ABSTRACT
BACKGROUND: Epstein-Barr virus (EBV) is associated to the etio-pathogenesis of an increasing number of tumors. Detection of EBV in pathology samples is relevant since its high prevalence in some cancers makes the virus a promising target of specific therapies. RNA in situ hybridization (RISH) is the standard diagnostic procedure, while polymerase chain reaction (PCR)-based methods are used for strain (EBV type-1 or 2) distinction. We performed a systematic comparison between RISH and PCR for EBV detection, in a group of childhood B-cell Non-Hodgkin lymphomas (NHL), aiming to validate PCR as a first, rapid method for the diagnosis of EBV-associated B-cell NHL. METHODS: EBV infection was investigated in formalin fixed paraffin-embedded tumor samples of 41 children with B-cell NHL, including 35 Burkitt's lymphoma (BL), from Rio de Janeiro, Brazil, by in situ hybridization of EBV-encoded small RNA (EBER-RISH) and PCR assays based on EBNA2 amplification. RESULTS: EBV genomes were detected in 68% of all NHL. Type 1 and 2 accounted for 80% and 20% of EBV infection, respectively. PCR and RISH were highly concordant (95%), as well as single- and nested-PCR results, allowing the use of a single PCR round for diagnostic purposes. PCR assays showed a sensitivity and specificity of 96% and 100%, respectively, with a detection level of 1 EBV genome in 5,000-10,000 EBV-negative cells, excluding the possibility of detecting low-number EBV-bearing memory cells. CONCLUSION: We describe adequate PCR conditions with similar sensitivity and reliability to RISH, to be used for EBV diagnostic screening in high grade B-NHL, in "at risk" geographic regions.
ABSTRACT
A interleucina 10 (IL-10) e o fator de necrose tumoral (TNF) são duas citocinas pleiotrópicas com papéis críticos na rede regulatória da resposta imune e no estabelecimento e resolução dos processos inflamatórios. Polimorfismos nos promotores destes genes determinam diferenças interindividuais na produção das moléculas. Para determinar se esses polimorfismos têm um papel na predisposição à doença ou na evolução clínica dos linfomas de Burkitt (LB), forma analisados o SNP (Single Nucleotide Polymorphism) IL-10 -1082(A/G), o microssatélite IL-10 G e o SNP TNF 308(G/A) em 71 pacientes com LNH, incluindo 52 crianças com LB. Os LNH foram caracterizados em relação a sua associação com vírus Epstein-Barr (EBV), determinando-se que a prevalência do EBV no grupo de LB infantil foi de 68%. Neste grupo, foi encontrada uma correlação significativa entre baixa idade e infecção por EBV (p=0.005). A freqüência do alelo IL-10 1082G nas crianças com LB foi maior que no grupo de controles sadios (p=0,036). Quando os pacientes foram agrupados em LB EBV- e EBV+, a frequência do alelo G, no grupo LB EBV foi significativamente maior em relação ao grupo controle (OR 2,82; CI 95% 1,22-6,507; p=0,013). Entre as crianças com LB que recaíram foi observada uma média maior de numero de repetições do microssatélite IL-10.G, embora com significância marginal (p=0,055). Não foram achadas diferenças estatisticamente significativas entre grupos de doenças, associação com o EBV ou evolução clínica e variantes alélicas ou genotípicas do polimorfismo de TNF. Nossos resultados sugerem que as variações genéticas interindividuais da IL-10 poderiam ter um papel na predisposição, na patogênese e na evolução do LB, particularmente no subgrupo EBV-. Estudos adicionais, abrangendo outros polimorfismos destas citocinas são necessários são para esclarecer a sua relevância como fator patogenético e prognóstico no linfoma de Burkitt.
Interleukin 10 (IL-I0) and Tumor Necrosis Factor (TNF) are two pleiotropic cytokines with critical roles in the regulatory net of the immune response and in the establishment and resolution of the inflammatory processes. Polymorphisms in IL-I0 and TNF genes promoters determine inter-individual differences in IL-I0 and TNF production. To determine if these polymorphisms have a role in the predisposition to, or in the clinical evolution of Burkitt's lymphomas (BL), IL-l0 -1082(A/G) Single Nucleotide Polymorphism (SNP), IL-I0.G microsatellite and TNF-308(G/A) SNP were analyzed in 71 patients with Non-Hodgkin Lymphoma (NHL), including 52 BL children. The patients were characterized in relation to EBV childhood BL group, was 68%. In this group, a significant correlation between low age and EBV infection was found (p = 0,005). The allele IL-10-1082G frequency in BL children was higher than in the healthy control group (p = 0,036). When the patients were grouped as BL EBV and EBV+, the frequency of allele G was significantly higher in the BL EBV- group. A higher mean number of repetitions of IL-10.G microsatellite was observed among BL children that relapsed, although with marginal significance (p = 0,055). No statistically significant differences between diseases' groups, EBV association or clinical evolution and allelic or genotypic TNF variants polymorphism were found. Our findings suggest that inter-individual genetic IL-10 variations could have a role in the predisposition, in the pathogenesis and in the BL evolution, particularly in EBV- sub-group. Additional studies, comprising other IL-10 and TNF polymorphisms, are necessary to elucidate their relevance as a pathogenic and prognostic factor in Burkitt's lymphoma.