Abstract A130: Metastatic melanoma patients responding to PD1 therapy have higher proportion of peripheral blood NKT-cells
Authors: Hakanen Henna H.E. , Hernberg Micaela , Mäkelä Siru , Yadav Bhagwan , Brück Oscar , Juteau Susanna , Kohtamäki Laura , Ilander Mette , Mustjoki Satu , Anna Kreutzman 
Published: February 2019

Abstract
Anti-PD1 therapy has proven to be effective in various cancer types, but not all patients benefit from the therapy. Further, no comprehensive immunologic monitoring during anti-PD1 treatment has yet been published. In this study, we aimed to discover the effects of anti-PD1 therapy on the immune system, especially on NK and NKT-cells, which are less studied, but known to be involved in antitumor immune events. Peripheral blood samples from immuno-oncology (IO) naive metastatic melanoma patients (n=20) were obtained before the first infusion of pembrolizumab or nivolumab (D0), then 1 and 3 months after the initiation of treatment. From each time-point, complete blood counts (CBC) were obtained, and comprehensive immunophenotyping of NK, NKT, and T-cells was performed with multicolor flow cytometry. Moreover, 92 different serum cytokines were measured using the Olink inflammation panel. The protein levels are presented as arbitrary units of normalized protein expression, NPX, on Log2 scale. The CBC revealed that the proportion of lymphocytes (mean D0 30.6% vs. 3mo 24.9%, p=0.02), decreased during the treatment but no changes were observed in absolute numbers or in other leukocytes. Immunophenotyping of lymphocyte subpopulations revealed that the frequency of NKT brighT-cells was increased (D0 1.8% vs. 1mo 2.3%, p=0.01) and that cytotoxic NK CD56dim cells expressed more CD25 (D0 19.5% vs. 1mo 23.7%, p=0.02) and CD45RO (D0 14.7% vs. 1mo 21.1%, p=0.03) surface markers after 1 month of therapy. The cytokine assay indicated that during anti-PD1 treatment the levels of CXC family cytokines were increased in the serum; CXCL9 (D0 470 vs. 1mo 1070, p=0.0003, D0 470 vs. 3mo 1227, p=0.0007), CXCL11 (D0 49.8 vs. 1mo 81.8, p=0.005), CXCL10 (D0 1000 vs. 1mo 2078, p=0.0003). Also, an increase in IL-12B (D0 31.6 vs. 1mo 41.2, p=0.003, D0 31.6 vs. 3mo 32.4, p=0.04) and TNFRSF9 (D0 126.4 vs. 1mo 181.0, p=0.01, D0 126.4 vs. 3mo 149.0, p=0.04) levels was observed.To further examine these results, patients were categorized into two cohorts: responders (R, n=6, PFS=17.0 months) and patients with progressive disease (PD, n=9, PFS=5.0 months) in terms of the duration of progression-free survival (PFS) and decrease in tumor burden. 5 patients were excluded due to challenging clinical evaluation of response. When examining the differences between these cohorts, CBC indicated a significant decrease in the mean frequency of lymphocytes in PD (D0 26.9% vs. 3mo 19.2%, p=0.04), but not in the R cohort. The responders had also higher frequency of lymphocytes at 1- and 3-month time-points (R 34.5% vs. PD 25.4%, p=0.02, R 32.4% vs. PD 19.2%, p=0.01, respectively) and lower frequency of neutrophils before initiation and after 1 and 3 months of treatment (R 50.8% vs. PD 58.6%, p=0.04, R 45.3% vs. PD 59.4%, p=0.01, R 49.8% vs. PD 64.5%, p=0.04, respectively). The CBC absolute counts revealed that the responders had less neutrophils (R 2.8 109/L vs. PD 4.9 109/L, p=0.04) and monocytes (R 0.4 109/L vs. PD 0.7 109/L, p=0.04) after 3 months of treatment when compared to PD. The immunophenotyping showed that responders had more NKT dim cells before (R 10.1% vs. PD 3.5%, p=0.03) and after 3 months of therapy (R 15.7% vs. PD 3.7%, p=0.03) and the increase in NKT bright frequency was observed only in R (D0 2.5% vs. 1mo 3.4%, p=0.04) and not in PD cohort. The cytokine assay indicated that the CXCL9 was increased in R cohort (D0 476.8 vs. 1mo 1480.2, p=0.01), but not in PD, making the cytokine levels greater in R (R 1480.2 vs. PD 639.3, p=0.01) after 1 month of therapy. Based on our preliminary results, we believe that high frequency of NKT-cells in blood is related to positive treatment response, and in addition to T-cells, also NK and NKT-cells may play a key role in the antitumor response induced by PD-1 inhibition. Hence, further research on the effect of anti-PD1 therapy on NK and NKT-cells is needed to better understand their role in positive therapy response.
Citation Format: Henna H.E. Hakanen, Micaela Hernberg, Siru Mäkelä, Bhagwan Yadav, Oscar Brück, Susanna Juteau, Laura Kohtamäki, Mette Ilander, Satu Mustjoki, Kreutzman Anna. Metastatic melanoma patients responding to PD1 therapy have higher proportion of peripheral blood NKT-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A130.