The Tumor Immune Landscape and Architecture of Tertiary Lymphoid Structures in Urothelial Cancer

Candidate immune biomarkers have been proposed for predicting response to immunotherapy in urothelial cancer (UC). Yet, these biomarkers are imperfect and lack predictive power. A comprehensive overview of the tumor immune contexture, including Tertiary Lymphoid structures (TLS), is needed to better understand the immunotherapy response in UC. We analyzed tumor sections by quantitative multiplex immunofluorescence to characterize immune cell subsets in various tumor compartments in tumors without pretreatment and tumors exposed to preoperative anti-PD1/CTLA-4 checkpoint inhibitors (NABUCCO trial). Pronounced immune cell presence was found in UC invasive margins compared to tumor and stroma regions. CD8+PD1+ T-cells were present in UC, particularly following immunotherapy. The cellular composition of TLS was assessed by multiplex immunofluorescence (CD3, CD8, FoxP3, CD68, CD20, PanCK, DAPI) to explore specific TLS clusters based on varying immune subset densities. Using a k-means clustering algorithm, we found five distinct cellular composition clusters. Tumors unresponsive to anti-PD-1/CTLA-4 immunotherapy showed enrichment of a FoxP3+ T-cell-low TLS cluster after treatment. Additionally, cluster 5 (macrophage low) TLS were significantly higher after pre-operative immunotherapy, compared to untreated tumors. We also compared the immune cell composition and maturation stages between superficial (submucosal) and deeper TLS, revealing that superficial TLS had more pronounced T-helper cells and enrichment of early TLS than TLS located in deeper tissue. Furthermore, superficial TLS displayed a lower fraction of secondary follicle like TLS than deeper TLS. Taken together, our results provide a detailed quantitative overview of the tumor immune landscape in UC, which can provide a basis for further studies

Multicenter Validation of Histopathologic Tumor Regression Grade After Neoadjuvant Chemotherapy in Muscle-Invasive Bladder Carcinoma

Response classification after neoadjuvant chemotherapy in muscle-invasive bladder carcinoma is based on the TNM stage at radical cystectomy. We recently showed that histopathologic tumor regression grades (TRGs) add prognostic information to TNM. Our aim was to validate the prognostic significance of TRG in muscle-invasive bladder cancer in a multicenter setting. We enrolled 389 patients who underwent cisplatin-based chemotherapy before radical cystectomy in 8 centers between 2010 and 2016. Median follow-up was 2.2 years. TRG was determined in radical cystectomy specimens by local pathologists. Central pathology review was conducted in 20% of cases, which were randomly selected. The major response was defined as ≤pT1N0. The remaining patients were grouped into partial responders (≥ypT2N0-3 and TRG 2) and nonresponders (≥ypT2N0-3 and TRG 3). TRG was successfully determined in all cases, and interobserver agreement in central pathology review was high (κ=0.83). After combining TRG and TNM, 47%, 15%, and 38% of patients were major, partial, and nonresponders, respectively. Combination of TRG and TNM showed significant prognostic discrimination of overall survival (major responder: reference; partial responder: hazard ratio 3.5 [95% confidence interval: 1.8-6.8]; nonresponder: hazard ratio 6.1 [95% confidence interval: 3.6-10.3]). This discrimination was superior compared with TNM staging alone, supported by 2 goodness-of-fit criteria (P=0.041). TRG is a simple, reproducible histopathologic measurement of response to neoadjuvant chemotherapy in muscle-invasive bladder cancer. Integrating TRG with TNM staging resulted in significantly better prognostic stratification than TNM staging alone

The clonal relation of primary upper urinary tract urothelial carcinoma and paired urothelial carcinoma of the bladder

The risk of developing urothelial carcinoma of the bladder (UCB) in patients treated by radical nephroureterectomy (RNU) for an upper urinary tract urothelial carcinoma (UTUC) is 22% to 47% in the 2 years after surgery. Subject of debate remains whether UTUC and the subsequent UCB are clonally related or represent separate origins. To investigate the clonal relationship between both entities, we performed targeted DNA sequencing of a panel of 41 genes on matched normal and tumor tissue of 15 primary UTUC patients treated by RNU who later developed 19 UCBs. Based on the detected tumor-specific DNA aberrations, the paired UTUC and UCB(s) of 11 patients (73.3%) showed a clonal relation, whereas in four patients the molecular results did not indicate a clear clonal relationship. Our results support the hypothesis that UCBs following a primary surgically resected UTUC are predominantly clonally derived recurrences and not separate entities

Radiation with concurrent radiosensitizing capecitabine tablets and single-dose mitomycin-C for muscle-invasive bladder cancer: A convenient alternative to 5-fluorouracil

Background and purpose: Chemoradiation (CRT) with mitomycin-C (MMC) and 5-fluorouracil (5-FU) has been shown to be superior to radiation alone in patients with muscle-invasive bladder cancer (MIBC). MMC/capecitabine is an effective replacement for 5FU as a radiosensitizer in other malignancies but has not been studied in bladder cancer. We evaluated the outcomes of MIBC patients treated with concurrent radiation and MMC/capecitabine. Materials and methods: MIBC patients treated with CRT (60 Gy in 5 weeks with single-dose MMC and capecitabine orally twice daily) between 2014 and 2019 were identified. Acute (<90 days) and late toxicity were registered. Endpoints were clinical complete response (cCR) in the bladder assessed by cystoscopy 3 months after CRT, locoregional disease-free survival (LDFS) and the number of salvage cystectomies. Results: We analysed 71 cT2-4aN0-2 M0 MIBC patients (median age 70 years). Twenty-one (30%) patients received neoadjuvant or induction chemotherapy and 14 (20%) patients underwent a pelvic lymph node dissection prior to CRT. All patients received the full dose of planned radiation. Seven (10%) patients experienced acute grade 3-4 toxicities and 2 (3%) patients experienced late grade 3-4 toxicities. Sixty-eight (96%) patients achieved cCR. Eight (11%) patients had a bladder recurrence, of whom 3 (4%) required salvage cystectomy. Two-year LDFS was 79% (95% CI: 68-88) at a median follow-up of 23 (95% CI: 17-28) months. Conclusion: Radiation with concurrent MMC/capecitabine is a well-tolerated bladder-sparing treatment. Severe toxicity is infrequent and locoregional tumor control and short-term disease free survival appear similar to previous studies with MMC/5FU. (c) 2020 Elsevier B.V. All rights reserved

Urothelial Carcinoma in Bladder Diverticula: A Multicenter Analysis of Characteristics and Clinical Outcomes

BACKGROUND Urothelial carcinoma arising in a bladder diverticulum (UCBD) is uncommon, and data on treatment and outcome are sparse. OBJECTIVE To analyze clinicopathological characteristics of UCBD and to compare outcome after radical cystectomy (RC) and partial cystectomy (PC). DESIGN, SETTING, AND PARTICIPANTS Data of 115 UCBD patients treated with RC (n=81) or PC (n=34) between 2000 and 2016 were collected from 11 institutional databases and were analyzed retrospectively. Median follow-up was 5.0yr (95% confidence interval [CI]: 4.0-6.2). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Upstaging of tumor stage at diagnostic transurethral resection (TUR) to the RC/PC specimen was investigated. Overall survival (OS) and metastasis-free survival (MFS) after RC and PC were analyzed using Kaplan-Meier estimates, and compared using the log-rank test. Intravesical recurrences after PC were reported. A multivariable Cox proportional-hazard model was used to identify factors associated with OS. RESULTS AND LIMITATIONS There were no statistically significant differences in clinicopathological characteristics between RC and PC groups. Fifty-five percent of patients with cTa/is/1 at diagnostic TUR had ≥pT2 tumors at RC/PC. Five-year OS and MFS were, respectively, 62% and 66% for RC and 66% and 55% for PC (p=0.9 and p=0.6). Intravesical tumor recurrence was seen in six of 34 (18%) PC patients. In multivariable analysis, positive surgical margins and extravesical disease (≥pT2) were associated with worse OS, whereas treatment modality was not (RC: reference; PC: hazard ratio 0.94, [95% CI: 0.47-1.90], p=0.9). CONCLUSIONS Upstaging of UCBD was frequent, indicating an inaccuracy in clinical staging. We found no differences in OS or MFS between PC and RC groups; therefore, PC may represent a feasible surgical alternative to RC in selected UCBD patients. PATIENT SUMMARY In this report, we looked at the treatment of urothelial carcinoma arising in a bladder diverticulum (UCBD). We found that bladder-sparing treatment by partial cystectomy may be an alternative to radical cystectomy in carefully selected UCBD patients

Prospective Evaluation of FDG-PET/CT for On-treatment Assessment of Response to Neoadjuvant or Induction Chemotherapy in Invasive Bladder Cancer

BACKGROUND: Neoadjuvant/induction chemotherapy (NAIC) improves survival in patients with muscle-invasive bladder carcinoma (MIBC). On-treatment response assessment may aid in decisions to continue or cease NAIC. OBJECTIVE: We investigated whether 18F-fluoro-2-deoxy-D-glucose-Positron Emission Tomography/Computed Tomography (FDG-PET/CT) could predict response to NAIC and compared to contrast-enhanced Computed Tomography (CECT). METHODS: We prospectively included 83 patients treated for MIBC (i.e. high-risk cT2-4N0M0 or cT1-4N+M0-1a) between 2014 and 2018. Response to NAIC was assessed after 2-3 cycles with FDG-PET/CT (Peter-Mac and EORTC criteria) and CECT (RECIST1.1 criteria). We assessed prediction of complete pathological response (pCR; ypT0N0), complete pathological down-staging (pCD;≤ypT1N0), any down-staging from baseline (ypTN < cTN) and progression (inoperable tumor/ypN+/M+). The reference standard was histopathological assessment or clinical follow-up. Sensitivity, specificity, and accuracy were calculated. RESULTS: Pathological response rates were 21% for pCR, 29% for pCD, and 10% progressed. All patients underwent FDG-PET/CT and 61 patients also underwent CECT (73%). Accuracy of FDG-PET/CT for prediction of pCR, pCD, and progression were 73%, 48%, and 73%, respectively. Accuracy of CECT for prediction of pCR, pCD, and progression were 78%, 65%, and 67%, respectively. Specificity of CECT was significantly higher than FDG-PET/CT for prediction of pCD and any down-staging (p = 0.007 and p = 0.022). In all other analyses, no significant differences between FDG-PET/CT and CECT were found. CONCLUSIONS: Routine FDG-PET/CT has insufficient predictive power to aid in response assessment compared to CECT

Staging 18F-fluorodeoxyglucose Positron Emission Tomography/Computed Tomography Changes Treatment Recommendation in Invasive Bladder Cancer

Given the high risk of systemic relapse following initial therapy for muscle-invasive bladder cancer (MIBC), improved pretreatment staging is needed. We evaluated the incremental value of 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) after standard conventional staging, in the largest cohort of MIBC patients to date. This is a retrospective analysis of 711 consecutive patients with invasive urothelial bladder cancer who underwent staging contrast-enhanced CT (chest and abdomen) and FDG-PET/CT in a tertiary referral center between 2011 and 2020. We recorded the clinical stage before and after FDG-PET/CT and treatment recommendation based on the stage before and after FDG-PET/CT. Clinical stage changed after FDG-PET/CT in 184/711 (26%) patients. Consequently, the recommended treatment strategy based on imaging changed in 127/711 (18%) patients. In 65/711 (9.1%) patients, potential curative treatment changed to palliative treatment because of the detection of distant metastases by FDG-PET/CT. Fifty (7.0%) patients were selected for neoadjuvant/induction chemotherapy based on FDG-PET/CT. Moreover, FDG-PET/CT detected lesions suspicious for second primary tumors in 15%; a second primary malignancy was confirmed in 28/711 (3.9%), leading to treatment change in ten (1.4%) patients. Contrarily 57/711 (8.1%) had false positive secondary findings. In conclusion, FDG-PET/CT provides important incremental staging information, which potentially influences clinical management in 18% of MIBC patients, but leads to false positive results as well. PATIENT SUMMARY: In this report, we investigated the impact of 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) scanning on treatment of bladder cancer patients. We found that FDG-PET/CT potentially influences the treatment of almost one-fifth of patients. We therefore suggest performing FDG-PET/CT as part of bladder cancer staging

The Tumor Immune Landscape and Architecture of Tertiary Lymphoid Structures in Urothelial Cancer

Candidate immune biomarkers have been proposed for predicting response to immunotherapy in urothelial cancer (UC). Yet, these biomarkers are imperfect and lack predictive power. A comprehensive overview of the tumor immune contexture, including Tertiary Lymphoid structures (TLS), is needed to better understand the immunotherapy response in UC. We analyzed tumor sections by quantitative multiplex immunofluorescence to characterize immune cell subsets in various tumor compartments in tumors without pretreatment and tumors exposed to preoperative anti-PD1/CTLA-4 checkpoint inhibitors (NABUCCO trial). Pronounced immune cell presence was found in UC invasive margins compared to tumor and stroma regions. CD8+PD1+ T-cells were present in UC, particularly following immunotherapy. The cellular composition of TLS was assessed by multiplex immunofluorescence (CD3, CD8, FoxP3, CD68, CD20, PanCK, DAPI) to explore specific TLS clusters based on varying immune subset densities. Using a k-means clustering algorithm, we found five distinct cellular composition clusters. Tumors unresponsive to anti-PD-1/CTLA-4 immunotherapy showed enrichment of a FoxP3+ T-cell-low TLS cluster after treatment. Additionally, cluster 5 (macrophage low) TLS were significantly higher after pre-operative immunotherapy, compared to untreated tumors. We also compared the immune cell composition and maturation stages between superficial (submucosal) and deeper TLS, revealing that superficial TLS had more pronounced T-helper cells and enrichment of early TLS than TLS located in deeper tissue. Furthermore, superficial TLS displayed a lower fraction of secondary follicle like TLS than deeper TLS. Taken together, our results provide a detailed quantitative overview of the tumor immune landscape in UC, which can provide a basis for further studies.Pattern Recognition and Bioinformatic

Staging 18F-fluorodeoxyglucose Positron Emission Tomography/Computed Tomography Changes Treatment Recommendation in Invasive Bladder Cancer

Given the high risk of systemic relapse following initial therapy for muscle-invasive bladder cancer (MIBC), improved pretreatment staging is needed. We evaluated the incremental value of 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) after standard conventional staging, in the largest cohort of MIBC patients to date. This is a retrospective analysis of 711 consecutive patients with invasive urothelial bladder cancer who underwent staging contrast-enhanced CT (chest and abdomen) and FDG-PET/CT in a tertiary referral center between 2011 and 2020. We recorded the clinical stage before and after FDG-PET/CT and treatment recommendation based on the stage before and after FDG-PET/CT. Clinical stage changed after FDG-PET/CT in 184/711 (26%) patients. Consequently, the recommended treatment strategy based on imaging changed in 127/711 (18%) patients. In 65/711 (9.1%) patients, potential curative treatment changed to palliative treatment because of the detection of distant metastases by FDG-PET/CT. Fifty (7.0%) patients were selected for neoadjuvant/induction chemotherapy based on FDG-PET/CT. Moreover, FDG-PET/CT detected lesions suspicious for second primary tumors in 15%; a second primary malignancy was confirmed in 28/711 (3.9%), leading to treatment change in ten (1.4%) patients. Contrarily 57/711 (8.1%) had false positive secondary findings. In conclusion, FDG-PET/CT provides important incremental staging information, which potentially influences clinical management in 18% of MIBC patients, but leads to false positive results as well. PATIENT SUMMARY: In this report, we investigated the impact of 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) scanning on treatment of bladder cancer patients. We found that FDG-PET/CT potentially influences the treatment of almost one-fifth of patients. We therefore suggest performing FDG-PET/CT as part of bladder cancer staging

Neoadjuvant treatment for muscle-invasive bladder cancer: The past, the present, and the future.

BACKGROUND Approximately half of patients who undergo radical cystectomy (RC) for muscle-invasive bladder cancer (MIBC) will succumb to metastatic disease. We summarize the evidence for neoadjuvant radiation (NAR), chemo (NAC), and immunotherapy (checkpoint inhibition) prior to RC for MIBC. MATERIALS AND METHODS Data were obtained by a search of PubMed, ClinicalTrials.gov, and Cochrane databases for English language articles published from 1925 up to 2017. RESULTS NAC usage has increased over the last decade, while NAR is rarely administered. Although NAR results in downstaging, its impact on survival is inconclusive. Based on level I evidence, cisplatin-based NAC (CB-NAC) is considered standard of care in cT2-4aN0M0 MIBC. NAC results in a 6% absolute 10-year overall survival (OS) benefit. In-depth analyses of key randomized controlled trials showed that failure to correct for uniform staging, surgical variation, and patient selection compromises the ability to identify factors predictive of response to NAC. The benefit appears to be restricted to patients downstaged to ypT1N0 or less. In these patients, 5-year OS is 80% to 90%. Regarding a number needed to treat of 17, most patients with cT2-4aN0M0 MIBC will be exposed to toxicity without benefit. Possible approaches to reduce overtreatment are suggested in this article and include patient selection, the chosen NAC regimen, and emerging molecular data to predict responsiveness to NAC. Neoadjuvant immunotherapy with checkpoint inhibitors is a promising future perspective currently under investigation. CONCLUSIONS Past studies on NAR show inconclusive results and NAR is rarely administered. Instead, CB-NAC is advised in eligible patients with cT2-4aN0M0 MIBC prior to RC. In the near future, predictive biomarkers will be the key to tailor the use of CB-NAC and reduce harm to nonresponders