients (69%) < pT2N0, including 16 (41%) pT0N0. No patient with < pT2N0 relapsed and four (11%) with ≥ pT2N0 relapsed with a median follow-up of 16.5 months (range: 7.0-33.7 months). One patient refused RC and two developed metastatic disease before RC; all were considered nonresponders. The most common grade 3-4 adverse event (AE) was neutropenia (n = 16; 36%). Grade 3 immune-related AEs occurred in five (11%) patients with two (5%) requiring systemic steroids. The median time from last dose of chemotherapy to surgery was 7.8 weeks (range: 5.1-17 weeks), and no patient failed to undergo RC because of AEs. Four of 39 (10%) patients had programmed death-ligand 1 (PD-L1)-positive tumors and were all < pT2N0. Of the patients with PD-L1 low or negative tumors, 23 of 34 (68%) achieved < pT2N0 and 11 of 34 (32%) were ≥ pT2N0 ( P = .3 for association between PD-L1 and < pT2N0)., Conclusion: Neoadjuvant GC plus A is a promising regimen for MIBC and warrants further study. Patients with < pT2N0 experienced improved relapse-free survival. The PD-L1 positivity rate was low compared with published data, which limits conclusions regarding PD-L1 as a predictive biomarker., Competing Interests: Samuel A. FuntEmployment: ByHeart (I)Stock and Other Ownership Interests: Kite, a Gilead company, Urogen pharma, Allogene Therapeutics, Neogene Therapeutics, Kronos Bio, Vida Ventures, IconOVir BioConsulting or Advisory Role: Merck, ImmunaiResearch Funding: Genentech/Roche (Inst), AstraZeneca (Inst), Decibel Therapeutics (Inst)Travel, Accommodations, Expenses: Bristol Myers Squibb, AstraZeneca/MedImmune Hikmat Al-AhmadieConsulting or Advisory Role: Bristol Myers Squibb, EMD Serono, AstraZeneca/MedImmune, Janssen Biotech, PAIGE.AI Min Yuen TeoConsulting or Advisory Role: Janssen OncologyResearch Funding: Bristol Myers Squibb (Inst), Clovis Oncology (Inst), Pharmacyclics (Inst) Chung-Han LeeHonoraria: Intellisphere, Research to Practice, American Institute of Continuing Medical EducationConsulting or Advisory Role: Eisai, Bristol Myers Squibb, Merck, Pfizer/EMD Serono, ExelixisResearch Funding: Eisai (Inst), Bristol Myers Squibb (Inst), Calithera Biosciences (Inst), Exelixis (Inst), Merck (Inst) David AggenConsulting or Advisory Role: Boehringer Ingelheim, Seattle Genetics, Astellas PharmaPatents, Royalties, Other Intellectual Property: University of Illinois—Urbana Champaign(OPTIONAL) Open Payments Link: https://openpaymentsdata.cms.gov/physician/4226107 Deaglan McHughConsulting or Advisory Role: Progenics Arlyn ApolloEmployment: Covera Health (I)Leadership: Covera Health (I)Stock and Other Ownership Interests: Covera Health (I)Consulting or Advisory Role: Covera Health (I)Travel, Accommodations, Expenses: Covera Health (I) Kaitlyn FranceseConsulting or Advisory Role: Seattle Genetics, Astellas Pharma Yuanquan YangStock and Other Ownership Interests: Bristol Myers Squibb, Pfizer, AstraZenecaConsulting or Advisory Role: The Whiteoak Group Eugene PietzakHonoraria: UpToDateConsulting or Advisory Role: Merck, Chugai Pharma, QED Therapeutics, Janssen Alvin C. GohConsulting or Advisory Role: MedtronicTravel, Accommodations, Expenses: Medtronic William C. HuangHonoraria: Urogen pharmaConsulting or Advisory Role: Intuitive SurgicalResearch Funding: SonaCare Medical, photocure, Storz, Storz, Merck (Inst), Intuitive Surgical (Inst)Travel, Accommodations, Expenses: Photocure Dean F. BajorinConsulting or Advisory Role: Merck, Dragonfly Therapeutics, Fidia Farmaceutici S. p. A, Bristol Myers Squibb FoundationResearch Funding: Novartis (Inst), Merck (Inst), Bristol Myers Squibb (Inst), AstraZeneca (Inst), Astellas Pharma (Inst), Seattle Genetics/Astellas (Inst)Travel, Accommodations, Expenses: Merck Gopa IyerConsulting or Advisory Role: Bayer, Janssen, Mirati Therapeutics, Basilea, Flare Therapeutics, Loxo/LillySpeakers' Bureau: Gilead Sciences, The Lynx GroupResearch Funding: Mirati Therapeutics (Inst), Novartis (Inst), Debiopharm Group (Inst), Bayer (Inst), Janssen (Inst), Seattle Genetics (Inst) Bernard H. BochnerConsulting or Advisory Role: Olympus Arjun V. BalarLeadership: GT BiopharmaStock and Other Ownership Interests: GT BiopharmaHonoraria: Merck, Genentech/Roche, AstraZeneca/MedImmuneConsulting or Advisory Role: Genentech/Roche, Merck, Cerulean Pharma, AstraZeneca/MedImmune, Pfizer/EMD Serono, Incyte, Seattle Genetics/Astellas, Nektar, Dragonfly Therapeutics, GlaxoSmithKline, Bristol Myers Squibb/CelgeneResearch Funding: Merck (Inst), Genentech/Roche (Inst), AstraZeneca/MedImmune (Inst), Seattle Genetics, Gilead Sciences (Inst) Amir MortazaviHonoraria: Motive Medical IntelligenceConsulting or Advisory Role: Seattle Genetics, Debiopharm Group, PfizerResearch Funding: Acerta Pharma (Inst), Genentech/Roche (Inst), Merck (Inst), Novartis (Inst), Seattle Genetics (Inst), Mirati Therapeutics (Inst), Bristol Myers Squibb (Inst), Roche (Inst), Astellas Pharma (Inst), Debiopharm Group (Inst), Debiopharm Group (Inst) Jonathan E. RosenbergHonoraria: UpToDate, Medscape, Peerview, Research To Practice, Intellisphere, Clinical Care Options, Physicans' Education Resource, MJH Life Sciences, EMD SeronoConsulting or Advisory Role: Lilly, Merck, Roche/Genentech, AstraZeneca/MedImmune, Bristol Myers Squibb, Seattle Genetics, Bayer, BioClin Therapeutics, QED Therapeutics, Adicet Bio, Pharmacyclics, western oncolytics, GlaxoSmithKline, Janssen Oncology, Astellas Pharma, Boehringer Ingelheim, Pfizer/EMD Serono, Mirati Therapeutics, Immunomedics, Tyra Biosciences, Infinity PharmaceuticalsResearch Funding: Genentech/Roche (Inst), Seattle Genetics (Inst), Bayer (Inst), AstraZeneca (Inst), QED Therapeutics (Inst), Astellas Pharma (Inst)Patents, Royalties, Other Intellectual Property: Predictor of platinum sensitivity (Inst)No other potential conflicts of interest were reported.

Purpose: Dedifferentiated liposarcoma (DDLS), one of the most common and aggressive sarcomas, infrequently responds to chemotherapy. DDLS survival and growth depend on underexpression of C/EBPα, a tumor suppressor and transcriptional regulator controlling adipogenesis. We sought to screen and prioritize candidate drugs that increase C/EBPα expression and may therefore serve as differentiation-based therapies for DDLS., Experimental Design: We screened known bioactive compounds for the ability to restore C/EBPα expression and inhibit proliferation selectively in two DDLS cell lines but not in normal adipose-derived stem cells (ASC). Selected hits' activity was validated, and the mechanism of the most potent, SN-38, was investigated. The in vivo efficacy of irinotecan, the prodrug of SN-38, was evaluated in DDLS xenograft models., Results: Of 3,119 compounds, screen criteria were met by 19. Validation experiments confirmed the DDLS selectivity of deguelin, emetine, and SN-38 and showed that they induce apoptosis in DDLS cells. SN-38 had the lowest IC 50 (approximately 10 nmol/L), and its pro-apoptotic effects were countered by knockdown of CEBPA but not of TP53 . Irinotecan significantly inhibited tumor growth at well-tolerated doses, induced nuclear expression of C/EBPα, and inhibited HIF1α expression in DDLS patient-derived and cancer cell line xenograft models. In contrast, doxorubicin, the most common treatment for nonresectable DDLS, reduced tumor growth by 30% to 50% at a dose that caused weight loss., Conclusions: This high-content screen revealed potential treatments for DDLS. These include irinotecan, which induces apoptosis of DDLS cells in a C/EBPα-dependent, p53-independent manner, and should be clinically evaluated in patients with advanced DDLS., (©2021 American Association for Cancer Research.)

In recent literature, the human microbiome has been shown to have a major influence on human health. To investigate this impact, scientists study the composition and abundance of bacterial species, commonly using 16S rRNA gene sequencing, among patients with and without a disease or condition. Methods for such investigations to date have focused on the association between individual bacterium and an outcome, and higher-order pairwise relationships or interactions among bacteria are often avoided due to the substantial increase in dimension and the potential for spurious correlations. However, overlooking such relationships ignores the environment of the microbiome, where there is dynamic cooperation and competition among bacteria. We present a method for identifying and ranking pairs of bacteria that have a differential dichotomized relationship across outcomes. Our approach, implemented in an R package PairSeek, uses the stability selection framework with data-driven dichotomized forms of the pairwise relationships. We illustrate the properties of the proposed method using a published oral cancer data set and a simulation study., Competing Interests: The authors have declared that no competing interests exist.

Objective: Recurrent genomic alterations in clear cell renal cell carcinoma (ccRCC) have been associated with treatment outcomes; however, current preoperative predictive models do not include known genetic predictors. We aimed to explore the value of common somatic mutations in the preoperative prediction of metastatic disease among patients treated for localized ccRCC., Materials and Methods: After obtaining institutional review board approval, data of 254 patients with localized ccRCC treated between 2005 and 2015 who underwent genetic sequencing was collected. The mutation status of VHL, PBRM1, SETD2, BAP1 and KDM5C were evaluated in the nephrectomy tumor specimen, which served as a proxy for biopsy mutation status. The Raj et al. preoperative nomogram was used to predict the 12-year metastatic free probability (MFP). The study outcome was MFP; the relationship between MFP and mutation status was evaluated with Cox-regression models adjusting for the preoperative nomogram variables (age, gender, incidental presentation, lymphadenopathy, necrosis, and size)., Results: The study cohort included 188 males (74%) and 66 females (26%) with a median age of 58 years. VHL mutations were present in 152/254 patients (60%), PBRM1 in 91/254 (36%), SETD2 in 32/254 (13%), BAP1 in 19/254 (8%), and KDM5C in 19/254 (8%). Median follow-up for survivors was 8.1 years. Estimated 12-year MFP was 70% (95% CI: 63%-75%). On univariable analysis SETD2 (HR: 3.30), BAP1 (HR: 2.44) and PBRM1 (HR: 1.78) were significantly associated with a higher risk of metastases. After adjusting for known preoperative predictors in the existing nomogram, SETD2 mutations remained associated with a higher rate of metastases after nephrectomy (HR: 2.09, 95% CI: 1.19-3.67, P = 0.011)., Conclusion: In the current exploratory analysis, SETD2 mutations were significant predictors of MFP among patients treated for localized ccRCC. Our findings support future studies evaluating genetic alterations in preoperative renal biopsy samples as potential predictors of treatment outcome., Competing Interests: Conflict of interest and disclosure Oguz Akin holds stock options and serves as a scientific advisor for Ezra AI, Inc., which is developing Artificial Intelligence algorithms and software unrelated to the research being reported., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Eosinophils influence antitumor immunity and may predict response to treatment with immune checkpoint inhibitors (ICIs). To examine the association between blood eosinophil counts and outcomes in patients with advanced or metastatic urothelial carcinoma (mUC) treated with ICIs, we identified 2 ICI-treated cohorts: discovery (n=60) and validation (n=111). Chemotherapy cohorts were used as comparators (first-line platinum-based chemotherapy, n=75; second-line or more pemetrexed, n=77). The primary endpoint was overall survival (OS). Secondary endpoints were time on treatment (ToT) and progression-free survival. Univariate and multivariate analyses were performed using Cox proportional hazard models. Associations between changes in eosinophil count at weeks 2/3 and 6 after the start of ICI treatment were analyzed using landmark analyses. Baseline characteristics of the ICI cohorts were similar. In the discovery cohort, an optimal cutoff for pretreatment eosinophil count was determined [Eos-Lo: <100 cells/µL; n=9 (15%); Eos-Hi: ≥100 cells/µL; n=51 (85%)]. Eos-Lo was associated with inferior outcomes [OS: hazard ratio (HR), 3.98; 95% confidence interval (CI), 1.85-8.56; P<0.013; ToT: HR, 2.45; 95% CI, 1.17-5.10; P=0.017]. This was confirmed in the validation cohort [Eos-Lo: n=17 (15%); Eos-Hi: n=94 (85%)] (OS: HR, 2.51; 95% CI, 1.31-4.80; P=0.006; ToT: HR, 2.22; 95% CI, 1.2-3.80; P=0.004), and remained significant after adjustment for other prognostic factors. Changes in eosinophil counts at weeks 2/3 and 6 were not clearly associated with outcomes. In chemotherapy cohorts, eosinophil counts were not associated with outcomes. In conclusion, low pretreatment eosinophil count was associated with poorer outcomes in patients with mUC treated with ICIs, and may represent a new predictive biomarker., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Intratumoral genetic heterogeneity (ITH) poses a significant challenge to utilizing sequencing for decision making in the management of cancer. Although sequencing of multiple tumor regions can address the pitfalls of ITH, it does so at a significant increase in cost and resource utilization. We propose a pooled multiregional sequencing strategy, whereby DNA aliquots from multiple tumor regions are mixed prior to sequencing, as a cost-effective strategy to boost translational value by addressing ITH while preserving valuable residual tissue for secondary analysis. Focusing on kidney cancer, we demonstrate that DNA pooling from as few as two regions significantly increases mutation detection while reducing clonality misattribution. This leads to an increased fraction of patients identified with therapeutically actionable mutations, improved patient risk stratification, and improved inference of evolutionary trajectories with an accuracy comparable to bona fide multiregional sequencing. The same approach applied to non-small-cell lung cancer data substantially improves tumor mutational burden (TMB) detection. Our findings demonstrate that pooled DNA sequencing strategies are a cost-effective alternative to address intrinsic genetic heterogeneity in clinical settings., Competing Interests: The rest of the authors have no conflicts to disclose.

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.