Frequent BRAF V600E Mutations Are Identified in CD207+ Cells in LCH Lesions, but BRAF Status does not Correlate with Clinical Presentation of Patients or Transcriptional Profiles of CD207+ Cells

Very little is known about the cell of origin or the pathogenesis of LCH. There remains debate regarding LCH as a malignant disorder or the result of immune dysregulation. While multiple studies in the past failed to identify significant genetic lesions, an activating mutation (V600E) in the serine/threonine kinase BRAF was recently described in LCH biopsy samples (Badalian-Very et al., 2010).

This study was designed to evaluate the frequency of BRAF mutations in LCH lesions, to identify the cells within the lesions carrying the mutation, and to evaluate the clinical and biological significance of the mutation.

Fresh LCH biopsy samples were collected, cells were sorted into CD3+ and CD207+ fractions, and RNA was purified then amplified into cDNA. Sanger sequencing as well as BRAF allele-specific PCR were performed for each sample. Categorical clinical data was compared to BRAF genotype to evaluate clinical significance of the mutation. Transcriptomes of CD207+ cells were also compared (wild-type BRAF vs V600E) with the Affymetrix U133Plus2.0 platform to determine the impact of the BRAF mutation on global gene expression.

The BRAF V600E mutation was consistently identified in cDNA generated from CD207+ cells in 17 of 32 (52%) LCH biopsy samples. Only the wild-type allele was detected in purified T (CD3+) cells from LCH lesions, control epidermal (CD207+) Langerhans cells, and control tonsil T (CD3+) cells. In two cases of recurrent disease, BRAF status was consistent in the presenting and the relapse CD207+ cells: wild-type BRAF in one case and V600E BRAF in another. However, mutation status did not correlate significantly with age (p=0.6), single lesion vs multifocal/systemic (p=1.0), or future recurrent/refractory disease (p=0.2) in this series. Furthermore, unsupervised clustering gene expression profiles CD207+ cells (wild-type BRAF vs V600E) did not segregate datasets based on BRAF status. Using standard statistical analysis, there were no genes identified as significantly up- or down-regulated as a result of the V600E mutation.

The BRAF V600E point mutation is the first reproducible molecular abnormality identified in LCH. In this study, we validate the observation that it occurs with high frequency, and definitively localize the pathologic CD207+ cell as the source of the mutation in LCH lesions. Interestingly, while the frequency of the mutation implies some functional significance, in this series there is no statistically significant clinical difference between patients with wild-type or mutated BRAF lesions, and the transcriptomes of LCH CD207+ cells with wild-type and V600E BRAF are indistinguishable. It is possible that the mutation affects LCH pathogenesis at earlier stages in tumorigenesis, or there may be other routes of Ras pathway activation in LCH lesions with wild-type BRAF. While the role for BRAF in LCH pathogenesis remains to be defined, this is an important molecular foothold from which to investigate the biology of LCH.

No relevant conflicts of interest to declare.