To explore the immune microenvironment of RAS‐mutated (RASmt) microsatellite stable (MSS) colon cancer (CC), we retrospectively performed whole exome sequencing, RNA sequencing, and robust digital pathology analyses and studied immune markers in a cohort of 161 patients treated with standard‐of‐care therapies with early stage disease (both fresh frozen and formalin‐fixed paraffin‐embedded [FFPE] samples) or 121 patients with metastatic setting (primary tumor FFPE samples). Only a small proportion of cases exhibited a highly infiltrated immune microenvironment, with a strong association between Immunoscore® (IS)‐high (13% of the samples) and Tumor Lymphocytes Infiltrating Score (TuLIS)‐high scores (25% of the samples). Immunoscore Immune‐Checkpoint (ISIC)‐high tumors (52% of the samples) shared a similar microenvironment composition to IS‐high and TuLIS‐like high tumors and displayed higher mutational burdens than ISIC‐low tumors. In conclusion, a substantial proportion of MSS RASmt CCs exhibit high ISIC scores, meriting evaluation in prospective trials of immunotherapy‐based combination regimens.

e12645 Background: NCIT efficacy remains limited in HR+ eBC. Given the role of cell interactions in antitumor immunity, we evaluated TIME spatial dynamics in this subtype. Methods: This is an exploratory analysis from trial NCT02999477 randomizing pts to a monotherapy run-in (A: nab-paclitaxel; B: pembrolizumab) followed by both in combination (NCIT). Biopsies were obtained at baseline (BL), after run-in (week 3, W3), and on NCIT (W7). At each timepoint, 4 tissue sections were stained with 4 mIF panels: T-cell (CD4, CD8a, FOXP3), activated T-cell (CD8a, granzyme B, Ki67), antigen presenting cells (APCs) (HLA-ABC, HLA-DR, CD8a), and myeloid (CD68, CD206, HLA-DR), all with pan-CK and nuclear staining. Objectives were to characterize immune cell densities (cell/mm2), changes from BL, tumor–immune nearest-neighbor distance (μm), and explore associations with clinicopathological features, residual cancer burden (RCB) (0/I vs II/III, Kruskal–Wallis) and event-free survival (EFS) (Cox model) (p < .05; FDR q < 0.2). Results: 27 pts were included. Interpatient heterogeneity in immune cell density was observed across timepoints. No significant BL density differences were seen by trial arm, age (≤50 vs > 50), race (White vs non-White), stage (II vs III). At BL, RCB-0/I pts had numerically higher density of helper T cells (Th) (CD4+; median 2154.1 vs 890.0; p = .06), cytotoxic T cells (Tc) (CD8+; 728.1 vs 367.9; p = .08), activated Tc (6.1 vs 1.3; p = .06), APCs (HLA-ABC+, HLA-DR+: 124.6 vs 51.3; p = .36), and macrophages (CD68+CD206+; 63.4 vs 26.7; p = .25) vs RCB-II/III. On treatment, RCB-0/I pts had numerically greater % increase in cell density at W3 from BL than RCB-II/III for Tc (median 52.7 vs −62.0; p = .64), activated Tc (269.5 vs −83.6; p = .13), APCs (417.6 vs 24.9; p = .60), and macrophages (780.2 vs −61.4; p = .24). At W7, APCs continued to show greater % increase in RCB-0/I vs RCB-II/III (1583.3 vs 121.3; p = .38). At BL, distance from tumor area was numerically shorter in RCB-0/I vs RCB-II/III for Tc (median 55.2 vs 79.3; p = .20), activated Tc (1015.4 vs 2274.1; p = .39), APCs (216.4 vs 370.4; p = .39), and macrophages (276.4 vs 517.8; p = .50). On treatment (W3, W7), % changes from BL in tumor-immune distance were heterogeneous in RCB-0/I vs RCB-II/III across most immune cells. At W3, APCs (−52.2 vs 27.6; p = .24) and macrophages (−37.7 vs 20.2; p = .18) got numerically closer to tumor vs BL in RCB-0/I. At BL, pts with RCB-0/I had a numerically shorter distance between HLA-ABC+ cells and CD8+ cells vs RCB-II/III (median 20.5 vs 30.8; p = .36). A continued numerical % decrease from BL was seen at W3 (−57.1 vs 5.3; p = .12) and W7 (−65.2 vs −16.5; p = .38). No density or distance thresholds predicted EFS. Conclusions: TIME remodeling was heterogeneous, trending toward greater Tc and APC density and proximity to the tumor area in RCB 0/I. Clinical trial information: NCT02999477 .

Supplementary Figure 1. CODEX panel and experimental procedure. CODEX panel used to identify tumor cell subpopulations, immune cell populations and key cellular markers. (B) Experimental workflow for 5 pre- and post-treatment FFPE samples. ROIs were selected for multiplexed imaging and downstream analysis. (C) U Map clusters show major cell populations. Immune populations showed similar clustering in pre- and post- treatment samples.

Supplementary Figure 3. Digital quantification from immunohistochemistry. Digital quantification from immunohistochemistry for T cell subsets and marker analysis from paired tumor samples for five patients enrolled to cohort A.

Supplementary Figure 2. Patient enrollment, treatment and disposition per cohort.

Nivolumab improved survival in patients with refractory metastatic clear-cell renal cell carcinoma (ccRCC), but no reliable biomarker of activity has been identified. We conducted a real-world phase 2 trial of nivolumab in patients progressing after one or more vascular endothelial growth factor (VEGF) receptor-directed therapies, which included an integrated translational programme. Candidate tissue and circulating biomarkers were assessed using immunoassays and gene expression profiling. Overall, 720 patients were treated, with activity and safety in line with pivotal trial data. Exploration of tissue architecture showed that the presence of tertiary lymphoid structures, CD8+ lymphocytes, and CD163+ macrophage infiltration at the invasive margin were all marginally associated with longer progression-free survival, similarly to PD-1 expression on immune cells. Expression of hypoxia-related marker VEGF on tumour cells was however strongly associated with shorter progression-free and overall survival. Recapitulation of microenvironment composition based on gene expression signatures showed that patients harbouring a high tumour lymphocyte infiltration, concomitantly to low infiltration of neutrophil and non-immune stromal cells, had improved response to nivolumab. Conversely, circulating cytokines related to protumoral inflammation interleukin (IL)-6 and IL-8 were independently associated with shorter progression-free and overall survival. Overall, immune and angiogenic features helped inform outcomes to nivolumab. Circulating factors were best potential predictors for immunotherapy activity in ccRCC.

PURPOSE:The phase II NIVOREN GETUG-AFU 26 study assessed the safety and activity of nivolumab in a real-world population with pretreated advanced clear-cell renal cell carcinoma. A comprehensive translational research program was conducted, including blood- and tissue-based analyses. In this study, we assessed circulating factors at baseline, their association with intratumoral immune contexture, and outcomes. EXPERIMENTAL DESIGN:Baseline blood samples were prospectively collected from 353 patients included in the NIVOREN trial. A cohort of 80 patients, including 40 responders and 40 with primary progressive disease, was used for biomarker discovery, with 14 soluble factors (VEGF, vascular cell adhesion molecule-1, IL-6, IL-7, IL-8, IL-10, APRIL, B-cell activating factor, 4-1BB, B-cell attracting chemokine, stromal cell-derived factor-1, macrophage-derived chemokine, IFN-γ, and TNF-α) assessed for association with overall survival (OS; discovery set). Candidate biomarkers were subsequently assessed in the remaining 273 patients for association with outcomes (validation set). Gene expression signatures were assessed on baseline tissue samples using RNA sequencing. RESULTS:Five candidate biomarkers, IL-6, IL-7, IL-8, VEGF, and 4-1BB, were significantly associated with OS in the discovery set. We confirmed in the validation set a significant adverse association between OS and higher baseline levels of IL-6, IL-8, and VEGF, with respective HR of 3.17 [95% confidence interval (CI), 2.29-4.40], 2.11 (95% CI, 1.60-2.80), and 1.36 (95% CI, 1.04-1.79). Higher IL-6 and IL-8 levels were associated with an intratumor IMmotion Myeloid gene expression signature (P = 0.004 and P = 0.041, respectively). CONCLUSIONS:Elevated baseline circulating IL-6, IL-8, and VEGF are associated with worse outcomes in patients with nivolumab-treated advanced clear-cell renal cell carcinoma. Circulating IL-6 and IL-8 showed the strongest association with outcomes and correlated with a myeloid tissue contexture, providing guidance for patient selection in future trials.

Purpose: Radiotherapy may enhance antitumor immune responses by several mechanisms, including induction of immunogenic cell death. We performed a phase 2 study of pembrolizumab with re-irradiation in patients with recurrent glioblastoma.Patients and Methods: Sixty patients with recurrent glioblastoma received pembrolizumab with re-irradiation alone (cohort A, bevacizumab-na & iuml;ve; n = 30) or with bevacizumab continuation (cohort B, n = 30). Dual primary endpoints, including the overall response rate and overall survival (OS) at either 12 (OS-12; cohort A) or 6 months (OS-6; cohort B), were assessed per cohort relative to historic benchmarks. Paired paraffin-embedded formalin-fixed tumor samples were assessed for immunologic biomarkers by IHC using digital quantification and co-detection-by-indexing (CODEX).Results: Study therapy was well tolerated, with most toxicities being grade <= 3. For cohort B, the primary endpoint of OS-6 was achieved (57%); however, survival was not improved for cohort A patients. The overall response rate was 3.3% and 6.7% for cohorts A and B, respectively. CODEX analysis of paired tumor samples from five patients revealed an increase of activated T cells in the tumor microenvironment after study therapy.Conclusions: Compared with historic controls, re-irradiation plus pembrolizumab seemed to improve survival among bevacizumab-refractory patients but not among bevacizumab-na & iuml;ve patients. CODEX revealed evidence of intratumoral infiltration of activated immune effector cells. A randomized, properly controlled trial of PD-1 blockade plus re-irradiation is warranted to further evaluate this regimen for bevacizumab-refractory patients, but alternative approaches are needed for bevacizumab-na & iuml;ve patients.

Supplementary Figure 6. Cell-cell interaction analyses. Cell-cell interaction comparison in pre- and post-treatment tumor samples using CODEX for patient #5. (A) All significant interacting cell types shown in the entire analyzed tumor region as annotated. (B) Example images of cell – cell interactions showing interaction of CD163+ macrophages with GZMB+ CD8 T cells, CD163+ macrophages with GZMB+ CD4 T cells, CD68+ macrophages with GZMB+ CD8 T cells. The plus sign indicates interactions that were in close proximity at a one-cell distance (20 pixels).

Supplementary Figure 7. Cellular Neighborhood Analyses. Comparison of annotated cellular neighborhoods in pre- and post-treatment tumor samples via CODEX (color code as in Figure 4).

Supplementary Figure 4. CODEX analysis of tumor PD-L1 expression patterns. (A) PD-L1 expression was detected in NCAM-1+ tumor cells surrounding by hypoxia region as detected by CA9 staining. (B) PD-L1 expression was also observed in GFAP+ tumor cells surrounded in the vicinity of apoptotic tumor cells (cleaved caspase-3 detection).

Natural killer (NK) cells play a pivotal role against cancer, both by direct killing of malignant cells and by promoting adaptive immune response though cytokine and chemokine secretion. In the lung tumor microenvironment (TME), NK cells are scarce and dysfunctional. By conducting single-cell transcriptomic analysis of lung tumors, and exploring pseudotime, we uncovered that the intratumoral maturation trajectory of NK cells is disrupted in a tumor stage-dependent manner, ultimately resulting in the selective exclusion of the cytotoxic subset. Using functional assays, we observed intratumoral NK cell death and a reduction in cytotoxic capacities depending on the tumor stage. Finally, our analyses of human public dataset on lung cancer corroborate these findings, revealing a parallel dysfunctional maturation process of NK cells during tumor progression. These results highlight additional mechanisms by which tumor cells escape from NK cell cytotoxicity, therefore paving the way for tailored therapeutic strategies.

AbstractOne billion people worldwide get flu every year, including patients with non–small cell lung cancer (NSCLC). However, the impact of acute influenza A virus (IAV) infection on the composition of the tumor microenvironment (TME) and the clinical outcome of patients with NSCLC is largely unknown. We set out to understand how IAV load impacts cancer growth and modifies cellular and molecular players in the TME. Herein, we report that IAV can infect both tumor and immune cells, resulting in a long-term protumoral effect in tumor-bearing mice. Mechanistically, IAV impaired tumor-specific T-cell responses, led to the exhaustion of memory CD8+ T cells and induced PD-L1 expression on tumor cells. IAV infection modulated the transcriptomic profile of the TME, fine-tuning it toward immunosuppression, carcinogenesis, and lipid and drug metabolism. Consistent with these data, the transcriptional module induced by IAV infection in tumor cells in tumor-bearing mice was also found in human patients with lung adenocarcinoma and correlated with poor overall survival. In conclusion, we found that IAV infection worsened lung tumor progression by reprogramming the TME toward a more aggressive state.

Immunotherapy is standard of care in mRCC but tools for patients' selection are lacking. The NIVOREN GETUG-AFU 26 study of nivolumab in mRCC included a translational program with assessment of circulating cytokines. We found IL-8 to be associated with overall survival (Carril-Ajuria et al. ASCO. 2022). Here, we aimed to evaluate the association between IL-8 and the mRCC immune landscape, including both circulating and tissue-based assessments. Out of 720 pts treated with nivo within the NIVOREN study, 353 with available cytokine quantification were included for analysis. Immunohistochemistry expression of VEGF and CD163 at the invasive margin (IM) and at the tumor core (TC) was assessed. Previously published tissue-based GS were analyzed, including IMmotion myeloid, IMmotion Teff, and JAVELIN 101 immuno. Circulating inflammatory markers included neutrophils, NLR (neutrophil lymphocyte ratio), LIPI (Lung Immune Prognostic Index, based on derived NLR>3 and LDH>upper limit of normal). The association of baseline levels of IL-8 with baseline levels of circulating inflammatory markers, and with VEGF/CD163 expression and tissue-based GS expression were analyzed. Baseline characteristics were similar to the overall trial population. Higher levels of baseline circulating IL-8 (cut-off:17.9 pg/ml) were associated with increased baseline circulating neutrophiles (p=0.046), and NLR (p=0.008) as well as with LIPI (p=0.005). Among 353 pts, only 64 had available information on GS expression. Baseline IL-8 significantly correlated with increased IMmotion myeloid expression (p=0.041). No significant correlations between baseline circulating IL-8 and the other two GS, VEGF or CD163 expression were observed. In our study, elevated baseline circulating IL-8 was not only significantly associated with higher levels of other circulating inflammatory markers, but also with increased expression of the myeloid inflammation gene signature at tumor level in mRCC pts.

607 Background: To date, no biomarker of efficacy of nivolumab+/-ipilimumab (N+/-I) or anti-VEGFR TKI has been prospectively validated in mRCC. The BIONIKK trial showed promising objective response rate (ORR) and progression-free survival (PFS) with these treatments in first line (L1) after selection by tumour molecular group. We report OS and efficacy results of the second-line (L2) treatment. Methods: BIONIKK is a French multicentre non-comparative phase II trial, randomising 199 mRCC pts to receive N (58), NI (101) or TKI (40) in L1 according to four molecular groups (ccrcc1-4). ORR and PFS were already reported. With an additional follow-up of ≥20 months, we report OS from randomization and from the start of L2, as well as ORR and PFS with a TKI in L2 by molecular group. Results: With a median follow-up of 42.1 months (40.5-45.2), 86 (43%) patients died: 27/58 (46.5%), 39/101 (39%) and 20/40 (50%) in the N, NI, and TKI arm, respectively. Median OS were 43.4 months (95%CI=31.4-NR) with N, 52.7 months (95%CI=46-NR) with NI and 38.1 months (95%CI=33.2-NR) with TKI (table). 175 (88%) patients discontinued first-line treatment, including 20 deaths, and 129 (74%) received a L2, 38/58 (65.5%), 64/101 (63%), and 27/40 (67.5%) after N, NI and TKI, respectively. The most frequent L2 received after N+/-I was a TKI in 96/102 (94%) pts, including cabozantinib in 49, sunitinib/pazopanib in 32, axitinib in 13, and lenvatinib in 2. N was the most frequent L2 after TKI, 20/27 (74%). ORR with TKI in L2 was 28.5% (10/35) after N, 39% (24/61) after NI and 80% (4/5) after TKI, with marked benefit in ccrcc2 pts (table). The mPFS with TKI in L2 was 8.2 (95%CI=6.9-19.3) after N, 11.4 (95%CI= 8.9-16.8) after NI, and 12.1 (95%CI =11.4-NR) months after TKI, with a higher benefit in ccrcc2 pts (vs. ccrcc1+4, p=0.04). Conversely, ORR and mPFS with N after TKI in ccrcc2-pts were 12.5% (2/16) and 5.4 (2.6-NR) months, respectively. Median OS L2 was reported in the table. The updated ORR and PFS in L1 will presented at the Meeting, as well as PFS2 and efficacy by TKI type in L2. Conclusions: We report for the first-time OS and L2 efficacy results by molecular group in a randomized trial. Molecular selection also has an impact on treatment efficacy in L2. These results, together with those reported in L1, can inform clinicians on the best treatment sequence in L1-2. Clinical trial information: NCT02960906 . [Table: see text]

Background Papillary renal cell carcinoma (pRCC) is the most common non-clear cell RCC, and associated with poor outcomes in the metastatic setting. In this study, we aimed to comprehensively evaluate the immune tumor microenvironment (TME), largely unknown, of patients with metastatic pRCC and identify potential therapeutic targets.Methods We performed quantitative gene expression analysis of TME using Microenvironment Cell Populations-counter (MCP-counter) methodology, on two independent cohorts of localized pRCC (n=271 and n=98). We then characterized the TME, using immunohistochemistry (n=38) and RNA-sequencing (RNA-seq) (n=30) on metastatic pRCC from the prospective AXIPAP trial cohort.Results Unsupervised clustering identified two “TME subtypes”, in each of the cohorts: the “immune-enriched” and the “immune-low”. Within AXIPAP trial cohort, the “immune-enriched” cluster was significantly associated with a worse prognosis according to the median overall survival to 8 months (95% CI, 6 to 29) versus 37 months (95% CI, 20 to NA, p=0.001). The two immune signatures, Teff and JAVELIN Renal 101 Immuno signature, predictive of response to immune checkpoint inhibitors (CPI) in clear cell RCC, were significantly higher in the “immune-enriched” group (adjusted p<0.05). Finally, five differentially overexpressed genes were identified, corresponding mainly to B lymphocyte populations.Conclusion For the first time, using RNA-seq and immunohistochemistry, we have highlighted a specific immune TME subtype of metastatic pRCC, significantly more infiltrated with T and B immune population. This “immune-enriched” group appears to have a worse prognosis and could have a potential predictive value for response to immunotherapy, justifying the confirmation of these results in a cohort of metastatic pRCC treated with CPI and in combination with targeted therapies.Trial registration number NCT02489695.

This file contains 6 supplementary figures and 5 supplementary Tables. Supplementary Figure S1 - Protein expression pattern of AXL in ccRCC specimens. Supplementary Figure S2 - Response rates, Survival and Biomarker analyses according to AXLneg, AXLlow and AXLhigh expression Supplementary Figure S3 - ORR and Progression-Free Survival according to PD-L1 Status alone or by grouping AXL expression plus PD-L1 status Supplementary Figure S4 - High AXL expression plus PD-L1 TC positivity is associated with worse OS in Nivolumab-treated patients in IMDC intermediate-risk/poor-risk group Supplementary Figure S5 - VHL status does not interfere with outcomes or AXL expression upon treatment with Nivolumab Supplementary Figure S6 - Biomarker distribution across AXL groups stratified based on VHL status

The generation of anti-tumor immunity in the draining lymph nodes is known as the cancer immunity cycle. Accumulating evidence supports the occurrence of such a cycle at tumor sites in the context of chronic inflammation. Here, we review the role of tertiary lymphoid structures (TLS) in the generation of T and B cell immunities, focusing on the impact of B cells that undergo full maturation, resulting in the generation of plasma cells (PCs) producing high-affinity IgG and IgA antibodies. In this context, we propose that antibodies binding to tumor cells induce macrophage or natural killer (NK)-cell-dependent apoptosis. Subsequently, released antigen-antibody complexes are internalized and processed by dendritic cells (DCs), amplifying antigen presentation to T cells. Immune complexes may also be fixed by follicular DCs (FDCs) in TLS, thereby increasing memory B cell responses. This amplification loop creates an intra-tumoral immunity cycle, capable of increasing sensitivity of tumors to immunotherapy even in cancers with low mutational burden.