A pilot study of next generation sequencing-liquid biopsy on cell-free DNA as a novel non-invasive diagnostic tool for Klippel-Trenaunay syndrome

Maria Palmieri, Anna Maria Pinto, Laura di Blasio, Aurora Currò, Valentina Monica, Laura Di Sarno, Gabriella Doddato, Margherita Baldassarri, Elisa Frullanti, Annarita Giliberti, Benedetta Mussolin, Chiara Fallerini, Francesco Molinaro, Massimo Vaghi, Alessandra Renieri, Luca Primo

Research output: Contribution to journalArticlepeer-review


OBJECTIVES: Somatic mosaicism of PIK3CA gene is currently recognized as the molecular driver of Klippel-Trenaunay syndrome. However, given the limitation of the current technologies, PIK3CA somatic mutations are detected only in a limited proportion of Klippel-Trenaunay syndrome cases and tissue biopsy remains an invasive high risky, sometimes life-threatening, diagnostic procedure. Next generation sequencing liquid biopsy using cell-free DNA has emerged as an innovative non-invasive approach for early detection and monitoring of cancer. This approach, overcoming the space-time profile constraint of tissue biopsies, opens a new scenario also for others diseases caused by somatic mutations.

METHODS: In the present study, we performed a comprehensive analysis of seven patients (four females and three males) with Klippel-Trenaunay syndrome. Blood samples from both peripheral and efferent vein from malformation were collected and cell-free DNA was extracted from plasma. Tissue biopsies from vascular lesions were also collected when available. Cell-free DNA libraries were performed using Oncomine™ Pan-Cancer Cell-Free Assay. Ion Proton for sequencing and Ion Reporter Software for analysis were used (Life Technologies, Carlsbad, CA, USA).

RESULTS: Cell-free circulating DNA analysis revealed pathogenic mutations in PIK3CA gene in all patients. The mutational load was higher in plasma obtained from the efferent vein at lesional site (0.81%) than in the peripheral vein (0.64%) leading to conclude for a causative role of the identified variants. Tissue analysis, available for one amputated patient, confirmed the presence of the mutation at the malformation site at a high molecular frequency (14-25%), confirming its causative role.

CONCLUSIONS: Our data prove for the first time that the cell-free DNA-next generation sequencing-liquid biopsy, which is currently used exclusively in an oncologic setting, is indeed the most effective tool for Klippel-Trenaunay syndrome diagnosis and tailored personalized treatment.

Original languageEnglish
Pages (from-to)1708538120936421
Publication statusE-pub ahead of print - Jun 26 2020


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