AbstractBackgroundKaposiform lymphangiomatosis (KLA) is a recently characterized systemic lymphatic anomaly. Activation of RAS/MAPK and PI3K/AKT/mTOR pathways may affect KLA pathogenesis, but the cellular basis of KLA is unclear. Abnormal‐spindle endothelial cells that express lymphatic endothelial cell (LEC) markers are characteristic of KLA histopathology. This study evaluated patient‐derived KLA cells to establish their morphological and biological characteristics.ProcedureWe established cell lines from primary KLA tissues of two patients with KLA and examined their morphological and functional characteristics, messenger RNA and protein expression profiles, gene mutations, and responses to inhibitors of the RAS/MAPK and PI3K/AKT/mTOR pathways.ResultsBoth KLA cell lines showed spindle‐shaped morphology, stained positive for podoplanin (PDPN), and exhibited impaired tube‐formation properties. They expressed LEC marker PDPN and mesenchymal stem cell markers (CD90, CD105) in the absence of endothelial cell markers (CD34, CD31, VWF), per real‐time polymerase chain reaction. Both mTOR inhibitor rapamycin and MEK inhibitor trametinib inhibited growth of the two cell lines. A NRAS p.Q61R variant was found in one of two independent KLA tissue samples, but not in the KLA cells (per targeted next‐generation sequencing); and KLA cells with this variant had elevated AKT phosphorylation levels. ERK phosphorylation levels were undetectable in both KLA cell lines.ConclusionsInhibition of the RAS/MAPK and PI3K/AKT/mTOR pathways may represent potential therapeutic targets in KLA. These patient‐derived KLA cell lines will be useful research tools to elucidate KLA etiology, and could pave the way for basic, translational, and preclinical studies of this disease.