February 2023 – Hematopathology Case Two

The following are H&E and immunohistochemical stains for a tonsil from a 20-year-old man.

Figure 1
Figure 2
Figure 3

What is the most important ancillary study that you would include in your workup for a diagnosis?

  • CD45.
  • FISH analysis for BCL2, BCL6 and MYC rearrangements.
  • EBER in situ hybridization.
  • Immunoglobulin gene rearrangement study.

The correct answer is ...

EBER in situ hybridization.

The H&E sections of tonsil show prominent paracortical expansion by heterogeneous population of lymphocytes, histiocytes, plasma cells, and numerous large immunoblasts including occasional Hodgkin-like cells. A few benign follicles are present. The differential diagnosis based on morphology includes reactive lymphoid proliferation, large cell lymphoma, and classic Hodgkin lymphoma (CHL). These large lymphocytes are B-cells highlighted by CD20 and PAX5. They are also positive for CD30, but negative for CD15. Plasma cells and immunoblasts are polytypic by kappa and lambda light chain immunostaining.

In a setting of a young patient with tonsillar hypertrophy, an acute EBV infection (infectious mononucleosis) needs to be considered. EBER in-situ hybridization stains many small and large lymphocytes. When reviewing this patient’s history, he had a positive Monospot test. A diagnosis of EBV-positive lymphadenitis was rendered.

The phenotype of large lymphocytes does not support of a diagnosis of CHL. Hodgkin cells are typically positive for CD30, CD15 (subset of cases), MUM1, and PAX5 (weak), and are negative for CD20 and CD45. In contrast to EBER positivity in variably sized B-cells of EBV-positive lymphadenitis, EBER expression is limited in Hodgkin cells of CHL. 

It is challenging to differentiate EBV-positive lymphadenitis from EBV-positive diffuse large B-cell lymphoma (EBV+DLBCL) due to morphologic and phenotypic overlap, such as extensive immunoblast proliferation, necrosis, and nodal architecture effacement.1,2,3 Clonal evidence and clinical correlation are required for EBV+DLBCL. Clonal evidence includes immunoglobulin light chain restriction on large cells, evident immunoglobulin gene rearrangements, and cytogenetics and molecular alterations. When encountering a biopsy from a young patient with tonsil and lymphadenopathy in head and neck region with paracortical expansion by increased immunoblasts, an acute EBV infection needs to be investigated before a diagnosis of EBV+DLBCL. 

Acute infectious mononucleosis typically occurs in adolescence and patients may present with asymmetrical lymphadenopathy or tonsilitis. EBV replicates in B-cells, inducing B-cell activation and proliferation. Morphological features that hint to a reactive EBV process include a polymorphous infiltrate consisting of histocytes, lymphocytes, and plasma cells and retained nodal architecture.4 No clonal evidence by flow cytometry, molecular, and cytogenetics/FISH studies is helpful to rule out a neoplastic process. Monospot test, EBV-specific antibodies, and quantitative detection of EBV DNA should be performed clinically.


  1. Salvador AH, Harrison Jr EG, Kyle RA. Lymphadenopathy due to infectious mononucleosis: its confusion with malignant lymphoma. Cancer 1971;27:1029–1040.
  2. Kojima M, Nakamura S, Itoh H, et al Lymph node lesion in infectious mononucleosis showing geographic necrosis containing cytologically atypically B-cells. A case report. Pathol Res Pract 2004;200:53–57.
  3. Childs CC, Parham DM, Berard CW. Infectious mononucleosis. The spectrum of morphologic changes simulating lymphoma in lymph nodes and tonsils. Am J Surg Pathol 1987;11:122–132.
  4. Louissaint, A., Ferry, J., Soupir, C. et al. Infectious mononucleosis mimicking lymphoma: distinguishing morphological and immunophenotypic features. Mod Pathol 25, 1149–1159 (2012).
Belinda Galeano profile picture square

Belinda Galeano, M.D., Ph.D.

Resident, Anatomic and Clinical Pathology
Mayo Clinic

Ji (Jane) Yuan, M.D., Ph.D.

Senior Associate Consultant, Hematopathology
Mayo Clinic
Associate Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science

MCL Education

This post was developed by our Education and Technical Publications Team.