p53 gene mutations are uncommon but p53 is commonly expressed in anaplastic large-cell lymphoma

Abstract

Anaplastic large-cell lymphoma (ALCL), as defined in the World Health Organization, is a heterogeneous category in which a subset of cases is associated with the t(2;5)(p23;q35) or variant translocations resulting in overexpression of anaplastic lymphoma kinase (ALK). p53 has not been assessed in currently defined subsets of ALCL tumors. In this study, we assessed ALK+ and ALK- ALCL tumors for p53 gene alterations using PCR, single-strand conformation polymorphism and direct sequencing methods. We also immunohistochemically assessed ALCL tumors for p53 expression. Three of 36 (8%) ALCL tumors (1/14 ALK+, 2/22 ALK-) with adequate DNA showed p53 gene mutations. By contrast, p53 was overexpressed in 36 of 55 (65%) ALCL tumors (16 ALK+, 20 ALK-). p21, a target of p53, was expressed in 15 of 31 (48%) ALCL tumors including seven of 15 (47%) p53-positive tumors. p21 expression in a subset of ALCL suggests the presence of functional p53 protein. Apoptotic rate was significantly higher in p53-positive than p53-negative tumors (mean 2.78 vs 0.91%, P = 0.0003). We conclude that the p53 gene is rarely mutated in ALK+ and ALK- ALCL tumors. Nevertheless, wild-type p53 gene product is commonly overexpressed in ALCL and may be functional in a subset of these tumors.

Full text

p53 gene mutations are uncommon but p53 is commonly expressed in anaplastic
large-cell lymphoma
GZ Rassidakis
1
, A Thomaides
1
, S Wang
1
, Y Jiang
2
, A Fourtouna
1
, R Lai
1
and LJ Medeiros
1
1
Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; and
2
Department of
Lymphoma-Myeloma, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
Anaplastic large-cell lymphoma (ALCL), as defined in the World
Health Organization, is a heterogeneous category in which a
subset of cases is associated with the t(2;5)(p23;q35) or variant
translocations resulting in overexpression of anaplastic lym-
phoma kinase (ALK). p53 has not been assessed in currently
defined subsets of ALCL tumors. In this study, we assessed
ALK þ and ALK ALCL tumors for p53 gene alterations using
PCR, single-strand conformation polymorphism and direct
sequencing methods. We also immunohistochemically as-
sessed ALCL tumors for p53 expression. Three of 36 (8%)
ALCL tumors (1/14 ALK þ , 2/22 ALK) with adequate DNA
showed p53 gene mutations. By contrast, p53 was over-
expressed in 36 of 55 (65%) ALCL tumors (16 ALK þ , 20 ALK).
p21, a target of p53, was expressed in 15 of 31 (48%) ALCL
tumors including seven of 15 (47%) p53-positive tumors. p21
expression in a subset of ALCL suggests the presence of
functional p53 protein. Apoptotic rate was significantly higher
in p53-positive than p53-negative tumors (mean 2.78 vs 0.91%,
P ¼ 0.0003). We conclude that the p53 gene is rarely mutated in
ALK þ and ALK ALCL tumors. Nevertheless, wild-type p53
gene product is commonly overexpressed in ALCL and may be
functional in a subset of these tumors.
Leukemia (2005) 19, 1663–1669. doi:10.1038/sj.leu.2403840;
published online 30 June 2005
Keywords: p53; MDM2; p21; anaplastic large-cell lymphoma; ALK;
apoptosis
Introduction
Anaplastic large-cell lymphoma (ALCL), as defined in the World
Health Organization (WHO) classification system, is a high-
grade lymphoma of T- or null-cell lineage with distinctive
histologic features. The neoplastic cells also strongly and
uniformly express CD30. In the WHO system, ALCL is
heterogeneous, with a substantial subset of tumors associated
with the t(2;5)(p23;q35) or variant translocations resulting in
overexpression of anaplastic lymphoma kinase (ALK).
1–3
Accu-
mulating evidence from in vitro studies in ALK þ ALCL has
shown activation of well-established oncogenic pathways
involved in cell proliferation and apoptosis.
4
Furthermore,
deregulation of cell cycle-controlling proteins and a high
proliferation rate have been reported previously in ALCL tumors
regardless of ALK status.
5–10
Nevertheless, the mechanisms of
ALCL oncogenesis are unclear.
The p53 tumor suppressor gene plays a crucial role in
response to various cellular stress conditions by inducing the
transcription of numerous genes controlling cell cycle arrest and
apoptotic cell death.
11
p53 is inactivated in many human
malignancies, and inactivating mutations of the p53 gene are the
most common genetic alteration in human cancer.
12,13
p53 also
can be inactivated by binding to oncogenic proteins.
14,15
In
non-Hodgkin’s lymphomas, previous studies have reported p53
overexpression in a subset of tumors, most frequently in high-
grade tumors.
16
However, the mechanisms of p53 overexpres-
sion are incompletely understood and functional status has been
rarely assessed in non-Hodgkin’s lymphomas.
16
p53 also has not
been systematically assessed in subsets of ALCL as they are
currently defined in the WHO classification.
In this study, we assessed ALCL tumors for p53 gene
mutations and expression. We also correlated the findings with
ALK status, apoptotic rate (AR), proliferation index and
expression of two proteins induced by wild-type p53 protein,
p21
WAF1
(p21) and MDM2.
14,17
Our results demonstrate that
p53 gene mutations are uncommon in both ALK þ and ALK
ALCL tumors. Nevertheless, variable levels of wild-type p53
protein are expressed in most ALCL tumors and p53 expression
correlates with AR. In addition, the expression of p21 in a subset
of ALCL tumors suggests that p53, at least in this subset, retains
its function.
Materials and methods
ALCL tumors
The study group included 55 ALCL tumors from previously
untreated patients accessioned at The University of Texas MD
Anderson Cancer Center. The diagnosis of ALCL was based on
morphologic and immunohistologic criteria as specified by the
WHO classification.
1
All ALCL tumors were uniformly positive
for CD30 and negative for CD15. None of the ALCLs expressed
B-cell antigens, including CD20, CD79a or PAX5/BSAP. A total
of 45 (82%) ALCL tumors expressed one or more T-cell or T-cell-
associated markers including CD3, CD5 or CD43. ALK was
assessed using the ALK-1 antibody (1:30; Dakocytomation,
Carpinteria, CA, USA) and was positive in 24 (44%) cases. Of
these ALK þ ALCL tumors, five (21%) showed only cytoplasmic
ALK-1 immunoreactivity, suggesting a variant abnormality
involving the alk locus (ie not NPM-ALK).
The median age of patients with ALK-positive tumors was 33
years compared with 54 years for patients with ALK-negative
tumors (P ¼ 0.0004 by Mann–Whitney U-test). All other
clinicopathologic parameters between the two groups were
comparable.
PCR-SSCP analysis
Genomic DNA was extracted from formalin-fixed, paraffin-
embedded tissues of 42 ALCL tumors using a QIAamp DNA
mini kit (QIAGEN, Valencia, CA, USA) according to the
Received 5 January 2005; accepted 10 May 2005; published online
30 June 2005
Correspondence: Dr LJ Medeiros, Department of Hematopathology,
The University of Texas MD Anderson Cancer Center, Box 72, 1515
Holcombe Boulevard, Houston, TX 77030, USA;
Fax: þ 1 713 745 0736; E-mail: jmedeiro@mail.mdanderson.org
This study was presented in part at the 93rd United States and
Canadian Academy of Pathology meeting, Vancouver, BC, Canada,
March 6–12, 2004
Leukemia (2005) 19, 1663–1669
& 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00
www.nature.com/leu

manufacturer’s protocol. We chose to analyze six exons of the
p53 gene, exons 4–9, as these exons have had virtually all p53
gene mutations reported in the literature.
18
The primers used for
exons 4–9 of the p53 gene and the size of PCR products are
shown in Table 1. We also chose the Pfu Turbo Hotstart DNA
Polymerase (Stratagene, La Jolla, CA, USA) for PCR because it is
believed to be the least mutagenic polymerase. The PCR
program included DNA denaturation at 951C (5 min), followed
by 35 cycles of 951C (30 s), 56–581C (30 s) and 721C (1 min),
and lastly extension at 721C (5 min). The presence and quality of
PCR products were tested using 1.5% agarose gels, UV light and
the Alpha-Imager system (Alpha Innotech Corporation, San
Leandro, CA, USA). The PCR products of 36 ALCL tumors (14
ALK þ , 22 ALK) that showed adequate amplification of all six
p53 exons were subsequently analyzed for possible p53 gene
mutations using a single-strand conformation polymorphism
(SSCP) method.
SSCP analysis was performed on the GenePhor electrophor-
esis unit (Amersham Biosciences, Piscataway, NJ, USA) using
appropriate gels, and PCR products were diluted in loading
buffer containing formamide, 1% xylene cyanol and 1%
bromophenol blue. Electrophoresis was performed using 80 V
for 20 min followed by 320 V for 60 min at 121C.
Subcloning and direct sequencing of PCR products
The PCR products of all ALCL tumors that showed mobility shifts
by SSCP were subcloned into pCR2.1-TOPO vector (Invitrogen,
Carlsbad, CA, USA) and sequenced. Sequencing was performed
using the GeneAmp PCR System 9600 (Perkin-Elmer, Norwalk,
CT, USA), fluorescently labeled M13 forward and reverse
primers and AmpliTaq-FS DNA polymerase (Perkin-Elmer,
Wellesley, MA, USA), according to the manufacturer’s directions.
Tissue microarray and immunohistochemical methods
Tissue sections of ALCL tumors, 4–5 mm thick, were cut from a
tissue microarray (43 tumors) or whole paraffin blocks (12
tumors). The tissue microarray included triplicate or quadruplet
tumor cores from 43 tumors and two reactive lymph nodes and
was constructed using a manual tissue arrayer (Beecher
Instruments, Silver Springs, MD, USA) as described previously.
19
The immunohistochemical methods used in this study were
described previously.
8
For all antibodies, heat-induced epitope
retrieval was performed. The panel of antibodies used in this
study included p53 (DO-7), p21 (SX118) (both from Dakocyto-
mation),
Ser15
phosphorylated p53,
Ser20
phosphorylated p53 (Cell
Signaling Technology, Beverly, MA, USA), MDM2 (N-20, Santa
Cruz Biotechnology, Santa Cruz, CA, USA) and MIB-1 (Ki-67,
Immunotech, Westbrook, ME, USA). The DO-7 antibody is
known to detect both wild-type and mutant p53. The slides were
incubated with the p53, p21 and MDM2 antibodies at 41C
overnight, and with MIB-1 at room temperature for 1 h. Reactive
small lymphocytes in all tissue sections served as internal
positive controls for p21 immunoreactivity. Slides cut from
formalin-fixed cell blocks of Karpas 299 cells served as positive
controls for p53, MDM2 and MIB-1.
Any nuclear staining of tumor cells was considered positive,
irrespective of intensity. Expression levels for p53, p21 and
MDM2 were determined by counting the percentage of positive
tumor cells and, for the purpose of statistical analysis, a 10%
cutoff was used to define positivity based on the distribution of
data (histogram shown in Results) and previously published
reports.
20
TUNEL assay
AR was assessed using a modified terminal deoxynucleotidyl
transferase (TdT)-mediated dUTP nick end-labeling (TUNEL)
assay and designated as the percentage of TUNEL-positive
tumor nuclei as described elsewhere.
8
Statistical analysis
The w
2
and Fisher’s exact tests were used to compare expression
of p53, p21 and MDM2 (positive vs negative) with various
parameters. The Mann–Whitney U-test and Kruskal–Wallis tests
were chosen for the nonparametric correlation of proliferation
index and AR between various groups. The Spearman R
correlation coefficient was used to assess correlations between
continuous variables. All computations were carried out
using the StatView statistical program (Abacus Concepts Inc.,
Berkeley, CA, USA).
Results
p53 gene mutations in ALK þ and ALK ALCL tumors
Out of 42 ALCL tumors, 36 (14 ALK þ and 22 ALK) had
adequate quality DNA to allow for assessment of p53 gene
mutations using PCR-SSCP methods. In six tumors, all exons of
Table 1 Primers used for p53 gene mutation analysis
p53 gene Primer sequences PCR product (bp)
Exon 4 Sense 5-TCC TCT GAC TGC TCT TTT CAC-3
0
348
Antisense 5
0
-TGA AGT CTC ATG GAA GCC AG-3
0
Exon 5 Sense 5
0
-CTT GTG CCC TGA CTT TCA ACT-3
0
266
Antisense 5
0
-CAA CCA GCC CTG TCG TCT-3
0
Exon 6 Sense 5
0
-TCT GAT TCC TCA CTG ATT GCT C-3
0
187
Antisense 5
0
-CCA CTG ACA ACC ACC CTT AAC-3
0
Exon 7 Sense 5
0
-TCA TCT TGG GCC TGT GTT ATC-3
0
169
Antisense 5
0
-AGT GTG CAG GGT GGC AAG-3
0
Exon 8 Sense 5
0
-AGG ACC TGA TTT CCT TAC TGC C-3
0
237
Antisense 5
0
-ATA ACT GCA CCC TTG GTC TCC-3
0
Exon 9 Sense 5
0
-ACT TTT ATC ACC TTT CCT TGC C-3
0
134
Antisense 5
0
-CAC TTG ATA AGA GGT CCC AAG AC-3
0
p53 gene in ALCL
GZ Rassidakis et al
1664
Leukemia

the p53 gene could not be amplified. Three of 36 (8%) ALCL
tumors (one ALK þ , two ALK) tested showed mobility shifts
indicating possible gene mutations and all three tumors over-
expressed p53 protein (Figure 1a and b). Subcloning and direct
sequencing of the PCR products revealed a frameshift mutation
and two point mutations in the three tumors (Figure 1c and d).
One of these tumors, an ALK-positive ALCL, showed a silent
point mutation (CAA-CAC) that resulted in no change of the
corresponding amino acid (Supplementary Table 1). A missense
point mutation of exon 7 (TGG-CGG) leading to an amino-
acid substitution (Thr-Ala) and a frameshift mutation were
found in the two ALK-negative ALCL cases (Supplementary
Table 1).
p53 expression in ALCL tumors
Using a 10% cutoff, p53 was overexpressed in 36 of 55 (65%)
ALCL, including 16 of 24 (67%) ALK þ and 20 of 31 (65%)
ALK tumors (P40.9, Fisher’s exact test; Figure 2a–c and
Table 2). In p53-positive tumors, the percentage of p53-positive
tumor cells ranged from 12 to 99% with a median of 70%
(Figure 3). In the three tumors with p53 gene mutations, virtually
all cells overexpressed p53 (Figure 2). However, p53 was also
overexpressed in most ALCL tumors without evidence of p53
gene mutation. In the unmutated cases, p53 was overexpressed
by a variable percentage of tumor cells (Figure 2). In many
tumors, the intensity of p53 staining was variable among the
tumor cells, ranging from weak to strong (Figure 2).
Phosphorylation of p53 on serine 15 and serine 20 was
assessed in a subset of 20 and 16 p53-positive ALCL tumors,
respectively, using phospho-specific antibodies. Using a 10%
cutoff, serine 15- and serine 20-phosphorylated p53 was
expressed in 13/20 (65%) and 8/16 (50%) tumors, respectively.
Staining intensity of phosphorylated p53 was generally weaker
compared with that of total p53 (Figure 2d and e). All ALCL
tumors positive for phosphorylated p53 were also positive for
MDM2.
Expression of MDM2 and p21 in ALCL tumors
Expression of MDM2, a p53-regulatory protein, was assessed in
43 ALCL tumors (17 ALK þ and 26 ALK). Of these tumors, 29
were p53-positive. MDM2 was expressed in 35 (81%) ALCL
(Figure 2d), 16 (94%) ALK þ and 19 (73%) ALK (P ¼ 0.1,
Fisher’s exact test). Among the MDM2-positive tumors, the
percentage of MDM2-positive tumor cells ranged from 15 to
100% with a median of 80%. MDM2 and p53 were frequently
coexpressed in ALCL (Table 2), as all but one p53-positive ALCL
tumor were also positive for MDM2 (P ¼ 0.0007, Fisher’s exact
test; Table 2). When analyzed as continuous variables, the
percentages of p53- and MDM2-positive tumor cells were
Figure 1 (a) Genomic DNA from ALCL tumors was amplified
using primers specific for exons 4–9 of the p53 gene. The quality of
PCR products was tested using 1.5% agarose gels and ultraviolet light,
which showed the presence of a single band corresponding to the
known size of each product. The PCR products (169 bp) of exon 7
amplification are shown. (b) PCR products with adequate amplifica-
tion of all six p53 exons were subsequently analyzed for possible p53
mutations using an SSCP method. Silver-stained gel from SSCP analysis
shows a PCR product (case #51, exon 7, arrow) with a mobility shift
indicating possible gene mutation. (c) PCR products with possible p53
gene mutations were subcloned using the pCR2.1 TOPO vector that
contains M13 sequences. Following transformation of Escherichia coli,
the positive colonies (white arrow) were selected based on their
resistance to ampicillin. The black arrow indicates the presence of the
vector DNA. The subcloned products containing the M13 were
subsequently sequenced. (d) Direct sequencing of the PCR products
(forward and reverse) using the GeneAmp PCR System 9600 confirmed
the presence of gene mutation. A nucleotide substitution (T-C) of
case # 51 is shown with the arrow.
p53 gene in ALCL
GZ Rassidakis et al
1665
Leukemia

significantly correlated (Spearman R ¼ 0.52, P ¼ 0.0008).
Among MDM2-positive tumors, p53 was absent in six tumors.
Absence of p53 and p21 expression was observed in three
MDM2-positive tumors.
Expression of p21 was assessed in a subset of 31 ALCL (12
ALK þ and 19 ALK). p21 was expressed in 15 (48%) ALCL
(Figure 4), eight (67%) ALK þ and seven (37%) ALK tumors.
The association between p21 and ALK expression was not
statistically significant (P ¼ 0.15, Fisher’s exact test). The
association between p21 and p53 expression was also not
statistically significant (Table 2). Of the 15 p21-positive ALCL
tumors, seven (47%) were also positive for p53.
Association of p53, MDM2 and p21 expression with
apoptotic rate and proliferation index
AR was available for 46 ALCL tumors.
8
In this group, the mean
AR was 2.8% in p53-positive ALCL compared with 0.9% in the
p53-negative ALCL (P ¼ 0.0003, Mann–Whitney test; Table 2
Figure 2 Expression of p53 and MDM2 in ALCL tumors. (a, b) p53 overexpression in cases of ALK þ (a, case #14) and an ALK (b, case #55)
ALCL. p53 gene was unmutated in both cases. (c) An ALK ALCL case that is p53-negative. (d, e) Expression of serine 15- (d) and serine 20-
(e) phosphorylated p53 in two cases of ALK þ ALCL tumors. (f) Expression of MDM2 in an ALK þ ALCL tumor.
Figure 3 Histogram showing the distribution of ALCL tumors
according to percentage of p53-positive tumor cells. Based on these
data, a cutoff of 10% was chosen to define a tumor as p53-positive.
x-axis, percentage of p53-positive tumor cells; y-axis, percentage
of ALCL tumors.
Table 2 Expression of p53, ALK, p21 and MDM2, and AR and
proliferation index in ALCL tumors
p53 expression P-value
p53+ P53
ALK expression 40.9
ALK+ 16/24 (67%) 8/24 (33%)
ALK 20/31 (65%) 11/31 (35%)
MDM2 expression 0.0007
MDM2+ 28/35 (80%) 7/35 (20%)
MDM2 1/8 (13%) 7/8 (87%)
p21 expression 40.9
p21+ 7/15 (47%) 8/15 (53%)
p21 9/16 (56%) 7/16 (44%)
Apoptotic rate (%)
(mean7s.d.)
2.7872.13 0.9170.64 0.0003
Proliferation index
(%) (mean7s.d.)
73.4719.1 69.4718.9 0.38
Figure 4 (a–d) p53 and p21 expression in ALK þ (a, b) and
ALK (c, d) ALCL tumors. (a–d, immunohistochemistry).
p53 gene in ALCL
GZ Rassidakis et al
1666
Leukemia

and Figure 5). As continuous variables, the percentage of p53-
positive tumor cells and the AR were positively correlated
(Spearman R ¼ 0.32, P ¼ 0.03). AR also correlated with MDM2
expression. The mean AR was 2.5% in MDM2-positive ALCL
compared with 0.8% in MDM2-negative ALCL (P ¼ 0.0019,
Mann–Whitney test). In addition, the percentage of MDM2-
positive tumor cells positively correlated with AR (Spearman
R ¼ 0.34, P ¼ 0.04). No significant association between p21
expression and AR was observed.
The mean proliferation index was 73.4% in p53-positive
ALCL compared with 69.4% in p53-negative ALCL (P ¼ 0.38,
Mann–Whitney test; Table 2). Proliferation index did not
correlate with expression of MDM2 or p21.
Clinical outcome
Complete follow-up data were available for 43 of 51 patients
with ALCL (18 ALK þ , 25 ALK) analyzed for p53 expression.
The median follow-up period was 36 months (2–159 months).
For the entire group, 5-year progression-free survival (PFS) was
57% for patients with p53-positive tumors compared with 80%
for patients with p53-negative tumors (P ¼ 0.2 by log rank).
Survival analysis was also performed separately for the ALK-
positive and ALK-negative groups. For 18 patients with
ALK-positive ALCL, 5-year PFS was 64% for patients with
p53-positive tumors compared with 100% for patients with p53-
negative tumors (P ¼ 0.12 by log rank). For 25 patients with ALK-
negative ALCL, 5-year PFS was 66% for patients with
p53-positive tumors compared with 71% for patients with
p53-negative tumors (P ¼ 0.6 by log rank). Similarly, overall
survival at 5 years did not differ significantly between patients
with p53-positive or p53-negative ALCL tumors.
Discussion
A large body of evidence accumulated over the past two
decades suggests that alterations of the p53 pathway play a
central role in tumorigenesis.
11
The p53 tumor suppressor gene
can be inactivated by a number of mechanisms including p53
gene mutation, the most frequent genetic alteration in human
cancer,
12
or defects in cell pathways that regulate p53 levels or
inhibit p53 function.
11
Although p53 gene alterations, most
commonly point mutations, are detected in more than 50% of
human cancers, their frequency is substantially lower in
lymphoid neoplasms compared with epithelial tumors.
16
The
presence of p53 gene alterations has been associated with
clinically aggressive lymphomas, usually of high cytologic
grade, or progression of low-grade non-Hodgkin’s lymphomas
to high-grade neoplasms.
16
However, the presence or absence
of p53 gene mutations has not been assessed in a group of ALCL
tumors, incorporating ALK status and classified using the criteria
of the current WHO classification system.
In this study, 8% of ALCL tumors (7% ALK þ , 9% ALK)
carried a mutated p53 gene and overexpressed p53 protein. In
these tumors, virtually all cells overexpressed p53 (Figure 1).
However, p53 was also overexpressed in most ALCL tumors
without evidence of p53 gene mutation. By contrast, in
unmutated cases, p53 was overexpressed by a variable
percentage of tumor cells (Figure 2). To our knowledge, only
one earlier study by Cesarman et al
21
has assessed p53 in ALCL.
This study included 17 cases classified as ALCL using out-of-
date classification criteria. In these 17 cases, ALK status was not
available, and this study included B-cell cases; the latter are no
longer considered ALCL.
1
Nevertheless, only one (6%) tumor in
that study was found to carry a mutated p53 gene.
21
Thus, our
results support the conclusion by Cesarman et al
21
that p53 gene
mutation is uncommon in ALCL, and extend their work by
showing that p53 gene mutation is uncommon in both ALK þ
and ALK ALCL.
Other studies assessing p53 gene and p53 expression in T-cell
lymphomas of various types have been performed, although p53
function has not been investigated. Matsushima et al
22
detected
p53 gene mutations in 9% of tumors although p53 was
overexpressed in 50%. Similarly, Quintanilla-Martinez et al
20
reported p53 gene mutations in 15% of a series of extranodal T/
NK lymphomas of nasal type whereas p53 was expressed in
60%. Others have not identified p53 gene mutations in
peripheral T-cell lymphomas.
23
The high frequency of p53
expression in high-grade T-cell lymphomas including ALCL, and
the low frequency of p53 gene mutations suggest that mechan-
isms other than p53 gene alterations stabilize the wild-type p53
gene product. The presence of wild-type p53 in most ALCL
tumors is further supported by our results showing that a p53-
induced gene, p21,
24
is expressed in a subset of ALCL tumors
(Table 2 and Figure 4), suggesting that p53 protein is capable of
inducing expression of target genes. As p53 gene mutations are
uncommon in ALCL, the mechanism of p53 overexpression in
these tumors is uncertain.
It is known that p53 functions as a transcriptional factor
through binding of its activation domain to DNA specific sites
inducing expression of multiple genes.
11
Thus, p53 can be
inactivated by binding to oncogenic proteins, such as MDM2,
that conceal the activation domain of p53.
14,15
In this study, p53
and MDM-2 expression levels were significantly correlated in
ALCL tumors (Table 2). It is likely that, at least in a subset of
these tumors, increased levels of MDM2 can inhibit p53
transcriptional activity resulting in cell cycle deregulation in
ALCL. MDM2 inhibition of p53 transcriptional activity is further
indicated by the absence of p21 in a subset of ALCL cases that
we assessed. Nevertheless, the detection of p21 in a subset of
tumors suggests that p53 is functional in some cases.
Apart from its p53 inhibitory activity, MDM2 is also an E3-
ubiquitin ligase that targets p53 for degradation through the
ubiquitin–proteasome system. Therefore, one would expect that
high MDM2 levels would lead to increased degradation of
p53.
14
There are several possible explanations for the inability of
MDM2 to degrade p53 in these tumors. One possibility is that
phosphorylation of p53 by ATM on serine 15,
25,26
or by Chk2 on
serine 20,
27,28
can impair the ability of MDM2 to bind p53
Figure 5 Box plots showing higher AR in p53-positive ALCL
tumors than in p53-negative ALCL tumors (P ¼ 0.0003).
p53 gene in ALCL
GZ Rassidakis et al
1667
Leukemia

resulting in p53 stabilization and increased p53-dependent
transactivation. In this study, we found that phosphorylated p53
on serine 15 and 20 is expressed in 65 and 50%, respectively, of
the ALCL tumors assessed; all of these phosphorylated p53-
positive tumors expressed MDM2 (Figure 2). Therefore, phos-
phorylation of p53 ALCL may explain, at least in part, p53
stabilization in the presence of MDM2 in ALCL. Other
possibilities include the presence of splice variants of MDM2
transcripts that may not contain the p53 binding site,
29
functional inactivation of MDM2 through phosphorylation
by ATM kinase on serine 395 or sequestration of MDM2 in
the nucleolus by p14
ARF
or promyelocytic leukemia (PML)
proteins.
30–32
AR was significantly higher in p53-positive than p53-negative
ALCL tumors in this study (Table 2). Apart from its cell cycle
regulatory function, p53 normally promotes apoptosis by
inducing expression of numerous apoptotic genes, including
genes involved in both the intrinsic and extrinsic apoptotic
pathways.
11
This is additional evidence suggesting that p53 in
ALCL is, at least in part, functional. In 35 ALCL tumors with
available data from a previous study,
8
p53 expression correlated
with BAX, a p53-induced proapoptotic protein (data not shown),
suggesting that wild-type p53 may induce expression of BAX,
and probably other apoptotic genes, resulting in a higher AR in
ALCL tumors.
In conclusion, p53 gene mutations are uncommon (o10%) in
ALK þ and ALK ALCL. Nevertheless, p53 is frequently
expressed in ALCL tumors at a variable level and appears to
be capable of inducing expression of target genes, such as p21,
in a subset of cases. p53 is serine phosphorylated in a subset of
ALCL tumors and, in part, phosphorylation may protect p53
from binding to MDM2 and subsequent degradation. Whether
or not high expression levels of MDM2 found in most cases of
ALCL might inhibit p53 transcriptional activity resulting in cell
cycle deregulation merits further investigation. The regulatory
p53–MDM2 system is being currently used as a target for
investigational therapy.
33
These approaches may result in
release of fully functional p53 capable of inducing cell cycle
arrest and apoptosis in tumor cells.
Acknowledgements
Dr GZ Rassidakis is a recipient of an Odyssey Program Special
Fellowship from The University of Texas MD Anderson Cancer
Center.
Supplementary Information
Supplementary Information accompanies the paper on the
Leukemia website (http://www.nature.com/leu).
References
1 Delsol G, Ralfkiaer E, Stein H, Wright D, Jaffe ES. Anaplastic large
cell lymphoma. In: Jaffe ES, Harris NL, Stein H, Vardiman JW (eds).
World Health Organization Classification of Tumours. Pathology
and Genetics of Tumors of Haematopoietic and Lymphoid Tissues.
Lyon, France: IARC Press, 2001, pp 230–235.
2 Morris SW, Kirstein MN, Valentine MB, Dittmer KG, Shapiro DN,
Saltman DL et al. Fusion of a kinase gene, ALK, to a nucleolar
protein gene, NPM, in non-Hodgkin’s lymphoma. Science 1994;
263: 1281–1284.
3 Morris SW, Naeve C, Mathew P, James PL, Kirstein MN, Cui X
et al. ALK, the chromosome 2 gene locus altered by the t(2; 5) in
non-Hodgkin’s lymphoma, encodes a novel neural receptor
tyrosine kinase that is highly related to leukocyte tyrosine kinase
(LTK). Oncogene 1997; 14: 2175–2188.
4 Duyster J, Bai RY, Morris SW. Translocations involving anaplastic
lymphoma kinase (ALK). Oncogene 2001; 20: 5623–5637.
5 Inghirami G, Macri L, Cesarman E, Chadburn A, Zhong J, Knowles
DM. Molecular characterization of CD30+ anaplastic large-cell
lymphoma: high frequency of c-myc proto-oncogene activation.
Blood 1994; 83: 3581–3590.
6 Chilosi M, Doglioni C, Magalini A, Inghirami G, Krampera M,
Nadali G et al. p21/WAF1 cyclin-kinase inhibitor expression in
non-Hodgkin’s lymphomas: a potential marker of p53 tumor-
suppressor gene function. Blood 1996; 88: 4012–4020.
7 Chilosi M, Doglioni C, Yan Z, Lestani M, Menestrina F, Sorio C
et al. Differential expression of cyclin-dependent kinase 6 in
cortical thymocytes and T-cell lymphoblastic lymphoma/leukemia.
Am J Pathol 1998; 152: 209–217.
8 Rassidakis GZ, Sarris AH, Herling M, Ford RJ, Cabanillas F,
McDonnell TJ et al. Differential expression of BCL-2 family
proteins in ALK-positive and ALK-negative anaplastic large
cell lymphoma of T/null-cell lineage. Am J Pathol 2001; 159:
527–535.
9 Rassidakis GZ, Claret FX, Lai R, Zhang Q, Sarris AH, McDonnell TJ
et al. Expression of p27(Kip1) and c-Jun activation binding
protein 1 are inversely correlated in systemic anaplastic large cell
lymphoma. Clin Cancer Res 2003; 9: 1121–1128.
10 Rassidakis GZ, Lai R, Herling M, Cromwell C, Schmitt-Graeff A,
Medeiros LJ. Retinoblastoma protein is frequently absent or
phosphorylated in anaplastic large-cell lymphoma. Am J Pathol
2004; 164: 2259–2267.
11 Vousden KH, Lu X. Live or let die: the cell’s response to p53. Nat
Rev Cancer 2002; 2: 594–604.
12 Levine AJ, Momand J, Finlay CA. The p53 tumour suppressor gene.
Nature 1991; 351: 453–456.
13 Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations
in human cancers. Science 1991; 253: 49–53.
14 Wu X, Bayle JH, Olson D, Levine AJ. The p53–mdm-2
autoregulatory feedback loop. Genes Dev 1993; 7: 1126–1132.
15 Oliner JD, Pietenpol JA, Thiagalingam S, Gyuris J, Kinzler KW,
Vogelstein B. Oncoprotein MDM2 conceals the activation domain
of tumour suppressor p53. Nature 1993; 362: 857–860.
16 Sanchez-Beato M, Sanchez-Aguilera A, Piris MA. Cell cycle
deregulation in B-cell lymphomas. Blood 2003; 101: 1220–1235.
17 Sherr CJ, Roberts JM. CDK inhibitors: positive and negative
regulators of G1-phase progression. Genes Dev 1999; 13:
1501–1512.
18 Soussi T, Beroud C. Assessing TP53 status in human tumours to
evaluate clinical outcome. Nat Rev Cancer 2001; 1: 233–240.
19 Rassidakis GZ, Jones D, Thomaides A, Sen F, Lai R, Cabanillas F
et al. Apoptotic rate in peripheral T-cell lymphomas: a study using
a tissue microarray with validation on full tissue sections. Am J Clin
Pathol 2002; 118: 328–334.
20 Quintanilla-Martinez L, Kremer M, Keller G, Nathrath M,
Gamboa-Dominguez A, Meneses A et al. p53 Mutations in nasal
natural killer/T-cell lymphoma from Mexico: association with large
cell morphology and advanced disease. Am J Pathol 2001; 159:
2095–2105.
21 Cesarman E, Inghirami G, Chadburn A, Knowles DM. High levels
of p53 protein expression do not correlate with p53 gene
mutations in anaplastic large cell lymphoma. Am J Pathol 1993;
143: 845–856.
22 Matsushima AY, Cesarman E, Chadburn A, Knowles DM. Post-
thymic T cell lymphomas frequently overexpress p53 protein but
infrequently exhibit p53 gene mutations. Am J Pathol 1994; 144:
573–584.
23 Pescarmona E, Pignoloni P, Santangelo C, Naso G, Realacci M,
Cela O et al. Expression of p53 and retinoblastoma gene in
high-grade nodal peripheral T-cell lymphomas: immuno-
histochemical and molecular findings suggesting different patho-
genetic pathways and possible clinical implications. J Pathol 1999;
188: 400–406.
24 Villuendas R, Pezzella F, Gatter K, Algara P, Sanchez-Beato M,
Martinez P et al. p21WAF1/CIP1 and MDM2 expression in non-
Hodgkin’s lymphoma and their relationship to p53 status: a p53+,
MDM2, p21 immunophenotype associated with missense p53
mutations. J Pathol 1997; 181: 51–61.
p53 gene in ALCL
GZ Rassidakis et al
1668
Leukemia

25 Shieh SY, Ikeda M, Taya Y, Prives C. DNA damage-induced
phosphorylation of p53 alleviates inhibition by MDM2. Cell 1997;
91: 325–334.
26 Canman CE, Lim DS, Cimprich KA, Taya Y, Tamai K,
Sakaguchi K et al. Activation of the ATM kinase by ionizing
radiation and phosphorylation of p53. Science 1998; 281:
1677–1679.
27 Chehab NH, Malikzay A, Appel M, Halazonetis TD. Chk2/hCds1
functions as a DNA damage checkpoint in G(1) by stabilizing p53.
Genes Dev 2000; 14: 278–288.
28 Hirao A, Kong YY, Matsuoka S, Wakeham A, Ruland J, Yoshida H
et al. DNA damage-induced activation of p53 by the checkpoint
kinase Chk2. Science 2000; 287: 1824–1827.
29 Bartel F, Taubert H, Harris LC. Alternative and aberrant splicing of
MDM2 mRNA in human cancer. Cancer Cell 2002; 2: 9–15.
30 Maya R, Balass M, Kim ST, Shkedy D, Leal JF, Shifman O et al.
ATM-dependent phosphorylation of Mdm2 on serine 395: role in
p53 activation by DNA damage. Genes Dev 2001; 15: 1067–1077.
31 Tao W, Levine AJ. P19(ARF) stabilizes p53 by blocking nucleo-
cytoplasmic shuttling of Mdm2. Proc Natl Acad Sci USA 1999; 96:
6937–6941.
32 Bernardi R, Scaglioni PP, Bergmann S, Horn HF, Vousden KH,
Pandolfi PP. PML regulates p53 stability by sequestering Mdm2 to
the nucleolus. Nat Cell Biol 2004; 6: 665–672.
33 Chene P. Inhibiting the p53–MDM2 interaction: an important
target for cancer therapy. Nat Rev Cancer 2003; 3: 102–109.
p53 gene in ALCL
GZ Rassidakis et al
1669
Leukemia