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

Objectives Somatic mosaicism ofPIK3CAgene is currently recognized as the molecular driver of Klippel-Trenaunay syndrome. However, given the limitation of the current technologies,PIK3CAsomatic 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 (TM) 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 inPIK3CAgene 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.

Automated summary

Our study conducted a comprehensive analysis of seven patients with Klippel-Trenaunay syndrome and found pathogenic mutations in the PIK3CA gene in all patients using cell-free DNA analysis. Tissue analysis confirmed the presence of the mutation at the malformation site in one amputated patient at a high molecular frequency, indicating its causative role. This study demonstrates that cell-free DNA-next generation sequencing-liquid biopsy can be an effective tool for diagnosis and personalized treatment of Klippel-Trenaunay syndrome.