Determination of allele burden of JAK2V617F mutation in genomic and plasma cell free DNA in mieloproliferative neoplasms

CARDOZO MA, GAYDOU L, FOLLONIER A, BOSQUIAZZO VL, RAMOS JG

Congreso; Reunión Conjunta de Sociedades de BioCiencias; 2017

Cell free DNA (cfDNA) in plasma has the clinical potential to be amore specific tumour marker for diagnosis and prognosis, as wellas for early disease detection. Currently, determination of the JAK 2 V617F mutation and measurement of its allele burden (AB) is performedin genomic DNA (gDNA) using both peripheral blood (PB)and bone marrow (BM) samples from patients with chronic myeloproliferativeneoplasms (MPNs). With this procedure, not all malignantalleles could be detected. In this work we compared the %ABof JAK2V617F (percentage of alleles JAK2V617F / total alleles JAK2)in gDNA and cfDNA samples using a quantitative rel time PCR methodology.For quantification we used a calibration curve made with amixture of gDNA from healthy individuals and gDNA derived froma homozygous JAK2V617F-Human-erythroleukemia cell line (ATCCHel 92.1.7). JAK2 status mutation was analyzed in 10 patients withMPNs, primary and secondary myelofibrosis (MF) [n: 4], Escentialthrombocytosis (ET) and Policitema Vera (PV) (n: 6), which werediagnosed according to the 2008 World Health Organization (WHO)classification. gDNA was obtained from peripheral blood using amodified Miller and Dykes technique, while cfDNA was obtainedfrom plasma using the QIAmp DNA Blood Mini kit. We showed ahighly significative correlation between the %AB of JAK2V617F incfDNA and gDNA (Spearman P=0.0028). Also, when we analyzedpaired samples, we observed a higher %AB in cfDNA (P=0.001)compared to the gDNA compartment. These data show that plasmais enriched with tumour-specific nucleic acid and could be the sampleof choice for testing JAK2 mutations.