Irina Ostrovnaya

Genome Sequencing Identifies a Basis for Everolimus Sensitivity

Tumor genome sequencing reveals the molecular basis of a patient’s unexpected and dramatic response to a cancer drug. Cancer drugs often induce dramatic responses in a small minority of patients. We used whole-genome sequencing to investigate the genetic basis of a durable remission of metastatic bladder cancer in a patient treated with everolimus, a drug that inhibits the mTOR (mammalian target of rapamycin) signaling pathway. Among the somatic mutations was a loss-of-function mutation in TSC1 (tuberous sclerosis complex 1), a regulator of mTOR pathway activation. Targeted sequencing revealed TSC1 mutations in about 8% of 109 additional bladder cancers examined, and TSC1 mutation correlated with everolimus sensitivity. These results demonstrate the feasibility of using whole-genome sequencing in the clinical setting to identify previously occult biomarkers of drug sensitivity that can aid in the identification of patients most likely to respond to targeted anticancer drugs.

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.

Adverse Outcomes in Clear Cell Renal Cell Carcinoma with Mutations of 3p21 Epigenetic Regulators BAP1 and SETD2 : A Report by MSKCC and the KIRC TCGA Research Network

Purpose: To investigate the impact of newly identified chromosome 3p21 epigenetic tumor suppressors PBRM1, SETD2, and BAP1 on cancer-specific survival (CSS) of 609 patients with clear cell renal cell carcinoma (ccRCC) from 2 distinct cohorts. Experimental Design: Select sequencing on 3p tumor suppressors of 188 patients who underwent resection of primary ccRCC at the Memorial Sloan-Kettering Cancer Center (MSKCC) was conducted to interrogate the genotype–phenotype associations. These findings were compared with analyses of the genomic and clinical dataset from our nonoverlapping The Cancer Genome Atlas (TCGA) cohort of 421 patients with primary ccRCC. Results: 3p21 tumor suppressors are frequently mutated in both the MSKCC (PBRM1, 30.3%; SETD2, 7.4%; BAP1, 6.4%) and the TCGA (PBRM1, 33.5%; SETD2, 11.6%; BAP1, 9.7%) cohorts. BAP1 mutations are associated with worse CSS in both cohorts [MSKCC, P = 0.002; HR 7.71; 95% confidence interval (CI)2.08–28.6; TCGA, P = 0.002; HR 2.21; 95% CI 1.35–3.63]. SETD2 are associated with worse CSS in the TCGA cohort (P = 0.036; HR 1.68; 95% CI 1.04–2.73). On the contrary, PBRM1 mutations, the second most common gene mutations of ccRCC, have no impact on CSS. Conclusion: The chromosome 3p21 locus harbors 3 frequently mutated ccRCC tumor suppressor genes. BAP1 and SETD2 mutations (6%–12%) are associated with worse CSS, suggesting their roles in disease progression. PBRM1 mutations (30%–34%) do not impact CSS, implicating its principal role in the tumor initiation. Future efforts should focus on therapeutic interventions and further clinical, pathologic, and molecular interrogation of this novel class of tumor suppressors. Clin Cancer Res; 19(12); 3259–67. ©2013 AACR.

Murine Anti-GD2 Monoclonal Antibody 3F8 Combined With Granulocyte-Macrophage Colony-Stimulating Factor and 13-Cis-Retinoic Acid in High-Risk Patients With Stage 4 Neuroblastoma in First Remission

PURPOSE Anti-GD2 monoclonal antibody (MoAb) combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) has shown efficacy against neuroblastoma (NB). Prognostic variables that could influence clinical outcome were explored. PATIENTS AND METHODS One hundred sixty-nine children diagnosed with stage 4 NB (1988 to 2008) were enrolled onto consecutive anti-GD2 murine MoAb 3F8 ± GM-CSF ± 13-cis-retinoic acid (CRA) protocols after achieving first remission (complete remission/very good partial remission). Patients enrolled in regimen A (n = 43 high-risk [HR] patients) received 3F8 alone; regimen B (n = 41 HR patients), 3F8 + intravenous GM-CSF + CRA, after stem-cell transplantation (SCT); and regimen C (n = 85), 3F8 + subcutaneous GM-CSF + CRA, 46 of 85 after SCT, whereas 28 of 85 required additional induction therapy and were deemed ultra high risk (UHR). Marrow minimal residual disease (MRD) was measured by quantitative reverse transcription polymerase chain reaction. Survival probability was calculated by the Kaplan-Meier method, and prognostic variables were analyzed by multivariate Cox regression model. RESULTS At 5 years from the start of immunotherapy, progression-free survival (PFS) improved from 44% for HR patients receiving regimen A to 56% and 62% for those receiving regimens B and C, respectively. Overall survival (OS) was 49%, 61%, and 81%, respectively. PFS and OS of UHR patients were 36% and 75%, respectively. Relapse was mostly at isolated sites. Independent adverse prognostic factors included UHR (PFS) and post-cycle two MRD (PFS and OS), whereas the prognostic factors for improved outcome were missing killer immunoglobulin-like receptor ligand (PFS and OS), human antimouse antibody response (OS), and regimen C (OS). CONCLUSION Retrospective analysis of consecutive trials from a single center demonstrated that MoAb 3F8 + GM-CSF + CRA is effective against chemotherapy-resistant marrow MRD. Its positive impact on long-term survival can only be confirmed definitively by randomized studies.

Radiogenomics of Clear Cell Renal Cell Carcinoma: Associations between CT Imaging Features and Mutations

PURPOSE To investigate associations between computed tomographic (CT) features of clear cell renal cell carcinoma (RCC) and mutations in VHL, PBRM1, SETD2, KDM5C, or BAP1 genes. MATERIALS AND METHODS The institutional review board approved this retrospective, hypothesis-generating study of 233 patients with clear cell RCC and waived the informed consent requirement. The study was HIPAA compliant. Three radiologists independently reviewed pretreatment CT images of all clear cell RCCs without knowledge of their genomic profile. One radiologist measured largest diameter and enhancement parameters of each clear cell RCC. Associations between CT features and mutations in VHL, PBRM1, SETD2, KDM5C, and BAP1 genes were tested by using the Fisher exact test. Associations between mutations and size and enhancement were assessed by using the independent t test. Interreader agreement was calculated by using the Fleiss κ. RESULTS Mutation frequencies among clear cell RCCs were as follows: VHL, 53.2% (124 of 233); PBRM1, 28.8% (67 of 233); SETD2, 7.3% (17 of 233); KDM5C, 6.9% (16 of 233); and BAP1, 6.0% (14 of 233). Mutations of VHL were significantly associated with well-defined tumor margins (P = .013), nodular tumor enhancement (P = .021), and gross appearance of intratumoral vascularity (P = .018). Mutations of KDM5C and BAP1 were significantly associated with evidence of renal vein invasion (P = .022 and .046, respectively). The genotype of solid clear cell RCC differed significantly from the genotype of multicystic clear cell RCC. While mutations of SETD2, KDM5C, and BAP1 were absent in multicystic clear cell RCC, mutations of VHL (P = .016) and PBRM1 (P = .017) were significantly more common among solid clear cell RCC. Interreader agreement for CT feature assessments ranged from substantial to excellent (κ = 0.791-0.912). CONCLUSION This preliminary radiogenomics analysis of clear cell RCC revealed associations between CT features and underlying mutations that warrant further investigation and validation.

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.

Genomic and Mutational Profiling to Assess Clonal Relationships Between Multiple Non–Small Cell Lung Cancers

Purpose: In cases of multiple non–small cell lung cancer, clinicians must decide whether patients have independent tumors or metastases and tailor treatment accordingly. Decisions are currently made using the Martini and Melamed criteria, which are mostly based on tumor location and histologic type. New genomic tools could improve the ability to assess tumor clonality. Experimental Design: We obtained fresh-frozen tumors specimens from patients who underwent surgery on at least two occasions for presumptively independent NSCLC. We did array comparative genomic hybridization (aCGH), mutational profiling of select genes, and detailed clinicopathologic review. Results: We analyzed a total of 42 tumors from 20 patients (6 patients with synchronous tumors, 14 patients with metachronous tumors, 24 potential tumor pair comparisons); 22 tumor pairs were evaluable by aCGH. Surprisingly, classification based on genomic profiling contradicted the clinicopathologic diagnosis in four (18%) of the comparisons, identifying independent primaries in one case diagnosed as metastasis and metastases in three cases diagnosed as independent primaries. Matching somatic point mutations were observed in these latter three cases. Another four tumor pairings were assigned an “equivocal” result based on aCGH; however, matching somatic point mutations were also found in these tumor pairs. None of the tumor pairs deemed independent primaries by aCGH harbored matching mutations. Conclusion: Genomic analysis can help distinguish clonal tumors from independent primaries. The development of rapid, inexpensive, and reliable molecular tools may allow for refinement of clinicopathologic criteria currently used in this setting. (Clin Cancer Res 2009;15(16):5184–90)

Synthetic Lethality in ATM-Deficient RAD50-Mutant Tumors Underlies Outlier Response to Cancer Therapy

UNLABELLED Metastatic solid tumors are almost invariably fatal. Patients with disseminated small-cell cancers have a particularly unfavorable prognosis, with most succumbing to their disease within two years. Here, we report on the genetic and functional analysis of an outlier curative response of a patient with metastatic small-cell cancer to combined checkpoint kinase 1 (CHK1) inhibition and DNA-damaging chemotherapy. Whole-genome sequencing revealed a clonal hemizygous mutation in the Mre11 complex gene RAD50 that attenuated ATM signaling which in the context of CHK1 inhibition contributed, via synthetic lethality, to extreme sensitivity to irinotecan. As Mre11 mutations occur in a diversity of human tumors, the results suggest a tumor-specific combination therapy strategy in which checkpoint inhibition in combination with DNA-damaging chemotherapy is synthetically lethal in tumor cells but not normal cells with somatic mutations that impair Mre11 complex function. SIGNIFICANCE Strategies to effect deep and lasting responses to cancer therapy in patients with metastatic disease have remained difficult to attain, especially in early-phase clinical trials. Here, we present an in-depth genomic and functional genetic analysis identifying RAD50 hypomorphism as a contributing factor to a curative response to systemic combination therapy in a patient with recurrent, metastatic small-cell cancer.

Genomic Predictors of Survival in Patients with High-grade Urothelial Carcinoma of the Bladder

UNLABELLED Urothelial carcinoma of the bladder (UCB) is genomically heterogeneous, with frequent alterations in genes regulating chromatin state, cell cycle control, and receptor kinase signaling. To identify prognostic genomic markers in high-grade UCB, we used capture-based massively parallel sequencing to analyze 109 tumors. Mutations were detected in 240 genes, with 23 genes mutated in ≥5% of cases. The presence of a recurrent phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutation was associated with improved recurrence-free survival (RFS) (hazard ratio [HR]: 0.35; p=0.014) and improved cancer-specific survival (CSS) (HR: 0.35; p=0.040) in patients treated with radical cystectomy (RC). In multivariable analyses controlling for pT and pN stages, PIK3CA mutation remained associated with RFS (HR: 0.39; p=0.032). The most frequent alteration, TP53 mutation (57%), was more common in extravesical disease (69% vs 32%, p=0.005) and lymph node-positive disease (77% vs 56%, p=0.025). Patients with cyclin-dependent kinase inhibitor 2A (CDKN2A)-altered tumors experienced worse RFS (HR: 5.76; p<0.001) and worse CSS (HR: 2.94; p=0.029) in multivariable analyses. Mutations in chromatin-modifying genes were highly prevalent but not associated with outcomes. In UCB patients treated with RC, PIK3CA mutations are associated with favorable outcomes, whereas TP53 and CDKN2A alterations are associated with poor outcomes. Genomic profiling may aid in the identification of UCB patients at highest risk following RC. PATIENT SUMMARY Using next-generation sequencing, we identified genomic subsets of high-grade urothelial bladder cancer associated with favorable and unfavorable outcomes. These findings may aid in the selection of patients most likely to benefit from novel combined modality approaches.

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.