Background
Epstein-Barr virus (EBV) is associated with the pathogenesis of several B-cell neoplasms, such as Burkitt lymphoma, plasmablastic lymphoma (PBL), primary effusion lymphoma (PEL), immunodeficiency-associated lymphoproliferative disorders, and some diffuse large B-cell lymphomas (DLBCL), which include lyphomatoid granulomatosis and DLBCL with chronic inflammation [
1,
2]. Most EBV-positive B-cell lymphomas, except for Burkitt lymphoma, develop in cases with systemic immunodeficiency or long-standing chronic inflammation, although B-cell lymphoma can infrequently develop in the absence of immunodeficiency or inflammatory conditions. These cases typically involve elderly patients, and some authors have described them as “EBV-positive DLBCL of the elderly” [
3]. Previous studies have revealed that 2–11% of DLBCL cases are positive for EBV [
4], and these cases are diagnosed based on the following findings: 1) diffuse infiltration of large lymphoid cells, 2) detection of B-cell markers during immunohistochemistry testing (IHC), and 3) the detection of EBV-encoded small RNAs using in situ hybridization. In this report, we describe an unusual case of EBV-positive DLBCL in an immunocompetent patient, which was difficult to diagnose due to the absence of many B-cell markers during routine IHC. Our final diagnosis was critically supported by the IHC identification of B-cell-specific transcription factors (Oct-2 and BOB.1) and findings from our gene rearrangement testing.
Discussion and conclusions
EBV-related lymphomas develop in various conditions, including systemic immunodeficiency and chronic inflammation [
1,
2]. Most of these lymphomas are derived from B-cells, and EBV-positive B-cell lymphomas usually express several B-cell surface markers, although they occasionally lack one or more markers. However, few studies have evaluated the frequency of B-cell marker loss in EBV-positive B-cell lymphomas. One flow cytometry-based study revealed that 4 of 25 cases (16%) of posttransplant lymphoproliferative disorder exhibited almost complete loss of CD20 expression (all 4 CD20-negative cases were EBV-positive), compared to only 8 of 334 cases (2%) of
de novo B-cell non-Hodgkin lymphoma [
5]. Another study revealed a higher incidence of decreased CD19 expression in cases of posttransplant lymphoproliferative disorder, compared to cases of common DLBCL (3 of 4 cases [75%] vs. 8 of 56 cases [14%]) [
6]. The frequency of CD20-negative tumors among HIV-positive DLBCL cases is variable (2–26%) [
7]. Nevertheless, to the best of our knowledge, there are no reported cases of EBV-positive B-cell lymphoma lacking a broad range of B-cell markers. McKelvie et al. reported a case of EBV-positive methotrexate-associated DLBCL that was negative for CD20 and CD79a [
8]. Although that case and the present case were both positive for CD30 and MUM-1, McKelvie et al.’s case was positive for Pax-5 and ours was negative for Pax-5.
Our histological findings included the diffuse proliferation of CD20-negative and CD3-negative large cells including immunoblastic cells, plasmacytic cells, and multinuclear cells, which was compatible with various differential diagnoses: ALCL, extracavitary PEL, ALK-positive large B-cell lymphoma (ALK-LBCL), and PBL. In histology of our case CD30-positive HRS-like cells appeared, which mimicked ALCL and support our preliminary diagnosis before EBV testing by EBERs in situ hybridization. However, ALCL is exclusively EBV-negative and does not exhibit clonal rearrangement of the immunoglobulin genes. Extracavitary PEL usually develops in cases of systemic immunodeficiency, such as HIV infection, and is almost exclusively positive for human herpesvirus-8. ALK-LBCL is positive for ALK, CD138, and EMA, but is negative for EBV. Our initial IHC findings and the subsequent detection of EBV using EBERs in situ hybridization excluded ALCL, extracavitary PEL, and ALK-LBCL. PBL is positive for CD138 in almost all cases and also frequently expresses CD38 and MUM-1 [
9,
10]. Although the results of IHC of our case could not clearly exclude PBL, diagnosis of PBL is unlikely because of lack of CD138 and CD38 expression. MUM-1 is one of markers for plasma cell differentiation, but previous studies showed that specificity of MUM-1 as a plasma cell marker is limited [
11]. Montes-Moreno et al. categorized tumors with plasmablastic morphology and atypical immunophenotype (CD138 low or positive, CD20 low, Pax-5 low, MUM-1 positive, Blimp1 positive, and XBP1 negative) as PBL with variant (faulty) plasmablastic phenotype, and our case might be classified into this category according to their scheme [
12]. Although we were initially unable to identify the lineage of tumor cells and histological type, the presence of necrotic foci and HRS-like cells in the tumor suggested an EBV-related disease, which prompted us to test for the expression of additional B-cell markers. Expression of B-cell-specific transcription factors detected by additional IHC and clonal rearrangement of the immunoglobulin genes confirmed our final diagnosis of EBV-positive DLBCL. Inadequate IHC using limited surface markers may lead to a misdiagnosis in cases of lymphomas with an unusual immunophenotype. It is important to miss a chance to perform further IHC in such cases, therefore detailed histological evaluation and accurate interpretation of results from a well-designed initial IHC panel are essential for reaching a correct diagnosis.
No previous studies have explained why B-cell markers are down-regulated in EBV-positive B-cell lymphomas, and the association of EBV infection with the suppression of B-cell markers remains unclear. Expression of latent EBV infection products influences the epigenetic status of the host cells [
13], which might be associated with the regulation of B-cell marker expression. Down-regulation of CD20 in B-cell neoplasms is often related to plasmablastic features and terminal B-cell differentiation of the tumor cells [
7]. Most CD20-negative B-cell lymphomas with plasmablastic features are positive for CD138, whereas tumor of the present case was negative for CD138. Therefore, plasmablastic features or plasmacytic differentiation is insufficient to explain the lack of these markers of our case. Moreover, the tumor did not express Pax-5, which is a critical B-cell lineage commitment factor and upregulates the expression of various B-cell differentiation markers [
14]. Thus, it is possible that down-regulation of Pax-5 or aberrancy of other factors that modulate Pax-5 might have caused the loss of B-cell markers in our case. Mutations and translocations that involve the Pax-5 gene have been reported in some cases of B-cell lymphoma and leukemia [
15‐
18]. In addition, the CD20-negative phenotype is observed in 26–27% of B-cell lymphomas after molecular-targeted therapy using rituximab [
19,
20]. Several mechanisms can cause the down-regulation of CD20 after rituximab treatment: 1) mutational changes in the CD20 gene [
21,
22], (2) aberrant transcriptional regulation of CD20 [
23‐
25], (3) degradation of the CD20 protein by the ubiquitin-proteasome system [
23], (4) other posttranscriptional or posttranslational changes in the regulation of CD20, and (5) genetic or transcriptional alterations in transcription factors that affect the expression of CD20 (e.g., PU.1 or Oct-2). Therefore, similar abnormalities involving Pax-5 or other common B-cell derivation factors may be responsible for the lack of B-cell markers in our case.
Previous studies have revealed that cases of DLBCL with reduced CD20 expression experience markedly inferior survival when they are treated using conventional CHOP (cyclophosphamide, hydroxydaunorubicin, Oncovin®, prednisolone) or rituximab-CHOP [
26,
27]. Furthermore, a study of CD20-negative DLBCL cases, in which PBL and ALK-LBCL were carefully excluded, revealed that CD20-negative cases had a poorer response to conventional treatment and a poorer prognosis, compared to CD20-positive cases [
7]. Moreover, CD20-negative cases had a higher proportion of the non-germinal center B-cell subtype, a higher proliferation index, and more frequent extranodal involvement, which might explain the biological aggressiveness of CD20-negative DLBCL [
7].
In conclusion, we encountered an unusual case of EBV-positive DLBCL that was lacking various B-cell markers. This type of unusual phenotype often leads to an incorrect diagnosis, which can only be avoided by detailed evaluation of histopathology and appropriate utility of ancillary diagnostic tools, such as IHC for lineage-specific markers and gene rearrangement testing.
Acknowledgements
The immunohistochemistry for CD38 was performed by SRL Inc. (Tokyo, Japan). The authors thank Ms Y. Tsuruta, S. Hara, Mr H. Ishimaru, K. Sugio, M. Yamane, and R. Yoshino (Kansai Rosai Hospital) for their technical assistance and Ms A. Fukuoka for her clerical works about our study.