Cell-free DNA next-generation sequencing liquid biopsy as a new revolutionary approach for arteriovenous malformation

Abstract

Objective Somatic mosaicism of KRAS gene is currently recognized as the only established molecular basis of arteriovenous malformations (AVM). However, given the limitations of the current technologies, KRAS somatic mutations are detected only in a limited proportion of AVMs and tissue biopsy remains an invasive high risky, sometimes life-threatening, diagnostic procedure. Next-generation sequencing liquid biopsy using cell-free DNA (cfDNA) has emerged as an innovative noninvasive approach for early detection and monitoring of cancer. This approach overcomes the space-time profile constraint of tissue biopsies opens a new scenario for vascular malformations owing to somatic mosaicism. Here, we propose a new approach as a fast noninvasive reliable tool in order to investigate the cfDNA coming from the AVMs. Methods A group of five patients suffering from AVM were selected. Blood samples from peripheral vein and efferent vein from vascular malformation were collected and cfDNA was extracted. The cfDNA libraries were performed using Oncomine Pan-Cancer Cell-Free Assay. We used Ion Proton for sequencing and Ion Reporter Software for analysis (Life Technologies, Carlsbad, Calif). Results In all cases, either G12D or G12V mutations in KRAS were identified. The mutational load was higher in the efferent vein than in peripheral blood, confirming the causative role of the identified mutation at a somatic level. Conclusions We demonstrate that cfDNA next-generation sequencing liquid biopsy is able to identify the KRAS mutation detected in affected tissues. Moreover, we have shown that blood sample withdrawal at the lesion site increases variant allele frequency with an order of magnitude above the limit of detection (usually 0.05%), decreasing the risk of a false negative. Finally, the noninvasiveness of the method avoids any risk of bleeding, being easily performed also in children. We propose this technique as the method of choice to better investigate AVMs and consequently to identify the therapy tailored to the genetic defect. Clinical Relevance This article highlights the importance of using liquid biopsy as a new method to investigate the molecular profile of AVMs. In view of the frequent inaccessibility of vascular tissues owing to the invasiveness of solid biopsy and the relative high incidence of biopsies with low diagnostic power, here we evaluated the efficacy of detecting cfDNA fragments released into the bloodstream from the affected tissue cells. Through a simple blood draw from the efferent vein at the vascular malformation site, the liquid biopsy allowed us to identify KRAS pathogenic mutations piloting a personalized therapeutic approach and opening a new scenario for new therapeutic strategies.

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.…”

ISSVA Classification of Vascular Anomalies and Molecular Biology

“…Mutations in the genes of the RAS pathway, including KRAS (e.g., p.G12D and p.G12V), MAP2K1 (e.g., p.F53L, p.Q56P, p.K57N, p.Q58del, and p.D67Y), and BRAF (e.g., p.V600E), have been detected [11][12][13]. Although the influence of these mutations on the expression and activation of RAS pathway molecules is not fully understood, downstream MEK/ERK signals have been found to be activated in the lesion [14].…”

Review of treatment and therapeutic targets in brain arteriovenous malformation

“…Additionally, next generation sequencing liquid biopsy using cell-free DNA may be a useful noninvasive approach to investigate KRAS mutations in bAVM patients. 52 Future studies are needed to determine whether blood-based markers can inform on KRAS somatic mutation burden and the relevance to bAVM hemorrhage.…”