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Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2977-2984
p16INK4a Gene Inactivation by Deletions, Mutations, and
Hypermethylation Is Associated With Transformed and Aggressive
Variants of Non-Hodgkin's Lymphomas
By
Magda Pinyol,
Francesc Cobo,
Silvia Bea,
Pedro Jares,
Iracema Nayach,
Pedro L. Fernandez,
Emilio Montserrat,
Antonio Cardesa, and
Elias Campo
From the Hematopathology Section, Laboratory of Pathology, and
Department of Hematology, Hospital Clinic, University of Barcelona,
Barcelona, Spain.
 |
ABSTRACT |
The molecular mechanisms underlying the pathogenesis of aggressive
lymphomas and the histological transformation of indolent variants are
not well known. To determine the role of p16INK4a gene
alterations in the pathogenesis of non-Hodgkin's lymphomas (NHLs) and
the histological progression of indolent variants, we have analyzed the
expression, deletions, and mutations of this gene in a series of 112 NHLs. Hypermethylation of the gene was also examined in a subset of
tumors with lack of protein expression but without mutations or
deletions of the gene. p16INK4a gene alterations were
detected in 3 out of 64 (5%) indolent lymphomas but in 16 out of 48 (33%) primary or transformed aggressive variants. In the low-grade
tumors, p16INK4a alterations were detected in 1 (4%)
chronic lymphocytic leukemia (hemizygous missense mutation), 1 (6%)
follicular lymphoma (homozygous deletion), and 1 (5%) typical mantle
cell lymphoma (homozygous deletion). The two later cases followed an
aggressive clinical evolution. In the aggressive tumors,
p16INK4a gene alterations were observed in 2 (29%)
Richter's syndromes (2 homozygous deletions), 3 (33%) transformed
follicular lymphomas (1 homozygous deletion and 2 nonsense mutations),
3 (43%) blastoid mantle cell lymphomas (2 homozygous and 1 hemizygous
deletions), 5 (28%) de novo large-cell lymphomas (1 homozygous
deletion and 4 hypermethylations), 2 lymphoblastic lymphomas (2 homozygous deletions), and 1 of 2 anaplastic large cell lymphomas
(hypermethylation). Protein expression was lost in all tumors with
p16INK4a alterations except in the typical chronic
lymphocytic leukemia (CLL) with hemizygous point mutation. Sequential
samples of the indolent and transformed phase of three cases showed the
presence of p16INK4a deletions in the Richter's syndrome
but not in the CLL component of two cases, whereas in a follicular
lymphoma the deletion was present in both the follicular tumor and in
the diffuse large-cell lymphoma. In conclusion, these findings indicate
that p16INK4a gene alterations are a relatively infrequent
phenomenon in NHLs. However, deletions, mutations, and hypermethylation
of the gene with loss of protein expression are associated with
aggressive tumors and they may also participate in the histological
progression of indolent lymphomas.
| |
INTRODUCTION |
LYMPHOID MALIGNANCIES are a heterogeneous
group of disease entities characterized by distinctive clinical,
morphological, immunophenotypical, and genetic features.1
These neoplasms can be generally divided into indolent and aggressive
tumors on the basis of the clinical presentation, histology, clinical
course, and response to therapy. The majority of aggressive
non-Hodgkin's lymphomas (NHLs) are primary tumors recognized at
diagnosis. In addition, indolent lymphomas are characterized by a
relatively frequent transformation to more aggressive variants. The
frequency of this transformation varies in different entities and,
thus, it may occur in 1% to 10% of chronic lymphocytic leukemias
(CLLs) small lymphocytic lymphomas2 but in 25% to 70% of
low grade follicular lymphomas.3 Aggressive variants of
mantle cell lymphomas (MCLs) are generally diagnosed at presentation.
However, progression of typical MCLs into more aggressive variants can
also occur in 24% to 39% of cases.4,5 Morphological
transformation of indolent lymphomas is associated with a rapidly
progressive clinical course and short survival of the patients.
Cytogenetic and molecular studies of NHLs have identified a series of
gene alterations usually associated with specific disease entities.
However, the molecular mechanisms responsible for the pathogenesis of
most primary high-grade tumors and the progression of indolent
lymphomas are not well known. p53 inactivation and c-myc rearrangements
have been implicated in the transformation of a number of indolent
lymphomas. Particularly, p53 mutations are relatively rare in low-grade
tumors, but they are found in 20% to 50% of high-grade B-cell
lymphomas.6 In addition, p53 mutations have also been
associated with progression in 25% to 40% of indolent lymphomas
including transformed CLLs,7 transformed follicular
lymphomas (FCLs),8,9 aggressive variants of
MCLs,4,10 and progressed mucosa-associated lymphoid tissue
lymphomas.11 These findings indicate that p53 inactivation
is an important pathway in the pathogenesis of a subset of primary
high-grade and transformed lymphomas. However, they also suggest that
other molecular mechanisms must be implicated in the development of these aggressive variants of tumors.
Cyclin-dependent kinase inhibitors (CDKIs) represent a class of
negative regulatory elements of cell growth that suppress the kinase
activity of the cyclin/CDK complexes. Among all these molecules,
p16INK4a has been implied as a tumor-suppressor gene.
Inactivation of this gene by homozygous deletions, mutations, and
hypermethylation occurs in a wide array of human
tumors.12-15 In hematologic disorders, homozygous deletions
are frequently found in acute lymphoblastic leukemias (ALL), mainly of
T-cell origin. In contrast, p16INK4a alterations in NHLs
seem to be rare, and its possible implication in the pathogenesis and
progression of these neoplasms is not well known. Some studies have
detected p16INK4a deletions in primary large-cell lymphomas
and sporadic transformed tumors.16-18 However, a clear
association between p16INK4a alterations and aggressive
variants of NHLs has not been observed in other
series.19,20 In addition, the role of these alterations in
the progression of indolent lymphomas has not been specifically addressed in previous studies.
To determine the possible implication of p16INK4a gene
alterations in the development and progression of aggressive variants
of NHLs, we have analyzed its gene structure and protein expression in
a large series of NHLs, including a number of histologically transformed cases. Our results indicated that p16INK4a gene
alterations are rare in low-grade lymphomas, but the inactivation of
the gene by homozygous deletions, point mutations, or hypermethylation with loss of protein expression mainly occurred in primary aggressive and transformed lymphomas.
| |
MATERIALS AND METHODS |
Case selection.
Tumor specimens from 112 NHLs were selected based on the availability
of frozen tissue for molecular analysis and classified according to the
Revised European-American Classification of Lymphoid Neoplasms.1 The tumors were grouped into indolent and
aggressive categories. Among indolent NHLs, we studied 24 CLLs, one
hairy cell leukemia (HCL), 18 FCLs, and 21 typical MCLs. Aggressive NHLs comprised 7 large-cell lymphomas evolved from CLLs (Richter's syndrome), 9 diffuse large-cell lymphomas (LCLs) transformed from FCLs,
7 blastoid variants of MCLs, 18 de novo B-cell diffuse LCLs, 4 lymphoblastic lymphomas (LBLs), 3 of B-cell and 1 of T-cell phenotype,
1 Burkitt's lymphoma (BL), and 2 anaplastic large cell lymphomas
(ALCLs). Data from 24 MCLs (18 typical and 6 blastoid variants) have
been reported elsewhere.21 Frozen material from sequential
samples were available in 2 CLLs and 1 FCL and their subsequent
transformed large-cell lymphoma.
Southern blot analysis.
Genomic DNA was extracted from frozen material in 105 cases (24 CLLs, 7 LCLs transformed from CLLs, 1 HCL, 16 FCLs, 9 LCLs evolved from FCLs,
21 MCLs, 7 blastoid MCLs, 14 de novo LCLs, 1 BL, 3 LBLs, and 2 ALCLs)
using proteinase K/RNAse treatment and phenol-chloroform extraction.
Southern blot analysis could be performed in 95 of these cases (Table
1). DNA from each case (15 µg) was
digested with EcoRI, HindIII, and BamHI and
analyzed as previously described.21,22 The
p16INK4a probe used was a 0.8-kb EcoRI-Xho
I fragment of the p16INK4a cDNA clone.23 The
 -actin probe was also used as a loading control. The probes were
radiolabeled using a random primer DNA labeling kit (Amersham
Life Science, Buckinghamshire, UK) with [ -32P]dCTP. The intensity of the autoradiographic
signals were quantified using a UVP-5000 video densitometer (UVP, San
Gabriel, CA).
Single-stranded conformation polymorphism (SSCP) analysis and DNA
sequencing.
SSCP analysis was used to screen for p16INK4a gene
mutations according to a previously described method.21,24
Exons 1 and 2 of the p16INK4a gene were amplified by
polymerase chain reaction (PCR) by using a simple set of flanking
intronic primers. Primers for exon 1 were 5 -GAAGAAAGAGGAGGGGCTG-3 and
5-GCGCTACCTGATTCCAATTC-3, and primers for exon 2 were
5-CTCTACACAAGCTTCCTTTCC-3 and 5-GGGCTGAACTTTCTGTGCTGG-3. We used a
"touch-down" PCR strategy for the amplification of both exons as
previously described.21 For the SSCP analysis, the PCR
products of both exons were digested with Sma I, 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 using a
silver staining procedure as previously described.21
Samples with an altered mobility were sequenced using a commercial
cycle sequencing kit (Perkin Elmer, Branchburg, NY) and -33P dATP as previously described.21 A total
of 0.5 µL of the p16INK4a gene PCR products were used as
template for sequencing. The primers described previously 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 µmol/L. The reaction was performed according to the instructions supplied by the manufacturer. 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 104 cases (Table 1). Protein extracts from the HeLa cell line were
used as positive control. In each case, 10 frozen sections of 30 µm
were incubated in 300 µL 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 µg/mL aprotinin, 1 µg/mL
leupeptin, and 1 µg/mL -1-antitrypsin for 20 minutes at 4°C. The
cell debris was sedimented by centrifugation at 14,000 revolutions per
minute at 4°C 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 were run per lane on a 15% SDS-polyacrylamide gel and
electroblotted to a nitrocellulose membrane (Amersham). The blocked
membrane was incubated with the monoclonal antibody
anti-p16INK4a, clone G175-405 (Pharmingen, San Diego, CA)
at a final concentration of 1 µg/mL for 1 hour and 30 minutes, washed
with phosphate-buffered saline 0.1% Tween-20, and exposed to sheep
antimouse conjugated to horseradish peroxidase (Amersham) at a 1:1000
dilution for 1 hour and 30 minutes. After washing, antibody binding was
detected by chemiluminescence detection procedures according to the
manufacturer's recommendations (ECL; Amersham).
Methylation analysis.
A PCR assay was performed to analyze the methylation status of the
first exon of the p16INK4a gene. Thirty units of
methylation-sensitive restriction enzymes Sac II (Bio-Labs,
Beverly, MA) and Sma I (Promega, Madison, WI) were
used to digest 0.2 µg of DNA. Digested DNA was then used as a
template in a multiplex PCR reaction for exon 1 of p16INK4a
and -globin. -globin was amplified as internal control of the PCR
reaction because the amplified fragment of this gene does not contain
Sma I or Sac II sites. The primers used for the
methylation analysis of p16INK4a exon 1 were described
previously. Primers for the -globin gene were
5-ACACAACTGTGTTCACTAGC-3 and 5-CAACTTCATCCACGTTCACC-3. We used a
touch-down 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
annealing temperature at 67°C; 35 cycles with annealing temperature
at 66°C; and a final step for 5 minutes at 72°C. PCR products were
resolved on 2% agarose gel. Only cases with methylated DNA were
expected to show amplified product of p16INK4a exon 1, whereas no amplification of this exon was obtained in nonmethylated
cases. HeLa and Raji cell lines were used as negative and positive
controls of methylation, respectively.
| |
RESULTS |
Analysis of p16INK4a gene deletions.
The p16INK4a gene was examined by Southern blot in 95 cases
including 55 indolent and 40 aggressive lymphomas in which genomic DNA
was available (Table 1 and Fig 1).p16INK4a homozygous deletions were detected in 10 tumors.
In one additional blastoid MCL, in which no DNA was available, the
cytogenetic analysis identified a hemizygous deletion affecting the
9p21 locus.21 Therefore, p16INK4a deletions
were present in 9 (22.5%) aggressive lymphomas but only in 2 (3.6%)
indolent tumors (Table 1). The two indolent lymphomas with
p16INK4a deletions were a grade II FCL (case 291) and a
typical MCL (case 8216; Fig 1A). In both cases, the
p16INK4a deletion was considered to be homozygous and it
was also associated with a homozygous deletion of the
p15INK4b gene. Interestingly, these two cases showed a very
aggressive clinical behavior. In fact, the FCL underwent a progression
to a diffuse LCL in 12 months and the patient died 14 months after transformation. This transformed phase also showed a homozygous deletion of the p16INK4a gene (Fig 1B, case 1106). The MCL
showed a relatively high proliferative index (2.6 mitosis × high
power field) and the patient died 19 months after the initial diagnosis
with no response to the therapy. The survival of these two patients was
much shorter than the median survival of other patients with FCLs and
typical MCLs in our institution, which were 102 and 48 months,
respectively.
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| Fig 1.
Southern blot analysis of 10 indolent (A) and 13 aggressive (B) NHLs. The indolent lymphoid neoplasms 321b (CLL), 577 (CLL), and 291 (FCL) in A have their subsequent sample of the
progressed lymphoma in B, cases 321 (Richter's syndrome), 8324 (Richter's syndrome), and 1106 (transformed FCL), respectively.
Genomic DNA was digested with HindIII restriction enzyme and
hybridized with the exon 2 of p16INK4a and -actin
probes. (A) Indolent lymphomas showed germline configuration except
cases 291 (FCL) and 8216 (typical MCL), which had a homozygous deletion
of the p16INK4a gene. (B) Analyses of transformed and
aggressive variants of NHLs showed homozygous deletions of both
p15INK4b and p16INK4a genes in cases 321 and
8324 (Richter's syndrome), 1106 (transformed FCL), and 9630 (diffuse
LCL). Case 2286 (lymphoblastic lymphoma) showed a homozygous deletion
of p16INK4a but no p15INK4b. The identification
number of these cases is the same as that in Figs 2, 3, and 4.
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The nine aggressive lymphomas with p16INK4a deletions were
two (29%) LCLs transformed from CLLs (Richter's syndrome), one (11%) LCL transformed from FCL, three (43%) blastoid MCLs, one (9%) de novo
diffuse B-cell LCL, and two (67%) LBLs, one of B- and one of T-cell
phenotype (Table 1). All p16INK4a deletions were associated
with p15INK4b deletions except in one LBL in which
p15INK4b was in germline configuration. No isolated
p15INK4b deletions were detected in any case. DNA from
sequential samples of the indolent tumor and the subsequent transformed
LCL were analyzed in the two CLLs and the FCL with p16INK4a
homozygous deletions. In the two CLLs, the p16INK4a
homozygous deletions were detected in the LCL but not in the CLL
component, whereas in the FCL (case 291 previously commented) it was
present in both the follicular tumor and in the diffuse LCL (Fig 1).
Mutational analysis of p16INK4a gene.
To determine whether mutations of the p16INK4a gene were
present in these lymphomas, we analyzed exon 1 and 2 of the
p16INK4a gene by PCR-SSCP. Cases with anomalous migrating
bands were sequenced (Table 1). Mutations were only found in one
indolent lymphoma (1.6%) and 2 aggressive tumors (5.5%). The mutated
indolent lymphoma was a typical CLL. This case showed a mutation at
codon 143 (GCC  ACC), resulting in a change of alanine by
threonine. This case showed a p16INK4a germline by Southern
blot analysis and normal residual bands in the SSCP and sequencing
analysis suggesting that this mutation was hemizygous. No differences
in the clinical course were observed in this case when compared with
the nonmutated CLLs. The other two mutated cases were two diffuse LCLs
progressed from FCLs. The two cases showed the same nonsense mutation
at codon 80 with the change CGA (Arginine) TGA (Stop) (Fig
2). Codon 80 is considered a mutational hot
spot within p16INK4a and it is frequently mutated in cell
lines and neoplasms.12,25 No signal of the wild allele was
observed in the SSCP and sequencing analysis and no protein expression
was detected in any of these two cases by Western blot (see later).
Five additional tumors, two FCLs, one typical MCL, and two LCLs showed
an abnormal migrating band in exon 2. This altered mobility was the
result of the known polymorphism at codon 148 with the change GCG
(Alanine) to ACG (Threonine).
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| Fig 2.
SSCP analysis of p16INK4a exon 2. The
abnormal mobility observed in two transformed FCLs (case 17000 and
3834) (A) is the result of the mutation in codon 80 with the change CGA
(Arg) TGA (Stop) (B). The western blot analysis of these two cases
is shown in Fig 3.
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P16INK4a gene expression.
To assess the possible alterations of p16INK4a gene
expression in these lymphomas, we examined the protein levels by
Western blot analysis in 104 cases. Some MCLs had also been studied
previously by Northern blot.21 Complete loss or very weak
p16 protein expression was observed only in 2 of the 59 (3.4%)
indolent lymphomas but in 14 of the 45 (32%) aggressive lymphomas
(Table 1 and Fig 3). Protein expression was
observed in all other tumors. The two indolent lymphomas with no
protein expression were the FCL (case 291) and the typical MCL (case
8216) with homozygous deletion of the gene. The CLL with a mutated
allele showed protein expression at similar levels than other
nonmutated CLLs. Nine of the 14 aggressive lymphomas with loss of
protein expression had shown genetic abnormalities in the Southern blot
or mutational analysis including biallelic p16INK4a gene
deletions in 7 cases and 2 homozygous nonsense mutations at codon 80 in
two transformed FCLs (Table 1). Interestingly, no gene deletions or
mutations were found in the 5 additional aggressive lymphomas, 4 de
novo LCLs and one ALCL, with lack of protein expression, suggesting
that other genetic alterations could be implicated in the inactivation
of the gene.
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| Fig 3.
Western blot analysis of p16INK4a in indolent
(A) and aggressive (B) lymphomas. Two transformed FCLs (3834 and 1700),
one LBL (2286), and two diffuse large B-cell lymphomas (9630 and 15136)
show a loss of protein expression. The Southern blot analysis of cases 17000 (T-FCL), 2286 (LBL), 9630 (DLCL), and 15136 (DLCL) is shown in
Fig 1. Tumors 2286 and 9630 showed a homozygous deletion of p16INK4a gene (Fig 1), whereas tumors 17000 and 15136 with
germline configuration in the Southern blot analysis had a stop codon
(Fig 2) and hypermethylation of the gene (Fig 4), respectively.
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Methylation analysis.
To know if the lack of protein expression in these later five
aggressive lymphomas with no evidence of deletions or mutations could
be caused by hypermethylation of the gene, DNA from these cases was
digested with the methylation sensitive restriction enzymes Sac
II and Sma I and amplified with a multiplex PCR including primers for p16INK4a exon 1 and -globin as an internal
control. All these cases showed the expected 340-bp band of exon 1 of
p16INK4a and the -globin band at 110 bp, indicating that
all these cases were methylated at exon 1 (Fig
4). To confirm these results, we also
studied the methylation status in five more cases (one FCL, two MCLs,
one blastoid-MCL, and one LCL progressed from a CLL) with p16 protein
expression. No hypermethylation of the gene could be shown in these
cases.
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| Fig 4.
Methylation status of the p16INK4a exon 1 in
NHL analyzed by PCR and comparison with the results of the protein
expression by Western blot analysis (WB). Products of
p16INK4a exon 1 and the internal control -globin are
indicated. The amplification of exon 1 after digestion with Sma
I indicates that this restriction site was methylated. Cases with
p16INK4a exon 1 amplification showed loss of protein
expression by Western blot whereas protein expression was detected in
cases in which p16INK4a exon 1 was not amplified.
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DISCUSSION |
The genetic basis underlying histological progression in NHLs is not
well known. p53 mutations are a common alteration in aggressive
tumors6 and they are also associated with morphological progression in different types of indolent lymphomas.4,7-11 In addition, c-myc rearrangements have been implicated in the transformation of occasional FCLs and CLLs.26,27 However,
alterations of these genes occur in 10% to 50% of the tumors
indicating that other mechanisms must be also involved in this process.
The strong inhibitory action of p16INK4a gene on cell cycle
progression,28 its frequent inactivation in advanced stages
of different solid tumors,29-31 and the spontaneous development of B-cell lymphomas with an aggressive morphology in the
INK4a knock-out mice32 suggest that this gene may also be a
target in the pathogenesis of aggressive and transformed lymphomas.
In hematologic malignancies, p16INK4a gene alterations have
been mainly described in lymphoid rather than myeloid
neoplasms.24,33-37 However, in contrast to the high number
of deletions found in acute lymphoblastic leukemias, the incidence of
p16INK4a homozygous deletions and mutations observed in
NHLs has been low, ranging from 0% to 15% of the cases. In most of
these studies,18,24,38-40 the NHLs examined were part of a
larger series of hematologic neoplasms with no further information on
the histological type of the lymphomas or the clinical evolution of the
patients. Some other studies have found p16INK4a deletions
in occasional diffuse lymphomas.16,17 However, the association between p16INK4a alterations and aggressive
variants of the tumors was not clear in other series in which the
incidence of p16INK4a alterations were similar in low- and
high-grade lymphomas.19,20 In this study, we have detected
p16INK4a gene alterations in 33% of primary high-grade and
33% of transformed lymphomas, but only in three (5%) histologically
indolent tumors indicating that inactivation of this gene may
participate in the development of primary aggressive and progressed
lymphomas. In addition, two of the indolent cases with
p16INK4a alterations were a typical MCL and a grade II FCL
that followed an aggressive clinical evolution. In a previous study on
MCLs, we found p16INK4a deletions and loss of gene
expression only in blastoid variants of this lymphoma.21 We
have now expanded the study and found p16INK4a homozygous
deletions with loss of protein expression in one case that was
morphologically a typical MCL. However, this patient had no response to
therapy and died in 19 months, an overall survival similar to that
observed in the blastoid variants of MCL. The FCL also had a homozygous
deletion of the p16INK4a gene with loss of protein
expression and followed an aggressive evolution with transformation to
an LCL. The third low-grade lymphoma with p16INK4a
alteration was a typical CLL with a hemizygous missense mutation, protein expression, and a clinical behavior similar to other typical CLLs. These findings suggested that complete abrogation of p16 expression in morphologically indolent lymphomas may confer to the
tumor a proliferative growth advantage similar to that observed in
aggressive lymphomas.
Whether p16INK4a gene alterations are involved in
histological progression of indolent lymphomas is not well known.
Biallelic loss of the p16INK4a gene has been reported in
isolated cases of lymphoblastic transformation of chronic myeloid
leukemias,41 chronic and acute phases of adult T-cell
leukemias,42 and sporadic peripheral
lymphomas.17,18 However, very few cases of transformed
lymphomas have been included in previous series. In this study, we have
observed that p16INK4a gene inactivation, either by
biallelic deletions or, less frequently, point mutations, is a
relatively common phenomenon in progressed NHLs. Specifically, we found
inactivation of the p16INK4a gene in 33% of transformed
CLLs, FCLs, and blastic MCLs but only in 5% of the indolent
counterparts. The incidence of p16INK4a alterations in
transformed lymphomas in our study is similar to the number of p53
alterations detected previously in other series of progressed lymphomas
and suggests that inactivation of this gene may also define a molecular
pathway in lymphoma progression.
In three of our progressed cases, two CLLs and one FCL, we were able to
study the indolent component and the subsequent transformed LCL. In the
two CLLs, a p16INK4a homozygous deletion was detected in
the LCL but not in the CLL phase, whereas in the FCL the homozygous
deletion was present in both components, the follicular and the
subsequent diffuse LCL. Although p53 mutations have been frequently
described in Richter's syndrome,7 they may be related to
the development of a new malignant clone rather than progression of
indolent CLLs.43 In our two CLLs, the immunoglobulin heavy
chain gene showed the same rearrangement pattern in the indolent and
aggressive component suggesting a clonal evolution. The finding of the
p16INK4a biallelic deletion in the progressed but not in
the low-grade phase in these two cases suggested that this alteration
was acquired during the transformation process and may have played a
role in its pathogenesis. In the FCL the homozygous deletion was
detected in both the indolent tumor and in the progressed LCL. The
clinical evolution in this case was more aggressive than in
conventional FCLs suggesting that this molecular alteration may have
had an influence in this evolution. Similarly to this case, c-myc
rearrangements and p53 overexpression have also been found in the
indolent component of FCLs that underwent morphological transformation
to LCLs8,9,26 indicating that these alterations may occur
early in the development of these lymphomas and may participate in
their progression.
The majority of inactivating alterations of the p16INK4a
gene in human tumors are homozygous deletions rather than point
mutations.12,25 Concordantly, p16INK4a gene
deletions, generally associated with deletions of the
p15INK4b gene, were the most frequent alteration detected
in our study. p16INK4a point mutations are rare in lymphoid
neoplasms, ranging from 0% to 7% of the cases.20,44,45
Similarly, we only detected three point mutations (3%) in our series,
a missense mutation at codon 143 in a CLL and two nonsense mutations at
codon 80 in two transformed FCLs. Nonsense mutations or microdeletions
and insertions leading to subsequent stop codons have been frequently described in hematologic neoplasms.20,44-46 In solid
tumors, nonsense mutations of the p16INK4a gene are also
relatively frequent (30%) compared with the number of these mutations
in the p53 gene (8%).25 It has been postulated that this
difference may reflect different mechanisms of inactivation of these
proteins. Missense mutations in the p53 gene frequently disrupt its
function, whereas it is possible that p16INK4a may tolerate
some missense mutations without impairing the normal function of the
protein.25,47,48 In this respect, the only missense
mutation in our study was seen in an indolent CLL, whereas the two
nonsense mutations were present in two transformed FCLs. This missense
mutation (codon 143) was outside the ankyrin repeat motifs of the gene
and located in a region that is not required for the protein to bind
and inhibit CDK4,47,48 suggesting that this mutation was
most probably not functionally significant. Interestingly, 5 out of 6 mutations previously described in indolent lymphomas (CLLs and an FCL)
were missense mutations,20,44 whereas 7 out of 10 mutations
in T-ALL or high-grade lymphomas were nonsense mutations
or microdeletions and insertions with subsequent stop codons.20,36,45,46
The p16INK4a gene expression in lymphoproliferative
disorders has been less well examined than the structure of the
gene.21,49 In this study we detected loss of p16 protein
expression in 16 of the 104 (15%) lymphomas examined. Loss of protein
expression in our tumors was clearly associated with disruptive
alterations of the gene in 11 cases, 9 with homozygous deletions and 2 with nonsense mutations. However, 5 additional aggressive lymphomas showed loss of protein expression without clear anomalies of the gene
in the Southern or SSCP analyses. These 5 tumors showed
hypermethylation of exon 1, suggesting that this mechanism was leading
to the inactivation of the gene in these tumors. Hypermethylation of 5
CpG islands of the p16INK4a gene has been recently
described as an alternative inactivating mechanism of this gene in
different human tumors including hematologic malignancies.14,19,34,50 In this respect, Herman et
al34 have recently shown hypermethylation of the
p16INK4a gene in 5 of 6 high-grade lymphomas but only in 1 of 6 low-grade tumors. These findings suggest that p16INK4a
hypermethylation may also be a mechanism involved in the development of
aggressive lymphomas. Hypermethylation of the p15INK4b gene
has also been described in acute lymphoblastic leukemias and rarely in
NHLs.34,49 However, the number of mature lymphomas included
in these studies is very scarce. It will be interesting to analyze
whether mutations and/or hypermethylation of this gene may also
be involved in the progression of indolent lymphomas.
In conclusion, our findings indicate that the p16INK4a gene
may be inactivated by homozygous deletions, point mutations, and
hypermethylation in NHLs. This inactivation is a relatively infrequent
phenomenon in low-grade tumors, but it is associated with aggressive
variants and may also define an alternative molecular pathway in the
histological transformation of indolent lymphomas.
| |
FOOTNOTES |
Submitted August 15, 1997;
accepted November 25, 1997.
M.P. and F.C. contributed equally to this study.
Supported by Grants SAF 96/61 from CICYT, 96SGR56 from CIRIT, and
Maraton-TV3 Cancer. M.P., F.C., S.B., and P.J. were fellows supported
by Maraton-TV3 Cancer (M.P.), Hospital Clinic (F.C.), and Spanish
Ministerio de Educacion y Cultura (S.B., P.J.).
Address reprint requests to Elias Campo, Laboratory of Pathology,
Hospital Clinic, 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.
| |
ACKNOWLEDGMENT |
The authors thank Dr Manuel Serrano for the gift of the
p16INK4a probe and his comments on the manuscript and Dr
Miguel A. Piris for the helpful discussions on the project.
| |
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Abstract
Introduction
Abstract