Abstract 569: Expression and modulation by IFN of HLA class I APM components in melanoma: Potential biomarkers of clinical response to checkpoint inhibitors
Authors: Giraudo Lidia , Gammaitoni Loretta , Valentina Bortolot , Iaia Ilenia , Cattaneo Giulia , Becco Paolo , Gallo Susanna , Zaccagna Alessandro , Pisacane Alberto , Crotto Luca , Ferrone Soldano , Aglietta Massimo , Schianca Fabrizio Carnevale , Sangiolo Dario 
Published: August 2019

Abstract
Purpose: The goals of this study are i) to analyze the expression of human leukocyte antigen class I (HLA-I) antigen processing machinery (APM) components in surgically removed melanoma tumors, ii) to assess their modulation by Interferons (IFNs) and iii) to determine their value as predictive biomarkers of the clinical response to immunotherapy with checkpoint inhibitors (CI).
Methods: The expression levels of HLA class I APM components [HLA-I (ABC) chains, beta(2)microglobulin (β2m), Calnexin, Calreticulin, ERp57, Tapasin, TAP1-2, LMP2, LMP7, LMP10] were evaluated by staining with mAbs and flow cytometry in melanoma cells isolated from surgical biopsies from 16 patients with advanced melanoma. In addition the modulation of these molecules by IFN-γ or IFN-α2 (IFNs) was evaluated.
Results: The frequency of cells expressing HLA-I/β2m was &gt;90% in 15/16 Mel tumors. In 1 case extracellular HLA-I/β2m molecules were not detected. The expression levels of extracellular HLA class I APM molecules were quite heterogeneous. Based on Mean Fluorescence Intensity (MFI) of HLA-I/β2m, Mel segregated into 2 groups with high (MFI &gt;200, n=8) and low (MFI&lt;200, n=8) expression levels. In 11 cases (5 from the high and 6 from the low expression groups) we could explore the modulatory activity of IFNs. In vitro exposure to IFNs globally enhanced the expression levels of HLA-I/β2m, allowing the transition from the low to the highexpression group in 1 case. The distribution of expression defects in the intracellular APM components resulted intensely heterogeneous. Highly defective (&lt;25%) expressions were detected notably for LMP2 (12/16), TAP1 (13/16), TAP2 (10/16) and Tapasin (14/16). In 10 cases we could explore the beneficial modulatory activity of IFNs on defective APM components. IFNs could restore the expression of LMP2 (4/10), TAP1 (6/10), and TAP2 (7/8), but not that of Tapasin. Seven patients were treated with CI (either anti-PD1 or anti-CTLA4). Four of them had clinical responses (PR or CR); in 3 of them Mel expressed high levels of HLA-I/β2m. Conversely, Mel from all 3 patients that did not respond to CI expressed low HLA-I/β2m levels which could not be upregulated by IFNs. Highly defective expression of APM components, not responsive to IFN restoration, was detected in all 3 Mel from non-responders.
Conclusions: We report a wide heterogeneous distribution of HLA-I/β2m expression levels and deficits of APM components in Melanoma, partially restorable by IFNs. Our data support the possibility that defects in HLA expression, not responsive to IFN restoration, may predict low responsiveness to CI. We provide rationale to incorporate the evaluation of HLA-I/β2m and APM in clinical immunotherapy studies with CI.
Citation Format: Lidia Giraudo, Loretta Gammaitoni, Bortolot Valentina, Ilenia Iaia, Giulia Cattaneo, Paolo Becco, Susanna Gallo, Alessandro Zaccagna, Alberto Pisacane, Luca Crotto, Soldano Ferrone, Massimo Aglietta, Fabrizio Carnevale Schianca, Dario Sangiolo. Expression and modulation by IFN of HLA class I APM components in melanoma: Potential biomarkers of clinical response to checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 569.