Identification of an Atypical Enzootic Bovine Leukosis in Japan by Using a Novel Classification of Bovine Leukemia Based on Immunophenotypic Analysis.

Bovine leukemia is classified into two types: enzootic bovine leukosis (EBL) and sporadic bovine leukosis (SBL). EBL is caused by infection with bovine leukemia virus (BLV), which induces persistent lymphocytosis and B-cell lymphoma in cattle after a long latent period. Although it has been demonstrated that BLV-associated lymphoma occurs predominantly in adult cattle of >3 to 5 years, suspicious cases of EBL onset in juvenile cattle were recently reported in Japan. To investigate the current status of bovine leukemia in Japan, we performed immunophenotypic analysis of samples from 50 cattle that were clinically diagnosed as having bovine leukemia. We classified the samples into five groups on the basis of the analysis and found two different types of EBL: classic EBL (cEBL), which has the familiar phenotype commonly known as EBL, and polyclonal EBL (pEBL), which exhibited neoplastic proliferation of polyclonal B cells. Moreover, there were several atypical EBL cases even in cEBL, including an early onset of EBL in juvenile cattle. A comparison of the cell marker expressions among cEBL, pEBL, and B-cell-type SBL (B-SBL) revealed characteristic patterns in B-cell leukemia, and these patterns could be clearly differentiated from those of healthy phenotypes, whereas it was difficult to discriminate between cEBL, pEBL, and B-SBL only by the expression patterns of cell markers. This study identified novel characteristics of bovine leukemia that should contribute to a better understanding of the mechanism underlying tumor development in BLV infection.

Classificação de 86 casos de linfoma em bovinos de acordo com a Working Formulation (WF) of Non-Hodgkin's Lymphomas for Clinical Usage e a Revised European-American Classification of Lymphoid Neoplasms (REAL) / Classification of 86 bovine lymphomas cases according to the Working Formulation (WF) of Non-Hodgkin's Lymphomas for Clinical Usage and The Revised European-American Classification of Lymphoid Neoplasms (REAL)

Por meio de um estudo retrospectivo, realizou-se avaliação fenotípica (histologia) e imunofenotípica (imuno-histoquímica [IHQ]) de 86 casos de linfoma bovino. Quanto ao padrão de distribuição, todos os linfomas foram incluídos como difusos. Em relação à dimensão dos linfócitos neoplásicos, 83,8% dos linfomas foram considerados como de grandes células e 11,6% como de pequenas células. Linfomas mistos, ou seja, constituídos por grandes e pequenas células, representaram apenas 4,6% dos casos. Quanto ao número de mitoses, 84,9% dos linfomas foram incluídos como de grau intermediário, 10,5% como de baixo grau e 4,6% como de alto grau. No que se refere à morfologia do núcleo, linfomas em que predominavam linfócitos não clivados (58,2%) ou linfócitos clivados (37,2%) foram mais frequentes do que aqueles em que havia uma mistura igualmente proporcional de linfócitos clivados e não clivados (4,6%). Com base nestes resultados, os 86 linfomas foram assim distribuídos utilizando-se a classificação proposta pela Working Formulation (WF) of Non-Hodgkin's Lymphomas for Clinical Usage: difuso de grandes células não clivadas (46,5%), difuso de grandes células clivadas (33,7%), difuso de pequenas e grandes células (4,6%), difuso de pequenas células - tipo plasmocitoide (7%), imunoblástico (3,5%), difuso de pequenas células - tipo intermediário (2,3%), difuso de pequenas células não clivadas (1,2%) e difuso de pequenas células não clivadas - tipo Burkitt (1,2%). Na imuno-histoquímica, 27 dos 86 (31,4%) linfomas foram positivos para o anticorpo monoclonal CD79αcy, utilizado para detecção de linfócitos B, e nenhum caso foi positivo para o anticorpo policlonal CD3, utilizado para detecção de linfócitos T. Com base nestes resultados, os 27 linfomas B foram assim distribuídos utilizando-se a Revised European-American Classification of Lymphoid Neoplasms (REAL): linfoma difuso de grandes células B (81,5%), linfomas imunoblásticos de grandes células (11,1%) e linfomas...

A retrospective study of 86 cases of bovine lymphoma classified as with diffuse pattern of distribution and verified by phenotypic (histology) and immunophenotypic (immunohistochemistry [IHC]) is presented. Regarding the size of the neoplastic lymphocytes, 83.8% was classified as large cells lymphoma and 11.6% as small cells lymphoma. Mixed lymphomas, i.e., formed by large and small cells simultaneously represented only 4.6% of all cases. Regarding their mitotic index, 84.9% of lymphomas was included in the intermediate-grade, 10.5% as low-grade and 4.6% as high-grade. Regarding the nucleus morphology, lymphomas with mostly non-cleaved cells (58.2%) or cleaved cells (37.2%) were the more frequent than those with a balanced mixed proportion of cleaved and non-cleaved cells (4.6%). Based on these results, the 86 lymphoma cases were classified by the Working Formulation (WF) of Non-Hodgkin's Lymphomas for Clinical Usage as: diffuse large non-cleaved cell (46.5%), diffuse large cleaved cell (33.7%), diffuse mixed small and large cell (4.6%), diffuse small cell - plasmacytoid (7%), immunoblastic (3.5%), diffuse small cell - intermediate (2.3%), diffuse small non-cleaved cell (1.2%), and diffuse small non-cleaved cell Burkitt's (1.2%). According to the IHC, 27 out of 86 (31.4%) lymphomas were positive to monoclonal antibody CD79αcy, used to detect B cells, and none were positive for polyclonal antibody CD3, used to detect T cells. Based on this, the 27 B-cell type lymphomas were distributed as follows: diffuse large B-cell lymphoma (81.5%), large cell immunoblastic lymphoma (11.1%), and lymphoplasmacytoid lymphoma...

Association of BoLA-DRB3.2 Alleles with BLV Infection Profiles (Persistent Lymphocytosis/Lymphosarcoma) and Lymphocyte Subsets in Iranian Holstein Cattle.

Major histocompatibility complex (MHC) is the best-characterized genetic region associated with resistance and susceptibility to a wide range of diseases. In cattle, the most important example of the relationship between the MHC and infectious diseases has been established by the resistance to Bovine leukemia virus (BLV) infection. The association of the bovine MHC class II BoLA-DRB3.2 alleles with BLV infection profiles was examined. BoLA-DRB3.2 allelic diversity was determined in 190 Iranian Holstein cattle using direct sequencing method. Association of the DRB3.2 alleles with BLV infection profiles was found as the odds ratio. Effects of the alleles on lymphocyte subsets were also evaluated by multivariate regression analysis and GLM procedures. The studied cattle were categorized into three groups: BLV seronegative, BLV seropositive with persistent lymphocytosis (PL), and BLV seropositive with lymphosarcoma (LS). The PL profile was significantly associated with the BoLA-DRB3.2*0101, *1101 and *4201 alleles, although the *3202 allele mediating resistance to PL was observed. Significant association was found between the BoLA-DRB3.2*1802, *3202, and *0901 alleles and susceptibility to LS, while the *0101 and *1101 alleles were associated with resistance to LS. BoLA-DRB3.2 alleles also showed a significant correlation with CD4, CD8, CD21 cells and CD4/CD8 ratio. Allelic differences influence the immune response to BLV infection and developing the disease profile. These differences also have important consequences for tumor resistance.

Morphometric analysis of bovine lymphomas classified according to the National Cancer Institute Working Formulation.

The National Cancer Institute Working Formulation (NCI-WF) for the subjective classification of human non-Hodgkin's lymphoma is readily applicable to the classification of bovine lymphomas. Forty-nine cases of bovine lymphoma were analysed morphometrically to see if nuclear size and cleavage were distributed continuously or discretely between different NCI-WF tumour cell types. The mean nuclear area (+/- standard error of the mean, SE) was significantly greater (P < 10(-6)) in cells from the different types of diffuse large-cell lymphoma than in cells from the different types of small-cell lymphoma (42.91 +/- 1.21 micron 2 vs 19.33 +/- 1.08 micron 2, respectively); there was no overlap between the two groups. The mean nuclear are (+/- SE) of cells from diffuse large-cell lymphomas was significantly greater (P < 10(-4)) than that of cells from small non-cleaved lymphomas (42.74 +/- 1.72 micron 2 vs 27.54 +/- 1.08 micron 2, respectively), and there was again no overlap between the two groups; these two cell types are difficult to distinguish by any criteria other than size. Additionally, the cell-to-cell variability in nuclear area within a given tumour was significantly greater (P < 0.001) for the diffuse large-cell type than for the small non-cleaved cell type. The mean nuclear form factor (+/- SE) and mean nuclear contour indices (+/- SE) of the diffuse large cleaved cell type (0.53 +/- 0.02 and 5.08 +/- 0.11, respectively) were significantly different (P < 0.01 to 10(-6)) from the same parameters in the diffuse large-cell type (0.82 +/- 0.01 and 3.94 +/- 0.04, respectively). Some of the major criteria of the NCI-WF used subjectively to discriminate between bovine lymphoma cell types were supported by morphometric measurements. The magnitude of the differences in nuclear morphological characteristics between bovine lymphoma cell types was such that there was no overlap.

The immunophenotypic characterization of bovine lymphomas.

A prospective analysis of 38 cases of bovine lymphoma was performed. Thirty-five of the lymphomas were considered enzootic because of serologic or molecular biologic association with the bovine leukemia virus (BLV). The three remaining cases were in cattle < 1 year of age and were not associated with BLV and hence were considered sporadic lymphomas. Utilizing the national cancer Institute Working Formulation for the morphologic classification of human non-Hodgkin's lymphoma, 54.3% (19/35) of the enzootic lymphomas were classified as the diffuse large cleaved cell type. The cells of the 35 enzootic lymphomas expressed a consistent immunophenotype represented by MHCII+, gamma-heavy chain+, and lambda-light chain+; this constellation of markers indicated a mature B-cell phenotype. The three sporadic lymphomas failed to express MHCII antigen despite the presence of other B-cell antigens. There was no correlation between morphologic cell type and immunophenotype in these bovine lymphomas.

Phenotypic analysis of neoplastic cells from calf, thymic, and intermediate forms of bovine leukosis.

Immunohistochemistry and flow cytometric analysis were used with monoclonal antibodies to examine the phenotype of neoplastic cells from cattle with sporadic bovine leukosis (three cases of calf form, two cases of thymic form, and three cases of intermediate form). Three cases of calf form and two cases of intermediate form were positive for B cell lineage in immunohistologic examination and in flow cytometric analysis for B-B2+, sIgM+, and major histocompatibility class II+. Two cases of thymic form and one case of intermediate form were CD2+, CD5+, CD6+, and CD8+ in immunohistologic examination and in flow cytometric analysis. The results show that neoplastic cells develop from B and T cell lineages in sporadic bovine leukosis.

Classification of 1,198 cases of bovine lymphoma using the National Cancer Institute Working Formulation for human non-Hodgkin's lymphomas.

A retrospective histologic study was made of 1,198 cases of bovine lymphoma using the National Cancer Institute Working Formulation for human non-Hodgkin's lymphoma. This classification scheme was found to be readily applicable to bovine lymphoma. Most of the cell types described in the National Cancer Institute Working Formulation occurred in this series of bovine lymphomas, but the distribution of cell types varied markedly compared to that of human beings. Eighty-nine percent (1,067/1,198) of bovine lymphomas were high-grade tumors. The diffuse large cell type and its cleaved variant comprised 65.9% of all bovine lymphomas. Similar to the dog, but in marked contrast to human beings where at least 34% of non-Hodgkin's lymphomas were follicular, follicular tumors were found to be extremely rare in cattle (0.3% or 4/1,198). The prevalence of cell types varied significantly between the enzootic and sporadic lymphomas. The cleaved variant of the diffuse large cell type constituted 38% (406/1,072) of enzootic lymphomas versus 14% (18/126) of sporadic lymphomas. The mitotic index (100 x oil immersion field, 175 microns in diameter) of enzootic lymphomas (3.72 +/- 0.06, mean +/- standard error) was significantly greater than the mitotic index of sporadic lymphomas (2.82 +/- 0.17). We concluded that the cleaved variant of the diffuse large cell type with high mitotic index is characteristic of enzootic lymphoma. This characteristic high-grade cell type may be a consequence of the viral etiology of the enzootic form of bovine lymphoma.