Cui, Haiyan

Gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II restricted presentation of multiple melanoma antigens. There is variable GILT protein expression in malignant melanocytes in melanoma specimens. High GILT mRNA expression in melanoma specimens is associated with improved overall survival, before the advent of immune checkpoint inhibitors (ICI). However, the association of GILT in metastatic melanoma with survival in patients treated with ICI and the cell type expressing GILT associated with survival have not been determined. Using RNA sequencing datasets, high GILT mRNA expression in metastatic melanoma specimens was associated with improved progression-free and overall survival in patients treated with ICI. A clinical dataset of metastatic melanoma specimens was generated and annotated with clinical information. Positive GILT immunohistochemical staining in antigen presenting cells and melanoma cells was observed in 100% and 65% of metastatic melanoma specimens, respectively. In the subset of patients treated with ICI in the clinical dataset, high GILT protein expression within melanoma cells was associated with improved overall survival. The association of GILT mRNA and protein expression with survival was independent of cancer stage. These studies support that high GILT mRNA expression in bulk tumor samples and high GILT protein expression in melanoma cells is associated with improved survival in ICI-treated patients. These findings support further investigation of GILT as a biomarker to predict the response to ICI.

Melanoma is the sixth most common cancer in the US. Immunotherapies, such as anti‐CTLA‐4 and anti‐PD‐1, have transformed the treatment of advanced melanoma; however, a subset of patients fail to respond to immunotherapy. Thus, improved predictors of the anti‐melanoma immune response and response to immunotherapy are needed. We have previously shown that 1) gamma‐interferon‐inducible lysosomal thiol reductase (GILT) is critical for MHC class II‐restricted presentation of a melanoma antigen, 2) there is variable GILT protein expression in melanoma cells from primary and cutaneous metastatic melanoma specimens, and 3) high GILT mRNA expression in melanoma specimens is associated with improved overall survival. Here, we created a database of n = 43 metastatic melanoma patients (stage III or IV) annotated with clinical outcome. Formalin‐fixed, paraffin‐embedded sections of pre‐treatment metastatic melanoma specimens were tested for GILT protein expression using immunohistochemistry and scored by a dermatopathologist and dermatologist. GILT expression in melanoma cells was detected in 65% of lymph node and distant organ metastases, consistent with our prior finding of GILT expression in 58% of cutaneous metastases. The frequency of GILT‐staining melanoma cells was determined by the ratio of the area of tumor cells with positive GILT staining to the total area of tumor cells. The frequency of GILT‐staining melanoma cells among specimens with positive GILT staining was 15.2% ± 24.7% (mean ± SD). The median frequency of GILT staining was used to divide the patients into no/low and high GILT expression groups. The median overall survival was 52.8 months in the no/low GILT expression group, while the median overall survival was not reached in the high GILT expression group (log‐rank, p = 0.67). For the n = 34 patients who were treated with immunotherapy targeting CTLA‐4 and/or PD‐1, the median overall survival was 38.2 months in the no/low GILT expression group, while the median overall survival was not reached in the high GILT expression group (log‐rank, p = 0.83). These data suggest that high GILT expression in melanoma cells may be associated with improved overall survival, although a statistically significant association of GILT with survival was not observed in the whole group or the subset of patients treated with immunotherapy. A limitation of the current analysis is that 65% of patients have incomplete clinical outcome data. This study demonstrates variable GILT protein expression in tumor cells in metastatic melanoma specimens. Updated clinical outcome data will allow us to better evaluate the association of GILT protein expression in melanoma cells with overall survival and response to immunotherapy in metastatic melanoma patients.

Abstract The MHC class I Ag presentation pathway in melanoma cells has a well-established role in immune-mediated destruction of tumors. However, the clinical significance of the MHC class II Ag presentation pathway in melanoma cells is less clear. In Ag-presenting cells, IFN-γ–inducible lysosomal thiol reductase (GILT) is critical for MHC class II–restricted presentation of multiple melanoma Ags. Although not expressed in benign melanocytes of nevi, GILT and MHC class II expression is induced in malignant melanocytes in a portion of melanoma specimens. Analysis of The Cancer Genome Atlas cutaneous melanoma data set showed that high GILT mRNA expression was associated with improved overall survival. Expression of IFN-γ, TNF-α, and IL-1β was positively associated with GILT expression in melanoma specimens. These cytokines were capable of inducing GILT expression in human melanoma cells in vitro. GILT protein expression in melanocytes was induced in halo nevi, which are nevi undergoing immune-mediated regression, and is consistent with the association of GILT expression with improved survival in melanoma. To explore potential mechanisms of GILT’s association with patient outcome, we investigated pathways related to GILT function and expression. In contrast to healthy skin specimens, in which the MHC class II pathway was nearly uniformly expressed and intact, there was substantial variation in the MHC class II pathway in the The Cancer Genome Atlas melanoma specimens. Both an active and intact MHC class II pathway were associated with improved overall survival in melanoma. These studies support a role for GILT and the MHC class II Ag presentation pathway in melanoma outcome.