Reappraisal Of The p16INK4A/Prb Pathway and Telomerase Activity, Markers Of Cell Activation and Aging, In Adult Acute Lymphoblastic Leukemia: A Graall Study
Authors: Ffrench Martine , Chien Wei W , Catallo Régine , Chebel Amel , Baranger Laurence , Thomas Xavier , Bene, PharmSciD Marie-Christine , Gerland Luc M , Schmidt Aline , Beldjord Kheira , Klein Nathalie , Escoffre-Barbe Martine , Leguay Thibault , Françoise Huguet , Larosa Fabrice , Hayette Sandrine , Plesa Adriana , Ifrah Norbert , Dombret Hervé , Salles Gilles Andre , Chassevent Agnès 
Published: October 2019

Abstract

Introduction
The treatment of Adult Acute Lymphoblastic Leukemia (ALL) has improved by the use of pediatric-like approaches, erasing the poor prognostic impact of numerous variables. New markers are however still needed to identify poor prognosis patients in prospective trials. The p16INK4A/CDK4-6/pRb pathway and telomerase activity (TA), markers of cell activation and aging, were analyzed in 175 adult ALLs, treated between November 2003 and January 2007 according to the GRAALL/GRAAPH trials, to investigate their prognostic value in this context.

Methods
The cohort comprised 105 males and 70 females aged between 16 and 59 years (median: 36). Immunophenotype by flow-cytometry allowed for the characterization of 123 B- and 52 T-lineage ALLs, 163 of them being subdivided according to EGIL criteria. Cytogenetic and/or molecular analyses allowed for the detection of BCR-ABL, MLL-AF4 or E2A-PBX1 fusion transcripts, NOTCH1 mutations in T-ALLs, and IKAROS deletions in BCR-ABL-negative B-ALLs. In all cases, cell samples were obtained at diagnosis, before any treatment, from bone marrow aspiration after the patients provided informed consent. Flow cytometric analysis of the DNA content was performed for evaluation of the percentages of cells in S/G2/M phases of the cell cycle. Cell cycle regulatory proteins were examined in 135 samples, by western blot. The TA assay was performed on 156 samples according to a telomeric repeat amplification protocol.
Furthermore, in vitro analyses of the p16INK4A/CDK4-6/pRb pathway and TA were carried out in normal peripheral blood lymphocytes before and after stimulation and during lymphocyte long-term culture.

Results
The p16INK4A/CDK4-6/pRb pathway and TA were analyzed according to the immunological phenotype and molecular characteristics of ALLs. Leukocytosis (p<0.0001), proliferating blast cell percentage (p=0.004), CDK6 (p = 0.011), and pRb phosphorylation (p-pRb) (p = 0.003) were significantly higher in T-ALLs compared to B-ALLs, while p16INK4A expression was significantly higher in the latter (p = 0.002). Enhanced p16INK4A significantly correlated with B- and T-maturation (p = 0.03) and MLL rearrangement (p < 0.003).
Among sub-groups defined by clinical or biological data, the most significant relationships between the p16INK4A/CDK4-CDK6/pRb pathway or TA and prognosis were observed for BCR-ABL1+ ALLs (31 cases). An above median expression of CDK4 was related to shorter disease-free survival (DFS) (p = 0.031) and overall survival (OS) when taking into account either death due to any cause (p = 0.005), or only disease-related death (p = 0.018). In spite of the small size of this series, this prognostic value remained when the analysis was restricted to patients who did not receive allogeneic stem-cell transplantation (non-Allo SCT) for DFS (p = 0.034), OS (p = 0.019) or disease-related OS (p = 0.05). A shorter DFS was also associated with the CDK4/6 mediated-phosphorylation of pRb (p-pRb/pRb ratio > 0) (p = 0.042), paradoxically, with high expression levels of p16INK4A (p = 0.029), and also with above median TA (p=0.040). Patients with the highest TA (over quartile 75) showed a significantly shorter OS (p = 0.018) especially when considering only disease-related death (p = 0.007). The poor prognostic value of above median TA for OS (p = 0.026) and disease–related OS (p = 0.009) was also confirmed in the non-Allo SCT group.
Considering that IKAROS is deleted in most of BCR-ABL1+ ALLs and since survival was similar in BCR-ABL1+ ALLs and BCR-ABL1-/IKAROSdel B-ALLs, these two groups of patients were pooled for analysis (42 cases). A shorter DFS was linked to pRb phosphorylation (p = 0.036), while both a shorter DFS (p = 0.026) and OS (p = 0.055) were noted for patients with high TA.
In vitro analyses in normal lymphocytes demonstrated that increased expression of p16INK4A, CDK4, p-pRb and TA is related to cell activation, suggesting that ALL blasts with these criteria could be in an activated stage.

Conclusions
These data bring new perspectives to the biological characterization of ALLs and associate a poor prognosis in BCR-ABL1+ ALLs with enhanced cell activation. Additional investigations could focus, in a prospective series, on the analysis of the cell activation markers described here and on the development of new therapeutic strategies by proposing the association of lymphocyte activation inhibitors.

Disclosures:
No relevant conflicts of interest to declare.