Hastings, Karen Taraszka

Abstract In human melanoma, tumor cell-specific MHC class II expression is associated with improved response to programmed death 1 and programmed death-ligand 1 blockade; yet the direct effect of melanoma cell-specific MHC class II expression on anti-melanoma T cell responses remains largely unknown. In the clinically relevant Yale University Mouse Melanoma Exposed to Radiation (YUMMER) cell lines, MHC class II expression was IFN-γ-inducible in a subset of cells. Transduction of melanoma cells with CIITA resulted in uniformly high, constitutive expression of MHC class II. YUMMER.G cells transduced with CIITA vector had increased in vivo tumor growth in wild-type mice compared to melanoma cells transduced with empty vector. YUMMER.G cells transduced with empty or CIITA vectors had the same in vivo tumor growth in RAG−/− mice, indicating that the difference in tumor growth between CIITA- and empty-transduced cells was dependent on an intact adaptive immune system. CIITA-transduced YUMMER.G tumors exhibited an increased frequency and infiltration depth of CD4+ T cells, an increased frequency of T regulatory cells, and a decreased frequency and infiltration depth of CD8+ T cells, compared to empty-transduced tumors. Antibody depletion revealed that CIITA- and empty-transduced YUMMER.G tumors were constrained by CD4+ and CD8+ T cells, but only tumor growth of CIITA-transduced cells was decreased by CD25 blockade, which primarily blocks T regulatory cell function. Overall, this work reveals a potential pro-tumorigenic role of melanoma cell-specific overexpression of MHC class II.

Abstract Description Evidence consistent with immunoediting in human cancer has been identified through associative studies. To provide direct evidence that differences in the neoantigen profile are immune-mediated, we compared the neoantigen profiles of cutaneous squamous cell carcinoma (cSCC) from immunocompetent and immunosuppressed patients. Despite consistency in the overall mutational burden and signature, immunocompetent patients have a lower clonal mutational burden compared to immunosuppressed patients. Furthermore, clonal mutations in cSCC from immunocompetent patients have a lower proportion of neoantigens predicted to bind MHC class I, supporting the role of the immune system in shaping the neoantigen profile. In single-cell RNA sequencing of intratumoral CD8+ T cells from a separate cohort of cSCC from immunocompetent and immunosuppressed patients, we observe an increase in exhausted CD8+ T cells in tumors from immunocompetent patients, suggesting an explanation for the persistence of binding neoantigens subclonally. Finally, we compare clonal and subclonal neoantigens as an unbiased screen for characteristics associated with neoantigen immunogenicity. Neoantigens with a large increase in predicted MHC class I binding relative to the normal peptide are enriched in subclonal neoantigens in immunocompetent patients. Overall, this work supports the immune system’s role in sculpting the neoantigen profile of cSCC. Funding Sources Supported by American Cancer Society - Simone Charitable Foundation - Discovery Boost Grant, AZ-DBG-23-1151311-01-IBCD; NIH F30CA281056; R35GM149235 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Background Challenges in predicting which neoantigens mediate tumor rejection limit the efficacy of neoantigen vaccines to treat cancers, especially for cancers with a high mutational burden like cutaneous squamous cell carcinoma (cSCC). Only a small portion of neoantigens prioritized by current methods elicit effective T cell responses, demonstrating the critical need for improved criteria for the prediction of tumor-rejecting neoantigens. Methods Publicly available human cSCC datasets were used to assess the frequency of shared mutations between patients. A transplantable ultraviolet light-induced mouse model of cSCC was generated. The mutational signature and driver mutations in the mouse model were compared with human tumors. Neoantigens were prioritized in the mouse model, and tumor-rejecting neoantigens were identified through (enzyme-linked immunosorbent spot (ELISpot) and in vivo prophylactic vaccination. Binding of the neoantigens and corresponding wild-type peptides to major histocompatibility complex (MHC) class I was determined. Structural modeling of peptide:MHC complexes was performed to assess for changes in structural characteristics of the neoantigens relative to the wild-type peptides. Results A minority of human cSCC tumors shared neoantigens. The mouse cSCC model recapitulated the mutational signature and driver mutations found in human disease and was constrained by CD8 T cells. Two MHC class I neoantigens were identified in the mouse model that constrained cSCC growth. One tumor-rejecting neoantigen exhibited improved MHC binding, and the other had increased solvent accessibility of the mutated residue, compared with wild-type. Across known neoantigens that do not impact MHC binding, increased exposure of the mutated residue distinguished tumor-rejecting from non-immunogenic neoantigens. Conclusions Given the paucity of shared mutations, this work supports the need for personalized neoantigen vaccines in cSCC. To facilitate further discovery, we provide a clinically relevant mouse cSCC model with two defined neoantigens that mediate tumor rejection. Structural changes in the exposure of features that promote T cell receptor recognition defined tumor-rejecting neoantigens. Incorporation of structural modeling to predict changes in T cell receptor accessibility is anticipated to improve the selection of neoantigens for inclusion in personalized cancer vaccines.

Abstract Background Various studies have revealed that there is a disproportionately low representation of skin of colour (SOC) in medical school dermatologic curriculum and board study resources. Methods First-year and second-year medical students were emailed an 18-question survey regarding (1) identifying correct diagnoses of dermatologic conditions on either White skin or SOC and (2) their confidence in identifying dermatologic conditions on SOC. Results 15% of the images of dermatologic conditions included in the institutional preclinical curriculum show images of patients with SOC. Regarding overall scores for diagnosing dermatologic diseases, students performed similarly on both the White image survey (61.73%) and SOC image survey (66.20%) with no statistically significant differences between surveys (p = 0.14). Second-year medical students performed better than first-year medical students overall (p = 0.01) and on White skin image survey scores (p = 0.02) but not on people of colour image survey scores (p = 0.09). Students largely agreed that they were more comfortable identifying dermatologic diagnoses on White skin and that their school could benefit from increased SOC dermatological resources. Conclusion The overall low scores for the diagnosis of common skin conditions on both the White image and SOC image survey by first- and second-year students are not surprising given the results of a prior study and support the need for re-exposure to dermatology presentations in all skin types during the preclinical curriculum. The low scores support the need for changes in the pre-clinical dermatology curriculum to improve diagnostic ability. Second-year students performed better at diagnosing dermatologic conditions overall and on White skin compared to SOC at the end of their didactic years, possibly due to an underrepresentation of SOC images in institutional and outside educational resources. Attitudes towards school-specific dermatologic SOC education demonstrated a clear desire amongst students for more exposure to dermatologic conditions in various skin colours throughout the curriculum.

Abstract The MHC class I antigen 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 antigen presentation pathway in melanoma cells is less clear. In antigen presenting cells, gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of multiple melanoma antigens. While 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 (TCGA) cutaneous melanoma dataset 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 the TCGA melanoma specimens. These cytokines were capable of inducing GILT expression in 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 analyzed the mRNA expression pattern in the MHC class II antigen presentation pathway. In contrast to healthy skin where the MHC class II pathway was nearly uniformly expressed and intact, there was substantial variation in the MHC class II pathway of the TCGA melanoma dataset. 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 antigen presentation pathway in melanoma outcome.

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.