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Pathologic specifications of analyzed tumor specimens (formal pathologist review).

Frequencies of mutations in genes previously reported6-8,10 as observed in our panel of bladder carcinoma patients.

Genes calculated to be significantly mutated by Genome MuSic algorithm (p

Clinical characteristics of the 81 patients, according to recurrence status.

Genes calculated to be significantly mutated by MutSigCV algorithm (p

Purpose: Because of suboptimal outcomes in muscle-invasive bladder cancer even with multimodality therapy, determination of potential genetic drivers offers the possibility of improving therapeutic approaches and discovering novel prognostic indicators.Experimental Design: Using pTN staging, we case-matched 81 patients with resected ≥pT2 bladder cancers for whom perioperative chemotherapy use and disease recurrence status were known. Whole-exome sequencing was conducted in 43 cases to identify recurrent somatic mutations and targeted sequencing of 10 genes selected from the initial screening in an additional 38 cases was completed. Mutational profiles along with clinicopathologic information were correlated with recurrence-free survival (RFS) in the patients.Results: We identified recurrent novel somatic mutations in the gene UNC5C (9.9%), in addition to TP53 (40.7%), KDM6A (21.0%), and TSC1 (12.3%). Patients who were carriers of somatic mutations in DNA repair genes (one or more of ATM, ERCC2, FANCD2, PALB2, BRCA1, or BRCA2) had a higher overall number of somatic mutations (P = 0.011). Importantly, after a median follow-up of 40.4 months, carriers of somatic mutations (n = 25) in any of these six DNA repair genes had significantly enhanced RFS compared with noncarriers [median, 32.4 vs. 14.8 months; hazard ratio of 0.46, 95% confidence interval (CI), 0.22–0.98; P = 0.0435], after adjustment for pathologic pTN staging and independent of adjuvant chemotherapy usage.Conclusion: Better prognostic outcomes of individuals carrying somatic mutations in DNA repair genes suggest these mutations as favorable prognostic events in muscle-invasive bladder cancer. Additional mechanistic investigation into the previously undiscovered role of UNC5C in bladder cancer is warranted. Clin Cancer Res; 20(24); 6605–17. ©2014 AACR.

Normal adjacent bladder tissue specimens (formal pathologist review).

Purpose Somatic mutations and copy number variation in the ERBB family are frequent in urothelial carcinoma (UC) and may represent viable therapeutic targets. We studied whether afatinib (an oral, irreversible inhibitor of the ErbB family) has activity in UC and if specific ERBB molecular alterations are associated with clinical response. Patients and Methods In this phase II trial, patients with metastatic platinum-refractory UC received afatinib 40 mg/day continuously until progression or intolerance. The primary end point was 3-month progression-free survival (PFS3). Prespecified tumor analysis for alterations in EGFR, HER2, ERBB3, and ERBB4 was conducted. Results The first-stage enrollment goal of 23 patients was met. Patient demographic data included: 78% male, median age 67 years (range, 36 to 82 years), hemoglobin < 10 g/dL in 17%, liver metastases in 30%, median time from prior chemotherapy of 3.6 months, and Eastern Cooperative Oncology Group performance status ≤ 1 in 100%. No unexpected toxicities were observed; two patients required dose reduction for grade 3 fatigue and rash. Overall, five of 23 patients (21.7%) met PFS3 (two partial response, three stable disease). Notably, among the 21 tumors analyzed, five of six patients (83.3%) with HER2 and/or ERBB3 alterations achieved PFS3 (PFS = 10.3, 7.0, 6.9, 6.3, and 5.0 months, respectively) versus none of 15 patients without alterations ( P < .001). Three of four patients with HER2 amplification and three of three patients with ERBB3 somatic mutations (G284R, V104M, and R103G) met PFS3. One patient with both HER2 amplification and ERBB3 mutation never progressed on therapy, but treatment was discontinued after 10.3 months as a result of depressed ejection fraction. The median time to progression/discontinuation was 6.6 months in patients with HER2/ERBB3 alterations versus 1.4 months in patients without alterations ( P < .001). Conclusion Afatinib demonstrated significant activity in patients with platinum-refractory UC with HER2 or ERBB3 alterations. The potential contribution of ERBB3 to afatinib sensitivity is novel. Afatinib deserves further investigation in molecularly selected UC.

Purpose: Because of suboptimal outcomes in muscle-invasive bladder cancer even with multimodality therapy, determination of potential genetic drivers offers the possibility of improving therapeutic approaches and discovering novel prognostic indicators. Experimental Design: Using pTN staging, we case-matched 81 patients with resected ≥pT2 bladder cancers for whom perioperative chemotherapy use and disease recurrence status were known. Whole-exome sequencing was conducted in 43 cases to identify recurrent somatic mutations and targeted sequencing of 10 genes selected from the initial screening in an additional 38 cases was completed. Mutational profiles along with clinicopathologic information were correlated with recurrence-free survival (RFS) in the patients. Results: We identified recurrent novel somatic mutations in the gene UNC5C (9.9%), in addition to TP53 (40.7%), KDM6A (21.0%), and TSC1 (12.3%). Patients who were carriers of somatic mutations in DNA repair genes (one or more of ATM, ERCC2, FANCD2, PALB2, BRCA1, or BRCA2) had a higher overall number of somatic mutations (P = 0.011). Importantly, after a median follow-up of 40.4 months, carriers of somatic mutations (n = 25) in any of these six DNA repair genes had significantly enhanced RFS compared with noncarriers [median, 32.4 vs. 14.8 months; hazard ratio of 0.46, 95% confidence interval (CI), 0.22–0.98; P = 0.0435], after adjustment for pathologic pTN staging and independent of adjuvant chemotherapy usage. Conclusion: Better prognostic outcomes of individuals carrying somatic mutations in DNA repair genes suggest these mutations as favorable prognostic events in muscle-invasive bladder cancer. Additional mechanistic investigation into the previously undiscovered role of UNC5C in bladder cancer is warranted. Clin Cancer Res; 20(24); 6605–17. ©2014 AACR.

Background The success of cancer immunotherapies has highlighted the potent ability of local adaptive immune responses to eradicate cancer cells by targeting neoantigens generated by somatic alterations. However, how these factors interact to drive the natural history of muscle-invasive bladder cancer (MIBC) is not well understood. Objective To investigate the role of immune regulation in MIBC disease progression, we performed massively parallel T-cell receptor (TCR) sequencing of tumor-infiltrating T cells (TILs), in silico neoantigen prediction from exome sequences, and expression analysis of immune-related genes. Design, setting, and participants We analyzed 38 MIBC tissues from patients who underwent definitive surgery with a minimum clinical follow-up of 2 yr. Outcome measurements and statistical analysis Recurrence-free survival (RFS) was determined. TCR diversity was quantified using Simpson's diversity index. The main analyses involved the Mann-Whitney U test, Kaplan-Meier survival analysis, and Cox proportional hazards models. Results and limitations Low TCRβ chain diversity, correlating with oligoclonal TIL expansion, was significantly correlated with longer RFS, even after adjustment for pathologic tumor stage, node status, and receipt of adjuvant chemotherapy (hazard ratio 2.67, 95% confidence interval 1.08–6.60; p =0.03). Patients with both a high number of neoantigens and low TCRβ diversity had longer RFS compared to those with fewer neoantigens and high TCR diversity (median RFS 275 vs 30 wk; p =0.03). Higher expression of immune cytolytic genes was associated with nonrecurrence among patients with low TCR diversity or fewer neoantigens. Limitations include the sample size and the inability to distinguish CD8 + and CD4 + T cells using TCR sequencing. Conclusions These findings are the first to show that detailed tumor immune-genome analysis at definitive surgery can identify molecular patterns of antitumor immune response contributing to better clinical outcomes in MIBC. Patient summary We discovered that clonal expansion of certain T cells in tumor tissue, possibly targeting cancer-specific antigens, contributes to prevention of bladder cancer recurrence.

Background Many current therapies for metastatic castration-resistant prostate cancer (mCRPC) are aimed at AR signaling; however, resistance to these therapies is inevitable. To personalize CRPC therapy in an individual with clinical progression despite maximal AR signaling blockade, it is important to characterize the status of AR activity within their cancer. Biopsies of bone metastases are invasive and frequently fail to yield sufficient tissue for further study. Evaluation of circulating tumor cells (CTCs) offers an alternative, minimally invasive mechanism to characterize and study late-stage disease. The goal of this study was to evaluate the utility of CTC interrogation with respect to the AR as a potential novel therapeutic biomarker in patients with mCRPC. Methods Fifteen mL of whole blood was collected from patients with progressive, metastatic mCRPC, the mononuclear cell portion was isolated, and fluorescence-activated cell sorting (FACS) was used to isolate and evaluate CTCs. A novel protocol was optimized to use ImageStreamX to quantitatively analyze AR expression and subcellular localization within CTCs. Co-expression of AR and the proliferation marker Ki67 was also determined using ImageStreamX. Results We found inter-patient and intra-patient heterogeneity in expression and localization of AR. Increased AR expression and nuclear localization are associated with elevated co-expression of Ki-67, consistent with the continued role for AR in castration-resistant disease. Despite intra-patient heterogeneity, CTCs from patients with prior exposure to abiraterone had increased AR expression compared to CTCs from patients who were abiraterone-naïve. Conclusions As our toolbox for targeting AR function expands, our ability to evaluate AR expression and function within tumor samples from patients with late-stage disease will likely be a critical component of the personalized management of advanced prostate cancer. AR expression and nuclear localization varies within patients and between patients; however it remains associated with markers of proliferation. This supports a molecularly diverse AR-centric pathobiology imparting castration-resistance. Electronic supplementary material The online version of this article (doi:10.1186/s12967-014-0313-z) contains supplementary material, which is available to authorized users.

459 Background: Metastatic UC has a dismal prognosis, with no FDA-approved second-line therapies. Somatic mutations and copy number (CN) variation in EGFR (ErbB1), HER2 (ErbB2), and ErbB3 are frequent in UC and may represent viable therapeutic targets. We studied whether afatinib, an oral, irreversible Erb family blocker, has activity in UC. Methods: In this single arm, phase II trial, patients (pts) with unresectable platinum-refractory UC received afatinib 40 mg/day continuously until progression or intolerance.The primary endpoint was 3-mo progression-free survival (PFS3) using a Simon two-stage design, with the trial proceeding to stage II if ≥ 30% pts in stage I had PFS3. Pre-specified analysis for EGFR, HER2, ErbB3, and ErbB4 was conducted using targeted next-generation sequencing (Life Technologies) and CN analysis (Taqman) of available archival tumor tissue. Results: The initial enrollment goal of 23 ptswas met: 18 M/5 F, median age 67 (36-82), ECOG 0 in 26%/1 in 74%, Hb < 10 g/dl in 17%, liver metastases in 30%, median time from prior chemotherapy = 3.6 mo. No unexpected toxicities were observed; 2 pts required dose-reduction (grade 3 fatigue, grade 3 rash). 5/23 pts (21.7%) had PFS3 (1 partial response (PR), 4 stable disease). Notably, 5/7 pts (71.4%) with ErbB molecular alterations achieved PFS3 (PFS = 10.3, 7.0, 6.9, 4.6 (ongoing) and 3.9 (ongoing) mo respectively) while 0/16 without alterations reached PFS3 (p < 0.001, Fisher’s exact). The 2 pts with alterations (both HER2 amplified) who did not reach PFS3 had liver metastases. All 3 pts with ErbB3 somatic mutations were responders. One pt with both HER2 amplification and R103G ErbB3 mutation never progressed on therapy, but discontinued after 10.3 mo due to depressed left ventricular ejection fraction. Average time to progression/discontinuation was 4.9 mo in pts with molecular alterations vs 1.7 mo for pts without alterations (p = 0.03). Conclusions: Afatinib demonstrates significant activity in platinum-refractory UC pts with HER2 and/or ErbB3 alterations. The potential contribution of ErbB3 to afatinib sensitivity is novel. A follow-on phase II study of afatinib in marker-selected refractory UC pts is underway. Clinical trial information: NCT02122172.

Background: Muscle-invasive bladder cancer (MIBC), unlike the more favorable low-grade papillary form, has a dismal prognosis with a significant risk of recurrence after surgical resection. Previous whole-exome sequencing of MIBC tumors performed by our lab demonstrated that carriers of mutations in any of six DNA mismatch repair genes (ATM, ERCC2, FANCD2, PALB2, BRCA1 or BRCA2) not only had a high somatic mutation burden, but also had enhanced recurrence-free survival compared to non-carriers of these mutations. We hypothesize that a high somatic mutation burden may generate a higher number of tumor-specific antigens that induce clonal expansion of tumor-infiltrating T-lymphocytes (TILs) to reduce a risk of recurrence and improve the survival. To investigate this hypothesis, we have sequenced the TCR cDNAs of TILs. Methods: Tumor and adjacent normal tissue (verified by a pathologist) were collected from patients whose tumors had previously been whole-exome sequenced. Library preparation was performed using previously described methods. Next-generation sequencing of TCRs was conducted on the Illumina platform (San Diego, CA) and CDR3 sequences were identified using a published algorithm. The diversity index of the TCRα and TCRβ chains was calculated using Simpson's diversity index, an ecological formula for calculating biodiversity based on evenness and richness. CD4+, CD8+, and FOXP3 gene expression levels were also examined with TaqMan Gene Expression Assays (Thermo Fisher Scientific, Carlsbad, CA). Results: TCR sequencing of ten tumors (5 non-recurrent (NR) with at least two years follow-up, 5 recurrent) has been performed. The average TCRα DI of five recurrent patients is 97±56.5 (SEM) compared to 23± 12.7 for five NR patients. In addition, we found an average DI of 13.8±0.77 in tumors with a high somatic mutation burden (average number of mutations 221), while it was 71.4±36.8 for tumors with a low somatic mutation burden (avg mutations 31). In samples with a relatively high DI, the three most abundant TCR clones represent less than 15% of the total TCR population, while in samples with a low DI, the top three clones comprise 35-90% of the total population. Finally, the CD8+/FOXP3 ratios were 10.1±2.1(SEM) and 5.6±2.9 for tumors with a low DI and those with a high DI, respectively (low vs. high defined by the average). Conclusions: We have elicited a correlating pattern where MIBC tumors with a higher number of somatic mutations have a lower TCR DI, indicating an oligoclonal expansion. Furthermore, in patients with recurrence, the TCR DI is higher, suggesting a lack of anti-tumor immune responses and non-specific T-cell expansion. The higher CD8+/FOXP3 ratio observed in tumors with low DI suggests that the clonal expansion is of CD8+ cells in tumors, which have been shown to be associated with favorable prognosis. Citation Format: Noura Choudhury, Kai Lee Yap, Kazuma Kiyotani, Poh Yin Yew, Alexa Campanile, Tatjana Antic, Gary Steinberg, Jae-Hyun Park, Peter H. O'Donnell, Yusuke Nakamura. Tumor T-cell receptor (TCR) diversity elucidates the immune response to genetic alterations of muscle-invasive bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4899. doi:10.1158/1538-7445.AM2015-4899

e16112 Background: XL184 (cabozantinib) is a small molecule inhibitor of receptor tyrosine kinases including VEGFR-2, c-MET. Trials of XL184 have shown significant improvements in bone pain and bon...

312 Background: Platinum-refractory UC has a dismal prognosis, with no FDA-approved second-line therapies. Somatic mutations and copy number (CN) variation in EGFR (ErbB1), HER2 (ErbB2), and ErbB3 are frequent in UC and may represent viable targets for novel therapies. We aimed to investigate whether afatinib, an oral, irreversible Erb family blocker, has clinical benefit in metastatic UC. Methods: In this single arm, phase II trial, patients (pts) with unresectable platinum-refractory UC who received ≤1 chemotherapy in the metastatic setting received afatinib 40 mg/day continuously until disease progression or intolerable toxicity.Primary endpoint was 3-month progression-free survival (PFS3), with drug deemed promising if ≥42% pts had PFS3. Pre-specified analysis for EGFR, HER2, ErbB3, and ErbB4 was conducted using targeted next-generation sequencing (Life Technologies) and CN analysis (Taqman). Results: 15 ptshave enrolled: median age 66 (44-78), 11 M/4 F, ECOG PS 0 in 20%, PS 1 in 80%, Hb