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Southern China has a high-incidence of nasopharyngeal carcinoma, sinonasal lymphoepithelial carcinoma and extranodal NK/T cell lymphoma (nasal type), all of which are closely associated with Epstein-Barr virus (EBV) infection.［1-3］ How many immunophenotypes can be classified as primary nasopharyngeal non-Hodgkin lymphoma (NPL) in Guangzhou? What is the relationship between Epstein-Barr virus (EBV) infection and immunophenotypes of NPL? What are the characteristics of gene expression of EBV existing in NPL cells? This paper discussed the issues mentioned above.
METHODS
Subjects
We reviewed all the biopsy slides taken from the nasopharynx in the Departments of Pathology affiliated to the Sun Yat-sen University, China, during the period of January 1, 1998 to December 31, 2000 and found 34 NPLs. In addition, 39 NPLs were collected from other hospitals in Guangzhou, China. Altogether, 73 NPLs were collected for this study. The ages of the 73 NPL patients ranged from 8 to 82 years old, with the mean of 45.5 years. The ratio of male to female was 2.17∶1 (50∶23).
All the tumor masses were located at the superior-posterior or lateral wall of the nasopharynx. Most of the 73 patients had symptoms and signs derived from the nasopharynx, including nasal obstruction, nasal discharge, hearing impairment and/or epistaxis. Thirty-five patients complained of having enlarged cervical lymph node(s) after the appearance of symptoms evoked by the nasopharyngeal lesion. None of the 73 NPL patients had skin, soft tissue, bone marrow or other visceral lesions. Forty-nine cases were categorized as clinically localized stage Ⅰ and Ⅱ, while the other 24 were advanced stage Ⅲ and Ⅳ.
Nucleic acid in situ hybridization
APES-(3-aminopropyl-triethoxy-saline) coated slides were used for preparing the paraffin embedded NPL sections. The DAKO hybridization detection kit (K 0046, DAKO NS, Glostrup, Denmark) and FITC-conjugated EBV (EBERs) probe (Y 0017, DAKO A/S, Glostrup, Denmark) were used for nucleic acid in situ hybridization. The procedure consisted of the following steps: ① deparaffinization and dehydration of sections using xylene and a series of graded ethanol; ② pretreatment with 0.1 mg/ml proteinase K; ③ hybridization with FITC-conjugated EBV (EBERs) probes at 37℃ for 2 h;④ alkaline phosphatase conjugated rabbit F (ab') anti-FITC and enzyme substrate BCIP/NBT were used for signal detection; ⑤ counterstaining the sections with 2% methyl green solution.
Immunohistochemistry
The DAKO universal LSAB kit (DAKO K 0681, DAKO NS, Glostrup, Denmark) was used for the detection of a variety of lymphocyte antigens and EBV encoded proteins. The primary antibodies and their working dilutions used in this study are listed in Table 1 . Besides routine HE staining, cytokeratin and leukocyte common antigen (CD45) immunohisto-chemically staining was first performed to exclude nasopharyngeal carcinoma. Three kinds of antibody cocktails (CD79α and CD20cy, CD3 and CD45RO, and CD56) for detecting B cells, peripheral T cells and NK/T cells were concomitantly used to differentiate the major immunophenotypes of NPL. CD4, CD8, CD21, CD30, LMP1, LMP2A, Zta (BZLF protein), VCA and MA were detected later on.
RESULTS
The lymphoma cells of 5 out of 44 B cell lineage NPLs (11.36%) were infected with EB virus confirmed by EBERs in situ hybridization. According to “The New World Health Organization Classification of Lymphomas”,［4,5］ two cases were identified as diffuse large B cell lymphoma, one as small lymphocytic lymphoma, one as lymphoplasmocytic lymphoma and one as Burkitt's lymphoma. The neoplastic cells of the primary Burkitt's lymphoma were latent infection and type Ⅰ, expressing EBNA1 and EBERs only; the neoplastic cells of the other 4 B cell lineage lymphomas were mainly latent infection and type Ⅱ, expressing EBNA1, EBERs, LMP1 and LMP2A. Furthermore, the expression of Zta and/or VCA and/or MA might be detected in a few lymphoma cells of these 4 cases. All neoplastic cells from 43 of the 44 B cell lineage NPLs were CD21-negative. A non-EBV-infected small lymphocytic B cell NPL with LCA+/CD20cy+/CD79α+ immunophenotype expressed CD21. A few CD30-positive neoplastic cells (about 5%) could be found in most of the 44 B cell lineage NPLs.
The neoplastic cells of 22 NPLs were positive for CD3 and CD45RO and negative for CD79α, CD20cy and CD56. These were categorized as peripheral T cell lymphoma. Morphologically, these 22 NPLs were “ peripheral T cell lymphoma, not otherwise characterized" according to the proposed WHO classification of lymphoid neoplasms.［4］ Depending on whether the peripheral T cell lymphoma cells expressed CD4 and/or CD8, these 22 primary nasopharyngeal peripheral T cell lymphomas could be further divided into 4 subtypes, namely, CD8-/CD4- (9), CD8+/CD4+ (2), CD8+/CD4- (2), CD8-/CD4+ (9). Table 2 shows that 9 peripheral T cell lymphomas (9/22, 40.9%) did not express either CD4 or CD8 (maybe equivalent to early thymocytes or a small population of T cells expressing the γδTCR in peripheral tissue and blood［6,7］), and 13 peripheral T cell neoplasms (13/22, 59.1%) expressed double CD4 and CD8 (possibly equivalent to common thymocytes or a large population of T cells expressing the αβ TCR in peripheral tissue and blood) or single CD8 or CD4.［6,7］ Eighteen of the 22 peripheral T cell NHLs were infected with EBV and most neoplastic cell nuclei showed EBERs-positivity. The EBV infection rate was 81.82% (18/22). All neoplastic cells of these 22 NPLs were CD21-negative, and a few CD30-positive lymphoma cells (about 5%) could be found in most of these 22 peripheral T cell NPLs.
Seven cases of CD3+CD56+ NPL, which might be NK cells with cytoplasmic CD3 (the ε chain) expression or T cells expressing NK-associated antigens (CD56), were deemed NK/T cell lymphoma. They could also be divided into 4 subtypes, namely CD8-/CD4- (1), CD8+/CD4+ (2), CD8+/CD4- (2), and CD8-/CD4+ (2). All 7 CD56-positive NHLs were EBERs-positive ( Fig. 1 ). The EBV infection rate of these cases was 100%. No NPL that were CD56-positive expressed CD21. Morphologically, these 7 CD3+CD56+ NPLs were of the “extranodal NK/T cell lymphoma, nasal type" according to the proposed WHO classification of lymphoid neoplasms.［4,5］ The neoplastic cells of this category always underwent angioinvasive growth and often were associated with ischemic necrosis, representing the histology of so-called “angiocentric lymphoma" previously ( Fig. 2 ). Cell morphology varied greatly. The lymphoma cells were polymorphic in 6 cases and had a small lymphocyte-like appearance in 1 case.
CD3+CD56+ (7), CD3+CD56-CD8-CD4- (9) and CD3+CD56-CD8+CD4- (2) cases were all infected with EBV. On the other hand, 2 cases with CD3+CD56-CD8+CD4+ were EBERs-negative. However, 9 of CD3+CD56-CD8-CD4+ peripheral T cell lymphomas were either EBERs-positive (7) or EBERs-negative (2). No NPLs with CD3-/CD56+ were observed in this study.
Twenty-five non-B cell lineage NPLs harboring EBV expressed EBNA1 and EBERs, while LMP1 was expressed in 18 (18/25, 72.0%) and LMP2A in 19 (19/25, 76.0%). Therefore, they were categorized mainly as EBV latent infection, type Ⅱ. A few neoplastic cells in these 25 cases also expressed Zta (15/25, 60.0%) and/or VCA (20/25, 80.0%) and/or MA (18/25, 72.0%), respectively. That is to say, the EBV lytic products of individual NHL cells in the non-B cell lineage lymphomas could often also be found.
DISCUSSION
Primary nasal lymphomas and nasopharyngeal lymphomas are uncommon extranodal lymphomas often reported together in literatures.［8,9］ Primary non-Hodgkin's lymphoma does not often occur in nasopharynx; therefore reports on that topic are rare in medical literature. However, we have collected over 182 cases of nasopharyngeal non-Hodgkin's lymphomas (NPLs) reported since 1983.［8,10-14］
Sugimoto et al［15］ reported that the ratio B cell lineage to non-B cell lineage of NPL was 1∶1 in Japan. Ye et al［11］ reported that the ratio of B cell lineage to non-B cell lineage of NPL was 1.06∶1 in Guangzhou. However, Lei et al［8］ reported that the ratio of B cell lineage to non-B cell lineage of NPL was 2.2∶1 in Hong Kong. We found that the number of B cell lineage was predominant over the non-B cell lineage in Guangzhou with a ratio of 1.52∶1 (44∶29). Recently, Norval et al［16］ reported that most of the NPLs were B cell lineage (72%). We found that the ratio of B cell lineage to non-B cell lineage of primary sinonasal non-Hodgkin's lymphoma was 1∶10 (data not shown). The ratio in the Lei's study was 1∶3.［8］We think the ratio of B cell to non-B cell lineage of non-Hodgkin's lymphoma varies differently in different sites, such as the nasopharynx and sinonasal region, although these anatomical sites closely approximate each other.
Furthermore, our results illustrated that there were three major immunophenotypes presented in NPLs as reported by Cheung et al in Hong Kong.［9］ Based on the results of CD56 immunostaining, Cheung et al subclassified the NPL and sinonasal non-Hodgkin's lymphoma into three immunophenotypes, namely B cell (CD79α+/CD3-/CD56-, 33.6%), peripheral T cell (CD79α-/CD3+/CD56-, 21.3%) and NK/T cell (CD79α-/CD3+/CD56+, 45.1%). The results of our study demonstrated that the percentages of these three immunophenotypes were 60.3%, 30.1% and 9.6%, respectively. Though the male-to-female ratio of NPL (2.17∶1) was similar to that of sinonasal non-Hodgkin's lymphoma (2.14∶1), the median age of 73 NPL patients (48 years) was higher than that of 44 patients with sinonasal non-Hodgkin's lymphoma (38 years) (data not shown). It should be pointed out that no CD3-CD56+ NPL was found in this study as reported by Drenou et al［17］ as well as Saito et al.［18］ That is to say, there were no cases of NPL derived from so-called distinct NK cell entity［19］ observed in this study.It is worthy to note that the EBV infection frequency of these three major immunophenotypes, namely, B cell, peripheral T cell and NK/T cell NPL gradually increased.
However, it should be emphasized that the neoplastic cells of the 7 CD56-positive lymphomas might be attributed to the NK cell lineage with CD3 expression or T cell receptor (TCR) gamma gene rearrangement. They might also be attributed to the T cell lineage with NK cell surface antigen expression.［20,21］ It is important to point out that T cell receptor rearrangement and surface CD3 immunohistochemistry were not performed in this study. However, as popularly recognized, the distinction between T cell and NK cell lineage might not be so sharp in cell differentiation orientation as imagined. We did not find a case of B cell lineage NPL that was CD3-positive, which means that expression of NK markers occurs exclusively in a proportion of T cell lymphomas, but not B cell or null cell lymphomas. Irrespective of either NK cell lineage or peripheral T cell lineage, the 7 NPLs that were CD3+/CD56+ were infected with EBV, which also confirms Cheung's viewpoint［9］ that the CD56 immunohistochemistry should be performed for every case of NPLs because the NK/T cell NPLs were consistently associated with EBV infection and their prognosis was always worst. The morphology of these 7 CD56-positive NPLs was similar to so-called “angiocentric lymphoma” or “EBV-associated extranodal natural killer NK/T cell non-Hodgkin lymphoma (NHL) of nasal type” as reported by Stadlmann et al.［22］ Tomita et al［23］ also found that the CD56-positive lymphomas were almost always EBV-positive in Japan. However, a limited number of EBV-negative cases have been reported. Matano et al［24］ had reported the existence of a CD56-positive NK/T cell lymphoma with no EBV infection. Worthy of note is that the 9 peripheral T cell NPLs without expression of either CD4 or CD8 in this study could be identified as peripheral immature T cell lymphoma and that they were all EBV-positive. Why peripheral immature T cell NPLs are consistently infected with EBV must be clarified by further studies.
As it is known, based on antigen expression patterns, there are three types of EBV latent infection, types Ⅰ, Ⅱ and Ⅲ, which may or may not be switched on to replicative or lytic infection. In this study, one case of nasopharyngeal Burkitt's lymphoma, which was infected with EBV, type Ⅰ, expressed EBNA1 and EBERs only; the other 29 cases of NPL infected with EBV were mainly of EBV latent infection type Ⅱ, and expressed EBNA1, EBERs, LMP1 and LMP2A. This type of EBV infection in NPLs is identical to that of nasopharyngeal carcinoma as reported by Chiang et al.［3］ However, a few lymphoma cells could express lytic products. Chan et al［25］ found that though the EBV genome presented in CD56-positive lymphoma cells was principally in an episomal form, it could also be switched to lytic viral replication. It should be pointed out that except for one CD21-positive nasopharyngeal small lymphocytic lymphoma, the neoplastic cells of the other 29 NPLs did not express CD21, which is a cell surface receptor for EBV. Therefore, how the EBV entered NPL cells remains investigation. In addition, a variable number of CD30-positive NPL cells could be found in most of the 30 EBV-infected NPLs. We assumed that the CD30 expression is only an indicator of activation of lymphoma cells, having no significant role in the tumorigenesis of NPL.
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