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From the Molecular Pathology Program,*
Centro Nacional
de Investigaciones Oncológicas Carlos III, Madrid; and the
Department of Genetics and Pathology,
Hospital
Virgen de la Salud, Toledo, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In lymphomas, the p53 gene has been found to be mutated in 19 to 35% of cases,10,11 whereas p16 has been found silenced in from 14 to 85% of cases, usually because of promoter hypermethylation or allelic loss, although rare mutations have been observed.12-15
p27/KIP1 (p27) is another CKI, which also acts at the restriction point, blocking Rb phosphorylation by cyclin E/CDK2 inactivation. p27 overexpression in a group of aggressive B-cell lymphomas with a high proliferative index and adverse clinical outcome has been shown to occur in previous studies.16-18 p27 overexpression has also been referred to in other types of tumors.19,20 This anomalous expression of p27 was described in association with the absence of p27/CDK2 complexes and the formation of p27/cyclin D3 complexes where p27 is inactive, stabilized, and detectable by immunohistochemical techniques.21,22
In vitro studies have demonstrated the existence of p27-cyclin D/CDK6 or p27-cyclin D/CDK4 complexes with Rb-kinase activity in cell lines of different types,23-26 suggesting that p27 is not active when joined to cyclin D/CDK4-6 complexes. A redistribution of p27 from cyclin E/CDK2 to cyclin D/CDK4 complexes, which precludes its negative regulator action on CDK2 activity, has also been described in growing cells, where p27 is associated with cyclin D/CDK4.21,22,27,28 p15 induction by transforming growth factor-ß displaces p27 from these complexes to cyclin E/CDK2, co-operating in inducing cell cycle arrest.23
The co-operation of the p16/Rb and p53/ARF pathways is suggested by evidence of the synergy found in in vitro analysis and the concomitant alteration in both pathways in many tumor types.29-37 Initial studies in non-Hodgkin’s lymphomas have shown hints of concerted inactivation in both pathways, as has also been described in virally -induced cell transformation.34 In mantle cell lymphomas it has recently been shown that simultaneous inactivation of the p16/Rb and p53/ARF pathways is associated with higher aggressivity,38 but this has not yet been fully explored in more frequent types of lymphoma, such as large B-cell lymphoma (LBCL) and Burkitt’s lymphoma (BL). We therefore explore the status and correlation of the p53 and p16 pathways and the possible relationship with anomalous p27 expression, to find if the accumulation of alterations in CKIs in the same tumor could give more aggressiveness to the tumor. To this end, we performed a molecular analysis for the p53 and p16 genes, together with an immunohistochemical study of p27 and Ki67 proliferation index, in a group of 62 cases of LBCLs and BLs.
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Materials and Methods |
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Sixty-two samples of tumor specimens from aggressive B-cell lymphoma, including 46 cases of LBCL and 16 BL, were obtained from the routine files of the Virgen de la Salud Hospital, and diagnosed using the criteria of the revised European-American Classification (REAL) classification.39
Cases were selected taking into account the existence of an adequate clinical follow-up and the availability of tissue for molecular studies. Some of the tumors in this series were included in previous studies of p53 mutations10,11 and p16 alterations.12
Molecular Studies
Allelic Loss Assays at the 9p21 Locus
Tumor and normal DNA were analyzed for loss of heterozygosity
(LOH) or homozygous deletion (HD) by amplification of dinucleotide
repeats containing sequence microsatellite markers,40
under conditions previously described by Villuendas and
colleagues.12
These markers are at the 9p21 region,
surrounding the p16 gene (IFN-
, D9S736, D9S1749, D9S1747, D9S974,
D9S1748, D9S1752, D9S171). Briefly, 100 ng of DNA was amplified in a
volume of 25 µl with 1x polymerase chain reaction (PCR) buffer, 200
µmol/L dNTPs, 10 pmol of each primer, 1 µCi of
32P-dCTP and 1 U of Taq polymerase.
Amplified products were separated by electrophoresis in denaturing 7
mol/L urea-6% acrylamide gels, followed by autoradiography. LOH was
considered to have occurred if an entire band was missing or was
reduced by >40% of the normal band. If the band was reduced by <40%
it was considered to be imbalanced.12,41
The presence of
HDs was assessed in all cases by comparative multiplex PCR assay, using
primer sets from loci outside the 9p21 region (D9S934 and D7S1824, in
chromosome 9q and 7q, respectively).
Methylation Studies
Methylation-specific PCR (MSP) assays were performed to analyze the methylation status of CpG islands of the first exon of p16, as described by Herman and colleagues.42 Briefly, 1 µg of denatured genomic DNA was modified by reaction with sodium bisulfite under conditions that convert all unmethylated cytosines to uracils. Modified DNA was purified using the Wizard DNA clean-up system (Promega, Madison, WI). Modification was completed by NaOH 0.3 mol/L treatment for 5 minutes at room temperature, followed by ethanol precipitation.
Bisulfite-modified DNA was amplified using p16 unmethylated-specific primers (U), methylated-specific primers (M), and unmodified or wild-type primers (W).42 Fifty ng of bisulfite-modified DNA was amplified using 1 U of AmpliTaq Gold (Applied Biosystems, Weiterstadt, Germany) under the following conditions: 30 seconds at 94°C, 30 seconds at 60°C (for U and W), or 65°C (for M) and 30 seconds at 72°C, for 35 cycles. Controls without DNA and positive controls for U and M reactions were performed for each set of PCRs. The PCR product was visualized under UV illumination in agarose gels stained with ethidium bromide. If a methylation-specific PCR product was detected, the whole procedure using sodium bisulfite and MSP was performed again to minimize the possible influence of contamination or incomplete bisulfite treatment.
DNA methylation was determined by the presence of a 150-pb fragment in those samples amplified with the M primers. DNA from nontumor samples (including peripheral blood lymphocytes from eight healthy donors and three reactive lymphoid nodes) was analyzed as a negative control. DNA from the Raji Burkitt cell line was used as a positive control of p16 methylation.43
Mutation Study
Mutations in exons 5 to 8 of p5310 and exon 2 of p1644 genes were analyzed by direct sequencing with an Automated DNA sequencer ABI PRISM 310 genetic analyzer (Applied Biosystems) according to the manufacturer’s procedures. The primers and conditions for this sequencing have been previously described.10,44
Immunostaining Techniques
All immunostaining techniques were performed in paraffin-embedded tissue sections, using a previous step of heat-induced antigen retrieval for all antibodies. Thus before incubation with the antibodies, the slides were heated in a pressure cooker for 3 minutes in a solution of 0.01 mol/L sodium citrate. After incubation with the primary antibodies, immunodetection was performed with biotinylated anti-mouse immunoglobulins, followed by peroxidase-labeled streptavidin (LSAB-DAKO, Glostrup, Denmark) with diaminobenzidine chromogen as substrate. All immunostaining was performed using the Techmate 500 (DAKO) automatic immunostaining device. Incubation omitting the specific antibodies was used as negative control of the technique.
p27 protein was detected with a monoclonal antibody, K25020 (1:1000 dilution; Transduction Laboratories, Lexington, KY), generated against the full-length mouse KIP1 protein. Expression in small lymphocytes was used as an internal control of immunostaining. p27 was scored in the following way: the mean value of the series, (17%), was chosen as a threshold for p27 reactivity. This is approximately equivalent to the level of expression of p27 within the germinal centers.
The proliferation index was evaluated using nuclear antigen Ki67 expression detected with MIB1 antibody from Novocastra (Newcastle on Tyne, UK). Cells showing nuclear staining were considered to be Ki67-positive. The Ki67 index was determined by scoring at least 300 tumor cells in well-preserved areas as a percentage of Ki67-positive cells. Ki67 reactivity was rounded to the nearest fifth percentile.
Statistical Analysis
Statistical study of the correlation between distributions was performed using either the chi-square test for categorical variables (Fisher’s exact test, Yates’ correction, or the Pearson test as appropriate) and the Kruskal-Wallis test for single ordered data. Survival curves were calculated by the Kaplan-Meier method and compared by log rank. Differences were considered significant if P < 0.05. We did not have appropriate clinical data for only 2 of 62 cases (two LBCL cases). The SPSS software package for Windows (SPSS, Inc., Chicago, IL) was used for all statistical analysis.
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Results |
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We searched for mutations in exons 5 to 8 of the p53 gene.
Fourteen mutations were identified in 13 cases (Table 1)
. Ten mutations were missense, and
there was one nonsense mutation. Three silent mutations were identified
in codon 213 (exon 6) in cases LB322, LB415, and BL324. In case BL324
two mutations were detected: one silent mutation in codon 213, and one
missense mutation in codon 215 (Table 1)
. In both codons, as well as in
the mutations detected in cases LB194 and LB271, the peak corresponding
to mutated nucleotide is higher than that corresponding to wild-type
(wt), suggesting that one allele is mutated and the other deleted. The
wt peak can be interpreted as being derived from the nontumoral cells
present in the sample.
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Analysis of p16 Alterations
9p21 Deletion
Forty-four cases, for which DNA from nontumor tissue was
available, were analyzed using markers for the microsatellites
surrounding the CDKN2A locus at 9p21. Allelic losses were detected in 7
of 44 cases (16%). The results of allelic loss analysis are shown in
Figure 1
, and representative cases are
shown in Figure 2
.
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loci and LOH at the D9S171 marker (Figure 2)
. In case BL323 a decrease in the intensity of both alleles of tumor
DNA at D9S1749 and D9S1748 markers was detected by multiplex PCR of
these markers with the control locus D9S934, so we considered this case
to show HD. Case LB8 showed HD of marker D9S1752, identified by
multiplex PCR (Figure 2)
.
Two cases (LB81 and LB66) showed instability at the D9S1749. Case LB1
showed imbalance at markers D9S1748 and IFN-, and another case
(LB54) showed imbalance at marker D9S1748. These four cases were
considered to have no deleted 9p21 locus.
p16 Methylation
The MSP technique was used to analyze the hypermethylation status
of the CpG island in the promoter region of the p16 gene.
Hypermethylation was found in 20 of 62 cases studied (32.2%) (Figure 3)
including 3 of 16 (18.7%) BLs and 17
of 46 (36.9%) LBCLs.
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p16 Mutation
To determine whether mutations of the p16 gene were present, we
analyzed exon 2 of this gene by direct sequencing in 62 cases. Five
mutations in exon 2 were identified in four different cases. Three of
them correspond to the change that substitutes
ACGThr for GCGAla, which
has previously been described as a polymorphism (Table 2)
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We investigated p27 expression by immunohistochemistry in 61 of 62
cases. Forty-two of 61 lymphoma cases showed absent or low reactivity,
below the chosen threshold. All other cases (19 of 61, 31%; 14
LBCLs and 5 BLs) were considered to be positive for p27 (Figure 4
and Table 3
).
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A summary of alterations of p16 and p53 together with p27
immunohistochemistry is shown in Table 3
. A total of 33 cases (24 of 46
LBCLs and 9 of 16 BLs) had inactivated p16 and/or p53 genes (53.2% of
cases). Five cases (5 LBCLs) showed concurrent p53 mutation and p16
inactivation (8.1%), all of which had p16 inactivated by methylation,
and one by mutation together with methylation. Remarkably, although it
is not statistically significant, none of the seven cases showing 9p21
locus deletion (where p14, a member of the p53/ARF pathway is located)
had p53 mutation. Another striking finding is the absence in the group
of BLs of double p53/p16 inactivation, only observed in the LBCL group.
The majority of the cases (11 of 14 LBCLs and 2 of 5 BLs) with a high level of expression of p27 have simultaneous p53 or p16 alterations. When the relationship between p27 overexpression and alterations in p53 and/or p16 were investigated in LBCLs, a significant association was found (chi-square, 4.202; P = 0.040; Yates’ correction test), which was absent in the BL group. Nevertheless, not all of the p53/p16 alterations were associated with p27 overexpression. Thus, there were 13 LBCLs and 6 BLs with p53 and/or p16 alteration, not showing p27 overexpression.
The Effect of the Simultaneous Inactivation of Different CKIs on Overall Survival
Survival analysis was performed to investigate the impact on the survival probability of the alterations in the p53, p16, and p27 gene/protein here analyzed. Additionally to p21 and p16 silencing, p27 overexpression was included here as an unfavorable marker, because it has been previously shown that, in aggressive lymphomas, it is the result of the formation of complexes with cyclin D3, where p27 is sequestered.21 In the group of BLs none of the alterations was significantly related with the survival probability, thus this group was not considered for this objective.
In the LBCL group, four different subgroups were compared: group A with
wt p53; group B, composed of those cases with p53 mutations; group C
with simultaneous p53 and p16 inactivation; and group D that included
cases with accumulation of p53, p16, and p27 alterations. Patients with
alteration in the three gene/proteins had a significantly worse
prognosis than those with p53 mutations or without alterations
(P = 0.0218) (Figure 5)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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p53 mutations and p16 inactivation have been described as the most frequent alterations in human cancer, having been found in approximately half of tumors studied.4,5 In this study, p53 mutations were detected in 17.7% (11 of 62) of the aggressive B-cell lymphomas analyzed [3 of 16 BL cases (18.8%) and 8 of 46 (17.4%) LBCL cases], this being approximately consistent with previous reports that have shown a percentage of p53 mutations in non-Hodgkin’s lymphoma ranging from 19 to 35% of cases, most of them in aggressive tumors.10,11 Alterations of p16 were observed in 43.5% of cases [6 of 16 BLs (37.5%) and 21 of 46 LBCLs (45.6%)]. In 20 of 62 (32.2%) we found promoter hypermethylation; 9p21 homozygous or hemizygous deletion were found in 7 of 44 cases (16%) and point mutations in 2 of 62 cases (3.2%). These results are consistent with previous reports on p16 alterations.12-15,45-49 p27 anomalous overexpression was detected in 19 of 61 cases (30.6%), 14 of 46 LBCLs (30.4%), and 5 of 15 BLs (31.3%), a percentage comparable to those described previously.16,21,50
A total of 33 cases (53.2%) showed inactivation of p16 and/or p53
(Table 3)
. Five of these (8.1%, all LBCLs) showed concurrent p53
mutation and p16 inactivation, and all of these cases had p16
inactivated by methylation (one case also shows a mutation in p16
together with promoter hypermethylation), thereby confirming the
existence of a subset of cases with simultaneous inactivation of the
p53 and Rb pathways, as was recently shown by Pinyol and
colleagues.38
Strikingly, none of the analyzed BLs showed
concurrent p53 and p16 alterations, and the frequency of loss of 9p21
was higher in this group, this showing that the inactivation of these
growth suppressor pathways (ARF/p53 and p16/Rb) is dependent of the
histological type and the underlying specific molecular alterations,
such as c-myc overexpression and others.
The INK4a/ARF locus in the 9p21 chromosomal region codifies for p14 and p16 proteins; this locus is the nexus between the two main pathways that control cell cycle progression, the Rb/p16 and p53/ARF pathways.51 None of the nine cases with LOH at 9p21 included in this series showed p53 mutation, which confirms previous observations that these two alterations may be mutually exclusive.52
Anomalous p27 protein overexpression, detected in 19 of 61 cases of aggressive B-cell lymphomas analyzed, tends to occur in cases with p16 and/or p53 alteration in aggressive lymphomas (11 of 14 LBCLs). This association is statistically significant (P = 0.040). The cause of the anomalous overexpression of p27 in lymphomas with a high growth fraction has previously been demonstrated to be the existence of cyclin D3/p27 complexes that lead to increased stabilization of the protein.21,22 In experimental assays it has also been shown that p15 induces p27 displacement from the cyclin D/CDK4 complex to cyclin E/CDK2 in response to transforming growth factor-ß.23 The presence of p16 has also been shown to modulate the reassortment of p27 from cyclin D/CDK4 to cyclin E/CDK2 complexes.26 The results of this series seem to support the hypothesis that in LBCLs p27 accumulation is secondary to loss of the p16 and p21 CKIs, and therefore extends the findings of in vitro observations to a specific subset of human tumors. The absence of p16 or p21 precludes the redistribution of p27 from complexes containing CDK4/cyclin D, where p27 is stabilized and probably inactive, to others containing CDK2/cyclin E, where p27 is active in arresting cell proliferation. However, there are 17 cases with p53 or p16 inactivation that do not show simultaneous p27 overexpression, thus indicating the existence of additional factors (c-myc or other possible targets) to explain p27 accumulation.53,54
Several in vitro studies have provided evidence of the synergy between p53 and p16, and concurrent alterations of p16 and p53 have been shown to confer a higher aggressiveness on different tumor types.35,38 This series analyzing LBCLs shows that the concurrent inactivation of these CKIs [p21 (provided by p53 mutations), p16, and p27], operating in different but co-operative pathways has an accumulative effect on overall survival of LBCL patients (P = 0.0218). Whether this phenomenon is valid only in LBCLs or could be extrapolated to other tumors is a debatable point. Thus, in this same series of data, BL patients seem to behave in a different way than LBCLs, p53 and p16 alteration being mutually exclusive and the association with p27 anomalous expression is not clinically significant. Nevertheless, more comprehensive studies in BLs and other tumoral types could bring new data concerning the existence and characteristics of the simultaneous inactivation of different CKIs, and clarify the role that c-myc or cyclin D could play in this complex interaction.
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Acknowledgements |
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Footnotes |
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This study was supported by a grant from the Fondo de Investigaciones Sanitarias (FIS 98/993), Ministerio de Sanidad y Consumo, and, from the Comisión Interministerial de Ciencia y Tecnología (1FD97-0431), Spain.
M. S.-B. and A. I. S. contributed equally to this work.
Accepted for publication March 21, 2001.
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2/3 cancer-prone mouse. Cell 1999, 97:515-525[Medline]
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, p53 wt;
· · · · · ·, p53 mut-p16 inact;
······, p53 mut;
– – –, p53 mut-p16 inact-p27 high.