Protein Expression and Methylation Status of the CKI p21, p15 and p16 in Adult Acute Lymphoblastic Leukemia (ALL) Patients: Prognostic Implications.
Authors: Gregorj Chiara , De Cave Fabiana , Petrucci Maria Teresa , Ricciardi Maria R. , Scerpa Maria C. , Santinelli Sara , Liso Vincenzo , Giustolisi Rosario , Camera Andrea , Gallo Eugenio , Ferrara Felicetto , Nobile Francesco , Cascavilla Nicola , Fabbiano Francesco , Leone Giuseppe , Perbellini Omar , Mazza Patrizio , Annino Luciana , Galieni Piero , Miraglia Eustachio , Martelli Massimo , Torelli Giuseppe , Cantore Nicola , Peta Antonio , D’Arco Alfonso M. , Tedeschi Alessandra , Fioritoni Giuseppe , Cuneo Antonio , Brugiatelli Maura , Gaidano Gianluca , Visani Giuseppe , Majolino Ignazio , Nieddu Rosa , Boccadoro Mario , Capussela Ernesto , Vitale Antonella , Meloni Giovanna , Fazzi Paola , Vignetti Marco , Foa Robert , Tafuri Agostino 
Published: October 2019

Abstract
Cyclin-dependent kinase inhibitors (CKI) regulate cell division resulting aberrantly expressed in many types of cancer. Alterations of CKI have been reported in acute leukemia, as the result of gene promoter methylation. Despite the common frequency of these alterations, little has been reported on the role of CKI aberrant protein expression and results are less clear, especially in acute lymphoblastic leukemia (ALL). The aim of this study was to analyze p21, p15 and p16 protein expression and their gene methylation status in primary cells from adult ALL cases enrolled in the LAL2000 GIMEMA protocol. Normal peripheral blood lymphocytes (PBL) and 91 primary samples from untreated ALL patients were evaluated in this study. The p21, p15 and p16 protein expression was analyzed by Western blot using the specifically MoAbs. The CKI gene methylation status was investigated using a widely accepted method based on bisulfite modification of DNA, followed by the use of the methylation-specific PCR assay (MSP). This assay was further validated in vitro by SSI methylase. Normal PBL from 10 healthy donors, as described, did not expressed all CKIs and resulted unmethylated. The p21 expression was found in 28/91 cases (30.8%); in contrast, samples were found constantly unmethylated. The p15 expression was found in 44/85 cases (51.8%) and its gene methylated in 41.7%; a significant correlation was found between absence of protein expression and gene methylation (P=0.040). The p16 resulted never expressed in adult ALL, while its promoter was found methylated in 8/42 cases (19.1%). A significant association (P=0.037) was observed between p21 expression and immunophenotype; in fact, 3/24 (12.5%) T-ALL and 24/65 (36.9%) B-lineage ALL expressed this protein. The p16 methylation was associated with T-ALL (P=0.082). Achievement of CR was not influenced by single protein expression, nor by gene methylation status. However, the co-expression of p15 and p21 was associated with failure to induction treatment; in fact, only 6/67 (9%) of patients co-expressing p15 and p21 achieved CR (P=0.021). In summary, in adult ALL p21 is not methylated and p16 is never found expressed, and CR achievement is adversely affected by the co-expression of p21 and p15. In conclusion, we report that in addition to CKI methylation, aberrant expression of CKI, namely p21 and p15, is associated with poor outcome in adult ALL, suggesting that chemotherapy resistance may be promoted in these cases by cell cycle arrest and/or abnormal survival.