Cellular and Molecular Mechanisms of Vascular Malformations

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Natural history study of lymphatic disorders We developed a prospective natural history study for patients with lymphatic anomalies to systematically evaluate the disease phenotypes and long-term outcomes. This study will allow us to provide improved prognostication to families, establish screening/monitoring guidelines, determine best practices for genetic diagnosis, and explore family opinions and explore fertility for those on long term medication management. This study will allow us to identify novel end points for future clinical trials. This study was IRB approved and started recruiting during this year. Cellular and molecular mechanism of lymphatic disorders Central conducting lymphatic anomaly (CCLA) due to congenital maldevelopment of the lymphatics can result in debilitating and life-threatening disease with limited treatment options. We identified 4 individuals with CCLA, lymphedema, and microcystic lymphatic malformation due to pathogenic, mosaic variants in KRAS. To determine the functional impact of these variants and identify a targeted therapy for these individuals, we used primary human dermal lymphatic endothelial cells (HDLECs) and zebrafish larvae to model the lymphatic dysplasia. Expression of the p.Gly12Asp and p.Gly13Asp variants in HDLECs in a 2dimensional (2D) model and 3D organoid model led to increased ERK phosphorylation, demonstrating these variants activate the RAS/MAPK pathway. Expression of activating KRAS variants in the venous and lymphatic endothelium in zebrafish resulted in lymphatic dysplasia and edema similar to the individuals in the study. Treatment with MEK inhibition significantly reduced the phenotypes in both the organoid and the zebrafish model systems. In conclusion, we present the molecular characterization of the observed lymphatic anomalies due to pathogenic, somatic, activating KRAS variants in humans. Our preclinical studies suggest that MEK inhibition should be studied in future clinical trials for CCLA due to activating KRAS pathogenic variants. Vascular anomalies are malformations or tumors of the blood or lymphatic vasculature and can be life-threatening. Although molecularly targeted therapies can be life-saving, identification of the molecular etiology is often impeded by lack of accessibility to affected tissue samples, mosaicism or insufficient sequencing depth. In a cohort of 356 participants with vascular anomalies, including 104 with primary complex lymphatic anomalies (pCLAs), DNA from CD31+ cells isolated from lymphatic fluid or cell-free DNA from lymphatic fluid or plasma underwent ultra-deep sequencing thereby uncovering pathogenic somatic variants down to a variant allele fraction of 0.15%. A molecular diagnosis, including previously undescribed genetic causes, was obtained in 41% of participants with pCLAs and 72% of participants with other vascular malformations, leading to a new medical therapy for 63% (43/69) of participants and resulting in improvement in 63% (35/55) of participants on therapy. Taken together, these data support the development of liquid biopsy-based diagnostic techniques to identify previously undescribed genotype-phenotype associations and guide medical therapy in individuals with vascular anomalies.