Protein Expression of p15 and p21 Plays an Unfavorable Prognostic Role in Adult Acute Lymphoblastic Leukemia (ALL) Patients Independently of Their Gene Promoter Methylation Status.
Authors: De Cave Fabiana , Petrucci Maria Teresa , Gregorj Chiara , Ricciardi Maria Rosaria , Decandia Samantha , Bergamo Paola , Santinelli Sara , Vitale Antonella , Specchia Giorgina , Di Raimondo Francesco , Camera Andrea , Gallo Eugenio , Ferrara Felicetto , Nobile Francesco , Cascavilla Nicola , Fabbiano Francesco , Leone Giuseppe , Perbellini Omar , Mazza Patrizio , Annino Luciana , Galieni Piero , Miraglia Eustachio , Meloni Giovanna , Fazi Paola , Vignetti Marco , Foa Robert , Tafuri Agostino 
Published: October 2019

Abstract
Epigenetic silencing of tumor suppressor (TS) genes is a hallmark in human leukemias, particularly through DNA methylation. Cyclin-dependent kinase inhibitors (CKI) are, among other genes, frequently found methylated in their promoter region. This epigenetic modification has been described also in acute lymphoblastic leukemia (ALL). However, the relationship between aberrant DNA methylation and protein expression of TS genes has not yet been extensively evaluated in adult ALL series. The aim of this study was to analyze in primary cells from newly diagnosed adult ALL patients, uniformly treated according to the LAL2000 GIMEMA protocol, the promoter methylation status of p73, p21, p15 and p16, evaluating in addition the p21, p15 and p16 protein expression. The DNA methylation status of promoter regions was investigated, according to cell availability, using a widely accepted method based on bisulfite modification of DNA, followed by methylation-specific PCR assay (MSP). Protein expression was evaluated by Western blot. Normal peripheral blood lymphocytes, as already described, resulted unmethylated for p73, p21, p15 and p16, and did not express the p21, p15 and p16 proteins. In ALL patients, in contrast, only the p21 promoter region was found constantly unmethylated. The p15, p16 and p73 promoter genes were found methylated in 15/37 (40.5%), 8/43 (18.6%) and 9/36 (25%) patients, respectively. Only 2/23 cases (8.6%) resulted simultaneously methylated for p15, p16 and p73. The p21 and p15 protein expression was found in 28/85 (32.9%) and 44/85 cases (51.8%), respectively. The p16 protein, in contrast, was never expressed. The p16 methylation was associated with the T-ALL (P=0.005) phenotype and with higher white blood cell (WBC) counts (P=0.027). Resistance to spontaneous induction of apoptosis was significantly associated with p21 protein expression (P=0.019) and its co-expression with p15 (P=0.049). Achievement of CR was not influenced by gene methylation status, nor by single protein expression. Interestingly, the co-expression of p15 and p21 was associated with failure to induction treatment: only 6/63 (9.5%) patients co-expressing p15 and p21 obtained a CR (P=0.027). Multivariate analysis confirmed the unfavorable role of this protein co-expression (P=0.059) on CR achievement. In contrast, once patients achieved remission, p21 protein expression was associated with a prolonged DFS, as confirmed by multivariate analysis for DFS (P=0.039). In conclusion, p15 and p21 protein expression plays an unfavorable prognostic role in adult ALL patients independently of the p73, p21, p15 and p16 gene promoter methylation status.