Garrett M. Dancik

A 20-gene model for molecular nodal staging of bladder cancer: development and prospective assessment

BACKGROUNDnNeoadjuvant chemotherapy before cystectomy confers a survival benefit in bladder cancer, but it has not been widely adopted since most patients do not benefit and we are at present unable to predict those that do. Since the most important predictor of recurrence after cystectomy is pathologically positive nodes, our aim was to assess techniques that define this stage for the selection of patients for neoadjuvant chemotherapy.nnnMETHODSnWe developed a gene expression model (GEM) to predict the pathological node status in primary tumour tissue from three independent cohorts of patients who were clinically node negative. From a subset of transcripts detected faithfully by microarrays from both paired frozen and formalin-fixed tissues (32 pairs), we developed both the GEM and cutoffs that identified patient strata with raised risk of nodal involvement by use of two separate training cohorts (90 and 66 patients). We then assessed the GEM and cutoffs to predict node-positive disease in tissues from a phase 3 trial cohort (AUO-AB-05/95; 185 patients).nnnFINDINGSnWe developed a 20-gene GEM with an area under the curve of 0·67 (95% CI 0·60-0·75) for prediction of nodal disease at cystectomy in AUO-AB-05/95. The cutoff system identified patients with high relative risk (1·74, 95% CI 1·03-2·93) and low relative risk (0·70, 95% CI 0·51-0·96) of node-positive disease. Multivariate logistic regression showed the GEM predictor was independent of age, sex, pathological stage, and lymphovascular space invasion (coefficient 9·81, 95% CI 1·64-18·00; p=0·019).nnnINTERPRETATIONnSelecting patients for neoadjuvant chemotherapy on the basis of risk of node-positive disease has the potential to benefit high-risk patients while sparing other patients toxic effects and delay to cystectomy.nnnFUNDINGnUS National Cancer Institute (R01CA143971).

Concurrent Alterations in TERT, KDM6A, and the BRCA Pathway in Bladder Cancer

Purpose: Genetic analysis of bladder cancer has revealed a number of frequently altered genes, including frequent alterations of the telomerase (TERT) gene promoter, although few altered genes have been functionally evaluated. Our objective is to characterize alterations observed by exome sequencing and sequencing of the TERT promoter, and to examine the functional relevance of histone lysine (K)–specific demethylase 6A (KDM6A/UTX), a frequently mutated histone demethylase, in bladder cancer. Experimental Design: We analyzed bladder cancer samples from 54 U.S. patients by exome and targeted sequencing and confirmed somatic variants using normal tissue from the same patient. We examined the biologic function of KDM6A using in vivo and in vitro assays. Results: We observed frequent somatic alterations in BRCA1 associated protein-1 (BAP1) in 15% of tumors, including deleterious alterations to the deubiquitinase active site and the nuclear localization signal. BAP1 mutations contribute to a high frequency of tumors with breast cancer (BRCA) DNA repair pathway alterations and were significantly associated with papillary histologic features in tumors. BAP1 and KDM6A mutations significantly co-occurred in tumors. Somatic variants altering the TERT promoter were found in 69% of tumors but were not correlated with alterations in other bladder cancer genes. We examined the function of KDM6A, altered in 24% of tumors, and show depletion in human bladder cancer cells, enhanced in vitro proliferation, in vivo tumor growth, and cell migration. Conclusions: This study is the first to identify frequent BAP1 and BRCA pathway alterations in bladder cancer, show TERT promoter alterations are independent of other bladder cancer gene alterations, and show KDM6A loss is a driver of the bladder cancer phenotype. Clin Cancer Res; 20(18); 4935–48. ©2014 AACR.

Biomarkers for prognosis and treatment selection in advanced bladder cancer patients.

Purpose of review Patients with locally ‘advanced’ or muscle invasive bladder cancer have higher mortality rates than patients with nonmuscle invasive (‘superficial’) bladder cancer. Biomarkers can stratify clinical outcomes and thus promise to more accurately prognosticate and thus help assign patients to the appropriate treatments. The aim of this review is to summarize biomarker developments in the past year and to discuss their implications in prognosis and treatment selection in locally advanced bladder cancer. Recent findings Prognostic biomarkers for bladder cancer are identified at the DNA, RNA and/or protein levels. Some are new markers, whereas others are established markers with new roles in bladder cancer. Markers can report on the risk of disease recurrence or metastasis, or treatment responsiveness and thus are useful in determining ‘who to treat’ and ‘what to treat with’. Summary The list of biomarkers for prognosis and treatment selection for advanced bladder cancer is growing. For most, their clinical relevance is unclear due to their lack of validation in external datasets. MicroRNAs and new techniques including next-generation sequencing offer additional opportunities for biomarker discovery, validation, and clinical applications.

A Framework to Select Clinically Relevant Cancer Cell Lines for Investigation by Establishing Their Molecular Similarity with Primary Human Cancers

Experimental work on human cancer cell lines often does not translate to the clinic. We posit that this is because some cells undergo changes in vitro that no longer make them representative of human tumors. Here, we describe a novel alignment method named Spearmans rank correlation classification method (SRCCM) that measures similarity between cancer cell lines and human tumors via gene expression profiles, for the purpose of selecting lines that are biologically relevant. To show utility, we used SRCCM to assess similarity of 36 bladder cancer lines with 10 epithelial human tumor types (N = 1,630 samples) and with bladder tumors of different stages and grades (N = 144 samples). Although 34 of 36 lines aligned to bladder tumors rather than other histologies, only 16 of 28 had SRCCM assigned grades identical to that of their original source tumors. To evaluate the clinical relevance of this approach, we show that gene expression profiles of aligned cell lines stratify survival in an independent cohort of 87 bladder patients (HR = 3.41, log-rank P = 0.0077) whereas unaligned cell lines using original tumor grades did not. We repeated this process on 22 colorectal cell lines and found that gene expression profiles of 17 lines aligning to colorectal tumors and selected based on their similarity with 55 human tumors stratified survival in an independent cohort of 177 colorectal cancer patients (HR = 2.35, log-rank P = 0.0019). By selecting cell lines that reflect human tumors, our technique promises to improve the clinical translation of laboratory investigations in cancer.

Pharmacogenomics in bladder cancer

Bladder cancer is a common cancer worldwide. For patients presenting with muscle-invasive disease, the 5-year survival rate is approximately 50%. Cisplatin-based combination chemotherapy is recommended in the neoadjuvant setting before cystectomy and is also the first line in the metastatic setting. However, the survival benefit of such therapy is modest. The identification of pharmacogenomic biomarkers would enable the rational and personalized treatment of patients by selecting those patients that would benefit most from such therapies sparing others the unnecessary toxicity. Conventional therapies would be recommended for an expected responder, whereas a nonresponder would be considered for alternative therapies selected on the basis of the individuals molecular profile. Although few effective bladder cancer therapies have been introduced in the past 30 years, several targeted therapies against the molecular drivers of bladder cancer appear promising. This review summarizes pharmacogenomic biomarkers that require further investigation or prospective evaluation or both, and publicly available tools for drug discovery and biomarker identification from in vitro data.

A 20 gene model for predicting nodal involvement in bladder cancer patients with muscle invasive tumors

Bladder cancer is the fourth most common cancer in males worldwide and also the most expensive cancer to treat. Approximately 25% of patients with muscle invasive disease are found to harbor occult lymph node involvement at the time of cystectomy and this finding is associated with a 5-year survival rate of <30%. If these patients could be identified pre-operatively, use of neoadjuvant chemotherapy may be advantageous because this approach has been shown to confer a small survival advantage in patients with muscle invasive disease. However, because only a few patients benefit from this approach it has not been used extensively in the United States with fewer than 2% of patients undergoing this treatment. This is largely due to concerns that since neoadjuvant therapy is beneficial for only a few patients, it has the potential to delay surgery in the majority who do not benefit. However, since neoadjuvant therapy is most likely to benefit those patients at highest risk for progression of disease, it follows that patients with lymph node metastases would constitute an ideal group for such treatment. Hence, if patients with occult node involvement prior to cystectomy could be identified, they would constitute an ideal group for application of neoadjuvant therapy as they are most likely to benefit. In this summary, we describe the first multi-analyte gene expression model developed for predicting occult nodal involvement at cystectomy in bladder cancer patients, for the purpose of making better informed decisions regarding neoadjuvant therapy. The 20 gene model, which was developed on Affymetrix Human Genome U133A and U133 Plus 2.0 arrays, identified individuals with high relative risk (RR) of nodal involvement (RR = 1.74, 95% CI, 1.03 – 2.93) intermediate risk (RR = 1.05, 95% CI, .45 – 2.41), and low risk (RR = 0.74, 95% CI, 0.51 – 0.96), when evaluated in an independent test dataset. The 20 gene model can be applied to formalin-fixed paraffin embedded tissue with sufficient tumor content, making implementation in routine diagnostic tissue highly feasible. Although a clinical assay for the gene panel has not undergone analytic validation in a clinical laboratory setting, multiple platforms are available which could be utilized for routine testing, including real-time reverse transcriptase PCR directed against individual analytes as well as microarray approaches.

Glycogen debranching enzyme (AGL) is a novel regulator of non-small cell lung cancer growth

Glycogen debranching enzyme (AGL) and Glycogen phosphorylase (PYG) are responsible for glycogen breakdown. We have earlier shown that AGL is a regulator of bladder tumor growth. Here we investigate the role of AGL in non-small cell lung cancers (NSCLC). Short hairpin RNA (shRNA) driven knockdown of AGL resulted in increased anchorage independent and xenograft growth of NSCLC cells. We further establish that an increase in hyaluronic acid (HA) synthesis driven by Hyaluronic Acid Synthase 2 (HAS2) is critical for anchorage independent growth of NSCLC cells with AGL loss. Using gene knockdown approach against HAS2 and by using 4-methylumbelliferone (4MU), an inhibitor of HA synthesis, we show that HA synthesis is critical for growth of NSCLC cells that have lost AGL. We further show NSCLC cells without AGL expression are dependent on RHAMM for HA signaling and growth. Analysis of NSCLC patient datasets established that patients with low AGL/high HAS2 or low AGL/high RHAMM mRNA expression have poor overall survival compared to patients with high AGL/low HAS2 or high AGL/low RHAMM expression. We show for the first time that loss of AGL promotes anchorage independent growth of NSCLC cells. We further show that HAS2 driven HA synthesis and signaling via RHAMM is critical in regulating growth of these cancer cells with AGL loss. Further patients presenting with low AGL and HAS2 or RHAMM over expressing tumors might present the ideal cohort who would respond to inhibitors of HA synthesis and signaling.