Nicolas Jacquelot, María Paula Roberti, David P

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Presentation transcript:

Immunophenotyping of Stage III Melanoma Reveals Parameters Associated with Patient Prognosis Nicolas Jacquelot, María Paula Roberti, David P. Enot, Sylvie Rusakiewicz, Michaela Semeraro, Sarah Jégou, Camila Flores, Lieping Chen, Byoung S. Kwon, Christophe Borg, Benjamin Weide, François Aubin, Stéphane Dalle, Holbrook Kohrt, Maha Ayyoub, Guido Kroemer, Aurélien Marabelle, Andréa Cavalcanti, Alexander Eggermont, Laurence Zitvogel Journal of Investigative Dermatology Volume 136, Issue 5, Pages 994-1001 (May 2016) DOI: 10.1016/j.jid.2015.12.042 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Immunotypes associated with the BRAFV600 mutation and the severity of lymph node invasion. Drop in (a) circulating CD4+ and (b) intratumoral CD8+PD-1+ T-cell proportions according to metastatic lymph node invasion. (c) Distribution in blood of CD137L+CD3–CD56– cells according to the number of invaded lymph nodes. Decreased CD137L expression in distinct circulating leukocyte subset in patients harboring BRAFV600 mutated tumors. (d, e) Flow cytometric analyses of blood leukocytes assessing CD137L expression on (d) CD8+ or (e) NK cells. (f) Increased percentages of CD45RA+CD4+ T cells in tumor-infiltrated lymphocytes from mutated tumors as assessed by flow cytometry. Each dot represents one patient. Wilcoxon Rank-Sum test: all P-values are indicated. Journal of Investigative Dermatology 2016 136, 994-1001DOI: (10.1016/j.jid.2015.12.042) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Naïve CD45RA+CD4+ T cells inversely correlated with intratumoral cytotoxic T lymphocytes and were associated with shorter progression-free survival (PFS). (a) Flow cytometric analyses of blood leukocytes and tumor-infiltrated lymphocytes (TILs) assessing the lineage-related marker CD45RA in CD4+ T cells in a paired manner. (b, c) Spearman correlations between the percentages of CD4+CD45RA+ TILs and CD8+ cytotoxic T lymphocytes or PD1+CD8+ TILs. Each dot represents one patient. Wilcoxon paired signed-rank and Spearman correlation tests: P-values are indicated. (d) Kaplan-Meier PFS curves segregating the cohort of metastatic melanoma patients according to the median value of CD4+CD45RA+ TILs retained in the Cox multivariate model in tumor analyses. Likelihood ratio tests from Cox regression modeling are used to assess the prognostic value for the marker with and without accounting for metastatic lymph node (mLN) invasion status (0–3 vs >3). Journal of Investigative Dermatology 2016 136, 994-1001DOI: (10.1016/j.jid.2015.12.042) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Regulatory T-cell (Treg) tumor-infiltrating lymphocytes (TILs) negatively impact on overall survival (OS) and overrule BRAF mutational status. (a) Flow cytometric analyses of blood leukocytes and tumor infiltrating cells assessing the CD127–CD25high cells in CD4+ TILs (Treg) in a paired manner. (b, c) Spearman correlations between the percentages of Treg and (b) CD4+CD69+ TILs and (c) CD8+PD1+TIM3+ TILs. Each dot represents one patient. Wilcoxon paired signed-rank and Spearman correlation tests: P-values are indicated. (d) Kaplan-Meier OS curves segregating the cohort of metastatic melanoma patients according to the median value of Treg TILs retained in the Cox multivariate model in tumor analyses. Likelihood ratio tests from Cox regression modeling are used to assess the prognostic value for the marker with and without accounting for BRAF mutational status (mutated [M] vs wild type [WT]). Journal of Investigative Dermatology 2016 136, 994-1001DOI: (10.1016/j.jid.2015.12.042) Copyright © 2016 The Authors Terms and Conditions

Figure 4 NKG2D and PDL-1 expression on CD8+ tumor-infiltrated lymphocytes and blood cells influenced the prognosis of stage III metastatic melanoma. (a). Flow cytometric analyses of CD8+ T cells for NKG2D expression and (b) prognosis value shown by Kaplan-Meier overall survival (OS) curves segregating the cohort of metastatic melanoma patients according to the median value of CD8+NKG2D+ tumor-infiltrated lymphocytes and BRAF mutational status (mutated [M] vs wild type [WT]) both retained in the Cox multivariate model. (c) Mean fluorescence intensity (MFI) of membrane expression of PD-L1 on CD8+ blood T cells compared with the isotype control antibody MFI (ratio of MFI, upper panel) and its Spearman correlation with the percentages of circulating PD-L1+CD8+ T cells (lower panel). Wilcoxon paired signed-rank and Spearman correlation tests: P-values are indicated. (d) Same as b but using the MFI ratio of PD-L1 positivity on CD8+ cytotoxic T lymphocytes for the Kaplan-Meier curve of OS. Journal of Investigative Dermatology 2016 136, 994-1001DOI: (10.1016/j.jid.2015.12.042) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Tumor CD3–CD56– cells inversely correlated with intratumoral cytotoxic T lymphocytes (CTLs) and negatively predicted progression-free survival (PFS). (a) Flow cytometric analyses of blood and tumor CD3–CD56– cells performed in a paired manner. (b, c) Spearman correlations between CD3–CD56– cells and (b) CD8+ CTL tumor-infiltrated lymphocytes and (c) CD4+CD45RA+ tumor-infiltrated lymphocytes. Each dot represents one patient. Wilcoxon paired signed-rank and Spearman correlation tests: P-values are indicated. (d) Kaplan-Meier PFS curves segregating the cohort of metastatic melanoma patients according to the median value of tumor CD3–CD56– cells retained in the Cox multivariate model in tumor analyses. Likelihood ratio tests from Cox regression modeling are used to assess the prognostic value for the marker with and without accounting for metastatic lymph node (mLN) invasion status (0–3 vs >3). Journal of Investigative Dermatology 2016 136, 994-1001DOI: (10.1016/j.jid.2015.12.042) Copyright © 2016 The Authors Terms and Conditions