Microbial profiling of urothelial carcinoma and benign bladder tissue from formalin-fixed specimens.

To evaluate the ability of bacille-Calmette Guèrin (BCG) to induce caspase-independent cell death and release the necrosis-associated chemokine high molecular group box protein 1 (HMGB1) from urothelial carcinoma (UC) cells; a correlative clinical trial determined if BCG treatment resulted in increased urinary levels of HMGB1. The human UC cell lines 253 J and T24 were pretreated with apoptosis inhibitors, exposed to BCG, and cell viability and ultrastructural changes measured. HMGB1 levels were assessed in cell culture supernatant after BCG treatment. The expression/function of HMGB1 receptors on the UC cell lines was determined by reverse transcription-polymer chain reaction and the ability of exogenous HMGB1 to activate nuclear factor (NF)-kappaB signalling assessed. An HMGB1 enzyme-linked immunosorbent assay was used to measure HMGB1 levels in urine obtained from BCG-treated patients. Inhibition of apoptotic pathways failed to inhibit BCG-induced cell death in UC cells. Electron microscopy showed BCG-dependent ultrastructural changes consistent with cellular necrosis. BCG exposure resulted in a binary increase in cell culture supernatant levels of HMGB1. UCs expressed multiple HMGB1 receptors. Treatment of UCs with HMGB1 activated NF-kappaB. In the clinical setting, six of seven patients had increased urinary levels of HMGB1 at 24 h after BCG treatment. BCG causes direct cytotoxicity in a subpopulation of UC cells. This cytotoxicity is caspase-independent and associated with ultrastructural changes and cellular protein release (HMGB1), characteristic of necrosis. Urinary levels of HMGB1 can be elevated in patients after BCG treatment. The expression and function of HMGB1 receptors in UC cells, coupled with the known role of HMGB1 on the host immune response, suggest a role for necrosis and HMGB1 release in the antitumour effect of BCG.

Background: Muscle invasive bladder cancer (MIBC) neoadjuvant responders as opposed to nonresponders demonstrated significantly improved 5-year cancer specific survival and reduced nodal positivity rates. Identification of chemo responsive cohorts would not only facilitate targeted delivery of chemotherapy to those most likely to benefit, but also avoid morbidity and delayed surgical intervention in patients unlikely to derive benefit. We evaluated the role of gene and miRNA expression profiles in initial TURBT (transurethral resection of bladder tumor) specimens of patients before undergoing neoadjuvant chemotherapy and subsequent radical cystectomy. Pathological response at time of cystectomy was used to classify initial TURBT specimens as responders (pT0) versus non-responders (≥pT2). Differential expression of gene and miRNA between the two groups may help stratify patients being considered for neoadjuvant chemotherapy. Methods: TURBT specimens from patients with MIBC were preserved in FFPE tissue blocks before patients subsequently received cisplatin-based neoadjuvant chemotherapy. A total of 13 pT0 and 17 ≥pT2 patients were selected and matched for age, gender and tumor stage. RNA was extracted from FFPE blocks using an Ambion total nucleic acid isolation kit. mRNA was sequenced using Illumina TruSeq RNA Access Library and NextSeq technology. Gene counts were assessed by ht-seq counts and differential expression using DESeq2. Pathway analysis was performed using GAGE. Differential expression of 754 miRNAs was analyzed using the TaqMan openarray miRNA panel. Results: In responders, a gene-set enrichment analysis revealed significant upregulation of genes in the hsa00190 pathway (implicated in oxidative phosphorylation), hsa03040(spliceosome function). In non-responders, there was significant upregulation of genes in the hsa04510 pathway (focal adhesion), hsa04512 (ECM-receptor interaction), hsa04310(Wnt signaling), hsa04350(TGF-beta signaling) and hsa04010 (MAPK signaling). For the miRNA analysis, significant upregulation of miR-23a, miR-27a, miR-135b, miR-145, miR-422a was seen in responders, and miR-93, miR-107, miR-339-3p, miR-532 in non-responders. Using a random forest classification, a significant proportion of non-responders were correctly identified compared to responders. Conclusion: Gene and miRNA expression in initial MIBC TURBT specimens may be used as an aid in predicting neoadjuvant non-responders. Increasing the sample size is needed to further validate our findings and may lead to a successful chemotherapy prediction model. Citation Format: Neal Murphy*, Paras Shah*, Annette Lee, Thomas Bradley, Manish Vira, Ilya Korsunsky, Andrew Shih, Oksana Yaskiv, Zachary Kozel, Anthony Liew, Houman Khalili, Xinhua Zhu. Determining neoadjuvant cisplatin-based chemosensitivity in muscle invasive bladder cancer through differential gene and miRNA expression analysis [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 3423.

Intravesical Bacillus Calmette-Guérin (BCG) is the standard therapy for non-muscle invasive bladder cancer (NMIBC); however, many patients experience recurrence or progression. We examined how urinary immune signals and the urinary microbiome change across BCG and are related to outcomes. In this single-center prospective cohort study, adults with NMIBC underwent transurethral resection of bladder tumor (TURBT), followed by BCG induction. Urine was collected before TURBT, before BCG, after BCG induction, and three months later. Urine sediment mRNA (PD-L1, PD-L2, CD33, and CD204) was quantified using TaqMan ΔCt. The urinary microbiome was profiled using 16S rRNA gene sequencing, and diversity, composition, and taxon balance were evaluated using nonparametric tests, PERMANOVA, repeated-measures correlations, and mixed-effects models. We analyzed the relationship between the urinary microbiome and prognosis. Twenty-three patients were analyzed; ten recurrences, eight progressions, and three deaths were observed. Relative to baseline, CD33 increased after BCG and after three months; PD-L2 increased immediately after BCG and returned to baseline by three months; PD-L1 and CD204 increased after BCG. Shannon alpha-diversity was unchanged, but total read count was higher at three months, with stable beta-diversity and dispersion. Higher PD-L1 expression was associated with lower Actinobacteria abundance in the bladder cancer microenvironment. A higher post-BCG Firmicutes/Bacteroidetes ratio was associated with worse prognosis, with the clearest signal for progression-free survival (PFS) observed in the univariate Cox models. Higher post-BCG Corynebacterium and Enterobacteriaceae abundance was associated with better PFS. BCG was associated with higher urinary PD-L1/PD-L2 and myeloid marker transcripts, while overall community structure remained stable. These exploratory data support that pre-BCG microbial features may be related to early response, and post-BCG profiles may reflect durability and survival. Urine immune-microbiome profiling could be a feasible, noninvasive adjunct for monitoring and risk stratification in NMIBC.

Objective: Bladder cancer is the commonest cancer of the urinary tract. Transurethral Resection of Bladder Tumour (TURBT) is the gold standard for diagnosis and treatment of non-muscle invasive bladder cancer. The absence of muscle in a TURBT specimen is associated with a significantly higher risk of residual disease, early recurrence and tumour under staging. Materials and methods: TURBT and bladder biopsy specimens were examined before and after the introduction of an open reporting system as a quality improvement exercise. All specimens from the 4th quarter (between 2010 and 2014) were examined to determine the effect of open reporting on our inadequate resection rates. Results: A total of 244 cases were performed under the care of 5 consultant urologists. Analysis revealed a significant improvement in quality of both T1 and Ta resections (p=0.04*; p=0.02*) after the introduction of open reporting. The total number of TURBT cases increased per year, however the percentage of inadequate resections has significantly decreased (p=0.02*). Conclusion: Individual reporting provided surgeons with direct, personal and timely feedback on their performance. It did not negatively impact on trainee participation, but led to improved training outcomes. We have demonstrated that our simple intervention has improved quality of patient care.

e15021 Background: Studies in breast and prostate surgery show reduced cancer recurrence after regional (RA) versus general anesthesia (GA). Mechanisms include RAÕs reduced post-operative opioid use and cortisol-mediated immunosuppression. RA may be used alone during transurethral resection of bladder tumors (TURBT) and in combination with GA during radical cystectomy (RC). We assess the impact of RA on short-term bladder urothelial cell carcinoma (UCC) recurrence after TURBT or RC. Methods: From 8/2001 and 6/2006 to 6/2011, 151 patients underwent RC and 488 patients underwent TURBT for bladder UCC, respectively. Those with incomplete resection on TURBT were excluded. Anesthesia included RA or GA for TURBT, and GA alone or GA + RA for RC. Multivariate logistic regression was performed to identify significant predictors of biopsy- or radiography-confirmed UCC recurrence. Results: TURBT. Of 252 patients, 211 received GA and 41 received RA during TURBT. Patient and operative characteristics were similar between groups. Recurrence was 56% at 12 months for GA and RA. Multivariate analysis revealed clinical stage to be the only predictor of UCC recurrence (HR=1.8, p<0.0001). Anesthesia had no affect on 6 or 12 month RFS, DSS or OS (see table). RC. GA was used in 114 patients and 37 patients had GA + RA at RC. There were no between group differences in patient or tumor characteristics. After follow-up of 18 months, 25.9% and 21.6% recurred in GA and GA+RA groups, respectively (p>0.05). There were no differences in RFS, DSS, or OS (see Table). Conclusions: Contrary to other malignancies, our data suggest anesthesia type at TURBT or RC does not affect bladder cancer outcomes. Anesthesia modality should be based on patient comorbidities and procedure type. [Table: see text]

Intravesical therapy for bladder cancer

295 Background: Randomized phase III clinical trial data (S8710) supports an overall survival (OS) advantage with neo-CTx for muscle-invasive urothelial carcinoma (miUC) patients (pts) prior to cystectomy. Recent S8710 subset analyses have demonstrated an OS for pts with both pure UC and VarHst. pCR to neo-CTx has been suggested as a surrogate endpoint for OS. The relationship between VarHist in TURBT specimens and pCR rates is uncertain. Methods: A retrospective review of the Indiana University Simon Cancer Center urology and medical oncology clinical databases was performed spanning the years 1991 – 2012. Subjects with miUC, pathology reports available for both TURBT and cystectomy procedures, and confirmed receipt of neo-CTx with regimen details were included in this analysis. Pts with clinically positive lymph nodes (LN+) were included provided they underwent cystectomy with curative intent and distant metastases were not present. Associations between pCR and pt baseline age, gender, race, clinical stage (T2N0 vs. T3/T4/N+), chemotherapy regimen received (cisplatin combination therapy (CisCTx) vs. non-cisplatin based), and presence of VarHst on TURBT sample were tested by multinomial logistic regression analysis with statistical significance set at p<0.05. Results: 72 miUC pts satisfying the inclusion criteria were identified. Cohort demographics included: age (median) – 59 yrs, 76% male, 93% Caucasian, 63% T2N0, 32% LN+, 81% CisCTx neo-CTx regimen, 42% VarHst on TURBT, pCR for entire cohort 18%. The presence of VarHst on TURBT sample was not associated with decreased rates of pCR (6/30 vs. 7/42) p=0.610. A trend toward significance with age over 59 was also observed. Conclusions: The presence of VarHst in TURBT specimen is not associated with decreased rates of pCR at cystectomy in miUC pts treated with neo-CTx. Further characterization of the amount of VarHst and reproducibility of its recognition in TURBT samples is warranted to determine its ultimate clinical value.

In the present study we tested the role of Human Epidermal Growth Factor Receptor-2 (HER-2) expression, as assayed by immunohistochemistry, in predicting recurrence and progression in 67 patients with T1G3 BC having undergone transurethral resection of bladder tumor (TURBT) alone (33) or TURBT + Bacillus Calmette Guerin (BCG) instillations (34). All patients had a negative restaging TURBT within 4 months after the first TURBT. At median follow-up of 75.7 months, the overall disease-free and progression-free rates were 35.8% and 73.0%, respectively. Univariate Kaplan-Meier survival analysis showed that traditional prognostic factors (sex, tumor number/size/recurrence) failed to predict disease-free and progression free survival (DFS, PFS). BCG treatment was a significant predictor of DFS (p=0.0231) but not of PFS (p=0.6901). HER-2 overexpression was a significant predictor of DFS (p=0.0013) and PFS (p=0.0322) in the overall patients population, but failed to predict PFS when patients were stratified for treatment (BCG: p=0.1290; no BCG: p=0.1696) probably due to the limited number of events. Multivariate Cox proportional-hazards regression analysis confirmed that BCG treatment was a significant predictor of DFS (p=0.012) but not of PFS (p=0.924), whereas HER-2 overexpression was a significant predictor of DFS (p=0.001) and PFS (p=0.041). These findings suggest that HER-2 status performs better than “traditional” prognostic factors as well as of BCG treatment in predicting the outcome of T1G3 BC, thus providing grounds for further testing this marker and possibly incorporating it in a panel of molecular markers that could reliably predict the behavior of this challenging disease.

Transurethral resection of bladder tumour (TURBT) is the standard approach for diagnosing and staging non-muscle invasive bladder cancer. Accurate staging depends on the presence of muscularis propria (MP) in resected tumour specimens, and inadequate MP sampling may necessitate repeat procedures. Non-linear microscopy (NLM), a laser-scanning, non-destructive imaging technique, enables real-time evaluation of fresh tissue and has the potential to improve staging accuracy intraoperatively. We retrospectively reviewed 94 TURBT pathology reports with high-grade urothelial carcinoma to assess MP sampling rates by tumour stage. MP was present in 55% (52/94) of cases, with variability across stages: 55% (23/42) in high-grade pTa, 39% (9/23) in pTis, 55% (11/20) in pT1 and 100% (9/9) in pT2. NLM was used to evaluate six fresh and 25 archived formalin-fixed, paraffin-embedded (FFPE) TURBT specimens. Fresh tissues were stained and imaged in real time, while thick sections from FFPE specimens were deparaffinised, imaged using NLM and converted to a digital format analogous to whole-slide images. NLM provided high-resolution imaging of MP as distinct, thick smooth muscle bundles in fresh specimens. Furthermore, NLM images of deparaffinised sections closely resembled conventional H&E histology, and a blinded reader achieved a sensitivity of 95% and specificity of 100% for MP detection. This proof-of-concept study supports the feasibility of NLM for intraoperative MP assessment during TURBT. By providing rapid, high-resolution and non-destructive tissue evaluation, NLM has the potential to improve staging accuracy, optimise intraoperative surgical decision-making and reduce the need for repeat TURBT.

To explore whether intravesical chemotherapy (IVC) between primary and second transurethral resection of bladder tumor (TURBT) affects the prognosis of non-muscle invasive bladder cancer (NMIBC) patients receiving Bacillus Calmette-Guérin (BCG) treatment. NMIBC patients who underwent a second TURBT and subsequent BCG treatment between 2012 and 2023 at the Affiliated Hospital of Xuzhou Medical University were retrospectively analyzed. These patients were divided into Group A, which received IVC between TURBT, and Group B, which did not. Recurrence-free survival (RFS) was compared among the different risk subgroups. A total of 292 NMIBC patients were included in this study. In the entire cohort, IVC treatment between the primary and second TURBT was associated with longer RFS (P = 0.009). When stratified by risk groups, in intermediate-risk patients, the difference in RFS between the groups was not statistically significant (P = 0.434). By contrast, for high-risk patients, the treated group exhibited a better prognosis compared to the non-treated group (85.6% vs. 77.6%, P = 0.007). In both univariate and multivariate COX regression analyses, after adjusting for clinical factors such as tumor stage and tumor grade, the IVC between the primary and second TURBT remained an independent prognostic factor for NMIBC patients (HR 0.571, 95% CI [0.380, 0.859], p = 0.007). IVC treatment administered between the primary and second TURBT has been demonstrated to enhance RFS of high-risk NMIBC patients undergoing BCG treatment, whereas it is not applicable to intermediate-risk patients.

The influence of immunocompromised status on recurrence and progression free survival among nonmuscle invasive bladder cancers (NMIBCs) undergoing transurethral resection of bladder tumor (TURBT) and adjuvant intravesical bacillus Calmette Guerin (BCG): Analysis of USA insurance claim data.

Intravesical Bacillus Calmette-Guérin (BCG) is an established adjuvant therapy for high-risk superficial bladder cancer, though its efficacy varies among patients. Recent interest in the urinary microbiome comprising microorganisms inhabiting the urinary tract stems from its potential impact on various urological conditions, including bladder cancer. Our study investigates the possible relationship between the bladder microbiome and BCG therapy outcomes in a preliminary and explorative analysis. We conducted a retrospective, descriptive study involving 31 high-risk bladder cancer (BC) patients treated with BCG. BC tissues were collected pre-treatment, and formalin-fixed paraffin-embedded (FFPE) samples were analyzed. DNA extracted from these samples underwent high-throughput 16S rRNA amplicon sequencing targeting the V1-V3 regions. Our cohort consisted of 15 BCG-resistant patients and 16 responders. Median instillation numbers were six (IQR: 6-9) for resistant patients and twelve (IQR: 14-15) for responders, with a median follow-up length of six months (IQR: 3.3-9.5) and 43 months (IQR: 24-55), respectively. Significant differences were observed in the microbiome: BCG responders showed higher median percentages of Firmicutes (1.1 vs. 0.3, P=0.0293) and Verrucomicrobiota (0.9 vs. 0.1, P=0.0285). Additionally, Fusobacteriota was more prevalent among responders (75% vs. 33.3%, P=0.0198), while Cyanobacteria were more common in resistant patients (73.3% vs. 31.3%, P=0.0191). Our preliminary findings illuminate the bladder microbiome's role in influencing BCG therapy outcomes, underscoring the complex microbial interplay affecting treatment efficacy in urological diseases. This explorative study sets the groundwork for ongoing data collection and future research pathways aimed at further delineating these relationships.

The Urinary Microbiome: Implications in Bladder Cancer Pathogenesis and Therapeutics

286 Background: Despite an improved overall survival with neo-CTx for muscle-invasive urothelial carcinoma (miUC) patients (pts) prior to cystectomy, the adoption of neo-CTx in suitable pts has been suboptimal. While associated with a more aggressive phenotype in early stage UC, the prognostic role of CIS in miUC is less clear. In particular, the effect of concurrent CIS in pts undergoing neo-CTx prior to cystectomy is unknown. Methods: A retrospective review of the Indiana University Simon Cancer Center urology and medical oncology clinical databases was performed spanning the years 1991 – 2012. Subjects with miUC, pathology reports available for both transurethral resection of bladder tumor (TURBT) and cystectomy procedures, and confirmed receipt of neo-CTx were included in this analysis. Pts with clinically positive lymph nodes (LN+) were included provided they underwent cystectomy with curative intent. Associations between pCR and pt baseline age, gender, race, clinical stage (T2N0 vs. T3/T4/N+), chemotherapy regimen received (cisplatin combination therapy (CisCTx) vs. non-cisplatin based), and presence of CIS on TURBT sample were tested by multinomial logistic regression analysis with statistical significance set at p<0.05. Results: 72 miUC pts satisfying the inclusion criteria were identified. Cohort demographics included: median age – 59 yrs, 76% male, 93% Caucasian, 63% T2N0, 32% LN+, 81% CisCTx neo-CTx regimen, 32% CIS on TURBT, pCR for entire cohort 18%. The presence of CIS on TURBT was significantly associated with decreased rates of pCR (1/23 vs. 12/49) p=0.002. TURBT CIS was not associated with decreased rates of less than pT2N0 (12/23 vs. 18/49) p=0.562. Seven pts (30%) with CIS on TURBT sample achieved pTisN0 at cystectomy. Conclusions: The presence of CIS in TURBT specimens is associated with decreased rates of pCR at cystectomy in miUC pts treated with neo-CTx. The finding of pTisN0 at cystectomy is common after neo-CTx in pts with CIS in preop TURBT specimens. Validation of the prognostic significance of TURBT CIS on pCR rates and characterization of pTisN0 clinical course in TURBT CIS pts after neo-CTx is warranted.

Objective: Transurethral resection of bladder tumor (TURBT) pathology specimens which lack muscle are associated with clinical upstaging and may necessitate repeat resections, potentially delaying curative treatment. This study evaluated whether resident involvement in TURBT is associated with suboptimal perioperative outcomes. Materials and methods: All TURBTs performed at a Canadian healthcare institution from November 2011 to June 2014 were reviewed. Multivariable logistic regression models assessed associations between intraoperative resident involvement and TURBT muscle presence. Among high-risk patients (high grade, ≥ T1 or carcinoma in situ) who underwent cystectomy, time from TURBT to cystectomy was compared between resident and attending urologists with the log-rank test. Results: In total, 463 TURBTs were identified. In multivariable analyses, residents were less likely to obtain muscle in specimens for all TURBTs [adjusted odds ratio (aOR) 0.59, p = 0.03] and the subset of 275 high-risk TURBTs (aOR 0.41, p = 0.006). Among patients who underwent cystectomy, time to cystectomy was delayed by a median of 23 days when residents were involved in the initial high-risk TURBT compared with attending urologists only (p = 0.024). Conclusions: In this single academic center series, intraoperative resident involvement was associated with a decreased rate of muscle presence in TURBT specimens and a prolonged time to cystectomy.