Hippocampal and neocortical BRAF mutant non-expansive lesions in focal epilepsies.

Objective: Mesial Temporal Lobe Epilepsy-associated Hippocampal Sclerosis (MTLE-HS) is a syndrome associated with various aetiologies. We previously identified CD34-positive extravascular stellate cells (CD34+ cells) possibly related to BRAF oncogenic variant in a subset of MTLE-HS. We aimed to identify the BRAF oncogenic variants and characterise the CD34+ cells.

Methods: We analysed BRAF oncogenic variant by digital droplet Polymerase Chain Reaction in 53 MTLE-HS samples (25 with CD34+ cells) and nine non-expansive neocortical lesions resected during epilepsy surgery (five with CD34+ cells). Ex vivo multi-electrode array recording, immunolabelling, methylation microarray and single nuclei RNAseq were performed on BRAF MTLE-HS and BRAF mutant non-expansive lesion of hippocampus and/or neocortex.

Results: We identified a BRAF oncogenic variant in five MTLE-HS samples with CD34+ cells (19%) and in five neocortical samples with CD34+ cells (100%). Single nuclei RNAseq of resected samples revealed two unique clusters of abnormal cells (including CD34+ cells) associated with senescence and oligodendrocyte development in both hippocampal and neocortical BRAF mutant samples. The co-expression of the oncogene-induced senescence marker p16 and the outer subventricular zone radial glia progenitor marker HOPX in CD34+ cells was confirmed by multiplex immunostaining. Pseudotime analysis showed that abnormal cells share a common lineage from progenitors to myelinating oligodendrocytes. Epilepsy surgery led to seizure freedom in eight of the 10 patients with BRAF mutant lesions.

Interpretation: BRAF underlies a subset of MTLE-HS and epileptogenic non-expansive neocortical focal lesions. Detection of the oncogenic variant may help diagnosis and open perspectives for targeted therapies.