背景:Klippel-Trenaunay综合征(KTS)是一种罕见的慢血流合并血管畸形伴肢体肥大。KTS被认为位于PIK3CA相关的过度生长谱上,但报道有限。PIK3CA编码p110α,磷脂酰肌醇3-激酶(PI3K)的催化亚基,在PI3K/AKT/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路中起重要作用。我们旨在证明靶向下一代测序(NGS)在识别KTS患者的福尔马林固定石蜡包埋(FFPE)组织中的PIK3CA镶嵌性方面的临床实用性。
结果:参与者是9名女性和5名男性KTS患者,他们被诊断为毛细血管静脉畸形(CVM)或毛细血管淋巴静脉畸形(CLVM)。切除时的中位年龄为14岁(范围,5-57岁)。从FFPE组织提取DNA前的中位存档期为5.4年(范围,3-7年)。PIK3CA的基于NGS的测序实现了119,000x的扩增子平均覆盖。14例患者中有12例出现PIK3CA错义突变(85.7%;6/8CVM和6/6CLVM),有8名患者表现出热点变异E542K,E545K,H1047R,和H1047L。非热点PIK3CA变体C420R,Q546K,在4例患者中发现了Q546R。总的来说,鉴定出的PIK3CA变异的平均变异等位基因频率为6.9%(范围,1.6-17.4%)。所有患有地理毛细血管畸形的患者,组织病理学淋巴畸形或足大指有PIK3CA变异。在热点和非热点PIK3CA变体之间没有发现基因型-表型关联。组织学上,病变的血管和脂肪组织显示PI3K/AKT/mTOR信号通路中的蛋白磷酸化,包括p-AKT,p-mTOR,和p-4EBP1。
结论:KTS患者间充质组织中PI3K/AKT/mTOR通路被激活。基于扩增子的靶向NGS可以从从FFPE组织中提取的低输入DNA中鉴定低水平的镶嵌性,可能为使用PI3K/AKT/mTOR信号通路抑制剂的个性化药物提供诊断选择。
Klippel-Trenaunay syndrome (KTS) is a rare slow-flow combined vascular malformation with limb hypertrophy. KTS is thought to lie on the PIK3CA-related overgrowth spectrum, but reports are limited. PIK3CA encodes p110α, a catalytic subunit of phosphatidylinositol 3-kinase (PI3K) that plays an essential role in the PI3K/AKT/mammalian target of rapamycin (mTOR) signaling pathway. We aimed to demonstrate the clinical utility of targeted next-generation sequencing (NGS) in identifying PIK3CA mosaicism in archival formalin-fixed paraffin-embedded (FFPE) tissues from patients with KTS.
Participants were 9 female and 5 male patients with KTS diagnosed as capillaro-venous malformation (CVM) or capillaro-lymphatico-venous malformation (CLVM). Median age at resection was 14 years (range, 5-57 years). Median archival period before DNA extraction from FFPE tissues was 5.4 years (range, 3-7 years). NGS-based sequencing of PIK3CA achieved an amplicon mean coverage of 119,000x. PIK3CA missense mutations were found in 12 of 14 patients (85.7%; 6/8 CVM and 6/6 CLVM), with 8 patients showing the hotspot variants E542K, E545K, H1047R, and H1047L. The non-hotspot PIK3CA variants C420R, Q546K, and Q546R were identified in 4 patients. Overall, the mean variant allele frequency for identified PIK3CA variants was 6.9% (range, 1.6-17.4%). All patients with geographic capillary malformation, histopathological lymphatic malformation or macrodactyly of the foot had PIK3CA variants. No genotype-phenotype association between hotspot and non-hotspot PIK3CA variants was found. Histologically, the vessels and adipose tissues of the lesions showed phosphorylation of the proteins in the PI3K/AKT/mTOR signaling pathway, including p-AKT, p-mTOR, and p-4EBP1.
The PI3K/AKT/mTOR pathway in mesenchymal tissues was activated in patients with KTS. Amplicon-based targeted NGS could identify low-level mosaicism from low-input DNA extracted from FFPE tissues, potentially providing a diagnostic option for personalized medicine with inhibitors of the PI3K/AKT/mTOR signaling pathway.