Spatial relationships in the urothelial and head and neck tumor microenvironment predict response to combination immune checkpoint inhibitors.

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.

Immune cells in mesothelioma microenvironment simplistic marker of response to nivolumab plus ipilimumab?

INTRODUCTION: Malignant pleural mesothelioma (MPM) is a malignant disease of the pleura which recently can be treated with immune checkpoint inhibitors (ICI). To optimize this treatment, a better understanding of the tumor micro environment is needed. We investigated subgroups of immune cells in subsequent tumor biopsies of patients treated with ICI. METHODS: Biopsies from MPM patients included in two clinical ICI trials (nivolumab alone and an ipilimumab/nivolumab combination) were examined. At baseline and after 6 weeks of treatment, pleural biopsies were taken to examine the tumor microenvironment (CD20+, CD4+, CD8+, FoxP3+ and PD-1+ ). Cell density was defined as the number of marker positive cells per mm2. Radiological responses were evaluated as partial response, stable disease or progressive disease according to modified RECIST criteria. RESULTS: Thirty-four and 36 patients were included in the nivolumab and ipiliumumab/nivolumab trial respectively. In the nivolumab trial, no significant differences in cell densities were seen in baseline biopsies of patients with partial response versus progressive disease. In contrast, in the ipilimumab/nivolumab trial, a higher cell density of CD4+, CD8+, FoxP3+ and PD-1+ cells at baseline was significantly correlated with partial responses. On-treatment biopsies of both trials did not show significant changes when compared to baseline biopsies. CONCLUSION: Biopsies from patients responding to nivolumab plus ipilimumab treatment show a significant higher cell density of CD4+, CD8+, FoxP3+ and PD-1+ cells, without a change after 6 weeks of treatment. This observation is a first step in exploring the tumor microenvironment as predictor of response in ICI treatment in MPM.

Neoadjuvant immunotherapy with nivolumab and ipilimumab induces major pathological responses in patients with head and neck squamous cell carcinoma.

Surgery for locoregionally advanced head and neck squamous cell carcinoma (HNSCC) results in 30‒50% five-year overall survival. In IMCISION (NCT03003637), a non-randomized phase Ib/IIa trial, 32 HNSCC patients are treated with 2 doses (in weeks 1 and 3) of immune checkpoint blockade (ICB) using nivolumab (NIVO MONO, n = 6, phase Ib arm A) or nivolumab plus a single dose of ipilimumab (COMBO, n = 26, 6 in phase Ib arm B, and 20 in phase IIa) prior to surgery. Primary endpoints are feasibility to resect no later than week 6 (phase Ib) and primary tumor pathological response (phase IIa). Surgery is not delayed or suspended for any patient in phase Ib, meeting the primary endpoint. Grade 3‒4 immune-related adverse events are seen in 2 of 6 (33%) NIVO MONO and 10 of 26 (38%) total COMBO patients. Pathological response, defined as the %-change in primary tumor viable tumor cell percentage from baseline biopsy to on-treatment resection, is evaluable in 17/20 phase IIa patients and 29/32 total trial patients (6/6 NIVO MONO, 23/26 COMBO). We observe a major pathological response (MPR, 90‒100% response) in 35% of patients after COMBO ICB, both in phase IIa (6/17) and in the whole trial (8/23), meeting the phase IIa primary endpoint threshold of 10%. NIVO MONO's MPR rate is 17% (1/6). None of the MPR patients develop recurrent HSNCC during 24.0 months median postsurgical follow-up. FDG-PET-based total lesion glycolysis identifies MPR patients prior to surgery. A baseline AID/APOBEC-associated mutational profile and an on-treatment decrease in hypoxia RNA signature are observed in MPR patients. Our data indicate that neoadjuvant COMBO ICB is feasible and encouragingly efficacious in HNSCC.

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.

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.

Androgen receptor signalling in macrophages promotes TREM-1-mediated prostate cancer cell line migration and invasion.

The androgen receptor (AR) is the master regulator of prostate cancer (PCa) development, and inhibition of AR signalling is the most effective PCa treatment. AR is expressed in PCa cells and also in the PCa-associated stroma, including infiltrating macrophages. Macrophages have a decisive function in PCa initiation and progression, but the role of AR in macrophages remains largely unexplored. Here, we show that AR signalling in the macrophage-like THP-1 cell line supports PCa cell line migration and invasion in culture via increased Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) signalling and expression of its downstream cytokines. Moreover, AR signalling in THP-1 and monocyte-derived macrophages upregulates IL-10 and markers of tissue residency. In conclusion, our data suggest that AR signalling in macrophages may support PCa invasiveness, and blocking this process may constitute one mechanism of anti-androgen therapy.

Tumour-infiltrating lymphocytes (TILs) and BRCA-like status in stage III breast cancer patients randomised to adjuvant intensified platinum-based chemotherapy versus conventional chemotherapy.

BACKGROUND: The prognostic value of tumour-infiltrating lymphocytes (TILs) differs by breast cancer (BC) subtype. The aim of this study was to evaluate TILs in stage III BC in the context of BRCA1/2-like phenotypes and association with outcome and benefit of intensified platinum-based chemotherapy. PATIENTS AND METHODS: Patients participated in a randomised controlled trial of adjuvant intensified platinum-based chemotherapy versus conventional anthracycline-based chemotherapy carried out between 1993 and 1999 in stage III BC. Stromal TILs were scored according to International guidelines in these human epidermal growth factor receptor 2 (HER2)-negative tumours. BRCA-profiles were determined using Comparative Genomic Hybridization. RESULTS: TIL levels were evaluated in 248 BCs. High TILs were associated with Triple Negative BC (TNBC). BRCA-like tumours harboured higher TILs compared to non-BRCA-like tumours (median TILs of 20% versus 10%, p < 0.01). TIL levels in BRCA1-like tumours were higher compared to BRCA2-like tumours (median TILs of 20% versus 10%, p < 0.001). These correlations remained significant within the oestrogen (ER)-positive subgroup, however not within the TNBC subgroup. In this stage III BC cohort, high TIL level was associated with favourable outcome (TILs per 10% increment, recurrence-free survival (RFS): multivariate hazard ratio (HR) 0.82, 95% confidence interval (CI) 0.71-0.94, p = 0.01; overall survival (OS): multivariate HR 0.80, 95% CI 0.68-0.94, p = 0.01). There was no significant interaction between TILs and benefit of intensified platinum-based chemotherapy. CONCLUSION: In this high-risk breast cancer cohort, high TILs were associated with TNBC and BRCA1-like status. Within the ER-positive subgroup, TIL levels were higher in BRCA1-like compared to BRCA2-like tumours. When adjusted for clinical characteristics, TILs were significantly associated with a more favourable outcome in stage III BC patients.