Clinical and demographic characteristics of Epstein-Barr virus-associated childhood Burkitt's lymphoma in Southeastern Brazil: epidemiological insights from an intermediate risk region

We studied a group of 54 children with Burkitt’s lymphomafrom Southeastern Brazil, where epidemiological status of Burkitt’slymphoma is poorly understood. Epstein-Barr virus associationshowed an intermediate frequency (~60%) between endemic andsporadic subtypes. Median age was five years. Epstein-Barr virusinfection was significantly associated to low age (Epstein-Barrvirus⁺ four years vs. Epstein-Barr virus^– eight years).Sex ratio (M:F) was 2:1, with a significantly higher numberof males in old age classes. Young age at diagnosis and excessof males at older ages, as well as a causal relationship betweenlow age, epstein-barr virus and Burkitt’s lymphoma risk,may characterize Burkitt’s lymphoma in Brazil.

Key words: Burkitt’s lymphoma, Epstein-Barr virus, clinico-demographic, Brazil.

Introduction

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Introduction

Burkitt’s lymphoma (BL) shows marked variation acrossdifferent geographical regions with respect to age-specificincidence, primary tumor site and association with Epstein-Barrvirus (EBV) infection,¹ which characterize the endemic (eBL)and sporadic (sBL) subtypes.² eBL is almost always EBV-associatedwhile sBL shows only 10%–30% EBV association.¹ Descriptionsof BL outside endemic and sporadic regions allow for a progressiveidentification of world areas where BL exhibits specific characteristicsand intermediate incidence.³

An alternative classification to the standard types, based onthree different epidemiological conditions, considers: (i) BLin areas of high risk or incidence (endemic), (ii) BL in areasof low risk or incidence (sporadic cases in USA, Northern andEastern Europe, and Japan), and (iii) BL in areas of intermediaterisk or incidence (Northern Africa, the Middle East, and northernand central regions of South America).³ BL in Brazil might beincluded in this third subtype,^4,5 although the epidemiologicalstatus of BL, in a geographically extensive and heterogeneouscountry like Brazil, remains poorly understood. We report astudy of 54 children with BL in Southeastern Brazil and analysetheir clinicopathological characteristics and EBV association.

Design and Methods

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Design and Methods

Patients and pathology samples
Fifty-four children (up to 16 years old), diagnosed with BLat the Instituto Nacional de Câncer (INCA), Brazil, between1992 and 2004, were included in this study based on availabilityof pathology specimens and clinical records, following approvalby INCA’s Ethics Committee. The clinical characteristicsof 29 patients have been previously described.⁵

Histopathological diagnosis was performed according to the WHOclassification.² Statistical analyses were based on non-parametricmethods and a p value <0.05 was considered significant.

EBV diagnosis and genotyping
Infection was diagnosed by EBERs in situ hybridization.⁶ DNAwas isolated from biopsies and from paraffin-embedded tissue.Genotyping was carried out by nested-PCR for the distinctiveEBV-1 and -2 EBNA2 regions.⁶ Detection of the polymorphic 30or 69 bp deletion in the carboxy-terminus of the LMP1 gene wascarried out by specific nested-PCR assays.⁷

Results and Discussion

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Results and Discussion

Demographic characteristics
BL was diagnosed in 50 immunocompetent patients and in 4 patientswith AIDS (AIDS-BL) (Table 1). Age distribution (median 5 years,range 2–14) showed 42.5% of the patients in the 0–4year age class, 42.5% in the 5–9 class, and 15% in the10–14 class, indicating predominance of young children,with 54% equal to or greater than the median age. A similarage distribution was observed in 52 children from Bahia (NortheasternBrazil),⁸ suggesting that it might be characteristic of pediatricBL in Brazil.

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Table 1. Characteristics of patients with Burkitt’s lymphoma, according to HIV and EBV association, age distribution and sex ratio.

In high risk areas, BL is a pediatric disease, with most casesin Equatorial Africa occurring in the 5–9 year age-classalthough in Europe, childhood cases are equally distributedamong different age classes.^9,10 A compilation of studies comparingBL median age from different regions showed an estimate of 6.1years in Africa against 19.2 years in North America.⁹ In theMiddle East and Brazil, median ages were similar to those observedin Africa,^4,8–13 indicating that factors associated withearly morbidity in these regions require investigation.

The sex ratio of the entire group was 2:1 (36M:18F) and a significantsex ratio variation in respect to age class was observed, withan increase of affected males in older age classes ( ${chi}$ ²= 10.96;p=0.004). The age of males was significantly higher than infemales, with a median of 6 years (range 3–14) vs. 4 years(2–9) (Mann-Whitney test, p=0.001). A positive correlationbetween median age per class and sex ratio per class was observed(Rho Spearman = ~1, p<0.01). Distortion of sex ratio in childhoodcancer is a known finding, representing a classical risk factor;childhood cancers showing an adjusted-by-age sex ratio of 1.2,with non-Hodgkin’s lymphomas showing the highest sex ratio(3:1).¹⁴ BL is more frequent in males, mainly in low risk areaslike Europe, contrary to early African reports where males andfemales were almost equally affected while, in intermediateareas, sex ratio was found to be variable.^3,9 In our patients,the positive correlation between sex ratio and age class wasthe same as the lower proportion of young males in high risk(endemic) areas. Interestingly, the opposite is not the casein the USA or the Middle East,^9,15 indicating that global sexratio estimates in endemic and sporadic areas do not representthe extremes of a single distribution. This reinforces the needto carry out genetic and environmental studies per age classto search for biological factors involved in age-specific riskin BL.

Clinical presentation
In 53 children with available clinical records, the abdomenwas the most frequently involved region, accounting for theprimary presentation in 38 cases (72%) (Table 2). Analysis of22 cases with available data on abdominal involvement showed7 tumors (32%) localized in the intestinal wall while 15 (68%)occurred at other sites (meso/epiplon 5 cases; retroperitoneum4; ovary 2; liver 1; stomach 1; and others sites 2 cases). Onepatient (2%) showed a mandibular primary involvement. Nodalpresentation (11%) was observed mainly in older children. Twelve(23%) patients showed bone marrow involvement (6 cases withFAB L3-ALL). The central nervous system (CNS) was involved in5 patients, 2 (4%) of whom were diagnosed as primary CNS-BL.Patients at advanced (III/IV) stages (75%) were younger thanstage I/II patients (median 4.5 vs. 7 years; Mann-Whitney test,p=0.03). This seems to be a characteristic of Brazilian BL,^4,5,8contrary to that observed in a large series in Kenya, wherestages I/II were predominant in childhood BL.¹⁶ Abdominal involvementwas unrelated to age class, while in older patients, a trendof nodal involvement was evident ( ${chi}$ ²=5.48; p=0.064). There wasno association between clinical characteristics and sex.

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Table 2. Clinical characteristics of children with Burkitt’s lymphoma.

Like sBL, our patients showed predominance of abdominal tumorsin all age classes, intestinal involvement characteristic ofsBL was observed only in approximately 32% of cases. This patternof intra-abdominal involvement is in agreement with anotherstudy from Brazil.⁸ The low frequency (2%) of head and necktumors in our patients also agreed with other reports, showingthe lowest world frequency of head and neck involvement despitethe young age of affected children.

EBV association
EBV was detected in 61% of BL cases, and in 58% of the HIV^–group. Remarkably, all HIV⁺ children were also EBV⁺. The frequencyof EBV association was intermediate between eBL (95%) and sBL(10–30%),^1,4 predominantly of type 1 (Table 1), as previouslyreported in Brazil.^5,8,17 In immunocompetent children, a significantassociation between EBV infection and lower age was observed( ${chi}$ ²=10.24; p=0.006) (Figure 1 A–B). EBV⁺ BL showed a medianage of 4 years, against a median of 8 years in EBV-negativecases (Mann-Whitney test; p=0.002). A high frequency of EBV⁺BL in younger children was also observed in Northeastern Brazil,⁸reinforcing the need for further investigation of the relationshipbetween low age of EBV seroconversion and BL risk in Brazil.In eBL, an early and severe EBV infection during the first monthsof life was shown to be a key event for subsequent BL development.¹⁸We could not assess the serological status of our patients.However, a study on EBV seroprevalence in healthy individualsfrom the same Brazilian region showed an intermediate curveof seroconversion age with respect to developed and underdevelopedcountries. It also found an earlier EBV seroconversion in low-incomegroups and, interestingly, associated to low maternal literacy.¹⁹

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Figure 1. Association of EBV with childhood Burkitt’s lymphoma in Southeastern Brazil. (A) Age distribution of EBV⁺ and EBV^– children (number of cases). (B) Frequency of EBV association per age class. Cases include 50 non-AIDS-related Burkitt’s lymphoma children.

We also investigated LMP1 C-terminal deletion polymorphisms,the pathogenic role of which in EBV-associated lymphomas iscontroversial.⁷ LMP1 nested-PCR resulted in a 175 bp, a 145bp or a 106 bp amplified fragment for wild type, del30 or del69variants respectively. Deletion variants were more frequentin BL (17/33, 52%) than in non-neoplastic controls (4/11, 36%)although the difference was not statistically significant (p=0.157).The del69 variant was detected in only one BL case. Our resultsin a large group of BL children do not support a pathogenicrole of del30/del69 LMP1 variants in BL, which had been proposedfor this region.¹⁷ However, in a group of Hodgkin’s lymphomas(HL) from our institution, a significant association of del30variants with lymphoma cases was observed.⁷ These differencesmay reflect the fact that LMP1 is expressed and therefore subjectedto selection in HL but not in BL, in which EBV expresses a restrictedpattern of gene expression (latency I). Children with AIDS-BLwere younger than immunocompetent BL children, as expected fromperinatal HIV infection, confirmed in 3 patients. All 4 caseswere EBV⁺ compared with 58% of HIV^– cases, in agreementwith EBV⁺ AIDS-related pediatric lymphomas vs. 10% EBV⁺ casesin immunocompetent BL from Argentina.²⁰ This differs from the6/11 EBV⁺, AIDS-BL from the USA.²¹ The scarcity of pediatricAIDS-BL reported to date does not allow us to determine whetherthese differences reflect characteristics of populations atrisk or socio-geographically determined pathogenic factors,mirroring epidemiological patterns of AIDS-non-related BL. However,the high frequency of EBV association in AIDS-related and -unrelatedyoung children with BL in this study suggested that EBV mightplay a pathogenic role in the development of BL in Brazil.

Footnotes

Funding: this work was supported by the Conselho Nacional dePesquisa (CNPq) Grant 478264–2003-8 (Brazil) and SwissbridgeFoundation (Switzerland). Manuscript received October 23, 2007.Manuscript accepted November 22, 2007.

Authorship and Disclosures

RH designed the study, performed ISH and developed molecularassays, analyzed data and wrote the manuscript. CEK took careof the patients, performed clinical review of the cases, andcontributed to the interpretation of data and the final versionof the manuscript. FEF, DMG and LRW performed ISH and molecularassays, interpreted data and contributed to the final versionof the manuscript. MHMB was involved in histopathology and immunohistochemistryanalyses. HNS and IZR contributed to the interpretation of dataand reviewed the final version of the manuscript. All authorsmade a substantial contribution, reviewed and approved the finalversion of the manuscript. The other authors reported no potentialconflicts of interest.

References

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