Abstract A154: Flow cytometric analysis of immune responses in the melanoma tissue biopsies before or during anti-PD1 immunotherapy
Authors: Shklovskaya Elena , Lee Jenny , Lim Su Yin , Alavi Sara , Thompson John , Saw Robyn , Carlino Matteo , Scolyer Richard , Menzies Alexander , Long Georgina , Kefford Richard , Rizos Helen 
Published: February 2019

Abstract
Antibodies directed against the programmed cell death protein 1 (PD-1) immune checkpoint have significantly improved survival of patients with advanced metastatic melanoma. Innate and acquired resistance to anti-PD-1 treatment represents a significant treatment obstacle, yet the mechanisms of resistance are poorly understood and remain difficult to model at the cellular level. Here we used multiparameter flow cytometry to examine the immune profiles of 39 tumor biopsies obtained from 31 patients with stage III-IV metastatic melanoma prior to (baseline, n = 21) or during (n = 18) single-agent anti-PD1 immunotherapy; twelve patients developed resistance to treatment. Samples were enzymatically dissociated and cryopreserved until thawed and stained with fluorescently labeled antibodies to enable a comprehensive analysis of melanoma cells and tumor infiltrating lymphocytes (TILs). Mean melanoma cell content was 57+4.7% (mean+s.e.m.) while mean immune infiltrate (CD45 positive cells) was 31.5+4.5%. Analysis of melanoma cell expression of antigen presenting molecules and PD-1 ligands revealed frequent downregulation of HLA-ABC expression in both baseline (9/21, 43%) and on-treatment tumors (10/18, 56%), including a complete HLA-ABC loss in 2/39 samples. HLA-ABC up-regulation was observed in 9/39 (23%) tumors. Expression of HLA-DR and PD-L1 strongly correlated with that of HLA-ABC (Spearman r=0.68 and =0.89, respectively), indicative of local interferon exposure. Unlike PD-L1, melanoma cell expression of PD-L2 was low in the biopsied tissue. In contrast, PD-L2 expression was readily induced by gamma interferon and correlated with HLA-ABC expression in matching melanoma cell lines established from the dissociated biopsies (9/39). We found multiple correlations between parameters characterizing the immune profiles of tumor cells and TILs in both baseline and on-treatment samples. Among significant treatment-associated changes, conventional T-cells bearing the αβ T-cell receptor were 1.4-fold higher in the on-treatment group compared to baseline (65+2.7% versus 46.2+4.1%, P&lt;0.001), while B cells were 7.7-fold lower (1.5+0.4%, versus 11.4+3.9%, P&lt;0.05). We have identified two unusual “hot melanoma, low TILs” samples (HLA-ABC high HLA-DR positive/high PD-L1 high melanoma cells, &lt;10% CD45 positive TILs) that warrant further investigation, in patients with acquired anti-PD-1 resistance. Our findings suggest that the dynamic downregulation of HLA-ABC expression on melanoma cells is more common than complete loss of HLA-ABC expression and is likely to be a key driver of both innate and acquired resistance to immunotherapy. Functional characterization of melanoma infiltrating T-cells is currently under investigation.
Citation Format: Elena Shklovskaya, Jenny Lee, Su Yin Lim, Sara Alavi, John Thompson, Robyn Saw, Matteo Carlino, Richard Scolyer, Alexander Menzies, Georgina Long, Richard Kefford, Helen Rizos. Flow cytometric analysis of immune responses in the melanoma tissue biopsies before or during anti-PD1 immunotherapy [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 A154.