Cisplatin-based neoadjuvant chemotherapy (NAC) followed by radical cystectomy is recommended for patients with muscle-invasive bladder cancer (MIBC). It has been shown that somatic deleterious mutations in ERCC2, gain-of-function mutations in ERBB2, and alterations in ATM, RB1, and FANCC are correlated with pathological response to NAC in MIBC. The objective of this study was to validate these genomic biomarkers in pretreatment transurethral resection material from an independent retrospective cohort of 165 patients with MIBC who subsequently underwent NAC and radical surgery. Patients with ypT0/Tis/Ta/T1N0 disease after surgery were defined as responders. Somatic deleterious mutations in ERCC2 were found in nine of 68 (13%) evaluable responders and two of 95 (2%) evaluable nonresponders (p = 0.009; FDR = 0.03). No correlation was observed between response and alterations in ERBB2 or in ATM, RB1, or FANCC alone or in combination. In an exploratory analysis, no additional genomic alterations discriminated between responders and nonresponders to NAC. No further associations were identified between the aforementioned biomarkers and pathological complete response (ypT0N0) after surgery. In conclusion, we observed a positive association between deleterious mutations in ERCC2 and pathological response to NAC, but not overall survival or recurrence-free survival. Other previously reported genomic biomarkers were not validated. Patient summary: It is currently unknown which patients will respond to chemotherapy before definitive surgery for bladder cancer. Previous studies described several gene mutations in bladder cancer that correlated with chemotherapy response. This study confirmed that patients with bladder cancer with a mutation in the ERCC2 gene often respond to chemotherapy.@en

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.@en

The authors regret that the following statement regarding author contributions was missed: Kristan van der Vos is currently a Scientific Editor for Cell Reports Medicine, which is published by Elsevier. Dr van der Vos was not involved in the peer-review process or editorial discussions about this manuscript. The authors would like to apologise for any inconvenience caused.@en

Immune checkpoint inhibitors (ICI) can achieve remarkable responses in urothelial cancer (UC), which may depend on tumor microenvironment (TME) characteristics. However, the relationship between the TME, usually characterized by immune cell density, and response to ICI is unclear. Here, we quantify the TME immune cell densities and spatial relationships (SRs) of 24 baseline UC samples, obtained before pre-operative combination ICI treatment, using multiplex immunofluorescence. We describe SRs by approximating the first nearest-neighbor distance distribution with a Weibull distribution and evaluate the association between TME metrics and ipilimumab+nivolumab response. Immune cell density does not discriminate between response groups. However, the Weibull SR metrics of CD8+ T cells or macrophages to their closest cancer cell positively associate with response. CD8+ T cells close to B cells are characteristic of non-response. We validate our SR response associations in a combination ICI cohort of head and neck tumors. Our data confirm that SRs, in contrast to density metrics, are strong biomarkers of response to pre-operative combination ICIs.@en