Plasma DNA Methylation Of P16and Shpl In Patients With B Cell Non-Hodgkin Lymphoma

ObjectiveTo investigate the methylation status of p16, shp1genes in plasma DNA from patients with B-NHL and discuss their possible applications in molecular diagnosis and prognosis evaluation of the disease.MethodsMethylation specific polymerase chain reaction (MSP) was used to detect the methylation status of p16, shp1genes in plasma, peripheral blood leukocytes (PBLs), with/without formaldehyde-fixed, paraffin-embedded (FFPE) tumor tissue samples derived from73patients with B-NHL. Six patients with benign lymphoid hyperplasia (BLH) and13healthy volunteers were selected as nonmalignant controls. Mann-Whitney U test was used to compare methylation frequencies of p16/shp1gene in different groups; Kappa measure of agreement was used to compare the concordance of p161shp1methylation in plasma or PBLs with that in FFPE tumor tissues; Spearman or Pearson correlation test was used to analyze the correlations of p16/shp1methylation status with clinicopathological characteristics.ResultsMethylation frequencies of p16in plasma, PBLs and FFPE tumor tissues from patients with B-NHL were37%(27/73),16%(12/73),39%(16/41); whereas those of shpl were47%(34/73),25%(18/73),63%(26/41); no methylation of p16or shp1was detected in any of the19nonmalignant controls. There was a high methylation consistency of p16/shp1between in plasma and in FFPE tumor tissues (the values of kappa were0.84,0.80), however, the methylation consistency was lower between in PBLs and in FFPE tumor tissues (the values of kappa were0.36,0.42). Methylation frequencies of p16and/or shp1in plasma, PBLs and FFPE tumor tissues derived from B-NHL were63%(46/73),38%(28/73),76%(31/41). In plasma and FFPE tumor tissues, there was a higher frequency of methylated p16in B-NHL patients who had later disease stage, while no similar association was observed in PBLs samples (P>0.05); there was a higher frequencies of methylated p16in B-NHL patients who had B symptoms and lower platelet count (<100×109/L) in all three samples. Higher frequencies of methylated shpl was observed in B-NHL patients who had higher serum LDH level in all three samples. Methylation of neither gene had association with the other clinicopathological parameters.ConclusionsMethylation of p16/shp1in plasma DNA can well represent p16/shp1methylation status in tumor tissues, and may be promising biomarkers in helping B-NHL diagnosis and prognosis evaluation.