PIK3CA mutations are specifically localized to lymphatic endothelial cells of lymphatic malformations

Abstract

Lymphatic malformations (LM) are characterized by the overgrowth of lymphatic vessels during pre- and postnatal development. Macrocystic, microcystic and combined forms of LM are known. The cysts are lined by lymphatic endothelial cells (LECs). Resection and sclerotherapy are the most common treatment methods. Recent studies performed on LM specimens in the United States of America have identified activating mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene in LM. However, whole tissue but not isolated cell types were studied. Here, we studied LM tissues resected at the University Hospitals Freiburg and Regensburg, Germany. We isolated LECs and fibroblasts separately, and sequenced the commonly affected exons 8, 10, and 21 of the PIK3CA gene. We confirm typical monoallelic mutations in 4 out of 6 LM-derived LEC lines, and describe two new mutations i.) in exon 10 (c.1636C>A; p.Gln546Lys), and ii.) a 3bp in-frame deletion of GAA (Glu109del). LM-derived fibroblasts did not possess such mutations, showing cell-type specificity of the gene defect. High activity of the PIK3CA—AKT- mTOR pathway was demonstrated by hyperphosphorylation of AKT-Ser473 in all LM-derived LECs (including the ones with newly identified mutations), as compared to normal LECs. Additionally, hyperphosphorylation of ERK was seen in all LM-derived LECs, except for the one with Glu109del. In vitro, the small molecule kinase inhibitors Buparlisib/BKM-120, Wortmannin, and Ly294002, (all inhibitors of PIK3CA), CAL-101 (inhibitor of PIK3CD), MK-2206 (AKT inhibitor), Sorafenib (multiple kinases inhibitor), and rapamycin (mTOR inhibitor) significantly blocked proliferation of LM-derived LECs in a concentration-dependent manner, but also blocked proliferation of normal LECs. However, MK-2206 appeared to be more specific for mutated LECs, except in case of Glu109 deletion. In sum, children that are, or will be, treated with kinase inhibitors must be monitored closely.

Characterization of lymphatic malformations using primary cells and tissue transcriptomes

“…This finding is consistent with the cell‐intrinsic hyper‐proliferative phenotype of the LM2. Activating mutations of PIK3CA and PIK3R3 have also been found in germ line and somatic cells of LM patients …”

“…Activating mutations of PIK3CA and PIK3R3 have also been found in germ line and somatic cells of LM patients. 12,[44][45][46]65,68 Only a few studies have addressed whether LEC proliferate in LMs; and two studies did not find prominent proliferation in vivo. 4,69 Our study was undertaken under the common hypothesis that lymphatic malformations result from an abnormal formation of lymphatic vessels because of uncontrolled growth of primitive/embryonic LEC.…”

Cellular and molecular mechanisms of PIK3CA-related vascular anomalies

“…Histological markers of LM are Prox-1 and VEGFR3, while Podoplanin levels can be variable (17). Somatic activating PIK3CA mutations have been identified in affected tissue resected from the majority (94%) of LM patients (18) and in cells isolated from LM tissue (19,20,21). Patient-derived lymphatic endothelial cells (LM-LEC) exhibit pro-angiogenic properties such as increased proliferation, sprout formation, and VEGF-C and VEGFR-3 overexpression.…”

“…Patient-derived lymphatic endothelial cells (LM-LEC) exhibit pro-angiogenic properties such as increased proliferation, sprout formation, and VEGF-C and VEGFR-3 overexpression. AKT that is downstream of PI3K is constitutively active in the mutant LM-LEC, while ERK1/2 phosphorylation levels are variable (17,19,20,22). A few animal models of LM have been reported and consist of xenografts generated by injection of LM-LEC (17) or LEC progenitor cells (23).…”

Microcystic lymphatic malformation in a child and his mother

“…Lymphatic malformations can occur as an isolated finding or as a part of syndromes such as Noonan syndrome, Turner syndrome, Down syndrome, and Klippel‐Trenaunay syndrome . Somatic (non‐inherited) activating mutations in the phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit alpha ( PIK3CA) gene have been found in many patients with LM, in the endothelial cells lining the malformed lymphatic vessels . PIK3CA plays a role in cell growth regulation by signaling via the PI3K/mTOR pathway, and five different point mutations in this gene have been identified in such patients .…”