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

Abstract

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.

Cell-free DNA from plasma as a promising alternative for detection of gene mutations in patients with Maffucci syndrome

“…K Zenner extracted cfDNA from the plasma and cystic fluid of patients with venous, lymphatic, and arteriovenous malformations, and variants were detected for the first time [ 13 ]. Subsequent studies of the application of cfDNA in arteriovenous malformations and Klippel–Trenaunay syndrome further validated the investigation of cfDNA-based molecular diagnostics and provided a noninvasive method to initiate targeted therapy for patients with vascular malformations [ 14 , 15 ]. However, there is currently no report of cfDNA genetic analysis on MS associated with SCH.…”

“…More sensitive and noninvasive diagnostic methods may provide adequate information for molecular characterization. In recent years, the clinical utility of cell-free DNA (cfDNA) has been explored in many kinds of vascular malformations [13][14][15][16]. However, there is currently no report of cfDNA genetic analysis on MS associated with SCH.…”

Clitoromegaly, Vulvovaginal Hemangioma Mimicking Pelvic Organ Prolapse, and Heavy Menstrual Bleeding: Gynecologic Manifestations of Klippel-Trénaunay Syndrome

“…A PIK3CA single gene test was reported for KTS [4,6] for determining a targeted molecular diagnosis through a biopsy of affected tissues [3,7]. Luks et al found that PIK3CA mutations were present at low frequencies (<10%) in many affected tissue samples [6].…”

“…Luks et al found that PIK3CA mutations were present at low frequencies (<10%) in many affected tissue samples [6]. Due to the inaccessibility or invasiveness of a solid biopsy, next-generation sequencing of cell-free DNA has recently been used as a non-invasive diagnostic tool for KTS [3]. In this study, we performed WGS to explain the difficulty in enriching only PIK3CA mutations and affected tissue from biopsies.…”

“…Accompanying heavy menstrual bleeding (HMB) was well-controlled with non-steroidal anti-inflammatory drugs (NSAIDs). KTS is a sporadic congenital disorder resulting from somatic mutations of the phosphatidylinositol-4,5-bisphospate 3-kinase, catalytic subunit alpha (PIK3CA) gene, which is involved in cell growth and proliferation [3]. We also performed whole-genome sequencing (WGS) to detect novel KTS variants responsible for the vascular phenotype.…”