Epstein barr virus-mediated deregulation of cell cycle pathways in malignant lymphomas: implication of NFKB,p53,p27 and the mutant p21 ras using in situ techniques

The mechanism(s) underlying the pathogenesis of malignant lymphomas in general and Hodgkin's lymphoma in particular is still largely unknown. Epstein Barr virus (EBV) has long been suggested to be causally related to the incidence of some of these lymphomas particularly Hodgkin's disease (HD). Recently, a defect in the p53 tumor suppressor pathway specifically implicating p!4ARF and Mdm2 regulatory proteins has been suggested as 'a candidate pathogenic mechanism in HD. One of the up-stream acting oncoproteins -Ras oncoprotein-has been previously shown to activate the. p53 pathway through p!4ARF protein. However, the role of Ras oncoprotein in the aberrant p53 pathway has not been evaluated in lymphomas before. Therefore a retrospective study was conducted to delineate the role EB viral and the cell cycle regulatory proteins, NFkB. p53, p27 proteins and the mutant p21Ras oncoprotein in various aspects of disease pathogenesis, specifically targeting HD. To that end, a total of 75 archival tissue biopsy samples were included, comprising 40 HD cases , 10 Burkitt lymphoma (BL) cases, 20 non-Hodgkin lymphomas (NHL, other than Burkitt) cases and 5 undifferentiated nasopharyngeal carcinoma (UNPC) cases. The study aimed at a retrospective clinico-epidemiologic assessment of the age, sex and HD subtype-specific incidence, evaluation of the prevalence of Epstein Barr virus in various types of lymphomas, evaluating the frequency of Hodgkin/Reed-Sternberg cells (H/RS) in lytic cycle of EBV infection, assessment of the frequency of expression of the cell cycle proteins including the mutant p21Ras in the malignant H/RS cells of HD per se on the one hand and in the context of the EBV status on the other hand, seeking correlation between p21Ras and each of the NFkB, p53 and p27 proteins, and finally to search for a link between Ras expression and the EB viral oncoprotein LMP-1 in H/RS cells. To fulfill these aims, the two standard In Situ techniques; In Situ Hybridization and Immunohistochemistry were employed for the detection of EBV in latently and lytically infected cells, and the expression in H/RS cells of EBV-LMP-lprotein,CD30, NFkB, p53, p21 and p27 cell cycle markers using the EB viral RNA-EBERs-specific probe and the immediate early Notl/Pstl fragment probe, the NFkB-specific-probe and marker-specific monoclonal antibodies, respectively. A Lymphoblastoid cell line (LCL) was established and used as EBV positive control. In addition, the following controls were used to validate the tests results; five cases of UNPC, one EBV-positive BL case, and LCL cyto-preparation slides. As a negative control, two normal lymph node post-mortem tissue biopsies were used. The results showed the following: • In HD, 27 cases out of 40 (67.5%) were below the age of 40 years versus 13 of 40 (32.5%) above 40 years. Bi-modal Age distribution was noted in this disease. Among the 30 total NHL cases, the corresponding numbers were 17 (56%) and 13 (43.3%), respectively, showing uni-modal age distribution. • No significant difference was noted in age-specific incidence between the two sexes in both HD and NHL [mean age was 39.15(+/_19.15) in males with HD versus 40.80(+/_ 9.54) in females with HD and 30.33(+/_4.74) in males with NHL versus 35.26(+/_ 6.01) in females with NHL]. Male to female sex ratio was ~ 1:1 in HD and 2:1 in NHL. Among the total 40 HD cases, 27 (67.5%) were of the MC subtype, 5 (12.5%) NS, 4 (10%) LP and 4 (10%) were of the LD subtype. Twenty five (62.5%) HD cases, 9 (30%) NHL cases, 4 (40%) BL cases, 5 (25%) non-BL NHL cases and 5 (100%) of UNPC cases were EBV-positive when tested with EBERs ISH and LMP-1IHC, with 100% concordance between the two tests. . All HD cases except one (39 out of 40) were positive for CD30 marker. • Among EBV-positive HD cases, 17(42.5%) were below and 8 (20%) were above 40 years. The corresponding numbers among EBV-positive NHL were 5 (16.6%) and 4 (13.3%). • EBV was detected in 19 (76%) of MC, 1 (4%) NS, 1 (4%) LP and 4 (16%) of LD subtypes. Significant difference in HD subtype distribution was found between EBV-positive and negative group (p<0.001), and a statistically significant association was observed between MC subtype and EBV positivity (X2= 26.01, p<0.05). • Only 5 out of 40 HD cases (12.5%) showed lytically-infected RS cells when tested with Notl/PstI probe ISH, with a frequency of 1-3 RS cells per high power field. • Staining of H/RS cells with EBV-EBERs and Notl/Pstl-specific probes gave nuclear pattern of staining, while LMP-1 and CD30-specific monoclonal gave cytoplasmic-membranous staining patterns. • The frequency of H/RS cells expression of NFkB, p53, p21 and p27 proteins in HD cases were 17 (85%), 19 (47%), 29 (72.5) and 9 (22.5%) cases, in that order. The expression was statistically significant [the corresponding Relative Risk (RR) value were 4.52, 5.43, 3.82, and 0.11]. • All cell cycle markers gave nuclear pattern of staining except p21Ras which gave clear cytoplasmic staining. • When cell marker expression was assessed by the EBV state in HD, a significant difference was found between the EBV positive and negative groups, where the number of cases showing positive expression for the four markers in each of the two groups respectively, were for the NFkB; 12 (82.5%) versus 5 (83.3%), for p53; 15 (60%) versus 4 (26.6%), for p21; 21 (84%) versus 9 (60%) and for p27; 1 (4%) versus 8 (53%). The corresponding RR values were, in the same order; 2.4 versus 0.53, 3.75 2.33 versus 0.61 and 0.13 versus 1.22. Higher expression of all markers except p27 was observed in EBV-positive HD cases. Correlation between the cell cycle marker revealed no correlation between expression of NFkB and any of the other markers, p53, p21 and p27 (p>0.05). p53 exhibited low but insignificant correlation with p21 expression (p>0.05), and no correlation with p27, while a highly significant but inverse correlation was found between p21 and p27 (p<0.01). The high expression of the mutant p21Ras and the low expression of p27 both contribute to cell growth and proliferation albeit through different pathways. The viral LMP-1 showed an intermediate correlation with NFkB, p53 and unexpectedly with CD30, which was short of the significance level. Similar correlation was found between CD30 and NFkB. On the other hand, significant correlation was observed between LMP-1 and each of the p21Ras and p27 proteins (p<0.01). In conclusion, this study highlighted the significant role of EBV in the pathogenesis of HD mainly through manipulating key cell cycle regulatory proteins each acting via different pathways that converge at the transcriptional machinery of the cell. An unprecedented finding in this study was the expression of the mutant form of Ras in this tumor. The correlation between EBV status and p21Ras expression suggests that both are important players in the pathogenesis of the disease, however, the causal relationship between the two remains to be determined.