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Deletions and Loss of Expression of P16 INK4a and P21 Waf1 Genes Are
Associated With Aggressive Variants of Mantle Cell Lymphomas
By Magda Pinyol, Luis Hernandez, Maite Cazorla, Milagros BalbıB n, Pedro Jares, Pedro L. Fernandez,
Emilio Montserrat, Antonio Cardesa, Carlos Lopez-OtıB n, and ElıB as Campo
Mantle cell lymphoma (MCL) is molecularly characterized
by bcl-1 rearrangement and cyclin D1 gene overexpression.
Some aggressive variants of MCL have been described with
blastic or large cell morphology, higher proliferative activity,
and shorter survival. The cyclin-dependent kinase inhibitors
(CDKIs) p21Waf1 and p16INK4a have been suggested as candidates for tumor-suppressor genes. To determine the role of
p21Waf1 and p16INK4a gene alterations in MCLs, we examined
the expression, deletions, and mutations of these genes in
a series of 24 MCLs, 18 typical, and 6 aggressive variants.
Loss of expression and/or deletions of p21Waf1 and p16INK4a
genes were detected in 4 (67%) aggressive MCLs but in none
of the typical variants. Two aggressive MCLs showed a loss
of p16INK4a expression. These cases showed homozygous deletions of p16INK4a gene by Southern blot analysis. An additional aggressive MCL in which expression could not be examined showed a hemizygous 9p12 deletion. Loss of p21Waf1
expression at both protein and mRNA levels was detected
in an additional aggressive MCL. No p21Waf1 gene deletions
or mutations were found in this case. The p21Waf1 expression
in MCLs was independent of p53 mutations. The two cases
with p53 mutations showed p21Waf1 and p16INK4a expression
whereas the 4 aggressive MCLs with p16INK4a and p21Waf1
gene alterations had a wild-type p53. p21Waf1 and p16INK4a
were expressed at mRNA and protein levels in all typical
MCLs examined. No gene deletions or point mutations were
found in typical variants. Two typical MCLs showed an
anomalous single-stranded conformation polymorphism
corresponding to the known polymorphisms at codon 148
of p16INK4a gene and codon 31 of p21Waf1 gene. These findings
indicate that p21Waf1 and p16INK4a alterations are rare in typical MCLs but the loss of p21Waf1 and p16INK4a expression, and
deletions of p16INK4a gene are associated with aggressive
variants of MCLs, and they occur in a subset of tumors with
a wild-type p53 gene.
q 1997 by The American Society of Hematology.
M
also shown that cyclin D1 may function as an oncogene
cooperating with other oncogenes in cellular transformation.14,15 However, the tumorigenic and transforming properties of cyclin D1 seem to be less effective than the conventional oncogenes. The possible additional oncogenic factors
cooperating with cyclin D1 in the pathogenesis of MCLs are
unknown.
In addition to cyclin D1, other cell-cycle regulatory molecules may participate in the development and progression
of human tumors. Particularly, an emerging group of small
proteins called cyclin-dependent kinase inhibitors (CDKIs)
are considered to be implicated in tumorigenesis as possible
tumor-suppressor genes.16,17 These proteins act as negative
regulatory elements of cell proliferation by inhibiting the
kinase activity of the cyclin/CDK complexes. Among all
these molecules, p21Waf1 and p16INK4a are strong candidates
to participate in tumor progression. p21Waf1 is a gene induced
by wild-type p53 but not mutant p53 and it is implicated in
the mechanisms of cell arrest that allow the cell for DNA
repair.18,19 p21Waf1 is also involved in mechanisms of cell
differentiation and senescence.20 The potential role for
p21Waf1 in tumorigenesis has been postulated on the basis of
its transcriptional control by p53. In addition, overexpression
of p21Waf1 may suppress tumor growth in different experimental models.21,22 However, p21Waf1 point mutations seem
to be very rare in human tumors.23 p16INK4a and p15INK4b
genes are located on 9p21, a chromosomal region frequently
deleted in many human tumors. p16INK4a gene has been found
to be deleted, mutated, or downregulated by hypermethylation at high frequency in different types of tumors.16,17,24,25 In
hematologic disorders, homozygous deletions are frequently
found in acute lymphoblastic leukemias but point mutations
seem to be rare.17 The spontaneous development of B-cell
lymphomas in the INK4a knock-out mice strongly supports
the role of this gene in the pathogenesis of lymphoproliferative disorders.26
The possible alterations of p21Waf1 and p16INK4a in MCLs
have not been previously examined. We and others have
ANTLE CELL lymphoma (MCL) is a malignant
lymphoproliferative disorder genetically characterized by chromosomal translocations involving the 11q13 region and its molecular counterpart the bcl-1 rearrangement.1-4
This translocation activates the cyclin D1 gene located 110
to 130 kb downstream from the major breakpoint of this
rearrangement.5-7 Cyclin D1 overexpression occurs in virtually all MCL independently of the detection of t(11;14)
translocation or bcl-1 rearrangements, and this overexpression is highly specific of this type of lymphoma.8-11
Cyclin D1 is a G1 cyclin that participates in the control of
the cell-cycle progression at the G1 to S phase transition.
Cyclin D1 binds to Retinoblastoma protein (pRb) and, coupled with cyclin-dependent kinases (CDKs), is capable of
its phosphorylation contributing to the inactivation of the
pRb growth suppressive effect.12,13 Different studies have
From the Departments of Anatomic Pathology and Postgraduate
School of Hematology ‘‘Farreras ValentıB ,’’ Hospital Clinic Provincial, University of Barcelona, Barcelona, Spain; the Department of
Basic Medical Sciences, School of Medicine, University of Lleida,
Lleida, Spain; and the Department of Biologia Funcional, University
of Oviedo, Oviedo, Spain.
Submitted May 15, 1996; accepted August 19, 1996.
Supported in part by Grants No. SAF 1195/93, SAF 96/61 from
CICYT, and F1596/236 Ministerio de Educación y Ciencia (E.C.).
M.P. and L.H. were fellows supported by a grant from the European
Union; M.C. and P.J. were fellows from the Spanish Ministerio de
Educación y Ciencia.
The first and second authors contributed equally to this study.
Address reprint requests to ElıB as Campo, MD, Laboratory of
Anatomic Pathology, Hospital Clinic Provincial, Villarroel 170,
08036-Barcelona, Spain.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
q 1997 by The American Society of Hematology.
0006-4971/97/8901-0035$3.00/0
Blood, Vol 89, No 1 (January 1), 1997: pp 272-280
272
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CDKIs IN MANTLE CELL LYMPHOMA
273
recently shown that p53 mutations are associated with aggressive variants of MCLs.27-29 Whether p53 mutations may
downregulate the transcription rate of p21Waf1 expression in
these aggressive MCLs is not known. On the other hand, it
has been suggested that p16INK4a, cyclin D1, and pRb are
interlinked in the same cell-cycle regulatory pathway.30-33
Oncogenic alterations of p16INK4a and cyclin D1 cooperate
synergistically to deregulate cell proliferation in cell lines
with normal pRb function.33 The constant cyclin D1 overexpression8 and the presence of an apparently functional
pRb34,35 in MCLs make these tumors an interesting in vivo
model to analyze the possible cooperating effects of p16INK4a
and p21Waf1 in the pathogenesis of these lymphomas.
To determine the possible role of p21Waf1 and p16INK4a gene
alterations in the development and progression of MCLs, we
have examined their gene structure and expression at both
mRNA and protein levels in a series of MCLs previously
characterized for cyclin D1 overexpression,8 retinoblastoma
status,34 and p53 gene mutations.28 Our results indicate that
p21Waf1 and p16INK4a alterations are rare in typical MCLs but
the loss of p21Waf1 and p16INK4a expression and deletions of
p16INK4a gene are associated with aggressive variants of
MCLs, and they occur in a subset of tumors with wild-type
p53 gene.
MATERIALS AND METHODS
Case selection. Tumor specimens from 24 MCLs were obtained
from the files of the Pathology Department of the Hospital Clinic
of Barcelona. The specimens included 18 lymph nodes, 4 spleens,
1 tonsil, and 1 skin biopsy. Eighteen cases were classified as typical
MCLs36,37 and 6 cases as aggressive variants of MCLs. These aggressive variants were further classified as blastic MCLs (4 cases) and
large cell (‘‘anaplastic’’) type of MCL (2 cases) defined according
to criteria previously described.38-40 Immunophenotype was analyzed
in all cases using immunohistochemistry on frozen tissue sections
and/or cell suspensions by flow cytometry. These studies included
Ig light and heavy chains, several of the B-cell, and T-cell markers,
CD10 and CD23. bcl-1 rearrangement at the major translocation
cluster (MTC) locus was detected by Southern blot and/or polymerase chain reaction (PCR) analysis in 6 typical and 2 aggressive cases
using previously described techniques.41 Cyclin D1 overexpression
was detected in all cases by Northern and/or Western blot analysis.8,42
All of these cases had also been examined for retinoblastoma protein
expression34 and p53 gene mutations28 in previous studies. Analysis
of p53 mutations of exon 5 to 9 had been examined by singlestranded conformation polymorphism (SSCP), denaturing gradient
gel electrophoresis, and direct sequencing as described.28 Cytogenetic analysis was performed in 5 patients (4 typical and 1 aggressive
MCL variants) with leukemic expression.34 The clinical and pathologic characteristics of the patients including the proliferative activity
of the tumors and the follow-up of the patients have been previously
reported.28,34 The mean Ki-67 proliferative index in aggressive variants of MCL was significantly higher (mean, 38.2%; SD, 5.6) than
in the typical MCL (mean, 10.3; SD, 5.6) (P õ .002). The median
overall survival of this series of MCL patients was 49.8 months
(range, 2 to 82 months). Patients with aggressive variants had an
overall survival (median, 18.3; n Å 6) significantly shorter than the
patients with typical MCL (median, 49.8, n Å 18) (P õ .001).
Southern and Northern blot analyses. Genomic DNA was extracted from frozen material in 23 (18 typical and 5 blastic) cases
using Proteinase K/RNAse treatment and phenol-chloroform extraction. DNA from each case (10 mg) was digested with EcoRI, HindIII,
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and BamHI, separated on 0.8% agarose gels, and transferred to
Hybond-N membranes (Amersham, Buckinghamshire, UK). The
membranes were prehybridized, hybridized with the p21Waf1 and
p16INK4a probes, and washed as previously described.8 The bcl-1
MTC probe was also used in all Southern blots. To evaluate the
intensity of the signals of the Southern blot analysis, the autoradiographic signals were quantified using a UVP-5000 video densitometer (UVP, San Gabriel, CA). Intensities of p16INK4a and p21Waf1 bands
were normalized to the bcl-1 MTC control bands.
Total RNA was isolated from frozen tissues in 17 cases (12 typical
and 5 aggressive variants) by guanidine isothiocyanate extraction
and cesium chloride gradient centrifugation.8 Eight (for p21Waf1 analysis) to 20 (for p16INK4a analysis) micrograms of total RNA were
electrophoresed on a denaturing 1.2% agarose formaldehyde gel and
transferred to Hybond-N membranes (Amersham). The membranes
were prehybridized, hybridized with the p21Waf1 and p16INK4a probes,
and washed as previously described.8
Probes. Probes were radiolabeled using a random primer DNA
labeling kit (Promega Corp, Madison, WI) with [a-32P]dCTP. Human p21Waf1 cDNA probe was obtained by reverse transcriptasePCR (RT-PCR) from poly A RNA and cloned using standard techniques. For PCR amplification, the following primers were used:
5*-GCGCCATGTCAGAACCGGCTG-3* and 5*-GCAGGCTTCCTGTGGGCGGAT-3*. The PCR conditions were 957C for 1 minute, 607C for 30 seconds, 757C for 2 minutes, and a total number
of 30 cycles. The identity of the amplified fragment was confirmed
by sequencing. The p16INK4a probes used were a 0.8-kb EcoRI-Xho
I fragment of the p16INK4a cDNA clone kindly provided by Dr M.
Serrano,43 and a fragment of exon 2 obtained by PCR using primers
previously described.44 The identity of the amplified fragment was
confirmed by sequencing.
SSCP analysis. SSCP analysis was used to screen for p16INK4a
and p21Waf1 gene mutations according to a modified protocol of a
previous described method.45 Exons 1 and 2 of p16INK4a gene were
amplified by PCR using a simple set of flanking intronic primers.
Primers for exon 1 were 5*- GAAGAAAGAGGAGGGGCTG-3*
and 5*- GCGCTACCTGATCCAATTC-3*, and primers for exon 2
were 5*- CTCTACACAAGCTTCCTTTCC-3* and 5*- GGGCTGAACTTTCTGTGCTGG-3*. Amplifications were performed with
0.2 mg of genomic DNA, 2.5 U of Taq polymerase (GIBCO-BRL,
Gaithersburg, MD), 0.5 mmol/L of each primer, 100 mmol/L of
dNTPs, 5% dimethyl sulfoxide, and PCR buffer in a final volume
of 50 mL. We used a ‘‘touch-down’’ PCR strategy for the amplification of both exons. Conditions were 1 cycle at 957C for 5 minutes,
4 cycles at 947C for 45 seconds, at 687C for 1 minute, and at 727C
for 1 minute; 4 cycles with annealing temperature at 647C; 4 cycles
with annealing temperature at 627C; and 30 cycles with annealing
temperature at 607C. Previously to SSCP analysis, the PCR product
of both exons were digested with Sma I.
Exon 2 of p21Waf1 gene contains 90% of the coding sequence.
This exon was amplified in two fragments using primers 5*-CATAGTGTCTAATCTCCGCCGT-3* and 5*-GCCTGCCTCCTCCCAACTCATC-3* for the 5* segment of exon 2 (fragment 2a), and
5*-TGCCCAAGCTCTACCTTCCCAC-3* and 5*-AGCCCTTGGACCATGGATTCTG-3* for the 3* segment of exon 2 (fragment 2b)
as previously described.23 The PCR mixture contained 0.2 mg of
genomic DNA, 2.5 U of Taq, 0.5 mmol/L each primer, 100 mmol/
L dNTPs, and PCR buffer in a final volume of 50 mL. The reaction
was performed for 30 cycles at 947C for 40 seconds, at 597C for 30
seconds, and at 727C for 2 minutes.23
The p16INK4a and p21Waf1 amplified products were diluted sixfold
in formamide-dye loading buffer, incubated for 3 minutes at 957C,
and immediately cooled on ice; 20 mL was loaded on a 15% nondenaturing polyacrylamide gel with or without 10% glycerol for p16INK4a,
and on a 10% nondenaturing polyacrylamide gel with or without
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274
PINYOL ET AL
7% glycerol for p21Waf1 samples. p16INK4a samples were electrophoresed at 150 V for 14 hours at room temperature, and p21Waf1 samples
at 150 V for 16 hours at room temperature. The gels were developed
using a silver staining procedure previously described.46
DNA sequencing. p16INK4a and p21Waf1 genes were sequenced
using a commercial cycle sequencing kit (Perkin Elmer, Branchburg,
NY) and 33P dATP. A total of 0.5 mL of the p16INK4a gene PCR
products was used as template for sequencing. The primers described
above and two internal primers for exon 2, 5*-ACTCTCACCCGACCCGTGCA-3* and 5*-AGCTCCTCAGCCAGGTCCA-3* were
used for the sequencing reaction at a final concentration of 0.5 mmol/
L. The reaction was performed according to the instructions supplied
by the manufacturer. The reaction was performed for 35 cycles at
947C for 30 seconds, 607C for 30 seconds, and 707C for 30 seconds.
The two coding exons of p21Waf1 gene were sequenced similarly.
Exon 2 was amplified with oligonucleotides 5*- GCGCCATGTCAGAACCGGC-3* and 5*- GAGAATCCTGGTCCCTTAC-3*, and exon
3 was amplified with oligonucleotides 5*-GCCCCCCACTGTCTTCCT-3* and 5*- GCGCTTCCAGGACTGCAGG-3*. PCR conditions
for amplification consisted of a 10-minute denaturalization step at
947C (without adding the enzyme), followed by 30 cycles of 30
seconds at 947C, 30 seconds at 557C, and 30 seconds at 727C. The
same primers were used for the sequencing reaction.
The final amplified products were diluted twofold in formamidedye loading buffer. Samples were denatured for 3 minutes at 957C
and 2 mL was analyzed in a denaturing 6% polyacrylamide/8 mol/L
urea sequencing gel for 2 or 3 hours at 55 W. The gels were dried
under vacuum at 857C and exposed to an x-ray film at room temperature for 3 days without intensifier screen. The presence of a mutation
was confirmed by sequencing the other DNA strand.
Western blot analysis. Protein extraction was obtained from additional frozen tissue available in 4 aggressive MCLs and 11 typical
MCLs. Protein samples were also extracted from HeLa and Jurkatt
cell lines to be used as positive and negative controls. In each case,
10 frozen sections of 30 mm were incubated in 300 mL of ice-cold
lysis buffer (50 mmol/L Tris-Cl, pH 8, 150 mmol/L NaCl, 0.4 mmol/
L EDTA, 10 mmol/L NaF, 0.02% sodium azide, 0.1% sodium dodecyl sulfate [SDS], 1% NP-40, and 0.5% sodium deoxycholate) containing 1 mg/mL aprotinin, 1 mg/mL leupeptin, and 1 mg/mL a-1antitrypsine for 20 minutes at 47C. The cell debris was sedimented
by centrifugation at 14,000 rpm at 47C for 25 minutes. The clarified
supernatants were collected, and the protein content of the lysate
was determined by the Lowry protein assay (Bio-Rad, Hercules,
CA). Fifty micrograms of total cellular protein was run per lane on
a 15% SDS-polyacrylamide gel and electroblotted to a nitrocellulose
membrane (Amersham). Membrane was blocked by overnight incubation in 5% dry milk and 0.1% Tween-20 at 47C. The blocked
membrane was then incubated with the monoclonal antibodies antip16INK4a, Clone G175-405 (Pharmingen, San Diego, CA), and antip21Waf1 clone EA10 (Oncogene Science, Cambridge, NY) at a final
concentration of 1 mg/mL for 1 hour and 30 minutes. The membrane
was then washed with phosphate-buffered saline (PBS) 0.1% Tween
20 and exposed to sheep antimouse conjugated to horseradish peroxidase (Amersham) at a 1:1,000 dilution for 1 hour and 30 minutes.
After washing, antibody binding was detected by chemiluminescence
a detection procedures according to the manufacturer’s recommendations (ECL; Amersham).
Immunohistochemical analysis. p21Waf1 protein expression was
immunohistochemically assessed in all cases on formalin fixed-paraffin embedded using the-anti-p21Waf1 clone EA10 monoclonal antibody (Oncogene Science). Before the application of the primary
antibody an antigen retrieval technique was performed. The deparaffinized and rehydrated slides were placed in 10 mmol/L citrate buffer,
pH 6, and heated in the microwave oven for 15 minutes at 700
W. The anti-p21Waf1 antibody was incubated overnight at 47C. The
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immunoreaction was detected by means of the streptavidin-biotinalkaline phosphatase (Biogenex, San Ramon, CA) technique using
Fast-Red (Biogenex) as chromogen and levamisole to inhibit endogenous alkaline phosphatase. The slides were counterstained with
hematoxylin. The quantification of positive cells was performed using a computerized digital analyzer (Microm Image Processing
IMCO; Kontron Electronik, Munich, Germany) to score the nuclei
of the tumor cells for the presence of p21Waf1 proteins. The value
measured was the percentage of the immunohistochemical stained
area against that of the total nuclear area. Nuclear boundary optical
density and antibody threshold were adjusted for each case examined. Five or more fields of each tumor were analyzed, until a minimum of 1,000 cells had been examined. The fields were selected in
each slide from the areas with greatest number of stained cells.
RESULTS
p16
gene analysis. p16INK4a gene was examined by
Southern blot and SSCP analysis in a series of 18 typical
MCLs and 5 aggressive variants in which genomic DNA
was available. The results are summarized in Tables 1 and
2, and in Fig 1. Southern blot analysis showed deletions
of p16INK4a gene in two aggressive MCLs with a blastic
morphology (cases 3858 and 15566) (Fig 1). These deletions
were judged to be homozygous because the DNA signal in
these cases was less than 80% of the placental DNA sample.
The slight signal observed in case 15566 could be caused
by contamination from normal cells. In fact, this sample was
a skin biopsy with normal epidermis and stromal tissues.
Deletions of the p16INK4a gene in these two cases were also
associated with homozygous deletions of the p15INK4b gene
(Fig 1). No deletions were found in any of the typical MCLs.
In one additional aggressive MCL (case 3747) in which
no DNA was available, the cytogenetic analysis showed a
complex karyotype with a hemizygous deletion of the short
arm of chromosome 9 -del(9)(p12)- affecting the 9p21 region
to which p16INK4a and p15INK4b genes have been mapped. No
alterations in chromosome 9 were detected in the other four
typical MCLs in which cytogenetic studies were performed.
To determine whether mutations of p16INK4a gene were
present in these lymphomas, we analyzed exon 1 and 2 of
the p16INK4a gene by PCR-SSCP. No abnormalities were detected in the 3 aggressive MCLs in which the gene was not
deleted and in 17 typical MCLs. In one typical MCL (case
2166), an abnormally migrating band was identified in exon
2. Sequencing of this exon showed that the altered mobility
was the result of the known polymorphism at codon 148
with the change GCG (alanine) r ACG (threonine) (Fig 2).
To confirm the SSCP analysis, exons 1 and 2 of the p16INK4a
gene were completely sequenced in the 3 aggressive MCLs
with no p16INK4a, and in 4 additional typical MCLs. No mutations were detected in any of these cases.
p16INK4a gene expression. To know the possible alterations of p16INK4a gene expression in these lymphomas, 5
aggressive MCLs and 17 typical variants were examined at
mRNA and/or protein levels by Northern and Western blot
analysis, respectively. Two of the 5 (40%) aggressive MCLs
(cases 3858 and 15566) showed a complete loss of p16INK4a
expression (Figs 3 and 4). In these two cases no protein was
detected by Western blot (Fig 3). In addition, the lack of
p16INK4a expression was confirmed by Northern blot in one
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CDKIs IN MANTLE CELL LYMPHOMA
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Table 1. Frequency of p53, p21Waf1, and p16INK4a Gene Alterations and Expression in MCLs
p21Waf1
p16INK4a
Diagnosis
MCL Variant
p53 Mutations
Deletions
Mutational
Analysis
Loss of
Expression
Aggressive
Typical
2/6
0/18
0/5
0/18
0/5
1/18‡
1/6
0/18
Total
2/24
0/23
1/23
1/24
Mutational
Analysis
Loss of
Expression
3/6*
0/18
0/3†
1/18‡
2/5
0/17
3/24
1/21
2/22
Deletions
* Two cases showed homozygous deletions by Southern blot analysis. One additional case had a hemizygous deletion of the 9p 0del (9)
(p12)0. No DNA was available in this case.
† The SSCP analysis was not performed in the cases with homozygous p16INK4a gene deletion.
‡ The two cases with anomalous SSCP had a known polymorphism in codons 31 of p21Waf1 gene and codon 148 of p16INK4a gene, respectively.
of the cases (case 3858) in which RNA was available (Fig
4). p16INK4a expression at either mRNA, protein, or both
protein and mRNA was detected in the other 3 aggressive
MCLs and in the 17 typical variants (Figs 3 and 4). Interestingly, two other aggressive MCLs analyzed by Western blot
showed a stronger overexpression of p16INK4a protein in comparison with the levels observed in the typical variants of
MCLs.
The two aggressive cases with lack of p16INK4a expression
were the two cases with gene deletions detected by Southern
blot. Unfortunately, p16INK4a expression could not be analyzed in the aggressive MCL with the 9p12 hemizygous
deletion detected by cytogenetic analysis because not enough
RNA was available (p16INK4a mRNA message was only
clearly shown in Northern blots with 20 mg of total RNA)
and no additional frozen material was available from this
patient.
p21Waf1 gene analysis. p21Waf1 gene structure was analyzed by Southern blot analysis and SSCP in the 18 typical
and 5 aggressive MCLs (Tables 1 and 2). No abnormalities
in p21Waf1 gene were detected by Southern blot analysis in
any of the typical or aggressive variants of MCLs (Fig 1).
Exon 2 of p21Waf1 gene was also examined by PCR-SSCP
to screen for possible mutations. No altered mobility was
observed in any of the 5 aggressive MCLs and in 17 typical
MCLs. In one typical case (case 9180) an abnormal band was
detected. Direct sequencing of the fragment with aberrant
mobility showed the known polymorphism in codon 31 with
the change AGC (serine) r AGA (arginine). To confirm the
SSCP analysis, the full coding sequence (exon 2 and 3) of
the p21Waf1 gene was completely sequenced in 4 aggressive
MCLs and 4 additional typical MCLs. No mutations were
detected in any of these cases.
p21Waf1 gene expression. p21Waf1 expression was analyzed by Northern and/or Western blot in the 6 aggressive
MCLs and in 18 typical variants. One aggressive MCL (case
441) showed a loss of p21Waf1 expression at both mRNA and
protein level (Figs 3 and 5). p21Waf1 protein and/or mRNA
expression was detected in the remaining 5 aggressive MCLs
and in all typical cases.
p21Waf1 could also be shown by immunohistochemical
staining of formalin-fixed and paraffin-embedded material
using an antigen retrieval technique. p21Waf1 expression was
detected in all typical MCLs with a nuclear pattern. No
cytoplasmic expression was observed in any case. The number of stained cells was usually scarce, varying from 1.5%
to 5.2% (2.8% { 1.4%, mean { SD) of the cells. In the
aggressive variants, immunostaining was similarly detected
in the nucleus of the cells. The number of positive cells was
higher than in typical variants (5.5 { 1.6; mean { SD).
However, no positive cells were observed in the aggressive
MCL in which no expression had been detected by Western
or Northern blot analysis.
Correlation between p53 mutations and p21Waf1/p16INK4a
Table 2. p21Waf1 and p16INK4a Gene Alterations and Expression in Aggressive MCLs
p21Waf1
Gene
Case
Histology
p53
SB
SSCP/Sequence
3858
15566
3747
441
1104
4958
Blastic
Blastic
Large cell
Blastic
Large cell
Blastic
WT
WT
WT
WT
M Codon 273
M Codon 278
///
///
ND
///
///
///
WT
WT
ND
WT
WT
WT
p16INK4a
Expresssion
mRNA
/
ND
/
0
/
/
Gene
Expression
Protein (WB and IHQ)
SB
SSCP/Sequence
/
/
/*
0
/
/
0/0
0/0
//0†
///
///
///
NA
NA
ND
WT
WT
WT
mRNA
Protein
0
ND
ND
/
//
/
0
0
ND
//
//
ND
Abbreviations: SB, Southern blotting; WB, Western blot; IHQ, immunohistochemistry; SB: /// no deletion; //0 hemizygous deletion; 0/0
homozygous deletion; M, mutated; WT, wild-type; ND, not done; NA, not applicable.
* In this case protein p21Waf1 protein expression was only detected by immunohistochemistry.
† In this case a hemizygous deletion of 9p arm 0del (9) (p12)0 was detected by cytogenetic analysis. No DNA was available for Southern
blotting.
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PINYOL ET AL
deletion of 9p12. No alterations in any of these genes were
observed in typical variants of MCLs.
DISCUSSION
Fig 1. Southern blot analysis of four typical and four aggressive
mantle cell lymphomas. HindIII-digested DNA was electrophoresed
on a 0.8% agarose gel, transferred to a nylon membrane, and hybridized with the exon 2 of p16INK4a, p21Waf1, and bcl-1 MTC probes. The
aggressive MCL 3858 and 15566 show homozygous deletions of
p16INK4a and p15INK4b genes. No alterations in the p21Waf1 signal was
seen in any of the cases. Case 4958 had a bcl-1 rearrangement detected with HindIII. Case 441 also had a bcl-1 rearrangement detected
with EcoRI and BamHI but not with HindIII. No rearrangements were
detected in the remaining cases.
gene alterations. The comparison between p53 mutations,
p16INK4a, and p21Waf1 gene alterations in the 6 aggressive
MCLs is summarized in Table 2. p53 gene mutations were
detected in two of the 6 aggressive MCLs. The mutations
were found at codon 278 (case 4958) and codon 273 (case
1104). Interestingly, p21Waf1 expression could be shown in
both cases at mRNA level. In one of these cases (case 1104),
p21Waf1 expression was also confirmed at protein level by
Western blot analysis. The only aggressive MCLs in which
no p21Waf1 expression could be detected showed a wild-type
p53. No p53 expression was detected in this case by immunohistochemistry. To confirm the SSCP-negative analysis in
this case, exons 4 to 10 were completely sequenced and no
mutations were detected. Wild-type p53 was also found in
the two aggressive MCLs with loss of p16INK4a expression
and in the aggressive MCL with hemizygous deletion of
9p12. Therefore, all aggressive MCLs in this series were
molecularly characterized by alterations in one of these 3
genes: p53 mutations in 2 cases, p16INK4a gene deletions and
loss of expression in 2 cases, and p21Waf1 loss of expression
in 1 case. One additional aggressive MCL had a hemizygous
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In this study we have examined the gene structure and
expression of the p16INK4a and p21Waf1 CDKIs genes in 18
typical and 6 aggressive variants of MCLs. All these cases
had been previously characterized for the expression of
cyclin D1, retinoblastoma status, and the presence of p53
mutations.8,28,34 Deletions and/or loss of expression of
p16INK4a and p21Waf1 genes were found only in 4 of the 24
(17%) MCLs. However, the four cases were in the histologically aggressive subgroups, further classified as blastic or
large cell variants of MCL. mRNA and/or protein expression
were detected in all typical MCLs with low proliferative
activity and longer survival. Concordantly, no gene deletions
were observed in any of these typical MCLs. These findings
indicate that alterations of the CDKI p16INK4a and p21Waf1
genes in MCLs are a relatively infrequent phenomenon (17%
of all the MCLs examined). However, the presence of deletions and loss of expression in 4 of the 6 histologically
aggressive cases (67%) with higher proliferative activity and
shortened survival indicates that inactivation of these CDKIs
genes may be involved in the pathogenesis of a subset of
aggressive MCLs. All of our aggressive MCLs were diagnosed at presentation, suggesting that these molecular alterations may occur in early phases of the evolution of these
lymphomas. Aggressive variants may also evolve as a pro-
Fig 2. SSCP analysis of p16INK4a exon 2. The abnormal mobility
observed in the typical mantle cell lymphoma 2166 (A) is the result
of the known polymorphism in codon 148 with the change GCG
(alanine) r ACG (threonine) (B).
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CDKIs IN MANTLE CELL LYMPHOMA
277
Fig 3. Western blot analysis of p21Waf1 and
p16INK4a in typical and aggressive mantle cell lymphomas. The aggressive MCL 441 shows a loss of p21Waf1
protein expression whereas MCL 3858 and 15566
have a loss of p16INK4a protein expression. These
genes are constantly expressed in the typical variants of MCLs.
gression of typical MCLs. It will be interesting to investigate
the possible implication of CDKIs genes in the aggressive
transformation of more indolent typical MCL variants.
Homozygous deletions of p16INK4a and p15INK4b CDKIs
genes are a common genetic event in acute lymphoblastic
leukemias (ALL), particularly of T-cell phenotype. Several
studies have reported homozygous deletions of the p16INK4a
gene in 18% to 83% of T-ALL and in 8% to 21% of BALL.44,47-50 The status of these genes in non-Hodgkin
lymphomas (NHLs) has been less well examined, but gene
deletions seem to be an infrequent phenomenon in these
neoplasms, ranging from 0% to 7% of the cases. However,
most of these deletions have been described in diffuse large
cell lymphomas de novo or in the transformed phase of a
low-grade NHL.50,51 Our findings in MCLs are concordant
with these studies in other NHL. Deletions of p16INK4a/
p15INK4b genes or the 9p21 chromosomal region where these
genes are mapped were found in 3 of the 6 aggressive variants of MCL (50%) but in none of the typical MCL. Koduru
et al51 have recently examined the presence of p16INK4a/
p15INK4b gene deletions in a large series of NHL including
Fig 4. Northern blot analysis of p16INK4a in typical and aggressive
mantle cell lymphomas. The aggressive MCL 3858 shows a loss of
p16INK4a mRNA expression. This finding is concordant with the lack
of protein expression and the homozygous deletion of the gene in
this case (shown in Figs 1 and 3, respectively). The blots were
stripped of the p16INK4a probe and rehybridized with the 28S probe
as a loading control.
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13 cases with the t(11;14)(q13;q32). A deleted p16INK4a/
p15INK4b gene was found in 4 of the 13 lymphomas with the
t(11;14) (31%), but only in 9 of 141 NHL (6%) with other
karyotypic abnormalities or normal karyotypes. In this study,
the NHLs were classified according to the Working Formulation and, consequently, the diagnosis of mantle cell
lymphoma was not considered in any of the cases. Interestingly, the four cases with the t(11;14) and deletions of
p16INK4a/p15INK4b genes were classified as diffuse large cell
lymphomas. The other 9 cases with the t(11;14) and no
deletions of the p16INK4a/p15INK4b genes were classified as
other low- or intermediate-grade categories of the Working
Formulation. Because the t(11;14) translocation is now recognized as a genetic alteration highly characteristic of MCL
and it is only very infrequently found in other NHLs,52 it is
possible that these cases with the t(11;14) and a deleted
p16INK4a/p15INK4b gene may represent aggressive variants of
MCLs. These observations and the findings in our study
would suggest that deletions of p16INK4a/p15INK4b genes may
be an important genetic event in the pathogenesis of aggressive variants of MCLs.
The p16INK4a gene expression in lymphoproliferative disorders has been less well analyzed than the structure of the
gene. In our study, p16INK4a expression was observed at
mRNA and/or protein levels in all typical MCLs. However,
Fig 5. Northern blot analysis of p21Waf1 in typical and aggressive
mantle cell lymphomas. The aggressive MCL 441 shows a loss of
p21Waf1 mRNA expression. This finding is concordant with the lack
of protein expression in this case (shown in Fig 3). The blots were
stripped of the p16INK4a probe and rehybridized with the 28S probe
as a loading control.
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278
PINYOL ET AL
no expression of the gene was detected in two (40%) aggressive variants with a blastic morphology. The Southern blot
analysis of these two cases showed a homozygous deletion
of p16INK4a gene. The loss of p16INK4a expression in the two
blastic MCLs confirms the implication of this gene in the
development of these aggressive variants of MCLs.
The levels of p16INK4a expression in two other aggressive
MCLs were much higher than the levels detected in the
typical MCLs. High levels of p16INK4a expression have been
detected in cell lines with lack of pRb function. Several
studies have provided evidences for a regulatory loop between p16INK4a expression, retinoblastoma protein, and cyclin
D/cdks complexes.53,54 In this model, transcription of p16INK4a
would be repressed by unphosphorylated pRb whereas phosphorylated pRb would activate p16INK4a transcription. Concordantly with this idea, we have shown in a previous study
that the two aggressive MCLs with p16INK4a overexpression
showed high levels of phosphorylated pRb whereas the typical MCLs constantly expressed low levels of pRb with negligible phosphorylated forms.34 The inefficient inhibitory activity of p16INK4a in these cases may be caused by alterations
in other inhibitory molecules. In fact, the two aggressive
cases with p16INK4a overexpression had a loss of p21Waf1
expression and a p53 mutation, respectively.
The p21Waf1 gene is regulated by p53 and can suppress
the growth of human normal and tumor cells by inhibiting
the activity of cyclins/CDKs complexes.18,19 Overexpression
of p21Waf1 has been shown to suppress proliferation and tumorigenicity in several human cell lines including Burkitt’s
lymphoma cells.21,22 The possible role of p21Waf1 gene alterations in the pathogenesis of human lymphoproliferative disorders has not been well examined. In this study, p21Waf1
protein and mRNA expression were detected in all typical
MCLs. However, one aggressive MCL with a blastic morphology showed a loss of both mRNA and protein expression, suggesting that abrogation of p21Waf1 expression may
be implicated in the higher proliferative activity of this tumor. The other 5 aggressive MCLs showed similar levels of
expression as the typical variants. p21Waf1 expression in these
lymphomas seems to be independent of the status of the p53
gene. No mutations were found in exons 4 to 10 in the blastic
MCL with no expression of p21Waf1. On the other hand, two
aggressive MCLs with p53 mutations showed similar levels
of p21Waf1 expression at mRNA and protein levels as the
tumors with wild-type p53. Several studies have now shown
p53-independent induction of p21Waf1 expression, particularly in the terminal differentiation process of several cell
lineages20,55,56 and in response to different growth factors.57
The presence of p21Waf1 expression in two aggressive MCLs
with p53 mutations suggests that other p53-responsive genes
may be involved in the pathogenesis of these tumors. No
p21Waf1 gene deletions or mutations were detected in our
blastic MCL with no p21Waf1 expression, indicating that other
mechanisms must be implicated in the inactivation of the
expression of this gene.
The comparative analysis of alterations in our series of
MCLs shows that these molecular abnormalities occur independently of each other. The independent occurrence of p53,
p16INK4a, and p21Waf1 gene alterations supports the idea that
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inactivation of any one of these genes is sufficient to induce
a higher proliferative activity of the tumors. p16INK4a, cyclin
D1, and retinoblastoma protein seem to participate in the
same cell-cycle regulatory pathway.30-33 Inactivation of
p16INK4a expression and overexpression of cyclin D1 cooperate synergistically to deregulate cell proliferation in cell lines
with normal pRb function.33 Our observations would suggest
that p53 mutations and p21Waf1 downregulation may also
cooperate with cyclin D1 overexpression in cell-cycle deregulation in tumors with a normal pRb. The constant overexpression of cyclin D1 in MCLs has been considered to play
an important role in the pathogenesis of these lymphomas.
The findings of this study would indicate that the additional
alteration in one of the negative cell-cycle regulatory genes
p53, p16INK4a, or p21Waf1 may lead to development of more
aggressive variants of these tumors with higher proliferative
activity and shorter survival of the patients.
ACKNOWLEDGMENT
The authors thank Dr M. Serrano for the gift of the p16INK4a probe
and his comments on the manuscript, and Iracema Nayach and Nerea
Peiro for excellent technical assistance.
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1997 89: 272-280
Deletions and Loss of Expression of P16INK4a and P21Waf1 Genes Are
Associated With Aggressive Variants of Mantle Cell Lymphomas
Magda Pinyol, Luis Hernandez, Maite Cazorla, Milagros Balbi?n, Pedro Jares, Pedro L. Fernandez,
Emilio Montserrat, Antonio Cardesa, Carlos Lopez-Oti?n and Eli?as Campo
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