B-RAF Mutant Alleles Associated with Langerhans Cell Histiocytosis, a Granulomatous Pediatric Disease

Background: Langerhans cell histiocytosis (LCH) features inflammatory granuloma characterised by the presence of CD1a+ dendritic cells or 'LCH cells'. Badalian-Very et al. recently reported the presence of a canonical B-V600E-RAF mutation in 57% of paraffin-embedded biopsies from LCH granuloma. Here we confirm their findings and report the identification of two novel B-RAF mutations detected in LCH patients.

Methods and Results: Mutations of B-RAF were observed in granuloma samples from 11 out of 16 patients using 'next generation' pyrosequencing. In 9 cases the mutation identified was B-V600E-RAF. In 2 cases novel polymorphisms were identified. A somatic B-600DLAT-RAF insertion mimicked the structural and functional consequences of the B-V600E-RAF mutant. It destabilized the inactive conformation of the B-RAF kinase and resulted in increased ERK activation in 293 T cells. The B-600DLAT-RAF and B-V600E-RAF mutations were found enriched in DNA and mRNA from the CD1a+ fraction of granuloma. They were absent from the blood and monocytes of 58 LCH patients, with a lower threshold of sequencing sensitivity of 1%-2% relative mutation abundance. A novel germ line B-T599A-RAF mutant allele was detected in one patient, at a relative mutation abundance close to 50% in the LCH granuloma, blood monocytes and lymphocytes. However, B-T599A-RAF did not destabilize the inactive conformation of the B-RAF kinase, and did not induce increased ERK phosphorylation or C-RAF transactivation.

Conclusions: Our data confirmed presence of the B-V600E-RAF mutation in LCH granuloma of some patients, and identify two novel B-RAF mutations. They indicate that B-V600E-RAF and B-600DLAT-RAF mutations are somatic mutants enriched in LCH CD1a+ cells and absent from the patient blood. Further studies are needed to assess the functional consequences of the germ-line B-T599A-RAF allele.