Association of Hodgkin ’ s Lymphoma with Epstein Barr Virus Infection

Th e role of Epstein Barr virus (EBV) in the onset of Hodgkin’s lymphoma has been a subject of ongoing research. However, confi rmation of EBV oncogenic involvement was not possible due to the small number of neoplastic cells characteristic for this type of tumor. Presence of EBV infection in neoplastic and non-neoplastic cells was analyzed in  cases of Hodgkin’s lymphoma. In neoplastic cells, using an immunohistochemical method, latent membrane protein  (LMP) was found in , of cases, while in situ hybridization results demonstrated the presence of EBER RNA in , of the cases. EBER RNA was found in non-neoplastic lymphocytes in , of cases. EBV is most frequently associated with Hodgkin’s lymphoma in the fi rst and seventh decade of life, specifi cally the nodular sclerosis subtype. No apparent diff erence was observed in the association of Hodgin’s lymphoma with EBV between genders, or in relation to clinical stage of the disease and average age of the patient. However, association with childhood age is signifi cantly greater in comparison to adults. EBV associated disease shows a signifi cantly greater prevalence in T lymphocytes. Slightly more abundant are cytotoxic T lymphocytes, which are also more frequently in contact with Reed-Sternberg cells, although there is no diff erence in number and positioning of histiocytes. Variations between the data on the association of EBV with Hodgkin’s lymphoma among studies from diff erent parts of the world suggest that factors of age, gender, ethnic background and social status might present biological modifi ers of EBV infl uence on the pathogenesis of this neoplasm. Th e diff erences in non-neoplastic infi ltrate EBV+ and EBVlymphoma indicate the eff ect of the virus on the immune interaction of tumor and host in this disease.


Introduction
Infectious agents as a cause of Hodgkin's lymphoma were extensively studied, mainly due to the clinical symptoms such as cycling fever and night sweats.Factors such as, morphological appearance of reactive tissue surrounding neoplastic cells, epidemiological fi ndings of bimodular curve of age incidence, geographic diversity of incidence in younger population, and diff erences in risk factors in children of diff erent social classes are also taken in consideration ().Th e role of Epstein Barr virus (EBV) in the onset of Hodgkin's lymphoma was discussed in the past, however the research on this subject was diffi cult to perform due to a small number of neoplastic cells characteristic for this type of tumor.Following the development of sensitive DNA methods, association of EBV with this neoplasm was confi rmed, along with the monoclonal presence of EBV genome (,).The association of EBV with specific types of Hodgkin's disease varies.A mixed cellularity type is EBV positive in up to  of cases, whereas nodular scleorsis is less frequently associated with this infection ().Lymphocyte depletion is rarely associated with EBV, while lymphocyte predominance type is practically never positive.There are significant regional differences in respect to this association.Higher rates of EBV associated Hodgkin's lymphoma were found in Peru and Honduras (over ), in comparison with those found in western Europe and USA (up to ) (,,,).Higher incidence of EBV associated Hodgkin's lymphoma was found in younger patients population (under  years of age) as well as older patients population (over  years of age) ().In AIDS patients with progressing Hodgkin's lymphoma, EBV is always present in Reed-Sternberg cells ().Some authors describe EBV receptor expression (CR, CD) on the surface of atypical cells in EBV positive cases of Hodgkin's lymphoma, thus explaining viral presence in the cells (,).Hodgkin's lymphoma associated with EBV infection shows expression of EBV gene in the form of latency type II.Reed-Sternberg cells and their variants exhibit strong expression of latent membrane protein  (LMP), although expression of Epstein Barr viral nuclear antigen  (EBNA) is absent ().Northern and Western blots of Hodgkin's lymphoma samples frequently reveal presence of latent membrane protein A (LMPA) and latent membrane protein B (LMPB) ().LMP is known to be a target for the attack by cytotoxic T lymphocytes in healthy individuals.High expression of LMP, LMPA, -B in tumor cells of Hodgkin's lymphoma is an indicator of some specifi c immunological defi ciencies in patients with this disease.
It is known that patients with Hodgkin's lymphoma have impaired immune response ().With regards to prognosis, variation among EBV positive and EBV negative cases was not found ().Numerous international studies done on this subject suggest that age, gender, ethnic background and social status factors might represent biological modifi ers of EBV eff ect on the pathogenesis of this neoplasm.Diff erences found among studies indicate epidemiologically complex relation of the virus and this disease, which can only partly be contributed to small samples and demographic diversity.Neoplastic cells of Hodgkin's lymphoma survive in the surroundings rich in infl ammatory cells which main role is prevention of their growth and proliferation.Even though they are resistant to endogenous apoptosis, neoplastic cells of Hodgkin lymphoma are mainly sensitive to therapeutic treatments that lead to cell death.It is not known which factors influence the balance between apoptotic and antiapoptotic mechanisms in untreated disease, or the predominance of apoptotic signals during treatment.EBV presence in neoplastic cells of Hodgkin disease might infl uence cytotoxic T cells' mechanism which main role is to destroy cells carrying pathogens, hence indirectly controlling programmed cell death.For better understanding of this enigmatic lymphoma it is necessary to identify factors involved in its emergence, and frequent association with EBV infection.

Objective
The objective of this study is to determine the presence of EBV infection in neoplastic cells of Hodgkin lymphoma and non-neoplastic infl ammatory infi ltrate.Th e study will classify EBV positive and negative cases of lymphoma according to the age, gender and clinical stage of the disease.Th e cell structure of a non-neoplastic inflammatory infiltrate and involvement of CD+ cytotoxic T lymphocytes in EBV positive and negative cases of Hodgkin's lymphoma will be analyzed.Association level of Hodgkin's lymphoma with EBV infection will be compared with the results of similar studies.

Materials and Methods
The study involved  cases of Hodgkin's lymphoma diagnosed at the Department of Pathology at the University Clinics Center in Tuzla, in the period from  to .Immunohistochemical method of three-step immunoperoxidase with streptavidin was used in the diagnostics.For the purpose of phenotyping neoplastic and background cells primary antibodies were used for CD(PC/A), CD(C/B), CD(CD-), CDcy(L), CD(Ber-H), CD(T/) and CD(PG-M).EBV infection detection was done using primary antibody for latent membrane protein LMP(CS -) (DAKO, Glostrup, Denmark).Deparaffi nized,  μm thick tissue slices, formalin fi xed and in paraffi n embedded Hodgkin's lymphoma tissue samples were incubated with primary antibodies for  hour.Prior to the incubation endogenous peroxidase activity was inhibited, and antigen de-masking performed, by cooking for  minutes in buff er pH ,.Incubation with biotin labeled secondary antibodies and streptavidine labeled peroxidase lasted  minutes.Chromogen used was , diaminobensidine hydrochloride, while contrast staining was done with hematoxilin.Epstein Barr virus ribonucleic acid (EBER RNA) detection was done with an in situ hybridization method, using FITC labeled peptide nucleic acid probes (PNA) (Dako, Glostrup, Denmark).Following standard procedure of deparaffi nization and rehydration, pretreatment of tissue slices was performed with proteinase K for  minutes.Next step was hybridization with PNA probe for  minutes at °C, and incubation with secondary anti-FITC antibody labeled with alkaline phosphatase for  minutes.Visualization was done with BCIP/NBT/levamisol substrate system for  minutes, followed by hematoxiline contrast staining.Statistical analysis included calculation of mean value, standard deviation, proportions testing, and hi-square test.

Results
Neoplastic Hodgkin's lymphoma cells were immunohistochemically positive for latent membrane protein  in  (,) .Lymphocytes of the background infiltrate in EBV negative group of subjects, did not show presence of EBV LMP by immunohistochemistry, while in situ hybridization confi rmed the presence of EBER RNA in lymphocytes of  (,) cases.Neoplastic cells of this group were positive for CD in  (,) cases.CD was positive in neoplastic cells in  (,) cases of this group, positive CD was not found, while CD was positive in one (,) case.CD was positive in Hodgkin's cells in  (,) cases of this group.

Discussion
Numerous findings indicate an etiological role of Epstein Barr virus, widely spread herpes virus, in numerous lymphoid and epithelial malignancies.Epidemiological studies have shown three times greater risk of acquiring Hodgkin's lymphoma in individuals who were diagnosed with infectious mononucleosis.Higher antibody titer for EBV antigens was found in patients with Hodgkin's lymphoma when compared with the control group ().Th e fact that EBV makes human B lymphocytes immortal, and in mouse neoplastic, that expression of EBV gene in immortalized cell lines of rodents leads to transformation in vitro and in vivo, as well as the tumorigenic eff ect of EBV in some primates, have led to biological basis of EBV association with Hodgkin lymphoma (,,,).Th e role of EBV in pathogenesis of Hodgkin's lymphoma is further supported by fi ndings of latent EBV infection in some of these neoplasms, using Southern blot DNA hybridization, in situ hybridization and antigen detection, as well as polymerase chain reaction (,,,).Lower association with EBV infection in comparison to present study was presented by the authors of study in Philippines (,), Costa Rica (), France ( and ), India ().Statistically signifi cantly lower association was found in studies done in Poland ()(p<,), Israel ()(p<,), and Sweden ()(p<,) (,,,,,,,). Higher association of EBV with Hodgkin's lymphoma was found in fifteen studies: in Turkey (), Japan ( and ), China (,), Malaysia (), Taiwan (,), and Algeria ().Statistically signifi cant increase in second study done in Algeria ()(p<,), Korea ( and )(p<,; p<,), Mexico ()(p<,), Kenya ( and )(p<,), and studies from Peru and Honduras showed association with EBV infection higher than  (,, ,,,,,,,,,,,).Th e association with EBV infection in  cases, similar to the present study was found in one study conducted in Italy ().With regards to the association of a particular Hodgkin's lymphoma subtypes with EBV infection, considerable increase in the frequency of EBV association with mixed cellularity subtype was determined by the authors of studies done in Poland (,), Philippines (,), Great Britain (), Taiwan (,), international studies from  instances of Hodgkin's disease (,), USA (), Korea ( and ), France (), Mexico (), Japan (), Malaysia (), Costa Rica (), China (,) and Hong Kong () (,,,,,,,, ,,,,,,).Less frequent association of EBV and this lymphoma subtype in relation to the present study group was found in studies from Sweden (,), Turkey (,) and Israel (,) (,,).Statistically signifi cant decrease in association (p<,) was found by authors from India with , EBV positive cases of this subtype ().Comparison of the association of a nodular sclerosis subtype with EBV infection between present study group and the above mentioned studies, demonstrated increased frequency of an EBV infection in nodular sclerosis subtype presented in studies from Mexico (,), Hong Kong (,), Korea (,), Turkey (,), and Taiwan (,), even though none of them showed statistically signifi cant diff erence (,,,,).Association frequency of a lymphocyte depletion subtype with EBV in the above listed studies spans from  in the study done in China, to  in the studies done in Hong Kong, India, Taiwan and Sweden (,,,,).Th e studies on the association of lymphocyte predominance with EBV diff er in fi ndings; in the studies done in India, Great Britain, Poland and Malaysia association was not found, while the study from Taiwan shows association in , cases, Sweden , Turkey , and international study of  cases of Hodgkin's disease , (,,,, ,,,).In relation to the present study group statistically signifi cant diff erence was not found.In the distribution of EBV associated cases according to the age in the present study group, the highest frequency of these cases is found in the fi rst (,) and seventh (,) decade of life.Statistically significant increase in the frequency of EBV positive Hodgkin's lymphoma (p<,) is found in the seventh in comparison to fourth decade of life.A frequency of EBV infection in the present study group was only slightly higher among female (,) than male gender (,).Distribution of the association of a particular lymphoma subtypes with EBV infection regarding patients gender did not show any considerable differences.Distribution of the association in a particular Hodgkin's lymphoma subtypes with EBV infection in regard to the patient gender and age, showed the highest association with EBV nodular sclerosis subtype (,) for males in the third decade of life.Half of the cases with nodular sclerosis subtype in the fi fth, sixth and seventh decade of life were associated with EBV infection.Mixed cellularity in the sixth decade of life in male was associated with EBV in , cases, and in the seventh decade in , cases.All cases of the lymphocyte depletion in males of the second and third decade of life were EBV associated.In males in the fourth decade of life association of Hodgkin's lymphoma with EBV infection was not found.In females, the subtype of nodular sclerosis associated with EBV infection in second decade was found in , cases, and in the seventh decade in , cases.In the EBV associated case of mixed cellularity for females in the second, third and seventh decade one case was found in each group, contributing to ,, ,, or more precisely , cases of this subtype were found in those decades.Absence of EBV associated Hodgkin's lymphoma in the eight decade of life is not possible to interpret due to small number of cases found in that age group.According to the data presented above, male population showed frequent association with EBV infection and nodular sclerosis subtype in the second decade, and mixed cellularity in the sixth and seventh decade of life.In female population nodular sclerosis subtype in the second and seventh decade of life showed the highest association with EBV infection.Analyzed with respect to the clinical stage of the disease, cases associated with EBV infection did not show statistically significant difference.Subtype of Hodgkin's lymphoma is usually considered as a basic factor of risk for the association of this disease with EBV infection.In the study of  cases of Hodgkin's disease it was found that , cases of mixed cellularity and , cases of nodular sclerosis are associated with EBV ().Th e diff erences between the subtypes of mixed cellularity and nodular sclerosis in the frequency and forms of association with EBV infection, along with clinical and other epidemiological indicators, are considered as a basis for the hypothesis that these two subtypes represent distinct entities ().Th e absence of statistically signifi cant diff erences in the frequency of EBV associated cases of mixed cellularity and nodular sclerosis (, in comparison to ,) in the present study group, does not support the above mentioned hypothesis.While the authors of the mentioned study found that the risk for development of Hodgkin's lymphoma associated with EBV is two times lower for young females than that for males of the same age, the present studied group for males of ages from  to  years resulted in , cases, and in females , cases associated with EBV infections ().Nodular sclerosis subtype was found to be rarely associated with EBV infection in young females in the above mentioned study, although the present study group found , of cases associated with this virus.Even though the association of Hodgkin lymphoma with EBV infection in young females of the present study group is larger in relation to the data of discussed studies, it is signifi cantly lower in respect to the association in childhood and in patients over  years of age.Th is could be in accordance with already assumed preventive eff ects in the reproductive age of a female ().Hodgkin lymphoma in children of present study group showed statistically signifi cant increase in the frequency of association with EBV infection (p<,) in comparison to adult group.In the age group up to , all of the six found cases showed EBV presence in the neoplastic cells.In a particular subtypes of Hodgkin's lymphoma in children, the frequency of association is found in  (/) cases of lymphocyte depletion, , (/) cases of nodular sclerosis and , (/) cases of mixed cellularity.All the male children had EBV positive neoplastic cells, while in , of female children Hodgkin's lymphoma was associated with the EBV infection.According to these data Hodgkin's lymphoma in children of the present study group is pronouncedly associated with an EBV infection, almost regularly in children up to  years of age, more often in male children, with association to the subtypes of lymphocyte depletion and nodular sclerosis.In the present study of children group Hodgkin's lymphoma showed greater association with EBV infection, hence contradicting the results of numerous other studies.In the study done in Australia  of EBV associated Hodgkin's lymphoma was found in chil-dren, in South Africa  and , in United Arab Emirates , in USA , in Brazil , in Greece , in France , in Argentina, Great Britain, Egypt and Jordan each  of EBV positive cases (,,,,,).Statistically signifi cant decrease in the association of this neoplasm with EBV infection in children, in comparison to present study, was found by the authors of two studies done in USA with ,(p<,) and ,(p<,) of these cases (,).Greater association of Hodgkin's lymphoma with EBV infection in children in comparison to the present study, was found in the studies done in Costa Rica (), China (,), Honduras () and Kenya () (,,).On the other hand, high frequency association of Hodgkin's lymphoma with EBV in children of the present study group, corresponds to the type of the disease also found in developing countries.Most of the authors presented association of a mixed cellularity subtype, and partly lymphocyte depletion with EBV in childhood (,,,, ,).Mean age of the patients with Hodgkin's lymphoma associated with EBV infection did not show statistically signifi cant diff erence from the mean age of patients suff ering from the disease not associated with this infection.A slightly higher mean age in patients with Hodgkin's lymphoma associated with EBV is a consequence of a signifi cant number of cases found in patients  years old or older.On the other hand, high frequency association of this disease with EBV in children (), could not have a signifi cant infl uence on the decrease of mean age in patients with EBV associated disease.Th is is due to a small number of children with this disease (,) in presently analyzed group.Characteristics of the infl ammatory infi ltrate in the group of patients with established association with EBV infection, in comparison to the group in which the association was not found, statistically significant higher abundance of T cells is noticed in the fi rst (,) when compared to the second group (,).B lymphoid cells were signifi cantly prevalent (p<,) in infi ltrate of the EBV negative when compared to EBV positive Hodgkin's lymphoma.Histiocytes positive for CD antigen were found in the infi ltrate of EBV positive disease in almost the same number as in EBV negative disease.Examination of cell structure in the infl ammatory infi ltrate lead to the conclusion that presence of EBV in neoplastic cells of Hodgkin's lymphoma has significant influence on the presence of CD positive T cells and CD positive B cells.However, a signifi cant infl uence on the number of CD positive histiocytes in background infi ltrate was not found.Th e use of in situ hybridization method in the studies of association of EBV with Hodgkin lymphoma demonstrated presence of the virus in certain number of non-neoplastic lymphoid cells (,).Presence of EBER RNA was found in non-neoplastic lymphocytes of the studied group in  (,) cases.Considerable majority (p<,) of these cases (,) showed presence of EBER RNA in neoplastic cells as well, while in , cases of neoplastic cases neoplastic cells were negative for EBV.In distribution of cases with EBER RNA positive non-neoplastic lymphocytes according to subtype of Hodgkin's lymphoma statistically signifi cant diff erence was not found.Th ey are more often associated with subtype of lymphocyte depletion (,), followed by mixed cellularity (,) and nodular sclerosis (,), while they are not found in the lymphocyte predominance type.Interestingly, in the group with EBV negative Hodgkin's lymphoma we found all three cases with EBV positive non-neoplastic lymphocytes to be of nodular sclerosis subtype.The presence of EBV in neoplastic and non-neoplastic cells of Hodgkin lymphoma could indicate coexistence of a clonal proliferation of EBV infected Reed Sternberg and Hodgkin's cells and nonspecifi c infection of non-neoplastic lymphocytes.Th e fi nding that speaks in favor to this hypothesis is that EBV positive neoplastic cells and non-neoplastic lymphocytes of Hodgkin lymphoma are infected with diff erent strains of EBV, contributing to diff erent oncogene abilities of this virus ().Viral infection of non-neoplastic lymphocytes could aff ect their function, thus impairing their defense mechanism against neoplastic tissue.

Conclusion
Th e presence of latent membrane protein , using an immunohistochemical method was determined in  (,) cases, while in situ hybridization results demonstrated presence of EBER and EBER RNK in  (,) studied cases of Hodgkin's lymphoma.EBV is associated with , cases of lymphocyte depletion subtype, , cases of mixed cellularity, , cases of nodular sclerosis and , cases of lymphocyte predominance.All cases of Hodgkin's lymphoma diagnosed in the fi rst decade of life are EBV positive, as well as , cases found in the seventh decade of life.In the eight decade, association with EBV infection has not been found, while it is , in the fourth decade.Th e frequency of Hodgkin's lymphoma association with EBV infection in children is .All male children and , of female children have EBV associated disease.Association of this neoplasm with EBV infection does not show signifi cant diff erence in respect to the clinical stage of disease, gender and mean age of patients.Variations between data on the association of EBV with Hodgkin's lymphoma among studies from diff erent parts of the world, suggests that factors of age, gender, ethnic background and social status may present biological modifi ers of EBV infl uence on pathogenesis of this neoplasm.Th e diff erences in the non-neoplastic infi ltrate between EBV+ and EBV-lymphomas suggest the eff ect of the virus on the immune interaction of tumor and host in this disease.
cases(Figure ).In situ hybridization results demonstrated positive EBER RNA in Reed-Sternberg, Hodgkin's and lacunar cells in (,)  cases(Figure ).Positive non-neoplastic lymphoid background cells were found in (,)  cases.Out of  cases of Hodgkin lymphoma, for which in situ hybridization confi rmed the association with EBV infection,  (,) cases were of male, and  (,) female gender.EBV associated Hodgkin's lymphoma in one (,) case was of lymphocyte predominance type, in  (,) cases nodular sclerosis type, in (,)  cases of mixed cellularity type, and in  (,) cases of lymphocyte depletion type.Th e youngest patient was , and the oldest  years old.Th e average age was , years, with standard deviation of , years.Distribution of EBV positive cases according to the gender and age is shown in Figure .Incidence of known specifi c clinical stages according to the Ann Arbor classifi cation, for EBV as-sociated Hodgkin's disease is presented in Table .In the group of subjects in which in situ hybridization demonstrated the presence of EBER RNA in neoplastic cells,  (,) cases were positive for EBV LMP with immunohistochemical method.Lymphocytes of the background infi ltrate in the EBV associated group of subjects showed no presence of EBV LMP immunohistochemically, while in situ hybridization showed the presence of EBER RNA in lymphocytes of  (,) cases.Neoplastic cells in this group were positive for CD in  (,) cases.CD was positive in neoplastic cells of  (,) cases in this group, while positive CD was not found.CD was positive in one (,) case.CD in Hodgkin's cells was positive in  (,) cases of this group.Table  presents the presence of CD positive T lymphocytes, CD positive B lymphocytes, CD positive T cytotoxic lymphocytes and CD positive histiocytes in the background infi ltrate of the EBER RNA positive Hodgkin's lymphoma cases.Cytotoxic CD positive T lymphocytes were found in close proximity of neoplastic cells of EBER RNA positive Hodgkin's lymphoma in  (,) cases.Histiocytes positive for CD antigen were found next to Hodgkin's cells in  (,) cases.Out of  cases of Hodgkin lymphoma, not associated with EBV infection,  (,) patients were male, and  (,) of female gender.EBV negative Hodgkin lymphoma in three (,) cases was of a lymphocyte predominance type, in  (,) cases of nodular sclerosis type, in  (,) cases of mixed cellularity type and in one (,) case of lymphocytic depletion type.Th e youngest patient was  years old, the oldest  years old, average age was , years, with standard deviation of , years.Incidence of known specifi c clinical stages according to the Ann Arbor classifi cation, for EBV negative Hodgkin's lymphoma is shown in Table