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Langerhans Cell Histiocytosis (LCH) is a neoplastic disorder of hematopoietic origin characterized by inflammatory lesions containing clonal histiocytes (LCH-cells) intermixed with various immune cells, including T cells. In 50–60% of LCH-patients, the somatic BRAF V600E driver mutation, which is common in many cancers, is detected in these LCH-cells in an otherwise quiet genomic landscape. Non-synonymous mutations like BRAF V600E can be a source of neoantigens capable of eliciting effective antitumor CD8+ T cell responses. This requires neopeptides to be stably presented by Human Leukocyte Antigen (HLA) class I molecules and sufficient numbers of CD8+ T cells at tumor sites. Here, we demonstrate substantial heterogeneity in CD8+ T cell density in n = 101 LCH-lesions, with BRAF V600E mutated lesions displaying significantly lower CD8+ T cell:CD1a+ LCH-cell ratios (p = 0.01) than BRAF wildtype lesions. Because LCH-lesional CD8+ T cell density had no significant impact on event-free survival, we investigated whether the intracellularly expressed BRAF V600E protein is degraded into neopeptides that are naturally processed and presented by cell surface HLA class I molecules. Epitope prediction tools revealed a single HLA class I binding BRAF V600E derived neopeptide (KIGDFGLAT E K), which indeed displayed strong to intermediate binding capacity to HLA-A*03:01 and HLA-A*11:01 in an in vitro peptide-HLA binding assay. Mass spectrometry-based targeted peptidomics was used to investigate the presence of this neopeptide in HLA class I presented peptides isolated from several BRAF V600E expressing cell lines with various HLA genotypes. While the HLA-A*02:01 binding BRAF wildtype peptide KIGDFGLAT V was traced in peptides isolated from all five cell lines expressing this HLA subtype, KIGDFGLAT E K was not detected in the HLA class I peptidomes of two distinct BRAF V600E transduced cell lines with confirmed expression of HLA-A*03:01 or HLA-A*11:01. These data indicate that the in silico predicted HLA class I binding and proteasome-generated neopeptides derived from the BRAF V600E protein are not presented by HLA class I molecules. Given that the BRAF V600E mutation is highly prevalent in chemotherapy refractory LCH-patients who may qualify for immunotherapy, this study therefore questions the efficacy of immune checkpoint inhibitor therapy in LCH. [ABSTRACT FROM AUTHOR]

Langerhans Cell Histiocytosis (LCH) is a neoplastic disorder of hematopoietic origin characterized by inflammatory lesions containing clonal histiocytes (LCH-cells) intermixed with various immune cells, including T cells. In 50-60% of LCH-patients, the somatic BRAF V600E driver mutation, which is common in many cancers, is detected in these LCH-cells in an otherwise quiet genomic landscape. Non-synonymous mutations like BRAF V600E can be a source of neoantigens capable of eliciting effective antitumor CD8 + T cell responses. This requires neopeptides to be stably presented by Human Leukocyte Antigen (HLA) class I molecules and sufficient numbers of CD8 + T cells at tumor sites. Here, we demonstrate substantial heterogeneity in CD8 + T cell density in n = 101 LCH-lesions, with BRAF V600E mutated lesions displaying significantly lower CD8 + T cell:CD1a + LCH-cell ratios ( p = 0.01) than BRAF wildtype lesions. Because LCH-lesional CD8 + T cell density had no significant impact on event-free survival, we investigated whether the intracellularly expressed BRAF V600E protein is degraded into neopeptides that are naturally processed and presented by cell surface HLA class I molecules. Epitope prediction tools revealed a single HLA class I binding BRAF V600E derived neopeptide (KIGDFGLAT E K), which indeed displayed strong to intermediate binding capacity to HLA-A * 03:01 and HLA-A * 11:01 in an in vitro peptide-HLA binding assay. Mass spectrometry-based targeted peptidomics was used to investigate the presence of this neopeptide in HLA class I presented peptides isolated from several BRAF V600E expressing cell lines with various HLA genotypes. While the HLA-A * 02:01 binding BRAF wildtype peptide KIGDFGLATV was traced in peptides isolated from all five cell lines expressing this HLA subtype, KIGDFGLAT E K was not detected in the HLA class I peptidomes of two distinct BRAF V600E transduced cell lines with confirmed expression of HLA-A * 03:01 or HLA-A * 11:01. These data indicate that the in silico predicted HLA class I binding and proteasome-generated neopeptides derived from the BRAF V600E protein are not presented by HLA class I molecules. Given that the BRAF V600E mutation is highly prevalent in chemotherapy refractory LCH-patients who may qualify for immunotherapy, this study therefore questions the efficacy of immune checkpoint inhibitor therapy in LCH., (Copyright © 2020 Kemps, Zondag, Steenwijk, Andriessen, Borst, Vloemans, Roelen, Voortman, Verdijk, van Noesel, Cleven, Hawkins, Lang, de Ru, Janssen, Haasnoot, Franken, van Eijk, Solleveld-Westerink, van Wezel, Egeler, Beishuizen, van Laar, Abla, van den Bos, van Veelen and van Halteren.)