Hematopoietic Stem Cells and Circulating Myelomonocytic Precursors With BRAF-V600E Are Identified In High-Risk Patients and Define LCH As a Myeloid Neoplasia
Authors: Phaik-Har Lim Karen , Peters Tricia L , Berres Marie-Luise , Price Jeremy , Takizawa Hitoshi , Lupo Philip , Hicks M. John , Shih Albert , Simko Stephen James , Heym Kenneth Matthew , Bigley Venetia , Collin Matthew P. , Manz Markus G. , McClain Kenneth , Merad Miriam , Allen Carl E. 
Published: October 2019

Abstract

Background
Langerhans Cell Histiocytosis (LCH) is a clonal lymphoproliferative disorder characterized by inflammatory lesions with characteristic CD207+ dendritic cells (DCs). LCH has variable clinical presentations ranging from single lesions to potentially fatal multi-system “High Risk” disease. The etiology of LCH remains elusive, with debate of LCH as an inflammatory versus malignant disorder unresolved. The first recurrent somatic genetic mutation in LCH, BRAF-V600E, was recently reported in 57% of LCH lesions (Badalian-Very et al., 2010). In this study, we investigate the clinical significance of BRAF-V600E and identify cells carrying the mutation to determine the origins of LCH.

Methods
Lesions, peripheral blood, peripheral monocyte/dendritic cell populations, and hematopoietic stem cells were genotyped for the BRAF-V600E mutation with real-time PCR. The presence of the BRAF-V600E mutation was correlated with clinical variables and analyzed with standard statistical methods. Colony-forming unit assays were used to test the hematopoietic potential of CD34+ cells purified from bone marrow aspirate.

Results
Lesions from 100 patients with LCH were genotyped, and 64% percent carried the V600E mutation, which localized to the infiltrating CD207+ DCs. In 16 patients with more than one lesion, BRAF status remained fixed, suggesting somatic mutation is an early event. BRAF-V600E did not define specific clinical risk groups or impact overall survival, but it was associated with approximately two-fold higher risk of relapse (p=0.04). Furthermore, the cellular compartment carrying the mutation correlated with disease severity: The ability to detect BRAF-V600E in circulating mononuclear cells defined High-Risk LCH with 100% sensitivity/87%specificity. The ability to detect BRAF-V600E in circulating blood cells in patients with High-Risk LCH defined clinically detectable disease with 97% sensitivity/100% specificity. Analysis of sorted populations localized the BRAF-V600E to CD11c+ and CD14+ fractions in peripheral blood, and to CD34+ cells in bone marrow. Potential of the CD34+ hematopoietic stem cells with the BRAF-V600E mutation to differentiate into myeloid precursors was verified with in vitrocolony-forming unit assays.

Conclusions
The molecular foothold of BRAF at the base of LCH pathogenesis will allow therapeutic strategies to move beyond empiric observation to risk-stratified and targeted approaches. Furthermore, effectiveness of therapy may be tested by following BRAF-V600E in peripheral blood cells as a marker of residual disease. We hypothesize that High-Risk LCH arises from somatic mutation of an immature myelomonocytic precursor cell, where Low-Risk disease arises from somatic mutation of tissue-restricted DC precursors. We therefore propose classifying LCH as a bone fide myeloid neoplasia in which BRAF-V600E expression in precursor versus mature dendritic cells defines clinically distinct risk-groups.

Disclosures:
No relevant conflicts of interest to declare.