Low-Frequency Mosaicism of the BRAF Mutation As the Origin of Langerhans Cell Histiocytosis
Authors: Takasugi Nao , Shirai Ryota , Osumi Tomoo , Sakamoto Kenichi , Kudo Ko , Tanaka Mariko , Uchiyama Toru , Tomizawa Daisuke , Ushiku Tetsuo , Yoshioka Takako , Shioda Yoko , Kato Motohiro 
Published: December 2024

Introduction:
Frequent relapses and late complications remain a clinical concern in Langerhans cell histiocytosis (LCH), although survival rates have been improved by risk-stratified chemotherapy. The BRAF V600E mutation, a genetic hallmark of LCH, is associated with high relapse rates and late complications such as neurodegenerative LCH (LCH-ND). Additionally, some patients develop characteristic melanocytic nevi after remission of LCH, linked to BRAF V600E. However, the detailed mechanisms underlying frequent relapses and late complications of LCH remain unclear. For better understanding of the unique pathogenesis of LCH, we investigated the origin of BRAF mutation in depth.
Methods:
We examined 8 patients who achieved long-term remission from BRAF mutation-positive LCH and subsequently developed complications like LCH-ND or melanocytic nevi. Among these, 4 patients exhibited both LCH-ND and melanocytic nevi, 3 had only LCH-ND, and 1 had only melanocytic nevi. To avoid false-positives from residual LCH, we selected patients in long-term remission, with no relapse for over 2 years post-sample collection. As controls, we included 3 LCH patients without detectable BRAF V600E mutations, including 1 patient who developed LCH-ND. DNA from normal tissue (peripheral blood, plasma, buccal swab, and nail) was analyzed using mutation-specific droplet digital PCR (ddPCR) to detect low-frequency mosaicism. For 4 cases with melanocytic nevi, skin biopsy samples from areas with nevi and normal areas were separately analyzed by ddPCR. We also examined these skin biopsy samples by RNA-based in situ hybridization, to assess not only the proportion of BRAF mutant cells but also their spatial distribution.
Results:
The BRAF V600E mutation was detected in tumor samples at diagnosis with a median variant allele frequency (VAF) of 13.5%. Among the 8 patients with LCH tumor with BRAF V600E mutation, a low frequency of BRAF V600E was also detected from normal tissue in 6 patients, particularly in 5 of the 7 patients with LCH-ND. The median of VAF was 0.08%, varying by tissue type. On the other hand, the variant was not detected from any samples in cases without BRAF V600E in LCH tumor. In 4 cases with melanocytic nevi, all the patients showed the low-frequency BRAF V600E variant in their normal tissues. A small proportion of BRAF mutations were identified, with a higher VAF in nevus areas (median 1.46%) compared to normal surrounding areas (median 0.26%). In situ hybridization assays confirmed the presence of BRAF V600E mutant cells in all 4 cases with melanocytic nevi. Notably, pigmented areas contained not only a higher concentration of mutant cells but also showed clustering, whereas normal areas showed only sparse positivity. This pattern suggests that BRAF mutant cells proliferate in pigmented areas, while serving as background cells in normal areas, supporting the presence of a mosaic variant, rather than residual LCH.
Conclusions:
Our findings suggest that the majority of BRAF V600E-positive LCH cases originate from low-frequency mosaicism. The unique clinical trajectory and symptoms of LCH can be better understood by recognizing that a subset of normal cells harbor BRAF mutations in a mosaic distribution across various organs, rather than solely attributing symptoms to infiltrating LCH cells with these mutations. Relapses, neurodegenerative diseases, and nevi may not arise from primary LCH cells but could be secondary manifestations originating from this mosaic variant. These findings may shift current understandings of LCH pathogenesis and revolutionize its treatment approaches. We further emphasize the importance of genetic background in cancer which may be overlooked by conventional genomic analyses.