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Purpose To assess the association of patient age with response to preoperative chemotherapy in patients with muscle-invasive bladder cancer (MIBC). Materials and methods We analyzed data from 1105 patients with MIBC. Patients age was evaluated as continuous variable and stratified in quartiles. Pathologic objective response (pOR; ypT0-Ta-Tis-T1N0) and pathologic complete response (pCR; ypT0N0), as well survival outcomes were assessed. We used data of 395 patients from The Cancer Genome Atlas (TCGA) to investigate the prevalence of TCGA molecular subtypes and DNA damage repair (DDR) gene alterations according to patient age. Results pOR was achieved in 40% of patients. There was no difference in distribution of pOR or pCR between age quartiles. On univariable logistic regression analysis, patient age was not associated with pOR or pCR when evaluated as continuous variables or stratified in quartiles (all p > 0.3). Median follow-up was 18 months (IQR 6–37). On Cox regression and competing risk regression analyses, age was not associated with survival outcomes (all p > 0.05). In the TCGA cohort, patient with age ≤ 60 years has 7% less DDR gene mutations (p = 0.59). We found higher age distribution in patients with luminal (p p = 0.002) compared to those with luminal papillary subtype. Conclusions While younger patients may have less mutational tumor burden, our analysis failed to show an association of age with response to preoperative chemotherapy or survival outcomes. Therefore, the use of preoperative chemotherapy should be considered regardless of patient age.

OBJECTIVE To assess the differential response to neoadjuvant chemotherapy (NAC) in patients with urothelial carcinoma of the bladder (UCB) compared to upper tract urothelial carcioma (UTUC) treated with radical surgery. PATIENTS AND METHODS Data from 1299 patients with UCB and 276 with UTUC were obtained from multicentric collaborations. The association of disease location (UCB vs UTUC) with pathological complete response (pCR, defined as a post-treatment pathological stage ypT0N0) and pathological objective response (pOR, defined as ypT0-Ta-Tis-T1N0) after NAC was evaluated using logistic regression analyses. The association with overall (OS) and cancer-specific survival (CSS) was evaluated using Cox regression analyses. RESULTS A pCR was found in 250 (19.2%) patients with UCB and in 23 (8.3%) with UTUC (P

Muscle-invasive bladder cancer (MIBC) is a sex-biased cancer with a higher incidence in men but worse outcomes in women. The root cause behind these observations remains unclear. To investigate whether sex-specific tumor biology could explain the differences in clinical behavior of MIBC, we analyzed the transcriptome profiles from transurethral resected bladder tumors of 1000 patients. Female tumors expressed higher levels of basal- and immune-associated genes, while male tumors expressed higher levels of luminal markers. Using molecular subtyping, we found that the rates of the basal/squamous subtype were higher in females than in males. Males were enriched with tumors of the luminal papillary (LumP) and neuroendocrine-like subtypes. Male MIBC tumors had higher androgen response activity across all luminal subtypes and male patients with LumP tumors were younger. Taken together, these data confirm differences in molecular subtypes based on sex. The role of the androgen response pathway in explaining subtype differences between men and women should be studied further. PATIENT SUMMARY: We explored the sex-specific biology of bladder cancer in 1000 patients and found that women had more aggressive cancer with higher immune activity. Men tended toward less aggressive tumors that showed male hormone signaling, suggesting that male hormones may influence the type of bladder cancer that a patient develops.

Background:: Cisplatin-based neoadjuvant chemotherapy (NAC) for muscle-invasive bladder cancer (MIBC) is associated with improved overall and cancer-specific survival. The post-NAC pathological stage has previously been reported to be a major determinant of outcome. Objective:: To develop a postoperative nomogram for survival based on pathological and clinical parameters from an international consortium. Design, setting, and participants:: Between 2000 and 2015, 1866 patients with MIBC were treated at 19 institutions in the USA, Canada, and Europe. Analysis was limited to 640 patients with adequate follow-up who had received three or more cycles of NAC. Outcome measurements and statistical analysis:: A nomogram for bladder cancer–specific mortality (BCSM) was developed by multivariable Cox regression analysis. Decision curve analysis was used to assess the model's clinical utility. Results and limitations:: A total of 640 patients were identified. Downstaging to non-MIBC (ypT1, ypTa, and ypTis) occurred in 271 patients (42 %), and 113 (17 %) achieved a complete response (ypT0N0). The 5-yr BCSM was 47.2 % (95 % confidence interval [CI]: 41.2–52.6 %). On multivariable analysis, covariates with a statistically significant association with BCSM were lymph node metastasis (hazard ratio [HR] 1.90 [95% CI: 1.4–2.6]; p < 0.001), positive surgical margins (HR 2.01 [95 % CI: 1.3–2.9]; p < 0.001), and pathological stage (with ypT0/Tis/Ta/T1 as reference: ypT2 [HR 2.77 {95 % CI: 1.7–4.6}; p < 0.001] and ypT3–4 [HR 5.9 {95 % CI: 3.8–9.3}; p < 0.001]). The area under the curve of the model predicting 5-yr BCSM after cross validation with 300 bootstraps was 75.4 % (95 % CI: 68.1–82.6 %). Decision curve analyses showed a modest net benefit for the use of the BCSM nomogram in the current cohort compared with the use of American Joint Committee on Cancer staging alone. Limitations include the retrospective study design and the lack of central pathology. Conclusions:: We have developed and internally validated a nomogram predicting BCSM after NAC and radical cystectomy for MIBC. The nomogram will be useful for patient counseling and in the identification of patients at high risk for BCSM suitable for enrollment in clinical trials of adjuvant therapy. Patient summary:: In this report, we looked at the outcomes of patients with muscle-invasive bladder cancer in a large multi-institutional population. We found that we can accurately predict death after radical surgical treatment in patients treated with chemotherapy before surgery. We conclude that the pathological report provides key factors for determining survival probability. We developed a nomogram predicting bladder cancer–specific mortality after neoadjuvant chemotherapy and radical cystectomy in a large, multi-institutional patient cohort.

Purpose Neoadjuvant chemotherapy (NAC) prior to radical cystectomy (RC) in patients with non-metastatic muscle-invasive bladder cancer (MIBC) confers an absolute survival benefit of 5-10%. There is evidence that molecular differences between tumors may impact response to therapy, highlighting a need for clinically validated biomarkers to predict response to NAC. Materials and methods Four bladder cancer cohorts were included. Inverse probability weighting was used to make baseline characteristics (age, sex, and clinical tumor stage) between NAC-treated and untreated groups more comparable. Molecular subtypes were determined using a commercial genomic subtyping classifier. Survival rates were estimated using weighted Kaplan Meier (KM) curves. Cox proportional hazards (PH) models were used to evaluate the primary and secondary study endpoints of overall survival (OS) and cancer-specific survival (CSS), respectively. Results A total of 601 patients with MIBC were included, where 247 had been treated with NAC and RC and 354 underwent RC without NAC. With NAC, the overall net benefit to OS and CSS at three years was 7% and 5%, respectively. After controlling for clinicopathologic variables, non-luminal tumors had greatest benefit from NAC with 10% greater OS at 3 years (71% vs 61%) while luminal tumors had minimal benefit (63% vs 65%) for NAC vs. non-NAC, respectively. Conclusions In patients with MIBC, a commercially available molecular subtyping assay revealed non-luminal tumors received the greatest benefit from NAC, while patients with luminal tumors experienced a minimal survival benefit. A genomic classifier may help identify patients with MIBC who would benefit most from NAC.

We investigated the pathological response rates and survival associated with 3 vs 4 cycles of cisplatin-based neoadjuvant chemotherapy (NAC) in patients with cT2-4N0M0 muscle invasive bladder cancer.In this cohort study we analyzed clinical data of 828 patients treated with NAC and radical cystectomy between 2000 and 2020. A total of 384 and 444 patients were treated with 3 and 4 cycles of NAC, respectively. Pathological objective response (pOR; ypT0-Ta-Tis-T1 N0), pathological complete response (pCR; ypT0 N0), cancer-specific survival and overall survival were investigated.pOR and pCR were achieved in 378 (45%; 95% CI 42, 49) and 207 (25%; 95% CI 22, 28) patients, respectively. Patients treated with 4 cycles of NAC had higher pOR (49% vs 42%, p=0.03) and pCR (28% vs 21%, p=0.02) rates compared to those treated with 3 cycles. This effect was confirmed on multivariable logistic regression analysis (pOR OR 1.46 p=0.008, pCR OR 1.57, p=0.007). On multivariable Cox regression analysis, 4 cycles of NAC were significantly associated with overall survival (HR 0.68; 95% CI 0.49, 0.94; p=0.02) but not with cancer-specific survival (HR 0.72; 95% CI 0.50, 1.04; p=0.08).Four cycles of NAC achieved better pathological response and survival compared to 3 cycles. These findings may aid clinicians in counseling patients and serve as a benchmark for prospective trials. Prospective validation of these findings and assessment of cumulative toxicity derived from an increased number of cycles are needed.

Purpose: After cisplatin-based neoadjuvant chemotherapy (NAC), 60% of patients with muscle-invasive bladder cancer (MIBC) still have residual invasive disease at radical cystectomy. The NAC-induced biological alterations in these cisplatin-resistant tumors remain largely unstudied. Experimental Design: Radical cystectomy samples were available for gene expression analysis from 133 patients with residual invasive disease after cisplatin-based NAC, of whom 116 had matched pre-NAC samples. Unsupervised consensus clustering (CC) was performed and the consensus clusters were investigated for their biological and clinical characteristics. Hematoxylin & Eosin and IHC on tissue microarrays were used to confirm tissue sampling and gene expression analysis. Results: Established molecular subtyping models proved to be inconsistent in their classification of the post-NAC samples. Unsupervised CC revealed four distinct consensus clusters. The CC1-Basal and CC2-Luminal subtypes expressed genes consistent with a basal and a luminal phenotype, respectively, and were similar to the corresponding established pretreatment molecular subtypes. The CC3-Immune subtype had the highest immune activity, including T-cell infiltration and checkpoint molecule expression, but lacked both basal and luminal markers. The CC4-Scar–like subtype expressed genes associated with wound healing/scarring, although the proportion of tumor cell content in this subtype did not differ from the other subtypes. Patients with CC4-Scar–like tumors had the most favorable prognosis. Conclusions: This study expands our knowledge on MIBC not responding to cisplatin by suggesting molecular subtypes to understand the biology of these tumors. Although these molecular subtypes imply consequences for adjuvant treatments, this ultimately needs to be tested in clinical trials.

Purpose: Neuroendocrine (NE) bladder carcinoma is a rare and aggressive variant. Molecular subtyping studies have found that 5% to 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 7 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 5 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 8 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 6.6% 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 pathologic factors, had a 6.4-fold increased risk of all-cause mortality (P = 0.001). IHC confirmed the neuronal character of these tumors. Conclusions: A single-patient classifier was developed that identifies patients with histologic urothelial cancer harboring a NE transcriptomic profile. These tumors represent a high-risk subgroup of MIBC, which may require different treatment.

Objective To assess the effect of patient's sex on response to neoadjuvant chemotherapy (NAC) in patients with clinically nonmetastatic muscle-invasive bladder cancer (MIBC). Methods Complete pathologic response, defined as ypT0N0 at radical cystectomy, and downstaging were evaluated using sex-adjusted univariable and multivariable logistic regression modeling. We used interaction terms to account for age of menopause and smoking status. The association of sex with overall survival and cancer-specific survival was evaluated using Cox regression analyses. Results A total of 1,031 patients were included in the analysis, 227 (22%) of whom were female. Female patients had a higher rate of extravesical disease extension (P = 0.01). After the administration of NAC, ypT stage was equally distributed between sexes (P = 0.39). On multivariable logistic regression analyses, there was no difference between the sexes or age of menopause with regards to ypT0N0 rates or downstaging (all P > 0.5). On Cox regression analyses, sex was associated with neither overall survival (hazard ratio 1.04, 95% confidence interval 0.75–1.45, P = 0.81) nor cancer-specific survival (hazard ratio 1.06, 95% confidence interval 0.71–1.58, P = 0.77). Conclusion Our study generates the hypothesis that NAC equalizes the preoperative disparity in pathologic stage between males and females suggesting a possible differential response between sexes. This might be the explanation underlying the comparable survival outcomes between sexes despite females presenting with more advanced tumor stage.

INTRODUCTION AND OBJECTIVE:Cisplatin-based neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC) is the standard and recommended treatment for clinical T2-T4aN0M0 bladder cancer. Howev...

Precision cancer medicine seeks to target the underlying genetic alterations of cancer; however, it has been challenging to use genetic profiles of individual patients in identifying the most appropriate anti-cancer drugs. This spurred the development of patient avatars; for example, patient-derived xenografts (PDXs) established in mice and used for drug exposure studies. However, PDXs are associated with high cost, long development time and low efficiency of engraftment. Herein we explored the use of microfluidic devices or microchambers as simple and low-cost means of maintaining bladder cancer cells over extended periods of times in order to study patterns of drug responsiveness and resistance. When placed into 75 µm tall microfluidic chambers, cancer cells grew as ellipsoids reaching millimeter-scale dimeters over the course of 30 days in culture. We cultured three PDX and three clinical patient specimens with 100% success rate. The turn-around time for a typical efficacy study using microchambers was less than 10 days. Importantly, PDX-derived ellipsoids in microchambers retained patterns of drug responsiveness and resistance observed in PDX mice and also exhibited in vivo-like heterogeneity of tumor responses. Overall, this study establishes microfluidic cultures of difficult-to-maintain primary cancer cells as a useful tool for precision cancer medicine.

Introduction The neutrophil-to-lymphocyte ratio (NLR) is an attractive marker because it is derived from routine bloodwork. NLR has shown promise as a prognostic factor in muscle invasive bladder cancer (MIBC) but its value in patients receiving neoadjuvant chemotherapy (NAC) before radical cystectomy (RC) is not yet established. Since NLR is related to an oncogenic environment and poor antitumor host response, we hypothesized that a high NLR would be associated with a poor response to NAC and would remain a poor prognostic indicator in patients receiving NAC. Methods A retrospective analysis was performed on patients with nonmetastatic MIBC (cT2-4aN0M0) who received NAC prior to RC between 2000 and 2013 at 1 of 19 centers across Europe and North America. The pre-NAC NLR was used to split patients into a low (NLR ≤ 3) and high (NLR > 3) group. Demographic and clinical parameters were compared between the groups using Student's t test, chi-squared, or Fisher's exact test. Putative risk factors for disease-specific and overall survival were analyzed using Cox regression, while predictors of response to NAC (defined as absence of MIBC in RC specimen) were investigated using logistic regression. Results Data were available for 340 patients (199 NLR ≤ 3, 141 NLR > 3). Other than age and rate of lymphovascular invasion, demographic and pretreatment characteristics did not differ significantly. More patients in the NLR > 3 group had residual MIBC after NAC than the NLR ≤ 3 group (70.8% vs. 58.3%, P = 0.049). NLR was the only significant predictor of response (odds ratio: 0.36, P = 0.003) in logistic regression. NLR was a significant risk factor for both disease-specific (hazard ratio (HR): 2.4, P = 0.006) and overall survival (HR:1.8, P = 0.02). Conclusion NLR > 3 was associated with a decreased response to NAC and shorter disease-specific and overall survival. This suggests that NLR is a simple tool that can aid in MIBC risk stratification in clinical practice.

BackgroundMuscle-invasive bladder cancer (MIBC) is a heterogeneous disease, and gene expression profiling has identified several molecular subtypes with distinct biological and clinicopathological characteristics. While MIBC subtyping has primarily been based on messenger RNA (mRNA), long non-coding RNAs (lncRNAs) may provide additional resolution.MethodsLncRNA expression was quantified from microarray data of a MIBC cohort treated with neoadjuvant chemotherapy (NAC) and radical cystectomy (RC) (n = 223). Unsupervised consensus clustering of highly variant lncRNAs identified a four-cluster solution, which was characterized using a panel of MIBC biomarkers, regulon activity profiles, gene signatures, and survival analysis. The four-cluster solution was confirmed in The Cancer Genome Atlas (TCGA) cohort (n = 405). A single-sample genomic classifier (GC) was trained using ridge-penalized logistic regression and validated in two independent cohorts (n = 255 andn = 94).ResultsNAC and TCGA cohorts both contained an lncRNA cluster (LC3) with favorable prognosis that was enriched with tumors of the luminal-papillary (LP) subtype. In both cohorts, patients with LP tumors in LC3 (LPL-C3) were younger and had organ-confined, node-negative disease. The LPL-C3 tumors had enhanced FGFR3, SHH, and wild-type p53 pathway activity. In the TCGA cohort, LPL-C3 tumors were enriched forFGFR3mutations and depleted forTP53andRB1mutations. A GC trained to identify these LPL-C3 patients showed robust performance in two validation cohorts.ConclusionsUsing lncRNA expression profiles, we identified a biologically distinct subgroup of luminal-papillary MIBC with a favorable prognosis. These data suggest that lncRNAs provide additional information for higher-resolution subtyping, potentially improving precision patient management.

Introduction: We identify the impact of neoadjuvant chemotherapy before open radical cystectomy on perioperative outcomes and identify actionable areas for improvement.Methods: The impact of neoadjuvant chemotherapy on perioperative outcomes after radical cystectomy for muscle invasive bladder cancer from 2003 to 2014 was assessed using an institutional database. Individual outcomes (venous thromboembolism, surgical site infection, cardiac event) and a composite score using the Clavien-Dindo classification were identified. Univariable and multivariable logistic regression models were used to identify predictors of perioperative complication and 30-day readmission rates.Results: A total of 241 patients were included in the study, of whom 175 underwent radical cystectomy alone (72.6%) and 66 were treated with neoadjuvant chemotherapy plus radical cystectomy (27.4%). The 30-day readmission rate for the neoadjuvant chemotherapy cohort was 30.5% compared to 17.2% for radical cystectomy alone. Multivari...

We assessed survival dependent on pathological response after neoadjuvant chemotherapy in a large multicenter patient cohort, with a particular focus on the difference between the absence of residual cancer (pT0) and the presence of only nonmuscle invasive residual cancer (pTa, pTis, pT1).We retrospectively reviewed records of patients with urothelial cancer who received neoadjuvant chemotherapy and underwent radical cystectomy at 19 contributing institutions from 2000 to 2013. Patients with cT2-4aN0M0 and eventual pN0 disease were selected for this analysis. Estimated overall survival was compared between patients with pT0 and pTa/Tis/T1 disease. A multivariable Cox proportional hazards regression model for overall survival was generated to evaluate hazard ratios for variables of interest.Of 1,543 patients treated with neoadjuvant chemotherapy and radical cystectomy during the study period 257 had pT0N0 and 207 had pTa/Tis/T1N0 disease. The Kaplan-Meier mean estimates of overall survival for pT0 and pTa/Tis/T1 cases were 186.7 months (95% CI 145.9-227.6, median 241.1) and 138 months (95% CI 118.2-157.8, median 187.4), respectively (p=0.58). In the Cox proportional hazards regression model for overall survival pTa/Tis/T1N0 status (HR 0.36, 95% CI 0.23-0.67) and pT0N0 status (HR 0.28, 95% CI 0.17-0.47) compared to pT2N0 pathology, positive surgical margin (HR 1.75, 95% CI 1.07-2.86), and receiving a methotrexate, vinblastine, doxorubicin and cisplatin regimen compared to an "other" regimen (HR 0.45, 95% CI 0.27-0.76) were predictors of overall survival.pTa/Tis/T1N0 and pT0N0 stage on the final cystectomy specimen are strong predictors of survival in patients treated with neoadjuvant chemotherapy and radical cystectomy. We did not discern a statistically significant difference in overall survival when comparing these 2 end points.

Background: Muscle-invasive bladder cancer (MIBC) is a heterogeneous disease and gene expression profiling has identified different molecular subtypes, each having distinct biologic and clinicopathologic characteristics. Subtyping MIBC has primarily been messenger RNA (mRNA)-based, although noncoding RNAs, including long noncoding RNAs (lncRNAs), have potential utility in providing additional resolution to current molecular subtyping models. Materials and Methods: The expression profiles of over 6,000 lncRNAs were quantified from whole-transcriptome microarray data of a MIBC patient cohort treated by neoadjuvant chemotherapy (NAC) and radical cystectomy (N=223). Unsupervised consensus clustering of the most highly variant lncRNAs identified a four-cluster solution, which was further characterized using a panel of MIBC biomarkers, gene signatures, and survival analysis. The four-cluster consensus was validated using the publicly available The Cancer Genome Atlas (TCGA) radical cystectomy cohort (N=405). A single-sample genomic classifier (GC) was trained using ridge-penalized logistic regression and then validated in two independent patient cohorts (N=255 and N=94). Results: In the NAC and TCGA cohorts, survival analysis of a lncRNA-based consensus cluster solution revealed a lncRNA-cluster (LC3) with strikingly good prognosis that was enriched for tumors of the luminal-papillary mRNA subtype. In both cohorts, the luminal-papillary tumors from this cluster (LPL-C3) were clinically less aggressive than other luminal-papillary tumors. Patients having LPL-C3 tumors were younger and had more frequent organ-confined, node-negative disease than other luminal-papillary tumors. LPL-C3 tumors were characterized by enhanced FGFR3 pathway activity, wild-type P53 expression, and robust SHH signaling. In the TCGA cohort, LPL-C3 tumors were also enriched for FGFR3 mutations and depleted for TP53 and RB1 mutations. A GC trained to identify these LPL-C3 patients showed robust performance in two validation cohorts. Conclusions: Using lncRNA expression profiling, we identified a biologically distinct subgroup of luminal-papillary MIBC with less-aggressive molecular characteristics and favorable prognosis. These data suggest that lncRNAs can provide additional information in resolving higher-resolution subtypes for more precise patient management strategies. Citation Format: Joep J. de Jong, Yang Liu, Roland Seiler, A. Gordon Robertson, Michiel S. van der Heijden, Jonathan L. Wright, James Douglas, Marc Dall'Era, Simon J. Crabb, Bas W.G. van Rhijn, Kim E.M. van Kessel, Elai Davicioni, Yair Lotan, Ellen C. Zwarthoff, Peter C. Black, Joost L. Boormans, Ewan A. Gibb. A long noncoding RNA-based genomic classifier identifies a subset of luminal muscle-invasive bladder cancer patients with favorable prognosis [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr B07.

© 2018 American Urological Association Education and Research, Inc. Introduction: Radical cystectomy with neoadjuvant chemotherapy is the standard of care for patients with localized muscle invasive urothelial carcinoma of the bladder. One of the strongest predictors of survival in these patients is pathological response to initial treatment. Our objective was to determine whether we could stratify the need for radical cystectomy based on pathological response to neoadjuvant chemotherapy. Methods: We present a cohort of patients with muscle invasive urothelial carcinoma of the bladder to whom surveillance and bladder preservation were offered if complete response was achieved following neoadjuvant chemotherapy. Descriptive statistics and survival analysis were performed to assess overall, cancer specific and metastasis-free survival. Patients were stratified based on pathological response to neoadjuvant chemotherapy. Results: A total of 60 patients were included in the cohort, of whom 32 (55%) had absence of residual disease on post-neoadjuvant chemotherapy transurethral resection and 27 (45%) had persistent disease. Of patients undergoing surveillance 52% maintained the bladder without evidence of recurrence. By comparison, of those with recurrence only 20% preserved the bladder and were without evidence of disease. Conclusions: Long-term followup shows a subset of patients achieving good outcomes while preserving the bladder. However, we also observed an inability to reliably identify this subset of patients given current clinical and pathological markers. Until we are able to achieve that goal, the safest oncologic approach remains neoadjuvant chemotherapy followed by radical cystectomy.

Cisplatin-based neoadjuvant chemotherapy (NAC) for muscle invasive bladder cancer improves all-cause and cancer specific survival. We aimed to evaluate whether the detection of carcinoma in situ (CIS) at the time of initial transurethral resection of bladder tumor (TURBT) has an oncological impact on the response to NAC prior to radical cystectomy. Patients were identified retrospectively from 19 centers who received at least three cycles of NAC or induction chemotherapy for cT2-T4aN0-3M0 urothelial carcinoma of the bladder followed by radical cystectomy between 2000 and 2013. The primary and secondary outcomes were pathological response and overall survival, respectively. Multivariable analysis was performed to determine the independent predictive value of CIS on these outcomes. Of 1213 patients included in the analysis, 21.8% had concomitant CIS. Baseline clinical and pathologic characteristics of the ‘CIS’ versus ‘no-CIS’ groups were similar. The pathological response did not differ between the two arms when response was defined as pT0N0 (17.9% with CIS vs 21.9% without CIS; p = 0.16) which may indicate that patients with CIS may be less sensitive to NAC or ≤ pT1N0 (42.8% with CIS vs 37.8% without CIS; p = 0.15). On Cox regression model for overall survival for the cN0 cohort, the presence of CIS was not associated with survival (HR 0.86 (95% CI 0.63–1.18; p = 0.35). The presence of LVI (HR 1.41, 95% CI 1.01–1.96; p = 0.04), hydronephrosis (HR 1.63, 95% CI 1.23–2.16; p = 0.001) and use of chemotherapy other than ddMVAC (HR 0.57, 95% CI 0.34–0.94; p = 0.03) were associated with shorter overall survival. For the whole cohort, the presence of CIS was also not associated with survival (HR 1.05 (95% CI 0.82–1.35; p = 0.70). In this multicenter, real-world cohort, CIS status at TURBT did not affect pathologic response to neoadjuvant or induction chemotherapy. This study is limited by its retrospective nature as well as variability in chemotherapy regimens and surveillance regimens.

Nanotechnology is being developed for the diagnosis and treatment of both nonmyoinvasive bladder cancer (NMIBC) and invasive bladder cancer. The diagnostic applications of nanotechnology in NMIBC mainly focus on tumor identification during endoscopy to increase complete resection of bladder cancer while nanotechnology to capture malignant cells or their components continues to be developed. The therapeutic applications of nanotechnology in NMIBC are to reformulate biological and cytotoxic agents for intravesical instillation, combine both diagnostic and therapeutic application in one nanoformulation. In invasive and advanced bladder cancer, magnetic resonance imaging with supraparamagnetic iron oxide nanoparticles can improve the sensitivity and specificity in detecting small metastasis to lymph nodes. Nanoformulation of cytotoxic agents can potentially decrease the toxicity while increasing efficacy.

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.

TPS4594 Background: Neoadjuvant cisplatin-based chemotherapy before radical cystectomy (RC) improves outcomes but ~50% of patients (pts) are cisplatin-unfit. Anti-PD(L)1 agents can prolong overall survival (OS) in platinum-resistant advanced BC and have shown high pathologic complete response rate (pCR) and safety as single agent in phase II trials in the neoadjuvant setting. The combination of anti-PD-1 and anti-KIR agents is feasible and very attractive based on complementary and non-overlapping roles in regulating adaptive and innate immune response as well as impacting the function CD8+ T and NK-cells. Higher CD8+ T cell density (TCD) at RC tissue correlates with longer OS. We hypothesize, that combining anti-PD1 (N) with anti-KIR (L) is safe and feasible as neoadjuvant therapy in cisplatin-unfit pts and results in high CD8+ TCD at RC. Methods: Phase Ib multi-institutional trial evaluating 2 doses (4 weeks apart) of N alone or N+L in 2 cohorts; pts will be assigned sequentially to N (Cohort 1), and if there is no negative safety signal after the first 12 pts, subsequent pts will be assigned to N+L (Cohort 2). Key eligibility: cT2-4aN0-1M0 stage, ≥20% tumor at TURBT, adequate organ function, no autoimmune disease within 2 years, no concurrent invasive upper urinary tract carcinoma or other active cancer. Primary endpoint: safety based on CTCAE v5.0 measured as the rate of ≥G3 treatment related adverse events (AE). Key secondary endpoints: CD8+ TCD absolute and % change between TURBT and RC, % of pts who do not get RC within 6 weeks after neoadjuvant treatment due to treatment-related AE, % pCR, recurrence-free survival, and evaluation of biomarkers in tumor tissue, blood, urine. Rates of ≥Grade 3 AE with neoadjuvant treatment will be reported along with 90% exact binomial CI. In Cohort 1, maximum CI width is 0.51; in Cohort 2, it is 0.36. Our hypothesis is that the change in CD8+ TCD between TURBT and RC will be about 3 CD8+ T cells / 100 tumor cells within HPF. Up to 43 pts will be enrolled for 36 eligible, treated pts (12:N, 24:N+L). Cohort 1 and 2 have 81% and 98% power, respectively, to detect the hypothesized difference with 1-sided type I error rate of 0.05. Trial is open to accrual in US. Clinical trial information: NCT03532451.

INTRODUCTION AND OBJECTIVES: Specific practices in the management of non-muscle invasive bladder cancer (NMIBC) have strong evidence supporting their benefit in reducing rates of recurrence and progression. These key practices include the utilization of intravesical chemotherapy following transurethral resection of bladder tumor (TURBT) for low grade (LG) disease, as well as re-staging TURBT and intravesical BCG therapy for high grade (HG) or T1 disease. However, recent studies, including the featured best poster at the AUA meeting in 2015, report alarmingly low adoption of such practices at a population level.(1) We report institutional rates of utilization of such practices following the implementation of quality improvement initiatives to determine reasonable expected benchmarks for implementation of these practices. METHODS: As part of a quality assessment and improvement initiative within our institution, data was prospectively collected for all patients with a new diagnosis of NMIBC undergoing TURBT over a 2 year period spanning 2014 and 2015. Summary data at the institution level was reported back to all providers to increase awareness and provide feedback. Data was collected on patient medical history, clinical and pathologic staging, and treatments performed. Reasons for failure to utilize specific practices in otherwise eligible patients were also assessed. RESULTS: A total of 153 patients were enrolled over a 2 year period across all stages of bladder cancer. Among incident cases of LG NMIBC, 77% received immediate intravesical chemotherapy following TURBT. Among patients with HG or T1 NMIBC, 76% underwent repeat TURBT within 3 months. When considering patients scheduled for restaging TURBT but unable to complete their surgery, rates increased to 92%. For patients with either HG or T1 disease, 91% underwent either induction intravesical therapy or radical cystectomy. The remaining 9% of patients who did not receive appropriate therapy either refused recommended treatment or were unable to receive due to the BCG shortage. CONCLUSIONS: In contrast to a wealth of data demonstrating shortcomings at the population level in the utilization of evidence based practices in NMIBC, we present encouraging results from a local quality initiative with a focus on maximizing such practices. We also report expected achievable rates of utilization with minimal intervention in the current practice environment. (1) Nabbout P, Slaton J. BCG and re-staging transurethral bladder tumor resection underused for T1 bladder cancer. AUA News. 20(10): 11-12, October 2015.

433 Background: After cisplatin-based neoadjuvant chemotherapy (NAC) almost two thirds of patients have residual muscle-invasive bladder cancer (MIBC) present at radical cystectomy (RC). The alterations induced by NAC in these cisplatin-resistant tumors remain largely unstudied. Here, we aim to investigate the characteristics of cisplatin-resistant tumors. Methods: RC samples were available for gene expression analysis from 133 patients with residual invasive disease after cisplatin-based NAC, of whom 116 had matched pre-NAC samples. In addition, the tumor bed (scar tissue) of 21 post-NAC RC specimens with no residual tumor was profiled. Unsupervised consensus clustering (CC) was performed and the CC were investigated for their biological and clinical characteristics. H&E and immunohistochemistry (KRT5/6, GATA3, KI67 and CD8) were used to confirm tissue sampling and gene expression analysis. Results: Unsupervised consensus clustering yielded four distinct consensus clusters (CC). Consistent basal-(CC1) and luminal-like (CC2) phenotype similar to pre-NAC subtyping was observed in 42% of cases. One third of cases became immune-infiltrated (CC3) in the post-NAC setting but lacked basal and luminal markers. These tumors expressed a strong T-cell signature, chemokine signaling and checkpoint molecules. Conversely, CC4 was associated with healing/scarring. This ‘scar-like’ character of CC4 was consistent with the scar samples. Despite being pathological non-responders, the relative risk of death for CC4 was 2.8 and 3 times less than CC2-Luminal (p = 0.038) and CC3-Infiltrated (p = 0.018), respectively. Luminal-like pre-NAC samples were more likely to adopt a scar-like character (CC4) in the post-NAC setting, while the basal-like tumors were more likely to develop luminal features (CC2). Conclusions: This study expands our knowledge of cisplatin-resistant MIBC by suggesting molecular subtypes to understand the biology of these tumors. Clinical trials are necessary to test the impact of these molecular subtypes with respect to selection of adjuvant and salvage treatments. Post-NAC immune infiltration could have implications for subsequent immunotherapy.

440 Background: Neuroendocrine (NE) carcinoma is a rare and aggressive variant of muscle invasive bladder cancer (MIBC). Molecular subtyping studies found 5-15% of bladder tumors had transcriptome profiles consistent with NE carcinoma but lacked NE histology (Robertson 2017, Sjödahl 2017). Identifying NE variants may have prognostic implications and modify treatment recommendations. In this study, we present a robust genomic classifier trained to identify NE carcinoma. Methods: Transcriptome-wide expression profiles were generated for 576 MIBC patients collected from seven institutions. Model training included profiles generated from TURBT and RC specimens from 320 patients prior to treatment with NAC or chemo-radiation. The validation cohort consisted of 256 RC specimens (no prior systemic treatment). Using 10 MIBC-related gene sets, a GLMNET model was built to predict patients with a NE carcinoma expression profile. Uni- and multi-variable survival analyses were used to characterize outcomes of the predicted NE tumors. Results: In the training set, hierarchical clustering using a panel of 54 genes showed a cluster of 17 patients (5.3%) with a NE carcinoma expression profile. These patients had significantly worse 1 year progression free survival (65% vs 82% for NE vs overall; p = 0.046). In the validation set, 7 tumors were classified (2.7%) as NE with 4 (57%) patients dying from the disease at 1 year after RC. Within 3 years of RC, 100% (7/7) of patients with NE tumors had died. After adjusting for various clinical and pathological factors, patients with predicted NE tumors had a 6.40 increased risk of all-cause mortality (p = 0.001). Conclusions: We have developed a gene expression signature that predicts a particularly high-risk group that may need treatment intensification, alternative chemotherapy or clinical trials. Validation will be required to assess the potential clinical utility of this NE carcinoma classifier.

Primary signet-ring cell carcinoma (SRCC) of the urinary bladder, a variant of adenocarcinoma, is exceedingly rare and as a result no gold standard exists for its management. We report a case of primary SRCC of the bladder with recurrent metastases; we utilized an innovative diagnostic approach and the patient exhibited a treatment response to palliative FOLFOX-6 chemotherapy.

© 2016 IOP Publishing Ltd. Chemotherapy commonly used in the treatment of advanced bladder cancer is only moderately effective and associated with significant toxicity. There has been no appreciable improvement in overall survival over the last three decades. The goal of this project is to develop and characterize bladder cancer-specific nanometer-scale micelles loaded with the chemotherapeutic drug paclitaxel (PTX) and determine the anti-tumor activity and toxicity. Micelle-building-material telodendrimers were synthesized through the stepwise conjugation of eight cholic acid units at one terminus of polyethylene glycol (PEG) and a bladder cancer-specific targeting peptide named PLZ4 at the other terminus. To synthesize disulfide-crosslinked PLZ4 nanomicelles (DC-PNM), cysteine was introduced between the cholic acid and PEG. DC-PNM-PTX was synthesized through the evaporation method by loading PTX in the core. The loading capacity of PTX in DC-PNM was 25% (W/W). The loading efficiency was over 99%. DC-PNM-PTX was spherical with the median size of 25 nm. The stability of DC-PNM-PTX was determined in a solution containing sodium docecyl sulfate (SDS). It was stable in a SDS solution, but dissolved within 5 min after the addition of glutathione at the physiological intracellular concentration of 10 mM. In vivo targeting and anti-tumor activity were determined in immunodeficient mice carrying patient-derived bladder cancer xenografts (PDXs). After intravenous administration, DC-PNM specifically targeted the bladder cancer PDXs, but very little to the lung cancer xenografts in the same mice (p < 0.001). DC-PNM loaded with PTX overcame cisplatin resistance, and improved the median survival from 55 d with free PTX to 69.5 d (p = 0.03) of mice carrying PDXs. In conclusion, DC-PNM remained stable in the SDS solution, specifically targeted the bladder cancer xenografts in vivo, and improved the anti-cancer efficacy of PTX.

The current standard treatment for muscle-invasive nonmetastatic bladder cancer is neoadjuvant platinum-based chemotherapy followed by radical cystectomy. However, neoadjuvant chemotherapy is not widely accepted even with level 1 evidence. Adjuvant chemotherapy should be discussed if patients have not received neoadjuvant chemotherapy before surgery and have high-risk pathologic features. Although not considered standard of care, bladder-sparing therapy can be considered for highly selected patients and for those medically unfit for surgery. Even though there are no level 1 data, the treatment outcomes for highly select patients given bladder-sparing therapy appear promising, with many patients retaining a functional bladder. Personalized chemotherapy is currently being actively pursued to target the underlying molecular changes and tailor to individual needs.

Purpose: DNA adduct formation is a critical step in platinum(Pt)–induced cell death. The goal of this project is to measure carboplatin-DNA adducts after a single non-toxic microdose of [14C]carboplatin, use this information to predict efficacy before cancer patients receive toxic chemotherapy and assess the underlying chemoresistance mechanisms, thus enhancing personalized therapy. Methods: Six non-small cell lung cancer (NSCLC) and six bladder cancer cell lines were treated with 1 μM (microdose) or 100 μM (therapeutic dose) carboplatin, but with equal amounts of [14C]carboplatin. Carboplatin-DNA monoadduct levels were determined by measuring 14C content in genomic DNA with accelerator mass spectrometry (AMS). Cellular sensitivity to carboplatin (IC50 values) was correlated with drug-DNA adduct levels. To validate the in vitro findings, a pilot Phase 0 trial with microdosing [14C]carboplatin was initiated to determine the recommended Phase II dose (RP2D) of [14C]carboplatin for optimal measurement of DNA monoadducts and pharmacokinetics (PK) in lung and bladder cancer. PK and DNA monoadduct levels in peripheral blood mononuclear cells and tumor tissues were determined by liquid scintillation counting and AMS, respectively, validated with inductively coupled plasma mass spectrometry (ICP-MS), and correlated with tumor response to chemotherapy. Results: DNA monoadduct levels in cell lines induced by microdose and therapeutic concentrations of carboplatin were highly linearly proportional (r2 = 0.95, p < 0.001) as measured by AMS, and correlated to the total Pt-DNA adduct levels induced by therapeutic carboplatin (r2 = 0.80, p < 0.001) as measured by ICP-MS. DNA adduct levels were superior in predicting resistance to carboplatin when compared to the mRNA expression of ERCC1, a DNA repair gene commonly used as a resistant biomarker of Pt-based chemotherapy. Nine patients were recruited at the pilot Phase 0 trial. The Level I dose of [14C]carboplatin at 107 dpm/Kg with total carboplatin at 1% of therapeutic dose (area under curve of 6) was determined as the RP2D. No clinical toxicity was observed as a result of microdosing. The radiation exposure from 14C was less than a chest X-ray per patient. DNA monoadduct levels and PK parameters of carboplatin at the microdose and therapeutic doses were highly correlated (p Conclusions: This novel microdosing approach predicts DNA monoadduct levels and PK of therapeutic carboplatin dosing; preliminary data suggest the feasibility of predicting chemosensitivity and the possibility of guiding personalized dosing. Citation Format: Chong-xian Pan, Sisi Wang, Miaoling He, Tao Li, Tzu-yin Lin, Hongyong Zhang, Michael Malfatti, David Gandara, Tianhong Li, Ken Yoneda, Joyce Lee, Marc Dall'Era, George Cimino, Ralph de Vere White, Kenneth Turteltaub, Paul Henderson. Phase 0 microdosing trial quantitating DNA adduct formation to identify platinum chemosensitivity: from bench to bedside. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1183. doi:10.1158/1538-7445.AM2013-1183

The efficacy of neoadjuvant chemotherapy (NAC) for muscle-invasive bladder cancer (BCa) was established primarily with methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC), with complete response rates (pT0) as high as 38%. However, because of the comparable efficacy with better tolerability of gemcitabine and cisplatin (GC) in patients with metastatic disease, GC has become the most commonly used regimen in the neoadjuvant setting.We aimed to assess real-world pathologic response rates to NAC with different regimens in a large, multicenter cohort.Data were collected retrospectively at 19 centers on patients with clinical cT2-4aN0M0 urothelial carcinoma of the bladder who received at least three cycles of NAC, followed by radical cystectomy (RC), between 2000 and 2013.NAC and RC.The primary outcome was pathologic stage at cystectomy. Univariable and multivariable analyses were used to determine factors predictive of pT0N0 and ≤pT1N0 stages.Data were collected on 935 patients who met inclusion criteria. GC was used in the majority of the patients (n=602; 64.4%), followed by MVAC (n=183; 19.6%) and other regimens (n=144; 15.4%). The rates of pT0N0 and ≤pT1N0 pathologic response were 22.7% and 40.8%, respectively. The rate of pT0N0 disease for patients receiving GC was 23.9%, compared with 24.5% for MVAC (p=0.2). There was no difference between MVAC and GC in pT0N0 on multivariable analysis (odds ratio: 0.89 [95% confidence interval, 0.61-1.34]; p=0.6).Response rates to NAC were lower than those reported in prospective randomized trials, and we did not discern a difference between MVAC and GC. Without any evidence from randomized prospective trials, the best NAC regimen for invasive BCa remains to be determined.There was no apparent difference in the response rates to the two most common presurgical chemotherapy regimens for patients with bladder cancer.