High prevalence of a 30-base pair deletion in the Epstein-Barr virus (EBV) latent membrane protein 1 gene and of strain type B EBV in Mexican classical Hodgkin's disease and reactive lymphoid tissue.

Depending on geographic location and patient age Hodgkin's disease (HD) is associated with Epstein-Barr virus (EBV), mostly type A EBV, in 20% to 100%. The highest prevalence occurs in children of developing countries. Molecular analysis of the oncogene coding for the latent membrane protein 1 (LMP-1) revealed a 30-base pair (bp) deletion in up to 46% of EBV-positive HD. We investigated the presence of EBV in a series of Mexican classical HD (n = 57) and reactive lymphoid tissues (n = 20) from a private and a public hospital with special emphasis on the prevalence of the 30-bp deletion and the type of EBV. EBV infection was analyzed at the cellular level by Epstein-Barr encoded early RNA transcripts (EBER) in situ hybridization (ISH) and by LMP-1 protein immunohistochemistry (IHC). Molecular analysis of the LMP-1 gene configuration was performed by polymerase chain reaction (PCR) with primers spanning the site of the deletion and subsequent Southern and/or dot blot hybridization using wild-type and deletion-specific probes. The prevalence of type A and type B EBV was investigated by PCR-analysis for divergence in the coding region of Epstein-Barr nuclear antigen (EBNA)-2. EBV was detected in Hodgkin- and Reed-Sternberg cells (H-RS) by LMP-1 IHC and/or EBER ISH in 35/57 (61%) Mexican HD including 18/32 (56%) with nodular sclerosis, 15/20 (75%) with mixed cellularity and 2/4 (50%) with lymphocyte depletion. In addition, LMP-1 gene sequences were detected by PCR in 9 cases of HD without LMP/EBER expression by H-RS cells and in 17/20 (85%) reactive lymph nodes, supposedly originating from rare latently infected B cells. Surprisingly, the 30-bp LMP-1 deletion was found in 28/35 (80%) EBV-positive HD. This deletion, however, was also found in all 9 (100%) HD with H-RS cells negative for EBV and in 10/17 (59%) reactive lymph nodes. Thus, the overall LMP-1 del prevalence in reactive tissue is 73% (19/26). Typing of EBV was successful in 26 cases of EBV-positive HD, 10 of these were infected by type B EBV (38%). Of the reactive lymphoid tissue, 9 (47%) were infected by type A, and 10 (53%) by type B; All 20 cases (100%) associated with type B, whether neoplastic or reactive, displayed the LMP-1 del variant compared with 18/25 (72%) infected by type A EBV. To our knowledge, this is the highest incidence for both the LMP-1 deletion variant and the infection by type B EBV in HD reported so far worldwide. Our data suggest that EBV infection contributes to the pathogenesis of the majority of Hodgkin's disease cases in Mexico. The specific tumorigenic role of the LMP-1 deletion variant, however, is doubtful with regard to its high frequency in nonneoplastic lesions. Moreover, type B infection frequently occurs in Mexican HD and reactive lymphoid tissue and is consistently associated with the deletion variant pointing to a pathogenetic role of this combined genotype.

Primary intestinal non-Hodgkin's lymphoma and Epstein-Barr virus: high frequency of EBV-infection in T-cell lymphomas of Mexican origin.

Epstein-Barr virus is universally associated with endemic Burkitt's lymphoma (BL) and undifferentiated nasopharyngeal carcinoma and can be detected in a significant proportion of cases of Hodgkin's disease (HD) and peripheral T-cell lymphoma, but only rarely in sporadic B-NHL. The frequency of EBV-positivity in certain neoplasms shows important geographic variations. Both HD and sporadic BL from Latin America have shown higher rates of EBV-association than cases from Western countries. In T-NHL, the frequency of EBV-positivity is influenced by the site of the primary tumor and the phenotype of the neoplastic cells. Nasal and nasal-type T-NHL, which show a T/NK-cell phenotype with expression of CD56 are virtually always EBV-associated, whereas only a proportion of nodal, gastrointestinal and pulmonary T-NHL are EBV-infected. A recent investigation of primary intestinal lymphomas of Mexican origin demonstrated EBV-positivity in all examined cases of T-NHL and BL and a proportion of other B-NHLs. The presence of EBV was independent of the presence or absence of enteropathy. Two of 6 cases studied showed CD56 expression. The high rate of EBV-positivity independent of histologic subtype is in contrast to the low to intermediate rates of EBV-positivity found in cases of intestinal T-NHL from Western countries and indicates that geographic differences in the frequency of EBV-association of lymphoid neoplasms might also extend to a fraction of peripheral T-cell lymphomas.

Post-transplantation lymphoproliferative disorders in Mexico: an aggressive clonal disease associated with Epstein-Barr virus type A.

Post-transplantation lymphoproliferative disorders (PT-LPDs) are a complication of immunosuppression with variable clinical behavior and frequent Epstein-Barr virus (EBV) association. There is geographic variation in the association of EBV with certain tumors and a lack of studies of PT-LPDs from developing countries, so we decided to study in detail a series of PT-LPDs from Mexico to identify similarities and differences between populations in Mexico and those in Europe and the United States. We used paraffin-embedded tissue from eight PT-LPDs (six from men, two from women) that arose after renal transplantation. Clinical data, morphologic features, and clonality on the basis of immunoglobulin (Ig) light chain restriction, as well as polymerase chain reaction (PCR) for Ig heavy chain genes, were studied. The presence of EBV was investigated with PCR, immunohistochemical analysis for latent membrane protein (LMP)-1, and in situ hybridization for EBV early RNA transcripts. In addition, the subtype of EBV based on the EBNA 2A and 2B genes and the presence of a 30-bp deletion in the LMP-1 gene were investigated by PCR. Seven (87.5%) of eight cases presented with gastrointestinal involvement; five patients died. Three cases were polymorphic PT-LPDs, four were monomorphic large cell lymphomas (one diffuse large cell, three immunoblastic), and one was unclassifiable. All showed a B-cell phenotype, with a clonal population demonstrated in seven of the eight cases. Tumor cells expressed EBERs in all of the cases and LMP-1 in six of seven evaluable cases. Seven of seven cases showed EBV subtype A. Two (25%) of eight cases had the 30-bp LMP-1 deletion. This study shows that PT-LPDs in Mexico are clonal disorders associated with EBV subtype A. In contrast to series from Europe and the United States, our cases showed a significantly higher incidence of gastrointestinal tract involvement (P < .001), and a lower incidence of the 30-bp LMP-1 deletion, although this was not statistically significant (P < .28).

Primary non-Hodgkin's lymphoma of the intestine: high prevalence of Epstein-Barr virus in Mexican lymphomas as compared with European cases.

Recent studies in Western European populations have shown that peripheral T-cell non-Hodgkin's lymphomas (T-NHLs) are associated with Epstein-Barr virus (EBV) in a higher percentage than sporadic B-cell NHL (B-NHLs), and that the frequency of EBV-positivity might be influenced by the primary site of the tumor. Because of the geographic differences in EBV expression in Burkitt's lymphoma (BL) and Hodgkin's disease (HD), and the lack of studies of sporadic NHL from developing countries, we decided to survey the presence of EBV in a series of primary intestinal lymphomas from patients in Mexico and in Western Europe, and to analyze whether EBV status is influenced by tumor phenotype, and geographic or ethnic determinants. Paraffin-embedded tissue from 43 primary intestinal NHLs (19 cases from Mexico and 24 from Western Europe) were examined, including 17 high grade B-NHLs, 9 low grade B-NHLs, and 17 T-NHLs; 6 of which were enteropathy associated T-cell lymphomas. The distribution of histologic subtypes was similar in both groups. The presence of EBV was investigated with a combined approach using a nested polymerase chain reaction technique as well as immunohistochemistry for latent membrane protein-1 and in situ hybridization for EBV early RNA transcripts (EBER 1/2) RNAs. The median age of the Mexican patients was significantly lower than the median age of the European patients (32 v 62 years). This difference was most pronounced in patients with T-cell lymphoma (24 v 63 years). EBER-positive tumor cells were detected in 13 of the 43 (30%) cases of primary intestinal lymphoma, including 5 of 26 sporadic B-NHL (3 high grade and 2 low grade), and 8 of 17 T-NHL, all of which were classified as pleomorphic, medium and large cell. The rates of EBV-positivity were markedly different for European and Mexican cases. Whereas 7 of 7 (100%) T-NHL and 5 of 12 (42%) sporadic B-NHL of Mexican origin were EBER-positive, only 1 of 10 T-NHL and 0 of 14 sporadic B-NHL from Europe showed EBER expression in tumor cells. Latent membrane protein was positive in only 2 of 43 cases, 1 of which was an EBER-negative high grade B-NHL from Mexico that showed intact total mRNA in control hybridization. CD30 expression was found in 4 of 8 EBV-positive T-NHL and in none of the EBV-positive B-NHL. In contrast to European cases, intestinal NHLs from Mexico show a very high frequency of EBV-positivity, which is not limited to T-NHL, but includes a significant proportion of B-NHL. This study strongly suggests that similar to HD and probably BL, there are important epidemiologic differences in EBV association in intestinal T-cell NHL between European and Mexican populations. These differences might be the result of environmental factors, for example, earlier contact with childhood viruses on intestinal lymphomagenesis.

Seminomas positive for Epstein-Barr virus by the polymerase chain reaction: viral RNA transcripts (Epstein-Barr-encoded small RNAs) are present in intratumoral lymphocytes but absent from the neoplastic cells.

A possible role of Epstein-Barr virus (EBV) in the pathogenesis of testicular germ cell neoplasms has been suggested repeatedly, but direct evidence for an association of testicular cancer with EBV is lacking. We examined 26 cases of classical seminoma, two spermatocytic seminomas, and 12 cases of nonseminomatous or combined germ cell tumors for the presence and cellular location of EBV with a combined approach using the polymerase chain reaction and nonradioactive in situ hybridization for EBV-encoded small RNAs (EBER1/2). After exclusion of cases without amplifiable DNA, 4/21 (19%) seminomas, but none of the other tumors, were positive for EBV by polymerase chain reaction. In situ hybridization for EBER1/2 showed rare positive lymphocytes, probably latently infected B-cells, in two of these four EBV-positive cases. No EBER-positive tumor cells were found in any of the analyzed tumors. The occurrence of EBV-positive lymphoid cells was not correlated to the frequency of intratumoral lymphocytes, including B-cells, which were present in seminomas in significant numbers. Our study demonstrates the absence of EBV from the neoplastic cells of testicular germ cell tumors and makes a direct role of EBV in the development of these malignancies improbable. Whether the presence of EBER-positive lymphocytes in some seminomas simply reflects the normal occurrence of latently infected cells in lymphoid tissue of seropositive individuals or is influenced by local factors remains to be determined.

Detection of monoclonal B-cell populations in decalcified, plastic-embedded bone marrow biopsies with the polymerase chain reaction.

Polymerase chain reaction (PCR) has been employed successfully for the detection of clonal immunoglobulin gene rearrangements in paraffin-embedded clinical samples. The authors examined whether this technique can also be applied to fixed, decalcified, and plastic-embedded bone marrow biopsies. DNA extracted from 66 glycolmethacrylate-embedded trephine biopsy samples was amplified for the detection of rearranged VDJ regions of the immunoglobulin heavy chain genes using both a single-step and a semi-nested PCR technique. After exclusion of samples with inadequate DNA, clonality was confirmed in 16 (67%) of 24 cases with B cell malignancy, whereas all 11 non-B cell neoplasms, and 6 of 9 cases with normal bone marrow showed evidence of a polyclonal B cell population. Patterns indicating oligo- or monoclonality were observed in three plastic-embedded samples of normal bone marrow, although control PCR of frozen bone marrow samples obtained in parallel showed no evidence of clonality. Repeated PCR of these cases revealed inconsistent bands, probably due to the amplification of rare templates from polyclonal B cells. Decalcified, plastic-embedded bone marrow biopsies are suitable for PCR-based determination of B-cell clonality. To exclude the possibility of false-positive results, monitoring of template DNA quality and independent control amplifications are mandatory.