We investigated the prognostic value of p16INK4aimmunocytochemistry (ICC) analysis in 126 cases of newly diagnosed childhood acute lymphoblastic leukemia (ALL). The incidence of negative p16INK4a ICC was 38.1% and was more frequent in T-lineage ALL. Overall survival (OS) and event-free survival (EFS) were significantly higher in patients with positive p16INK4a ICC than in patients with negative ICC (6 years OS, 90% versus 63%,P = .0014; 6 years EFS, 77.8% versus 55%,P = .0033). The p16INK4a ICC remained a significant prognostic factor within the subgroup of B-precursor ALL. Multivariate analysis showed that negative p16INK4a ICC was an independent prognostic factor for OS (relative risk [RR], 3.38;P = .02) and EFS (RR, 2.49; P = .018). Sequential study showed that p16INK4a expression remained stable during first relapse in most patients. These findings indicate that p16INK4a ICC is an independent factor of outcome in childhood ALL.

The p15INK4b and p16INK4aproteins are cell-cycle regulators involved in the inhibition of G1 phase progression. The p16INK4aand p15INK4b genes are homozygously deleted in many tumor types, including childhood acute lymphoblastic leukemia (ALL), but p16INK4a expression in leukemic cells may vary in the absence of gene deletion or point mutation.1-12 We recently reported the results of p16INK4a protein analysis by immunocytochemistry (ICC) in a limited cohort of adult ALL patients.13 The technique of p16INK4a ICC requires only bone marrow or blood smears; many samples can be processed in the same day, and this technique allows direct identification of leukemic cells, leading to an easier interpretation. We observed that negative p16INK4a ICC conferred an adverse outcome in adult ALL with standard-risk karyotype, but the small number of samples did not allow us to perform multivariate analysis. The low rate of high-risk karyotypic abnormalities in childhood ALL allowed us to expect a specific prognostic value of p16INK4a ICC in childhood ALL.

In a large, homogenously treated cohort of childhood ALL patients, we investigated the influence of negative p16INK4a ICC on overall survival (OS) and event-free survival (EFS) in univariate and multivariate analysis. Additionally, we sequentially investigated p16INK4a ICC in a cohort of those patients at relapse.

We analyzed p16INK4a gene expression by p16INK4a ICC in 126 childhood ALLs (89 B-precursor ALLs, 28 T-ALLs, and 9 acute undifferentiated leukemias). Patient bone marrow (n = 87) or blood (n = 39) smears were collected from May 1992 to December 2000. Median age was 4.9 years (range, 0.5-15). There were 73 males and 53 females. Of these patients, 80 were treated according to the European Organization for Research and Treatment of Cancer (EORTC) protocol 58881 between 1991 and December 1998, and 46 were treated by EORTC protocol 58951 between January 1998 and December 2000.14 The median observation time for all patients was 4.8 years (range, 1.4-9.2 years). There were 31 other patients studied by p16INK4a ICC in first relapse. Among them, 20 were also studied sequentially (14 at diagnosis and first relapse, 6 in first and second or third relapse).

Immunocytochemical detection of p16INK4a protein was performed with the immunoglobulin G1-κ mouse monoclonal antibody antihuman p16INK4a (clone DCS-50.1/H4) (Oncogene Research Products, Cambridge, United Kingdom), as previously described.13 The ICC reaction was performed with avidin-biotin-peroxidase technique by means of Vector reagents (Vector Laboratories, Burlingame, CA). Positive cells appeared with brownish granules. Samples were considered ICC-positive when more than 5% of cells showed p16INK4a protein, according to our previous study in adult ALL.13 

The chi-square and Fisher exact test were used for comparison between initial parameters. OS and EFS were estimated according to the Kaplan-Meier method. Events were defined as induction death, relapse, and death in complete remission (CR). Multivariate analysis was based on the Cox proportional hazards regression model. Statistical analyses were performed on SPSS 9.1 analysis software (SPSS, Chicago, IL).

As previously observed in adult ALL, a great variation in the percentage of p16INK4a ICC-positive cells was seen (median, 20%; range, 0-100).13 We found 51 samples (38.1%) to be p16INK4a ICC–negative. All patients achieved CR. No difference for sex, white blood cell count, presence of a bulky mass, central nervous system disease, hemoglobin level, chromosome 9p abnormalities, or karyotype could be observed between p16INK4a ICC–positive and p16INK4aICC–negative cases. However, positive p16INK4a ICC was found significantly more frequently in B-precursor ALL (70.7%) than in T-ALL (43%) (P = .006). Positive p16INK4a ICC ALL patients were also more likely to be older than age 9 years than were negative p16INK4a ICC ALL patients, but significance (P = .046) disappeared after stratification by phenotype (P = .205).

Increased sensitivity threshold for glucocorticoid-induced apoptosis induced by forced p16INKa expression in lymphoblastic leukemia cell line has been reported.15 However, we did not find significant association in our cohort of childhood ALL patients between p16INK4a ICC status and prednisone response at day 8 (P = .434). Hence, it remains unclear whether p16INK4a expression influences in vivo glucocorticoid-induced apoptosis of ALL cells.

Univariate analysis showed significantly better OS and EFS in patients with positive p16INK4a ICC at diagnosis (Figure1). OS estimates at 6 years for patients with or without positive p16INK4a ICC at diagnosis were 90% (SE = 3.8%) and 63% (SE = 8.7%), respectively (P = .0014, log-rank test). EFS estimates at 6 years were 78% (SE = 5.8%) and 54% (SE = 8.4%) for p16INK4aICC–positive and p16INK4a ICC–negative subgroups, respectively (P = .0033, log-rank test). When cutoff values of 0% and 10%, rather than 5%, for negative versus positive p16INK4a ICC were used, results were less significant. These findings indicate that, as in our previous analysis in adult ALL, 5% is a valid cutoff value for prognostic studies in ALL.13 Despite the association between negative ICC and T phenotype at diagnosis, p16INK4a ICC remained a significant prognostic factor in the subgroup of B-precursor ALLs for both OS (P = .0044) and EFS (P = .011) (Figure 1). This was not the case for T-ALL (OS, P = .23; EFS,P = .19).

Fig. 1.

Survival of childhood ALL patients according to their p16INK4a ICC status.

OS (panel A) and EFS (panel B) of the 126 childhood ALL patients. OS (panel C) and EFS (panel D) of childhood B-precursor ALL patients according to their p16INK4a ICC status.

Fig. 1.

Survival of childhood ALL patients according to their p16INK4a ICC status.

OS (panel A) and EFS (panel B) of the 126 childhood ALL patients. OS (panel C) and EFS (panel D) of childhood B-precursor ALL patients according to their p16INK4a ICC status.

Close modal

Final models of multivariate analysis showed that p16INK4a ICC remained an independent prognostic factor for both OS (P = .02) and EFS (P = .0188) in the whole cohort (Table1). However, karyotype remained the main prognostic factor for OS (relative risk = 3.92 versus 3.38). Incorporation of immunophenotype into the model did not modify the results. It has been suggested in previous studies that the significance of p16INK4a gene deletions would disappear within the subgroups of T-ALL and B-precursor ALL.16,17Our data show that T phenotype does not account for the poorer outcome of p16INK4a ICC–negative patients. However, the results of gene deletion studies and protein expression analyses by ICC may differ. Indeed, several studies have shown that p16INK4aprotein expression in leukemic cells is a complex phenomenon and can be altered not only by gene deletion but also by promoter methylation and other, unknown mechanisms.18-21 We also observed, both in adult ALL and in the present pediatric study, that a few samples of leukemic cells with no detectable p16INK4a protein at diagnosis showed p16INK4a expression at relapse (Table2, patient I120).13 These findings might explain why p16INK4a ICC provides prognostic information distinct from that derived throughp16INK4a gene deletion analysis. The lower proportion of high-risk karyotypes such as t(9;22)(q34;q11) in children may explain why p16INK4a ICC has a specific prognostic value in childhood ALL as compared with adult ALL.

Of the children with ALL analyzed at first relapse, 11 (35.5%) showed negative p16INK4a ICC (Table 2). Previous studies had shown that p16INK4a gene deletion could be acquired during evolution of the disease, but these findings have not been observed by other groups.3,5,16,22-24 In our study, ICC status became negative between diagnosis and first relapse in one patient (I119), and between first and second relapse in two others (I102, I103). Substantial variations of the percentage of p16INK4a-positive cells were also observed in 5 patients, suggesting that p16INK4a expression varies widely among both patients and varieties of disease progression. Thus, p16INK4a ICC should be tested when drugs targeting p16INK4a protein are tested.20 These findings indicate that absence of p16INK4a expression is an early phenomenon in the evolution of ALL. Some patients may lose p16INK4a expression during further relapse, but the impact of p16INK4a inactivation in those leukemic cells, which probably have accumulated numerous other gene alterations, remains to be determined.

Thus, the simple and reproducible p16INK4a ICC method should provide important prognostic information in large-scale prospective therapeutic studies.

Supported by the Ligue Contre le Cancer (Comité du Nord and Comité du Pas de Calais).

J.H.D. and M.F. contributed equally to this work as first authors.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

1
Quesnel
B
Preudhomme
C
Philippe
N
et al
p16 gene homozygous deletions in acute lymphoblastic leukemia.
Blood.
85
1995
657
663
2
Iolascon
A
Faienza
MF
Coppola
B
della Ragione
F
Santoro
N
Schettini
F
High frequency of homozygous deletions of CDK4I gene in childhood acute lymphoblastic leukaemia.
Br J Haematol.
91
1995
647
651
3
Ohnishi
H
Kawamura
M
Ida
K
et al
Homozygous deletions of p16/MTS1 gene are frequent but mutations are infrequent in childhood T-cell acute lymphoblastic leukemia.
Blood.
86
1995
1269
1275
4
Takeuchi
S
Bartram
CR
Seriu
T
et al
Analysis of a family of cyclin-dependent kinase inhibitors: p15/MTS2/INK4B, p16/MTS1/INK4A, and p18 genes in acute lymphoblastic leukemia of childhood.
Blood.
86
1995
755
760
5
Takeuchi
S
Bartram
CR
Wada
M
et al
Allelotype analysis of childhood acute lymphoblastic leukemia.
Cancer Res.
55
1995
53775382
6
Zhou
M
Gu
L
James
CD
et al
Homozygous deletions of the CDKN2 (MTS1/p16ink4) gene in cell lines established from children with acute lymphoblastic leukemia.
Leukemia.
9
1995
1159
1161
7
Iolascon
A
Faienza
MF
Coppola
B
della Ragione
F
Schettini
F
Biondi
A
Homozygous deletions of cyclin-dependent kinase inhibitor genes, p16(INK4A) and p18, in childhood T cell lineage acute lymphoblastic leukemias.
Leukemia.
10
1996
255
260
8
Nakao
M
Yokota
S
Kaneko
H
et al
Alterations of CDKN2 gene structure in childhood acute lymphoblastic leukemia: mutations of CDKN2 are observed preferentially in T lineage.
Leukemia.
10
1996
249
254
9
Ohnishi
H
Hanada
R
Horibe
K
et al
Homozygous deletions of p16/MTS1 and p15/MTS2 genes are frequent in t(1;19)–negative but not in t(1;19)–positive B precursor acute lymphoblastic leukemia in childhood.
Leukemia.
10
1996
1104
1110
10
Pui
CH
Acute leukemia in children.
Curr Opin Hematol.
3
1996
249
258
11
Kees
UR
Burton
PR
Lu
C
Baker
DL
Homozygous deletion of the p16/MTS1 gene in pediatric acute lymphoblastic leukemia is associated with unfavorable clinical outcome.
Blood.
89
1997
4161
4166
12
Rubnitz
JE
Behm
FG
Pui
CH
et al
Genetic studies of childhood acute lymphoblastic leukemia with emphasis on p16, MLL, and ETV6 gene abnormalities: results of St Jude Total Therapy Study XII.
Leukemia.
11
1997
1201
1206
13
Soenen
V
Lepelley
P
Gyan
E
et al
Prognostic significance of p16INK4a immunocytochemistry in adult ALL with standard risk karyotype.
Leukemia.
15
2001
1054
1059
14
Vilmer
E
Suciu
S
Ferster
A
et al
Long-term results of three randomized trials (58831, 58832, 58881) in childhood acute lymphoblastic leukemia: a CLCG-EORTC report. Children Leukemia Cooperative Group.
Leukemia.
14
2000
2257
2266
15
Ausserlechner
MJ
Obexer
P
Wiegers
GJ
Hartmann
BL
Geley
S
Kofler
R
The cell cycle inhibitor p16(INK4A) sensitizes lymphoblastic leukemia cells to apoptosis by physiologic glucocorticoid levels.
J Biol Chem.
276
2001
10984
10989
16
Carter
TL
Watt
PM
Kumar
R
et al
Hemizygous p16(INK4A) deletion in pediatric acute lymphoblastic leukemia predicts independent risk of relapse.
Blood.
97
2001
572
574
17
Graf Einsiedel
H
Taube
T
Hartmann
R
et al
Prognostic value of p16(INK4a) gene deletions in pediatric acute lymphoblastic leukemia.
Blood.
97
2001
4002
4004
18
Nakamura
M
Sugita
K
Inukai
T
et al
p16/MTS1/INK4A gene is frequently inactivated by hypermethylation in childhood acute lymphoblastic leukemia with 11q23 translocation.
Leukemia.
13
1999
884
890
19
Wong
IH
Ng
MH
Huang
DP
Lee
JC
Aberrant p15 promoter methylation in adult and childhood acute leukemias of nearly all morphologic subtypes: potential prognostic implications.
Blood.
95
2000
1942
1949
20
Omura-Minamisawa
M
Diccianni
MB
Batova
A
et al
In vitro sensitivity of T-cell lymphoblastic leukemia to UCN-01 (7-hydroxystaurosporine) is dependent on p16 protein status: a Pediatric Oncology Group study.
Cancer Res.
60
2000
6573
6576
21
Omura-Minamisawa
M
Diccianni
MB
Batova
A
et al
Universal inactivation of both p16 and p15 but not downstream components is an essential event in the pathogenesis of T-cell acute lymphoblastic leukemia.
Clin Cancer Res.
6
2000
1219
1228
22
Ogawa
S
Hangaishi
A
Miyawaki
S
et al
Loss of the cyclin-dependent kinase 4-inhibitor (p16; MTS1) gene is frequent in and highly specific to lymphoid tumors in primary human hematopoietic malignancies.
Blood.
86
1995
1548
1556
23
Maloney
KW
McGavran
L
Odom
LF
Hunger
SP
Acquisition of p16(INK4A) and p15(INK4B) gene abnormalities between initial diagnosis and relapse in children with acute lymphoblastic leukemia.
Blood.
93
1999
2380
2385
24
Carter
TL
Reaman
GH
Kees
UR
INK4A/ARF deletions are acquired at relapse in childhood acute lymphoblastic leukaemia: a paired study on 25 patients using real-time polymerase chain reaction.
Br J Haematol.
113
2001
323
328

Author notes

Bruno Quesnel, Service des Maladies du Sang, CHU Lille, 1 Place de Verdun, 59037, Lille, France; e-mail:bquesnel@nordnet.fr.

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