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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 Laı̈, Francis Bauters, Pierre Fenaux, and
Bruno Quesnel
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 ⴝ .0014; 6 years EFS, 77.8% versus
55%, P ⴝ .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 ⴝ .02) and EFS (RR, 2.49; P ⴝ .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.
(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-biotinperoxidase 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 KaplanMeier 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
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
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.
Submitted August 31, 2001; accepted November 30, 2001.
Supported by the Ligue Contre le Cancer (Comité du Nord and Comité du Pas
de Calais).
2620
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,
J.H.D. and M.F. contributed equally to this work as first authors.
Reprints: Bruno Quesnel, Service des Maladies du Sang, CHU Lille, 1 Place
de Verdun, 59037, Lille, France; e-mail: [email protected].
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.
© 2002 by The American Society of Hematology
BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
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BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
p16INK4a IS AN INDEPENDENT FACTOR IN CHILDHOOD ALL
2621
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).
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
Table 1. Multivariate Cox model analysis for overall and event-free survival of
the 126 childhood acute lymphoblastic leukemia patients
Univariate analysis
P
Variable
Multivariate analysis
P
RR
95% CI
OS
p16INK4aICC*
.0014
.0204
3.38
1.20-9.48
Karyotype†
.0032
.0227
3.92
1.21-12.72
Age‡
.0165
.12
2.30
0.81-6.61
WBC§
.0425
.0719
2.55
0.92-7.09
Prednisone response day 8㛳
.069
—
—
—
Hemoglobin¶
.964
—
—
—
Bulky tumor mass
.27
—
—
—
Immunophenotype#
.25
—
—
—
Sex
.8425
—
—
—
p16INK4aICC*
.0033
.0188
2.49
1.16-5.32
Karyotype†
.0609
—
—
—
Age‡
.0374
.0735
2.06
0.93-4.55
WBC§
.297
—
—
—
Prednisone response day 8㛳
.4
—
—
—
Hemoglobin¶
.73
—
—
—
Bulky tumor mass
.77
—
—
—
Immunophenotype#
.28
—
—
—
Sex
.52
—
—
—
EFS
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.
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
RR indicates relative risk; CI, confidence interval; OS, event-free survival; ICC,
immunocytochemistry; WBC, white blood count; EFS, event-free survival.
*Greater than 5% acute lymphoblastic leukemia cells; 5% or fewer acute
lymphoblastic leukemia cells.
†High risk, including t(9; 22), t(4; 11), t(1; 19) and hypodiploid; intermediate and
low risk.
‡Younger than 1 and older than 9 years; 1 year or older and 9 years or younger.
§Lower than 50 000/␮L; 50 000/␮L or higher.
㛳Circulating blasts at 1000/␮L or more; fewer than 1000/␮L circulating blasts.
¶More than 11 g/dL; lower than 11 g/dL.
#T-acute lymphoblastic leukemia; B-precursor acute lymphoblastic leukemia.
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2622
BLOOD, 1 APRIL 2002 䡠 VOLUME 99, NUMBER 7
DALLE et al
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.
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.
Table 2. Sequential analysis of p16INK4a immunocytochemistry in patients with
acute lymphoblastic leukemia
Positive leukemic cells by p16INK4a ICC, %
Patient no.
Immunophenotype
Diagnosis
First
relapse
Second
relapse
Third
relapse
I101
B
ND
80
95
100
I102
B
ND
90
0
—
I103
B
ND
100
0
—
I104
B
70
100
—
—
I105
B
100
25
—
—
I106
AUL
3
2
—
—
I107
T
0
1
0
0
I108
B
ND
100
ND
100
I109
T
75
40
I110
B
2
2
—
—
I111
B
16
100
—
—
I112
B
95
20
—
—
I113
AUL
100
30
—
—
I114
T
ND
2
0
—
I115
B
1
1
ND
—
I116
B
ND
1
0
—
I117
B
100
70
—
—
I118
T
0
1
—
—
I119
B
80
1
0
—
I120
B
16
—
—
I121
T
ND
40
—
—
I122
B
ND
35
—
—
I123
B
ND
25
—
—
I124
B
ND
50
—
—
I125
B
ND
100
—
—
I125
AUL
ND
100
—
—
I127
B
ND
1
—
—
I128
B
ND
2
—
—
I129
T
ND
1
—
—
I130
B
ND
35
—
—
I131
B
ND
7
—
—
2.5
—
ICC indicates immunocytochemistry; AUL, acute undifferentiated leukemia; ND,
not done.
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2002 99: 2620-2623
doi:10.1182/blood.V99.7.2620
p16INK4a immunocytochemical analysis is an independent prognostic factor
in childhood acute lymphoblastic leukemia
Jean Hughes Dalle, Martine Fournier, Brigitte Nelken, Françoise Mazingue, Jean-Luc Lai?, Francis Bauters,
Pierre Fenaux and Bruno Quesnel
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