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Background: Locally advanced oral cavity squamous cell carcinoma (OCSCC) presents a significant clinical challenge despite being partially responsive to standard treatment modalities. This study investigates the prognostic implications of programmed death-ligand 1 (PD-L1) expression in these tumors, focusing on its association with treatment outcomes and the immune microenvironment., Methods: We assessed tumor-infiltrating lymphocytes (TILs) in 132 patients with OCSCC to evaluate their impact on survival. Multiplex immunohistochemistry staining for CD3, CD68, CD11c, PD-L1, and P40 was used to explore correlations with clinical outcomes in patients with early-stage (n=22) and locally advanced (n=36) OCSCC. These initial findings were validated through differential gene expression analysis, gene set enrichment, and immune cell deconvolution in a The Cancer Genome Atlas cohort of 163 locally advanced OCSCC tumors. Additionally, single-cell RNA sequencing (scRNA-seq) on a smaller cohort (n=10) further characterized the PD-L1 hi or PD-L1 lo cancer cells in these tumors., Results: Elevated PD-L1 expression was associated with poor outcomes in patients with locally advanced OCSCC undergoing standard adjuvant therapy, irrespective of "hot" or "cold" classification based on TILs assessment. PD-L1 hi tumors exhibited an active immune response phenotype, enriched with M1 macrophages, CD8 + T cells and T regulatory cells in the tumor microenvironment. Notably, the negative impact of PD-L1 expression on outcomes was primarily attributed to its expression by cancer cells, rather than immune cells. Furthermore, scRNA-seq revealed that immune interactions were not essential for PD-L1 upregulation in cancer cells, instead, complex regulatory networks were involved. Additionally, PD-L1 lo locally advanced tumors exhibited more complex pathway enrichment and diverse T-cell populations compared with those in the early-stage., Conclusion: Our findings underscore the prognostic significance of PD-L1 expression in locally advanced OCSCC, and unveil the complex interplay between PD-L1 expression, immune responses, and molecular pathways in the tumor microenvironment. This study provides insights that may inform future therapeutic strategies, including the possibility of tailored immunotherapeutic approaches for patients with PD-L1 hi locally advanced OCSCC., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Vaginal melanoma (VM) is a rare cancer and has a poor response to immune checkpoint blockade (ICB). CD8 + Tissue Resident Memory (TRM) T cells proliferate in response to ICB and correlate with longer survival in metastatic cutaneous melanoma. However, their capacity to respond to VM and their neoantigens is not known., Methods: Using longitudinal samples, we explored the evolution of VM mutations by whole-exome sequencing and RNAseq, we also defined the immune context using multiplex immunohistochemistry and nanostring pan cancer immune profile. Then using fresh single cell suspensions of the metastatic samples, we explored VM T cells via mass cytometry and single cell RNAseq and T cell receptor sequencing (TCRseq). Finally, we investigated TRM, pre-TRM and exhausted T cell function against melanoma neo-antigens and melanoma differentiation antigens in vitro., Results: Primary VM was non-inflamed and devoid of CD8 + TRM cells. In contrast, both metastases showed proliferating CD8 + TRM were clustered at the tumor margin, with increased numbers in the second ICB-refractory metastasis. The first metastasis showed dense infiltration of CD8 + T cells, the second showed immune exclusion with loss of melanoma cell Major histocompatibility complex (MHC)-I expression associated with downregulation of antigen presentation pathway gene expression. CD8 + TRM from both metastases responded to autologous melanoma cells more robustly than all other CD8 + T cell subsets. In addition, CD8 + TRM shared TCR clones across metastases, suggesting a response to common antigens, which was supported by recognition of the same neoantigen by expanded tumor infiltrating lymphocytes., Conclusions: In this study, we identified TRM clusters in VM metastases from a patient, but not primary disease. We showed TRM location at the tumor margin, and their superior functional response to autologous tumor cells, predicted neoantigens and melanoma differentiation antigens. These CD8 + TRM exhibited the highest tumor-responsive potential and shared their TCR with tumor-infiltrating effector memory T cells. This suggests VM metastases from this patient retain strong antitumor T cell functional responses; however, this response is suppressed in vivo. The loss of VG MHC-I expression is a common immune escape mechanism which was not addressed by anti-PD-1 monotherapy; rather an additional targeted approach to upregulate MHC-I expression is required., Competing Interests: Competing interests: AP, NK and SPK were supported by imCORE, F. Hoffmann-La Roche. MP is an employee of F. Hoffmann-La Roche. DEG has been on advisory board for Amgen, Q Biotics, Provectus and Bayer. SS has been receiving advisory board fees, paid to her institution, and grant support from AstraZeneca and Merck Sharp & Dohme, and grant support from Amgen, Endocyte, and Genentech., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Patients with BRAF -mutant and wild-type melanoma have different response rates to immune checkpoint blockade therapy. However, the reasons for this remain unknown. To address this issue, we investigated the precise immune composition resulting from BRAF mutation in treatment-naive melanoma to determine whether this may be a driver for different response to immunotherapy., Methods: In this study, we characterized the treatment-naive immune context in patients with BRAF -mutant and BRAF wild-type ( BRAF -wt) melanoma using data from single-cell RNA sequencing, bulk RNA sequencing, flow cytometry and immunohistochemistry (IHC)., Results: In single-cell data, BRAF -mutant melanoma displayed a significantly reduced infiltration of CD8 + T cells and macrophages but also increased B cells, natural killer (NK) cells and NKT cells. We then validated this finding using bulk RNA-seq data from the skin cutaneous melanoma cohort in The Cancer Genome Atlas and deconvoluted the data using seven different algorithms. Interestingly, BRAF -mutant tumors had more CD4 + T cells than BRAF -wt samples in both primary and metastatic cohorts. In the metastatic cohort, BRAF -mutant melanoma demonstrated more B cells but less CD8 + T cell infiltration when compared with BRAF -wt samples. In addition, we further investigated the immune cell infiltrate using flow cytometry and multiplex IHC techniques. We confirmed that BRAF -mutant melanoma metastases were enriched for CD4 + T cells and B cells and had a co-existing decrease in CD8 + T cells. Furthermore, we then identified B cells were associated with a trend for improved survival (p=0.078) in the BRAF -mutant samples and Th2 cells were associated with prolonged survival in the BRAF -wt samples., Conclusions: In conclusion, treatment-naive BRAF -mutant melanoma has a distinct immune context compared with BRAF -wt melanoma, with significantly decreased CD8 + T cells and increased B cells and CD4 + T cells in the tumor microenvironment. These findings indicate that further mechanistic studies are warranted to reveal how this difference in immune context leads to improved outcome to combination immune checkpoint blockade in BRAF -mutant melanoma., Competing Interests: Competing interests: RAS has received fees for professional services from Qbiotics, Novartis, Merck Sharp & Dohme, NeraCare, AMGEN, Bristol-Myers Squibb, Myriad Genetics, GlaxoSmithKline. GL has received professional consulting fees from Aduro Biotech, Amgen, Array Biopharma, Boehringer Ingelheim International GmbH, Bristol-Myers Squibb, Highlight Therapeutics SL, Merck Sharpe & Dohme, Novartis Pharma AG, Pierre Fabre, QBiotics Group, Regeneron Pharmaceuticals, SkylineDX BV (all unrelated to this work). PJN received research funding from Roche/Genentech, BMS, MSD, Allergan, Compugen, Crispr Therapeutics. The other authors declare no conflicts of interest., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Melanoma brain metastases (MBMs) are a challenging clinical problem with high morbidity and mortality. Although first-line dabrafenib-trametinib and ipilimumab-nivolumab have similar intracranial response rates (50%-55%), central nervous system (CNS) resistance to BRAF-MEK inhibitors (BRAF-MEKi) usually occurs around 6 months, and durable responses are only seen with combination immunotherapy. We sought to investigate the utility of ipilimumab-nivolumab after MBM progression on BRAF-MEKi and identify mechanisms of resistance., Methods: Patients who received first-line ipilimumab-nivolumab for MBMs or second/third line ipilimumab-nivolumab for intracranial metastases with BRAF V600 mutations with prior progression on BRAF-MEKi and MRI brain staging from March 1, 2015 to June 30, 2018 were included. Modified intracranial RECIST was used to assess response. Formalin-fixed paraffin-embedded samples of BRAF V600 mutant MBMs that were naïve to systemic treatment (n=18) or excised after progression on BRAF-MEKi (n=14) underwent whole transcriptome sequencing. Comparative analyses of MBMs naïve to systemic treatment versus BRAF-MEKi progression were performed., Results: Twenty-five and 30 patients who received first and second/third line ipilimumab-nivolumab, were included respectively. Median sum of MBM diameters was 13 and 20.5 mm for the first and second/third line ipilimumab-nivolumab groups, respectively. Intracranial response rate was 75.0% (12/16), and median progression-free survival (PFS) was 41.6 months for first-line ipilimumab-nivolumab. Efficacy of second/third line ipilimumab-nivolumab after BRAF-MEKi progression was poor with an intracranial response rate of 4.8% (1/21) and median PFS of 1.3 months. Given the poor activity of ipilimumab-nivolumab after BRAF-MEKi MBM progression, we performed whole transcriptome sequencing to identify mechanisms of drug resistance. We identified a set of 178 differentially expressed genes (DEGs) between naïve and MBMs with progression on BRAF-MEKi treatment (p value <0.05, false discovery rate (FDR) <0.1). No distinct pathways were identified from gene set enrichment analyses using Kyoto Encyclopedia of Genes and Genomes, Gene Ontogeny or Hallmark libraries; however, enrichment of DEG from the Innate Anti-PD1 Resistance Signature (IPRES) was identified (p value=0.007, FDR=0.03)., Conclusions: Second-line ipilimumab-nivolumab for MBMs after BRAF-MEKi progression has poor activity. MBMs that are resistant to BRAF-MEKi that also conferred resistance to second-line ipilimumab-nivolumab showed enrichment of the IPRES gene signature., Competing Interests: Competing interests: PKHL has received honoraria (Bristol-Myers Squibb, Pfizer) and support for meeting attendance (Merck Sharp and Dohme). DKe has received honoraria (Merck Sharp and Dohme, and Bristol Myers Squibb), support for meeting attendance (MSD) and fees for advisory board (Novartis and Merck). PB has received honoraria (Novartis) and received support for meeting attendance (Merck Sharp and Dohme). DKo has received support for meeting attendance (Merck and Pfizer). PS has received grants (Bristol-Myers Squibb, Roche/Genentech, Allergan, Compugen, Beigene and Crispr Therapeutics), support for meeting attendance (Bristol-Myers Squibb, Roche/Genentech and Astra Zeneca) and advisory board (Bristol-Myers Squibb). BS has received honoraria and fees for advisory board (Bristol-Myers Squibb, Merck Sharp and Dohme, AstraZeneca, Pfizer, Roche/Genentech, Amgen, Lilly, Amgen and Sanofi/Regeneron) SS has received grants (Novartis, Advanced Accelerator Applications, AstraZeneca, Merck Sharp and Dohme, Amgen and Genentech) and personal fees donated to the institution (AstraZeneca, Merck Sharp and Dohme, Bristol Myers Squibb and AstraZeneca) outside the submitted work. GAM has been a consultant or advisor (Provectus), received research funding from (Pfizer, Celgene, and Ventana) and has had travel accommodation and expenses paid (Roche and Novartis). No stated competing interests: BF, LS, AL, RM, KS, AW, CA, BL, RJY, AI, HAY, IAV, KD, KES and TP., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Prostate cancer (PCa) has a profoundly immunosuppressive microenvironment and is commonly immune excluded with few infiltrative lymphocytes and low levels of immune activation. High-dose radiation has been demonstrated to stimulate the immune system in various human solid tumors. We hypothesized that localized radiation therapy, in the form of high dose-rate brachytherapy (HDRBT), would overcome immune suppression in PCa., Methods: To investigate whether HDRBT altered prostate immune context, we analyzed preradiation versus postradiation human tissue from a cohort of 24 patients with localized PCa that received HDRBT as primary treatment (RadBank cohort). We performed Nanostring immune gene expression profiling, digital spatial profiling, and high-throughput immune cell multiplex immunohistochemistry analysis. We also resolved tumor and nontumor zones in spatial and bioinformatic analyses to explore the immunological response., Results: Nanostring immune profiling revealed numerous immune checkpoint molecules (eg, B7-H3, CTLA4, PDL1, and PDL2) and TGFβ levels were increased in response to HDRBT. We used a published 16-gene tumor inflammation signature (TIS) to divide tumors into distinct immune activation states (high: hot , intermediate and low: cold ) and showed that most localized PCa are cold tumors pre-HDRBT. Crucially, HDRBT converted 80% of these 'cold'-phenotype tumors into an 'intermediate' or 'hot' class. We used digital spatial profiling to show these HDRBT-induced changes in prostate TIS scores were derived from the nontumor regions. Furthermore, these changes in TIS were also associated with pervasive changes in immune cell density and spatial relationships-in particular, between T cell subsets and antigen presenting cells. We identified an increased density of CD4 + FOXP3 + T cells, CD68 + macrophages and CD68 + CD11c + dendritic cells in response to HDRBT. The only subset change specific to tumor zones was PDL1 - macrophages. While these immune responses were heterogeneous, HDRBT induced significant changes in immune cell associations, including a gained T cell and HMWCK + PDL1 + interaction in tumor zones., Conclusion: In conclusion, we showed HDRBT converted "cold" prostate tumors into more immunologically activated "hot" tissues, with accompanying spatially organized immune infiltrates and signaling changes. Understanding and potentially harnessing these changes will have widespread implications for the future treatment of localized PCa, including rational use of combination radio-immunotherapy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)