Angioimmunoblastic T-cell lymphoma (AITL) belongs to the mature T/NK cell classification of non-Hodgkin lymphoma and accounts for about 1∼2% [1]. AITL is a lymphoproliferative disorder of mature T follicular helper cells characterized by advanced-stage disease, generalized lymphadenopathy, systemic symptoms, and polyclonal hypergammaglobulinemia [1]. In most cases, proliferation of Epstein-Barr virus (EBV)-positive B-cells is observed, where B-cells may show either polyclonal or clonal proliferation [1]. Additionally, since the occurrence of secondary B cell lymphoma such as diffuse large B-cell lymphoma (DLBCL) after AITL treatment has been reported, it is important to confirm the existence of a clonal cell population [2, 3]. Identification of the clonal cell population is mainly performed by flow cytometry, but when non-specific binding exists, it becomes difficult to differentiate from normal cells. Here, we describe an AITL case in which a patient with a neck mass who was referred for bone marrow (BM) study was almost misdiagnosed with involvement of B-cell lymphoma since the BM B-cell population showed abnormal immunophenotypes by flow cytometry. This report suggests how to interpretate several results whether clonal B cells are present when the flow cytometry results are ambiguous. This study was approved by the Institutional Review Board of Soonchunhyang University Hospital, Seoul, Korea (IRB File No. 2020-12-008).

An 80-year-old woman presented with a neck mass, fever, edema, and general weakness. Multiple enlarged lymph nodes were detected by a physical examination and computed tomography. Complete blood count parameters were as follows: hemoglobin, 6.1 g/dL; MCHC, 32.1 g/dL; platelets, 44×10⁹/L; and, white blood cells, 12.8×10⁹/L, with 3% abnormal lymphoid cells.

Protein electrophoresis revealed polyclonal IgA and oligoclonal IgG gammopathy and a BM aspirate showed 22.4% large-sized abnormal lymphoid cells with basophilic cytoplasm (Figure 1A). The flow cytometric analysis revealed that the lymphocytes were T-cells (12% of all nucleated cells [ANCs]) that were CD3-positive with a normal CD4: CD8 ratio and B-cells (18% of ANCs) that were CD19-positive but CD20- and CD22-negative with lambda light chain restriction (Figure 1B, 1C). Further, biopsies of the left axillary lymph node (LN) and BM were performed. Immunohistochemical (IHC) staining revealed small-to-medium-sized lymphocytes that were positive for CD3, CD5, PD-1, and CD23 in follicular dendritic cell meshwork but negative for CD10, CD30 and Bcl-6. Ki-67 was positive for 30∼40% of lymphocytes and shows loss of polarity.

Fig. 1. Microscopic findings at diagnosis and immunophenotypic features of the bone marrow aspirate by flow cytometric analysis at the first time. (A) Several pleomorphic immunoblastic large cells are evident on this bone marrow aspirate smear (red arrow) (Wright–Giemsa ×1,000). (B) The T-cells are positive for CD3 (shown in blue) with no skewing of the CD4: CD8 ratio. (C) The B-cells are positive for CD19 (shown in red) with surface lambda light chain restriction. (D) T-cell receptor (TCR) gene clonality assays for the patient’s bone marrow showed positive results.

When immunoglobulin (IGH, IGK, and IGL) and T-cell receptor (TCR) gene clonality assays (IdentiClone; Invivoscribe Technologies, USA) were performed with a BM sample, monoclonal TCR beta and delta rearrangements were detected that were negative for immunoglobulin gene rearrangements (Figure 1D). Consequently, the patient was diagnosed with AITL with BM involvement (stage IV). The patient was subsequently treated with a cyclophosphamide, hydroxyldoxorubicin, vincristine, and prednisone regimen after which she expired due to tumor lysis syndrome one month after chemotherapy.

AITL involves the BM in more than 50% of the cases [1]. In our case, atypical lymphoid cells were observed in the BM, and flow cytometric immunophenotypes of the B-cells showed unusual results, such as CD19+, CD20-, CD22-, and lambda light chain restriction. Initially, we suspected BM involvement of B-cell lymphoma based on the abnormal B-cells revealed by flow cytometry. However, the patient was diagnosed with AITL involving the BM, since both her LN and BM biopsy samples were positive for PD-1 and TCR gene rearrangements in the following BM molecular studies showed positive results.

A re-analysis of the flow cytometric immunophenotyping revealed a minor, aberrant T-cell popu-lation with dim CD3 and CD4 (about 2% of ANCs) (Figure 2A). AITL has often been reported to be accompanied by B-cell lymphoma [4] however, since there was no immunoglobulin gene rearrangement, B-cell lymphoma was clearly absent in this case. Immunophenotypically, CD20-negative B-cells, assumed to be abnormal cells, were identified to actually be normal plasma cells after adding markers for CD38 and CD138 in the flow cytometric assay (Figure 2B). Polyclonal B-cell or plasma cell proliferation, as in this case, has been frequently reported in AITL [5].

Fig. 2. Re-analysis of the immunophenotypic features of the bone marrow aspirate by flow cytometric analysis. (A) A minor, aberrant T-cell population with dim CD3 and CD4 is observed (approximately 2% of ANCs). (B) The abnormal B-cell population was confirmed to be reactive plasma cells with no cytoplasmic light chain restriction.

Since no restriction was observed in the cytoplasmic light chain, the lambda light chain restriction seen in the surface stain was considered as a non-specific binding. Non-specific binding of antibodies can occur through the adherence of antibodies to damaged or dying cells [6].

In the morphological examination of the BM with differential counts, typical plasma cells and abnormal lymphoid cells were observed in 1% and 22% of all nucleated cells, respectively. The abnormal lymphoid cells were plasma cells that accounted for 10% of the cells in flow cytometry. The abnormal cells did not have a typical plasma cell-like morphology, and plasma cells with typical morphology accounted for 1%, making it difficult to recognize the abnormal B-cell population observed in flow cytometry as plasma cells. Since this discrepancy often occurs due to the broad morphological spectrum of plasma cells including atypical lymphocytes, plasmacytoid lymphocytes, and lymphocytoid plasma cells, it is important to consider the flow cytometry results while interpreting the results [7].

B-cells in patients with EBV-positive T-cell lymphoma may exhibit immunophenotypes similar to those of plasma cells. Although there have been few previous reports on both CD20 and CD22 losses in EBV-infected B-cells, EBV can cause B-cells to show abnormal immunophenotypes, including huge losses of B-cell markers [8-10]. EBV interferes with the cellular transcription factor paired box protein 5, which is essential for B-cell differentiation and antigen expression for viral gene expression [11, 12].

To conclude, the patient was diagnosed with AITL with BM involvement and polyclonal plasma cell proliferation. The current case emphasized that plasma cells could be misinterpreted as abnormal B-cells in EBV-infected conditions. The proliferation of these B-cells or plasma cells could also potentially mask underlying T-cell lymphoma. A more integrated approach is required that includes clinical, histopathological, flow cytometric, and molecular studies, which can often be challenging.

An 80-year-old woman presented with a neck mass, fever, edema, and general weakness. Multiple enlarged lymph nodes were detected by a physical examination and computed tomography. Complete blood count parameters were as follows: hemoglobin, 6.1 g/dL; MCHC, 32.1 g/dL; platelets, 44×10⁹/L; and, white blood cells, 12.8×10⁹/L, with 3% abnormal lymphoid cells.

Protein electrophoresis revealed polyclonal IgA and oligoclonal IgG gammopathy and a BM aspirate showed 22.4% large-sized abnormal lymphoid cells with basophilic cytoplasm (Figure 1A). The flow cytometric analysis revealed that the lymphocytes were T-cells (12% of all nucleated cells [ANCs]) that were CD3-positive with a normal CD4: CD8 ratio and B-cells (18% of ANCs) that were CD19-positive but CD20- and CD22-negative with lambda light chain restriction (Figure 1B, 1C). Further, biopsies of the left axillary lymph node (LN) and BM were performed. Immunohistochemical (IHC) staining revealed small-to-medium-sized lymphocytes that were positive for CD3, CD5, PD-1, and CD23 in follicular dendritic cell meshwork but negative for CD10, CD30 and Bcl-6. Ki-67 was positive for 30∼40% of lymphocytes and shows loss of polarity.

Fig. 1. Microscopic findings at diagnosis and immunophenotypic features of the bone marrow aspirate by flow cytometric analysis at the first time. (A) Several pleomorphic immunoblastic large cells are evident on this bone marrow aspirate smear (red arrow) (Wright–Giemsa ×1,000). (B) The T-cells are positive for CD3 (shown in blue) with no skewing of the CD4: CD8 ratio. (C) The B-cells are positive for CD19 (shown in red) with surface lambda light chain restriction. (D) T-cell receptor (TCR) gene clonality assays for the patient’s bone marrow showed positive results.

When immunoglobulin (IGH, IGK, and IGL) and T-cell receptor (TCR) gene clonality assays (IdentiClone; Invivoscribe Technologies, USA) were performed with a BM sample, monoclonal TCR beta and delta rearrangements were detected that were negative for immunoglobulin gene rearrangements (Figure 1D). Consequently, the patient was diagnosed with AITL with BM involvement (stage IV). The patient was subsequently treated with a cyclophosphamide, hydroxyldoxorubicin, vincristine, and prednisone regimen after which she expired due to tumor lysis syndrome one month after chemotherapy.

AITL involves the BM in more than 50% of the cases [1]. In our case, atypical lymphoid cells were observed in the BM, and flow cytometric immunophenotypes of the B-cells showed unusual results, such as CD19+, CD20-, CD22-, and lambda light chain restriction. Initially, we suspected BM involvement of B-cell lymphoma based on the abnormal B-cells revealed by flow cytometry. However, the patient was diagnosed with AITL involving the BM, since both her LN and BM biopsy samples were positive for PD-1 and TCR gene rearrangements in the following BM molecular studies showed positive results.

A re-analysis of the flow cytometric immunophenotyping revealed a minor, aberrant T-cell popu-lation with dim CD3 and CD4 (about 2% of ANCs) (Figure 2A). AITL has often been reported to be accompanied by B-cell lymphoma [4] however, since there was no immunoglobulin gene rearrangement, B-cell lymphoma was clearly absent in this case. Immunophenotypically, CD20-negative B-cells, assumed to be abnormal cells, were identified to actually be normal plasma cells after adding markers for CD38 and CD138 in the flow cytometric assay (Figure 2B). Polyclonal B-cell or plasma cell proliferation, as in this case, has been frequently reported in AITL [5].

Fig. 2. Re-analysis of the immunophenotypic features of the bone marrow aspirate by flow cytometric analysis. (A) A minor, aberrant T-cell population with dim CD3 and CD4 is observed (approximately 2% of ANCs). (B) The abnormal B-cell population was confirmed to be reactive plasma cells with no cytoplasmic light chain restriction.

Since no restriction was observed in the cytoplasmic light chain, the lambda light chain restriction seen in the surface stain was considered as a non-specific binding. Non-specific binding of antibodies can occur through the adherence of antibodies to damaged or dying cells [6].

This work was supported by the Soonchunhyang University Research Fund.

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Shin WY, M.D.; Bang HI, Professor; Kim JA, M.D.; Kim J, Professor; Park R, Professor.