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From the Laboratory of Pathology*
and
Department of Hematology,
Hospital
Clínic, Institut d’Investigacions Biomediques August Pi i
Sunyer, University of Barcelona, Barcelona, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and 2 of the
INK4a/ARF locus and p53 gene aberrations
in 97 tumors previously characterized for
p16INK4a alterations. p53 alterations were
detected in four of 51 (8%) indolent lymphomas but in 15 of 46 (33%)
aggressive tumors. Inactivation of p14ARF
was always associated with p16INK4a
alterations. Exon 1ß was concomitantly deleted with exon 1 and 2
in eight tumors. One additional lymphoblastic lymphoma showed deletion
of exon 1 and 2 but retained exon 1ß. No mutations were detected
in exon 1 and 1ß in any case. Two of the three mutations detected
in exon 2 caused a nonsense mutation in the
p16INK4a reading frame and a missense
mutation in the ARF reading frame involving the nucleolar transport
domain of the protein. The third mutation was a missense mutation in
the p16INK4a reading frame, but it
was outside the coding region of p14ARF.
Aggressive lymphomas with p14ARF
inactivation and p53 wild type showed a significantly
lower p53 protein expression than tumors with no alteration in any of
these genes. In this series of tumors, inactivation of the
INK4a/ARF locus mainly occurred in tumors with a wild-type
p53 gene because only two lymphomas showed simultaneous
aberrations in these genes. Tumors with concomitant alterations of
p16INK4a and
p14ARF/p53 genes seem to exhibit a worse
clinical behavior than lymphomas with no alterations or isolated
inactivation of any of these genes. These findings indicate that
p14ARF genetic alterations occur in a subset
of aggressive NHLs, but they are always associated with
p16INK4a aberrations. Concomitant disruption
of p16INK4a and
p14ARF/p53 regulatory pathways may have a
cooperative effect in the progression of these tumors.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The recent understanding of the peculiar genomic organization of the
INK4a locus has provided strong evidence for a common
genetic link between the p53 and
p16INK4a/Rb pathways. In addition to
p16INK4a, this locus codifies for a second
transcript, called p14ARF in humans and
p19ARF in mice (ARF for alternative
reading frame), which is derived from a different exon 1 (exon 1ß)
centromeric to the first exon of p16INK4a
(exon 1).6,7
Exon 1ß is spliced to the same exon 2
used by exon 1 but produces a different open reading frame coding
for a protein that is structurally different from
p16INK4a. p14/p19ARF
overexpression induces a cell cycle arrest in both
G1 and G2 without direct
interaction with cyclin-dependent kinases (CDKs).7
However, its inhibitory effect is dependent on p53, indicating that
p14/p19ARF may act as an upstream regulator of
p53 function.7,8
Recent studies have shown that
p14/p19ARF interacts physically with MDM2 and
stabilizes p53 protein in the nucleus of the cell by blocking its
cytoplasmic transport and MDM2-mediated degradation.9-14
These observations have suggested that inactivation of
p14/19ARF may lead to an increased degradation of
p53, and, consequently, it may function as an alternative mechanism to
p53 mutations in the pathogenesis of tumors. Therefore,
genetic alterations of the INK4a/ARF locus may impair
both p14ARF/p53 and
p16INK4a/Rb pathways, providing a selective
advantage in the development and progression of tumors.
Different studies have suggested a possible role of p14/19ARF in tumorigenesis, independently of the inactivation of p16INK4a. Knockout mice lacking exon 1ß develop a spectrum of malignant neoplasms similar to those described in animals deficient for INK4a exon 2,8,15,16 as well as to the tumors described in p53 null mice.17 In addition, selective losses of exon 1ß have been observed in other experimental murine lymphomas,18 occasional human melanomas,19 and acute lymphoblastic leukemias.20,21 However, the presence of p14ARF alterations in human NHLs and their possible alternative or coordinate inactivation with p16INK4a and p53 genes are not well known. To determine the potential role of p14ARF in the pathogenesis of NHLs and its relationship with p16INK4a and p53 inactivation, we have investigated the possible p14ARF and p53 gene alterations in a large series of NHLs previously characterized for p16INK4a abnormalities.22 Our results indicate that p14ARF alterations in human NHLs are always associated with p16INK4a aberrations and occur mainly in tumors with wild-type p53 gene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tumor specimens from 97 NHLs were obtained from the Department of
Pathology of the Hospital Clinic, University of Barcelona, on the basis
of the availability of frozen samples for molecular studies. These
cases were classified according to the Revised European-American
Classification of Lymphoid Neoplasms.23
The tumors were
grouped into indolent and aggressive categories for statistical
purposes. Indolent tumors included 18 chronic lymphocytic leukemias
(CLLs), two hairy cell leukemias (HCLs), 14 follicular lymphomas (FLs),
and 17 typical mantle cell lymphomas (MCLs). Aggressive NHLs comprised
seven large cell lymphomas evolved from CLLs (Richter’s Syndrome), 10
diffuse large-cell lymphomas (LCLs) transformed from FLs, nine blastoid
variants of MCLs, 15 de novo diffuse B-cell LCLs, two
lymphoblastic lymphomas (LBLs), one Burkitt’s lymphoma (BL), and two
anaplastic large cell lymphomas (ALCLs). All of these tumors had
previously been analyzed for p16INK4a exon
2 deletions, exon 1 and 2 mutations, and hypermethylation of the
gene.22
Sixteen MCLs had been included in a previous study
of exon 5–9 mutations of the p53 gene.24
p53 Gene Analysis
Mutational analysis of exons 5–9 was performed using single-stranded conformation polymorphism (SSCP) and direct sequencing as previously described.24 In addition, exon 4 was amplified by polymerase chain reaction (PCR), using the primers 5'-TTTTCACCCATCTACAGTCCC-3' and 5'-CTCAGGGCAACTGACCGTG-3'. Amplifications were performed with 0.5 µg of genomic DNA, 1.75 U of Taq polymerase (Boehringer-Mannheim, Mannheim, Germany), 0.5 µmol/L of each primer, 100 µmol/L of deoxynucleoside triphosphate, and PCR buffer in a final volume of 50 µl. The reaction was performed for 35 cycles at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute. Two microliters of amplified products were diluted sixfold in formamide dye loading buffer, incubated for 3 minutes at 95°C, and immediately cooled on ice. Twenty microliters was electrophoresed at 130 V for 20 hours at room temperature on a 12% nondenaturing polyacrylamide gel and at 4°C on a 10% nondenaturing polyacrylamide gel. The gels were developed using a previously described silver staining procedure.25
To confirm the possible p53 gene mutations, the samples with
altered migration in the SSCP analysis were sequenced using a
commercial cycle sequencing kit (Perkin Elmer, Branchburg, NY) and
[-33P]deoxyadenosine triphosphate. A total
of 0.5 µl of the p53 gene PCR product was used as a
template for sequencing. The primers and conditions used for exon 5–9
sequencing were described elsewhere.24
The primers for
exon 4 were the same as those used in the PCR reaction described above.
Amplification conditions included an annealing temperature of 68°C.
Southern blot analysis was performed with genomic DNA from frozen material in 55 cases, using proteinase K/RNase treatment and phenol-chloroform extraction. DNA from each case (10 µg) was digested with EcoRI and HindIII, separated on 0.8% agarose gels, and transferred to Quiabrane Nylon Plus (Quiagen, Hilden, Germany). The membranes were prehybridized, hybridized with a cDNA p53 probe, and washed as previously described.26
INK4a Locus Analysis
Deletions of exon 1, exon 1ß, and exon 2 of the
INK4a/ARF locus were analyzed by multiplex PCR, using
the amplification of the ß-actin gene as an internal
control. The primers used for ß-actin were
5'-TCCTTAATGTCACGCACGATTTC-3' and 5'-GTCACCCACACTGTGCCCATCTA-3', the
primers for exon 1ß were 5'-GGAGGCGGCGAGAACAT-3' and
5'-GGGCCTTTCCTACCTG-GTCTT-3', the primers for exon 1 were
5'-GAAGAAAG-AGGAGGGGCTG-3' and
5'-GCGCTACCTGATTCC-AA-TTC-3', and the primers for exon 2 were
5'-CTCTACA-CAAGCTTCCTTTC-3' and 5'-CGGGCTGAACTTTCTGT-GCTGG3'.
We used a "touchdown" PCR strategy for the amplification.
Conditions were one cycle at 95°C for 5 minutes; four cycles at
94°C for 45 seconds, at 68°C for 1 minute, and at 72°C for 1
minute; four cycles with an annealing temperature of 67°C; and 30
cycles with an annealing temperature of 66°C. PCR products were
resolved on a 2% agarose gel. DNA from laryngeal tumors previously
analyzed for homozygous deletions by microsatellite amplification were
used as positive and negative controls.27
Homozygous
deletions were considered in our samples when the normalized intensity
was less than 60% of the intensity of a normal control case.
Exon 1ß mutational analysis was also performed by PCR-SSCP, using the same primers as described above. The PCR products were diluted in formamide-dye loading buffer and electrophoresed on a 15% nondenaturing polyacrylamide gel with or without 10% glycerol at 150 V for 14 hours at room temperature. The gels were developed with a previously described silver staining procedure.25
p14ARF mRNA Expression
Total RNA was obtained from 20 tumors by guanidine isothiocyanate extraction and cesium chloride gradient centrifugation. The reverse transcription reaction was performed with the SuperScript preamplification kit (Gibco BRL, Gaithersburg, MD) from 1 µg of total RNA, following the manufacturer specifications for a cDNA synthesis using an oligo (dT) primer. A 259-bp fragment of p14ARF was amplified by PCR, using the primers 5'-AGTGGCGCTGCTCACCTC-3' and 5'-CTAGGAAGCGGCTGCTGC-3'. In each case the RPS14 ribosomal mRNA (143 bp) was amplified as positive control, using the primers 5'-GGCAGACCGAGATGAATCCTCA-3' and 5'-CAGGTCCAGGGGTCTTGGTCC-3'. The amplification conditions were four cycles at 95°C for 45 seconds, 68°C for 1 minute, and 72°C for 1 minute, and 30 cycles with an annealing temperature of 66°C, with an additional elongation step at 72°C for 10 minutes. The amplified products were separated by electrophoresis in 2% agarose gel and visualized by ethidium bromide staining.
p53 Immunohistochemical Analysis
p53 protein expression was assessed immunohistochemically on fixed and paraffin-embedded material, using the anti-p53 clone BP53–12 monoclonal antibody (Novocastra, Newcastle on Tyne, UK) at a final dilution of 1:50. The deparaffinized and rehydrated slides were placed in Dako ChemMate citrate solution (Dako, Glostrup, Denmark) in a pressure cooker before incubation with the primary antibody. p53 antibody was incubated for 1 hour at room temperature. The immunoreaction was developed in an automated TechMate 500 (Dako), using the Dako EnVision+ System, peroxidase (DAB) technique, with diaminobenzidine as the chromogen. The slides were counterstained with Mayer’s hematoxylin. Quantification of positive cells was performed with a computerized digital analyzer (MicroImage; Olympus Europe, Hamburg, Germany). To score the positive nuclei, the percentage of the immunohistochemically stained area was measured against the total nuclear area. Five or more fields of each tumor were analyzed, until a minimum of 1000 cells had been examined. The fields in each slide were selected from the areas with greatest number of stained cells. p53 expression was considered as negative, low, or high when less than 5%, 5–30%, or more than 30% of the cells showed nuclear immunostaining.
Statistical Analysis
Categorical data were compared using the Fisher’s exact test (two-sided P value), whereas for ordinal data nonparametric tests were used. The actuarial survival analysis was performed according to the method described by Kaplan and Meier, and the curves were compared by the log-rank test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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p53 gene mutations in exons 4–9 were analyzed by
PCR-SSCP, and cases with anomalous migrating bands were sequenced. Gene
alterations were found in four (8%) indolent lymphomas and 15 (33%)
aggressive tumors (indolent versus aggressive;
P = 0.01) (Table 1)
. The
mutated indolent lymphomas were three CLLs and one typical MCL. The
CLLs showed two missense mutations at exon 5 and one microdeletion of
four nucleotides at exon 4, with a frameshift in the sequence leading
to a stop codon (Table 2)
. The typical
MCL had a missense mutation in exon 5 (Table 2)
. The 15 aggressive
lymphomas with p53 alterations were two LCLs transformed from CLL
(Richter’s syndrome), three LCLs transformed from FL, five blastoid
MCLs, three de novo diffuse B-cell LCLs, one Burkitt’s
lymphoma, and one T-cell lymphoblastic lymphoma (Tables 1 and 2)
.
Twelve of these tumors showed missense mutations, two at exon 4, two at
exon 5, four at exon 7, and four at exon 8 (Table 2)
. A LCL transformed
from a CLL showed a deletion of five nucleotides at exon 8 with a
frameshift in the sequence causing a subsequent stop codon. The PCR
amplification signal of the p53 gene was very low in two
blastoid MCLs (cases 441 and 15855). Southern blot analysis of these
two cases showed a homozygous deletion of the p53 gene
(Table 2)
. The very faint band obtained in the PCR analysis could be
due to amplification of the gene from the scarce normal or reactive
cells present in the sample. No p53 gene alterations were
detected in 53 additional tumors that were also examined by Southern
blot.
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Deletions of exons 1, 1ß, and 2 of the
INK4a/ARF locus were analyzed by multiplex PCR. Exon 2
deletions were detected in nine cases, two indolent and seven
aggressive lymphomas. The two indolent tumors were a typical MCL and a
follicular lymphoma. The seven aggressive lymphomas were two LCLs
transformed from CLL (Richter’s syndrome), one LCL transformed from
FL, two blastoid MCLs, one de novo diffuse B-cell LCL, and
one lymphoblastic lymphoma of T-cell phenotype (Table 1)
. The results
obtained in this PCR analysis of exon 2 were concordant with those
obtained in the previous analysis of exon 2 by Southern
blot.22
p16INK4a exon 1 and
p14ARF exon 1ß deletions were always
associated with exon 2 losses. However, exon 1ß was retained in one
lymphoblastic lymphoma in which exon 1 and exon 2 were both deleted
(case 2286) (Figure 1)
. Interestingly,
the Southern blot analysis of this case showed a deletion of
p16INK4a exon 2, but
p15INK4b was in germ line
configuration.22
p15INK4b was
also deleted in other seven tumors with exon 1ß, 1, and 2
deletions but was in germ line configuration in one Richter’s syndrome
with deletion of exons 1ß, 1 and 2.22
No isolated
deletions of exon 1ß were detected in any case.
|
Mutations of exons 1ß and 2 were analyzed by PCR-SSCP followed
by sequencing of cases with anomalous migrating bands. No mutations
were detected in exon 1ß in any case (Table 1)
. Exon 2 mutations had
been detected in one CLL and two transformed follicular
lymphomas.22
In the open reading frame of
p16INK4a, the CLL mutation was at codon
143 (GCC 
ACC), resulting in a change of alanine to threonine. This
mutation was located outside the open reading frame of
p14ARF. The two transformed FLs showed the
same mutation. In the open reading frame of
p16INK4a, this change was a nonsense
mutation at codon 80 CGA (Arg) TGA (Stop). The same change in the
p14ARF open reading frame was at codon 94,
producing a missense mutation, CCG (Pro) CTG (Leu). Western blot
analysis of these two cases had shown a lack of p16 protein
expression.22
Correlation between p53, p14ARF, and p16INK4a Gene Alterations
The correlation between p53,
p14ARF, and
p16INK4a gene alterations is shown in
Table 3
. p53 inactivation was
detected in 19 (20%) tumors: 15 missense mutations, two microdeletions
leading to stop codons, and two homozygous deletions.
p14ARF gene alterations were detected in
11 (11%) tumors: eight showed deletions of exon 1ß and exon 2, one
case showed deletion of exon 2 but retained exon 1ß, and two tumors
had point mutations in exon 2 involving the nucleolar transport domain
of the protein. p16INK4a alterations were
detected in 17 (18%) tumors, including nine homozygous deletions
involving both exons 1 and 2, five hypermethylations, and three
point mutations.22
|
. Seventeen cases with p53 gene alterations had a
wild-type INK4a/ARF locus, whereas 15 tumors with genetic
alterations in this locus had a wild-type p53 gene.
Concomitant alterations in the p53 gene and the
INK4a/ARF locus were detected in only two cases (Table 2)
.
Interestingly, one of these cases was the lymphoblastic lymphoma in
which the p14ARF exon 1ß was retained,
whereas p16INK4a exons 1 and 2 were
deleted. p53 and p14ARF Gene Expression
Experimental studies have shown that p14ARF stabilizes p53 protein in the nucleus of the cell by inhibiting its cytoplasmic transport and the MDM2-mediated degradation.9-14 To determine whether p14ARF gene alterations may be associated with lower p53 levels in human tumors, p53 protein expression was examined by immunohistochemistry, and its expression levels were compared to the status of the p53 and p14ARF genes.
p53 protein expression in indolent lymphomas was usually negative (<5%). Similarly, the CLL with a nonsense mutation in exon 4 was negative for p53 protein. The two CLLs and the typical MCL with p53 gene missense mutations showed a high p53 protein expression. The typical MCL and the FL with p14ARF deletion showed negative levels similar to those of the remaining tumors with wild-type p14ARF gene.
Contrary to the negative immunostaining in indolent lymphomas,
aggressive tumors with wild-type p53 and
p14ARF genes showed a broad range of p53
protein expression (mean 17% SD ± 25) (Table 4)
. Tumors with p53 missense
mutations, including the two cases with concomitant
INK4a/ARF exon 2 deletions, showed high levels of p53
protein expression, whereas the two tumors with homozygous deletions of
p53 were negative (Tables 2 and 4)
. Interestingly, protein
expression in aggressive tumors with
p14ARF altered and wild-type
p53 was significantly lower than in tumors with no
alteration in any of these genes (mean 4%, SD ± 7
versus mean 17%, SD ± 25; P = 0.04)
(Table 4
and Figure 2
).
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The p53/p14ARF and
p16INK4a gene alterations according to the
histological subgroup (indolent versus aggressive) are
detailed in Table 5
. p53 and
p14ARF genes are considered to be in the
same regulatory pathway. Therefore, patients with alterations in either
of these genes were included in the same group. Patients with
aggressive lymphomas showed one impaired pathway (either
p53/p14ARF or
p16INK4a) (39% versus 8%) and
two impaired pathways (both p53/p14ARF and
p16INK4a) more frequently (20%
versus 4%; P < 0.001) than indolent
lymphomas.
|
. In the whole series, patients of
group A showed a trend for better survival than those of groups B and C
(median survival: 60, 24, and 17 months, respectively;
P = 0.08).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study we have examined the status of p53 and
p14ARF genes simultaneously in a large
series of human NHLs previously analyzed for
p16INK4a. The frequency of alterations in
the p53 and p16INK4a genes was
similar to that observed in other series of
lymphomas.29-33
Thus p53 aberrations were
detected in 8% of indolent lymphomas but in 33% of aggressive tumors,
whereas p16INK4a alterations were found in
6% and 30% of low- and high-grade lymphomas, respectively.
Inactivation of p14ARF was always
associated with p16INK4a alterations. Exon
1ß was concomitantly deleted with exon 1 and 2 in almost all
cases. Only one lymphoblastic lymphoma, which harbored exon 1 and 2
deletions, retained exon 1ß. In addition, no mutations were detected
in exon 1ß in any case. Although some studies in murine
models8,16,18
and human acute lymphoblastic
leukemias20,21
have suggested that
p14ARF may be selectively inactivated in
lymphoproliferative disorders, our findings suggest that independent
inactivation of p14ARF in human NHLs is a
rare phenomenon.
p14/19ARF is considered to be an upstream regulator of p53 function. Experimental studies have shown that p14/19ARF interacts physically with MDM2, thus preventing p53 degradation and promoting p53 stabilization and accumulation.9,14,34,35 These observations suggest that tumors with inactivation of the ARF locus may have an increased degradation of p53 protein and, consequently, a defective regulation of the p53 regulatory pathway. Consistent with these experimental studies, we observed that p53 protein expression in aggressive lymphomas with p14ARF exon 1ß deletions was significantly lower than in other aggressive tumors with a wild-type INK4a/ARF locus.
Interestingly, two transformed FLs with mutations in exon 2 were also negative for p53 protein. Initial studies of the functional domains of p19ARF showed that exon 1ß was necessary and sufficient to produce cell-cycle arrest, whereas exon 2 mutations seemed to specifically inactivate the p16INK4a gene but not p19ARF.9,36 However, recent studies on human p14ARF have identified a C-terminal domain between residues 83 and 101 of exon 2 that are required for its localization in the nucleolus and for the formation of stable p14ARF-MDM2-p53 nuclear complexes.13 Mutations in these residues disrupt the nucleolar localization of p14ARF, leading to a cytoplasmic diffusion of the protein and a reduced stabilization of p53 protein.13 Interestingly, in contrast to human p14ARF, the nucleolar localization domain of murine p19ARF seems to reside in a region coded by exon 1ß.34 These observations suggest that mutations in the human exon 2 involving this nucleolar domain may target both the p14ARF and p16INK4a genes. The exon 2 mutation detected in our two transformed FLs was found in codon 80 of INK4a and codon 94 of ARF reading frames, leading to a stop codon in p16INK4a and a missense mutation within the nucleolar localization domain of p14ARF. In fact, these two tumors showed a lack of expression of both p16INK4a and p53 protein, suggesting that this mutation may target p14ARF and p16INK4a genes simultaneously.
We have also detected one exon 2 mutation in an indolent CLL. This mutation was identified in p16INK4a codon 143, which is outside the ankyrin repeat motifs in a region that is not required for the protein to bind and inhibit CDK4,37 and it is outside the open reading frame of p14ARF. Therefore, it is possible that this mutation was not interfering with the function of either of these proteins. Concordantly, the clinical behavior of this patient was similar to that of other indolent CLL patients with an INK4a/ARF wild-type locus. In a review of the literature of the INK4a/ARF alterations in lymphoproliferative disorders, we have identified 21 mutations involving the p16INK4a gene, with eight occurring in indolent disorders (six CLLs and two FLs) and 13 in aggressive tumors (nine acute lymphoblastic leukemias, three LCLs, and one Burkitt’s lymphoma).20,22,29 Six of the 13 mutations in aggressive tumors, but only one of the eight indolent disorders, led to a change in the ARF open reading frame. The remaining changes were downstream of the ARF stop codon or did not cause an amino acid change in the sequence. These observations suggest that exon 2 mutations involving both genes may play a role in the development of aggressive tumors.
The recent understanding of the INK4a/ARF locus as a regulatory region of both p16INK4a/Rb and p14ARF/p53 pathways has suggested that tumors with inactivation of this locus may harbor fewer p53 mutations than tumors with wild-type INK4a/ARF genes.10 In our study p53 mutations and INK4a/ARF alterations appeared to occur in different tumors because only two aggressive lymphomas, one lymphoblastic lymphoma and one Richter’s syndrome, harbored simultaneous p53 mutation and homozygous deletion of the INK4a/ARF locus. Reciprocal alterations between p53 mutations and p16INK4a alterations have been observed in some tumors.25,30,38 However, mutual exclusion between these alterations has not been detected in leukemia-lymphoma cell lines39 and other human tumors, including large B-cell lymphomas.40,41 It has been suggested that the pattern of these alterations in tumors may depend on the order of events.12 If deletion of the INK4a/ARF locus occurs early in the development of the tumor, the neoplastic cells may remain wild type for p53. In contrast, an initial mutation in the p53 gene may be followed by a strong pressure against p16INK4a that would lead to a concomitant deletion of p14ARF. However, tumors with inactivation of both p53 and p14ARF may have a selective growth advantage because there is no complete overlap between the ARF and p53 functions.40 In fact, p14ARF does not seem to participate in the apoptotic response to DNA damage, and, consequently, cells with p53 loss may tolerate higher levels of genomic damage than tumor cells with wild-type p53.8,12
Different studies have demonstrated the prognostic value of p53 mutations in NHLs. However, the prognostic significance of INK4a/ARF alterations in these tumors is not well known. Garcia-Sanz et al have indicated that p16INK4a deletions and rearrangements are associated with poor prognosis in B-cell NHLs, but this study did not include p14ARF and p53 analysis.42 Similarly, we have recently shown that DNA losses in 9pter are associated with a poor prognosis in MCL patients.26 Genetic alterations in the INK4a/ARF locus target both p14ARF/p53 and p16INK4a/Rb pathways and, therefore, may provide a selective growth advantage in the progression of tumors. In our study, although the series was heterogeneous and the number of patients relatively small, there was a trend indicating a poor prognosis for patients with inactivation of both p16INK4a and p14ARF/p53 genes. In the homogeneous subset of MCLs, patients with genetic alterations in the INK4a/ARF locus had a significantly worse prognosis than patients with tumors with only p53 mutations or with no aberrations in any of these genes.
In this study, independent inactivation of p16INK4a only occurred by hypermethylation. Similar to observations of other tumors, in our lymphomas hypermethylation of the p16INK4a gene does not affect the expression of p14ARF.43,44 On the other hand, different studies have already shown that inactivation of p14ARF by hypermethylation is extremely rare.44,45 The number of patients with isolated inactivation of p16INK4a in our series was too small to discern whether hypermethylation of p16INK4a may have any particular influence on the survival of the patients.
In conclusion, our findings indicate that genetic alterations in p14ARF occur in a subset of aggressive NHLs, but they are always associated with p16INK4a aberrations. Alterations in the INK4a/ARF locus seem to develop frequently in tumors with a wild-type p53 gene. The shorter survival of patients with lymphomas harboring concomitant alterations of the p16INK4a and p14ARF/p53 genes suggests that simultaneous inactivation of these two regulatory pathways may have a cooperative effect on the progression of the tumors.
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Acknowledgements |
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Footnotes |
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Supported by the Comision Interministerial de Ciencia y Tecnologia (CICYT) (SAF 99/20), Asociación Española Contra el Cancer, and CIRIT, Generalitat de Catalunya (98SGR21).
The first two authors contributed equally to this study.
Accepted for publication February 16, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L. Hernandez, S. Bea, M. Pinyol, G. Ott, T. Katzenberger, A. Rosenwald, F. Bosch, A. Lopez-Guillermo, J. Delabie, D. Colomer, et al. CDK4 and MDM2 Gene Alterations Mainly Occur in Highly Proliferative and Aggressive Mantle Cell Lymphomas with Wild-type INK4a/ARF Locus Cancer Res., March 15, 2005; 65(6): 2199 - 2206. [Abstract] [Full Text] [PDF]
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Y.-C. Wang, R.-K. Lin, Y.-H. Tan, J.-T. Chen, C.-Y. Chen, and Y.-C. Wang Wild-Type p53 Overexpression and Its Correlation With MDM2 and p14ARF Alterations: An Alternative Pathway to Non-Small-Cell Lung Cancer J. Clin. Oncol., January 1, 2005; 23(1): 154 - 164. [Abstract] [Full Text] [PDF]
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S. Bea, L. Colomo, A. Lopez-Guillermo, I. Salaverria, X. Puig, M. Pinyol, S. Rives, E. Montserrat, and E. Campo Clinicopathologic Significance and Prognostic Value of Chromosomal Imbalances in Diffuse Large B-Cell Lymphomas J. Clin. Oncol., September 1, 2004; 22(17): 3498 - 3506. [Abstract] [Full Text] [PDF]
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 T. Watanabe, Y. Katayama, A. Yoshino, C. Komine, and T. Yokoyama Deregulation of the TP53/p14ARF Tumor Suppressor Pathway in Low-Grade Diffuse Astrocytomas and Its Influence on Clinical Course Clin. Cancer Res., October 15, 2003; 9(13): 4884 - 4890. [Abstract] [Full Text] [PDF]
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Y. Huang, T. Tyler, N. Saadatmandi, C. Lee, P. Borgstrom, and R. A. Gjerset Enhanced Tumor Suppression by a p14ARF/p53 Bicistronic Adenovirus through Increased p53 Protein Translation and Stability Cancer Res., July 1, 2003; 63(13): 3646 - 3653. [Abstract] [Full Text] [PDF]
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M. Nakamura, M. Kishi, T. Sakaki, H. Hashimoto, H. Nakase, K. Shimada, E. Ishida, and N. Konishi Novel Tumor Suppressor Loci on 6q22-23 in Primary Central Nervous System Lymphomas Cancer Res., February 15, 2003; 63(4): 737 - 741. [Abstract] [Full Text] [PDF]
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 M. Sanchez-Beato, A. Sanchez-Aguilera, and M. A. Piris Cell cycle deregulation in B-cell lymphomas Blood, February 15, 2003; 101(4): 1220 - 1235. [Abstract] [Full Text] [PDF]
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F. Tort, S. Hernandez, S. Bea, A. Martinez, M. Esteller, J. G. Herman, X. Puig, E. Camacho, M. Sanchez, I. Nayach, et al. CHK2-decreased protein expression and infrequent genetic alterations mainly occur in aggressive types of non-Hodgkin lymphomas Blood, December 15, 2002; 100(13): 4602 - 4608. [Abstract] [Full Text] [PDF]
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 L Leoncini, S Lazzi, C Bellan, and P Tosi Cell kinetics and cell cycle regulation in lymphomas J. Clin. Pathol., September 1, 2002; 55(9): 648 - 655. [Abstract] [Full Text] [PDF]
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 S. Bea, A. Lopez-Guillermo, M. Ribas, X. Puig, M. Pinyol, A. Carrio, L. Zamora, F. Soler, F. Bosch, S. Stilgenbauer, et al. Genetic Imbalances in Progressed B-Cell Chronic Lymphocytic Leukemia and Transformed Large-Cell Lymphoma (Richter's Syndrome) Am. J. Pathol., September 1, 2002; 161(3): 957 - 968. [Abstract] [Full Text] [PDF]
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J. F. Garcia, R. Villuendas, M. Sanchez-Beato, A. Sanchez-Aguilera, L. Sanchez, I. Prieto, and M. A. Piris Nucleolar p14ARF Overexpression in Reed-Sternberg Cells in Hodgkin's Lymphoma : Absence of p14ARF/Hdm2 Complexes Is Associated with Expression of Alternatively Spliced Hdm2 Transcripts Am. J. Pathol., February 1, 2002; 160(2): 569 - 578. [Abstract] [Full Text] [PDF]
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E. Camacho, L. Hernandez, S. Hernandez, F. Tort, B. Bellosillo, S. Bea, F. Bosch, E. Montserrat, A. Cardesa, P. L. Fernandez, et al. ATM gene inactivation in mantle cell lymphoma mainly occurs by truncating mutations and missense mutations involving the phosphatidylinositol-3 kinase domain and is associated with increasing numbers of chromosomal imbalances Blood, January 1, 2002; 99(1): 238 - 244. [Abstract] [Full Text] [PDF]
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M. Nakamura, T. Sakaki, H. Hashimoto, H. Nakase, E. Ishida, K. Shimada, and N. Konishi Frequent Alterations of the p14ARF and p16INK4a Genes in Primary Central Nervous System Lymphomas Cancer Res., September 1, 2001; 61(17): 6335 - 6339. [Abstract] [Full Text] [PDF]
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A. Calogero, A. Arcella, G. De Gregorio, A. Porcellini, D. Mercola, C. Liu, V. Lombari, M. Zani, G. Giannini, F. M. Gagliardi, et al. The Early Growth Response Gene EGR-1 Behaves as a Suppressor Gene That Is Down-Regulated Independent of ARF/Mdm2 but not p53 Alterations in Fresh Human Gliomas Clin. Cancer Res., September 1, 2001; 7(9): 2788 - 2796. [Abstract] [Full Text] [PDF]
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S. A. Nicholson, N. T. Okby, M. A. Khan, J. A. Welsh, M. G. McMenamin, W. D. Travis, J. R. Jett, H. D. Tazelaar, V. Trastek, P. C. Pairolero, et al. Alterations of p14ARF, p53, and p73 Genes Involved in the E2F-1-mediated Apoptotic Pathways in Non-Small Cell Lung Carcinoma Cancer Res., July 1, 2001; 61(14): 5636 - 5643. [Abstract] [Full Text] [PDF]
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M. Sanchez-Beato, A. I. Saez, I. C. Navas, P. Algara, M. Sol Mateo, R. Villuendas, F. Camacho, A. Sanchez-Aguilera, E. Sanchez, and M. A. Piris Overall Survival in Aggressive B-Cell Lymphomas Is Dependent on the Accumulation of Alterations in p53, p16, and p27 Am. J. Pathol., July 1, 2001; 159(1): 205 - 213. [Abstract] [Full Text] [PDF]
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S. Beà, F. Tort, M. Pinyol, X. Puig, L. Hernández, S. Hernández, P. L. Fernández, M. van Lohuizen, D. Colomer, and E. Campo BMI-1 Gene Amplification and Overexpression in Hematological Malignancies Occur Mainly in Mantle Cell Lymphomas Cancer Res., March 1, 2001; 61(6): 2409 - 2412. [Abstract] [Full Text]
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