Detailed spatial immunophenotyping of primary melanomas reveals immune cell subpopulations associated with patient outcome.

While the tumor immune microenvironment (TIME) of metastatic melanoma has been well characterized, the primary melanoma TIME is comparatively poorly understood. Additionally, although the association of tumor-infiltrating lymphocytes with primary melanoma patient outcome has been known for decades, it is not considered in the current AJCC melanoma staging system. Detailed immune phenotyping of advanced melanoma has revealed multiple immune biomarkers, including the presence of CD8+ T-cells, for predicting response to immunotherapies. However, in primary melanomas, immune biomarkers are lacking and CD8+ T-cells have yet to be extensively characterized. As recent studies combining immune features and clinicopathologic characteristics have created more accurate predictive models, this study sought to characterize the TIME of primary melanomas and identify predictors of patient outcome. We first phenotyped CD8+ T cells in fresh stage II primary melanomas using flow cytometry (n = 6), identifying a CD39+ tumor-resident CD8+ T-cell subset enriched for PD-1 expression. We then performed Opal multiplex immunohistochemistry and quantitative pathology-based immune profiling of CD8+ T-cell subsets, along with B cells, NK cells, Langerhans cells and Class I MHC expression in stage II primary melanoma specimens from patients with long-term follow-up (n = 66), comparing patients based on their recurrence status at 5 years after primary diagnosis. A CD39+CD103+PD-1- CD8+ T-cell population (P2) comprised a significantly higher proportion of intratumoral and stromal CD8+ T-cells in patients with recurrence-free survival (RFS) ≥5 years vs those with RFS <5 years (p = 0.013). Similarly, intratumoral B cells (p = 0.044) and a significantly higher B cell density at the tumor/stromal interface were associated with RFS. Both P2 and B cells localized in significantly closer proximity to melanoma cells in patients who remained recurrence-free (P2 p = 0.0139, B cell p = 0.0049). Our results highlight how characterizing the TIME in primary melanomas may provide new insights into how the complex interplay of the immune system and tumor can modify the disease outcomes. Furthermore, in the context of current clinical trials of adjuvant anti-PD-1 therapies in high-risk stage II primary melanoma, assessment of B cells and P2 could identify patients at risk of recurrence and aid in long-term treatment decisions at the point of primary melanoma diagnosis.

Prevalence and Cellular Distribution of Novel Immune Checkpoint Targets Across Longitudinal Specimens in Treatment-naïve Melanoma Patients: Implications for Clinical Trials.

PURPOSE: Immunotherapies targeting costimulating and coinhibitory checkpoint receptors beyond PD-1 and CTLA-4 have entered clinical trials. Little is known about the relative abundance, coexpression, and immune cells enriched for each specific drug target, limiting understanding of the biological basis of potential treatment outcomes and development of predictive biomarkers for personalized immunotherapy. We sought to assess the abundance of checkpoint receptors during melanoma disease progression and identify immune cells enriched for them.Experimental Design: Multiplex immunofluorescence staining for immune checkpoint receptors (ICOS, GITR, OX40, PD-1, TIM-3, VISTA) was performed on 96 melanoma biopsies from 41 treatment-naïve patients, including patient-matched primary tumors, nodal metastases, and distant metastases. Mass cytometry was conducted on tumor dissociates from 18 treatment-naïve melanoma metastases to explore immune subsets enriched for checkpoint receptors. RESULTS: A small subset of tumor-infiltrating leukocytes expressed checkpoint receptors at any stage of melanoma disease. GITR and OX40 were the least abundant checkpoint receptors, with <1% of intratumoral T cells expressing either marker. ICOS, PD-1, TIM-3, and VISTA were most abundant, with TIM-3 and VISTA mostly expressed on non-T cells, and TIM-3 enriched on dendritic cells. Tumor-resident T cells (CD69+/CD103+/CD8+) were enriched for TIGIT (>70%) and other coinhibitory but not costimulatory receptors. The proportion of GITR+ T cells decreased from primary melanoma (>5%) to lymph node (<1%, P = 0.04) and distant metastases (<1%, P = 0.0005). CONCLUSIONS: This study provides the first comprehensive assessment of immune checkpoint receptor expression in any cancer and provides important data for rational selection of targets for trials and predictive biomarker development.

Integrated molecular and immunophenotypic analysis of NK cells in anti-PD-1 treated metastatic melanoma patients.

Purpose: Anti-PD-1 therapy has revolutionized the treatment and improved the survival of stage IV melanoma patients. However, almost half of the patients fail to respond due to immune evasive mechanism. A known mechanism is the downregulation of major histocompatibility complex (MHC) class I expression, which prevents T cell recognition of the tumor. This study determined the relationship between natural killer (NK) cell numbers and clinical response to anti-PD-1 therapy in metastatic melanoma. Experimental Design: Twenty-five anti-PD-1 treated metastatic melanoma patients were categorized into responders (complete response (CR)/partial response (PR)/stable disease (SD) ≥ 6 mo, n = 13) and non-responders (SD < 6 days/progressive disease (PD), n = 12) based on RECIST response. Whole transcriptome sequencing and multiplex immunofluorescent staining were performed on pre-treatment and on a subset of early during treatment tumor samples. Spatial distribution analysis was performed on multiplex immunofluorescent images to determine the proximity of NK cells to tumor cells. Flow cytometry was used to confirm NK phenotypes in lymph node metastases of treatment naïve melanoma patients (n = 5). Cytotoxic assay was performed using NK cells treated with anti-PD-1 or with isotype control and co-cultured with 3 different melanoma cell lines and with K562 cells (leukemia cell line). Results: Differential expression analysis identified nine upregulated NK cell specific genes (adjusted p < 0.05) in responding (n = 11) versus non-responding patients (n = 10). Immunofluorescent staining of biopsies confirmed a significantly higher density of intra- and peri-tumoral CD16+ and granzyme B + NK cells in responding patients (p < 0.05). Interestingly, NK cells were in closer proximity to tumor cells in responding PD-1 treated patients compared to non-responding patients. Patients who responded to anti-PD-1 therapy, despite MHC class I loss had higher NK cell densities than patients with low MHC class I expression. Lastly, functional assays demonstrated PD-1 blockade induces an increase in NK cells' cytotoxicity. Conclusions: A higher density of tumoral NK cells is associated with response to anti-PD-1 therapy. NK cells may play an important role in mediating response to anti-PD-1 therapy, including in a subset of tumors downregulating MHC class I expression.

Distinct Immune Cell Populations Define Response to Anti-PD-1 Monotherapy and Anti-PD-1/Anti-CTLA-4 Combined Therapy.

Cancer immunotherapies provide survival benefits in responding patients, but many patients fail to respond. Identifying the biology of treatment response and resistance are a priority to optimize drug selection and improve patient outcomes. We performed transcriptomic and immune profiling on 158 tumor biopsies from melanoma patients treated with anti-PD-1 monotherapy (n = 63) or combined anti-PD-1 and anti-CTLA-4 (n = 57). These data identified activated T cell signatures and T cell populations in responders to both treatments. Further mass cytometry analysis identified an EOMES+CD69+CD45RO+ effector memory T cell phenotype that was significantly more abundant in responders to combined immunotherapy compared with non-responders (n = 18). The gene expression profile of this population was associated with longer progression-free survival in patients treated with single agent and greater tumor shrinkage in both treatments.

CD103+ Tumor-Resident CD8+ T Cells Are Associated with Improved Survival in Immunotherapy-Naïve Melanoma Patients and Expand Significantly During Anti-PD-1 Treatment.

Purpose: Therapeutic blockade of immune checkpoints has revolutionized cancer treatment. Durable responses, however, occur in less than half of those treated, and efforts to improve treatment efficacy are confounded by a lack of understanding of the characteristics of the cells that initiate antitumor immune response.Patients and Methods: We performed multiparameter flow cytometry and quantitative multiplex immunofluorescence staining on tumor specimens from immunotherapy-naïve melanoma patients and longitudinal biopsy specimen obtained from patients undergoing anti-PD-1 therapy.Results: Increased numbers of CD69+CD103+ tumor-resident CD8+ T cells were associated with improved melanoma-specific survival in immunotherapy-naïve melanoma patients. Local IL15 expression levels strongly correlated with these tumor-resident T-cell numbers. The expression of several immune checkpoints including PD-1 and LAG3 was highly enriched in this subset, and these cells significantly expanded early during anti-PD-1 immunotherapy.Conclusions: Tumor-resident CD8+ T-cell numbers are more prognostic than total CD8+ T cells in metastatic melanoma. In addition, they are likely to initiate response to anti-PD-1 and anti-LAG-3 treatments. We propose that the immune profile of these cells prior to treatment could inform strategies for immune checkpoint blockade. Clin Cancer Res; 24(13); 3036-45. ©2018 AACR.