BRAF-V600E in Peripheral Mononuclear Cells and Microglia-like Brain Cells Suggest Hematopoietic Origin of Langerhans Cell Histiocytosis-Associated Neurodegeneration

Introduction: Langerhans cell histiocytosis (LCH) is a myeloid neoplasia characterized by inflammatory lesions with pathologic CD207+ myeloid dendritic cells (DC). Activation of ERK occurs in all patients with the most common somatic driver mutation being BRAF-V600E being found in up to 60% of patients.Mononuclear cells harboring the BRAF-V600E mutation have been found in peripheral blood of patients with involvement of the liver, spleen, or bone marrow, known as high risk organs. LCH involvement of the brain may include mass lesions in 25% of patients, most commonly the pituitary gland, or a progressive neurodegenerative syndrome (LCH-ND) that arises in approximately 5% of patients. LCH-ND may arise years after the initial LCH episode was presumed to have been cured and may lead to permanent neurologic deficits. The pathogenesis of LCH-ND is poorly understood, but is believed to be due to an autoimmune or paraneoplastic process. No standard approaches to surveillance or therapy exist. In order to define the etiology of LCH-ND, develop clinical tools for identifying patients at risk for LCH-ND, and to identify potential targets for novel therapies, we evaluated cerebral spinal fluid (CSF) proteins, extracellular BRAF- V600E DNA in CSF, and BRAF-V600E + cells in peripheral blood and brain biopsies.

Methods: CSF biomarkers, including 121 unique proteins associated with inflammation and/or neurodegeneration and extracellular BRAF- V600E, were evaluated in 40 patients with LCH brain lesions and/or LCH-ND. CSF samples from 29 patients with acute lymphoblastic leukemia in remission and 25 patients with pediatric brain tumors represented control samples. Peripheral mononuclear cells were evaluated for BRAF-V600E by qPCR in blood samples collected pre-therapy (14 LCH-ND, 140 non-ND LCH), post-therapy at the time of relapse (14 LCH-ND, 28 non-ND LCH) and post-therapy with no evidence of active disease (35 LCH-ND, 25 non-ND LCH). Brain biopsy specimens were tested for the presence of cells harboring BRAF-V600E and relative expression of osteopontin.

Results: Osteopontin was significantly elevated and S100B was decreased in CSF from patients with LCH compared to patients with brain tumors and other neurodegenerative conditions. While extracellular BRAF-V600E was detected in CSF of only 2/23 patients with LCH CNS lesions or LCH-ND, peripheral blood mononuclear cells with the BRAF- V600E mutation were more frequently found in patients with LCH-ND. In pre-therapy samples, mononuclear cells harboring the BRAF-V600E mutation were identified in 10/17 LCH-ND patients (3/9 with LCH limited to the CNS and 7/8 with active LCH beyond the CNS) vs 21/140 non-ND LCH patient samples. Comparing pre-therapy samples from patients with active LCH outside of the CNS, circulating BRAF-V600E was associated with LCH-ND with a sensitivity of 0.875 and specificity of 0.85 (P=<0.0001). In post-therapy blood collected at a time of relapsed non-CNS LCH, BRAF-V600E was detected in 6/14 LCH-ND samples and 2/28 non-ND LCH samples (sensitivity 0.4286, specificity 0.9286, P=0.0105). In post-therapy blood collected in patients with no active non-CNS LCH, circulating BRAF-V600E was detected in 8/29 LCH-ND samples vs 0/43 non-ND LCH samples (sensitivity 0.2162, specificity 1, P=0.0013). Brain biopsies of patients with LCH-ND demonstrate diffuse infiltration by BRAF-V600E + cells with a transformed microglial phenotype and elevated osteopontin expression, surrounded by CD3+ T cells. Three of four patients with LCH-ND treated with BRAF inhibitors experienced significant clinical and radiologic improvement.

Conclusions: Prior theories of LCH-ND pathogenesis include autoimmune or inflammatory mechanisms. Our results support a model of LCH lesions and LCH-ND arising from a common hematopoietic precursor: whereas ERK-activated myeloid precursors differentiate into CD207+ DCs in non CNS LCH lesions, they may migrate to the brain and differentiate into hyper activated inflammatory microglia-like cells in LCH-ND. This revised model of pathogenesis supports evaluation for LCH mutations in serial blood samples prospectively along with long-term clinical surveillance to identify patients at risk for LCH-ND who may benefit from early initiation of therapy directed against the clonal reservoir of myeloid precursors with activated ERK.