1 Département de Pathologie, 2 Service dHématologie and 3 Unité dInformatique Médicale, Hôpital Henri Mondor, AP-HP, Créteil; 4 Service dAnatomie Pathologique and 9 Service dHématologie, Centre Hospitalier Lyon-Sud, Pierre-Bénite; 5 Service dAnatomie Pathologique, Hôpitaux de Brabois, CHU Nancy; 6 Service dAnatomie Pathologique and 10 Institut dHématologie, Hôpital Saint-Louis, AP-HP, Paris; 7 Service dAnatomie Pathologique, Hôpital Dupuytren, Limoges; 8 Laboratoire de Génétique Oncologique, Centre Henri Becquerel, Rouen, France
Received 9 November 2001; revised 21 January 2002; accepted 12 February 2002
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Abstract |
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p53 alterations have been associated with a poor prognosis in aggressive B-cell lymphoma. We investigated the clinical relevance of p53 status in diffuse large B-cell lymphoma (DLBCL), focusing on patients who belong to lower risk groups of the international prognostic index and were uniformly treated. We aimed to determine whether this biological marker could identify among such patients those with a pejorative outcome who could benefit from a distinct therapeutic approach.
Patients and methods:
We studied 69 patients presenting with no, one (low-risk, n = 40) or two (low-intermediate risk, n = 29) risk factors treated with an anthracyclin-containing induction regimen. p53 exons 58 mutations were screened for using denaturing gradient gel electrophoresis and confirmed by direct sequencing. Immunohistochemical detection of p53 protein and of its downstream target p21 were also evaluated in 60 of 69 cases.
Results:
p53 mutations were detected in 16 of 69 (23%) lymphoma samples. The presence of a p53 gene mutation affected survival (P = 0.01), with a 6-year survival rate estimated to be 44% in mutated patients, compared with 79% in non-mutated ones. Using a stepwise Cox model, p53 mutation constituted the only parameter affecting survival (relative risk = 2.7, P = 0.03). A p53+/p21 immunohistochemical pattern (n = 15), suggestive of a disrupted p53 function, strongly correlated with p53 gene status and was associated with a lower 6-year survival rate when compared with a p53 or p53+/p21+ phenotype (47% versus 74%, P = 0.05).
Conclusions:
p53 alterations constitute a pejorative biological indicator able to discriminate among clinically defined lower risk patients with DLBCL.
Key words: diffuse large B-cell lymphomas, p53, survival
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Introduction |
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The p53 gene encodes a 393 amino acid phosphoprotein that regulates the transcription of several genes [8]. It is now considered that the main function of the p53 protein is to integrate cellular responses to stress [9]. In response to various internal or external signals, such as hypoxia, DNA damage or oncogenic stimulus, the wild-type p53 protein undergoes post-translational modifications, accumulates within the nucleus of the cells and induces cell cycle arrest or apoptosis [10]. Alterations of the p53 tumor suppressor gene may be considered as a model for carcinogenesis, since this gene is mutated and/or deleted in >50% of human cancers [9], its germinal mutations predispose to neoplasia [11], its somatic mutation pattern can be used as an epidemiological marker of environmental mutagens [12], and last but not least, its protein product regulates both cell growth and cell death [9]. In addition, several studies have shown that wild-type p53 function was required for apoptosis induced by genotoxic damage [13, 14], suggesting that p53 functional status could be correlated with treatment response. Despite a large amount of data in the field, the clinical relevance of p53 alterations is still a matter for debate in the case of solid tumors [15], holding true for some cancers [16] but not others [17, 18]. As for lymphoid proliferations, p53 gene mutations are associated with the histological transformation of follicular lymphoma [19, 20] and B-cell chronic lymphocytic leukemia [21], and with blastic variants of mantle cell lymphoma [22]. In B-cell chronic lymphocytic leukemia, as well as in multiple myeloma, p53 deletions have been associated with shorter survival and poor response to chemotherapy [23, 24]. A few studies have reported that p53 mutations are associated with drug resistance, shorter progression-free survival and decreased overall survival in aggressive B-cell lymphomas [2527]. However, since these studies were conducted on patients who were heterogeneous with regard to histological subtypes, clinical risk factors and treatment regimens, the clinical impact of p53 mutations remains unclear.
This prompted us to investigate the clinical relevance and the predictive value of p53 gene mutations in a series of 69 patients with DLBCL, a low or low-intermediate risk of disease according to the IPI and who were treated uniformly. Indeed, we hypothesized that among these lower risk patients, those with a p53 mutation would benefit from more intensive therapies and that higher risk group patients might have accumulated numerous genetic changes and confounding adverse clinical factors, which could obscure the impact of p53 on outcome. All the patients studied were enrolled in the LNH87 and LNH93 trials of the Groupe dEtude des Lymphomes de lAdulte. p53 mutations in exons 58 were screened for using denaturing gradient gel electrophoresis (DGGE) and confirmed by direct sequencing. In addition, some authors suggested that the combined immunohistochemical analysis of p53 and one of its downstream target p21/WAF1 [8]a cyclin-dependent kinase inhibitorcould be a valuable means to assess p53 functional status in lymphomas [28, 29]. These surrogate markers, which could readily be used in routine laboratory practice, were evaluated in 60 of these patients and correlated with p53 mutations and outcome.
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Patients and methods |
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DNA samples
High molecular weight DNA was prepared from frozen tumoral cryostat sections (60 cases), and from tumoral cell pellets (nine cases) using proteinase K digestion, phenolchloroform extraction and ethanol precipitation, as described previously [31]. An histological or cytological control was performed in each case.
Denaturing gradient gel electrophoresis
DGGE screening for p53 mutations located in exons 58 was performed essentially as described previously [32]. Five PCRs are required to explore the presence of mutations in these exons. We used the primer pairs described by Hamelin et al. [32], with a GC clamp associated with the antisense primer for DGGE analysis of exon 8. DNAs were amplified in a Perkin Elmer 2400 thermocycler (Applied Biosystems, Foster City, CA, USA) in 25 µl reaction mixture containing 200 ng of tumoral genomic DNA, 0.3 µM of each primer, 200 µM of each deoxynucleotide and 0.5 U Taq Gold DNA polymerase (Applied Biosystems) in GeneAmp PCR buffer II (Tris 10 mM pH 8.3, KCl 50 mM) with MgCl2 at a final concentration of 1.2 mM (PCR exon 8) or 2 mM (PCR exon 5p, 5d, 5+6 and 7). The reactions were heated at 95°C for 10 min, amplified by 38 cycles of 94°C for 30 s, 55°C (PCR exon 5p, 8) or 62°C (PCR exon 5d, 5+6, 7) for 1 min and 72°C for 90 s, followed by a final 7 min elongation step. Heteroduplex formation was enhanced by: (i) a round of denaturation (98°C for 10 min) and hybridization (55°C or 62°C for 30 min) at the end of the PCR program; and (ii) by adding 50 ng of normal liver genomic DNA to each reaction. In our experience, this avoids misinterpretation due to the fact that mutated homoduplexes gel migration is sometimes very close to, and undistinguishable from, that of normal homoduplexes, without significantly lowering the sensitivity of this method. In this study, PCR amplifications were performed both with and without the addition of normal liver genomic DNA in 20 cases, with similar results.
Samples were loaded onto 16 x 22 cm, 0.75-mm thick, 6.5% polyacrylamide gels (acrylamidebisacrylamide 29:1) containing a denaturant gradient (100% denaturant; 7 M urea, 40% v/v deionized formamide) parallel to the direction of electrophoresis. Gels were run in a DGGE system (C.B.S., Del Mar, CA, USA) in 1x TAE buffer (40 mM Tris acetate, 20 mM sodium acetate, 1 mM EDTA pH 7.4) maintained at 60°C. Electrophoresis was performed in a 50% to 100% gradient for 15 h (exon 5p) or for 4 h (exon 5d), a 30% to 100% gradient for 6 h (exons 5+6), a 30% to 80% gradient for 5 h (exon 7) or a 20% to 70% gradient for 7 h (exon 8). After electrophoresis, gels were stained with ethidium bromide and photographed using a UV transilluminator. Control DNAs (from human leukemic cell lines HUT78 and CEM, and colon tumors) exhibiting known mutations in the various PCR fragments explored were systematically amplified and run in parallel with the lymphoma samples studied.
Sequencing
Tumoral DNAs exhibiting an abnormal profile upon DGGE analysis were PCR amplified with p53 primer pairs containing M13 reverse or 21M13 sequences at their 5' end. Amplicons used for sequencing corresponded to fragments spanning nucleotides 13 00413 457, 13 94714 140 and 14 40314 804 of p53 human gene locus (GenBank locus name HSP53G, GI: 35214). PCR products were run in 1.5% agarose gels containing ethidium bromide to ascertain both the amount and the quality of PCR products obtained, diluted 1:5 to 1:10 in water and used for direct sequencing on both strands with Dye-Primer Ready-Reaction mixes (Applied Biosystems). Excising the abnormal DGGE fragments from the acrylamide gel, followed by re-amplification and direct sequencing, was required for one sample (R1397) harboring a codon 248 missense mutation that was thus qualified as minor. The intron 6 mutations were identified upon direct sequencing of the P8-Pa9 PCR products using a Dye-Terminator Ready-Reaction mix, and the 5'-TCAGCATCTTATCCGAGTGG-3' oligonucleotide located in p53 exon 6, 80 nucleotides upstream from the splice site. Sequencing reactions were loaded on a 4.25% acrylamide7 M urea denaturing gel using a ABI 377 (Applied Biosystems) sequencing apparatus, and mutation analysis performed with Sequence navigator software (Applied Biosystems).
Immunohistochemical detection of p53 and p21 proteins
Sixty cases were studied for p53 expression by immunohistochemistry (IHC), performed on deparaffinized tissue sections using an indirect immunoperoxidase method with a Nexes automat (Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturers instructions. p21 expression was also evaluated in the cases showing p53 overexpression. The slides were pre-treated using microwave oven heating (three cycles of 5 min in 0.01 M citrate buffer, pH 7.3), and incubated for 30 min at 37°C with monoclonal antibodies directed against p53 protein (DO-7; Dako SA, Glostrup, Denmark; dilution 1:20) or p21/WAF1 protein (EA-10; Oncogene Sciences, Uniondale, NY, USA; dilution 1:50). Positive control slides were included in all tests. Each slide was examined by two authors (K.L., P.G.) without any knowledge of the clinical and molecular data. A semi-quantitative evaluation of p53 and p21 protein expression was performed by estimating the percentage of positive tumor cells: , 0% to 10%; +, 10% to 25%; ++, 25% to 50%; and +++, 50% to 100%, with a cut-off point of 10% positive cells as reported by several authors [28, 29].
Statistical analysis
Patient characteristics and CR rates were compared using the 2 test. Overall survival time was calculated from the date of diagnosis until death, last follow-up examination or stopping date, 1 December 1999. Survival curves were estimated using the KaplanMeier product-limit method [33] and were compared using the log-rank test. Univariate analyses were made using the
2 test and the log-rank test. A multivariate regression analysis according to Coxs proportional hazards regression model [34], with overall survival as the dependent variable, was used to adjust the effect of p53 mutations, as well as p53 expression and
p53 (p53+/p21) immunophenotype for potential independent prognostic factors. A P value of <0.05 (two-sided test) was considered to indicate statistical significance. All calculations were performed with SAS software, version 6.10 (SAS Institute, Cary, NC, USA).
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Results |
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Discussion |
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The frequency of p53 gene mutations that was observed in this series of patients (23%), their typei.e. mostly missense mutationsand their location, are overall in agreement with previous reports describing the p53 mutation pattern in this disease [25, 26, 28, 29]. Obviously, we cannot rule out the possibility of mutations outside the exons 58, which were not explored in this study, especially in the three cases presenting an IHC p53+/p21 phenotype. However, >90% of p53 mutations reported so far affect the central core domain involved in DNA binding, and mutations outside exons 58 appear to be rare [12]. Two lymphoma samples presented the same unusual mutation, located in intron 6. One of these two samples also displayed a minor missense mutation of codon 248, and both exhibited p53 protein accumulation. The deleterious effect associated with such intronic alterations cannot be formally assessed. However, nucleotide alterations in p53 intron 6 have been reported to be associated with cancer predisposition syndromes, p53 protein accumulation and decreased in vitro chemotherapy-induced apoptosis [3840], suggesting that intron 6 mutations might somehow dysregulate p53 function and promote tumor formation.
We were also interested to explore the relationship between the location of the different mutations identified and the clinical course. Quite interestingly, it appeared that mutations occurring in the p53 protein domains in close contact with DNA [41] (i.e. those located in loop L3, sheet S10 and helix H2) were associated with a dramatic clinical course. Indeed, six of seven patients harboring such mutations (see Table 1) died within 2 years, whereas only three of nine patients with a p53 mutation located outside these regions died, which represents a death rate similar to that observed in non-mutated patients (12 deaths among 43 patients). The limited number of mutated cases precludes a statistical analysis of the prognostic load associated with p53 mutation location in this series of patients. However, a poor prognosis and/or poor chemotherapy response specifically associated with L3 domain mutations have already been reported in the case of colorectal [42] and breast cancers [43].
In this series, we observed that p53 gene mutations were more frequent in patients presenting with a bulky disease. This association was not reported in previous studies concerning p53 alterations in B-cell lymphomas, but it might have been obscured by the heterogeneity of the lymphomas studied with regard to histological types and clinical stage. The higher frequency of p53 gene mutations in bulky disease might either reflect an increased risk of secondary genetic events parallel to tumoral growth or be related to an increased resistance to hypoxia of p53 mutated cells [44], as it has been suggested in the case of solid tumors [45].
In contrast to previous studies [25, 27], we failed to demonstrate a significant relationship between the presence of a p53 mutation and response to chemotherapy in this series of patients, but it should be stressed that the response was evaluated at the end of the induction regimen in this study, and much later (after chemotherapy completion) in the previous reports. Contrary to what could be expected from in vitro studies demonstrating cell killing resistance associated with p53 mutations [13, 46], it might be that p53 alterations do not, per se, prevent the initial tumoral burden reduction induced by chemotherapy. Since patients with a p53-mutated lymphoma frequently experienced short-term relapse, one might hypothesize that p53 mutations favor the rapid emergence of resistant clones, as a consequence of the increased genomic instability that has been linked to p53 alterations [47, 48].
The evaluation of p53 status with molecular techniques cannot easily be carried out in routine laboratory practice. Several investigators have attempted to use the IHC analysis of p53 protein accumulation or both p53 and p21 proteins, as a surrogate for p53 mutations [28, 29]. This combined evaluation, which relies on the definition of a p53 phenotype (p53+/p21), was recently reported to be a predictor of treatment failure and poor prognosis in a B- and T-cell NHL population-based study [49]. In our series of patients, p53 IHC analysis appeared to be slightly less efficient than the combined p53 and p21 analysis in discriminating between p53 mutated and non-mutated lymphomas, and use of the
p53 immunophenotype did show clinical relevance with respect to overall survival. However, considering that p53 and p21 IHC staining may vary between laboratories, the definition and validation of optimal cut-off values are needed to routinely apply these techniques.
Altogether, this study allowed us to assess the poor prognosis associated with p53 mutations in a homogenous series of patients with low and low-intermediate risk DLBCL. The clinical use of this prognostic marker should be useful to identify those among these patients who require distinct therapeutic approaches. Moreover, further studies would be helpful to evaluate the prognostic significance of the genomic location of the mutation and confirm the clinical relevance of the p53 immunophenotype.
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Acknowledgements |
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Footnotes |
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K.Leroy and C.Haioun contributed equally to this work.
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References |
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