Alternative genetic mechanisms of BRAF activation in Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is characterized by inflammatory lesions containing pathologic CD207+ dendritic cells with constitutively activated ERK. Mutually exclusive somatic mutations in MAPK pathway genes have been identified in approximately 75% of LCH cases, including recurrent BRAF -V600E and MAP2K1 mutations. In order to elucidate mechanisms of ERK activation in the remaining 25% of patients, we performed whole exome sequencing (WES, n=6), targeted BRAF sequencing (n=19) and/or whole transcriptome sequencing (RNA-seq, n=6) on 24 LCH patient samples lacking BRAF -V600E or MAP2K1 mutations. WES and BRAF sequencing identified in-frame BRAF deletions in the β3-αC loop in 6 lesions. RNA-seq revealed one case with an in-frame FAM73A - BRAF fusion lacking the BRAF autoinhibitory regulatory domain but retaining an intact kinase domain. High levels of phospho-ERK were detected in vitro in cells overexpressing either BRAF fusion or deletion constructs and ex vivo in CD207+ cells from lesions. ERK activation was resistant to BRAF -V600E inhibition, but responsive to both a second-generation BRAF inhibitor and a MEK inhibitor. These results support an emerging model of universal ERK-activating genetic alterations driving pathogenesis in LCH. A personalized approach in which patient-specific alterations are identified may be necessary to maximize benefit from targeted therapies for LCH patients.