Leukemia (1999) 13, 181–189 1999 Stockton Press All rights reserved 0887-6924/99 $12.00 http://www.stockton-press.co.uk/leu Enhanced expression of p16ink4a is associated with a poor prognosis in childhood acute lymphoblastic leukemia Y Mekki1, R Catallo1, Y Bertrand1,2, AM Manel1,3, P Ffrench3, N Baghdassarian1, P Duhaut4, PA Bryon1,3 and M Ffrench1,3 1Laboratoire de Cytologie Analytique, Universite´ Claude Bernard, MESRT JE 1879; 3Laboratoire Central d’He´matologie et de Cytoge´ne´tique, Hoˆpital E Herriot; 2Service d’He´matologie Pe´diatrique, Hoˆpital Debrousse; and 4RECIF, Universite´ Claude Bernard, Lyon, France The tumor suppressor gene p16ink4a is homozygously deleted p16ink4a overexpression leads to a G1 arrest in the presence of in numerous T as well as in some B lineage acute lymphoblas- a functional pRb.5 When present in high levels, the inhibitor tic leukemia (ALL). We therefore analyzed the clinical and bio- ink4a competes with D cyclins for binding to CDKs, which are logical implications of this feature by studying p16 6 ink4a expression in 58 cases of childhood ALL. mRNA and protein sequestered in inactive protein complexes. p16 mRNA is, were significantly correlated and both appeared more highly at least in part, negatively controlled by pRb. However, it has expressed in B than in T lineage ALLs: 13 out of the 15 T cell also been shown that p16ink4a mRNA and protein accumulate ALLs did not show any p16ink4a expression. The main result of in senescent fibroblasts, and an apparent overexpression ink4a this study is the strong prognostic value of p16 expression. could be caused by an increase in the number of When stratifying the patients in three groups according to 7 p16ink4a expression, we observed in univariate analysis: (1) the population doublings. shortest disease-free survival for patients presenting a high The INK4a gene was mapped to chromosome 9p21, and a p16ink4a level; (2) contrasting with the good prognosis in the high frequency of homozygous deletions or rearrangements of group of patients expressing p16ink4a at low level; (3) while the p16ink4a locus was reported in numerous human cancer cases without any expression of the inhibitor were associated cell lines, including leukemic cell lines.8 Homozygous INK4a = with a medium course of the disease (P 0.0165). This prog- deletions are less frequent in primary leukemic cells than in nostic value was confirmed by the multivariate analysis show- ink4a cell lines, but may occur without any detectable cytogenetic ing therapeutic regimen and p16 protein expression as the 9 only variables retained in the model. A specific metabolic pro- changes in 9p. This deletion is observed more frequently in file related to cellular survival and proliferation was observed T acute lymphoblastic leukemia (ALL) than in B lineage in each of the three p16ink4a expression groups. Among the cell ALL.10–15 The comparison of clinical features at presentation cycle-related proteins we analyzed, only p21waf1 bcl-2 and CDK4 in p16ink4a−/− and in p16ink4a germ line ALL cases showed a ink4a were significantly and positively correlated to p16 . Further- greater leukemic cell mass and a higher white blood cell more, CDK6 was also strongly expressed in the group of cases 13 ink4a with high p16ink4a level. An enhancement of p16ink4a, p21waf1 and count in the first group. However, it is unlikely that p16 bcl-2 was previously described in prolonged cellular survival, gene deletion could represent a clinically prognostic factor in while aging cells showed a decrease in CDK4 expression. The childhood ALL.14,15 concomitant high expression of the oncogenic protein CDK4 Results of treatment in childhood ALL remain incompletely (and of CDK6), we observed, may amplify the leukemic advan- satisfactory because of relapses observed even with high-dose tage of prolonged lifespan blast cells by favoring cell pro- chemotherapy. In order to better understand the regulation of gression through G1 phase. These data suggest that at least two mechanisms may be associated in the oncogenesis of very mitotic activity of lymphoblastic cells in childhood ALL and its aggressive ALLs, ie deregulation of cell multiplication and relationship to treatment outcome, we turned to the analysis of prolonged blast lifespan. p16ink4a mRNA and protein expression and their relationships Keywords: childhood acute lymphoblastic leukemia; p16ink4a; to the other cell cycle regulatory or related proteins. We show p21waf1; cyclin; CDK; prognosis the poor influence of an enhanced expression of p16ink4a on prognosis and the possible linkage of a p16ink4a high level to prolonged blast survival. Introduction The intra-cellular control of cell proliferation is mainly Materials and methods effected by a group of serine-threonine kinases associated, when active, with cyclins and called cyclin-dependent kinases Patients (CDK). CDK4 (or CDK6) associated with cyclin D (D1, D2 or D3), their regulatory subunits, are involved in G1 pro- Fifty-eight cases of childhood ALL were studied from January gression.1,2 These kinases phosphorylate and inactivate the 1994 to July 1997. Thirty patients were male and 28 were retinoblastoma susceptibility gene product (pRb), enabling female, with ages ranging from 0.24 to 17.95 years (median: cells to continue the cell cycle. The kinasic activity is mainly 6.6). Cell samples were obtained, at diagnosis, from bone regulated by: (1) the association with cyclins; (2) several steps marrow aspiration in all cases. Selection for evaluation was of phosphorylation and dephosphorylation; and (3) binding of based on the availability of cell sample; consequently, specific inhibitors called CDK inhibitors (CKI).3 p16ink4a patients having high initial blast-cell counts are numerous in inhibits the kinasic activity of the CDK4 (or CDK6)/cyclin D this study (WBC over 100 G/l in 26% cases). The ALLs were enzymes and represents a major CKI for the G1 phase.4 classified according to the French–American–British (FAB) recommendations16 after cytological examination and cyto- chemical assays (peroxidase and naphthyl acetate esterase) as Correspondence: M Ffrench, Laboratoire de Cytologie Analytique, L1 (50 cases), L2 (five cases), L3 (one case) and unclassified Faculte´ de Me´decine Rockefeller, 8 Av Rockefeller, 69 373 Lyon Cedex 08, France; Fax: 04 72 11 73 05 (one case). All cell samples contained at least 80% blast cells. The first and second authors contributed equally to this work. Mononuclear cells were isolated by Ficoll–Hypaque density Received 24 August 1998; accepted 2 November 1998 gradient (d = 1.077) centrifugation. In all cases, immunolog- p16ink4a and prognosis of ALL Y Mekki et al 182 ical phenotype, cytogenetics and cell cycle variables were gel(7.5% SDS polyacrylamide gel for pRb) and transferred to determined on cell suspension at diagnosis and a specimen nitrocellulose membrane in a semi-dry system as previously of a neoplastic tissue was frozen at −80°C pending protein described.17 Upon completion of transfer, Ponceau S staining and mRNA studies. was performed to verify that an equal amount of protein was Immunological tumor cell phenotype was determined as present in all lanes.20 To ensure independence of expected previously described.17 Lymphoblast populations were div- variations between different ALL groups from differences ided into four categories: (1) early early pre-B (CD10−) ALL resulting from gel and blot preparations, we ran a sample of (six cases), (2) early pre-B (CD10+) (33 cases) and pre-B-ALL Reh-6 cell line as an internal control. For p16ink4a, the HeLa (IgM+) (three cases), (3) B ALL (one case) and (4) T-ALL (15 cell line was used as internal control. cases). The membranes were probed with the following antibodies: Chromosome analysis of bone marrow was performed in mouse monoclonal anti-p16ink4a (1:2000), anti-pRb (1:500), all cases by short-term cultures without phytohemaglutinin. mouse monoclonal IgE anti-cyclin A (1:1000) from Phar- Banding analysis was based on RHG banding. 20 banded Mingen (San Diego, CA, USA); mouse monoclonal anti- metaphases were karyotyped per patient. A t(9;22)(q34;q11) p34cdc2 (1:500), anti-cyclin B (1:500), anti-cyclin E (1:250), translocation was observed in two cases and a anti-p53 (1:500) and rabbit polyclonal anti-cdk2 (1:500), anti- t(4;11)(q21;q23) translocation in two other cases. cdk4 (1:2000), anti-cdk6 (1:1000), anti-cyclin D3 (1:250), Patients were treated according to the EORTC 58881 CLCG anti-p27Kip1 (1:1000) from Santa Cruz Biotechnology (Santa trial18 in the same institution (Debrousse Hospital, Lyon, Cruz, CA, USA); mouse monoclonal anti-p21Waf1 (1;250) from France). Patients were stratified according to their risk factor. Amersham (Little Chalfont, UK), anti-Bcl2 (1:500) from Dako- A so-called very high risk group (VHR) was defined by the patts (Copenhagen, Denmark). The membranes were then presence of at least one of the following criteria regardless of incubated with biotinylated rabbit anti-mouse immuno- the initial blast cell count: (1) blast cell count over 1 G/1 at globulin antibody (Dakopatts), biotinylated goat anti-rabbit the end of the glucocorticoid pre-treatment; (2) non-achieve- immunoglobulin antibody (Sigma) or biotinylated rat anti- ment of complete remission at the end of induction; (3) poor mouse IgE antibody (Southern Biotechnology Associates, cytogenetic features, ie t(9;22), t(4;11), t(1;19) or near hap- Birmingham, AL, USA). In order to confirm the similarity of loidy. A resistance to glucocorticoid was found in 16 cases. specimen quality a labeling of ␤-actin (monoclonal antibody Thirty-five patients were treated with the standard risk sched- from Serotec) was realized. The final step was incubation with ule, 20 with the VHR protocol. Two patients received another streptavidin-biotinylated horseradish peroxidase complex treatment: the unique L3 ALL of this series, and a patient (Amersham) and development with an enhanced chemo- treated in another hospital.