Institute of Anatomy and Cell Biology, University Medical School Göttingen, UMG, Göttingen, Germany.
Institute of Human Genetics, University Medical School Göttingen, UMG, Göttingen, Germany.
PLoS One. 2018 Jul 9;13(7):e0200343. doi: 10.1371/journal.pone.0200343. eCollection 2018.
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.
淋巴管畸形（LM）的特征是在产前和产后发育过程中淋巴管过度生长。已知存在大囊型、小囊型和混合性 LM。这些囊肿由淋巴管内皮细胞（LEC）组成。切除和硬化疗法是最常见的治疗方法。最近在美国对 LM 标本进行的研究表明，在 LM 中发现了磷脂酰肌醇-4,5-二磷酸 3-激酶催化亚单位α（PIK3CA）基因的激活突变。然而，研究的是整个组织而不是分离的细胞类型。在这里，我们研究了德国弗莱堡大学医院和雷根斯堡大学医院切除的 LM 组织。我们分别分离了 LEC 和成纤维细胞，并对 PIK3CA 基因的常见受影响外显子 8、10 和 21 进行了测序。我们在 6 个 LM 衍生的 LEC 系中确认了 4 个典型的单等位基因突变，并描述了两个新的突变 i.) 在exon 10（c.1636C>A；p.Gln546Lys），和 ii.) 一个 GAA 的 3 个碱基的框内缺失（Glu109del）。LM 衍生的成纤维细胞没有这种突变，显示出基因缺陷的细胞类型特异性。与正常 LEC 相比，所有 LM 衍生的 LEC（包括新发现突变的 LEC）中 AKT-Ser473 的过度磷酸化表明 PIK3CA-AKT-mTOR 通路的高活性。此外，除了 Glu109del 之外，所有 LM 衍生的 LEC 中都可见 ERK 的过度磷酸化。在体外，小分子激酶抑制剂 Buparlisib/BKM-120、Wortmannin 和 Ly294002（PIK3CA 的所有抑制剂）、CAL-101（PIK3CD 的抑制剂）、MK-2206（AKT 抑制剂）、Sorafenib（多种激酶抑制剂）和 rapamycin（mTOR 抑制剂）以浓度依赖性方式显著阻断了 LM 衍生的 LEC 的增殖，但也阻断了正常 LEC 的增殖。然而，MK-2206 似乎对除了 Glu109 缺失之外的突变 LEC 更具特异性。总之，正在接受或将要接受激酶抑制剂治疗的儿童必须密切监测。
