Expression of Epstein-Barr virus lytically related genes in African Burkitt's lymphoma: correlation with patient response to therapy.

A study on the Epstein-Barr virus (EBV)-associated malignancy (endemic) Burkitt's lymphoma (BL) was initiated on fine-needle-aspiration biopsies from 46 proven BL cases in Malawi. Gene expression that might correlate with patient serology (where high levels of antibodies to lytically related genes are commonly observed) was explored. In two-thirds of the cases, we identified the EBV BZLF1 replication activator intermediate early protein ZEBRA in varying quantities and to varying extents in cells by immuno-cytochemistry. The early lytic-cycle gene transcript BHLF1 was assessed positively by solid-phase hybridisation in over half of the same tumours. Evidence of transcription of these genes was confirmed on a smaller number of surgically removed fresh biopsies by RT-PCR. We asked whether our findings, which are generally counter to the established notion that EBV gene expression in BLs is restricted to the latent function, EBNA1, might offer some explanation for the differential responses to chemotherapy observed among African patients. Where the duration of follow-up was sufficient to assign the cases (37 in number) to one of 3 categories, namely, complete, partial or no response, a significant correlation between expression of the viral function ZEBRA and a positive patient response to treatment was found. Lack of this was associated with poor prognosis. Clinical data and EBV gene expression results support the postulate of subgroups of African BLs, the intermediate early antigen providing a marker of potential use in patient management.

Epstein-Barr virus in epithelial cell tumors: a breast cancer study.

The human herpes virus Epstein-Barr (EBV) is clearly associated with African Burkitt's lymphoma and the undifferentiated from of nasopharyngeal carcinoma, although its role in oncogenesis is still poorly defined. Recently EBV has been implicated in other types of lymphomas, as well as in some nonlymphomatous neoplastic processes. Its possible association with human breast cancer has been investigated here. DNA from 91 cases of breast carcinoma and blood samples from the same patients were amplified with the PCR over a region in the EBV BamHIW major repeat sequence following a single-step amplification protocol. Nineteen samples (21%) were found to be positive; 10 samples of blood (only 3 of them from patients with EBV-positive tumors) were found by the adopted protocol to contain EBV DNA. Another series of PCR amplifications using primers covering a unique (nonreiterated) fragment in BamHIC encoding the EBERs (two short nonpolyadenylated RNAs generally highly expressed in cells latently infected with EBV) confirmed these findings. A good correlation between the two sets of experiments was observed, and only five differences in results were obtained on samples tested. In situ hybridization was carried out using BamHIW biotinylated DNA probes or EBER-1 digoxigenin-labeled riboprobes with the aim of confirming as well as localizing the signal to the epithelial cell. Twelve sections (63%) among the PCR-positive samples were found positive by in situ hybridization with the DNA probe, and six (31.5%) sections were found with the RNA probe. Twenty-one samples from benign breast tumors or normal breast tissue were used as controls for PCR amplification in this study, none of which was found positive. This is the first known report showing positive results for EBV in breast cancer. No statistical association was found in these studies between the presence of EBV and the histological type of the tumor, however. Its role therefore remains for the moment unknown, as well as does the significance of the association of EBV with only a subset of the cases.

Correlation between cytopathological results and in situ hybridisation on needle aspiration biopsies of suspected African Burkitt's lymphomas.

Burkitt's lymphoma (BL) is a very high-incidence malignancy in sub-Saharan Africa, where it targets mainly young children. This lymphoma is closely associated with Epstein-Barr virus (EBV). Diagnosis of BL relies on clinical presentation as well as histological results obtained from biopsies. In this report, 66 new patients from Malawi (one of the southernmost African countries with high-incidence BL), suspected on clinical grounds to present with BL, had fine needle aspiration biopsies taken, smeared on slides and used for May-Grünwald Giemsa staining. Duplicate slides were independently assessed for EBV presence and expression by DNA-DNA and/or RNA-RNA in situ hybridisation (ISH), using respectively the repetitive viral BamHIW DNA fragment in a biotinylated probe and the small EBV-encoded RNA EBER I in a digoxigenin-labelled riboprobe. There was very good correlation between the various techniques in the diagnosis of the lymphomas, showing 67% of clinically suspect cases to be BL. Our report, presenting data on BL in Malawi, illustrates the usefulness of a simple aspiration biopsy in the diagnosis of this malignancy by Giemsa staining and also in both types of ISH.

In situ hybridisation of EBV DNA-DNA hybrids using wet heat in polypropylene containers.

AIMS: To explore procedures designed to optimise DNA-DNA in situ hybridisation, using cells infected with Epstein-Barr virus (EBV) and tissues and subfragments of the EBV DNA as probes. METHODS: The denaturation step occurred in a polypropylene container, using wet heat generated by a hot water container, the pressure cooker, or the microwave oven, without coverslips, reaching a temperature of 121 degrees C or more in these two last systems. Two different visualisation systems were used. RESULTS: Fixed cells and tumours harbouring a high and medium to low copy number (a few hundreds to 33 copies per cell), were clearly labelled, using a simple reiterated subfragment (BamW) of the EBV DNA, and fresh frozen cells, harbouring a very low copy number (one to two on average) labelled using BamW as well as BamH (single non-reiterated 6 kilobase subfragment). CONCLUSION: This is a valuable alternative technique for DNA-DNA ISH that can be used in fresh frozen samples as well as fixed samples.