
==== Front
Sao Paulo Med J
Sao Paulo Med J
Sao Paulo Med J
São Paulo Medical Journal
1516-3180
1806-9460
Associação Paulista de Medicina - APM

11723536
10.1590/S1516-31802001000600006
Original Article
p53 gene analysis in childhood B non -Hodgkin's lymphoma
Klumb Claudete Esteves Nogueira Pinto *
de Resende Lídia Maria Magalhães *
Tajara Eloísa Helena *
Bertelli Erika Cristina Pavarino *
Rumjanek Vivian Mary *
Maia Raquel Ciuvalschi *
Address for correspondence Claudete Esteves Nogueira Pinto Klumb PraçadaCruzVermelha, 23-Centro RiodeJaneiro/RJ- Brasil-CEP20230-130 Tel:(+5521)506-6198 E-mail:klumb@uol.com.br
Conflict of interest: Not declared.

01 11 2001
2001
119 6 212215
12 7 2001
25 7 2001
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons license.
ABSTRACT

CONTEXT:

Mutations or deletions in the tumor-suppressor gene p53 are among the commonest genetic changes found in human neoplasms including breast, lung and bowel cancers. In hematological malignancies, p53 is most often mutated in Burkitt's lymphoma, with p53 mutations present in 30 to 40% of tumor samples and in 70% of cell lines.

OBJECTIVE:

To analyze the p53 gene alterations in child patients with B non-Hodgkin's lymphoma.

DESIGN:

Descriptive study.

SETTING:

Tertiary oncology care center.

PARTICIPANTS:

The study investigated 12 patients with childhood B non-Hodgkin's lymphoma (Burkitt's lymphoma). Screening for p53 mutations was done by polymerase chain reaction - single strand conformational polymorphism(PCR-SSCP)analysis of exon 5 to 8/9 of the gene.

RESULTS:

Abnormal polymerase chain reaction - single strand conformational polymorphism migration pattern was observed in 4 patients (33.3%), one on exon 6 and three on exon 7. Positive cases included 2 patients who died from disease.

CONCLUSION:

These preliminary results suggest that p53 mutations are quite frequent in children with Burkitt's lymphoma and may play a role in lymphoma genesis or disease progression.

RESUMO

CONTEXTO:

Alterações do gene supressor de tumor p53, como mutações e deleções, são lesões genéticas encontradas com maior freqüência nas neoplasias humanas, incluindo câncer de mama, pulmão e cólon. Entre as malignidades hematológicas, o gene 53 é freqüentemente mutado no linfoma de Burkitt, sendo detectadas mutações em 3040% das amostras tumorais e em 70% das linhagens celulares.

OBJETIVO:

Analisar as alterações do gene p53 em crianças com linfoma não-Hodgkin de origem B.

TIPO DE ESTUDO:

Estudo descritivo. LOCAL: Centro de Oncologia Terciário. PARTICIPANTES: O estudo analisou 12 pacientes com linfoma não-Hodgkin B classificados como linfoma de Burkitt. A análise de possíveis mutações do gene p53 foi realizada pela técnica de PCR-SSCP dos exons 5, 6, 7 e 8/9 do gene.

RESULTADOS:

Um padrão anormal de migração foi observado em quatro pacientes (33.3%), em um paciente no exon 6 e em três no exon 7. Os casos positivos incluíam dois pacientes que evoluíram para o óbito por progressão da doença.

CONCLUSÃO:

Esses resultados preliminares sugerem que as alterações do gene p53 são freqüentes em crianças com linfoma de Burkitt e podem contribuir para patogênese ou progressão da doença.

KEY WORDS:

p53 mutation
B non-Hodgkin's lymphoma
Burkitt's lymphoma
PALAVRAS-CHAVE:

Mutação do gene p53
Linfoma não-Hodgkin
Linfoma de Burkitt
==== Body
pmcINTRODUCTION

p53 is a tumor-suppressor gene whose disruption or loss is implicated in development or progression of several types of human cancer.1-3 Its inactivation is mainly caused by point mutations in the coding sequence of exons 5 to 9 in one allele, with or without loss of the other allele.4 Burkitt's lymphoma is a subgroup of nonHodgkin's lymphoma (NHL) with distinct epidemiological, clinical-pathological, immunological, and molecular features. It occurs as an Epstein-Barr virus associated with B nonHodgkin's lymphoma with high incidence among children, in the malaria belt of equatorial Africa (endemic Burkitt's lymphoma) and sporadically in other geographical areas, when it is named sporadic Burkitt's lymphoma.5 The characteristic genetic marker of Burkitt's lymphoma is a reciprocal translocation involving the c-myc gene on chromosome 8 and one of three immunoglobulin gene loci, most commonly the heavy chain gene locus on chromosome 14 and less frequently the light chain gene loci on chromosome 2 and 22.6 Evidence from several studies has led to the conclusion that c-myc translocation is not the only event in the pathogenesis of Burkitt's lymphoma. Occurrence of additional pathogenic steps like tumor cell infection with Epstein-Barr virus and mutations in the regulatory and coding regions of c-myc might contribute to the oncogenic process.7, 8

Experimental studies suggest that alterations in the p53 gene might be relevant to the development of lymphomas in transgenic mice carrying mutant p53 alleles.9 A very strong correlation between p53 status and tumor-proneness among nude mice has been observed for Burkitt's lymphoma cell lines but not for other cell types.10

p53 protein is necessary to induce the response to many chemotherapeutic drugs used for treatment of Burkitt's lymphoma. The present chemotherapeutic protocols are highly effective, although they are not free from adverse effects. Therefore, in the context of resistance/sensitivity to treatment, absence of p53 mutation could be an important finding for selecting patients that could be treated with a less intensive chemotherapeutic schedule.

In order to establish the frequency of p53 alterations among B non-Hodgkin's lymphoma in children, especially in Burkitt's lymphoma, we studied 12 newly diagnosed patients.

METHODS

Samples

Twelve cases of untreated B non-Hodgkin's lymphoma (all of which were Burkitt's lymphoma) were evaluated. The cases were classified according to the non-Hodgkin's Lymphoma Pathologic Classification Project.11 B-cell phenotype was confirmed by immunostaining with CD20 monoclonal antibody L26.

DNA was obtained from paraffin-embedded tumor tissue and extracted using previously published methods.12, 13

Polymerase chain reaction - single strand conformational polymorphism (PCR-SSCP) of the p53 gene

Single strand conformational polymorphism analysis was based on the method reported by Orita et al.14 Polymerase chain reaction was performed with 100-500 ng of genomic DNA, 25 pmol of each primer exon 5, 6, 7 and 8/9, 0.2 mM dNTP, 1 U Taq polymerase (GIBCO-BRL) in a final volume of 45 mL (exon 6, 25 mL). Thirty-four cycles of denaturation (94 °C), annealing (annealing temperatures were optimized for each pair of primers), and extension (72 °C) were performed in a thermocycler (MinicyclerTM, MJ Research). The four exons were amplified separately. After amplification, 5mL of reaction mixture was mixed with 10 mL 98% formamide / 10 mM EDTA, 0.025% bromophenol blue, and 0.025% xylene cyanol. Samples were heated at 95 °C for 5 min, chilled on ice and immediately loaded on a 7.5% polyacrylamide - TBE gel. We performed the electrophoresis at room temperature, with or without the addition of glycerol, or at 4 °C in a cold room. Gels were run at 3 W for 4 h, stained with silver nitrate and air-dried.

RESULTS

Clinic pathological data of patients are listed in the Table. The patients ranged in age from 4 to 9 years (median = 5.5 years). Male/female ratio was 2:1. According to the St. Jude staging system,15 16.6%% of patients had stage I/II disease, 66.6% stage III and 16.6% stage IV disease. All patients had a histological diagnosis corresponding to Burkitt's lymphoma.

Table Clinical pathological data and outcome for patients with Burkitt's lymphoma

Patient	Sex	Age (years)	Primary Site of tumor	Disease Stage	HIV	Survival (months)	DFS (months)	Outcome	
									
1	M	5	Cervical node	I	Neg	28	26	Alive	
2	M	6	Nasopharynx	I	Neg	4	0	Dead*	
3	F	4	Abdomen	III	Neg	7	2	Dead*	
4	M	5	Abdomen	III	Neg	5	0	Dead¶	
5	M	8	Abdomen	III	Neg	21	19	Alive	
6	M	4	Abdomen	III	Neg	21	18	Alive	
7	F	4	Abdomen/pelvis	II	Neg	38	36	Alive	
8	M	7	Abdomen	III	Neg	4	0	Dead*	
9	M	6	Abdomen	III	Neg	54	49	Alive	
10	M	5	Abdomen	III	ND	84	82	Alive	
11	F	9	Abdomen/BM	IV	Neg	64	62	Alive	
12	F	6	Pelvis/CNS	IV	Neg	21	19	Alive	
Abbreviations: M, male;F, female;BL, Burkitt's lymphoma;BM, bone marrow;DFS, disease free survival;Neg, negative;ND, not determined.

* Death related to refractory disease;¶Death(quit treatment).

The outcome for all patients is shown in the Table. Eleven patients achieved complete remission, 8/12 remain alive. Four patients died because of failure to achieve complete remission, relapse or non-adherence to treatment.

Upon polymerase chain reaction-single strand conformational polymorphism analysis, four p53 gene alterations were demonstrated. These four alterations consisted of one on exon 6 and three on exon 7.

The figure shows the results of polymerase chain reaction-single strand conformational polymorphism analysis for exons 6 and 7. The polymerase chain reaction products of exon 6 (lane 5) and exon 7 (lanes 2, 4, 9) showed mobility shifts upon single strand conformational polymorphism analysis. Positive cases included 2 patients who died from disease.

Figure Polymerase chain reaction-single strand conformotional polymorphism analysis of p53 gene. Amplified genomic DNA fragments corresponding to exons 5-9 were denatured by heating and electrophoresis was performed in 7.5% polyacrylamide gel. DNA samples suggestive of having p53 mutations show shifts in electrophoretic mobility compared to other DNA samples on the same gel. Arrows show shifts in electrophoretic mobility (A): Exon 6. Lane 5 corresponds to patient 1 (B): Exon 7. Lanes 2,4,9 correspond to patients 3,5 and 8, respectively.

The polymerase chain reaction products of exon 5 and exons 8/9 showed no mobility shifts. To confirm that our experiments were free from contamination or some other artifacts, all experiments were repeated using another section cut from the same block of paraffin embedded tissue.

DISCUSSION

We examined 12 cases of childhood B nonHodgkin's lymphoma for alterations of p53 gene using polymerase chain reaction-single strand conformational polymorphism analysis, which is a simple detection system for point mutations.16,17 An abnormal single strand conformational polymorphism migration pattern was detected in four samples (33.3%). Polymerase chain reaction-single strand conformational polymorphism analysis is a technique based on the three-dimensional conformation taken by a single-stranded DNA in a non-denaturing environment where any change in the sequence can result in a variation of the electrophoretic mobility. The specificity of polymerase chain reaction-single strand conformational polymorphism is more than 95% for 100 to 300 bp polymerase chain reaction fragments.17 In practice, not all of the changes can be resolved but the modification of the migration conditions can improve the detectability of the mutations along the sequence. The polymerase chain reaction-single strand conformational polymorphism protocol is a very useful screening method for detecting mutations in a short region of a gene. However, the samples with abnormal single strand conformational polymorphism require nucleotide sequencing studies to assert whether the abnormal migration represents a mutation or a polymorphism.

Several groups have found that p53 is usually mutated in Burkitt's lymphoma cell lines.18- 20 The frequency of p53 mutations in fresh tumor samples has previously been reported as 40%. 18,21 We found that 33.3% of paraffin embedded samples from children studied at diagnosis of Burkitt's lymphoma had p53 alterations suggestive of gene mutation. Our results were similar to those reported by other authors,18,21 who found 33% to 37%. However, a recent study has shown 19% of p53 mutations in newly diagnosed adult and child patients with Burkitt's lymphoma.22

In our study, all the Burkitt's patients were children studied at diagnosis. In the study by Preudhomme et al.,22 there were only 2/12 children with p53 alterations among the patients studied (16%). Bathia et al.21 found 37% of p53 mutations in patients with Burkitt's lymphoma from Argentina and Brazil, although there was no reference to how many children were included and whether they represented newly diagnoses or relapse of disease. Higher percentages of p53 mutations would be expected in relapse situations.

In a previous report studying a large series of childhood lymphoid malignancies, p53 mutations were detected in 2/8 B non-Hodgkin's lymphoma.23 The total number of cases examined by that group was too small to determine the frequency of p53 mutation in B non-Hodgkin's. Among all the types of lymphoid neoplasm, the most frequent targets for p53 mutations are Burkitt's lymphoma and its leukemic counterpart L3-type B-cell acute lymphoblastic leukemia.18

The use of p53 gene mutations as a potential prognostic marker in lymphoid childhood malignancies is limited by its low frequency at diagnosis. Nevertheless, the higher frequency of p53 in Burkitt's lymphoma patients observed by Gaidano et al18 and others21 suggests that p53 mutations can be found in children with Burkitt's lymphoma.

In our study, four Burkitt's patients with abnormal p53 migration were found. Of those, two died as a result of lack of response to treatment and consequent disease progression, but two are still alive.

In a large panel of Burkitt's lymphoma cell lines,18,24the normal allele had been lost in a large number of lines or both alleles present had undergone point mutations. Burkitt's lymphoma cell lines carrying a p53 mutation are more radioresistant when they lose the normal p53 allele than when they retain it.25 Preudhomme et al.22 found persistence of the normal p53 allele in most newly diagnosed Burkitt's lymphoma patients with mutations. The loss of the normal allele in cell lines suggests that this is a late event in the evolution of Burkitt's lymphoma, especially in relapsed patients and may be responsible for the resistance to chemotherapy observed in those tumors upon relapse. Hence, particular forms of mutant p53 may directly enhance the resistance of tumor cells to anticancer agents and confer a selective survival advantage during chemotherapy.26The relevance of these findings should be extended to cells lines and patients with Burkitt's lymphoma.

These observations suggest that p53 gene alteration in tumors carrying c-myc protooncogene activation in children can be detected in newly diagnosed patients and play a role in lymphoma genesis or disease progression.

Burkitt's lymphoma includes two pathogenically distinct forms, namely the endemic African type characterized by Epstein-Barr virus infection and the sporadic American type characterized by Epstein-Barr virus infection in only 30% of cases.27 Higher frequencies of Epstein-Barr virus infection (71 to 87%) have been found in Brazil.28, 29

A common denominator among all these diseases is the consistent presence of chromosomal translocation leading to c-myc activation.6 On the other hand, environmental factors or lifestyle acts on the molecular subtypes of Burkitt's lymphoma.30 It may possible that ethnic and/or geographical factors might account for different frequencies of p53 alterations between Brazilian Burkitt's lymphoma and sporadic Burkitt's lymphoma patients from others countries.

Claudete Esteves Nogueira Pinto Klumb, MD. Laboratory of Cellular and Molecular Hematology, HematologyService, HospitaldoCâncer/NationalCancer Institute, RiodeJaneiro, Brazil.

Lídia Maria Magalhães de Resende, MD. Pathology Service, Hospital doCâncer/NationalCancer Institute, Rio deJaneiro, Brazil.

Eloísa Helena Tajara, PhD. Department of Biology, Instituto de Biociências Letras e Exatas, Universidade Estadual Paulista, SãoJosédo Rio Preto, São Paulo, Brazil.

Erika Cristina Pavarino Bertelli, PhD. Department of Molecular Biology, FacultyofMedicine, SãoJosédo Rio Preto, São Paulo, Brazil.

Vivian Mary Rumjanek, PhD. Laboratory of Tumoral Immunology, DepartmentofBiochemistry, Universidade Federal do Rio deJaneiro, Rio deJaneiro, Brazil.

Raquel Ciuvalschi Maia, MD, PhD. Laboratory of Cellular and Molecular Hematology, Hematology Service, Hospital do Câncer/National Cancer Institute, Rio de Janeiro, Brazil

Research grant support: This workwas supported by the Ministryof Health, Fundação deAmparo à Pesquisa do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Programa de Apoio aos Núcleos de Excelência (FINEP/ PRONEX/FUJB-Convênio: 76.97.1000.00).

Hospital do Câncer/National Cancer Institute, Rio de Janeiro, Brazil
==== Refs
REFERENCES

1 Hollstein M Soussi T Thomas G p53 gene alterations in human tumors: perspectives for cancer control Recent Results Cancer Res 1997 143 369 389 8912433
2 Hollstein M Sidransky D Vogelstein B Harris CC p53 mutations in human cancers Science 1991 253 49 53 1905840
3 Wang XW Harris CC p53 tumor suppressor gene: clues to molecular carcinogenesis J Cell Physiol 1997 173 247 255 9365531
4 Sidransky D Clinical implications of the p53 gene Ann Rev Med 1996 47 285 301 8712782
5 Dalla-Favera R Erilkson J Patterson D Translocations and rearrangements of c-myc oncogene locus in human undifferentiated B-cell lymphomas Proc Natl Acad Sci USA 1982 79 7824 7827 6961453
7 Henderson S Rowe M Gregory C Induction of bcl-2 expression by Epstein–Barr virus latent membrane protein I protects infected B cells from programmed cell death Cell 1991 65 1 20 2013090
8 Bathia K Huppi K Splangler G Point mutations in the c-myc transactivation domain are common in Burkitt's lymphoma and mouse plasmocytomas Nat Genet 1993 5 56 61 8220424
9 Hooper ML The role of p53 and Rb-1 genes in cancer, development and apoptosis J Cell Sci 1994 8 13 17
10 Bathia GK Sgadari C Role of the p53 tumor suppressor gene in tumorigenicity of Burkitt's lymphoma cells Cancer Res 1997 57 2508 2515 9192833
11 The non-Hodgkin's Lymphoma Pathologic Classification Project: National Cancer Institute sponsored study of classifications of non-Hodgkin's lymphomas: summary and description of working formulation for clinical usage Cancer 1982 49 2112 2135 6896167
12 Wright DK Manos MM Sample preparation from paraffin-embedded tissues Innis MA Gelfand DH Sninsky JJ White TJ PCR Protocols: a guide to methods and applications New York Academic Press 1990 153 158
13 Banerjee SK Makdisi WF Weston AP Microwave based DNA extraction from paraffin embedded tissue for PCR amplification Biotechnics 1995 18 769 773
14 Orita M Iwahana H Kanazawa H Detection of polymorphisms of human DNA by gel electrophoresis as singlestrand conformation polymorphism Proc Natl Acad Sci USA 1989 86 2766 2770 2565038
15 Murphy SB Fairclough DL Hutchison RE Non-Hodgkin's lymphoma of childhood: an analysis of histology, staging, and response to treatment of 338 cases at a single institution J Clin Oncol 1989 7 186 193 2915234
16 Murakami Y Hayashi K Skiya T Detection of aberrations of p53 alleles and the gene transcript in human tumor cell lines by single-strand conformation polymorphism analysis Cancer Res 1991 51 3356 3361 2054775
17 Hayashi K PCR-SSCP: a simple and sensitive method for detection in genomic DNA PCR Methods and Appl 1991 1 34 38 1842918
18 Gaidano G Ballerini P Gong JZ p53 mutation in human lymphoid malignancies associated with Burkitt's lymphoma and chronic lymphocytic leukemia Proc Natl Acad Sci USA 1991 88 5413 5417 2052620
19 Farrel PJ Allan GJ Shanahan F p53 is frequently mutated in Burkitt's lymphoma cells lines EMBO J 1991 10 2879 2887 1915267
20 Wiman KG Magnisson KP Ramqvist T Mutated p53 detected in a majority of Burkitt lymphoma cell lines by monoclonal antibody Pab 2490 Oncogene 1991 6 1633 1639 1923530
21 Bathia KS Gutierrez MI Huppi K The pattern of p53 mutations in Burkitt's lymphoma differs from that of solid tumors Cancer Res 1992 52 4273 4276 1638540
22 Preudhomme C Dervite I Wattel E Clinical significance of p53 mutations in newly diagnosed Burkitt's lymphoma and acute lymphoblastic leukemia: a report of 48 cases J Clin Oncol 1995 13 812 820 7707106
23 Wada M Bartram CR Nakamura H Analysis of p53 mutations in a large series of lymphoid hematological malignancies of childhood Blood 1993 82 3163 3169 8219205
24 Duthu A Debuire B Romano J p53 mutations in Rajii cells: Characterization and localization relative to other Burkitt's lymphomas Oncogene 1992 7 2161 2167 1437144
25 O’Connor PM Jackman J Jondle D Role of the p53 tumor suppressor gene in cell cycle arrest and radio-sensitivity of Burkitt's lymphoma cell lines Cancer Res 1993 53 4776 4780 8402660
26 Blandino G Levine JA Oren M Mutant p53 gain of function: differential effects of different p53 mutants on resistance of cultured cells to chemotherapy Oncogene 1999 18 477 485 9927204
27 Shiramizu B Barriga F Neequaye J I. Patterns of chromosomal breakpoint locations in Burkitt's lymphoma: relevance to geography and EBV association Blood 1991 77 1516 1526 1849033
28 Bacchi MM Bacchi CE Alvarenga M Burkitt's lymphoma in Brazil: strong association with Epstein-Barr virus Mod Pathol 1995 9 1 63 67
29 Araujo I Foss HD Bittencourt A Expression of Epstein-Barr virus gene products in Burkitt's lymphoma in Northeast Brazil Blood 1996 87 5279 5286 8652843
30 Gutierrez M Hamdy N Bathia K Geographic variation in t(8;14) chromosomal breakpoint locations and EBV association in Burkitt's lymphoma Int J Pediat Hematol Oncol 1999 6 161 168
