Jordan Kardos

Impact of Molecular Subtypes in Muscle-invasive Bladder Cancer on Predicting Response and Survival after Neoadjuvant Chemotherapy

BACKGROUND An early report on the molecular subtyping of muscle-invasive bladder cancer (MIBC) by gene expression suggested that response to neoadjuvant chemotherapy (NAC) varies by subtype. OBJECTIVE To investigate the ability of molecular subtypes to predict pathological downstaging and survival after NAC. DESIGN, SETTING, AND PARTICIPANTS Whole transcriptome profiling was performed on pre-NAC transurethral resection specimens from 343 patients with MIBC. Samples were classified according to four published molecular subtyping methods. We developed a single-sample genomic subtyping classifier (GSC) to predict consensus subtypes (claudin-low, basal, luminal-infiltrated and luminal) with highest clinical impact in the context of NAC. Overall survival (OS) according to subtype was analyzed and compared with OS in 476 non-NAC cases (published datasets). INTERVENTION Gene expression analysis was used to assign subtypes. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Receiver-operating characteristics were used to determine the accuracy of GSC. The effect of GSC on survival was estimated by Cox proportional hazard regression models. RESULTS AND LIMITATIONS The models generated subtype calls in expected ratios with high concordance across subtyping methods. GSC was able to predict four consensus molecular subtypes with high accuracy (73%), and clinical significance of the predicted consensus subtypes could be validated in independent NAC and non-NAC datasets. Luminal tumors had the best OS with and without NAC. Claudin-low tumors were associated with poor OS irrespective of treatment regimen. Basal tumors showed the most improvement in OS with NAC compared with surgery alone. The main limitations of our study are its retrospective design and comparison across datasets. CONCLUSIONS Molecular subtyping may have an impact on patient benefit to NAC. If validated in additional studies, our results suggest that patients with basal tumors should be prioritized for NAC. We discovered the first single-sample classifier to subtype MIBC, which may be suitable for integration into routine clinical practice. PATIENT SUMMARY Different molecular subtypes can be identified in muscle-invasive bladder cancer. Although cisplatin-based neoadjuvant chemotherapy improves patient outcomes, we identified that the benefit is highest in patients with basal tumors. Our newly discovered classifier can identify these molecular subtypes in a single patient and could be integrated into routine clinical practice after further validation.

Claudin-low bladder tumors are immune infiltrated and actively immune suppressed

We report the discovery of a claudin-low molecular subtype of high-grade bladder cancer that shares characteristics with the homonymous subtype of breast cancer. Claudin-low bladder tumors were enriched for multiple genetic features including increased rates of RB1, EP300, and NCOR1 mutations; increased frequency of EGFR amplification; decreased rates of FGFR3, ELF3, and KDM6A mutations; and decreased frequency of PPARG amplification. While claudin-low tumors showed the highest expression of immune gene signatures, they also demonstrated gene expression patterns consistent with those observed in active immunosuppression. This did not appear to be due to differences in predicted neoantigen burden, but rather was associated with broad upregulation of cytokine and chemokine levels from low PPARG activity, allowing unopposed NFKB activity. Taken together, these results define a molecular subtype of bladder cancer with distinct molecular features and an immunologic profile that would, in theory, be primed for immunotherapeutic response.

MYC activation cooperates with Vhl and Ink4a/Arf loss to induce clear cell renal cell carcinoma

Renal carcinoma is a common and aggressive malignancy whose histopathogenesis is incompletely understood and that is largely resistant to cytotoxic chemotherapy. We present two mouse models of kidney cancer that recapitulate the genomic alterations found in human papillary (pRCC) and clear cell RCC (ccRCC), the most common RCC subtypes. MYC activation results in highly penetrant pRCC tumours (MYC), while MYC activation, when combined with Vhl and Cdkn2a (Ink4a/Arf) deletion (VIM), produce kidney tumours that approximate human ccRCC. RNAseq of the mouse tumours demonstrate that MYC tumours resemble Type 2 pRCC, which are known to harbour MYC activation. Furthermore, VIM tumours more closely simulate human ccRCC. Based on their high penetrance, short latency, and histologic fidelity, these models of papillary and clear cell RCC should be significant contributions to the field of kidney cancer research.

Intrinsic Genomic Differences Between African American and White Patients With Clear Cell Renal Cell Carcinoma.

Importance There are well-documented racial disparities in outcomes for African American patients with clear cell renal cell carcinoma (ccRCC). Despite a dramatic improvement in overall survival in white patients since the advent of targeted therapy, survival for African Americans with advanced ccRCC has not changed. There is little known about potential racial differences in tumor biology of ccRCC. Objective To determine if there are racial differences in the somatic mutation rate and gene expression of ccRCC tumors from white and African American patients. Design, Setting, and Participants Overall, 438 patients with ccRCC were identified through The Cancer Genome Atlas (TCGA) clear cell kidney (KIRC) dataset (419 white and 19 African American patients). The GSE25540 dataset containing 135 patients (125 white and 10 African American patients) was used for validation. Tumor samples were collected from numerous cancer centers and were examined for racial differences in somatic mutation rates and RNA expression. Racial differences in somatic mutation rates and RNA expression were examined. Main Outcomes and Measures The comparison of somatic mutation rates and differences in RNA expression in white and African American patients with ccRCC. Results Overall, 419 ccRCC tumor data sets from non-Hispanic white patients and 19 from non-Hispanic African American patients were identified through the publically available TCGA KIRC data set, and a validation set of 125 white and 10 African American ccRCC patient tumors was identified from the publicly available GSE25540 data set. African American patients were significantly less likely than white patients to have VHL mutations (2 of 12 [17%] vs 175 of 351 [50%], respectively; P = .04) and were enriched in the ccB molecular subtype (79% in African American vs 45% in white patients ; P = .005), a molecular subtype that carries a worse prognosis. It was found that RNA expression analysis revealed relative down-regulation of hypoxia-inducible factor (HIF) and vascular endothelial growth factor (VEGF)-associated pathways in African American patients compared with white patients. Conclusions and Relevance African American patients have less frequent VHL inactivation, are enriched in the ccB molecular subtype, and have decreased up-regulation of HIF-associated gene signatures than white patients. These genomic differences would predict decreased responsiveness to VEGF-targeted therapy and are a biologically plausible contributing factor to the worse survival of African American patients with ccRCC, even in the targeted therapy era.

Molecular subtype-specific immunocompetent models of high-grade urothelial carcinoma reveal differential neoantigen expression and response to immunotherapy

High-grade urothelial cancer contains intrinsic molecular subtypes that exhibit differences in underlying tumor biology and can be divided into luminal-like and basal-like subtypes. We describe here the first subtype-specific murine models of bladder cancer and show that Upk3a-CreERT2; Trp53L/L; PtenL/L; Rosa26LSL-Luc (UPPL, luminal-like) and BBN (basal-like) tumors are more faithful to human bladder cancer than the widely used MB49 cells. Following engraftment into immunocompetent C57BL/6 mice, BBN tumors were more responsive to PD-1 inhibition than UPPL tumors. Responding tumors within the BBN model showed differences in immune microenvironment composition, including increased ratios of CD8+:CD4+ and memory:regulatory T cells. Finally, we predicted and confirmed immunogenicity of tumor neoantigens in each model. These UPPL and BBN models will be a valuable resource for future studies examining bladder cancer biology and immunotherapy.Significance: This work establishes human-relevant mouse models of bladder cancer. Cancer Res; 78(14); 3954-68. ©2018 AACR.

Abstract 1654: Development of subtype specific mouse models of bladder cancer

Introduction: High-grade, muscle-invasive bladder cancer has recently been shown to harbor intrinsic molecular subtypes with distinct biologic features. Current murine models of bladder cancer, including the prominent carcinogen induced model MB49, do not account for subtype specific characteristics, leaving a gap in available tools for understanding subtype specific differences in bladder cancer. We have developed and validated immunocompetent, subtype specific models of bladder cancer, and we have used these models to assess differential responses to immune checkpoint inhibition. Methods: Two distinct models of murine bladder cancer were developed in a C57BL/6 background. The UPPL models were generated through Pten/Trp53 conditional knockout in Uroplakin3a expressing cells. BBN models were generated through exposure of wild-type C57BL/6 mice to the carcinogen N-Butyl-N-(4-hydmoxybutyl)nitrosamine and subsequent generation of cell lines from spontaneous tumors. RNAseq was performed on several BBN and UPPL tumors and cell lines, with findings validated with flow cytometry and T/B cell receptor (TCR/BCR) amplicon sequencing of tumor infiltrating lymphocytes (TILs). Results: BBN and UPPL models reflected characteristics of human basal and luminal bladder cancers, respectively. BBN (basal) models demonstrated higher immune gene signature expression, with concordantly higher numbers of TILs compared to the UPPL (luminal) model (p Discussion: We have developed two unique classes of murine bladder cancer lines, UPPL and BBN, with gene expression and TIL profiles that closely correlate with human luminal and basal bladder cancers, respectively. The BBN and UPPL subtype specific models can serve as a tool for elucidating bladder cancer responses to immunotherapy. The mixed response of BBN963 tumors to PD-1 blockade should be an asset for assessing pathways mediating response to checkpoint blockade as well as the value of combination therapy. [C.S., R.S, B.V, W.K contributed equally to this work] Citation Format: Christof C. Smith, Ryoichi Saito, Lisa M. Bixby, Takanobu Utsumi, Jordan Kardos, Shengjie Chai, Sara E. Wobker, Bhavani Krishnan, Jeffrey S. Damrauer, Jonathan S. Serody, David Darr, Benjamin G. Vincent, William Y. Kim. Development of subtype specific mouse models of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1654. doi:10.1158/1538-7445.AM2017-1654

The Cancer/Testes (CT) Antigen HORMAD1 promotes Homologous Recombinational DNA Repair and Radioresistance in Lung adenocarcinoma cells

The Cancer/Testes (CT) Antigen HORMAD1 is germ cell-restricted and plays developmental roles in generation and processing of meiotic DNA Double Strand Breaks (DSB). Many tumors aberrantly overexpress HORMAD1 yet the potential impact of this CT antigen on cancer biology is unclear. We tested a potential role of HORMAD1 in genome maintenance in lung adenocarcinoma cells. We show that HORMAD1 re-distributes to nuclear foci and co-localizes with the DSB marker γH2AX in response to ionizing radiation (IR) and chemotherapeutic agents. The HORMA domain and C-term disordered oligomerization motif are necessary for localization of HORMAD1 to IR-induced foci (IRIF). HORMAD1-depleted cells are sensitive to IR and camptothecin. In reporter assays, Homologous Recombination (HR)-mediated repair of targeted ISce1-induced DSBs is attenuated in HORMAD1-depleted cells. In Non-Homologous End Joining (NHEJ) reporter assays, HORMAD1-depletion does not affect repair of ISce1-induced DSB. Early DSB signaling events (including ATM phosphorylation and formation of γH2AX, 53BP1 and NBS1 foci) are intact in HORMAD1-depleted cells. However, generation of RPA-ssDNA foci and redistribution of RAD51 to DSB are compromised in HORMAD1-depleted cells, suggesting that HORMAD1 promotes DSB resection. HORMAD1-mediated HR is a neomorphic activity that is independent of its meiotic partners (including HORMAD2 and CCDC36. Bioinformatic analysis of TCGA data show that similar to known HR pathway genes HORMAD1 is overexpressed in lung adenocarcinomas. Overexpression of HR genes is associated with specific mutational profiles (including copy number variation). Taken together, we identify HORMAD1-dependent DSB repair as a new mechanism of radioresistance and a probable determinant of mutability in lung adenocarcinoma.

Phase II trial of palbociclib in patients with metastatic urothelial cancer after failure of first-line chemotherapy

BackgroundThe majority of urothelial cancers (UC) harbor alterations in retinoblastoma (Rb) pathway genes that can lead to loss of Rb tumour suppressor function. Palbociclib is an oral, selective inhibitor of CDK 4/6 that restores Rb function and promotes cell cycle arrest.MethodsIn this phase II trial, patients with metastatic platinum-refractory UC molecularly selected for p16 loss and intact Rb by tumour immunohistochemistry received palbociclib 125 mg p.o. daily for 21 days of a 28-day cycle. Primary endpoint was progression-free survival at 4 months (PFS4) using a Simon’s two-stage design. Next-generation sequencing including Rb pathway alterations was conducted.ResultsTwelve patients were enrolled and two patients (17%) achieved PFS4 with insufficient activity to advance to stage 2. No responses were seen. Median PFS was 1.9 months (95% CI 1.8–3.7 months) and median overall survival was 6.3 months (95% CI 2.2–12.6 months). Fifty-eight percent of patients had grade ≥3 hematologic toxicity. There were no CDKN2A alterations found and no correlation of Rb pathway alterations with clinical outcome.ConclusionsPalbociclib did not demonstrate meaningful activity in selected patients with platinum-refractory metastatic UC. Further development of palbociclib should only be considered with improved integral biomarker selection or in rational combination with other therapies.

MP88-20 ESTABLISHMENT OF NOVEL MOUSE BLADDER CANCER CELL LINES MIMICKING INTRINSIC SUBTYPE OF HUMAN INVASIVE BLADDER CANCER

experiments such as Western blotting analysis(WB), Hoechst33342 staining and immunostaining. The evaluation of targeting KLF4 by miR145 was performed by luciferase assay. Moreover, the effects of knockdown of KLF4 on the various phenotypes of BC cells were also examined. Furthermore, the networks involving KLF4/PTBP1/PKMs in the Warburg effect relatedmolecules were examined by WB even in clinical BC samples. Finally, we examined immunohistochemical staining to evaluate KLF4 expression. RESULTS: The expression levels of miR-145 were significantly down-regulated in clinical BC samples and BC cells compared with those in normal tissues and HUC. Luciferase assay showed that miR-145 directly targeted KLF4. Also, the Warburg related affectgenes were modulated by the transfection of miR-145 or siR-KLF4 in BC cells. Moreover, the expression levels of KLF4, PTBP1, and PKM2 is up-regulated in all clinical samples by WB. Finally, we observed KLF4-positive staining in the tumor by immunohistochemical staining. CONCLUSIONS: In this study, we indicated that miR-145 affect the Warburg effect through targeting KLF4/PTBP1/PKMs axis in BC cells, which exhibited a significant cell growth inhibition.

MP34-01 MOLECULAR SUBTYPES OF MUSCLE INVASIVE BLADDER CANCER ARE RELATED TO BENEFIT FROM NEOADJUVANT CHEMOTHERAPY: DEVELOPMENT OF A SINGLE SAMPLE PATIENT ASSAY

Roland Seiler*, Hussam Al Deen Ashab, Nicholas Erho, Vancouver, Canada; Bas W. G. van Rhijn, Amsterdam, Netherlands; Brian Winters, Seattle, WA; James Douglas, Hampshire, United Kingdom; Kim Van Kessel, Rotterdam, Netherlands; Elisabeth E. Fransen van de Putte, Amsterdam, Netherlands; Matthew Sommerlad, Hampshire, United Kingdom; Qiqi Wang, Voleak Choeurng, Ewan A. Gibb, Beatrix PalmerAronsten, Lucia L. Lam, Christine Buerki, Elai Davicioni, Vancouver, Canada; Gottfrid Sj€ odahl, Malmo, Sweden; Jordan Kardos, Katherine A. Hoadley, Chapel Hill, NC; Seth P. Lerner, David J. McConkey, Woonyoung Choi, Houston, TX; William Y. Kim, Chapel Hill, NC; Bernhard Kiss, George N. Thalmann, Bern, Switzerland; Tilman Todenh€ ofer, Vancouver, Canada; Simon J. Crabb, Hampshire, United Kingdom; Scott North, Edmonton, Canada; Ellen C. Zwarthoff, Joost L. Boormans, Rotterdam, Netherlands; Jonathan Wright, Seattle, WA; Marc Dall’Era, Sacramento, CA; Michiel S. van der Heijden, Amsterdam, Netherlands; Peter C. Black, Vancouver, Canada