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P53-MDM2 and RB expression in HD
ABSTRACTS 53 1:100) and IF-2 anti-MDM2 McAb (IgG2b, dilution 1:50) (Oncogene Science), using sensitive immunohistochemical techniques (LSAB-peroxidase APAAP Dako), in a single- and double marker combinations (MDM2/ CD30, p53/CD30, MDM2/p53). Paraffin sections were pre-treated with microwave irradiation to increase the sensitivity and reproducibility of immunostaining. Results. In all normal tissues (thymus, lymph node, spleen, bone marrow) used as controls MDM2 immunostaining was negative. In the larger majority of cases HD (64/76) we observed nuclear accumulation of MDM2 in a variable but significant proportion (range 10-90%) of cells recognized as H-RS by morphology and CD30 expression in double marker preparations. In 47/76 cases the numbers of p53+ and MDM2+ H-RS were comparable on serial sections as also were the intensities of nuclear staining. In most HD cases of the nodular sclerosis (47/62) and mixed cellularity type (9/11) both p53 and MDM2 were abnormally expressed. Coexpression of MDM2 and 1)53 could be often documented in the same H-RS cells in double-staining preparations. Nevertheless, a clear correspondence of MDM2 and p53 was not always evident since a proportion of NS cases exhibited divergent expression of the two proteins: p53+, MDM2(3/62); p53- MDM2+ (8/62). Heterogeneous figures were also obtained when MDM2 and I)53 immunostainings were compared with the presence of EBV as defined by LMP-I and EBER analysis. 3/3 cases of HD of the lymphocyte predominance type did not express either I)53 or MDM2. Our data strongly suggest that derangement of the feedback loop that regulates both the expression and activity of i)53 and MDM2 can exert a significant role in the pathogenesis of HD. P53-MDM2 and RB expression in HD
M. A. Piris, J. C. Martinez-Montero, R. Villuendas, M. Sanchez-Beato, J. F. Garcia, J. L. Orradre, P.
Martinez, Department of Pathology, HosT)ital Virgen de la Salud, Toledo, Spain p53 expression has been found in 83% of cases of HD, always labelling Sternberg-Reed and large mononuclear cells, and having found a significant relation between 1)53 immunostaining and amount of SR cells. Its expression has been accompanied by MDM2 expression in all the studied cases, suggesting p53 and MDM2 proteins are regulating each other in HD by a self-regulatory loop. The status of MDM2 gene was studied using RTPCR, which showed a weak constant presence of MDM2 MRNA in cases of HD, confirming the absence of MDM2 amplification and suggesting that MDM2 immunohistological detection is secondary to its stabilization by complexing with 1)53. As a way of assessing p53 functional status, p21 (WAF1) expression was analyzed in cases of HD, having found a constant presence of this protein in SR cells. MDM2 and WAFI protein expression were not found in cases of NHL with i953 mutation, suggesting that both MDM2 and WAF1 expression could be a marker of wild 1)53. its presence in HD support
that at least some SR cells have a wild p53 conformation, which is consistent with the absence of mutations in p53 gene in cell lines studies (L248, DEV). Rb expression has also been found to be nearly constant feature in SR cells (present in 97% of cases). Taking into account all cases, a weak relation of Rb with MIBI values was also found. This relation could be dependent of the known high growth fraction of SR and Hodgkin cells, and mirrors the oscillation of Rb values along the cell cycle found in non-tumoral cells. The result suggest absence of Rb expression is not a frequent lesion in HD. Indeed, the most striking results obtained is the strong intensity of Rb expression, also found in other types of non-Hodgkin's lymphoma. Differences in the expression of both Rb and p53 between LP, NS and MC suggest that the mechanism of disregulation of these tumour suppressor genes could vary in between types of HD. The association of EBV with Hodgkin's disease (HD) and related diseases. An overview
G. Pallesen, Laboratory of lmmuno1)athology, UniversiO, Institute of Pathology, Kommunehospitalet, Finsensgade 12, DK-8000, Aarhus C, Denmark Since 1987, EBV has obtained renewed interest as a possible aetiological factor in the development of HD (for review, see Pallesen G et al: Adv. Cancer Res. 62, 179-239, 1993). In situ hybridization (ISH) studies for EBV DNA and RNA (EBERI and 2) and immuno histological studies demonstrating EBV encoded latent membrane protein 1 (LMP1) have revealed EBV gene products in the Hodgkin and Reed Sternberg (HRS) cells in 45-50% of cases in most studies. The highest incidence (75-95~) is found in mixed cellularity whereas only 20-30% of nodular sclerosis contain EBV in HRS cells. In nodular paragranuloma, the association with EBV is low and similar to that found in spontaneous large-cell B-lymphomas in non-immunocompromised patients (5-7%). Using microwave pretreatment of paraffin sections for detection of LMP1, immunohistochemistry is not less sensitive than EBER ISH, the latter method being more resource consuming. However, detection of EBV in the non-neoplastic (small) lymphocyte population is HD tissues can only be demonstrated with the EBER ISH method. Because LMP1 is often not expressed by infected tumour cells in EBV-associated disorders other than HD, EBER ISH should be applied for the in situ detection in such cases. Immunohistochemical and molecular genetic studies have shown a characteristic pattern of EBV gene expression in HRS cells: EBNAI+, EBNA2-6-, LMPI+, LMP2A and B+, and EBERI and 2+, with the EBNA1 transcripts initiated at the Fp promoter but not at the Cp or Wp promoters. This type of EBV latent gene expression (latency type II) resembles that found in nasopharyngeal carcinoma *This study has been performedthanks to the help of the Spanish Collaborative Group for the study of Hodgkin'sDisease.
M. A. Piris, J. C. Martinez-Montero, R. Villuendas, M. Sanchez-Beato, J. F. Garcia, J. L. Orradre, P.
Martinez, Department of Pathology, HosT)ital Virgen de la Salud, Toledo, Spain p53 expression has been found in 83% of cases of HD, always labelling Sternberg-Reed and large mononuclear cells, and having found a significant relation between 1)53 immunostaining and amount of SR cells. Its expression has been accompanied by MDM2 expression in all the studied cases, suggesting p53 and MDM2 proteins are regulating each other in HD by a self-regulatory loop. The status of MDM2 gene was studied using RTPCR, which showed a weak constant presence of MDM2 MRNA in cases of HD, confirming the absence of MDM2 amplification and suggesting that MDM2 immunohistological detection is secondary to its stabilization by complexing with 1)53. As a way of assessing p53 functional status, p21 (WAF1) expression was analyzed in cases of HD, having found a constant presence of this protein in SR cells. MDM2 and WAFI protein expression were not found in cases of NHL with i953 mutation, suggesting that both MDM2 and WAF1 expression could be a marker of wild 1)53. its presence in HD support
that at least some SR cells have a wild p53 conformation, which is consistent with the absence of mutations in p53 gene in cell lines studies (L248, DEV). Rb expression has also been found to be nearly constant feature in SR cells (present in 97% of cases). Taking into account all cases, a weak relation of Rb with MIBI values was also found. This relation could be dependent of the known high growth fraction of SR and Hodgkin cells, and mirrors the oscillation of Rb values along the cell cycle found in non-tumoral cells. The result suggest absence of Rb expression is not a frequent lesion in HD. Indeed, the most striking results obtained is the strong intensity of Rb expression, also found in other types of non-Hodgkin's lymphoma. Differences in the expression of both Rb and p53 between LP, NS and MC suggest that the mechanism of disregulation of these tumour suppressor genes could vary in between types of HD. The association of EBV with Hodgkin's disease (HD) and related diseases. An overview
G. Pallesen, Laboratory of lmmuno1)athology, UniversiO, Institute of Pathology, Kommunehospitalet, Finsensgade 12, DK-8000, Aarhus C, Denmark Since 1987, EBV has obtained renewed interest as a possible aetiological factor in the development of HD (for review, see Pallesen G et al: Adv. Cancer Res. 62, 179-239, 1993). In situ hybridization (ISH) studies for EBV DNA and RNA (EBERI and 2) and immuno histological studies demonstrating EBV encoded latent membrane protein 1 (LMP1) have revealed EBV gene products in the Hodgkin and Reed Sternberg (HRS) cells in 45-50% of cases in most studies. The highest incidence (75-95~) is found in mixed cellularity whereas only 20-30% of nodular sclerosis contain EBV in HRS cells. In nodular paragranuloma, the association with EBV is low and similar to that found in spontaneous large-cell B-lymphomas in non-immunocompromised patients (5-7%). Using microwave pretreatment of paraffin sections for detection of LMP1, immunohistochemistry is not less sensitive than EBER ISH, the latter method being more resource consuming. However, detection of EBV in the non-neoplastic (small) lymphocyte population is HD tissues can only be demonstrated with the EBER ISH method. Because LMP1 is often not expressed by infected tumour cells in EBV-associated disorders other than HD, EBER ISH should be applied for the in situ detection in such cases. Immunohistochemical and molecular genetic studies have shown a characteristic pattern of EBV gene expression in HRS cells: EBNAI+, EBNA2-6-, LMPI+, LMP2A and B+, and EBERI and 2+, with the EBNA1 transcripts initiated at the Fp promoter but not at the Cp or Wp promoters. This type of EBV latent gene expression (latency type II) resembles that found in nasopharyngeal carcinoma *This study has been performedthanks to the help of the Spanish Collaborative Group for the study of Hodgkin'sDisease.