Stroma-infiltrating T cell spatiotypes define immunotherapy outcomes in adolescent and young adult patients with melanoma.

The biological underpinnings of therapeutic resistance to immune checkpoint inhibitors (ICI) in adolescent and young adult (AYA) melanoma patients are incompletely understood. Here, we characterize the immunogenomic profile and spatial architecture of the tumor microenvironment (TME) in AYA (aged ≤ 30 years) and older adult (aged 31-84 years) patients with melanoma, to determine the AYA-specific features associated with ICI treatment outcomes. We identify two ICI-resistant spatiotypes in AYA patients with melanoma showing stroma-infiltrating lymphocytes (SILs) that are distinct from the adult TME. The SILhigh subtype was enriched in regulatory T cells in the peritumoral space and showed upregulated expression of immune checkpoint molecules, while the SILlow subtype showed a lack of immune activation. We establish a young immunosuppressive melanoma score that can predict ICI responsiveness in AYA patients and propose personalized therapeutic strategies for the ICI-resistant subgroups. These findings highlight the distinct immunogenomic profile of AYA patients, and individualized TME features in ICI-resistant AYA melanoma that require patient-specific treatment strategies.

Interferon-gamma signature as prognostic and predictive marker in macroscopic stage III melanoma.

BACKGROUND: A substantial proportion of patients with macroscopic stage III melanoma do not benefit sufficiently from adjuvant anti-PD-1 therapy, as they either recur despite therapy or would never have recurred. To better inform adjuvant treatment selection, we have performed translational analyses to identify prognostic and predictive biomarkers. PATIENTS AND METHODS: Two cohorts of patients with macroscopic stage III melanoma from an ongoing biobank study were included. Clinical data were compared between an observation cohort (cohort 1) and an adjuvant intention cohort (cohort 2). RNA sequencing for translational analyses was performed and treatment subgroups (cohort 1A and cohort 2A) were compared for possible biomarkers, using a cut-off based on the treatment-naïve patients. In addition, two validation cohorts (Melanoma Institute Australia (MIA) and University Medical Centre Utrecht (UMCU)) were obtained. RESULTS: After a median follow-up of 26 months of the 98 patients in our discovery set, median recurrence-free survival (RFS) was significantly longer for the adjuvant intention cohort (cohort 2, n=49) versus the observation cohort (cohort 1, n=49). Median overall survival was not reached for either cohort, nor significantly different. In observation cohort 1A (n=24), RFS was significantly longer for patients with high interferon-gamma (IFNγ) score (p=0.002); for adjuvant patients of cohort 2A (n=24), a similar trend was observed (p=0.086). Patients with high B cell score had a longer RFS in cohort 1A, but no difference was seen in cohort 2A. The B cell score based on RNA correlated with CD20+ cells in tumor area but was not independent from the IFNγ score. In the MIA validation cohort (n=44), longer RFS was observed for patients with high IFNγ score compared with low IFNγ score (p=0.046), no difference in RFS was observed according to the B cell score. In both the observation (n=11) and the adjuvant (n=11) UMCU validation cohorts, no difference in RFS was seen for IFNγ and B cell. CONCLUSIONS: IFNγ has shown to be a prognostic marker in both patients who were and were not treated with adjuvant therapy. B cell score was prognostic but did not improve accuracy over IFNγ. Our study confirmed RFS benefit of adjuvant anti-PD-1 for patients with macroscopic stage III melanoma.

CD4+ T cell immunity against cutaneous melanoma encompasses multifaceted MHC II-dependent responses.

Whereas CD4+ T cells conventionally mediate antitumor immunity by providing help to CD8+ T cells, recent clinical studies have implied an important role for cytotoxic CD4+ T cells in cancer immunity. Using an orthotopic melanoma model, we provide a detailed account of antitumoral CD4+ T cell responses and their regulation by major histocompatibility complex class II (MHC II) in the skin. Intravital imaging revealed prominent interactions of CD4+ T cells with tumor debris-laden MHC II+ host antigen-presenting cells that accumulated around tumor cell nests, although direct recognition of MHC II+ melanoma cells alone could also promote CD4+ T cell control. CD4+ T cells stably suppressed or eradicated tumors even in the absence of other lymphocytes by using tumor necrosis factor-α and Fas ligand (FasL) but not perforin-mediated cytotoxicity. Interferon-γ was critical for protection, acting both directly on melanoma cells and via induction of nitric oxide synthase in myeloid cells. Our results illustrate multifaceted and context-specific aspects of MHC II-dependent CD4+ T cell immunity against cutaneous melanoma, emphasizing modulation of this axis as a potential avenue for immunotherapies.

Classification of the tumor immune microenvironment and associations with outcomes in patients with metastatic melanoma treated with immunotherapies.

BACKGROUND: Tumor microenvironment (TME) characteristics are potential biomarkers of response to immune checkpoint inhibitors in metastatic melanoma. This study developed a method to perform unsupervised classification of TME of metastatic melanoma. METHODS: We used multiplex immunohistochemical and quantitative pathology-derived assessment of immune cell compositions of intratumoral and peritumoral regions of metastatic melanoma baseline biopsies to classify TME in relation to response to anti-programmed cell death protein 1 (PD-1) monotherapy or in combination with anti-cytotoxic T-cell lymphocyte-4 (ipilimumab (IPI)+PD-1). RESULTS: Spatial profiling of CD8+T cells, macrophages, and melanoma cells, as well as phenotypic PD-1 receptor ligand (PD-L1) and CD16 proportions, were used to identify and classify patients into one of three mutually exclusive TME classes: immune-scarce, immune-intermediate, and immune-rich tumors. Patients with immune-rich tumors were characterized by a lower proportion of melanoma cells and higher proportions of immune cells, including higher PD-L1 expression. These patients had higher response rates and longer progression-free survival (PFS) than those with immune-intermediate and immune-scarce tumors. At a median follow-up of 18 months (95% CI: 6.7 to 49 months), the 1-year PFS was 76% (95% CI: 64% to 90%) for patients with an immune-rich tumor, 56% (95% CI: 44% to 72%) for those with an immune-intermediate tumor, and 33% (95% CI: 23% to 47%) for patients with an immune-scarce tumor. A higher response rate was observed in patients with an immune-scarce or immune-intermediate tumor when treated with IPI+PD-1 compared with those treated with PD-1 alone. CONCLUSIONS: Our study provides an automatic TME classification method that may predict the clinical efficacy of immunotherapy for patients with metastatic melanoma.

Lag-3 expression and clinical outcomes in metastatic melanoma patients treated with combination anti-lag-3 + anti-PD-1-based immunotherapies.

Lymphocyte-activation gene-3 (LAG-3), an immune checkpoint receptor, negatively regulates T-cell function and facilitates immune escape of tumors. Dual inhibition of LAG-3 and programmed cell death receptor-1 (PD-1) significantly improved progression-free survival (PFS) in metastatic melanoma patients compared to anti-PD-1 therapy alone. Investigating the utility of LAG-3 expression as a biomarker of response to anti-LAG-3 + anti-PD-1 immunotherapy is of great clinical relevance. This study sought to evaluate the association between baseline LAG-3 expression and clinical outcomes following anti-LAG-3 and anti-PD-1-based immunotherapy in metastatic melanoma. LAG-3 immunohistochemistry (clone D2G4O) was performed on pre-treatment formalin-fixed, paraffin-embedded metastatic melanoma specimens from 53 patients treated with combination anti-LAG-3 + anti-PD-1-based therapies. Eleven patients had received prior anti-PD-1-based treatment. Patients were categorized as responders (complete/partial response; n = 36) or non-responders (stable/progressive disease; n = 17) based on the Response Evaluation Criteria in Solid Tumours (RECIST). Tumor-infiltrating lymphocytes (TILs) were scored on hematoxylin and eosin-stained sections. LAG-3 expression was observed in 81% of patients, with staining in TILs and dendritic cells. Responders displayed significantly higher proportions of LAG-3+ cells compared to non-responders (P = .0210). LAG-3 expression positively correlated with TIL score (P < .01). There were no significant differences in LAG-3 expression between different sites of metastases (P > .05). Patients with ≥ 1% LAG-3+ cells in their tumors had significantly longer PFS compared to patients with < 1% LAG-3 expression (P = .0037). No significant difference was observed in overall survival between the two groups (P = .1417). Therefore, the assessment of LAG-3 expression via IHC warrants further evaluation to determine its role as a predictive marker of response and survival in metastatic melanoma.

Molecular and clinical correlates of HER3 expression highlights its potential role as a therapeutic target in melanoma.

Overexpression of the epidermal growth factor receptor family member HER3 (erbB3) has been implicated in several types of cancer and recently drugs targeting HER3 have shown promising clinical activity. In melanoma, HER3 overexpression has been linked to both metastasis formation and resistance to drug therapy in cell culture models. Here, we sought to characterise the expression of HER3 in 187 melanoma biopsies (149 cutaneous, 38 mucosal) using immunohistochemistry, as well as to analyse the association between HER3 expression and molecular, clinical and pathological variables. A subset of the cutaneous melanoma specimens was taken prior to treatment with immune checkpoint blockade therapy (pre-ICB) (n=79). HER3 expression (≥1+) was observed in 136 of 187 samples (∼73%). HER3 expression was found to be markedly lower in the mucosal melanomas, with 17 of the 38 tumours (∼45%) demonstrating no HER3 expression. In cutaneous melanomas, there was a negative association between HER3 expression and mutational load, a positive association with NRAS mutational status, and a trend of negative association with PD-L1 expression. In the pre-ICB cohort, an association was found between high HER3 expression (≥2+) and overall survival after anti-PD-1-based immunotherapy. Overall, our results indicate that HER3 is a promising therapeutic avenue in cutaneous melanoma worthy of further clinical evaluation.

InterMEL: An international biorepository and clinical database to uncover predictors of survival in early-stage melanoma.

INTRODUCTION: We are conducting a multicenter study to identify classifiers predictive of disease-specific survival in patients with primary melanomas. Here we delineate the unique aspects, challenges, and best practices for optimizing a study of generally small-sized pigmented tumor samples including primary melanomas of at least 1.05mm from AJTCC TNM stage IIA-IIID patients. We also evaluated tissue-derived predictors of extracted nucleic acids' quality and success in downstream testing. This ongoing study will target 1,000 melanomas within the international InterMEL consortium. METHODS: Following a pre-established protocol, participating centers ship formalin-fixed paraffin embedded (FFPE) tissue sections to Memorial Sloan Kettering Cancer Center for the centralized handling, dermatopathology review and histology-guided coextraction of RNA and DNA. Samples are distributed for evaluation of somatic mutations using next gen sequencing (NGS) with the MSK-IMPACTTM assay, methylation-profiling (Infinium MethylationEPIC arrays), and miRNA expression (Nanostring nCounter Human v3 miRNA Expression Assay). RESULTS: Sufficient material was obtained for screening of miRNA expression in 683/685 (99%) eligible melanomas, methylation in 467 (68%), and somatic mutations in 560 (82%). In 446/685 (65%) cases, aliquots of RNA/DNA were sufficient for testing with all three platforms. Among samples evaluated by the time of this analysis, the mean NGS coverage was 249x, 59 (18.6%) samples had coverage below 100x, and 41/414 (10%) failed methylation QC due to low intensity probes or insufficient Meta-Mixed Interquartile (BMIQ)- and single sample (ss)- Noob normalizations. Six of 683 RNAs (1%) failed Nanostring QC due to the low proportion of probes above the minimum threshold. Age of the FFPE tissue blocks (p<0.001) and time elapsed from sectioning to co-extraction (p = 0.002) were associated with methylation screening failures. Melanin reduced the ability to amplify fragments of 200bp or greater (absent/lightly pigmented vs heavily pigmented, p<0.003). Conversely, heavily pigmented tumors rendered greater amounts of RNA (p<0.001), and of RNA above 200 nucleotides (p<0.001). CONCLUSION: Our experience with many archival tissues demonstrates that with careful management of tissue processing and quality control it is possible to conduct multi-omic studies in a complex multi-institutional setting for investigations involving minute quantities of FFPE tumors, as in studies of early-stage melanoma. The study describes, for the first time, the optimal strategy for obtaining archival and limited tumor tissue, the characteristics of the nucleic acids co-extracted from a unique cell lysate, and success rate in downstream applications. In addition, our findings provide an estimate of the anticipated attrition that will guide other large multicenter research and consortia.

Cross-platform comparison of immune signatures in immunotherapy-treated patients with advanced melanoma using a rank-based scoring approach.

BACKGROUND: Gene expression profiling is increasingly being utilised as a diagnostic, prognostic and predictive tool for managing cancer patients. Single-sample scoring approach has been developed to alleviate instability of signature scores due to variations from sample composition. However, it is a challenge to achieve comparable signature scores across different expressional platforms. METHODS: The pre-treatment biopsies from a total of 158 patients, who have received single-agent anti-PD-1 (n = 84) or anti-PD-1 + anti-CTLA-4 therapy (n = 74), were performed using NanoString PanCancer IO360 Panel. Multiple immune-related signature scores were measured from a single-sample rank-based scoring approach, singscore. We assessed the reproducibility and the performance in reporting immune profile of singscore based on NanoString assay in advance melanoma. To conduct cross-platform analyses, singscores between the immune profiles of NanoString assay and the previous orthogonal whole transcriptome sequencing (WTS) data were compared through linear regression and cross-platform prediction. RESULTS: singscore-derived signature scores reported significantly high scores in responders in multiple PD-1, MHC-1-, CD8 T-cell-, antigen presentation-, cytokine- and chemokine-related signatures. We found that singscore provided stable and reproducible signature scores among the repeats in different batches and cross-sample normalisations. The cross-platform comparisons confirmed that singscores derived via NanoString and WTS were comparable. When singscore of WTS generated by the overlapping genes to the NanoString gene set, the signatures generated highly correlated cross-platform scores (Spearman correlation interquartile range (IQR) [0.88, 0.92] and r2 IQR [0.77, 0.81]) and better prediction on cross-platform response (AUC = 86.3%). The model suggested that Tumour Inflammation Signature (TIS) and Personalised Immunotherapy Platform (PIP) PD-1 are informative signatures for predicting immunotherapy-response outcomes in advanced melanoma patients treated with anti-PD-1-based therapies. CONCLUSIONS: Overall, the outcome of this study confirms that singscore based on NanoString data is a feasible approach to produce reliable signature scores for determining patients' immune profiles and the potential clinical utility in biomarker implementation, as well as to conduct cross-platform comparisons, such as WTS.

Spatial mapping reveals granuloma diversity and histopathological superstructure in human tuberculosis.

The hallmark of tuberculosis (TB) is the formation of immune cell-enriched aggregates called granulomas. While granulomas are pathologically diverse, their tissue-wide heterogeneity has not been spatially resolved at the single-cell level in human tissues. By spatially mapping individual immune cells in every lesion across entire tissue sections, we report that in addition to necrotizing granulomas, the human TB lung contains abundant non-necrotizing leukocyte aggregates surrounding areas of necrotizing tissue. These cellular lesions were more diverse in composition than necrotizing lesions and could be stratified into four general classes based on cellular composition and spatial distribution of B cells and macrophages. The cellular composition of non-necrotizing structures also correlates with their proximity to necrotizing lesions, indicating these are foci of distinct immune reactions adjacent to necrotizing granulomas. Together, we show that during TB, diseased lung tissue develops a histopathological superstructure comprising at least four different types of non-necrotizing cellular aggregates organized as satellites of necrotizing granulomas.

Genomic Profiling of Metastatic Basal cell Carcinoma Reveals Candidate Drivers of Disease and Therapeutic Targets.

Basal cell carcinomas (BCCs) are human beings' most common malignant tumors. Most are easily managed by surgery or topical therapies, and metastasis is rare. Although BCCs can become locally advanced, metastatic BCCs are very uncommon and may be biologically distinct. We assessed the clinicopathologic characteristics of 17 patients with metastatic BCC and pursued whole-exome sequencing of tumor and germline DNA from 8 patients. Genomic profiling revealed aberrant activation of Hedgehog signaling and alterations in GLI transcriptional regulators and Notch and Hippo signaling. Matched local recurrences of primary BCCs and metastases from 3 patients provided evidence of a clonal origin in all cases. Mutations associated with YAP inhibition were found exclusively in 2 hematogenously-spread lung metastases, and metastatic BCCs were enriched for mutations in the YAP/TAZ-binding domain of TEAD genes. Accordingly, YAP/TAZ nuclear localization was associated with metastatic types and Hippo mutations, suggesting an enhanced oncogenic role in hematogenously-spread metastases. Mutations in RET, HGF, and phosphatidylinositol 3­kinase (PI3K)/protein kinase B (AKT) signaling were enriched compared with a cohort of low clinical-risk BCCs. Our results implicate Hippo and PI3K/AKT dysregulation in metastatic progression of BCCs, making these potential therapeutic targets in metastatic disease. The common clonal origin of matched recurrent and metastatic BCCs suggests that molecular profiling can assist in determining the nature/origin of poorly differentiated metastatic tumors of uncertain type. Genes and pathways enriched for mutations in this cohort are candidate drivers of metastasis and can be used to identify patients at high risk of metastasis who may benefit from aggressive local treatment and careful clinical follow-up.

Intratumoral CD16+ Macrophages Are Associated with Clinical Outcomes of Patients with Metastatic Melanoma Treated with Combination Anti-PD-1 and Anti-CTLA-4 Therapy.

PURPOSE: This study characterizes intratumoral macrophage populations within baseline melanoma biopsies from patients with advanced melanoma who received either anti-PD-1 monotherapy or a combination with anti-CTLA-4. Particularly, FcγRIIIa (CD16)-expressing macrophage densities were investigated for associations with response and progression-free survival. EXPERIMENTAL DESIGN: Patients with advanced melanoma who received either anti-PD-1 monotherapy or combination anti-PD-1 and anti-CTLA-4 were retrospectively identified. Macrophage populations were analyzed within baseline melanoma biopsies via multiplex IHC in relation to treatment outcomes. RESULTS: Patients who responded to combination immune checkpoint inhibitor contained higher CD16+ macrophage densities than those who did not respond (196 vs. 7 cells/mm2; P = 0.0041). There was no diffidence in CD16+ macrophage densities in the PD-1 monotherapy-treated patients based on response (118 vs. 89 cells/mm2; P = 0.29). A significantly longer 3-year progression-free survival was observed in combination-treated patients with high intratumoral densities of CD16+ macrophages compared with those with low densities (87% vs. 42%, P = 0.0056, n = 40). No association was observed in anti-PD-1 monotherapy-treated patients (50% vs. 47%, P = 0.4636, n = 50). Melanoma biopsies with high densities of CD16+ macrophages contained upregulated gene expression of critical T-cell recruiting chemokines (CXCL9, CXCL10, and CXCL11). CONCLUSIONS: Our data demonstrate that tumor microenvironments enriched with CD16+ macrophages are favorable for response to combination anti-PD-1 and anti-CTLA-4 therapy but not anti-PD-1 monotherapy. These data provides a potential biomarker of response for combination immunotherapies in patients with metastatic melanoma. See related commentary by Smithy and Luke, p. 2345.

Melanomas in children and adolescents: Clinicopathologic features and survival outcomes.

BACKGROUND: Melanomas in the first 2 decades of life are uncommon and poorly understood. OBJECTIVE: To assess clinicopathologic features and survival of children (≤11 years) and adolescents (12-19 years) diagnosed with melanoma. METHODS: A pooled cohort of 514 patients was analyzed (397 Dutch, 117 Australian; 62 children, 452 adolescents). Pathology reports were reevaluated to determine melanoma subtypes. Multivariable Cox models were generated for recurrence-free survival (RFS) and overall survival (OS). RESULTS: Melanoma subtypes were conventional melanoma (superficial spreading, nodular, desmoplastic, and acral lentiginous), spitzoid melanoma, and melanoma associated with a congenital nevus in 428, 78, and 8 patients, respectively. Ten-year RFS was 91.5% (95% confidence interval [CI], 82.4%-100%) in children and 86.4% (95% CI, 82.7%-90.3%) in adolescents (P = .32). Ten-year OS was 100% in children and 92.7% (95% CI, 89.8%-95.8%) in adolescents (P = .09). On multivariable analysis possible only for the adolescent cohort due to the small number of children, ulceration status, and anatomic site were associated with RFS and OS, whereas age, sex, mitotic index, sentinel node status and melanoma subtype were not. Breslow thickness >4 mm was associated with worse RFS. LIMITATIONS: Retrospective study. CONCLUSIONS: Survival rates for children and adolescents with melanomas were high. Ulceration, head or neck location and Breslow thickness >4 mm predicted worse survival in adolescents.

Clinical Features Associated with Outcomes and Biomarker Analysis of Dabrafenib plus Trametinib Treatment in Patients with BRAF-Mutant Melanoma Brain Metastases.

PURPOSE: This study aimed to identify baseline clinical features associated with the outcomes of patients enrolled in the COMBI-MB phase II study of dabrafenib and trametinib treatment in patients with V600 BRAF-mutant metastatic melanoma with melanoma brain metastases (MBM). Exploratory biomarker analysis was also conducted as part of the synergistic COMBI-BRV trial (BRV116521), to identify molecular and immunologic changes associated with dabrafenib in MBMs and extracranial metastases (ECM). PATIENTS AND METHODS: Post hoc analysis was performed for baseline features of patients (n = 125) enrolled in COMBI-MB. Analyses were performed to identify baseline clinical features associated with intracranial response rate (ICRR), progression-free survival (PFS), and overall survival (OS).Exploratory biomarker analysis was performed on biospecimen collected in the COMBI-BRV trial in which patients with BRAF-mutant, resectable MBM were treated with dabrafenib for 10 to 14 days prior to craniotomy. Accessible ECM were resected or biopsied at the time of craniotomy. Biospecimens underwent molecular and immunologic profiling for comparative analyses. RESULTS: In COMBI-MB baseline treatment with corticosteroids was independently associated with lower ICRR [39% vs. 63%; OR, 0.323; 95 % confidence interval (CI), 0.105-0.996; P = 0.049] and shorter PFS (HR, 1.93; 95% CI, 1.06-3.51; P = 0.031). Additional significant associations identified in the multivariate analysis were improved PFS in patients with a BRAFV600E genotype (HR, 0.565; 95% CI, 0.321-0.996; P = 0.048) and improved OS in patients with Eastern Cooperative Oncology Group 0 (HR, 0.44; 95% CI, 0.25-0.78; P = 0.005). CONCLUSIONS: Corticosteroid treatment was associated with reduced ICRR and PFS in COMBI-MB, similar to results with immunotherapy for MBMs. Baseline corticosteroid treatment is a key factor to consider in MBM patient management and clinical trial design/interpretation.

Hypoxia Controls the Glycome Signature and Galectin-8-Ligand Axis to Promote Protumorigenic Properties of Metastatic Melanoma.

The prognosis for patients with metastatic melanoma (MM) involving distant organs is grim, and treatment resistance is potentiated by tumor-initiating cells (TICs) that thrive under hypoxia. MM cells, including TICs, express a unique glycome featuring i-linear poly-N-acetyllactosamines through the loss of I-branching enzyme, ß1,6 N-acetylglucosaminyltransferase 2. Whether hypoxia instructs MM TIC development by modulating the glycome signature remains unknown. In this study, we explored hypoxia-dependent alterations in MM glycome‒associated genes and found that ß1,6 N-acetylglucosaminyltransferase 2 was downregulated and a galectin (Gal)-8-ligand axis, involving both extracellular and cell-intrinsic Gal-8, was induced. Low ß1,6 N-acetylglucosaminyltransferase 2 levels correlated with poor patient outcomes, and patient serum samples were elevated for Gal-8. Depressed ß1,6 N-acetylglucosaminyltransferase 2 in MM cells upregulated TIC marker, NGFR/CD271, whereas loss of MM cell‒intrinsic Gal-8 markedly lowered NGFR and reduced TIC activity in vivo. Extracellular Gal-8 bound preferentially to i-linear poly-N-acetyllactosamines on N-glycans of the TIC marker and prometastatic molecule CD44, among other receptors, and activated prosurvival factor protein kinase B. This study reveals the importance of hypoxia governing the MM glycome by enforcing i-linear poly-N-acetyllactosamine and Gal-8 expression. This mechanistic investigation also uncovers glycome-dependent regulation of pro-MM factor, NGFR, implicating i-linear poly-N-acetyllactosamine and Gal-8 as biomarkers and therapeutic targets of MM.

Obesity Is Associated with Altered Tumor Metabolism in Metastatic Melanoma.

PURPOSE: Overweight/obese (OW/OB) patients with metastatic melanoma unexpectedly have improved outcomes with immune checkpoint inhibitors (ICI) and BRAF-targeted therapies. The mechanism(s) underlying this association remain unclear, thus we assessed the integrated molecular, metabolic, and immune profile of tumors, as well as gut microbiome features, for associations with patient body mass index (BMI). EXPERIMENTAL DESIGN: Associations between BMI [normal (NL < 25) or OW/OB (BMI ≥ 25)] and tumor or microbiome characteristics were examined in specimens from 782 patients with metastatic melanoma across 7 cohorts. DNA associations were evaluated in The Cancer Genome Atlas cohort. RNA sequencing from 4 cohorts (n = 357) was batch corrected and gene set enrichment analysis (GSEA) by BMI category was performed. Metabolic profiling was conducted in a subset of patients (x = 36) by LC/MS, and in flow-sorted melanoma tumor cells (x = 37) and patient-derived melanoma cell lines (x = 17) using the Seahorse XF assay. Gut microbiome features were examined in an independent cohort (n = 371). RESULTS: DNA mutations and copy number variations were not associated with BMI. GSEA demonstrated that tumors from OW/OB patients were metabolically quiescent, with downregulation of oxidative phosphorylation and multiple other metabolic pathways. Direct metabolite analysis and functional metabolic profiling confirmed decreased central carbon metabolism in OW/OB metastatic melanoma tumors and patient-derived cell lines. The overall structure, diversity, and taxonomy of the fecal microbiome did not differ by BMI. CONCLUSIONS: These findings suggest that the host metabolic phenotype influences melanoma metabolism and provide insight into the improved outcomes observed in OW/OB patients with metastatic melanoma treated with ICIs and targeted therapies. See related commentary by Smalley, p. 5.

The effect of organ-specific tumor microenvironments on response patterns to immunotherapy.

Immunotherapy, particularly immune checkpoint inhibitors, have become widely used in various settings across many different cancer types in recent years. Whilst patients are often treated on the basis of the primary cancer type and clinical stage, recent studies have highlighted disparity in response to immune checkpoint inhibitors at different sites of metastasis, and their impact on overall response and survival. Studies exploring the tumor immune microenvironment at different organ sites have provided insights into the immune-related mechanisms behind organ-specific patterns of response to immunotherapy. In this review, we aimed to highlight the key learnings from clinical studies across various cancers including melanoma, lung cancer, renal cell carcinoma, colorectal cancer, breast cancer and others, assessing the association of site of metastasis and response to immune checkpoint inhibitors. We also summarize the key clinical and pre-clinical findings from studies exploring the immune microenvironment of specific sites of metastasis. Ultimately, further characterization of the tumor immune microenvironment at different metastatic sites, and understanding the biological drivers of these differences, may identify organ-specific mechanisms of resistance, which will lead to more personalized treatment approaches for patients with innate or acquired resistance to immunotherapy.

Causes, consequences and clinical significance of aneuploidy across melanoma subtypes.

Aneuploidy, the state of the cell in which the number of whole chromosomes or chromosome arms becomes imbalanced, has been recognized as playing a pivotal role in tumor evolution for over 100 years. In melanoma, the extent of aneuploidy, as well as the chromosomal regions that are affected differ across subtypes, indicative of distinct drivers of disease. Multiple studies have suggested a role for aneuploidy in diagnosis and prognosis of melanomas, as well as in the context of immunotherapy response. A number of key constituents of the cell cycle have been implicated in aneuploidy acquisition in melanoma, including several driver mutations. Here, we review the state of the art on aneuploidy in different melanoma subtypes, discuss the potential drivers, mechanisms underlying aneuploidy acquisition as well as its value in patient diagnosis, prognosis and response to immunotherapy treatment.

Unveiling the tumor immune microenvironment of organ-specific melanoma metastatic sites.

BACKGROUND: The liver is a known site of resistance to immunotherapy and the presence of liver metastases is associated with shorter progression-free and overall survival (OS) in melanoma, while lung metastases have been associated with a more favorable outcome. There are limited data available regarding the immune microenvironment at different anatomical sites of melanoma metastases. This study sought to characterize and compare the tumor immune microenvironment of liver, brain, lung, subcutaneous (subcut) as well as lymph node (LN) melanoma metastases. METHODS: We analyzed OS in 1924 systemic treatment-naïve patients with AJCC (American Joint Committee on Cancer) stage IV melanoma with a solitary site of organ metastasis. In an independent cohort we analyzed and compared immune cell densities, subpopulations and spatial distribution in tissue from liver, lung, brain, LN or subcut sites from 130 patients with stage IV melanoma. RESULTS: Patients with only liver, brain or bone metastases had shorter OS compared to those with lung, LN or subcutaneous and soft tissue metastases. Liver and brain metastases had significantly lower T-cell infiltration than lung (p=0.0116 and p=0.0252, respectively) and LN metastases (p=0.0116 and p=0.0252, respectively). T cells were further away from melanoma cells in liver than lung metastases (p=0.0335). Liver metastases displayed unique T-cell profiles, with a significantly lower proportion of programmed cell death protein-1+ T cells compared to all other anatomical sites (p<0.05), and a higher proportion of TIM-3+ T cells compared to LN (p=0.0004), subcut (p=0.0082) and brain (p=0.0128) metastases. Brain metastases had a lower macrophage density than subcut (p=0.0105), liver (p=0.0095) and lung (p<0.0001) metastases. Lung metastases had the highest proportion of programmed death ligand-1+ macrophages of the total macrophage population, significantly higher than brain (p<0.0001) and liver metastases (p=0.0392). CONCLUSIONS: Liver and brain melanoma metastases have a significantly reduced immune infiltrate than lung, subcut and LN metastases, which may account for poorer prognosis and reduced immunotherapy response rates in patients with liver or brain metastases. Increased TIM-3 expression in liver metastases suggests TIM-3 inhibitor therapy as a potential therapeutic opportunity to improve patient outcomes.

Distinct pretreatment innate immune landscape and posttreatment T cell responses underlie immunotherapy-induced colitis.

Immune-related adverse events represent a major hurdle to the success of immunotherapy. The immunological mechanisms underlying their development and relation to antitumor responses are poorly understood. By examining both systemic and tissue-specific immune changes induced by combination anti-CTLA-4 and anti-PD-1 immunotherapy, we found distinct repertoire changes in patients who developed moderate-severe colitis, irrespective of their antitumor response to therapy. The proportion of circulating monocytes were significantly increased at baseline in patients who subsequently developed colitis compared with patients who did not develop colitis, and biopsies from patients with colitis showed monocytic infiltration of both endoscopically and histopathologically normal and inflamed regions of colon. The magnitude of systemic expansion of T cells following commencement of immunotherapy was also greater in patients who developed colitis. Importantly, we show expansion of specific T cell subsets within inflamed regions of the colon, including tissue-resident memory CD8+ T cells and Th1 CD4+ T cells in patients who developed colitis. Our data also suggest that CD8+ T cell expansion was locally induced, while Th1 cell expansion was systemic. Together, our data show that exaggerated innate and T cell responses to combination immunotherapy synergize to propel colitis in susceptible patients.