Molecular subtype concordance and metastatic patterns in muscle-invasive bladder cancer.

Genomic Differences Between “Primary” and “Secondary” Muscle-invasive Bladder Cancer as a Basis for Disparate Outcomes to Cisplatin-based Neoadjuvant Chemotherapy

Muscle invasive bladder cancer (MIBC) is a heterogeneous disease with a high recurrence rate and poor clinical outcomes. Molecular subtype provides a new framework for the study of MIBC heterogeneity. Clinically, MIBC can be classified as basal and luminal subtypes; they display different clinical and pathological characteristics, but the molecular mechanism is still unclear. Lipidomic and metabolomic molecules have recently been considered to play an important role in the genesis and development of tumors, especially as potential biomarkers. Their different expression profiles in basal and luminal subtypes provide clues for the molecular mechanism of basal and luminal subtypes and the discovery of new biomarkers. Herein, we stratified MIBC patients into basal and luminal subtypes using a MIBC classifier based on transcriptome expression profiles. We qualitatively and quantitatively analyzed the lipids and metabolites of basal and luminal MIBC subtypes and identified their differential lipid and metabolite profiles. Our results suggest that free fatty acids (FFAs) and sulfatides (SLs), which are closely associated with immune and stromal cell types, can contribute to the diagnosis of basal and luminal subtypes of MIBC. Moreover, we showed that glycerophosphocholine (GCP)/imidazoles and nucleosides/imidazoles ratios can accurately distinguish the basal and luminal tumors. Overall, by integrating transcriptomic, lipidomic, and metabolomic data, our study reveals specific biomarkers to differentially diagnose basal and luminal MIBC subtypes and may provide a basis for precision therapy of MIBC.

BackgroundThere might be differential sensitivity to neoadjuvant chemotherapy (NAC) in patients with primary muscle-invasive bladder cancer (MIBC) in comparison to patients with secondary MIBC after a history of non–muscle-invasive disease. ObjectiveTo investigate pathologic response rates and survival associated with primary versus secondary MIBC among patients treated with cisplatin-based NAC for cT2–4N0M0 MIBC. Design, setting, and participantsOncologic outcomes were compared for 350 patients with primary MIBC and 64 with secondary MIBC treated with NAC and radical cystectomy between 1992 and 2021 at 11 academic centers. Genomic analyses were performed for 476 patients from the Memorial Sloan Kettering/The Cancer Genome Atlas cohort. Outcome measurements and statistical analysisThe outcome measures were pathologic objective response (pOR; ≤ypT1 N0), pathologic complete response (pCR; ypT0 N0), overall mortality, and cancer-specific mortality. Results and limitationsThe primary MIBC group had higher pOR (51% vs 34%; p = 0.02) and pCR (33% vs 17%; p = 0.01) rates in comparison to the secondary MIBC group. On multivariable logistic regression analysis, primary MIBC was independently associated with both pOR (odds ratio [OR] 0.49, 95% confidence interval [CI] 0.26–0.87; p = 0.02) and pCR (OR 0.41, 95% CI 0.19–0.82; p = 0.02). However, on multivariable Cox regression analysis, primary MIBC was not associated with overall mortality (hazard ratio 1.70, 95% CI 0.84–3.44; p = 0.14) or cancer-specific mortality (hazard ratio 1.50, 95% CI 0.66–3.40; p = 0.3). Genomic analyses revealed a significantly higher ERCC2 mutation rate in primary MIBC than in secondary MIBC (12.4% vs 1.3%; p < 0.001). ConclusionsPatients with primary MIBC have better pathologic response rates to NAC in comparison to patients with secondary MIBC. Chemoresistance might be related to the different genomic profile of primary versus secondary MIBC. Patient summaryWe investigated the treatment response to neoadjuvant chemotherapy (NAC; chemotherapy received before the primary course of treatment) and survival for patients with a primary diagnosis of muscle-invasive bladder cancer (MIBC) in comparison to patients with a history of non–muscle-invasive bladder cancer that progressed to MIBC. Patients with primary MIBC had a better response to NAC but this did not translate to better survival after accounting for other tumor characteristics.

497 Background: Neoadjuvant chemotherapy followed by cystectomy in MIBC is associated with improved survival compared to cystectomy alone. Recent retrospective studies indicated that secondary MIBC (non-MIBC progressed to MIBC) had worse outcome with cisplatin based neoadjuvant chemotherapy when compared to primary MIBC. To further evaluate this observation, we queried our database to assess the differential response and determine if prior use of intravesical therapy diminishes the effect of cisplatin based neoadjuvant chemotherapy. Methods: A total of 387 patients diagnosed with T2-4 or N0-3 and M0 from 2000-2018 and underwent cystectomy were retrospectively chart reviewed for demographics, treatment and outcomes at University of Iowa Holden Comprehensive Cancer Center. Cox regression models were utilized to assess differences in recurrence-free survival (RFS) and overall survival (OS). Time was calculated from cystectomy to recurrence or death due to any cause for RFS and OS, respectively. Results: Of the 387 patients, 324 patients had primary MIBC and 63 had secondary MIBC. Median follow up was 25.8 months. Intravesical therapy was administered to 98% (62/63) of secondary MIBC patients. Neoadjuvant chemotherapy was administered to 38% (122/324) of primary MIBC patients and 21% (13/63) of secondary MIBC patients. NAC had no difference in response rates (CR and PR) for primary vs secondary MIBC (p=0.73). Additionally, there was no difference between the primary and secondary MIBC with regards to RFS (p=0.54) and OS (p=0.12) on univariate analysis. The effect of neoadjuvant chemotherapy did not differ based on prior use of intravesical therapy in terms of RFS (p=0.61) or OS (p=0.40). Additionally, neoadjuvant chemotherapy irrespective of prior use of intravesical therapy was not associated with RFS (p=0.66) or OS (p=0.15). Conclusions: Prior intravesical therapy was not associated with differential efficacy of neoadjuvant chemotherapy in secondary MIBC when compared to primary MIBC.

Introduction: Using RNA-seq (HiSeq) transcriptome data in The Cancer Genome Atlas (TCGA) bladder cancer (BCa) dataset two molecular subtypes, basal and luminal, were identified in muscle invasive BCa (MIBCa). Recurring reported markers of the basal subtype are KRT5, KRT6A, KRT14 high and KRT20, GATA3, UPK3A, FOXA1 low. The luminal subtype is the opposite pattern of expression. Double-Negative (DN) subtype was defined as no expression of the 7 markers. Luminal, basal and DN subtypes were reported as predictors of better, poor and worst prognosis, respectively, based on overall survival (OS). EGFR is defined as a squamous differentiation marker associated with poor prognosis. The objective was to assess the clinical significance of these subtypes in TCGA data with validation in datasets from Oncomine. Methods: TCGA dataset containing 407 MIBCa patients was accessed through Xena Browser. The dataset included patient demographics, clinical parameters, OS, recurrence-free survival (RFS) and the transcript levels (log2(nor._count+1) of basal and luminal markers. All available follow-up data was included (27.9±28.49; max: 166 months). 25 BCa datasets (n=360 MIBCa patients) containing these same variables were accessed through Oncomine. High and low levels were stratified by median. Association of individual basal and luminal markers and the subtypes with clinical and outcome variables was analyzed by univariate and multivariate analyses. Kaplan-Meier analysis was performed to stratify patients into risk groups for OS and RFS. Results: In TCGA data, neither basal nor luminal markers levels significantly correlated with metastasis or lymphovascular invasion (LVI); P = 0.2 to 0.9. Only KRT5 significantly but inversely correlated with lymph node (LN) positivity; P=0.011. EGFR levels did not correlate with metastasis, LVI, or LN positivity; P &amp;gt; 0.05. In TCGA dataset 77 (18.9%), 50 (12.3%) and 14 (3.4%) expressed basal, luminal and DN subtypes, respectively; 266 (66.4%) patients did not conform to any group. In univariate or multivariate analyses, the subtypes also did not correlate with metastasis, LVI or LN status. Only luminal subtype associated with better OS; P=0.003. However no subtype significantly correlated with RFS. In KM analysis no subtype stratified patients regarding RFS (P&amp;gt;0.2). Oncomine BCa datasets validated these results. Conclusion: TCGA and Oncomine datasets show that the majority of MIBCa tissues express a mixed pattern of basal and luminal markers. Furthermore, basal, luminal or DN subtypes do not associate with clinical parameters or prognosis of MIBCa patients. Citation Format: Daley S. Morera, Daniel Belew, Andre R. Jordan, Vinata B. Lokeshwar. Molecular subtypes in muscle invasive bladder cancer: Evaluation of clinical significance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4614.

PD36-10 SURVIVAL DIFFERENCES BETWEEN PRIMARY VERSUS SECONDARY MUSCLE-INVASIVE BLADDER CANCER PATIENTS TREATED WITH RADICAL CYSTECTOMY

443 Background: Defining the role of MIBC molecular subtypes and immune expression in determining clinical outcomes is an area of active investigation. However, changes in these transcriptomic profiles pre- and post-NAC have not been well characterized. Methods: This retrospective study reviewed 53 pts with MIBC treated with NAC, of whom 12 pts without complete pathological response had both pre- and post-NAC samples of sufficient quality. Post-NAC staging was &gt; = pT2 in 11 pts and pT1 in 1 pt. We performed RNA expression analysis of matched pre-NAC transurethral resection of bladder tumor specimens and post-treatment radical cystectomy primary bladder tumor specimens. We used a customized NanoString panel incorporating previously reported immune signatures (Ayers, JCI 2017; O’Donnell, ASCO 2017) and additional genes to assign basal ( CD14, CD44, PDGFC, KRT14, KRT5) and luminal ( GATA3, PPARG, SHH, CD24, FOXA1, WNT7B, ERBB2) molecular subtypes. Results: We first classified the bladder cancer cohort of The Cancer Genome Atlas into basal and luminal subtypes using the BASE47 signature (Damrauer, PNAS 2014) and the NanoString panel and there was good agreement (Rand Index = 0.72). We then assigned subtypes using the NanoString panel on matched pre- and post-NAC samples and found marked subtype shift (Table). We identified two robust clusters of samples according to immune expression with a 3-fold change of immune expression between them (FDR = 0.0008). We found that 4 pts switched from the low to the high cluster, while 2 switched from the high to the low cluster after NAC (Table). Conclusions: MIBC molecular subtype membership is dynamic and is influenced by NAC. NAC can induce both enhanced and suppressed immune activity. These findings have implications on future studies exploring the predictive value of RNA expression patterns for bladder cancer therapies as well as post-NAC immunotherapy. [Table: see text]

Intrinsic molecular subtypes may explain marked variation between bladder cancer patients in prognosis and response to therapy. Complex testing algorithms and little attention to more prevalent, early‐stage (non‐muscle invasive) bladder cancers (NMIBCs) have hindered implementation of subtyping in clinical practice. Here, using a three‐antibody immunohistochemistry (IHC) algorithm, we identify the diagnostic and prognostic associations of well‐validated proteomic features of basal and luminal subtypes in NMIBC. By IHC, we divided 481 NMIBCs into basal (GATA3−/KRT5+) and luminal (GATA3+/KRT5 variable) subtypes. We further divided the luminal subtype into URO (p16 low), URO‐KRT5+ (KRT5+), and genomically unstable (GU) (p16 high) subtypes. Expression thresholds were confirmed using unsupervised hierarchical clustering. Subtypes were correlated with pathology and outcomes. All NMIBC cases clustered into the basal/squamous (basal) or one of the three luminal (URO, URO‐KRT5+, and GU) subtypes. Although uncommon in this NMIBC cohort, basal tumors (3%, n = 16) had dramatically higher grade (100%, n = 16, odds ratio [OR] = 13, relative risk = 3.25) and stage, and rapid progression to muscle invasion (median progression‐free survival = 35.4 months, p = 0.0001). URO, the most common subtype (46%, n = 220), showed rapid recurrence (median recurrence‐free survival [RFS] = 11.5 months, p = 0.039) compared to its GU counterpart (29%, n = 137, median RFS = 16.9 months), even in patients who received intravesical immunotherapy (p = 0.049). URO‐KRT5+ tumors (22%, n = 108) were typically low grade (66%, n = 71, OR = 3.7) and recurred slowly (median RFS = 38.7 months). Therefore, a simple immunohistochemical algorithm can identify clinically relevant molecular subtypes of NMIBC. In routine clinical practice, this three‐antibody algorithm may help clarify diagnostic dilemmas and optimize surveillance and treatment strategies for patients.

MP58-02 GENOMIC DIFFERENCES BETWEEN “PRIMARY” AND “SECONDARY” MUSCLE INVASIVE BLADDER CANCER: IMPLICATIONS FOR NEOADJUVANT CHEMOTHERAPY

BackgroundMuscle-invasive bladder carcinomas (MIBCs) exhibit significant heterogeneity, with diverse histopathological features associated with varied prognosis and therapeutic response. Although genomic profiling studies have identified several molecular subtypes of MIBC, two basic molecular subtypes are identified - luminal and basal, differing in biological behaviour and response to treatment. As molecular subtyping is complex, surrogate immunohistochemical (IHC) markers have been used to determine the molecular subtypes with good correlation to genomic profiling.MethodsWe analysed the clinicopathological features of 66 cases of MIBCs received over a 5-year study period. IHC expression was determined using GATA3 and CK5/6 to classify MIBC into luminal, basal and double-negative subtypes. The association between clinicopathologic variables and molecular subtypes were analysed using Chi-square test.ResultsThe mean age at diagnosis of MIBC was 65.91 years with a male predominance. Based on IHC expression of GATA3 and CK5/6, MIBCs were classified into luminal, basal and double negative subtypes in 62.1%, 30.3% and 7.6% respectively. The luminal subtype occurred at an older age and showed predominantly conventional urothelial carcinoma with papillary morphology. Basal subtype occurred at earlier age, showed greater association with smoking and was more commonly associated with urothelial carcinoma with non -papillary morphology and exhibiting divergent differentiation as well as pure squamous cell carcinoma on histopathological examination. The double-negative subtype was found exclusively in males and exhibited a non-papillary morphology. Notably, all diagnosed neuroendocrine carcinomas were classified as double-negative type. While there was no statistically significant difference in tumour stage in cystectomy specimens between the molecular subtypes, lympho-vascular invasion and lymph node metastasis was more commonly associated with the basal type (p < 0.05) There was no significant difference in recurrence rates, metastasis and death between luminal and basal subtypes.ConclusionA simple two-antibody panel using GATA3 and CK5/6 could help in classifying MIBC into basic molecular subtypes of MIBC with distinctive histopathological features that can provide insights into the corresponding molecular subtype. Greater association of lymphovascular invasion and lymph nodal involvement in cystectomy specimens in basal type and distant metastasis in the double-negative subtype suggests a more aggressive clinical behaviour of these, necessitating more intensive treatment.

BackgroundMore than one third of breast cancer patients including those that are diagnosed in early stages will develop distant metastasis. Patterns of distant metastasis and the associated risks according to the molecular subtypes are not completely revealed particularly in populations of patients with delayed diagnosis and advanced stages.MethodsBreast cancer patients (n = 1304) admitted to our institute (2014–2017) were evaluated to identify the metastatic patterns and the associated risks. Metastatic breast cancers at diagnosis were found in 245 patients (18.7%), and 1059 patients were then grouped into non-metastatic and metastatic groups after a median follow-up of 3.8 years.ResultsInfiltration of the tumor to the skin and chest wall prevailed as the most powerful predictor for distant metastasis (OR 2.115, 95% CI 1.544–2.898) particularly in the luminal A-like subtype (OR 2.685, 95% CI 1.649–4.371). Nodal involvement was also significantly associated with the risk of distant metastasis (OR 1.855, 95% CI 1.319–2.611), and the risk was higher in the Luminal A-like subtype (OR 2.572, 95% CI 1.547–4.278). Luminal A-like subtype had a significant higher risk of bone metastasis (OR 1.601, 95% CI 1.106–2.358). In respect to treatment, a combination of anthracyclines and taxanes-based chemotherapy was significantly associated with lower distant organ spread in comparison with anthracycline-based chemotherapy (OR 0.510, 95% CI 0.355–0.766) and the effect was stronger in Luminal A-like subtype (OR 0.417, 95% CI 0.226–0.769). Classification into Luminal and non-Luminal subtypes revealed significant higher risks of bone metastasis in the Luminal subtype (OR 1.793, 95% CI 1.209–2.660) and pulmonary metastasis in non-Luminal breast cancer (OR 1.445, 95% CI 1.003–2.083).ConclusionIn addition to guiding the treatment plan, a comprehensive analysis of clinicopathological variables including the molecular subtypes could assist in the determination of distant metastasis risks of breast cancer patients. Our study offers new perspectives concerning the risks of distant metastasis in breast cancer subtypes in order to plan intensive surveillance or escalation of treatment particularly in a setting where patients are predominantly diagnosed in late stages.

Background: Stage IV breast cancer at initial diagnosis (BCID) can be recognized in approximately 5% of all breast neoplasms. Clinical outcomes of these patients (pts) are highly variable and depend on tumor biology and pt characteristics. The prognostic influence of metastatic pattern (MP) at initial presentation and factors associated with specific organ involvement have been understudied. The primary aim of this study was to analyze the influence of MP compared with other biologic and clinical factors in the survival of pts with stage IV BCID. The secondary aim was to evaluate factors associated with specific sites of metastatic spread. Methods: We evaluated women with microscopically confirmed stage IV BCID with known metastatic sites, reported to the Surveillance, Epidemiology and End Results (SEER) 18 registries program from 2010 to 2013. Pts with other primary tumor either before or after breast cancer were excluded. MP was categorized as bone only, visceral (lung, liver or brain), bone and visceral and other. Pt characteristics were compared between MP. Univariate and multivariate analyses determined the effects of each variable on overall survival (OS). Logistic regression examined factors associated with specific sites of metastases. Results: We included 9143 pts. Median age was 61 years (range 19-102). Median OS for the entire cohort was 28 months (95% CI 27-29 months). At diagnosis, bone only metastases represented 37.5% of pts, visceral 21.9%, bone and visceral 28.8% and other 11.9%. Median OS by MP was: bone only 38 months, visceral 21 months, bone and visceral 19 months and other 33 months (p Conclusions: To our knowledge, this is the largest study of MP in stage IV BCID. There were substantial differences in prognosis according to MP, bone only was the most common MP and had the best OS, whereas bone and visceral MP had the worst prognosis. We observed significant differences in pt characteristics according to MP. Independent predictors of OS included age at diagnosis, race, marital status, tumor grade, tumor subtype and MP. There was a clear influence of tumor subtype among other factors on specific sites of metastases. Our study identified several prognostic factors that could guide therapy selection in treatment naive pts.Background: Stage IV breast cancer at initial diagnosis (BCID) can be recognized in approximately 5% of all breast neoplasms. Clinical outcomes of these patients (pts) are highly variable and depend on tumor biology and pt characteristics. The prognostic influence of metastatic pattern (MP) at initial presentation and factors associated with specific organ involvement have been understudied. The primary aim of this study was to analyze the influence of MP compared with other biologic and clinical factors in the survival of pts with stage IV BCID. The secondary aim was to evaluate factors associated with specific sites of metastatic spread. Methods: We evaluated women with microscopically confirmed stage IV BCID with known metastatic sites, reported to the Surveillance, Epidemiology and End Results (SEER) 18 registries program from 2010 to 2013. Pts with other primary tumor either before or after breast cancer were excluded. MP was categorized as bone only, visceral (lung, liver or brain), bone and visceral and other. Pt characteristics were compared between MP. Univariate and multivariate analyses determined the effects of each variable on overall survival (OS). Logistic regression examined factors associated with specific sites of metastases. Results: We included 9143 pts. Median age was 61 years (range 19-102). Median OS for the entire cohort was 28 months (95% CI 27-29 months). At diagnosis, bone only metastases represented 37.5% of pts, visceral 21.9%, bone and visceral 28.8% and other 11.9%. Median OS by MP was: bone only 38 months, visceral 21 months, bone and visceral 19 months and other 33 months (p Conclusions: To our knowledge, this is the largest study of MP in stage IV BCID. There were substantial differences in prognosis according to MP, bone only was the most common MP and had the best OS, whereas bone and visceral MP had the worst prognosis. We observed significant differences in pt characteristics according to MP. Independent predictors of OS included age at diagnosis, race, marital status, tumor grade, tumor subtype and MP. There was a clear influence of tumor subtype among other factors on specific sites of metastases. Our study identified several prognostic factors that could guide therapy selection in treatment naive pts. Citation Format: Leone BA, Vallejo CT, Romero AO, Machiavelli MR, Perez JE, Leone J, Leone JP. Prognostic impact of metastatic pattern in stage IV breast cancer at initial diagnosis [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-09-21.

Molecular assessments of muscle-invasive bladder cancer (MIBC) have yielded several molecular categorizations associated with basal and luminal subtypes or tumor-associated immune cell status (TAICs). However, the histological relationships among histological subtypes, molecular subtypes, and TAICs and their clinical implications remain unclear. Thus, we aimed to evaluate the histological associations among these factors and their clinicopathological outcomes. We retrospectively analyzed 106 patients with MIBC who underwent radical cystectomy. The histological subtypes and TAICs were evaluated with hematoxylin and eosin staining, while the basal and luminal molecular subtypes were determined by immunohistochemical expression of cytokeratin (CK) 5/6, CK14, CK20, GATA3 and uroplakin II. Urothelial carcinoma with squamous differentiation and the sarcomatoid variant were highly associated with the basal subtype (P < 0.001 and P = 0.04, respectively). Additionally, high TAICs were significantly correlated with the basal subtype (P < 0.001). Although there was no significant difference in the cancer-specific survival (CSS) rate between molecular subtypes (P = 0.295), TAICs significantly discriminated CSS rates (P < 0.001). Furthermore, the combination of molecular subtypes and TAICs significantly stratified cancer-specific mortality rates. In conclusion, a comprehensive pathological evaluation of histological subtypes, molecular subtypes, and TAICs is feasible and can influence the oncological outcome.

Comprehensive transcriptome expression analyses of bladder cancer revealed distinct lncRNA clusters with differential molecular and clinical characteristics. In this study, pivotal lncRNAs were assessed for their impact on survival and their differential expression between the molecular bladder cancer subtypes. FFPE samples from chemotherapy-naïve patients with muscle invasive bladder cancer (MIBC) were analyzed on the Nanostring nCounter platform for absolute quantification. An established 36-gene panel was used for molecular subtype classification into basal, luminal and infiltrated MIBC. In a second step, 14 pivotal lncRNAs were assessed for their molecular subtype attribution, and their predictive value in disease-specific survival. In silico validation was performed on a total of 487 MIBC patients (MDA, TGCA and Chungbuk cohort). Several pivotal lncRNAs showed a distinct molecular subtype attribution: e.g. MALAT1 showed a downregulation in the basal subtype (p = 0.009), TUG1 and CBR3AS1 showed an upregulation in the luminal subtype (p ≤ 0.001). High transcript levels of SNHG16, CBR3AS1 and H19 appeared to be predictive for a shorter disease-specific survival. Patients overexpressing putative oncogenes MALAT1 and TUG1 in MIBC tissue presented prolonged survival, suggesting tumor suppressive effects of both lncRNAs. The Nanostring nCounter proved to be a valid platform for the quantification of low-abundance transcripts including lncRNAs.

828 Background: Consensus molecular subtypes of UC/BC include basal/squamous (Ba/Sq), stroma-rich (SR), luminal unstable (lumU), luminal papillary (lumP), luminal nonspecified (lumNS), and neuroendocrine (NE)-like. Nectin-4 and HER2 are targets for novel antibody-drug conjugates. We report exploratory analyses of molecular subtypes, including NECTIN4 and HER2 RNA expression, in pts with aUC or MIBC from the JAVELIN Bladder 100 phase 3 trial and Tempus real-world database. Methods: JAVELIN Bladder 100 (NCT02603432) enrolled pts with aUC without progression after first-line platinum-based chemotherapy. Pts with MIBC or aUC were identified in the Tempus database of deidentified pt data from US clinical practice. Whole-transcriptome profiles in tumor samples were generated using RNA sequencing. NECTIN4 and HER2 transcript levels were quantified using Personalis ACE technology (JAVELIN Bladder 100) or kallisto (Tempus). Results: In evaluable aUC tumors from the JAVELIN cohort (n=560), NECTIN4 and HER2 RNA expression was heterogenous across molecular subtypes and was highest in lumU/lumP/lumNS subtypes and lowest in the NE-like subtype (Table); 57% of tumors had high (≥ median) expression of both NECTIN4 and HER2 RNA. Variations in NECTIN4 / HER2 RNA expression across molecular subtypes were similar in MIBC/aUC tumors from the Tempus cohort (n=501); 39% of tumors had high NECTIN4/HER2 RNA expression. In both cohorts, a strong correlation between NECTIN4 and HER2 RNA expression was observed overall (R=0.6-0.655), although correlation was not observed within all subtypes. In pts treated with avelumab in JAVELIN Bladder 100 (n=283; excluding NE-like [n=1]), no significant difference in overall survival was noted by molecular subtype (median [95% CI], mo: Ba/Sq, 27.2 [18.2-not estimable (NE)]; LumNS, 34.4 [20.8-NE]; LumP, 20.8 [18.2-31.4]; LumU, 24.9 [18.6-43.1]; SR, 28.8 [19.3-NE]). Limitations include exploratory analyses and potential selection bias. Conclusions: In these analyses, including the first analysis of consensus molecular subtypes in a phase 3 trial in aUC, NECTIN4 and HER2 RNA expression was heterogeneous across molecular subtypes of aUC and MIBC tumors and was highest in luminal subtypes. No significant difference in overall survival was noted across subtypes in pts treated with avelumab first-line maintenance. Clinical trial information: NCT02603432 . Subtype JAVELIN (n=560) Tempus (n=501) n (%) Expression level* n (%) Expression level* NECTIN4 HER2 NECTIN4 HER2 Ba/Sq 95 (17.0) 0.812 0.779 177 (35.3) 0.828 0.769 LumNS 30 (5.4) 1.029 0.905 30 (6.0) 0.990 0.926 LumP 135 (24.1) 0.984 0.888 89 (17.8) 0.962 0.925 LumU 95 (17.0) – – 99 (19.8) – – NE-like 3 (0.5) 0.152 0.460 37 (7.4) 0.311 0.613 SR 202 (36.1) 0.833 0.862 69 (13.8) 0.884 0.879 *Fold difference in transcripts per million vs LumU.