Blood, 1 April 2002, Vol. 99, No. 7, pp. 2620-2623
BRIEF REPORT
p16INK4a immunocytochemical analysis is an
independent prognostic factor in childhood acute lymphoblastic leukemia
Jean Hughes Dalle,
Martine Fournier,
Brigitte Nelken,
Françoise Mazingue,
Jean-Luc Laï,
Francis Bauters,
Pierre Fenaux, and
Bruno Quesnel
From Service des Maladies du Sang, Centre Hospitalier
et Universitaire Lille; 2-Service d'Hématologie
Pédiatrique, Centre Hospitalier et Universitaire Lille;
Laboratoire d'Hématologie, Centre Hospitalier et Universitaire
Lille; Laboratoire de Cytogénétique, Centre Hospitalier
et Universitaire Lille; Unité INSERM 524, Institut de Recherche
sur le Cancer de Lille, Lille, France.
 |
Abstract |
We investigated the prognostic value of p16INK4a
immunocytochemistry (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.
(Blood. 2002;99:2620-2623)
© 2002 by The American Society of Hematology.
| |
Introduction |
The p15INK4b and p16INK4a
proteins are cell-cycle regulators involved in the inhibition of
G1 phase progression. The p16INK4a
and 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.
| |
Study design |
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-
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).
| |
Results and discussion |
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 p16INK4a
ICC-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 (Figure
1). 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 p16INK4a
ICC-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).
View larger version (16K):
[in this window]
[in a new window]
| Figure 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.
|
|
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 (Table
1). 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.16,17
Our 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 p16INK4a
protein 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 (Table
2, patient I120).13 These
findings might explain why p16INK4a ICC provides prognostic
information distinct from that derived through
p16INK4a 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.
View this table:
[in this window]
[in a new window]
|
Table 1.
Multivariate Cox model analysis for overall and event-free
survival of the 126 childhood acute lymphoblastic leukemia patients
|
|
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.
| |
Footnotes |
Submitted August 31, 2001; accepted November 30, 2001.
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.
Reprints: Bruno Quesnel, Service des Maladies du Sang, CHU
Lille, 1 Place de Verdun, 59037, Lille, France; e-mail:
bquesnel{at}nordnet.fr.
| |
References |
1.
Quesnel B, Preudhomme C, Philippe N, et al.
p16 gene homozygous deletions in acute lymphoblastic leukemia.
Blood.
1995;85:657-663[Abstract/Free Full Text].
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.
1995;91:647-651[Medline]
[Order article via Infotrieve].
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.
1995;86:1269-1275[Abstract/Free Full Text].
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.
1995;86:755-760[Abstract/Free Full Text].
5.
Takeuchi S, Bartram CR, Wada M, et al.
Allelotype analysis of childhood acute lymphoblastic leukemia.
Cancer Res.
1995;55:53775382.
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.
1995;9:1159-1161[Medline]
[Order article via Infotrieve].
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.
1996;10:255-260[Medline]
[Order article via Infotrieve].
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.
1996;10:249-254[Medline]
[Order article via Infotrieve].
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.
1996;10:1104-1110[Medline]
[Order article via Infotrieve].
10.
Pui CH.
Acute leukemia in children.
Curr Opin Hematol.
1996;3:249-258[Medline]
[Order article via Infotrieve].
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.
1997;89:4161-4166[Abstract/Free Full Text].
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.
1997;11:1201-1206[CrossRef][Medline]
[Order article via Infotrieve].
13.
Soenen V, Lepelley P, Gyan E, et al.
Prognostic significance of p16INK4a immunocytochemistry in adult ALL with standard risk karyotype.
Leukemia.
2001;15:1054-1059[Medline]
[Order article via Infotrieve].
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.
2000;14:2257-2266[CrossRef][Medline]
[Order article via Infotrieve].
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.
2001;276:10984-10989[Abstract/Free Full Text].
16.
Carter TL, Watt PM, Kumar R, et al.
Hemizygous p16(INK4A) deletion in pediatric acute lymphoblastic leukemia predicts independent risk of relapse.
Blood.
2001;97:572-574[Abstract/Free Full Text].
17.
Graf Einsiedel H, Taube T, Hartmann R, et al.
Prognostic value of p16(INK4a) gene deletions in pediatric acute lymphoblastic leukemia.
Blood.
2001;97:4002-4004[Free Full Text].
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.
1999;13:884-890[CrossRef][Medline]
[Order article via Infotrieve].
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.
2000;95:1942-1949[Abstract/Free Full Text].
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.
2000;60:6573-6576[Abstract/Free Full Text].
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.
2000;6:1219-1228[Abstract/Free Full Text].
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.
1995;86:1548-1556[Abstract/Free Full Text].
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.
1999;93:2380-2385[Abstract/Free Full Text].
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.
2001;113:323-328[CrossRef][Medline]
[Order article via Infotrieve].
Top
Abstract
Introduction
