Novel genomic alterations in BRAF in patients with LCH results in constitutive ERK activation, which is responsive to second-generation BRAF and MEK inhibitors but not to a BRAF-V600E inhibitor. (A) Illustration of the FAM73A-BRAF fusion identified in this study. (B) Illustration of the BRAF exon 12 in-frame deletions identified in this study. (C) HEK293 cells were transiently transfected with expression plasmids encoding the indicated BRAF wild-type, fusion, and mutant cDNAs, and corresponding lysates from cells maintained in serum were subjected to immunoblotting with the indicated antibodies. Where indicated, cells were treated with BRAF or MEK inhibitor for 4 hours before harvest. (D) LCH lesion biopsy cell suspension harboring indicated BRAF mutations or fusion were treated with BRAF or MEK inhibitor for 4 hours and corresponding lysates were subjected to immunoblotting with the indicated antibodies. (E) LCH lesion biopsy cell suspension harboring indicated BRAF mutations (BRAF-V600E or in-frame exon 12 deletions [indel]) or fusion (FAM73A-BRAF) were treated with BRAF or MEK inhibitor for 4 hours. Median fluorescent intensity (MFI) of p-ERK1/2 in CD207⁺ cells were determined through IFC analyses post treatment with various MAPK pathway inhibitors. (F) Pie chart showing the estimated distribution of genetic alterations identified in MAPK pathway genes in patients with LCH from an institutional cohort, including the novel BRAF fusion and deletions. The frequencies of MAPK pathway mutations were estimated based on previously reported data and the current study (supplemental Methods).^2,4  The asterisk indicates single cases.