Immunotherapy and Sonpavde score validation in advanced upper tract urothelial carcinoma: a retrospective study by the Italian Network for Research in Urologic-Oncology (Meet-URO group).

Background: Few data are available regarding the effectiveness of immune checkpoint inhibitors in advanced upper tract urothelial carcinoma (UTUC) patients. Methods: To provide a real-world experience with anti-PD-1/PD-L1-based therapy in UTUC patients, we involved an Italian network in a multicenter retrospective analysis. Results: A total of 78 UTUC patients were enrolled. The median follow-up was 25.1 months. The median progression-free survival (mPFS) was 2.2 months (95% CI 1.8-2.6), and the median OS (mOS) was 6.0 months (95% CI 3.6-8.4). The Sonpavde score (including performance status > 0, hemoglobin < 10 g/dl, liver metastases, time from prior chemotherapy ≥ 3 months) split the patients into three groups (0 vs 1 vs 2-4 factors), efficiently predicting the OS and PFS outcome at the multivariate analyses (p < 0.0001). Conclusion: The prognosis of unselected UTUC patients is still unsatisfactory. The Sonpavde score was validated for the first time in an UTUC population, as a useful tool for the treatment decision-making process.

BCG invokes superior STING-mediated innate immune response over radiotherapy in a carcinogen murine model of urothelial cancer.

Radiation and bacillus Calmette-Guérin (BCG) instillations are used clinically for treatment of urothelial carcinoma, but the precise mechanisms by which they activate an immune response remain elusive. The role of the cGAS-STING pathway has been implicated in both BCG and radiation-induced immune response; however, comparison of STING pathway molecules and the immune landscape following treatment in urothelial carcinoma has not been performed. We therefore comprehensively analyzed the local immune response in the bladder tumor microenvironment following radiotherapy and BCG instillations in a well-established spontaneous murine model of urothelial carcinoma to provide insight into activation of STING-mediated immune response. Mice were exposed to the oral carcinogen, BBN, for 12 weeks prior to treatment with a single 15 Gy dose of radiation or three intravesical instillations of BCG (1 × 108 CFU). At sacrifice, tumors were staged by a urologic pathologist and effects of therapy on the immune microenvironment were measured using the NanoString Myeloid Innate Immunity Panel and immunohistochemistry. Clinical relevance was established by measuring immune biomarker expression of cGAS and STING on a human tissue microarray consisting of BCG-treated non-muscle-invasive urothelial carcinomas. BCG instillations in the murine model elevated STING and downstream STING-induced interferon and pro-inflammatory molecules, intratumoral M1 macrophage and T-cell accumulation, and complete tumor eradication. In contrast, radiotherapy caused no changes in STING pathway or innate immune gene expression; rather, it induced M2 macrophage accumulation and elevated FoxP3 expression characteristic of immunosuppression. In human non-muscle-invasive bladder cancer, STING protein expression was elevated at baseline in patients who responded to BCG therapy and increased further after BCG therapy. Overall, these results show that STING pathway activation plays a key role in effective BCG-induced immune response and strongly indicate that the effects of BCG on the bladder cancer immune microenvironment are more beneficial than those induced by radiation. © 2021 The Pathological Society of Great Britain and Ireland.

Survival Prediction by Baseline Systemic Immune-inflammation Index (SII) and its Changes During First-line Platinum-based Treatment in a Caucasian Population of Patients With Metastatic Urothelial Carcinoma (MUC).

BACKGROUND/AIM: Systemic immune-inflammation index (SII) predicts survival of patients with various malignancies. This study explored the prognostic value of SII in metastatic urothelial carcinoma (MUC) subjects. PATIENTS AND METHODS: We evaluated 181 consecutive MUC patients treated with first-line platinum-based therapy. Karnofsky performance status <80% and visceral metastasis were present in 18.2% and 46.4% of patients, respectively. SII was based on platelet × neutrophil/lymphocyte counts. Study population was dichotomized by median into high and low SII groups before the initiation of chemotherapy and at week 6. Progression-free survival (PFS) and overall survival (OS) were estimated by the Kaplan-Meier method and compared with the log-rank test. RESULTS: At median follow-up of 9.6 months, 174 patients experienced disease progression and 173 died. Patients with low SII at baseline and at week 6 had significantly better PFS (HR=0.58; p=0.0002 and HR=0.55; p<0.0001) and OS (HR=0.54; p<0.0001 and HR=0.54; p<0.0001) compared to patients with high SII. Independent prognostic value of SII was confirmed in a multivariate analysis. CONCLUSION: High SII before chemotherapy that persists at week 6 negatively affects survival. SII at baseline can be used in the stratification of patients within clinical trials and in clinical practice.

Pparg signaling controls bladder cancer subtype and immune exclusion.

Pparg, a nuclear receptor, is downregulated in basal subtype bladder cancers that tend to be muscle invasive and amplified in luminal subtype bladder cancers that tend to be non-muscle invasive. Bladder cancers derive from the urothelium, one of the most quiescent epithelia in the body, which is composed of basal, intermediate, and superficial cells. We find that expression of an activated form of Pparg (VP16;Pparg) in basal progenitors induces formation of superficial cells in situ, that exit the cell cycle, and do not form tumors. Expression in basal progenitors that have been activated by mild injury however, results in luminal tumor formation. We find that these tumors are immune deserted, which may be linked to down-regulation of Nf-kb, a Pparg target. Interestingly, some luminal tumors begin to shift to basal subtype tumors with time, down-regulating Pparg and other luminal markers. Our findings have important implications for treatment and diagnosis of bladder cancer.

Metformin strengthens uroepithelial immunity against E. coli infection.

Urinary tract infection frequently caused by E. coli is one of the most common bacterial infections. Increasing antibiotic resistance jeopardizes successful treatment and alternative treatment strategies are therefore mandatory. Metformin, an oral antidiabetic drug, has been shown to activate macrophages in the protection against certain infecting microorganisms. Since epithelial cells often form the first line of defense, we here investigated the effect on uroepithelial cells during E. coli infection. Metformin upregulated the human antimicrobial peptides cathelicidin LL-37 and RNase7 via modulation of the TRPA1 channel and AMPK pathway. Interestingly, metformin stimulation enriched both LL-37 and TRPA1 in lysosomes. In addition, metformin specifically increased nitric oxide and mitochondrial, but not cytosolic ROS. Moreover, metformin also triggered mRNA expression of the proinflammatory cytokines IL1B, CXCL8 and growth factor GDF15 in human uroepithelial cells. The GDF15 peptide stimulated macrophages increased LL-37 expression, with increased bacterial killing. In conclusion, metformin stimulation strengthened the innate immunity of uroepithelial cells inducing enhanced extracellular and intracellular bacterial killing suggesting a favorable role of metformin in the host defense.

First-line immune-checkpoint inhibitor combination therapy for chemotherapy-eligible patients with metastatic urothelial carcinoma: A systematic review and meta-analysis.

INTRODUCTION: Platinum-based combination chemotherapy is the standard treatment for patients with chemotherapy-eligible metastatic urothelial carcinoma (mUC). Immune-checkpoint inhibitors (ICIs) are currently assessed in this setting. This review aimed to assess the role of ICIs alone or in combination as first-line treatment in chemotherapy-eligible patients with mUC. METHODS: Multiple databases were searched for articles published until November 2020. Studies were deemed eligible if they compared overall survival (OS), progression-free survival (PFS), objective response rates (ORRs), complete response rates (CRRs), durations of response (DORs) and adverse events (AEs) in chemotherapy-eligible patients with mUC. RESULTS: Three studies met our eligibility criteria. ICI combination therapy was associated with significantly better OS and PFS, higher CRR and longer DOR than chemotherapy alone (hazard ratio [HR]: 0.85, 95% confidence interval [CI]: 0.76-0.94, P = 0.002; HR: 0.80, 95% CI: 0.71-0.90, P = 0.0002; odds ratio [OR]: 1.48, 95% CI: 1.12-1.96, P = 0.006; and mean difference: 1.39, 95% CI: 0.31-2.46, P = 0.01, respectively). ICI-chemotherapy combination therapy was also associated with significantly better OS and PFS, higher ORR and CRR and longer DOR than chemotherapy alone. Although OS and PFS benefits of ICI combination therapy were larger in patients with high expression of programmed death-ligand 1 (PD-L1), PD-L1 low expression patients also had a benefit; HR for OS (high PD-L1: HR 0.79 versus low PD-L1: HR 0.89) and PFS (high PD-L1: HR 0.74 versus low PD-L1: HR 0.82). ICI monotherapy was not associated with better oncological outcomes but was associated with better safety outcomes than chemotherapy alone. CONCLUSIONS: Our analysis indicates a superior oncologic benefit to first-line ICI combination therapies in patients with chemotherapy-eligible mUC over standard chemotherapy. In contrast, ICI monotherapy was associated with favorable safety outcomes compared with chemotherapy but failed to show its superiority over chemotherapy in oncological benefits. PD-L1 status alone cannot help guide treatment decision-making. However, caution should be exercised in interpreting the conclusions drawn from this study, given that there is the heterogeneity of the population of interest, risk of bias and the nature of the studies evaluated whose data remain immature or unpublished.

TERT promoter mutations and other prognostic factors in patients with advanced urothelial carcinoma treated with an immune checkpoint inhibitor.

BACKGROUND: Immune checkpoint inhibitors (ICI) can achieve durable responses in a subset of patients with locally advanced or metastatic urothelial carcinoma (aUC). The use of tumor genomic profiling in clinical practice may help suggest biomarkers to identify patients most likely to benefit from ICI. METHODS: We undertook a retrospective analysis of patients treated with an ICI for aUC at a large academic medical center. Patient clinical and histopathological variables were collected. Responses to treatment were assessed for all patients with at least one post-baseline scan or clear evidence of clinical progression following treatment start. Genomic profiling information was also collected for patients when available. Associations between patient clinical/genomic characteristics and objective response were assessed by logistic regression; associations between the characteristics and progression-free survival (PFS) and overall survival (OS) were examined by Cox regression. Multivariable analyses were performed to identify independent prognostic factors. RESULTS: We identified 119 aUC patients treated with an ICI from December 2014 to January 2020. Genomic profiling was available for 78 patients. Overall response rate to ICI was 29%, and median OS (mOS) was 13.4 months. Favorable performance status at the start of therapy was associated with improved OS (HR 0.46, p=0.025) after accounting for other covariates. Similarly, the presence of a TERT promoter mutation was an independent predictor of improved PFS (HR 0.38, p=0.012) and OS (HR 0.32, p=0.037) among patients who had genomic profiling available. Patients with both a favorable performance status and a TERT promoter mutation had a particularly good prognosis with mOS of 21.1 months as compared with 7.5 months in all other patients (p=0.03). CONCLUSIONS: The presence of a TERT promoter mutation was an independent predictor of improved OS in a cohort of aUC patients treated with an ICI who had genomic data available. Most of the clinical and laboratory variables previously shown to be prognostic in aUC patients treated with chemotherapy did not have prognostic value among patients treated with an ICI. Genomic profiling may provide important prognostic information and affect clinical decision making in this patient population. Validation of these findings in prospective patient cohorts is needed.

Pembrolizumab alone or combined with chemotherapy versus chemotherapy as first-line therapy for advanced urothelial carcinoma (KEYNOTE-361): a randomised, open-label, phase 3 trial.

BACKGROUND: PD-1 and PD-L1 inhibitors are active in metastatic urothelial carcinoma, but positive randomised data supporting their use as a first-line treatment are lacking. In this study we assessed outcomes with first-line pembrolizumab alone or combined with chemotherapy versus chemotherapy for patients with previously untreated advanced urothelial carcinoma. METHODS: KEYNOTE-361 is a randomised, open-label, phase 3 trial of patients aged at least 18 years, with untreated, locally advanced, unresectable, or metastatic urothelial carcinoma, with an Eastern Cooperative Oncology Group performance status of up to 2. Eligible patients were enrolled from 201 medical centres in 21 countries and randomly allocated (1:1:1) via an interactive voice-web response system to intravenous pembrolizumab 200 mg every 3 weeks for a maximum of 35 cycles plus intravenous chemotherapy (gemcitabine [1000 mg/m2] on days 1 and 8 and investigator's choice of cisplatin [70 mg/m2] or carboplatin [area under the curve 5] on day 1 of every 3-week cycle) for a maximum of six cycles, pembrolizumab alone, or chemotherapy alone, stratified by choice of platinum therapy and PD-L1 combined positive score (CPS). Neither patients nor investigators were masked to the treatment assignment or CPS. At protocol-specified final analysis, sequential hypothesis testing began with superiority of pembrolizumab plus chemotherapy versus chemotherapy alone in the total population (all patients randomly allocated to a treatment) for the dual primary endpoints of progression-free survival (p value boundary 0·0019), assessed by masked, independent central review, and overall survival (p value boundary 0·0142), followed by non-inferiority and superiority of overall survival for pembrolizumab versus chemotherapy in the patient population with CPS of at least 10 and in the total population (also a primary endpoint). Safety was assessed in the as-treated population (all patients who received at least one dose of study treatment). This study is completed and is no longer enrolling patients, and is registered at ClinicalTrials.gov, number NCT02853305. FINDINGS: Between Oct 19, 2016 and June 29, 2018, 1010 patients were enrolled and allocated to receive pembrolizumab plus chemotherapy (n=351), pembrolizumab monotherapy (n=307), or chemotherapy alone (n=352). Median follow-up was 31·7 months (IQR 27·7-36·0). Pembrolizumab plus chemotherapy versus chemotherapy did not significantly improve progression-free survival, with a median progression-free survival of 8·3 months (95% CI 7·5-8·5) in the pembrolizumab plus chemotherapy group versus 7·1 months (6·4-7·9) in the chemotherapy group (hazard ratio [HR] 0·78, 95% CI 0·65-0·93; p=0·0033), or overall survival, with a median overall survival of 17·0 months (14·5-19·5) in the pembrolizumab plus chemotherapy group versus 14·3 months (12·3-16·7) in the chemotherapy group (0·86, 0·72-1·02; p=0·0407). No further formal statistical hypothesis testing was done. In analyses of overall survival with pembrolizumab versus chemotherapy (now exploratory based on hierarchical statistical testing), overall survival was similar between these treatment groups, both in the total population (15·6 months [95% CI 12·1-17·9] with pembrolizumab vs 14·3 months [12·3-16·7] with chemotherapy; HR 0·92, 95% CI 0·77-1·11) and the population with CPS of at least 10 (16·1 months [13·6-19·9] with pembrolizumab vs 15·2 months [11·6-23·3] with chemotherapy; 1·01, 0·77-1·32). The most common grade 3 or 4 adverse event attributed to study treatment was anaemia with pembrolizumab plus chemotherapy (104 [30%] of 349 patients) or chemotherapy alone (112 [33%] of 342 patients), and diarrhoea, fatigue, and hyponatraemia (each affecting four [1%] of 302 patients) with pembrolizumab alone. Six (1%) of 1010 patients died due to an adverse event attributed to study treatment; two patients in each treatment group. One each occurred due to cardiac arrest and device-related sepsis in the pembrolizumab plus chemotherapy group, one each due to cardiac failure and malignant neoplasm progression in the pembrolizumab group, and one each due to myocardial infarction and ischaemic colitis in the chemotherapy group. INTERPRETATION: The addition of pembrolizumab to first-line platinum-based chemotherapy did not significantly improve efficacy and should not be widely adopted for treatment of advanced urothelial carcinoma. FUNDING: Merck Sharp and Dohme, a subsidiary of Merck, Kenilworth, NJ, USA.

Urothelial Carcinoma In Situ of the Bladder: Correlation of CK20 Expression With Adaptive Immune Resistance, Response to BCG Therapy, and Clinical Outcome.

Immunohistochemical stains have been suggested to aid in diagnostically challenging cases of urothelial carcinoma in-situ (CIS). Although full thickness immunostaining for CK20 is supportive of CIS, a subset of CIS cases is CK20(-), the clinical significance of which was unknown. This study included 43 patients with primary diagnosis of bladder CIS including 32 with only CIS, 5 with CIS and separate noninvasive high-grade papillary urothelial carcinoma, and 6 with CIS and separate high-grade urothelial carcinoma with lamina propria invasion. Digital morphometric image analysis showed that the average nuclear areas of enlarged nuclei were similar in CK20(+) and CK20(-) CIS (26.9 vs. 24.5 µM2; P=0.31). Average Ki67 index for CK20(+) CIS was higher than CK20(-) CIS (31.1% vs. 18.3%; P=0.03). Patients with CK20(+) CIS [28 (65%)] and patients with CK20(-) CIS [15 (35%)] had the same rates of Bacillus Calmete-Guerin (BCG) failure but patients with CK20(-) CIS had higher stage progression [3 CK20(+) (11%) vs. 6 CK20(-) (40%); P=0.02]. Given recent approval of immune checkpoint inhibitors in patients with CIS refractory to BCG, programmed death ligand-1 expression and colocalization with CD8(+) lymphocytes was investigated as signature of adaptive immune response and was seen in 8 patients regardless of CK20 status and exclusively among patients who failed BCG. Our results confirm that negative CK20 IHC does not exclude CIS and that those patients have similar clinical outcomes as patients with CK20(+) CIS. Programmed death ligand-1 and CD8 colocalization seen among patients who failed BCG therapy is an easy assay to perform to identify patients who could potentially benefit from combined BCG therapy and immune checkpoint inhibition.

Autoimmunity to urothelial antigen causes bladder inflammation, pelvic pain, and voiding dysfunction: a novel animal model for Hunner-type interstitial cystitis.

Recent evidence revealed that Hunner-type interstitial cystitis (HIC) is a robust inflammatory disease potentially associated with enhanced immune responses and histologically characterized by epithelial denudation and lymphoplasmacytic infiltration with frequent clonal expansion of infiltrating B cells. To date, few animal models that reproduce the histological and clinical correlates of HIC have yet been established. In the present study, we aimed to develop a novel animal model for HIC via autoimmunity to the bladder urothelium using the transgenic mouse model (URO-OVA) that expresses the membrane form of the model antigen ovalbumin (OVA) as a self-antigen on the bladder urothelium. OVA-specific lymphocytes (splenocytes) were generated by immunization of C57BL/6 mice with OVA protein and injected intravenously into URO-OVA mice. The splenocytes from OVA-immunized C57BL/6 mice showed increased interferon (IFN)-γ production in response to OVA stimulation in vitro. URO-OVA mice adoptively transferred with OVA-primed splenocytes developed cystitis exhibiting histological chronic inflammatory changes such as remarkable mononuclear cell infiltration predominantly composed of T and B lymphocytes, increased vascularity, and mucosal hyperemia in the bladder at days 7-28 with a peak at day 21 tested. No systemic inflammation was found in cystitis-induced URO-OVA mice, nor was any inflammation found in wild-type C57BL/6 mice adoptively transferred with OVA-primed splenocytes. Along with bladder inflammation, URO-OVA mice demonstrated significantly increased pelvic nociceptive responses, voiding dysfunction, and upregulated mRNA expression levels for IFN-γ, tumor necrosis factor-α (TNF-α), and substance P precursor in the bladder. This model reproduces the histological and clinical features of human HIC, providing a novel model for HIC research.

Shionone alleviates NLRP3 inflammasome mediated pyroptosis in interstitial cystitis injury.

Shionone is a triterpenoid component derived from the herbal medicine Aster tataricus, and it has been reported to possess marked anti-inflammatory properties. The activation of NLRP3 inflammasome plays an important role in cystitis, and the effect of Shionone on NLRP3 inflammasome-dependent pyroptosis remains unclear. In this study, we established an interstitial cystitis (IC) rat model and SV-HUC-1 cell model with CYP or LPS + ATP treatment to mimic inflammation response and induce NLRP3 inflammasome activation. Shionone treatment significantly attenuated the bladder wet weight, score of edema and hemorrhage, enhanced the viability of SV-HUC-1 cell, decreased the rate of pyroptosis. Moreover, Shionone reduced the expression of NF-κB, NLRP3, ASC, Pro-caspase-1, Caspase-1, GSDMD, GSDMD-N at the mRNA and protein levels both in rat and SV-HUC-1 cell model, demonstrating NLRP3 inflammasome pathway was blocked and pyroptosis degree was reduced. These results indicated that Shionone could alleviate interstitial cystitis in Rat model and enhancing the viability of SV-HUC-1 cells via NF-κB/NLRP3/GSDMD-N pathway, which illustrated that Shionone could be used as a drug candidate for the treatment of interstitial cystitis.

FGFR3 Alterations in the Era of Immunotherapy for Urothelial Bladder Cancer.

FGFR3 is a prognostic and predictive marker and is a validated therapeutic target in urothelial bladder cancer. Its utility as a marker and target in the context of immunotherapy is incompletely understood. We review the role of FGFR3 in bladder cancer and discuss preclinical and clinical clues of its effectiveness as a patient selection factor and therapeutic target in the era of immunotherapy.

Preoperative ipilimumab plus nivolumab in locoregionally advanced urothelial cancer: the NABUCCO trial.

Preoperative immunotherapy with anti-PD1 plus anti-CTLA4 antibodies has shown remarkable pathological responses in melanoma1 and colorectal cancer2. In NABUCCO (ClinicalTrials.gov: NCT03387761 ), a single-arm feasibility trial, 24 patients with stage III urothelial cancer (UC) received two doses of ipilimumab and two doses of nivolumab, followed by resection. The primary endpoint was feasibility to resect within 12 weeks from treatment start. All patients were evaluable for the study endpoints and underwent resection, 23 (96%) within 12 weeks. Grade 3-4 immune-related adverse events occurred in 55% of patients and in 41% of patients when excluding clinically insignificant laboratory abnormalities. Eleven patients (46%) had a pathological complete response (pCR), meeting the secondary efficacy endpoint. Fourteen patients (58%) had no remaining invasive disease (pCR or pTisN0/pTaN0). In contrast to studies with anti-PD1/PD-L1 monotherapy, complete response to ipilimumab plus nivolumab was independent of baseline CD8+ presence or T-effector signatures. Induction of tertiary lymphoid structures upon treatment was observed in responding patients. Our data indicate that combined CTLA-4 plus PD-1 blockade might provide an effective preoperative treatment strategy in locoregionally advanced UC, irrespective of pre-existing CD8+ T cell activity.

Neoadjuvant PD-L1 plus CTLA-4 blockade in patients with cisplatin-ineligible operable high-risk urothelial carcinoma.

Immune checkpoint therapy is being tested in the neoadjuvant setting for patients with localized urothelial carcinoma1,2, with one study reporting data in cisplatin-ineligible patients who received anti-PD-L1 monotherapy2. The study reported that patients with bulky tumors, a known high-risk feature defined as greater than clinical T2 disease, had fewer responses, with pathological complete response rate of 17%2. Here we report on the first pilot combination neoadjuvant trial ( NCT02812420 ) with anti-PD-L1 (durvalumab) plus anti-CTLA-4 (tremelimumab) in cisplatin-ineligible patients, with all tumors identified as having high-risk features (n = 28). High-risk features were defined by bulky tumors, variant histology, lymphovascular invasion, hydronephrosis and/or high-grade upper tract disease3-5. The primary endpoint was safety and we observed 6 of 28 patients (21%) with grade ≥3 immune-related adverse events, consisting of asymptomatic laboratory abnormalities (n = 4), hepatitis and colitis (n = 2). We also observed pathological complete response of 37.5% and downstaging to pT1 or less in 58% of patients who completed surgery (n = 24). In summary, we provide initial safety, efficacy and biomarker data with neoadjuvant combination anti-PD-L1 plus anti-CTLA-4, which warrants further development for patients with localized urothelial carcinoma, especially cisplatin-ineligible patients with high-risk features who do not currently have an established standard-of-care neoadjuvant treatment.

B7-H3 expression in upper tract urothelial carcinoma associates with adverse clinicopathological features and poor survival.

B7-H3, a member of the B7 superfamily, is an immune checkpoint molecule. An association between B7-H3 expression and poor survival has been reported in many types of cancer. However, its prognostic value in patients with upper tract urothelial carcinoma (UTUC) has not yet been reported. The aim of this study was to examine the clinical significance of tumor B7-H3 expression in UTUC. B7-H3 positivity was observed in 36 of 271 cases (13 %) by immunohistochemistry and was significantly associated with several adverse clinicopathological features such as tumor grade, tumor stage, and lymph node metastasis. In addition, B7-H3 positivity was significantly associated with shorter metastasis-free survival and cancer-specific survival. We also found that B7-H3/programmed cell death ligand-1 (PD-L1) co-positivity was significantly associated with worse prognosis. These results suggest the utility of B7-H3 positivity and B7-H3/PD-L1 co-positivity as novel prognostic biomarkers in UTUC, and the potential usefulness of B7-H3 targeted therapy for patients with UTUC, the effect of which may be enhanced by combination with programmed cell death-1 /PD-L1 blockade.

Biomarkers of the Response to Immune Checkpoint Inhibitors in Metastatic Urothelial Carcinoma.

The mechanisms underlying the resistance to immune checkpoint inhibitors (ICIs) therapy in metastatic urothelial carcinoma (mUC) patients are not clear. It is of great significance to discern mUC patients who could benefit from ICI therapy in clinical practice. In this study, we performed machine learning method and selected 10 prognostic genes for constructing the immunotherapy response nomogram for mUC patients. The calibration plot suggested that the nomogram had an optimal agreement with actual observations when predicting the 1- and 1.5-year survival probabilities. The prognostic nomogram had a favorable discrimination of overall survival of mUC patients, with area under the curve values of 0.815, 0.752, and 0.805 for ICI response (ICIR) prediction in the training cohort, testing cohort, and combined cohort, respectively. A further decision curve analysis showed that the prognostic nomogram was superior to either mutation burden or neoantigen burden for overall survival prediction when the threshold probability was >0.35. The immune infiltrate analysis indicated that the low ICIR-Score values in mUC patients were significantly related to CD8+ T cell infiltration and immune checkpoint-associated signatures. We also identified differentially mutated genes, which could act as driver genes and regulate the response to ICI therapy. In conclusion, we developed and validated an immunotherapy-responsive nomogram for mUC patients, which could be conveniently used for the estimate of ICI response and the prediction of overall survival probability for mUC patients.

Nivolumab in Patients with Advanced Platinum-resistant Urothelial Carcinoma: Efficacy, Safety, and Biomarker Analyses with Extended Follow-up from CheckMate 275.

PURPOSE: We report efficacy and safety with extended follow-up, and exploratory biomarker analyses from the phase II CheckMate 275 trial to identify biomarkers of response to nivolumab in platinum-resistant metastatic or unresectable urothelial carcinoma (mUC). PATIENTS AND METHODS: Patients received nivolumab 3 mg/kg once every 2 weeks until disease progression, unacceptable toxicity, or other protocol-defined reasons. The primary endpoint was objective response rate (ORR) per blinded independent review committee (BIRC; using RECIST v1.1) in all treated patients and by tumor PD-L1 expression. Key secondary endpoints were progression-free survival (PFS) per BIRC using RECIST v1.1 and overall survival (OS) in all patients and by PD-L1 expression. Exploratory endpoints included safety and biomarker analyses of tumor mutational burden (TMB), PD-L1, and previously identified mutational signatures. RESULTS: Of 270 treated patients, 139 had evaluable TMB. With 33.7 months' minimum follow-up, ORR per BIRC, median PFS, and median OS [95% confidence interval (CI)] in all treated patients were 20.7% (16.1-26.1), 1.9 months (1.9-2.3), and 8.6 months (6.1-11.3), respectively. No new safety signals were identified. Higher TMB was associated (P < 0.05) with improved ORR [OR (95% CI): 2.13 (1.26-3.60)], PFS [HR: 0.75 (0.61-0.92)], and OS [HR: 0.73 (0.58-0.91)]. TMB combined with PD-L1 better predicted ORR, PFS, and OS than PD-L1 alone. Higher mutational signature 2 score was associated with better OS but did not improve the predictive value of TMB. CONCLUSIONS: These results support the durable antitumor activity of nivolumab and suggest that TMB may enrich for better response in mUC. Future studies of TMB/PD-L1 as biomarkers for response to nivolumab in randomized trials are warranted.See related commentary by Swami et al., p. 5059.

Radiation of the urinary bladder attenuates the development of lipopolysaccharide-induced cystitis.

In the present study we assessed how ionizing radiation affects TLR4-stimulated immune activation in lipopolysaccharide (LPS)-induced cystitis. LPS or saline was administered intravesically to female rats followed by urinary bladder irradiation (20 Gy) 24 h later or sham treatment. Presence in the urinary bladder of inflammatory cells (mast cells, CD3+, ionized calcium-binding adapter molecule 1 (Iba-1)+, CD68+, CD40+, CD80+, CD11c + and CD206 + cells) and expression of oxidative stress (8-OHdG), hypoxia (HIF1α) and anti-oxidative responses (NRF2, HO-1, SOD1, SOD2, catalase) were assessed 14 days later with western blot, qPCR and/or immunohistochemistry. LPS stimulation resulted in a decrease of Iba-1 + cells in the urothelium, an increase in mast cells in the submucosa and a decrease in the bladder protein expression of HO-1, while no changes in the bladder expression of 8-OHdG, NRF2, SOD1, SOD2, catalase and HIF1α were observed. Bladder irradiation inhibited the LPS-driven increase in mast cells and the decrease in Iba1 + cells. Combining LPS and radiation increased the expression of 8-OHdG and number of CD3-positive cells in the urothelium and led to a decrease in NRF2α gene expression in the urinary bladder. In conclusion, irradiation may attenuate LPS-induced immune responses in the urinary bladder but potentiates LPS-induced oxidative stress, which as a consequence may have an impact on the urinary bladder immune sensing of pathogens and danger signals.

Spatial proteomics revealed a CX3CL1-dependent crosstalk between the urothelium and relocated macrophages through IL-6 during an acute bacterial infection in the urinary bladder.

The urothelium of the urinary bladder represents the first line of defense. However, uropathogenic E. coli (UPEC) damage the urothelium and cause acute bacterial infection. Here, we demonstrate the crosstalk between macrophages and the urothelium stimulating macrophage migration into the urothelium. Using spatial proteomics by MALDI-MSI and LC-MS/MS, a novel algorithm revealed the spatial activation and migration of macrophages. Analysis of the spatial proteome unravelled the coexpression of Myo9b and F4/80 in the infected urothelium, indicating that macrophages have entered the urothelium upon infection. Immunofluorescence microscopy additionally indicated that intraurothelial macrophages phagocytosed UPEC and eliminated neutrophils. Further analysis of the spatial proteome by MALDI-MSI showed strong expression of IL-6 in the urothelium and local inhibition of this molecule reduced macrophage migration into the urothelium and aggravated the infection. After IL-6 inhibition, the expression of matrix metalloproteinases and chemokines, such as CX3CL1 was reduced in the urothelium. Accordingly, macrophage migration into the urothelium was diminished in the absence of CX3CL1 signaling in Cx3cr1gfp/gfp mice. Conclusively, this study describes the crosstalk between the infected urothelium and macrophages through IL-6-induced CX3CL1 expression. Such crosstalk facilitates the relocation of macrophages into the urothelium and reduces bacterial burden in the urinary bladder.

Kdm6a Deficiency Activates Inflammatory Pathways, Promotes M2 Macrophage Polarization, and Causes Bladder Cancer in Cooperation with p53 Dysfunction.

PURPOSE: Epigenetic deregulation is deeply implicated in the pathogenesis of bladder cancer. KDM6A (Lysine (K)-specific demethylase 6A) is a histone modifier frequently mutated in bladder cancer. However, the molecular mechanisms of how KDM6A deficiency contributes to bladder cancer development remains largely unknown. We hypothesized that clarification of the pathogenic mechanisms underlying KDM6A-mutated bladder cancer can help in designing new anticancer therapies. EXPERIMENTAL DESIGN: We generated mice lacking Kdm6a in the urothelium and crossed them with mice heterozygous for p53, whose mutation/deletion significantly overlaps with the KDM6A mutation in muscle-invasive bladder cancer (MIBC). In addition, BBN (N-butyl-N-(4-hydroxybutyl) nitrosamine), a cigarette smoke-like mutagen, was used as a tumor-promoting agent. Isolated urothelia were subjected to phenotypic, pathologic, molecular, and cellular analyses. The clinical relevance of our findings was further analyzed using genomic and clinical data of patients with MIBC. RESULTS: We found that Kdm6a deficiency activated cytokine and chemokine pathways, promoted M2 macrophage polarization, increased cancer stem cells and caused bladder cancer in cooperation with p53 haploinsufficiency. We also found that BBN treatment significantly enhanced the expression of proinflammatory molecules and accelerated disease development. Human bladder cancer samples with decreased KDM6A expression also showed activated proinflammatory pathways. Notably, dual inhibition of IL6 and chemokine (C-C motif) ligand 2, upregulated in response to Kdm6a deficiency, efficiently suppressed Kdm6a-deficient bladder cancer cell growth. CONCLUSIONS: Our findings provide insights into multistep carcinogenic processes of bladder cancer and suggest molecular targeted therapeutic approaches for patients with bladder cancer with KDM6A dysfunction.