Epstein-Barr virus latent membrane protein-1 expression in Hodgkin lymphoma

Expression of latent membrane protein-1 of Epstein-Barr virus is documented in Hodgkin lymphoma indicating its relationship in disease process. A total of 50 cases of Hodgkin lymphoma were analyzed for latent membrane protein-1 expression by immunohistochemistry. The mean age was 28.7 years with male predominance. Mixed cellularity classical Hodgkin lymphoma was the commonest subtype (50% cases). Out of 50 cases, 48% cases were found positive for latent membrane protein. Of the five histologic subtypes, all cases of lymphocyte depleted classical Hodgkin lymphoma showed positivity which was followed by 60% positivity of mixed cellularity classical Hodgkin lymphoma. Association of latent membrane protein-1 expression in relation to different age group, site of involvement and subtypes of Hodgkin lymphoma were found statistically insignificant while latent membrane protein-1 expression was significantly higher in male than female.


Introduction
Hodgkin lymphoma is an unusual malignancy because only a small number of cells constitute the tumor mass. This small fraction of cells are admixed with abundant heterogeneous population of non-neoplastic inflammatory and accessory cells. As a result, the study on these cells is challenging. Regarding pathogenesis of the Hodgkin lymphoma, it is found that Epstein -Barr virus is associated with some fractions which are causal. 1 Several studies were performed to see the association of Hodgkin lymphoma with Epstein -Barr virus infection using immunohistochemical method in neoplastic cells of Hodgkin lymphoma 2 The detection of Epstein-Barr virus nucleic acids and proteins in neoplastic cells of Hodgkin lymphoma defines Epstein-Barr virus positive Hodgkin lymphoma. Viral RNAs referred to as EBERs detection by in situ hybridization technique and immunohistochemical detection for the latent membrane protein-1 are widely used in the diagnostic laboratories. Its association is dependent on various factors such as geographic location, histological subtypes, sex, ethnicity and age. Epstein-Barr virus infection is associated with various malignancy such as nasopharyngeal carcinoma, peripheral T cell lymphoma, nasal natural killer (NK) and T-cell lymphoma. [3][4][5] As the histological features of Hodgkin lymphoma sometimes overlap with anaplastic large cell lymphoma and reactive lymphoid hyperplasia, Epstein-Barr virus detection tests also help to arrive accurate diagnosis. Latent membrane protein-1 is expressed in the cytoplasm and surface membrane of Reed-Sternberg/Hodgkin cells which can be detected by immunohistochemical stain. 4 This study is planned to explore the latent membrane protein-1 detection to see its overall positivity in Hodgkin lymphoma, its pattern of positivity in different subtypes of Hodgkin lymphoma along with their correlation with demographics of 50 cases of Hodgkin lymphoma in terms of age and gender.

Materials and Methods
A total of 50 cases of Hodgkin lymphoma were obtained from the Pathology Department from January 2013 to December 2016. The clinical parameters like age and gender were recorded from the patient's requisition form.
All hematoxillin and eosin stained slides were reexamined and classified according to WHO Classification 2008 into to five subtypes viz. nodular sclerosis classical Hodgkin lymphoma, mixed cellularity classical Hodgkin lymphoma, lymphocyte rich classical Hodgkin lymphoma, lymphocyte depleted classical Hodgkin lymphoma and nodular lymphocyte predominant Hodgkin lymphoma.
All cases were examined for latent membrane protein-1 detection by immunohistochemical stain. Thin sections (4.0 µm) were taken on poly-L-lysin coated slides from the selected paraffin blocks. The slides were air-dried and incubated for 16 hours at 37°C in an incubator. Dewaxing of slides was done by subsequent two changes in xylene for 5 min duration each. The slides were dehydrated in decreasing strength of isopropyl alcohol (100, 90, 80, 70%) for 10 min in each. Then the slides were treated with Dako target retrieval solution (Code No. S1700). Retrieval solution was taken in coplin jar and preheated in the water bath at 65°C. the slides were kept in this solution and heated in the water bath at 95-99°C for 30-40 min. After 20 min, each slide was washed with deionized water for 5 min. Peroxidase-blocking solution DakoReal TM (Code No S2023) applied for 10 min followed by wash in Tris buffer saline for 5 min. Monoclonal mouse anti-Epstein-Barr virus, LMP clones CS, 1-4 (Code No M0897) was used as the primary antibody with a dilution of 1:200 and incubated for 30 min. This was followed by Tris buffer saline wash 5 min twice. The incubation in Envision TM Detection System, HRP, rabbit/mouse, (code K5007) was done for 30 min followed by wash in Tris buffer saline for 5 min twice. Application of 3,3-diaminobenzidine substrate (DAB) was used as chromogen and kept for 10 min. This step was followed by deionized water wash. Counter-staining of nucleus was performed by Mayer's Hematoxillin for 2 min.
Dehydration was done by increasing strength of isopropyl alcohol (70, 80, 90 and 100%), each step for 10 min. Clearing was done by subsequent two changes in xylene for 5 min duration each. The slide was mounted with DPX. Immunostaining procedure was followed according to Dako, Denmark product Envision TM Detection System, HRP, rabbit/ mouse, (code K5007). Each case was run with control slide (diagnosed positive case of Hodgkin lymphoma). Cytoplasmic and membrane staining of the latent membrane protein-1 were interpreted as positive. Hematoxillin was used as counter stain ( Figure 1).

Statistical analysis
Statistical analysis was done using SPSS version 20. Chi-square test of independence was used to find out the association of latent membrane protein-1 expression between/among different age group, gender and subtypes of Hodgkin lymphoma along with latent membrane protein-1 positivity.

Results
Among the 50 studied cases of Hodgkin lymphoma, 48% cases were positive for Epstein-Barr virus latent membrane protein-1 by immunohistochemistry (Table I).
The age of studied cases ranged from 4 to 75 years with the mean age of 28.7 years (SD ± 16.95). Male cases outnumbered the female (76/24%).
Among the subtypes of Hodgkin lymphoma, latent membrane protein-1 was found 100% positivity in lymphocyte depleted classical Hodgkin lymphoma followed by 60% in mixed cellularity classical Hodgkin lymphoma, 41.7% in classical Hodgkin lymphoma, unclassifiable, 30% in nodular sclerosis classical Hodgkin lymphoma and 0% each for lymphocyte rich classical Hodgkin lymphoma and nodular lymphocyte predominant Hodgkin lymphoma (Table I).

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BSMMU J 2018; 11: 203-208 The association of latent membrane protein-1 expression in relation to different age group and subtypes of Hodgkin lymphoma was found statistically insignificant. However, the mentioned association was found statistically significant in respect to gender (Table I).

Discussion
This study shows 48% positivity in Hodgkin lymphoma. Only the positivity of latent membrane protein-1 in male patients (55.3%) were significantly higher than the females, however the association of latent membrane protein-1 expression in relation to different age group and subtypes of Hodgkin lymphoma was found statistically insignificant. The most common subtype was mixed cellularity classical Hodgkin lymphoma with 60% latent membrane protein-1 positivity, however lymphocyte depleted classical Hodgkin lymphoma showed 100% positivity (Table I). In addition, each case of nodular lymphocyte predominance Hodgkin lymphoma and lymphocyte rich classical lymphoma showed negativity to the latent membrane protein-1. Hodgkin lymphoma is presented with bimodal age incidence. In developing countries, the first age incidence peak occurs in childhood and the second peak occurs in elderly patients. However in developed countries the first peak delayed until young adulthood. 20 Dinand et al. found statistically significant association between Epstein-Barr viruslatent membrane protein-1 detection in younger age group of Indian children. This study also described statistically significant association between Epstein-Barr virus-latent membrane protein-1 detection and middle-low socioeconomic status as compared with the higher socioeconomic status. 21 There is no statistically significant differences found between <15 years age group, 15-50 years age group and >50 years age group in our series.
The mean age of Hodgkin lymphoma cases of the present study is similar to the study performed by others 12, 22 but lower than Cickusic et al. (2007). 17 However, a retrospective analysis of diagnosed hematological malignancies in Bangladesh over The present study showed 60% positive cases which is lower in comparison to others data. One explanation could be due to inclusion of 24% cases of unclassifiable classical Hodgkin lymphoma in our series.
In this study, Epstein-Barr virus latent membrane protein-1 positive staining was seen in 100% cases of lymphocyte depleted classical Hodgkin lymphoma subtype. A study carried out in Iran found 100% positive cases of latent membrane protein-

Conclusion
Forty eight percentage cases of the present study series of Hodgkin lymphoma expressed latent membrane protein-1 positivity. Among other parameters male patients showed significant positivity (55.3% cases) of latent membrane protein-1 than the female. Though the mixed cellularity classical Hodgkin lymphoma was found to the most common subtype (50% cases), their association with latent membrane protein-1 showed 60% positivity while 100% cases of lymphocyte depleted classical Hodgkin lymphoma showed positivity for latent membrane protein-1.