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Background <p>Vascular malformations originate from congenital abnormalities in vascular differentiation and formation, and their subtypes exhibit diverse overlapping phenotypes that make differential diagnosis challenging. Recent advances in next-generation sequencing (NGS) technology for tissue specimens have enabled the identification of somatic variants in genes that are associated with vascular malformations, such as those in the RAS/MEK/ERK and phosphoinositide 3-kinase/AKT/mTOR pathways. Liquid biopsy using cell-free DNA (cfDNA) is a minimally invasive alternative method that has recently been applied to vascular malformations. This study aimed to apply ultra-deep targeted NGS using liquid biopsy to detect somatic variants in vascular malformations.</p> Methods <p>A total of 10 mL peripheral blood was collected from 40 patients with vascular malformations, and cfDNA was extracted for liquid biopsy. Ultra-deep NGS (mean coverage: 104,000×) with unique molecular identifier error correction was performed using a custom panel of five genes: <i>BRAF</i>, <i>KRAS</i>, <i>MAP2K1</i>, <i>PIK3CA</i>, and <i>TEK/TIE2</i>.</p> Results <p>The diagnoses included arteriovenous malformation (AVM, <i>n</i> = 11), <i>PIK3CA</i>-related overgrowth spectrum (PROS, <i>n</i> = 9), and venous malformation (VM, <i>n</i> = 20). Somatic variants were identified in 23 patients, even at a low variant allele frequency of 0.05%. Hotspot <i>BRAF</i> and <i>KRAS</i> variants and a non-hotspot <i>MAP2K1</i> variant were detected in patients with AVM. Fifteen variants were detected in seven patients with PROS, including hotspot <i>PIK3CA</i> variants in helical domains. Three novel <i>PIK3CA</i> and four novel <i>TEK</i> variants were identified in patients with VM.</p> Conclusions <p>Liquid biopsy of cfDNA from the peripheral blood enables safe, outpatient-compatible molecular diagnosis, to potentially guide tailored treatment.</p>

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Ultra-deep targeted next-generation sequencing with peripheral blood in patients with vascular malformations: a cohort of 40 patients

  • 期刊论文

  • 发表日期：

    2025年12月19日

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  • Shintaro Mitamura,
  • Kosuke Ishikawa,
  • Kanako C. Hatanaka,
  • Tatsuro Saito,
  • Yuki Sasaki,
  • Takahiro Miura,
  • Taku Maeda,
  • Emi Funayama,
  • Naoki Murao,
  • Satoru Sasaki,
  • Yutaka Hatanaka

摘要

Background

Vascular malformations originate from congenital abnormalities in vascular differentiation and formation, and their subtypes exhibit diverse overlapping phenotypes that make differential diagnosis challenging. Recent advances in next-generation sequencing (NGS) technology for tissue specimens have enabled the identification of somatic variants in genes that are associated with vascular malformations, such as those in the RAS/MEK/ERK and phosphoinositide 3-kinase/AKT/mTOR pathways. Liquid biopsy using cell-free DNA (cfDNA) is a minimally invasive alternative method that has recently been applied to vascular malformations. This study aimed to apply ultra-deep targeted NGS using liquid biopsy to detect somatic variants in vascular malformations.

Methods

A total of 10 mL peripheral blood was collected from 40 patients with vascular malformations, and cfDNA was extracted for liquid biopsy. Ultra-deep NGS (mean coverage: 104,000×) with unique molecular identifier error correction was performed using a custom panel of five genes: BRAF, KRAS, MAP2K1, PIK3CA, and TEK/TIE2.

Results

The diagnoses included arteriovenous malformation (AVM, n = 11), PIK3CA-related overgrowth spectrum (PROS, n = 9), and venous malformation (VM, n = 20). Somatic variants were identified in 23 patients, even at a low variant allele frequency of 0.05%. Hotspot BRAF and KRAS variants and a non-hotspot MAP2K1 variant were detected in patients with AVM. Fifteen variants were detected in seven patients with PROS, including hotspot PIK3CA variants in helical domains. Three novel PIK3CA and four novel TEK variants were identified in patients with VM.

Conclusions

Liquid biopsy of cfDNA from the peripheral blood enables safe, outpatient-compatible molecular diagnosis, to potentially guide tailored treatment.