| Objective To detect the expression of LMP1 and EBER in 100 cases of lymphoma patients for screening EBV-positive lymphoma cases and preparing for EBNA1 gene sequencing;To observe the correlation between LMP1 and EBER and that between the expression of them and the type of lymphoma for providing the basis for the diagnosis of lymphoma.EBNA1 carboxy-terminal sequencing analysis was performed on EBV-positive cases to see if there was variation for the mutation hotspots AA487 and AA466-527.Methods The protein expression of LMP1 was detected in 100 cases of lymphomas by using two step method of immunohistochemistry(Envision).The expression of EBER was detected in 100 cases of lymphomas by in situ hybridization.EBV-positive cases were screened,and the sensitivity of the two methods were compared at the same time.Polymerase chain reaction(PCR)method was used to amplify the carboxy terminal of EBNA1 gene in EBV-positive cases,and the amplified products were sent to the company for sequencing analysis.The relationship between EBV expression and prognosis in lymphoma was also analyzed.Results(1)From the expression rates of LMP1 and EBER,only classical Hodgkin’s lymphoma(HL)and diffuse large B-cell lymphoma express LMP1 in different types of lymphoma.LMP1-positive expression rate of classical Hodgkin’s lymphoma(47%)is higher than that of diffuse large B cell lymphoma(1.79%)(P<0.05),with statistical significance;Among the different types of lymphoma,only NK/T cell lymphoma,classical Hodgkin’s lymphoma(HL),and diffuse large B-cell lymphoma express EBER.Non-tumor cells can be scattered to express EBER in the remaining types of lymphoma.The positive expression rate of EBER in NK/T cell lymphoma was the highest(8/9,about 89%),and the positive expression rate of EBER in Hodgkin’s lymphoma was the second highest(10/17,about 59%),both of which were significantly higher than the expression rate of EBER in large B-cell lymphoma(3/56,5.36%)(χ2=32.770,χ2=21.948;both P<0.05),with statistical significance;The positive expression rate of EBER in patients with total lymphoma was detected by in situ hybridization(21%),which was higher than that of LMP1 detected by immunohistochemical method(9%)(χ2=5.647,P<0.05),with statistical significance.(2)From the expression patterns of LMP1 and EBER,the positive expression of LMP1 is located in the cell membrane,cytoplasm and paranuclear,and the positive signal of EBER is located in the nucleus.LMP1-positive tumor cells are usually scattered,and EBER-positive tumor cells are usually clustered or flaky,even diffusely distributed,and EBER-positive non-tumor cells are also present.(3)This experiment successfully amplified the partial fragmentthe of carboxy terminus of EBNA1 gene in 15 cases of lymphomawith EBER-positive cells(including tumor cells and non-tumor cells),including 7 cases of classical Hodgkin’s lymphoma,3 cases of diffuse large B-cell lymphoma,3 cases of NK/T-cell lymphoma,1 case of follicular lymphomaand 1 case of T lymphoblastic lymphoma.Among them,diffuse large B-cell lymphoma,NK/T-cell lymphoma and Hodgkin’s lymphoma have more amplification products,which is basically consistent with the positive results of EBER.(4)By sequencing the carboxy terminal of EBNA1 gene in 15 cases of lymphoma,the encoded product was AA439-555.Compared with the standard strain B95-8,sequence variation occurred in the C-terminus of EBNA1 in 15 EBV-positive lymphoma tissue specimens.Several lymphomas with EBER-positive non-tumor cells also showed the same common mutations as in previous studies(the variation of mutation hotspots AA487 and AA466-527).In addition to some common mutations,lymphoma samples with EBER-positive tumor cells showed non-common mutations.(5)Due to the small number of cases,the correlation between EBV and prognosis needs to be further analyzed in large samples.Conclusion The expression of EBV in lymphoma is closely related to NK/T cell lymphoma and classic Hodgkin’s lymphoma,and can be used as an auxiliary diagnosis basis of the two kinds of lymphoma.And diffuse large B-cell lymphoma can be associated with a certain proportion of EBV infection.The detection method of in situ hybridization is sensitive and the expression pattern of EBER contributes to the determination of lymphoma type.The amount of carboxy-terminal amplification product of EBNA1 gene was basically consistent with the positive expression of EBER,and that indicates that EBER is usually accompanied by EBNA1 gene.In addition to some common mutations,lymphoma samples with EBER-positive tumor cells showed non-common mutations.This is different from previous studiesand may be related to geographical differences,provides some value for exploring the pathogenesis of EBV,and needs further research.Furthermore,Due to the small number of cases,the correlation between EBV and prognosis needs to be further analyzed in large samples. |
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