Shizhen Zhang

Identification of Distinct Basal and Luminal Subtypes of Muscle-Invasive Bladder Cancer with Different Sensitivities to Frontline Chemotherapy

Muscle-invasive bladder cancers (MIBCs) are biologically heterogeneous and have widely variable clinical outcomes and responses to conventional chemotherapy. We discovered three molecular subtypes of MIBC that resembled established molecular subtypes of breast cancer. Basal MIBCs shared biomarkers with basal breast cancers and were characterized by p63 activation, squamous differentiation, and more aggressive disease at presentation. Luminal MIBCs contained features of active PPARγ and estrogen receptor transcription and were enriched with activating FGFR3 mutations and potential FGFR inhibitor sensitivity. p53-like MIBCs were consistently resistant to neoadjuvant methotrexate, vinblastine, doxorubicin and cisplatin chemotherapy, and all chemoresistant tumors adopted a p53-like phenotype after therapy. Our observations have important implications for prognostication, the future clinical development of targeted agents, and disease management with conventional chemotherapy.

Frequent truncating mutations of STAG2 in bladder cancer

Here we report the discovery of truncating mutations of the gene encoding the cohesin subunit STAG2, which regulates sister chromatid cohesion and segregation, in 36% of papillary non-invasive urothelial carcinomas and 16% of invasive urothelial carcinomas of the bladder. Our studies suggest that STAG2 has a role in controlling chromosome number but not the proliferation of bladder cancer cells. These findings identify STAG2 as one of the most commonly mutated genes in bladder cancer.

Meta-Analysis of the Luminal and Basal Subtypes of Bladder Cancer and the Identification of Signature Immunohistochemical Markers for Clinical Use

Background It has been suggested that bladder cancer can be divided into two molecular subtypes referred to as luminal and basal with distinct clinical behaviors and sensitivities to chemotherapy. We aimed to validate these subtypes in several clinical cohorts and identify signature immunohistochemical markers that would permit simple and cost-effective classification of the disease in primary care centers. Methods We analyzed genomic expression profiles of bladder cancer in three cohorts of fresh frozen tumor samples: MD Anderson (n = 132), Lund (n = 308), and The Cancer Genome Atlas (TCGA) (n = 408) to validate the expression signatures of luminal and basal subtypes and relate them to clinical follow-up data. We also used an MD Anderson cohort of archival bladder tumor samples (n = 89) and a parallel tissue microarray to identify immunohistochemical markers that permitted the molecular classification of bladder cancer. Findings Bladder cancers could be assigned to two candidate intrinsic molecular subtypes referred to here as luminal and basal in all of the datasets analyzed. Luminal tumors were characterized by the expression signature similar to the intermediate/superficial layers of normal urothelium. They showed the upregulation of PPARγ target genes and the enrichment for FGFR3, ELF3, CDKN1A, and TSC1 mutations. In addition, luminal tumors were characterized by the overexpression of E-Cadherin, HER2/3, Rab-25, and Src. Basal tumors showed the expression signature similar to the basal layer of normal urothelium. They showed the upregulation of p63 target genes, the enrichment for TP53 and RB1 mutations, and overexpression of CD49, Cyclin B1, and EGFR. Survival analyses showed that the muscle-invasive basal bladder cancers were more aggressive when compared to luminal cancers. The immunohistochemical expressions of only two markers, luminal (GATA3) and basal (KRT5/6), were sufficient to identify the molecular subtypes of bladder cancer with over 90% accuracy. Interpretation The molecular subtypes of bladder cancer have distinct clinical behaviors and sensitivities to chemotherapy, and a simple two-marker immunohistochemical classifier can be used for prognostic and therapeutic stratification. Funding U.S. National Cancer Institute and National Institute of Health.

Gene Expression Profile of the Clinically Aggressive Micropapillary Variant of Bladder Cancer.

BACKGROUND Progression of conventional urothelial carcinoma of the bladder to a tumor with unique microscopic features referred to as micropapillary carcinoma is coupled with aggressive clinical behavior signified by a high propensity for metastasis to regional lymph nodes and distant organs resulting in shorter survival. OBJECTIVE To analyze the expression profile of micropapillary cancer and define its molecular features relevant to clinical behavior. DESIGN, SETTING, AND PARTICIPANTS We retrospectively identified 43 patients with micropapillary bladder cancers and a reference set of 89 patients with conventional urothelial carcinomas and performed whole-genome expression messenger RNA profiling. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS The tumors were segregated into distinct groups according to hierarchical clustering analyses. They were also classified according to luminal, p53-like, and basal categories using a previously described algorithm. We applied Ingenuity Pathway Analysis software (Qiagen, Redwood City, CA, USA) and gene set enrichment analysis for pathway analyses. Cox proportional hazards models and Kaplan-Meier methods were used to assess the relationship between survival and molecular subtypes. The expression profile of micropapillary cancer was validated for selected markers by immunohistochemistry on parallel tissue microarrays. RESULTS AND LIMITATIONS We show that the striking features of micropapillary cancer are downregulation of miR-296 and activation of chromatin-remodeling complex RUVBL1. In contrast to conventional urothelial carcinomas that based on their expression can be equally divided into luminal and basal subtypes, micropapillary cancer is almost exclusively luminal, displaying enrichment of active peroxisome proliferator-activated receptor γ and suppression of p63 target genes. As with conventional luminal urothelial carcinomas, a subset of micropapillary cancers exhibit activation of wild-type p53 downstream genes and represent the most aggressive molecular subtype of the disease with the shortest survival. The involvement of miR-296 and RUVBL1 in the development of micropapillary bladder cancer was identified by the analyses of correlative associations of genome expression profiles and requires mechanistic validation. CONCLUSIONS Micropapillary cancer evolves through the luminal pathway and is characterized by the activation of miR-296 and RUVBL1 target genes. PATIENT SUMMARY Our observations have important implications for prognosis and for possible future development of more effective therapies for micropapillary bladder cancer.

Aurora Kinase A is a Biomarker for Bladder Cancer Detection and Contributes to its Aggressive Behavior

The effects of AURKA overexpression associated with poor clinical outcomes have been attributed to increased cell cycle progression and the development of genomic instability with aneuploidy. We used RNA interference to examine the effects of AURKA overexpression in human bladder cancer cells. Knockdown had minimal effects on cell proliferation but blocked tumor cell invasion. Whole genome mRNA expression profiling identified nicotinamide N-methyltransferase (NNMT) as a downstream target that was repressed by AURKA. Chromatin immunoprecipitation and NNMT promoter luciferase assays revealed that AURKA’s effects on NNMT were caused by PAX3-mediated transcriptional repression and overexpression of NNMT blocked tumor cell invasion in vitro. Overexpression of AURKA and activation of its downstream pathway was enriched in the basal subtype in primary human tumors and was associated with poor clinical outcomes. We also show that the FISH test for the AURKA gene copy number in urine yielded a specificity of 79.7% (95% confidence interval [CI] = 74.2% to 84.1%), and a sensitivity of 79.6% (95% CI = 74.2% to 84.1%) with an AUC of 0.901 (95% CI = 0.872 to 0.928; P < 0.001). These results implicate AURKA as an effective biomarker for bladder cancer detection as well as therapeutic target especially for its basal type.

Dysregulation of EMT Drives the Progression to Clinically Aggressive Sarcomatoid Bladder Cancer

The sarcomatoid variant of urothelial bladder cancer (SARC) displays a high propensity for distant metastasis and is associated with short survival. We report a comprehensive genomic analysis of 28 cases of SARCs and 84 cases of conventional urothelial carcinomas (UCs), with the TCGA cohort of 408 muscle-invasive bladder cancers serving as the reference. SARCs showed a distinct mutational landscape with enrichment of TP53, RB1, and PIK3CA mutations. They were related to the basal molecular subtype of conventional UCs and could be divided into epithelial/basal and more clinically aggressive mesenchymal subsets based on TP63 and its target genes expression levels. Other analyses revealed that SARCs are driven by downregulation of homotypic adherence genes and dysregulation of cell cycle and EMT networks, and nearly half exhibited a heavily infiltrated immune phenotype. Our observations have important implications for prognostication and the development of more effective therapies for this highly lethal variant of bladder cancer.

Detection of Bladder Cancer in Urine Sediments by a Novel Multicolor Fluorescence in Situ Hybridization (Quartet) Test

BACKGROUND Bladder cancer is among the common human malignancies that show a heavy mutational load and copy number variations of numerous chromosomes, which makes them a target for diagnostic explorations. OBJECTIVE We aimed to design a multicolor fluorescence in situ hybridization (FISH) test referred to as the quartet test for the detection of bladder cancer in urine. DESIGN, SETTING, AND PARTICIPANTS We performed genome-wide copy number variation analysis on cohorts from the University of Texas MD Anderson Cancer Center (n=40) and The Cancer Genome Atlas (n=129), and identified the most frequently amplified chromosomal regions. These data were used to select four of the amplified regions to design a multicolor FISH test, referred to as the quartet test. Assay validation was performed on urine samples from 98 patients with bladder cancer: 56 with low-grade papillary, 42 with high-grade invasive disease, and 48 benign controls. INTERVENTION The quartet test can be used in clinical practice for noninvasive detection of bladder cancer. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS We initially analyzed samples using a fraction of abnormal cell scores and then by the quantitative score, which included not only the proportion of cells with abnormal copy numbers, but also the proportion of cells with numbers of altered copies and degree of amplification. We used receiver operator characteristic (ROC) curves to identify cutoff values for the scores at which performances of sensitivity and specificity were maximized. RESULTS AND LIMITATIONS The copy number status assessed by probes detected in voided urine reflected the amplification status of the primary tumor. An ROC curve summarizing the proportion of assayed cells with any abnormal copy numbers gave specificity of 93.8% and sensitivity of 78.6% using the proportion of cells with abnormal copy numbers. The quantitative score giving extra weight to cells with multiple simultaneous amplifications provided 95.8% specificity and 76.8% sensitivity. Both percentage of abnormal cells and quantitative scores were highly effective for assessing the grade of the tumor. The full spectrum of potential clinical applications was not explored in the current study, and further validation studies are needed. CONCLUSIONS The quartet test shows promising specificity and sensitivity results, but it requires validation on a larger multi-institutional cohort of samples. PATIENT SUMMARY The quartet test can be used for noninvasive detection of bladder cancer in voided urine. It can also be used to assess the grade of the tumor and tumor recurrence as well as post-treatment effects.

Abstract 4809: Validation of molecular subtypes of urothelial carcinoma of bladder in multi-institutional and public cohorts

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA BACKGROUND: Recently we have proposed a molecular sub-classification of conventional urothelial carcinoma which could be divided into two major categories designated as basal and luminal similar to the ones defined for breast cancer. In addition, luminal cancers contain a group of cases characterized by upregulation of p53 regulatory genes and is referred to as p53-like. The purpose of this project is to verify whether such subtypes can be identified in several large independent cohorts of bladder cancers. DESIGN: Gene expression profiles and molecular subtyping were performed by using previously described analytical algorithms on the following bladder cancer patient cohorts: (1) The Cancer Genome Atlas (TCGA) cohort containing tumor tissues from muscle invasive high grade urothelial carcinomas of 129 patients; (2) the MD Anderson Cancer Center (MDA) cohort of fresh frozen tumor tissues which included 65 superficial papillary carcinomas and 77 invasive carcinomas; (3) the Swedish cohort containing fresh frozen tumor samples of 116 superficial papillary tumors and 192 invasive tumors. RESULTS: The three distinct molecular subtypes defined as luminal, p53-like and basal were identified in all three cohorts. The distribution of molecular subtypes in individual cohorts was as follows: the TCGA cohort contained 50 (39%) luminal, 30 (23%) p53-like and 49 (38%) basal tumors; the MDA cohort contained 54 (38%) luminal, 61 (43%) p53-like and 27 (19%) basal; the Swedish cohort contained 102 (33%) luminal, 129 (42%) p-53 like and 77 (25%) basal tumors. Interestingly, in the MDA and Swedish cohorts virtually all superficial tumors, of either low or high grade, were of luminal type. CONCLUSION: The conventional bladder cancer can be separated into three distinct molecular subtypes with luminal phenotype defining the vast majority of superficial bladder cancers while muscle invasive tumors can be separated into luminal and basal forms. A subset of luminal cancers is characterized by upregulation of p53 regulatory genes. The identification of subtypes may help to stratify the patients for targeted therapies. Citation Format: Vipulkumar Dadhania, Charles G. Guo, Tadeusz Majewski, Li Zhang, Jolanta Bondaruk, Shizhen Zhang, Woonyoung Choi, David McConkey, Colin Dinney, Keith Baggerly, Bogdan A. Czerniak. Validation of molecular subtypes of urothelial carcinoma of bladder in multi-institutional and public cohorts. [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 4809. doi:10.1158/1538-7445.AM2015-4809

Frequent truncating mutations of the cohesin complex gene STAG2 in urothelial carcinoma of the bladder.

290 Background: We have recently identified the cohesin complex subunit STAG2 as a gene that is somatically mutated in human cancer and whose inactivation leads directly to chromosomal instability and aneuploidy (Solomon et al, Science, 2011 Aug 19). However, the complete tumor spectrum harboring STAG2 mutations and the clinical significance of STAG2 inactivation in cancer remain undefined. Methods: Immunohistochemistry was used to screen 2,214 tumors from each of the major tumor types for somatic loss of STAG2 expression. Sequencing of the STAG2 gene was performed on 111 urothelial carcinomas, and Affymetrix CytoscanHD Arrays were performed on STAG2 mutant tumors. Clinical data from 34 patients with non-muscle invasive urothelial carcinoma treated with transurethral resection and 349 patients with invasive urothelial carcinoma treated with radical cystectomy was correlated with tumor STAG2 status. Results: Complete loss of STAG2 expression was discovered in 52/295 urothelial carcinomas (18%), ranging fro...

Abstract 5256: Mechanism of field cancerization

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We developed a strategy that combines histologic and genetic mapping that permits interrogation of the chronology of genetic changes associated with cancer development on a whole-organ scale. By using this approach, we analyzed the sequence of genetic alterations contiguous to the tumor suppressor RB1 and identified a set of alternative target genes that we term “forerunner” (FR) genes whose silencing was associated with development of clonal plaque-like mucosal field effects initiating bladder carcinogenesis. Expression and methylation studies identified five candidate FR genes (ITM2B, LPAR6, MLNR, CAB39L, and ARL11). In vitro mechanistic studies demonstrated that three of these genes (ITM2B, LPAR6 and ARL11) control cell survival and proliferation consistent with their loss of function being contributory to tumorigenesis, and in vivo genetic ablation of Lpar6 in a mouse model resulted in activation of basal/stem cell transcriptional programs involving STAT3 and NFκB causing proliferative expansion of the basal layer of the urothelium. Whole genome analyses of the field effects in a representative whole-organ map revealed a sequential accumulation of alterations in RHO/RAC/Cdc42 pathways that control invasion and cell motility. These studies identify FR genetic alterations in tissue fields as one of the earliest events in carcinogenesis and provide comprehensive description of field cancerization in a primary human tumor. The results have important implications for the development of novel detection markers and chemoprevention therapeutic strategies. Citation Format: Bogdan A. Czerniak, Sangkyou Lee, Jolanta Bondaruk, Sooyong Lee, Tadeusz Majewski, Shizhen Zhang, Li Shen, Yuexin Liu, Charles Guo, Colin Dinney, H. Barton Grossman, Wei Zhang, Menashe Bar-Eli, Keith Baggerly, Richard Behringer, David McConkey, David McConkey, Margaret Knowles, Woonbok Chung, Jean-Pierre Issa. Mechanism of field cancerization. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5256. doi:10.1158/1538-7445.AM2014-5256