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Background The phosphatidyl 3-inositol kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) pathway frequently is activated in patients with urothelial carcinoma (UC). In the current study, the authors performed a phase 2 study evaluating the efficacy of the pan-isoform class I PI3K inhibitor buparlisib in patients with platinum-refractory metastatic UC. Methods Two cohorts were recruited: an initial genetically unselected cohort and a subsequent expansion cohort of patients with PI3K/Akt/mTOR pathway-altered tumors. The primary endpoint was the 2-month progression-free survival rate. A rate of ≥80% was considered promising using a Simon 2-stage minimax design. Secondary endpoints included safety and correlation of markers of PI3K pathway activation with outcome. Results Six of 13 evaluable patients within the initial cohort demonstrated stable disease and 1 demonstrated a partial response, which was below the cutoff of 9 patients required to proceed to stage 2. Three of the patients with stable disease and the patient with a partial response harbored somatic TSC1 alterations. Four patients subsequently were recruited onto an expansion cohort: 3 patients with TSC1 alterations and 1 patient with a PIK3CA-activating mutation. No patient achieved disease control at 8 weeks and accrual was halted. Of the 19 patients evaluable for toxicity, 17 demonstrated treatment-related toxicities, 2 of whom had to discontinue therapy. Conclusions Buparlisib was found to demonstrate modest activity in patients with metastatic UC whose tumors harbored TSC1 loss of function alterations; however, this was not a robust predictor of response to buparlisib. The pattern of genetic coalterations likely influences drug sensitivity. Given the modest clinical activity and substantial toxicity of buparlisib, future trials of PI3K inhibitors in patients with UC should focus on isoform-selective PI3K inhibitors in genomically selected patients. Lay summary The phosphatidyl 3-inositol kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling pathway frequently is upregulated in patients with metastatic urothelial carcinoma (UC). This trial explored buparlisib, an inhibitor of the pathway, in patients with heavily pretreated metastatic UC. Although the drug was found to have modest efficacy, with 6 patients experiencing stable disease and 1 patient achieving a partial response at 8 weeks on therapy, significant side effects also were observed. Patients with specific genetic alterations responded to treatment. Further studies of PI3K pathway inhibition are warranted using newer agents that have superior toxicity profiles and are more selective inhibitors of the pathway.

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.

Background Despite the rapid adoption of immunotherapies in advanced non–small cell lung cancer (advNSCLC), knowledge gaps remain about their real‐world (rw) performance. Methods This retrospective, observational, multicenter analysis used the Flatiron Health deidentified electronic health record‐derived database of rw patients with advNSCLC who received treatment with PD‐1 and/or PD‐L1 (PD‐[L]1) inhibitors before July 1, 2017 (N = 5257) and had ≥6 months of follow‐up. The authors investigated PD‐(L)1 line of treatment and PD‐L1 testing rates and the relationship between overall survival (OS) and rw intermediate endpoints: progression‐free survival (rwPFS), rw time to progression (rwTTP), rw time to next treatment (rwTTNT), and rw time to discontinuation (rwTTD). Results First‐line PD‐(L)1 inhibitor use increased from 0% (in the third quarter of 2014 [Q3 2014]) to 42% (Q2 2017) over the study period. PD‐L1 testing also increased (from 3% in Q3 2015 to 70% in Q2 2017). The estimated median OS was 9.3 months (95% CI, 8.9‐9.8 months), and the estimated rwPFS was 3.2 months (95% CI, 3.1‐3.3 months). Longer OS and rwPFS were associated with ≥50% PD‐L1 percentage staining results. Correlations (⍴) between OS and intermediate endpoints were ⍴ = 0.75 (95% CI, 0.73‐0.76) for rwPFS and ⍴ = 0.60 (95% CI, 0.57‐0.63) for rwTTP, and, for treatment‐based intermediate endpoints, correlations were ⍴ = 0.60 (95% CI, 0.56‐0.64) for rwTTNT (N = 856) and ⍴ = 0.81 (95% CI, 0.80‐0.82) for rwTTD. Conclusions The use of first‐line PD‐(L)1 inhibitors and PD‐L1 testing has substantially increased, with better outcomes for patients who have ≥50% PD‐L1 percentage staining. Intermediate rw tumor‐dynamics estimates were moderately correlated with OS in patients with advNSCLC who received immunotherapy, highlighting the need for optimizing and standardizing rw endpoints to enhance the understanding of patient outcomes outside clinical trials., Immunotherapy adoption in non–small cell lung cancer has been rapid. The evaluation of progression endpoints in large cohorts of real‐world patients is feasible and can assess real‐world immunotherapy performance.

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 effect 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.

Purpose Neoadjuvant chemotherapy followed by radical cystectomy (RC) is a standard of care for the management of muscle-invasive bladder cancer (MIBC). Dose-dense cisplatin-based regimens have yielded favorable outcomes compared with standard-dose chemotherapy, yet the optimal neoadjuvant regimen remains undefined. We assessed the efficacy and tolerability of six cycles of neoadjuvant dose-dense gemcitabine and cisplatin (ddGC) in patients with MIBC. Patients and Methods In this prospective, multicenter phase II study, patients received ddGC (gemcitabine 2,500 mg/m2 on day 1 and cisplatin 35 mg/m2 on days 1 and 2) every 2 weeks for 6 cycles followed by RC. The primary end point was pathologic downstaging to non–muscle-invasive disease (< pT2N0). Patients who did not undergo RC were deemed nonresponders. Pretreatment tumors underwent next-generation sequencing to identify predictors of chemosensitivity. Results Forty-nine patients were enrolled from three institutions. The primary end point was met, with 57% of 46 evaluable patients downstaged to < pT2N0. Pathologic response correlated with improved recurrence-free survival and overall survival. Nineteen patients (39%) required toxicity-related dose modifications. Sixty-seven percent of patients completed all six planned cycles. No patient failed to undergo RC as a result of chemotherapy-associated toxicities. The most frequent treatment-related toxicity was anemia (12%; grade 3). The presence of a presumed deleterious DNA damage response (DDR) gene alteration was associated with chemosensitivity (positive predictive value for < pT2N0 [89%]). No patient with a deleterious DDR gene alteration has experienced recurrence at a median follow-up of 2 years. Conclusion Six cycles of ddGC is an active, well-tolerated neoadjuvant regimen for the treatment of patients with MIBC. The presence of a putative deleterious DDR gene alteration in pretreatment tumor tissue strongly predicted for chemosensitivity, durable response, and superior long-term survival.

443 Background: Defining the role of MIBC molecular subtypes and immune expression in determining clinical outcomes is an area of active investigation. However, changes in these transcriptomic profiles pre- and post-NAC have not been well characterized. Methods: This retrospective study reviewed 53 pts with MIBC treated with NAC, of whom 12 pts without complete pathological response had both pre- and post-NAC samples of sufficient quality. Post-NAC staging was > = pT2 in 11 pts and pT1 in 1 pt. We performed RNA expression analysis of matched pre-NAC transurethral resection of bladder tumor specimens and post-treatment radical cystectomy primary bladder tumor specimens. We used a customized NanoString panel incorporating previously reported immune signatures (Ayers, JCI 2017; O’Donnell, ASCO 2017) and additional genes to assign basal ( CD14, CD44, PDGFC, KRT14, KRT5) and luminal ( GATA3, PPARG, SHH, CD24, FOXA1, WNT7B, ERBB2) molecular subtypes. Results: We first classified the bladder cancer cohort of The Cancer Genome Atlas into basal and luminal subtypes using the BASE47 signature (Damrauer, PNAS 2014) and the NanoString panel and there was good agreement (Rand Index = 0.72). We then assigned subtypes using the NanoString panel on matched pre- and post-NAC samples and found marked subtype shift (Table). We identified two robust clusters of samples according to immune expression with a 3-fold change of immune expression between them (FDR = 0.0008). We found that 4 pts switched from the low to the high cluster, while 2 switched from the high to the low cluster after NAC (Table). Conclusions: MIBC molecular subtype membership is dynamic and is influenced by NAC. NAC can induce both enhanced and suppressed immune activity. These findings have implications on future studies exploring the predictive value of RNA expression patterns for bladder cancer therapies as well as post-NAC immunotherapy. [Table: see text]

Background:Inhibition of programmed death-ligand one (PD-L1) with atezolizumab can induce durable clinical benefit (DCB) in patients with metastatic urothelial cancers, including complete remissions in patients with chemotherapy refractory disease. Although mutation load and PD-L1 immune cell (IC) staining have been associated with response, they lack sufficient sensitivity and specificity for clinical use. Thus, there is a need to evaluate the peripheral blood immune environment and to conduct detailed analyses of mutation load, predicted neoantigens and immune cellular infiltration in tumors to enhance our understanding of the biologic underpinnings of response and resistance.Methods and Findings:The goals of this study were to (1) evaluate the association of mutation load and predicted neoantigen load with therapeutic benefit, and (2) determine whether intratumoral and peripheral blood T cell receptor (TCR) clonality inform clinical outcomes in urothelial carcinoma treated with atezolizumab. We hypothesized that an elevated mutation load in combination with T cell clonal dominance among intratumoral lymphocytes prior to treatment or among peripheral T cells after treatment would be associated with effective tumor control upon treatment with anti-PD-L1 therapy. We performed whole exome sequencing (WES), RNA sequencing (RNA-seq), and T cell receptor sequencing (TCR-seq) of pre-treatment tumor samples as well as TCR sequencing of matched, serially collected peripheral blood collected before and after treatment with atezolizumab. These parameters were assessed for correlation with DCB (defined as progression free survival (PFS) > 6 months), PFS, and overall survival (OS), both alone and in the context of clinical and intratumoral parameters known to be predictive of survival in this disease state.Patients with DCB displayed a higher proportion of tumor infiltrating T lymphocytes (TIL) (n=24, Mann-Whitney p=0.047). Pre-treatment peripheral blood TCR clonality below the median was associated with improved PFS (n=29, log-rank p=0.048) and OS (n=29, log-rank p=0.011). Patients with DCB also demonstrated more substantial expansion of tumor-associated TCR clones in the peripheral blood 3 weeks after starting treatment (n=22, Mann-Whitney p=0.022). The combination of high pre-treatment peripheral blood TCR clonality with elevated PD-L1 IC staining in tumor tissue was strongly associated with poor clinical outcomes (n=10, HR (mean)=89.88, HR (median)=23.41, 95% CI (2.43, 506.94), p(HR>1)=0.0014). Marked variations in mutation loads were seen with different somatic variant calling methodologies, which in turn impacted associations with clinical outcomes. Missense mutation load, predicted neoantigen load and expressed neoantigen load did not demonstrate significant association with DCB (n=25, Mann-Whitney p=0.22, n=25, Mann-Whitney p=0.55, and n=25, Mann-Whitney p=0.29 respectively). Instead, we found evidence of time-varying effects of somatic mutation load on progression-free survival in this cohort (n=25, p=0.044). A limitation of our study is its small sample size (n=29), a subset of the patients treated on IMvigor 210 (NCT02108652). Given the number of exploratory analyses performed, we intend for these results to be hypothesis-generating.Conclusions:These results demonstrate the complex nature of immune response to checkpoint blockade and the compelling need for greater interrogation and data integration of both host and tumor factors. Incorporating these variables in prospective studies will facilitate identification and treatment of resistant patients.

Background Inhibition of programmed death-ligand 1 (PD-L1) with atezolizumab can induce durable clinical benefit (DCB) in patients with metastatic urothelial cancers, including complete remissions in patients with chemotherapy refractory disease. Although mutation load and PD-L1 immune cell (IC) staining have been associated with response, they lack sufficient sensitivity and specificity for clinical use. Thus, there is a need to evaluate the peripheral blood immune environment and to conduct detailed analyses of mutation load, predicted neoantigens, and immune cellular infiltration in tumors to enhance our understanding of the biologic underpinnings of response and resistance. Methods and findings The goals of this study were to (1) evaluate the association of mutation load and predicted neoantigen load with therapeutic benefit and (2) determine whether intratumoral and peripheral blood T cell receptor (TCR) clonality inform clinical outcomes in urothelial carcinoma treated with atezolizumab. We hypothesized that an elevated mutation load in combination with T cell clonal dominance among intratumoral lymphocytes prior to treatment or among peripheral T cells after treatment would be associated with effective tumor control upon treatment with anti-PD-L1 therapy. We performed whole exome sequencing (WES), RNA sequencing (RNA-seq), and T cell receptor sequencing (TCR-seq) of pretreatment tumor samples as well as TCR-seq of matched, serially collected peripheral blood, collected before and after treatment with atezolizumab. These parameters were assessed for correlation with DCB (defined as progression-free survival [PFS] >6 months), PFS, and overall survival (OS), both alone and in the context of clinical and intratumoral parameters known to be predictive of survival in this disease state. Patients with DCB displayed a higher proportion of tumor-infiltrating T lymphocytes (TIL) (n = 24, Mann-Whitney p = 0.047). Pretreatment peripheral blood TCR clonality below the median was associated with improved PFS (n = 29, log-rank p = 0.048) and OS (n = 29, log-rank p = 0.011). Patients with DCB also demonstrated more substantial expansion of tumor-associated TCR clones in the peripheral blood 3 weeks after starting treatment (n = 22, Mann-Whitney p = 0.022). The combination of high pretreatment peripheral blood TCR clonality with elevated PD-L1 IC staining in tumor tissue was strongly associated with poor clinical outcomes (n = 10, hazard ratio (HR) (mean) = 89.88, HR (median) = 23.41, 95% CI [2.43, 506.94], p(HR > 1) = 0.0014). Marked variations in mutation loads were seen with different somatic variant calling methodologies, which, in turn, impacted associations with clinical outcomes. Missense mutation load, predicted neoantigen load, and expressed neoantigen load did not demonstrate significant association with DCB (n = 25, Mann-Whitney p = 0.22, n = 25, Mann-Whitney p = 0.55, and n = 25, Mann-Whitney p = 0.29, respectively). Instead, we found evidence of time-varying effects of somatic mutation load on PFS in this cohort (n = 25, p = 0.044). A limitation of our study is its small sample size (n = 29), a subset of the patients treated on IMvigor 210 (NCT02108652). Given the number of exploratory analyses performed, we intend for these results to be hypothesis-generating. Conclusions These results demonstrate the complex nature of immune response to checkpoint blockade and the compelling need for greater interrogation and data integration of both host and tumor factors. Incorporating these variables in prospective studies will facilitate identification and treatment of resistant patients., Alexandra Snyder and colleagues reveal the complex nature of the immune response to checkpoint blockade in metastatic urothelial cancer patients. Time-varying effects of somatic mutation load on survival are reported., Author summary Why was this study done? A new type of cancer treatment called checkpoint blockade therapy activates the immune system to fight cancer. When these therapies work, patients with advanced disease can experience long-lasting disease control or even cures. However, most patients will not experience these benefits, and it is crucial to identify these patients in advance so that we can develop better treatments for them. What did the researchers do and find? In this study, we studied 29 patients with advanced bladder cancers treated with a checkpoint blockade drug called atezolizumab. We examined features of the tumor and the immune system, as well as clinical features. We found that these features were related to each other, and to the success of therapy, in various ways. Patients who had a diverse repertoire of T cells in their blood tended to survive longer. Patients who had poor clinical prognostic factors, like having cancer that had traveled to their liver, tended to have worse survival. What did the research findings mean? This study demonstrates that we need to take the tumor, immune system, and clinical picture into account if we are to improve the efficacy of immune-mobilizing therapies in cancer. Some patients may be too sick to benefit from checkpoint blockade therapy, despite, in some cases, having biomarkers in their tumors that would predict benefit.

Background: Cisplatin-based combination chemotherapy is standard first-line treatment for patients with advanced urothelial carcinoma (UC). Molecular profiling studies reveal that the PI3K/AKT/mTOR pathway is altered in a significant percentage of UCs. Objective: We conducted a phase I trial to evaluate the feasibility of combining the mTOR inhibitor everolimus with gemcitabine and split-dose cisplatin (GC) in advanced UC in the first-line setting. Methods: Patients received gemcitabine 800 mg/m2 and cisplatin 35 mg/m2 on days 1 and 8 of 21-day cycles for a total of 6 cycles in combination with everolimus at increasing dose levels (DL1:5 mg QOD, DL2:5 mg daily, DL3:10 mg daily) following a standard 3+3 design. Responses were assessed every 2 cycles. Patients with at least stable disease (SD) continued everolimus until progression. Goals were to establish dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) for the combination. Results: 12 patients were enrolled, 3 at DL1, 3 at DL2, and an additional 6 at DL1 *(DL1 following de-escalation). 3/3 patients at DL2 had DLTs during cycle 1. 2/8 evaluable patients at DL1/DL1 * had DLTs during cycle 1. DLTs were primarily hematologic. Further toxicities, also primarily hematologic, were observed during later treatment cycles, leading to 8 chemotherapy dose reductions overall. Partial responses were observed in 4/10 evaluable patients, and SD in 5/10. Median overall survival was 10.8 months (95% CI 6.9, not reached). Conclusions: The maximum tolerated dose was reached at the lowest dose level, 5 mg QOD, for everolimus in combination with gemcitabine and split-dose cisplatin in advanced UC. The regimen was limited by hematologic toxicity.

with the BSCC group, while the incidence of lymph node metastasis in the BSCC group was significantly higher than that in the UC group. Of the 12 EMT markers, N-cadherin expression in the UC group was significantly greater than that in the BSCC group, while the expression levels of MMP-9 and ZEB1 in the BSCC group were significantly greater than those in the UC group. Both recurrencefree and overall survivals in the BSCC group were significantly poor compared with those in the UC group. In the UC group, multivariate analyses identified pathological stage, lymph node metastasis and N-cadherin expression as independent predictors of recurrence-free survival (RFS), and age, pathological stage, lymph node metastasis and N-cadherin expression as those of overall survival (OS). In the BSCC group, pathological stage and lymph node metastasis were shown to be independently associated with RFS, while pathological stage, lymph node metastasis, MMP-9 expression and ZEB1 expression appeared to be independent predictors of OS. CONCLUSIONS: These findings suggest the important roles of EMT in the process of disease progression following radical cystectomy for both UC and BSCC; however, different molecules associated with EMT may be involved in this process between UC and BSCC.

Background: Mutational signatures of exposure to DNA damaging agents such as UV irradiation, tobacco, and temozolomide have been described; however a cisplatin mutational signature has not been identified. We analyzed mutational changes in matched pre- and post-cisplatin based neoadjuvant chemotherapy (NAC)-treated muscle-invasive bladder cancer (MIBC) samples to identify a cisplatin mutational signature. Methods: Whole exome sequencing and mutation calling was performed on matched germline and pre- and post-cisplatin based NAC (MVAC and GC) tumor samples from 46 MIBC patients with gross residual disease (≥ pT2) at cystectomy. After quality control, samples from 30 patients were analyzed. For each tumor, we characterized single nucleotide mutations (C>A, C>T, C>G, T>A, T>C, T>G) within a tri-nucleotide context, and used an optimized non-negative matrix factorization (NMF) to discover signatures of mutational processes in pre-NAC tumors, post-NAC tumors, and among mutations unique to the post-NAC samples. We compared our discovered signatures to previously described human mutational signatures and to a signature of cisplatin exposure in a DT40 (chicken lymphoblast) cell line (normalized for a human exome context). We also performed strand asymmetry analysis to search for evidence of transcription-coupled repair. Results: In both pre and post-NAC tumors we identified mutational signatures matching those attributed to APOBEC activity and nucleotide excision repair pathway defects, consistent with past studies in MIBC. In post-NAC tumors we identified an additional novel mutational signature, with modest overall cosine similarity (0.61) to the DT40 cisplatin signature, but similar C>A and T>A motifs. There was a strong correlation in inferred mutational activity (Pearson R = 0.98) when we replaced the novel signature with the DT40 signature in the post-NAC tumors, which was highly unlikely to be due to chance (p < 0.001, empiric null distribution). Further, we found evidence of transcriptional strand bias in C>A (p = 0.00025) and T>A (p = 4.2e-06) motifs with depletion of coding strand mutations, consistent with transcription coupled repair of platinum crosslinks at GpG and ApG motifs in the non-coding strand. Finally, we were able to rediscover the novel signature when limiting analyses to mutations unique to post-NAC tumors, consistent with mutational activity during chemotherapy. Conclusions: Analysis of matched pre- and post- cisplatin treated MIBC identified a novel signature in post-cisplatin treated samples that (1) has mutational activity similar to a preclinical cisplatin mutational signature; (2) has a transcription strand bias consistent with known repair characteristics of platinum-induced DNA damage; and (3) arises in tumors following cisplatin chemotherapy. This may represent a cisplatin-induced mutational signature in human tumors. Citation Format: David Liu, Daniel Keliher, Philip Abbosh, Kent Mouw, Diana Miao, Mariel Boyd, Jean Hoffman-Censits, Gopa Iyer, Sara Tolaney, Jaegil Kim, Gad Getz, Scott Carter, Joaquim Bellmunt, Elizabeth R. Plimack, Jonathan E. Rosenberg, Eliezer M. Van Allen. Analysis of matched pre and post cisplatin-treated muscle-invasive bladder cancer reveals a candidate cisplatin mutational signature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2918. doi:10.1158/1538-7445.AM2017-2918

356 Background: M-MDSCs promote tumor progression through complex mechanisms, including immunosuppression and the production of mediators of angiogenesis and invasion. M-MDSCs are associated with poor outcomes in a number of malignancies. We conducted an exploratory analysis enumerating M-MDSCs (Lin-CD14+/HLA-DRlow/-) in the blood of pts with mUC and assessed for assay variability and correlation with clinical outcomes. Methods: Whole blood was collected at a single time point for each pt and stabilized in Cyto-Chex tubes. M-MDSC% was calculated by flow cytometric analysis of HLA-DR expression on CD14+ monocytes using an MSKCC developed computational algorithm-based approach (Kitano et al. 2014). Individual samples were run in quadruplicate at MSKCC. The mean MDSC% was used in subsequent analyses and the replicate standard deviation (SD) was considered a reflection of intra-assay variability. Clinical variables were collected and pts followed for OS, which was calculated from time of sample collection. Results: 21 pts with mUC who progressed after prior chemotherapy were included. At the time of collection, pts were on clinical trials with immune checkpoint blockade (n=13) or targeted therapy (n=1), receiving salvage chemotherapy (n=2), or awaiting treatment on clinical trials with immune checkpoint blockade (n=4) or targeted therapy (n=1). The median follow up from the time of collection in surviving patients (n=15) was 11.1 months (range: 10.5-11.5). Median OS was not reached. Mean (SD) M-MDSC% was 29.2 (4.92). The range of replicate SD was 0.13-1.05. M-MDSC% was not higher in pts with visceral mets (p=0.109). Pts with above median M-MDSC% had worse OS (p=0.01; 6 patients died during follow up, all with above median M-MDSC%). Conclusions: In a heterogeneous cohort of pts with mUC, the enumeration of M-MDSCs in stabilized whole blood was feasible and demonstrated marked inter-patient but not intra-assay variability. Pts with higher M-MDSC% values had worse OS. Additional characterization of M-MDSCs in larger cohorts and in pts receiving immunotherapy is ongoing and may have important prognostic and therapeutic implications in mUC.

4518Background: The majority of patients with muscle-invasive bladder cancer (MIBC) treated with neoadjuvant cisplatin-based chemotherapy (NAC) have residual disease at time of cystectomy. Changes ...

5011Background: Individual DDR genetic alterations have been reported to be predictive for neo chemotherapy response in UC. We sought to identify whether alterations in a DDR gene set correlate wit...

e23219Background: Genomic instability related to DDR defects leads to accumulation of somatic alterations that promote tumorigenesis, may confer sensitivity to platinum-based chemotherapy and may p...

3005Background: The anti-programmed death-ligand 1 (PD-L1) antibody atezolizumab is active in patients (pts) with advanced mUC. We hypothesized that the TCR repertoire in tumors and blood may corre...

4532 Background: Platinum-based chemotherapy (PBC) is standard of care in the neoadjuvant and metastatic UC settings. Recent work identified mutations in ERCC2, RB1, FANCC and ATM as potential pred...

324 Background: PI3 kinase (PI3K) pathway alterations are found in 39% of urothelial carcinoma (UC) (TCGA). Prior reports have shown significant responses in metastatic UC patients (pts) whose tumors harbor PI3K pathway alterations treated with mTOR inhibitors, providing a rationale to investigate BKM120 in mUC. Methods: This phase II study enrolled mUC pts progressing on platinum-based chemotherapy (up to 4 prior agents). The primary and secondary endpoints were proportion of pts progression-free at 2 months and response rate (RR) by Response Criteria in Solid Tumors (RECIST) v1.1, respectively. A Simon 2-stage design was used to discriminate between a 2 month PFS rate of 80%. Pts received 100 mg drug once daily. To identify predictors of response/resistance to BKM120, targeted exon capture sequencing was performed to define the mutation status of PIK3CA, PTEN, AKT1, TSC1, and additional genes within tumors from all treated pts. Results: 13 of 15 enrolled pts were eligible for the primary endpoint. Median age was 65 (53-82). Pts had received an average of 3 agents (2-4) before enrollment. The median progression-free survival (PFS) was 2.77 months (95% CI: 1.83-3.71) with 6 pts displaying stable disease (SD) and 1 partial response (PR) at 2 months (PFS rate 54%). Sequencing identified 2 pts with PIK3CA mutations (E542K, H1047R) who experienced progression as best RECIST response. Tumor from the pt with a 16 month PR harbored a TSC1 R500* nonsense mutation. 1 pt with SD lasting 3.7 months had a TSC1 L330fs truncation. Conclusions: While BKM120 therapy did not display a significant improvement in 2-month PFS rate compared to standard chemotherapy in the second-line setting, 2 pts, one with a durable PR and one with SD, had tumors with inactivating mutations in TSC1. Based upon these results, an expansion cohort is accruing in which select mUC pts whose tumors harbor PI3K pathway alterations receive BKM120. Clinical trial information: NCT01551030.

322 Background: Gemcitabine and split-dose cisplatin (GC) is standard first-line therapy for patients with advanced urothelial cancer (UC). Everolimus is an inhibitor of the PI3K/AKT/mTOR pathway, dysregulated in ~35% of UCs. The drug has been shown to have single-agent activity in phase II trials of advanced UC. The safety of combining everolimus with GC in the first-line setting for UC is unknown. Methods: Previously untreated cisplatin-eligible patients with advanced UC were enrolled. Patients received gemcitabine 800 mg/m2 and cisplatin 35mg/m2 on days 1 and 8 with everolimus at dose levels (DL1: 5 mg QOD, DL2: 5 mg daily or DL3: 10 mg daily) for six 21-day cycles. Patients with at least SD continued maintenance everolimus until progression. Restaging evaluations were performed every 2 cycles. The primary objective was to establish the dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of the three-drug combination. Results: 12 patients were enrolled, 7 with bladder and 5 with upper tract primaries. 4/12 patients completed at least 6 cycles. 0/3 patients at DL1 had DLTs. 3/3 patients at DL2 had DLTs (pancytopenia, hypersensitivity reaction to everolimus and anemia). Following de-escalation to DL1, 2/6 patients in a new DL1 cohort had DLTs (neutropenia and diarrhea, 1/6 patients inevaluable). Grade 3/4 events included anemia (67%), neutropenia (42%), lymphopenia (42%), thrombocytopenia (25%), urinary tract infection (25%), hypomagnesemia (17%), and hypophosphatemia (17%). Tumor responses included: 4 PR (33%), 5 SD (42%), 1 PD (8%), and 2 inevaluable (17%). 2 patients with PR underwent consolidative surgery but eventually progressed. Responses by MSKCC Risk Scores of 0, 1, and 2 were seen in 3/5 (60%), 1/6 (17%), and 0/1 patients, respectively. Median PFS was 3.9 months (95% CI, 3.7-no reached) and median OS was 10.8 months (95% CI, 6.9-not reached). Conclusions: MTD was reached at DL1 for combination gemcitabine, split-dose cisplatin and everolimus, with 2/8 evaluable patients exhibiting DLTs at this dose level. Clinical trial information: NCT01182168.

298 Background: The type and frequency of recurrent, targetable alterations in muscle-invasive, treatment-naïve urothelial cancer (UC) have been well-catalogued through the bladder TCGA. We sought to validate these findings within a prospective cohort of patients (pts) with progressive UC treated at our institution. This cohort typifies the UC patient population managed by medical oncologists. Methods: Pts with a diagnosis of UC were enrolled onto an IRB-approved protocol between 1/14/14 and 7/29/14, which allowed for sequencing of all exons in 341 oncogenes and tumor suppressor genes using an exon capture next generation sequencing assay (NGS) platform (MSK-IMPACT) in a CLIA lab from tumor and matched germline DNA. Somatic point mutations, truncations, copy number alterations, and insertions/deletions were detected. Results: 49 UC pts were sequenced, 5 (10%) having upper tract disease. 33 (67%) tumors were pure UC histology with the remainder containing divergent differentiation. 17 samples (24%) were metastatic in origin, and 40 samples were analyzed from pts who had received prior chemotherapy. The most common alteration identified was point mutations within the TERT promoter (71%), a region not sequenced by TCGA. Additional alteration frequencies were similar between both sets. FGFR3 mutations overlapped with both PIK3CA and TSC1 alterations and co-alterations were observed between FGFR3 and CDKN2A as well as PIK3CA and CCND1. Conclusions: TERT promoter mutations were found at high frequency in the MSK-IMPACT tumor cohort. The frequency and type of alterations identified in the bladder TCGA are similar to the MSK-IMPACT cohort, suggesting that TCGA data can be used to guide clinical trials in the metastatic, pre-treated population. Overlap of alterations within and across core signaling pathways underscores the need for functional validation of alterations and for rational combinations of targeted therapies to effectively treat advanced UC. [Table: see text]