Ilana Rebecca Garcia-Grossman

Prevalence and Co-Occurrence of Actionable Genomic Alterations in High-Grade Bladder Cancer

PURPOSE We sought to define the prevalence and co-occurrence of actionable genomic alterations in patients with high-grade bladder cancer to serve as a platform for therapeutic drug discovery. PATIENTS AND METHODS An integrative analysis of 97 high-grade bladder tumors was conducted to identify actionable drug targets, which are defined as genomic alterations that have been clinically validated in another cancer type (eg, BRAF mutation) or alterations for which a selective inhibitor of the target or pathway is under clinical investigation. DNA copy number alterations (CNAs) were defined by using array comparative genomic hybridization. Mutation profiling was performed by using both mass spectroscopy-based genotyping and Sanger sequencing. RESULTS Sixty-one percent of tumors harbored potentially actionable genomic alterations. A core pathway analysis of the integrated data set revealed a nonoverlapping pattern of mutations in the RTK-RAS-RAF and phosphoinositide 3-kinase/AKT/mammalian target of rapamycin pathways and regulators of G1-S cell cycle progression. Unsupervised clustering of CNAs defined two distinct classes of bladder tumors that differed in the degree of their CNA burden. Integration of mutation and copy number analyses revealed that mutations in TP53 and RB1 were significantly more common in tumors with a high CNA burden (P < .001 and P < .003, respectively). CONCLUSION High-grade bladder cancer possesses substantial genomic heterogeneity. The majority of tumors harbor potentially tractable genomic alterations that may predict for response to target-selective agents. Given the genomic diversity of bladder cancers, optimal development of target-specific agents will require pretreatment genomic characterization.

Phase II study of everolimus in metastatic urothelial cancer

No recent advances have been made in the treatment of patients with advanced bladder cancer and, to date, targeted therapies have not resulted in an improvement in outcome. The mammalian target of rapamycin pathway has been shown to be up‐regulated in bladder cancer and represents a rational target for therapeutic intervention. In the present phase II study of everolimus, one near‐complete response, one partial response and several minor responses suggest that everolimus possesses biological activity in a subset of patients with bladder cancer. To maximize benefit from targeted agents such as everolimus, the preselection of patients based on molecular phenotype is required.

Phase II Study of Gemcitabine, Carboplatin, and Bevacizumab in Patients With Advanced Unresectable or Metastatic Urothelial Cancer

PURPOSE Although gemcitabine and carboplatin (GCa) is a standard option for patients with advanced urothelial cancer (UC) who are ineligible for cisplatin, outcomes remain poor. This trial evaluated the efficacy and safety of bevacizumab with GCa in advanced UC. PATIENTS AND METHODS Patients with Karnofsky performance status of 60% to 70%, creatinine clearance less than 60 mL/min, visceral metastasis, or solitary kidney were eligible and received a lead-in dose of bevacizumab 10 mg/kg followed 2 weeks later by gemcitabine 1,000 mg/m(2) on days 1 and 8 and carboplatin at area under the [concentration-time] curve (AUC) 5.0 or 4.5 and bevacizumab 15 mg/kg on day 1 every 21 days for six cycles. Patients achieving at least stable disease (SD) continued bevacizumab 15 mg/kg every 21 days for 18 additional cycles. The study was powered to detect a 50% improvement in median progression-free survival (PFS) over a historical control. RESULTS Fifty-one patients, median age 67 years (range, 42 to 83 years), were enrolled onto the study and were evaluable for toxicity. Twenty (39%) experienced grade 3 to 4 toxicity, and 10 (20%) had thromboembolic events (deep venous thrombosis or pulmonary embolism). Four received one or fewer cycles leaving 47 evaluable for outcomes. Twenty-three (49%) achieved response (three complete; 20 partial), and 11 had SD. Median PFS was 6.5 months (95% CI, 4.7 to 7.8 months); PFS was greater in the carboplatin AUC 5.0 group (P = .04). Median overall survival (OS) was 13.9 months. CONCLUSION The 95% one-sided lower confidence bound of 4.77 months for median PFS did not meet the predesignated PFS of more than 4.8 months considered sufficient for further study. Median OS was greater than expected. An ongoing phase III trial in patients who are eligible for therapy with cisplatin will define the role of bevacizumab in UC.

The Safety and Efficacy of Single-Agent Pemetrexed in Platinum-Resistant Advanced Urothelial Carcinoma: A Large Single-Institution Experience

BACKGROUND Pemetrexed is a commonly used treatment for platinum-resistant advanced urothelial carcinoma (UC) based on objective response rates of 8% and 28% in two small phase II studies. To address the discrepancy in reported response rates and to assess efficacy and toxicity outside of a clinical trial setting, we performed a large retrospective analysis of pemetrexed use at Memorial Sloan Kettering Cancer Center. We also investigated candidate prognostic factors for overall survival in this setting to explore whether the neutrophil-lymphocyte ratio (NLR) had independent prognostic significance. PATIENTS AND METHODS Patients receiving pemetrexed for platinum-resistant advanced UC between 2008 and 2013 were identified. The Response Evaluation Criteria in Solid Tumors (RECIST, version 1.1) were used to determine response rate. Kaplan-Meier and Cox regression analyses were used to examine the association of various factors with efficacy and survival outcomes. Hematologic toxicity and laboratory abnormalities were recorded. RESULTS One hundred and twenty-nine patients were treated with pemetrexed. The objective response rate was 5% (95% confidence interval: 1%-9%), and the median duration of response was 8 months. Median progression-free survival (PFS) was 2.4 months, and the 6-month PFS rate was 14%. There was no significant difference in response rate by age, Eastern Cooperative Oncology Group (ECOG) performance status, or number of prior therapies. On multivariable analysis, ECOG performance status (p < .01), liver metastases (p = .02), and NLR (p < .01) had independent prognostic significance for overall survival. CONCLUSION This 129-patient series is the largest reported data set describing pemetrexed use in advanced UC. Activity was modest, although discovery of molecular biomarkers predictive of response would be valuable to identify the small subset of patients who do gain significant benefit. Overall, the data highlight the urgent need to develop novel therapies for these patients.

Sunitinib in urothelial cancer: clinical, pharmacokinetic, and immunohistochemical study of predictors of response.

BACKGROUND Sunitinib has activity in patients with metastatic urothelial cancer (UC), but most patients do not respond. OBJECTIVE To identify predictors of response to sunitinib. DESIGN, SETTING, AND PARTICIPANTS Seventy-seven patients with advanced UC received sunitinib on one of two schedules at a single institution. Blood pressure (BP), immunohistochemistry (IHC), and pharmacokinetic (PK) results were correlated with response to sunitinib. MEASUREMENTS BP was assessed on day 1 and 28 of each cycle and on day 14 of cycle 1. IHC was performed on 55 samples from 38 cases using mammalian target of rapamycin and hypoxia-inducible factor (HIF) pathway marker antibodies. Blood samples for PK analysis were collected from 15 patients at three time points. Response was assessed using Response Evaluation Criteria in Solid Tumors criteria. RESULTS AND LIMITATIONS Sunitinib-induced hypertension predicted improved response when hypertension was categorized as a discrete (p = 0.02) or continuous variable (p = 0.005 [systolic BP] and p = 0.007 [diastolic BP]). The odds ratio of response was 12.5 (95% confidence interval, 1.95-246.8) for grade 3/4 hypertension compared with grade 0. Response was associated with low HIF-1α expression in primary (p = 0.07) tissue. A nonstatistically significant trend was seen for an association between greater drug concentration and best response. A correlation between expression markers within the same pathways was identified, phosphorylated-4EBP1 and phosphorylated-S6 (p = 6.5 × 10(-9)), and vascular endothelial growth factor receptor 2 and HIF-1α (p = 0.008). Results are limited by small numbers. CONCLUSIONS Clinical and molecular biomarkers of response to sunitinib may have clinical relevance and require prospective validation. There is an urgent need for predictive biomarkers to guide the management of UC.

Germline single nucleotide polymorphisms associated with response of urothelial carcinoma to platinum-based therapy: the role of the host.

BACKGROUND Variations in urothelial carcinoma (UC) response to platinum chemotherapy are common and frequently attributed to genetic and epigenetic variations of somatic DNA. We hypothesized that variations in germline DNA may contribute to UC chemosensitivity. PATIENTS AND METHODS DNA from 210 UC patients treated with platinum-based chemotherapy was genotyped for 80 single nucleotide polymorphisms (SNPs). Logistic regression was used to examine the association between SNPs and response, and a multivariable predictive model was created. Significant SNPs were combined to form a SNP score predicting response. Eleven UC cell lines were genotyped as validation. RESULTS Six SNPs were significantly associated with 101 complete or partial responses (48%). Four SNPs retained independence association and were incorporated into a response prediction model. Each additional risk allele was associated with a nearly 50% decrease in odds of response [odds ratio (OR) = 0.51, 95% confidence interval 0.39-0.65, P = 1.05 × 10(-7)). The bootstrap-adjusted area under the curves of this model was greater than clinical prognostic factors alone (0.78 versus 0.64). The SNP score showed a positive trend with chemosensitivity in cell lines (P = 0.115). CONCLUSIONS Genetic variants associated with response of UC to platinum-based therapy were identified in germline DNA. A model using these genetic variants may predict response to chemotherapy better than clinical factors alone.

Alterations in the PI3K/Akt signaling pathway and association with outcome in invasive high-grade urothelial cancer (UC).

277 Background: Activating mutations in PIK3CA have been associated with improved outcomes in patients (pts) with breast cancer (Kalinsky, Clin Cancer Res 2009). Molecular profiling of invasive high-grade UC demonstrates that PI3K/Akt pathway alterations occur in up to 15% of cases. The prognostic value of PI3K/Akt pathway alterations in invasive UC is unknown. METHODS Clinically annotated archival frozen surgical specimens from 95 pts (94 cystectomies, 1 nephroureterectomy) with high-grade invasive UC were genotyped for mutations in all coding exons of PIK3CA, PIK3R1, TSC1, PTEN and the AKT isoforms using high-throughput Sanger sequencing. Copy number alterations were examined using an Agilent 1M oligonucleotide array. Clinical variables including time to recurrence (TTR) and overall survival (OS) were correlated with the presence of mutations or copy number events in PIK3CA, PIK3R1, TSC1, PTEN and the AKT isoforms. RESULTS Specimens from 95 pts (71 M; 24 F) with a median age of 71 years (41-88) were evaluated. 34 (36%) received neoadjuvant chemotherapy and 75 (79%) were prior smokers. 4 pts (4%) had Stage 0, 11 (12%) had Stage I, 15 (16%) had Stage II, 33 (35%) had Stage III and 32 (34%) had Stage IV disease at surgery. The median follow-up was 31.5 months. Alterations (mutations or copy number gains/losses) in PI3K/Akt signaling pathway genes were identified in 26 (27%) specimens and were associated with a trend toward longer TTR, hazard ratio 0.53 (95%CI 0.24, 1.14) (p=0.08). CONCLUSIONS Alterations in PI3K/Akt signaling in pts with invasive UC may be associated with an improvement in outcome similar to that observed in breast cancer. Further analysis in a large independent tumor set is ongoing.

Clinical evaluation of cisplatin sensitivity germ line polymorphisms in neoadjuvant chemotherapy (NAC) for urothelial cancer (UC).

342 Background: Cisplatin-based NAC in UC confers a survival benefit, but prediction of which patients (pts) will benefit is not possible. We attempted to validate pharmacogenomic (PGx) markers of cisplatin sensitivity derived from a cell-based model and previously associated with response in a population of pts receiving NAC. Methods: Three germline single nucleotide polymorphisms (SNPs) were previously associated with pathologic response at cystectomy in a single-institution discovery set of 59 cT2 UC pts. These SNPs were tested for association with NAC response in a prospectively-identified, multi-institution, independent cohort. The primary analysis tested for association between rs244898 and rs7937567 and pathologic complete response (pT0). A replication set of 134 pts would provide 80% power to detect effects of both SNPs at p=0.05 independently using a multivariate logistic model. Results: N=146 pts with ≥cT2 N0 disease were identified from three institutions. All pts received ≥3 cycles of cisplati...

Prediction of pathologic complete response to neoadjuvant chemotherapy in urothelial cancer by genetic determinants of platinum sensitivity.

290 Background: Despite level 1 evidence demonstrating survival benefit for cisplatin-based neoadjuvant chemotherapy in urothelial cancer, its utilization remains low, likely due to the modest benefit and potentially significant toxicities. To improve selection of patients (pts) most likely to benefit, we evaluated the association of pharmacogenomic markers of cisplatin sensitivity with neoadjuvant therapy outcome. METHODS Pts receiving standard neoadjuvant cisplatin-based chemotherapy for muscle-invasive disease were recruited to provide a germline DNA sample. Nine single nucleotide polymorphisms (SNPs) selected for their associations with platinum sensitivity in various clinical and pre-clinical studies were tested for association with complete pathologic response (pT0) rate at cystectomy. RESULTS Sixty-three Caucasian pts were analyzed (median age=64; 67% male; all bladder primary and cN0). The overall pT0 rate was 27%; 52% were <pT2. Using a multivariate, recessive genetic model, rs244898 in RARS (odds ratio [OR] 6.8 [95% CI 1.6-32.6], P=0.01) and rs7937567 in GALNTL4 (OR 5.2 [95% CI 1.1-26.2], P=0.03) were associated with likelihood of achieving pT0. For each SNP, pts carrying the favorable genotype achieved pT0 in >58% of cases. Demonstrating the apparent independent nature of the two SNPs, pts carrying either favorable genotype had pT0 OR=8.5 (95% CI 2.5-31.8, P=0.0008). The combined effect of detecting both SNPs was most informative, as 2/3 pts with both favorable genotypes achieved pT0 (67%), compared to 9/15 pts with one favorable genotype (60%), and only 6/43 lacking both favorable genotypes (14%). The negative predictive value considering both SNPs was 86%. CONCLUSIONS Two germline SNPs having prior evidence of governing platinum sensitivity in pre-clinical and clinical models were demonstrated to confer strong associations with complete response to cisplatin-based neoadjuvant chemotherapy. Validation testing in an independent, multi-institutional cohort of urothelial cancer pts is underway. If reproducible, these SNPs deserve consideration as predictive clinical biomarkers to inform the use of neoadjuvant chemotherapy in this disease.

Association of single-nucleotide polymorphisms in BCL2L1 and TACC3 with response to bacillus Calmette-Guérin intravesical therapy in non-muscle-invasive bladder cancer.

260 Background: Bacillus Calmette-Guérin intravesical therapy (BCG) has an important role in the management of high risk non-muscle-invasive bladder cancer (NMIBC). This study examines the association between germline single nucleotide polymorphisms (SNPs) and response to BCG therapy. METHODS Saliva or blood was collected from pts with NMIBC treated at a single center and diagnosed between 1984 and 2010. SNPs were selected based on reported associations with bladder cancer and BCG response, and genotyped using the Sequenom MassARRAY iPLEX system. No response to BCG was defined as the presence at 6 months of pathologically documented tumor in the bladder. Univariate logistic regression was used to test the association between the outcome of interest (no response to BCG at 6 months) and clinical variables (stage, grade and multifocality), or individual SNPs. RESULTS The cohort consisted of 158 pts with a median age of 65 years who received intravesical BCG for NMIBC (35.2% stage T1, 32.7% stage Ta and 31.4% stage Tis) with 93% having high-grade disease. At 6 months follow up, 22 (13.9%) patients showed no response to BCG. We successfully genotyped 80 of the 88 selected SNPs. Of these, 2 SNPs were associated with lack of response to BCG; rs798766 is an intronic SNP in TACC3 (OR for no response is 2.4 for each T allele relative to CC genotype, P=0.01), and rs1994251 an intronic SNP in BCL2L1 (OR for no response is 3.2 for each C allele relative to AA, p= 0.0008). Both SNPs remained significantly associated with lack of response after adjusting for predictive clinical variables. CONCLUSIONS Single nucleotide polymorphisms of BCL2L1 and TACC3 may be predictive of BCG refractory bladder cancer. Future validation studies on independent datasets are needed to determine the clinical utility of these findings.