Molecular characterization of neuroendocrine-like bladder ... · ABSTRACT (250 max) Purpose: Neuroendocrine (NE) bladder carcinoma is a rare and aggressive variant. Molecular subtyping

Molecular characterization of neuroendocrine-like bladder cancer José Batista da Costa1, Ewan A. Gibb2, Trinity J. Bivalacqua3, Yang Liu2, Htoo Zarni Oo1, David T. Miyamoto4, Mohammed Alshalalfa2, Elai Davicioni2, Jonathan Wright5, Marc A Dall’Era6, James Douglas7, Joost L. Boormans8, Michiel S. Van der Heijden9, Chin-Lee Wu10, Bas W.G. van Rhijn11, Shilpa Gupta12, Petros Grivas13, Kent W. Mouw14, Paari Murugan15, Ladan Fazli1, Seong Ra16, Badrinath R. Konety17, Roland Seiler18, Siamak Daneshmand19, Omar Y. Mian20, Jason A. Efstathiou4, Yair Lotan21 and Peter C. Black1*

1. Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada 2. GenomeDx Inc., Vancouver, British Columbia, Canada 3. Department of Urology, Johns Hopkins Medical Institution, Baltimore, USA 4. Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, USA 5. Department of Urology, University of Washington School of Medicine, Seattle, Washington, USA 6. Department of Urology, UC Davis Comprehensive Cancer Center, Sacramento, CA 95817, USA 7. Department of Urology, University Hospital of Southampton, Hampshire, UK 8. Department of Urology, Erasmus MC Cancer Institute, Erasmus Medical Center, Rotterdam, The Netherlands 9. Department of Medical Oncology, Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital, Amsterdam,

Netherlands 10. Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, USA 11. Department of Surgical Oncology (Urology), Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital,

Amsterdam, Netherlands 12. Hematology, Oncology and Transplantation Division, University of Minnesota, Minnesota, USA 13. Department of Oncology, University of Washington, Seattle, Washington, USA 14. Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, USA 15. Department of Laboratory Medicine and Pathology, University of Minnesota, Minnesota, USA 16. San Diego Pathologist Medical Group, San Diego, USA 17. Department of Urology, University of Minnesota, Minnesota, USA 18. Department of Urology, University of Bern, Bern, Switzerland 19. Urology Department, University of Southern California, Los Angeles, California, USA 20. Department of Translational Hematology and Oncology Research, Cleveland Clinic, Cleveland, Ohio, USA 21. Department of Urology, University of Texas Southwestern Medical Center, Dallas, Texas

Keywords: Muscle-invasive bladder cancer, neuroendocrine, small cell bladder cancer, molecular subtypes, gene expression analysis Running title: Characterizing neuroendocrine-like bladder cancer Additional information: *Corresponding Author

Peter C. Black MD, FACS, FRCSC Vancouver Prostate Centre University of British Columbia Level 6, 2775 Laurel St Vancouver, BC V5Z 1M9 Canada Tel: (604) 875-4301 Email: [email protected] Conflict of interest: Four authors (Ewan Gibb, Yang Liu, Mohammed Alshalalfa, Elai Davicioni) are employees of GenomeDx Biosciences, which funded the original gene expression analysis of the patient tissue and assisted with data analysis. The remaining authors have no direct or indirect commercial financial incentive associated with publishing the article.

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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 5, 2019; DOI: 10.1158/1078-0432.CCR-18-3558

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Acknowledgements: José Batista da Costa’s salary was partially funded by the Association Française d'Urologie (AFU). We are grateful for the insightful comments provided by A. Gordon Robertson on this manuscript. Manuscript word count: 4471, Table: 5, Figures: 5, Abstract word count: 372 Translational relevance: (91 words) Neuroendocrine bladder cancer is an aggressive variant of bladder cancer with significant metastatic potential and high risk of mortality. Diagnosis of these tumors is currently dependent on morphological criteria and staining for neuroendocrine markers. Using machine learning and multiple validation cohorts in different clinical settings, we developed a model that identifies tumors with transcriptomic profiles consistent with NE bladder cancers with an absence of NE features by morphological criteria. Early and accurate identification of these patients by genomic analysis may improve outcomes through treatment intensification and adaptation of standard treatment regimens.




ABSTRACT (250 max) Purpose: Neuroendocrine (NE) bladder carcinoma is a rare and aggressive variant. Molecular subtyping studies have found that 5-15% of muscle-invasive bladder cancer (MIBC) have transcriptomic patterns consistent with NE bladder cancer in the absence of NE histology. The clinical implications of this NE-like subtype have not been explored in depth. Experimental design: Transcriptome-wide expression profiles were generated for MIBC collected from seven institutions and clinical-use of Decipher Bladder. Using unsupervised clustering, we generated a clustering solution on a prospective training cohort (PTC; n=175), developed single-sample classifiers to predict NE tumors, and evaluated the resultant models on a testing radical cystectomy (RC) cohort (n=225). A random forest model was finalized and applied to five validation cohorts (n=1302). Uni- and multivariable survival analyses were used to characterize clinical outcomes. Results: In the training cohort (PTC), hierarchical clustering using an 84 gene panel showed a cluster of eight patients (4.6%) with highly heterogeneous expression of NE markers in the absence of basal or luminal marker expression. NE-like tumors were identified in 1% to 7.1% of cases in validation cohorts. Patients with NE-like tumors had significantly worse 1-year progression free survival (65% NE-like vs 82% overall; p=0.046) and, after adjusting for clinical and pathological factors, had a 6.4-fold increased risk of all-cause mortality (p=0.001). IHC confirmed the neuronal character of these tumors. Conclusion: A single patient classifier was developed that identifies patients with histological urothelial cancer harboring a NE transcriptomic profile. These tumors represent a high-risk subgroup of MIBC which may require different treatment.




INTRODUCTION Neuroendocrine (NE) bladder carcinoma is a rare variant of muscle-invasive bladder cancer (MIBC) representing between 0.5% to 1.2% of newly diagnosed cases.1–4 These tumors can be classified into small cell (SC) and large cell subtypes based on morphology.4 Both are very aggressive tumors, characterized by rapid progression with high metastatic potential. Patients with NE carcinoma have worse outcomes compared to conventional urothelial carcinoma (UC), with median survival ranging between 9-20 months after curative intent in the non-metastatic setting. 2,3,5–7 Molecular subtyping has revealed that MIBC can be coarsely divided into basal-like and luminal-like subtypes, although additional subtypes have been described in more granular models. 8–10 Previously, The Cancer Genome Atlas (TCGA) and Lund groups have independently identified NE-like tumors in 5-15% of patients with predominantly histological UC.11,12 Consistent with their small cell / NE biology, these patients were found to have inferior outcomes.11,13 Importantly, the majority of these NE-like cases had morphologic characteristics of conventional UC associated with a NE genotype, although both cohorts included a small number of histological NE tumors. Given the high rate of occult metastases and the poor survival associated with SC/NE tumors, neoadjuvant chemotherapy followed by either radical cystectomy or radiation therapy has become the standard of care in non-metastatic cases.14–17 The chemotherapy regimen is extrapolated from experience with small cell carcinoma of the lung and includes cisplatin and etoposide.18 However, given the rarity of NE carcinoma of the bladder, there is little direct evidence to define a standardized therapeutic strategy. The best treatment of patients with tumors that have the appearance of histological UC, but NE-like gene expression profiles, has yet to be defined. These patients may be at higher risk of progression and stand to gain significantly from treatment intensification or alternative therapies. In this study, we used machine learning to develop a single-sample gene expression classifier designed to identify tumors with a NE-like transcriptomic signature in the absence histological features of NE carcinoma. MATERIALS AND METHODS Patient populations The prospective training cohort (PTC) consisted of the de-identified and anonymized gene expression profiles of 175 MIBC patients from the clinical use of the Decipher Bladder transurethral bladder resection test that were available in the Decipher GRID registry (NCT02609269). Pathological TNM staging as well as clinical outcome data were not available for this cohort (Table 1). The radical cystectomy cohort 1 (RC1) used for model testing consisted of 225 patients who underwent radical cystectomy without any neoadjuvant therapy and had pathologic pT1 disease with nodal involvement or MIBC.19 We have specifically used only tumors with conventional urothelial histology to develop the NE-like classifier, since the objective is to identify NE-like gene expression in tumors lacking true NE histology. The radical cystectomy cohort 2 (RC2) consisted of 256 consecutive patients with pT2-4N0-3M0/pT1N1-3M0 urothelial carcinoma of the bladder who underwent radical cystectomy with extended pelvic lymphadenectomy at Vancouver General Hospital (118 cases from 2005 to 2013), UT Southwestern (94 cases from 2007 to 2013) or Johns Hopkins University (44 cases from 1994 to 2002)In both cohorts, radical cystectomy tissue was analyzed. The TMT cohort consisted of 136 patients with cT2-T4aN0M0 UC who were treated with TMT at the Massachusetts General Hospital from 1986 to 2013. Overall 475 patients were treated in this



https://clinicaltrials.gov/ct2/show/NCT02609269


time period, but only 136 had adequate TURBT tissue available for analysis. Patient selection criteria for TMT and the standard treatment regimen at this institution have been reported previsouly20. The NAC cohort consisted of 223 patients from seven institutions who received induction or neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC) for cT2-4aN0-3M0 UC.21 In both cohorts, pre-treatment TURBT tissue was analyzed. The true SC/NE cohort consisted of five tumors collected at the Vancouver Prostate Centre, two clinical-use cases and one collected at the University of Minnesota. The retrospective patient cohorts (RC1, RC2, NAC, TMT) were designed to include only cases lacking any component of variant histology other than mixed squamous or glandular differentiation based on the original pathology report at the treating institution. However, the NAC cohort contained two patients with a minor component of micropapillary carcinoma who were included in the analysis. A central pathology review was not conducted. All tumors from the PTC cohort were predominantly conventional UC, and all tissue was reviewed to ensure that only conventional UC was sampled. However, a minor portion of variant histology elsewhere in the tumor cannot be excluded. There were four cases in the prospective cohort with a secondary neuroendocrine variant reported at the source institution. The human ethics board of each institution approved this study and all patients provided written consent to analysis of their tumor tissues when required by each instutiion. The studies were conducted in accordance with the ethical guidelines of the Declaration of Helsinki. Array files for profiled bladder tumors are available under GEO accession codes GSE128701 and 128702 (http://www.ncbi.nlm.nih.gov/geo/). Transcriptome profiling: Whole transcriptome analysis was performed on formalin-fixed, paraffin-embedded tumor tissue with GeneChip1 Human Exon 1.0 ST Array (Affymetrix) in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory (GenomeDx Inc, San Diego, CA).22 Microarray data were normalized and genes summarized using single-channel array normalization.23 Datasets from the public domain The TCGA RNA-seq data was previously analyzed as part of the TCGA consortium and was available for 408 patients with MIBC treated with RC in the absence of NAC11. The Lund microarray data was available for 307 patients with NMIBC and MIBC.13 The Cleveland Clinic SC / NE cohort utilized the HTG EdgeSeq Oncology Biomarker Panel Assay (HTG Molecular Diagnostics, Tucson AZ) and included 39 pathologic and IHC confirmed small cell / NE cases, one metastatic tumor and six matched normal.24 Expression data from primary bladder tumors (n=195) in IMvigor210, a phase II trial investigating the efficacy of the PD-L1 inhibitor atezolizumab in the second line metastastic setting, was downloaded .25,26 Discovery and validation of the NE-like Classifier: The prospective cohort was used for initial model training. Genes with high variance (S.D.>0.15 and bimodal distribution) were selected by comparing the Bayesian information criterion (BIC) of a two-component to a one-component Gaussian mixture model.27,28 Hierarchal clustering was used to cluster genes and sequentially remove clusters of genes without clear biological functions in UC. The selection of genes was based on overlap with TCGA 2017, gene enrichment analysis, literature review and expert opinion. This resulted in a list of 72 genes corresponding to four biological categories: luminal (21 genes), basal (16 genes), immune (17 genes), and stromal (18 genes). As tumors with SC/NE features are typically rare in most cohorts, neuronal genes were not initially selected by our algorithms. As such, we




manually added 12 neuronal genes curated from the TCGA11 (8 features; MS1, PLEKHG4B, GNG4, PEG10, RND2, APLP1, SOX2, TUBB2B) and the literature (4 features; CHGB, ENO2, SV2A, SYP). All neuronal genes, including those used to highlight the neuronal subtype in the TCGA, were carefully reviewed for additional literature support and an association with neuroendocrine tumors. There was a total of 84 genes on the final gene list (Table S1). The final set of 84 genes and consensus clustering was used to generate a clustering solution with eight unique clusters.29 One of these eight clusters had high neuronal gene expression, but was depleted in all other genes, including genes associated with basal and luminal tumors. The clustering labels were further adjusted by PAMR (R-package) to ensure the tightness of each cluster. A random forest classification model with 2000 trees was then trained to predict the cluster labels. For this particular study, we focused exclusively on evaluating the NE cluster. The performance of this classifier was first evaluated in the testing cohort (RC1) where it was shown to be better than other classifier choices, such as PAMR or logistic regression.30 The finalized model was then locked and evaluated on a set of seven true SC/NE cases and in four independent validation cohorts of conventional UC. Additionally, in order to test the performance of the model a cohort of true small cell/NE tumors was used.

Model porting to RNA-seq platforms

The NE model was ported to the RNA-seq platform (TCGA) using quantile normalization and imputation. For the 82 out 84 genes shared by the RNA-seq and microarray platforms, their expression values from RNA-seq were normalized by quantile-quantile matching with the expression values in our training cohort (PTC, n=175) as implemented in R package preprocess Core)31. For the two genes not captured by the RNA-seq platform, linear regression models with lasso penalty were discovered on the training cohort and applied to the quantile normalized RNA-seq data to impute their expression values. IHC staining and antibodies: Immunohistochemistry (IHC) was performed on a subset of 10 NE-like tumors from the TMT (n=7) and RC2 (n=3) cohorts, and 5 true retrospective NE tumors. A tissue microarray (TMA) was created from 8 tumors (3 NE-like and 5 true NE tumors) and individual sections were used for the other 7 tumors (TMT cohort) in which not enough tissue was available for a TMA. The positive control was neuroendocrine prostate cancer and the negative control was benign kidney. Immunohistochemistry was performed with a panel of antibodies against 6 classical NE markers (CgA, SYP, CD56, PEG10, SOX2, ENO2; Table S2). All stained slides were digitalized with the SL801 autoloader and Leica SCN400 scanning system (Leica Microsystems; Concord, Ontario, Canada) at magnification equivalent to 20X. The images were subsequently stored in the SlidePath digital imaging hub (DIH; Leica Microsystems) of the Vancouver Prostate Centre. Values on a four-point scale were assigned to each image. Descriptively, 0 represents no staining, 1 represents low, but detectable degree of staining, 2 represents clearly positive cases, and 3 represents strong expression. IHC was quantified for staining intensity (0-3). Drug response score: Using in vitro drug sensitivity and microarray data from the NCI-60 panel, we generated gene signatures predicting tumor sensitivity to four drugs used in the treatment of bladder cancer, including cisplatin, etoposide, gemcitabine and ifosamide.32 For each drug, we used the CellMiner tool33 to identify drug response related genes correlated to the IC50 value of the NCI-60 cell lines. The most significantly correlated genes were selected and the expression of the corresponding genes in the prospective cohort were extracted for drug response score (DRS)




calculations. A patient specific DRS was calculated using these correlation coefficients (Cor) as weighting factors of the corresponding gene expression normalized by the sum of Cor. Statistical analysis: The Kaplan-Meier method was used for estimates of overall survival. Overall survival was defined as the moment of first diagnosis of MIBC till death. Patients who were lost to follow-up were censored at the date of last contact. The differences between the overall survival curves of the NE-like patients compared to all other patients was tested using the log-rank test. Multi-variable Cox proportional hazard models were used to demonstrate the effect of NE-like subtype on overall survival adjusting common clinical variables, including age, sex and stage. All model training and statistical analyses were performed in R (R Core Team (2018). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/). RESULTS Development of a single-sample classifier to identify neuroendocrine-like tumors To develop the NE classifier, we leveraged both prospective and retrospective MIBC genome-wide expression data sets for model training and testing, respectively (Figure 1). The clinical and pathological features of the patient cohorts are described in Table 1. The initial model training used a set of 84 genes that were selected from the intersection of literature-curated, MIBC-associated genes (Table S1) and the MIBC subtyping model described by the TCGA11 to identify NE-like tumors. In the prospective training cohort (PTC) of 175 patients with phenotypical UC, we identified 8 (4.2%) NE-like tumors with gene expression profiles consistent with histological small cell / NE tumors (Figure S1). These patients showed robust, but heterogeneous, expression of various neuronal markers (i.e. NESTIN, TUBB2B, PEG10). Conversely, these patients showed low or no expression of either basal (i.e. KRT5/6, KRT14) or luminal (i.e. PPARG, KRT20) markers. While the NE-like model includes genes capturing immune and stromal infiltration, here we focused specifically on the characterization of NE-like tumors, using the established basal and luminal axis as a reference. We evaluated the classifier on a testing cohort of 225 patients who underwent radical cystectomy (RC1), classifying eight patients as NE-like. Histologically small cell tumors validate feature selection in NE-like classifier The model performance was evaluated on a set of 7 MIBC tumors with SC/NE histology (Figure S2) where 100% of the cases were identified as NE-like by the model, supporting the accuracy of the classifier. We also evaluated an independent single institution cohort of histologically confirmed NE tumors (n=39) and matched adjacent normal bladder tissue (n=6) profiled on a targeted RNA-seq platform.24 Despite incomplete feature overlap as a consequence of cross platform comparison, we found that a subset of NE markers was strongly upregulated relative to matched normal tissues, while conventional UC markers were downregulated (Figure S3). Furthermore, four patients in the PTC had a histologic component of NE tumor separate from the urothelial component that was sampled for transcriptomic profiling. Three of these four cases demonstrated NE-like gene expression. These data provide external supporting evidence of appropriate feature selection for the NE classifier. Neuroendocrine-like tumors are defined by unique biological characteristics




To further validate the NE-like classifier, we applied our model to three additional cohorts of patients with phenotypical UC who underwent different treatments, including a radical cystectomy only cohort (RC2, n=256), a neoadjuvant chemotherapy (plus radical cystectomy) cohort (NAC; n=223) and a trimodal therapy cohort (TMT; n=136). In all three cohorts, we identified NE-like tumors ranging in frequency from 1.3% to 7.9% (Table 1). Consistent with the observations in the training and testing sets, the NE-like tumors showed weak or no expression of basal or luminal markers, although the non-NE-like tumors in the same cohorts expressed these genes robustly. In contrast, the NE-like tumors robustly expressed a suite of neuronal markers, although there was heterogeneity in the expression of individual markers between tumors (Figure 2). In general, across all cohorts, the expression of these neuronal markers was much higher in the NE-like tumors, compared to tumors with basal- or luminal-like profiles (Figure 2). Deletion of CDKN2A and amplification of CCND1 are commonly found in conventional UC, while the inverse has been reported for phenotypic NE cancers.34–36 The NE-like cases in our study had profiles consistent with these latter reports, including higher expression of CDKN2A and lower expression of CCDN1 relative to basal or luminal cases (Figure S4). We next evaluated the performance of our NE-like classifier in the TCGA (n=408) and Lund (n=307) cohorts (Figure 3). In the TCGA cohort we identified 9/20 patients of the TCGA neuronal subtype to be NE-like (Figure 3A). The 11 cases not classified as NE by our model expressed lower levels of neuronal markers compared to the nine NE-like cases (Figure S5). In addition, 4 of these tumors strongly expressed either basal (n=3) or luminal markers (n=1). As the NE-like classifier was trained to exclude non-NE subtypes and enrich for robust neuronal marker expression, with only minimal basal and/or luminal marker expression, this was an expected observation and illustrates the stringency of the NE-like classifier. Despite efforts to exclude non-UC cases, three small cell bladder cases were included in the TCGA cohort. All three of these cases were identified as NE-like by our classifier. In the Lund cohort we identified 11/24 cases flagged as Sc/NE - Sc/NE-like by the LundTax model, with no overlap with the Sc/NE - GU class (Figure 3B). One patient was identified as NE-like by our model, but not by the LundTax model. As was observed for the TCGA cohort, the non-overlapping NE-like cases tended to have lower expression of neuronal genes and / or co-expressed basal or luminal markers. When comparing the NE-like calls to the LundTax IHC results, we find that 8/11 of the NE-like calls had strong staining for several of the profiled markers, including CD56 (NCAM1), SYP and TUBB2B (Table S3). However, when comparing to the LundTax tumor cell phenotype assessment of Sc/NE, our model identified 5/11 cases as NE-like (Table S3). In the IMvigor210 cohort we identified 2/195 cases as being NE-like. Consistent with the other validation cohorts, the gene expression profile of these two patients (Figure 3C) showed high neuronal gene expression with low basal and luminal gene expression. The identification of NE-like tumors with unique gene expression profiles (high neuronal genes, low basal and luminal genes) across six independent cohorts strongly supported the identification of a NE-like tumor subtype, independent of SC/NE histology. The clinical characteristics of all NE-like tumors in the testing cohort and six validation cohorts are summarized in Table S4. Confirmation of neuronal gene expression in NE-like tumors by IHC staining




To confirm the neuronal character of the NE-like cases at the protein level, we selected tumors classified as NE-like by our model in the retrospective cohorts that had tissue available for immunohistochemical (IHC) analysis (RC2: 3, TMT: 7). As these tumors were selected for retrospective studies exploring conventional UC, we did not expect to observe variant histology. Consistent with this expectation, these tumors were confirmed as conventional UC by histologic criteria, with no obvious SC or NE features identified. IHC staining was performed with a panel of antibodies against six classical NE markers (Chromogranin A (CgA), Synaptophsin (SYP), CD56, PEG10, SOX2 and ENO2; Figure S2). In Figure 4, we show a representative subset of tumors, including one true NE tumor and two NE-like tumors. The NE tumor (Figure 4A) had nests of small round malignant cells distributed evenly with “salt and pepper chromatin’, as well as the typical crush artefact often observed in these stroma-poor tumors. These histological characteristics were absent in the NE-like tumors, which instead show irregular clusters of larger tumor cells with nuclear pleomorphism invading deep into the wall. The true NE tumor had strong staining for most markers, while the NE-like cases were positive for a subset of neuronal markers, with reduced staining intensity. In a broader panel of tumors (Figure S2, panel B), some NE-like tumors had IHC profiles that were similar to true NE tumors, while others show more modest staining intensity for various NE markers. Like the transcript-level data, the neuronal marker staining was heterogeneous across the 10 NE-like patients, and no specific pattern observed. Neuroendocrine-like tumors have poor prognosis In general, patients with histological SC/NE tumors have aggressive disease and poor outcomes.2,3,5,14 To determine if NE-like tumors have a similarly poor prognosis, we performed both univariable and multivariable overall survival analysis stratified by NE-like status in all four retrospective cohorts. In the cohort of patients who underwent cystectomy without preceding chemotherapy (RC2), NE-like patients had significantly worse outcomes, with 1-year progression free survival of 65% compared to 82% in the rest of the cohort (p=0.046) (Figure 5B). We did not capture which adjuvant and salvage therapies these patients received. All eight NE-like patients had died within 30 months. NE-like classification correlated strongly with death on multivariable analysis adjusted for age, sex, T-stage and lymph node involvement (adjusted HR 6.03 (2.68-13.58, p<0.001)) (Table 2). There were only 3 NE-like patients in the NAC cohort; two were treated with gemcitabine/cisplatin (Gem/Cis) and one with methotrexate/vinblastine/adriamycin/cisplatin (MVAC). NE-like classification correlated with death (adjusted HR: 58.18 (10.94-309.28, p<0.001)) in this cohort (Table 3). Two patients died within two months of surgery (Figure 5C), including one elderly patient who died of metastatic progression shortly after surgery without any salvage therapy and one who died due to post-operative complications. In contrast to the cystectomy-only and NAC cohorts, the NE-like patients in the trimodal therapy setting showed no significant difference with respect to 1-year progression-free survival compared to patients with UC (Figure 5D). While in the other cohorts, all the NE-patients had died within 30 months of follow-up, in the TMT cohort (Table 4) 7/9 NE-like patients were still alive at 30 months (adjusted HR of 0.87 (0.35 – 2.15, p=0.458). The stage of NE-like tumors was comparable to the UC patients. In this cohort, two of nine patients with NE-like tumors underwent salvage cystectomy, compared to 30 of 127 with non-NE-like tumors. The outcomes for NE-like patients in the TCGA cohort, who also underwent cystectomy without neoadjuvant chemotherapy, were equally poor, with 1-year progression free survival rates of 57%, compared to 80% for the rest of the cohort (Figure 5E). None of the nine NE-like patients




in the TCGA cohort survived beyond 30 months of follow-up (adjusted HR 3.80 (1.76-8.23, p<0.001)) (Figure 5E, Table 5). In the IMvigor210 cohort (Figure 5F), the outcomes of the two NE-like patients was similar to the non-NE patients (p=0.931). Overall, the NE-like patients showed significantly worse outcomes in the surgical cohorts which is consistent with the poor prognostic features that have been described for pathologically diagnosed NE tumors. Predicting treatment response to chemotherapy for NE-like tumors Since conventional UC is usually treated with Gem/Cis or MVAC, but SC/NE bladder cancer is typically treated with cisplatin/etoposide (EP/PE)19, we sought to explore the potential drug sensitivities of the NE-like tumors. Using the CellMiner analysis tool that contains gene expression and IC50 data for the NCI-60 cancer cell lines treated with a panel of oncology drugs, we generated drug response scores (DRS) to establish a numerical metric for predicting sensitivity for each tumor. Applying this to our training (PTC) and retrospective cohorts (RC2, NAC, TMT) we observed that NE-like patients had higher predicted sensitivity scores to drugs relevant to MIBC treatment, including cisplatin, gemcitabine and etoposide when compared to basal and luminal patients (Figure S6). Consistent with reports of basal and luminal sensitivity to cisplatin, basal tumors were predicted to have higher sensitivity to cisplatin compared to luminal tumors. To confirm these results, we applied the DRS model to the TCGA and Lund datasets and observed similar patterns (Figure S7, S8).

DISCUSSION Transcriptome profiling of MIBC has revealed several unique molecular subtypes with distinct biological and clinical features. In two recent studies, a neuronal or Sc/NE subtype has been defined, having robust neuronal marker expression and poor outcomes.11,12 Notably, the majority of the tumors classified as NE-like did not have histological features consistent with this tumor type but were phenotypically similar to conventional UC. This suggests NE-like gene expression profiles, in conjunction with pathological examination, can provide additional information for identifying tumors with NE biology. Here, we present a single-sample NE-like classifier capable of identifying patients with NE-like biology regardless of their underlying pathological presentation. We applied this classifier to multiple unique patient cohorts and identified NE-like tumors in similar proportions in all cohorts. Our classifier proved to be more stringent than the TCGA and Lund subtyping models, categorizing 9/20 of the TCGA neuronal cases and 11/24 of the Lund Sc/NE cases as NE-like (Table S2). The NE classifier was constructed with a strict definition of NE, particularly in having robust expression of neuronal markers with minimal expression of basal and luminal markers. This was done to maximize high confidence calls to identify the tumors most likely to harbour NE-like biology. Moreover, porting a model from one platform (microarray) to another very different platform (RNA-seq) can introduce confounders which may impact the model performance and therefore these results should be applied judiciously. The NE-like tumors identified by our classifier demonstrated poor overall survival across cohorts, similar to the clinical outcomes of patients with pathologically confirmed NE tumors. The NE-like tumors in the TMT cohort were a noteworthy exception, as they had similar overall survival to conventional UC. Although the clinical and pathologic parameters of these patients were comparable to the NE-like patients in the surgical cohorts, selection of more favorable patients for TMT cannot be excluded. The NE-like patients in the TMT cohort received radiosensitizing chemotherapy, but not at a dose that would have been expected to affect




disease outside the field of radiation. The NAC cohort fared poorly despite receiving neoadjuvant chemotherapy, but this cohort contained only 3 NE-like patients, and one died of post-operative complications, so it is unclear what impact the addition of chemotherapy had to the better outcome in the TMT cohort. The NE-like patients in the NAC cohort also presented with more advanced stage. Such comparisons across different cohorts are problematic and are limited by the very small number of cases in each cohort. Since the primary risk of recurrence after loco-regional therapy for SC/NE carcinoma of the bladder is systemic, one could postulate that the same will be true for NE-like tumors. This would make systemic chemotherapy a particularly important component of the treatment algorithm, but loco-regional treatment with either TMT or RC is likely to lead to similar outcomes.37,38 Systemic chemotherapy in the form of cisplatin and etoposide is usually favored for patients with histologic NE tumors.15 Therefore, the question must now be posed whether NE-like tumors based on gene expression should receive the same treatment. Preliminary analysis using DRS metrics suggested that NE-like tumors are more likely to respond to cisplatin, gemcitabine and/or etoposide than conventional UC. However, predicted response does not necessarily translate into a durable response with survival benefit, and the DRS model did not suggest that cisplatin/etoposide would be superior to cisplatin/gemcitabine. Regardless, the relative rarity of this tumor entity will make it difficult to study this clinical question. The detection of tumors that resemble a specific histologic subtype by gene expression but are classified as conventional UC by morphologic criteria, suggests that morphology alone may be inadequate in identifying variant biology. It is well described in the pathology literature that identification of variant histologic subtypes is subjective and highly susceptible to inter-observer variability.39,40 While the NE-like gene expression pattern is particularly striking and therefore straightforward to identify in a larger patient cohort, it remains to be determined how many other variant histologic subtypes can also be readily identified by gene expression profiling. Early evidence suggests that the clinically aggressive micropapillary variant of bladder cancer has a unique gene expression profile driven by the activation of miR-296 and RUVBL1 target genes.19 Moreover, sarcomatoid transformation may be driven by the initiation of an EMT transcriptional program, potentially offering a signature to capture this variant as well.19,41 The different histologic subtypes of bladder cancer frequently occur together with conventional UC, and the usual dictum is that these subtypes share a common origin with the concurrent UC.34,35 It is therefore conceivable that the NE-like tumors with conventional urothelial morphology but a NE transcriptional program, represent an intermediate stage in the evolution towards a histologic NE phenotype. An analogous transdifferentiation pathway has been described for the sarcomatoid variant of bladder cancer.42 In this study, rather than expressing classic ‘endpoint’ markers of terminal NE differentiation (i.e. CgA, SYP), the NE-like tumors expressed intermediate neuronal markers (i.e. SOX2, PEG10) while demonstrating urothelial differentiation. Taken together, these data suggest the NE-like tumors are differentiating towards a neuroendocrine phenotype. With early detection, it is conceivable that timely intervention may interrupt this transition towards histological NE carcinoma. The concept that the NE-like tumors represent a transition state is supported by the finding that the conventional urothelial component of three of four PTC cases with a secondary NE variant region was classified as NE-like by our classifier. We know from a recent molecular characterization of small cell bladder cancer43 that UC and NE are genomically (ie mutational and copy number changes) similar but transcriptomically different, as has also been observed in prostate and lung cancer.44 The transition towards NE is supported by the loss of RB1 and TP53 in NE tumors45, but the transcriptional regulators that lead to the transcriptomic and ultimately the phenotypic shift are poorly understood. The plasticity of this transition appears to go in both




directions, since local recurrences in the bladder after chemoradiation for muscle-invasive small cell carcinoma are frequently conventional UC.46 This study is limited by the lack of central pathology review of all cases, although the majority of cases were assessed by dedicated genitourinary pathologists at the respective tertiary care centers and the NE-like cases were centrally reviewed. The prospectively collected clinical-use cases underwent stringent sample processing with only the muscle-invasive urothelial component of the tumor being selected and profiled. Four of these PTC cases had secondary NE variant histology in other regions of the tumor that were not sampled. An additional limitation is the retrospective nature of the study. Prospective validation will be necessary before this classification can be used clinically. In summary, we report the development and validation of a robust single-sample genomic classifier trained to identify NE-like patients, regardless of histological presentation.

FIGURE LEGENDS

Figure 1: Summary flowchart of neuroendocrine (NE) classifier development. A cohort of 175 prospective cases was used for initial model training using a set of 84 genes selected from machine learning approaches and literature review. A clustering solution was used to train a random forest model, which was tested on an additional cohort (RC1) and retrained if necessary. The final model was locked and proceeded to validation on six additional cohorts, including the TCGA MIBC cohort and a set of pathological NE MIBC cases. Candidate NE-like cases were stained with neuronal IHC markers. See main text for additional details.

Figure 2: Forced-order heatmap for three biological categories (luminal, basal and neuronal) of selected MIBC marker genes in (A) RC2 cohort, (B) NAC cohort and (C) TMT cohort. Cases identified as NE-like are boxed.

Figure 3: Forced-order heatmap for three biological categories (luminal, basal and neuronal) of selected MIBC marker genes in the (A) TCGA cohort, (B) Lund cohort and (C) Imvigor210 cohort. Cases identified as NE-like are boxed.

Figure 4: H&E and IHC images (SYP, CgA, CD56, ENO2, SOX2, PEG10) of (A) neuroendocrine bladder cancer and (B and C) two cases of neuroendocrine-like bladder cancer. Dotted line boxes represent low magnified images of corresponding adjacent high magnification view. Scale bar represents 100 µm. The IHC staining scores and gene expression values are indicated above each image.

Figure 5: Prognosis of NE-like patients in different treatment settings. KM plots for (A) RC1 cohort (n=225), (B) RC2 cohort (n=255), (C) NAC cohort (n=223), (D) TMT cohort (n=136), (E) TCGA radical cystectomy cohort (n=402) and (F) Imvigor210 cohort.







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Table 1: Patient clinical characteristic table of the prospective and retrospective cohorts.

Training Testing Validation

Variable PTC RC1 RC2 NAC21 TMT TCGA

11 LUND12

Imvigor21025

n 175 225 256 223 136 405 307 195

NE-like n (%) 8 (4.6) 4 (1.8) 8 (3.1) 3 (1.4) 9 (6.6) 9 (2.2%) 11 (3.6) 2 (1)

Age Median [IQR] 70 [47, 90]**

69 [63, 75] 70 [63, 77] 62 [56, 71] 70 [62, 77] 69 [60,76] NA NA

Gender Female 41 (23.4%) 41 (18.2) 48 (18.8) 69 (30.9) 27 (20.0) 106 (26.2) NA 42 (21.5)

Male 134 (76.6%) 184 (81.8) 208 (81.2) 154 (69.1) 108 (80.0) 299 (73.8) NA 153 (78.5)

Clinical lymph node

stage (%)

cN0 NA 0 (0.0) 231 (90.2) 140 (62.8) 60 (44.2) NA NA NA

cN1-3 NA 0 (0.0) 20 (7.8) 83 (37.2) 2 (1.4) NA NA NA

cNx NA 225 (100.0) 5 (2.0) 0 (0.0) 74 (53.2) NA NA NA

Clinical tumor stage

(%)

cTis NA 0 (0.0) 1 (0.4) 0 (0.0) 0 (0.0) NA NA NA

cTa NA 0 (0.0) 7 (2.7) 0 (0.0) 0 (0.0) NA NA NA

cT1 NA 0 (0.0) 22 (8.6) 0 (0.0) 0 (0.0) NA NA NA

cT2 NA 0 (0.0) 162 (63.3) 90 (40.4) 96 (70.6) NA NA NA

cT3 NA 0 (0.0) 42 (16.4) 90 (40.4) 33 (24.6) NA NA NA

cT4 NA 0 (0.0) 21 (8.2) 43 (19.3) 6 (4.4) NA NA NA

cTx NA 225 (100.0) 1 (0.4) 0 (0.0) 1 (0.7) NA NA NA

Pathological tumor stage

(%)*

pT0 NA 0 (0.0) 0 (0.0) 80 (35.9) 4 (2.9) 0 (0) 0 (0.0) NA

pTa NA 0 (0.0) 1 (0.4) 3 (1.3) 1 (0.7) 0 (0) 13 (4.2) NA

pT1 NA 9 (4.0) 0 (0.0) 13 (5.8) 2 (1.5) 0 (0) 44 (14.3) NA

pTis NA 0 (0.0) 0 (0.0) 7 (3.1) 1 (0.7) 0 (0) 1 (0.3) NA

pT2 NA 68 (30.2) 72 (28.1) 42 (18.8) 6 (4.4) 131 (32.5) 241 (78.5)

NA

pT3 NA 102 (45.3) 122 (47.7) 50 (22.4) 11 (8.1) 139 (34.2) 1 (0.3) NA

pT4 NA 46 (20.4) 61 (23.8) 24 (10.8) 2 (1.5) 133 (32.8) 1 (0.3) NA

pTx NA 0 (0.0) 0 (0.0) 4 (1.8) 109 (80.2) 2 (0.5) 6 (2.0) NA

Pathological lymph node

stage (%)

pN0 NA 145 (64.4) 142 (55.5) 157 (70.4) 14 (10.3) 231 (57) NA NA

pN1-3 NA 80 (35.6) 105 (41.0) 52 (23.3) 4 (2.9) 128 (31.6) NA NA

pNx NA 0 (0.0) 9 (3.5) 14 (6.3) 118 (86.8) 46 (11.4) NA NA

* NAC: ypT, ** age> 90 censored to protect patient privacy




Table 2: Multivariable analysis of OS on the RC2 surgery only cohort.

Variable Unadjusted HR p-value Adjusted HR p-value

Age 1.02 (1.00-1.04) 0.023* 1.02 (1.01-1.04) 0.004*

Gender: Male 0.97 (0.66-1.45) 0.9 0.91 (0.61-1.36) 0.644

>pT2 1.34 (0.94-1.90) 0.107 1.14 (0.79-1.65) 0.493

pN1-3 2.35 (1.70-3.26) <0.001* 2.45 (1.76-3.41) <0.001*

NE-like 3.88 (1.81-8.34) <0.001* 6.03 (2.68-13.58) <0.001*

Table 3: Multivariable analysis of OS on the NAC cohort


Age 1.02 (1.00-1.05) 0.026* 1.03 (1.01-1.06) 0.005*

Gender: Male 0.97 (0.60-1.56) 0.899 0.83 (0.50-1.39) 0.485

>pT2 1.56 (0.98-2.49) 0.062 1.84 (1.12-3.03) 0.017*

pN1-3 4.06 (2.55-6.47) <0.001* 4.91 (3.05-7.91) <0.001*

NE-like 58.18 (10.94-309.28) <0.001* 105.32 (18.78-590.71) <0.001*

Table 4: Multivariable analysis of OS on the TMT cohort.


Age 1.02 (1.00-1.04) 0.058 1.02 (1.00-1.04) 0.015*

Gender: Male 1.19 (0.66-2.18) 0.562 1.40 (0.75-2.60) 0.289

>cT2 2.23 (1.42-3.50) <0.001* 2.24 (1.41-3.55) <0.001*

Hydrophr. 1.62 (0.92-2.87) 0.096 1.55 (0.85-2.81) 0.15

NE-like 0.87 (0.35-2.15) 0.759 0.70 (0.28-1.78) 0.458

Table 5: Multivariable analysis of OS on the TCGA RC cohort.


Age 1.04 (1.02-1.05) <0.001* 1.04 (1.02-1.05) <0.001*

Gender: Male 0.89 (0.64-1.23) 0.48 0.88 (0.63-1.22) 0.44

>pT2 2.14 (1.48-3.09) <0.001* 1.61 (1.07-2.43) 0.023*

pN1-3 2.10 (1.56-2.83) <0.001* 1.70 (1.22-2.38) 0.002*

NE-like 2.67 (1.25-5.71) 0.011* 3.80 (1.76-8.23) <0.001*






















Published OnlineFirst April 5, 2019.Clin Cancer Res José Batista da Costa, Ewan A Gibb, Trinity J. Bivalacqua, et al. cancerMolecular characterization of neuroendocrine-like bladder

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Molecular characterization of neuroendocrine-like bladder ... · ABSTRACT (250 max) Purpose: Neuroendocrine (NE) bladder carcinoma is a rare and aggressive variant. Molecular subtyping