Purpose: In NABUCCO, the safety and efficacy of preoperative ipilimumab plus nivolumab were assessed in stage III urothelial cancer. Encouraging responses were achieved, and ipilimumab 3 mg/kg (ipilimumab-high) seemed more effective than ipilimumab 1 mg/kg (ipilimumab-low). We explored ipilimumab plus nivolumab response biomarkers and tumor microenvironment (TME) treatment dynamics. Patients and Methods: Baseline formalin-fixed, paraffinembedded tumor tissue was analyzed using PD-L1 IHC (n ¼ 51) and whole-exome and transcriptome sequencing (both n ¼ 53) and correlated with response. Baseline infiltration of CD8⁺ T cells (n ¼ 51) and at cystectomy (n ¼ 42) was examined. Single-cell RNA sequencing (scRNA-seq) of CD3⁺ T cells was conducted on on-treatment resection tissue of two responders to ipilimumab-high to explore the characteristics of CD8⁺ T cells within the TME. Results: High tumor mutational burden and PD-L1 positivity were associated with response to ipilimumab plus nivolumab. Nonresponding patients exhibited increased expression of a TGFβ signature. We observed increased transcription of the g2m checkpoint and e2f target in responders to ipilimumab-high and enhanced transcription of IFN-α and IFN-γ hallmarks in responders to ipilimumab-low. CD8⁺TCF7⁺ T cells accumulated in the TME of responders to ipilimumab-high. scRNA-seq of CD8A⁺TCF7⁺ T cells demonstrated enhanced expression of IL7R, CCR7, GPR15, XCL1, SELL, and LEF1. Conclusions: Our data indicate that tumor mutational burden, PD-L1, and TGFβ are potential biomarkers for response to ipilimumab plus nivolumab in stage III urothelial cancer. An inflammatory TME might be relevant for responding to ipilimumab-low. We found that in responders to ipilimumabhigh, TCF7⁺CD8⁺ T cells accumulated in the TME. scRNA-seq in two responders suggested that TCF7⁺CD8A⁺ T cells express genes associated with immunologic memory formation and T-cell homing.

PURPOSE: Platinum-based chemotherapy and immune checkpoint inhibitors are key components of systemic treatment for muscle-invasive and advanced urothelial cancer. The ideal integration of these two treatment modalities remains unclear as clinical trials have led to inconsistent results. Modulation of the tumor-immune microenvironment by chemotherapy is poorly characterized. We aimed to investigate this modulation, focusing on potential clinical implications for immune checkpoint inhibitor response. EXPERIMENTAL DESIGN: We assessed immune cell densities, spatial relations, and tumor/stromal components from 116 patients with urothelial bladder cancer (paired data for 95 patients) before and after platinum-based chemotherapy. RESULTS: Several published biomarkers for immunotherapy response changed upon chemotherapy treatment. The intratumoral CD8+ T-cell percentage increased after treatment and was associated with increased TNFα-via-NF-κB signaling. The percentage of PDL1+ immune cells was higher after chemotherapy. An increase in chemo-induced changes that potentially inhibit an antitumor immune response was also observed, including increased fibroblast-based TGFβ signaling and distances from immune cells to the nearest cancer cell. The latter two parameters correlated significantly in posttreatment samples, suggesting that TGFβ signaling in fibroblasts may play a role in spatially separating immune cells from cancer cells. We examined specific chemotherapy regimens and found that treatment with methotrexate, vinblastine, doxorubicin, and cisplatin was associated with an increase in the macrophage cell percentage. Gemcitabine-containing chemotherapy was associated with upregulation of fibroblast TGFβ signaling. CONCLUSIONS: The opposing effects of platinum-based chemotherapy on the immune cell composition and stromal context of the tumor-immune microenvironment may explain the inconsistent results of clinical trials investigating chemotherapy and immune checkpoint inhibitor combinations in bladder cancer.

PURPOSE: Patients with postpartum breast cancer diagnosed after cessation of breastfeeding (postweaning, PP-BCPW) have a particularly poor prognosis compared with patients diagnosed during lactation (PP-BCDL), or to pregnant (Pr-BC) and nulliparous (NP-BC) patients, regardless of standard prognostic characteristics. Animal studies point to a role of the involution process in stimulation of tumor growth in the mammary gland. However, in women, the molecular mechanisms that underlie this poor prognosis of patients with PP-BCPW remain vastly underexplored, due to of lack of adequate patient numbers and outcome data. EXPERIMENTAL DESIGN: We explored whether distinct prognostic features, common to all breast cancer molecular subtypes, exist in postpartum tumor tissue. Using detailed breastfeeding data, we delineated the postweaning period in PP-BC as a surrogate for mammary gland involution and performed whole transcriptome sequencing, immunohistochemical, and (multiplex) immunofluorescent analyses on tumor tissue of patients with PP-BCPW, PP-BCDL, Pr-BC, and NP-BC. RESULTS: We found that patients with PP-BCPW having a low expression level of an immunoglobulin gene signature, but high infiltration of plasma B cells, have an increased risk for metastasis and death. Although PP-BCPW tumor tissue was also characterized by an increase in CD8+ cytotoxic T cells and reduced distance among these cell types, these parameters were not associated with differential clinical outcomes among groups. CONCLUSIONS: These data point to the importance of plasma B cells in the postweaning mammary tumor microenvironment regarding the poor prognosis of PP-BCPW patients. Future prospective and in-depth research needs to further explore the role of B-cell immunobiology in this specific group of young patients with breast cancer.