PB2191 ANALYSIS OF SALIVA SAMPLES AND CD3+ LYMPHOCYTES AS A SOURCE OF GERMINAL DNA IN MYELOPROLIFERATIVE NEOPLASMS
Authors: N. Garcia‐Gisbert, L. Camacho, L. Fernandez‐Ibarrondo, M. C. Fernandez‐Rodriguez, R. Longaron, J. Gibert, A. Angona, C. Besses, B. Bellosillo
Journal: HemaSphere (2019)

Background: The widespread use of genetic studies of patients with myeloproliferative neoplasms (MPN) by next generation sequencing has led to the identification of new genetic variants not previously reported. In order to establish their potential pathogenic role, it is important to verify if they are somatic or germline variants. In this context, germinal DNA is required, which is frequently obtained from saliva, buccal mucosa, CD3+ lymphocytes, fibroblasts or hair. Saliva has generally been considered as an accessible source of non‐hematopoietic DNA, however, the presence of neutrophils may misrepresent data interpretation. Aims: To ascertain if DNA obtained from saliva samples and CD3+ lymphocytes is a suitable source of germinal DNA for molecular studies of patients with Ph negative myeloproliferative neoplasms. Methods Paired samples of saliva and granulocytes of 191 patients with myeloproliferative neoplasms (76 PV, 109 ET, 5 PMF, 1 no classifiable MPN) mutated for one of the three MPN driver genes: JAK2 (n = 159), CALR (n = 26) and MPL (n = 6) were analyzed. Saliva DNA was obtained using the Oragene‐DNA kit (DNA Genotek). CD3+ T lymphocytes fraction from 64 patients whose saliva sample was positive for the JAK2 V617F mutation were isolated. The mutational status of JAK2 gene was determined by real time PCR (Levine et al., 2006), CALR gene by fragment analysis electrophoresis (3500 Genetic Analyzer, Applied Biosystems) and MPL gene by Digital PCR (QuantStudio 3D, Applied Biosystems). Results: From the 191 saliva samples analyzed, 69/191 (36.1%) were excluded because no human DNA was found, as neither the wild type nor the mutated form of the JAK2, CALR or MPL gene was detected. From the remaining 122 saliva DNA samples, somatic mutations in one of these driver genes were detected in 89/122 cases (73.0%). In the cases with positive saliva, there was a strong, positive correlation between the variant allele frequency (VAF) of the mutation in granulocytes and saliva (r = 0.772, p &lt; 0.001, Spearman). Moreover, in 75/122 samples (61.5%) the VAF was higher than 15%. Being 15% the limit of detection of Sanger sequencing, which is the gold standard for validating germinal mutations, saliva DNA would be positive for the driver mutation in most cases, due to the infiltration of the mutated clone in oral mucosa. Leucocyte count in peripheral blood was higher in patients with positive saliva (p = 0.025, T‐test). Searching for an alternative sample type for validating germinal mutations, we isolated CD3+ T lymphocytes of 64 JAK2 V617F positive patients (40 PV, 20 ET, 3 PMF, 1 no classifiable MPN). DNA extracted from CD3+ lymphocytes was analyzed by real time PCR, and only 2/64 samples (3.12%) had a VAF of JAK2 V617F higher than 15%. Although the CD3+ fraction was not completely pure and still had positivity in some cases for the JAK2 V617F, the presence of the mutated clone was significantly lower than in saliva samples, with a median VAF of 3.39% in CD3+ and a median VAF of 35.1% in saliva (p &lt; 0.001, Wilcoxon)(Figure 1). Summary/Conclusion: In patients with myeloproliferative neoplasms, saliva samples are not a reliable source of germinal DNA. The use of CD3 + lymphocytes is a better option than saliva for the study of germline DNA in myeloproliferative neoplasms. image