Clinical History
This is a 26 year-old man who presented with a sore
throat and an asymmetrically enlarged right tonsil (although both tonsils appeared enlarged). He had no
significant past medical history. He noted a 3 pound weight loss and low-grade fever for the past week.
Physical examination revealed several enlarged cervical nodes and a palpable liver edge.
A CBC showed mild normochromic, normocytic anemia (HCT=36.2%) and thrombocytopenia (platelets
79,000/microliter). The patient's reticulocyte count was 3.8% and liver function tests were mildly
elevated (AST=85 IU/L). A tonsillectomy was performed.
Case 1 - Figure 1 - Low power photomicrograph of the right tonsil showing partial effacement of architecture and prominent large cell infiltrate, H&E X100
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Case 1 - Figure 2 - High power photomicrograph of right tonsil demonstrating large cell infiltrate and Reed-Sternberg-like cell, H&E 400X
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Case 1 - Figure 3 - High power photomicrograph showing diversity of interfollicular infiltrate and "mummified" apoptotic large cells, H&E 400X
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Case 1 - Figure 4 - Immunohistochemical stain for CD20 and CD3, 400X
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Case 1 - Figure 5 - Immunohistochemical staining for CD30, 400X
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Case 1 - Figure 6 - LMP (latent membrane protein) immunohistochemical staining and EBER in-situ hybridization demonstrating EBV virus, 400X
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Histology
Section of the enlarged right tonsil show partial effacement of
the nodal architecture by a paracortical infiltrate containing numerous immunoblasts and large
transformed cells. In areas the large cells appear to form confluent sheets. Admixed with the large
cells are occasional multinucleate large cells with prominent nucleoli that resemble Reed-Sternberg cells
as well as apoptotic "mummified" cells. Definitive reactive germinal centers are also identified. A
reticulin stain highlights the germinal centers and demonstrates that many of the irregularly shaped
sheets of large cells are expanded germinal centers. There are large areas of necrosis and focally the
lymphoid infiltrate extends into the tonsillar epithelium.
Immunophenotypic Studies
Immunoperoxidase staining performed on the
formalin-fixed tonsil showed prominent follicular structures that were highlighted by the CD20 and
included irregular large sheets of cells. The paracortical infiltrate showed the large cells and
immunoblasts to be a mixture of B- and T-cells as they stained with both CD20 and CD3. A CD30 stain
showed strong cytoplasmic and membranous staining in many of the large cells. A CD15 stain and ALK-1
stain were negative. Staining for EBV-LMP (latent membrane protein) was positive in a spectrum of large
and small lymphoid cells.
In-Situ Hybridization
In-situ hybridization with the EBER (Epstein-Barr
virus early RNA) oligonucleotide probe that recognizes early RNA transcripts for EBV infection that
accumulate in infected cells showed strong staining of both large and small lymphoid cells throughout the
specimen.
Diagnosis
Infectious Mononucleosis (EBV infection)
Treatment and Follow-up
The patient had EBV titers drawn that showed a
markedly elevated EBV titer that was predominantly IgM and developed a positive MonoSpot test two days
after surgery. He recovered over the period of one month from infectious mononucleosis and has been
healthy over the past three years.
Discussion
Infectious mononucleosis or acute Epstein-Barr virus infection is a common viral infection by a member
of the Herpesviridae family. It is an enveloped double-stranded DNA virus. Infection with EBV is
ubiquitous, although there are epidemiologic differences with infections in underdeveloped countries
happening very early in life (nearly 100% positive serology by age 3) when more developed countries tend
to have later infections (peak age between 15-20 years). In the US, only about 50% of patients exposed
to the virus will develop an active infection. In the US, the clinical syndrome usually affects
teenagers and young adults, although atypical cases may be seen in older adults. Most patients will
present (as did this patient) with fever, pharyngitis and cervical or generalized adenopathy.
Splenomegaly, hepatomegaly and tonsillitis are less commonly seen, and may be associated with more severe
infections. Occasionally patients may develop full-blown hepatitis, splenic rupture, encephalitis or
skin rashes.
The EBV virus infects lymphocytes in the oropharynx through the C3d complement receptor. During an
active infection, the virus will replicate in perifollicular B-cells, triggering a robust cellular and
humoral immune response. The antibodies induced are initially IgM, with later production of IgG that
provides life-long immunity in the immunocompetent host. The immune response also produces an
agglutinating antibody that forms the basis for the rapid screening MonoSpot test. The cellular immune
response is evidenced by the appearance of large immunoblastic cells in the interfollicular areas.
Blood Findings
The blood findings are often characteristic with a rise in the leukocyte count over the first two
weeks of infection, often to >10,000/mm3. Many of the cells will be lymphocytes with
atypical features including a larger size than normal with indented or horseshoe-like nuclei that are
eccentrically placed. The atypical lymphocytes will have abundant cytoplasm that is characteristically
basophilic (ranging from slightly to deeply basophilic) and appears pleated or billowing. Often the
cytoplasm will appear to wrap around red cells in the blood film (ballerina skirt). The lymphoid
population is heterogeneous, with a spectrum of cells ranging from small lymphoid cells to the large,
atypical or activated lymphocytes. Flow cytometry and other analysis demonstrate that the atypical
lymphoid population is of activated T-cell origin, and are often CD8 positive T-cells. Often the
reactive cells may show down-modulation of some pan-T-cell antigens, such as CD7, although antigen
deletion is unusual. In infectious mononucleosis, the number of atypical or activated lymphocytes is
usually increased to approximately 10% of leukocytes. Usually patients with EBV infections will have a
mild leukocytosis (usually around 10,000 WBCs/microliter) and an increase in mononuclear cells (i.e.
lymphocytosis of 50-60% of cells). Mild anemia and thrombocytopenia may also be seen in some patients,
probably reflecting generalized activation of the reticuloendothelial system and spleen, leading to
premature removal from the circulation or low-level hemolysis.
Histologic Findings
EBV infection in lymph nodes or tonsils can show a range of histologic appearances that can elicit a
differential diagnosis that ranges from a benign reactive process to lymphoma. Usually there is regional
distortion, but not effacement, of normal architecture by a follicular expansion and a paracortical
expansion. Small capillaries are usually increased. Often the lymph node sinuses are dilated and filled
with reactive cells and eosinophilic, proteinaceous fluid.
The involvement of both follicular and paracortical areas gives rise to a mixed hyperplasia pattern,
although one component may predominant in some cases. Follicular hyperplasia ranges from moderately to
markedly hyperplastic follicles that are irregular in shape. There is often active proliferation and
numerous tingible body macrophages in the germinal center. The paracortex is expanded and often appears
"moth-eaten" due to increased numbers of paler staining imunoblasts. Occasionally the immunoblasts may
form confluent sheets. The immunoblasts usually show a range of sizes, but are generally 3-4 times the
size of a resting lymphocyte with abundant cytoplasm and one or more prominent nucleoli. Sometimes the
cells may appear polynucleated and cells may strongly resemble mononuclear or classic Reed-Sternberg
cells, although the cells typically do not have the eosinophilic macronucleoli that are seen in Hodgkin's
cells. The mixed inflammatory background (including eosinophils and plasma cells) seen in Hodgkin's
lymphoma is not seen. Single cell apoptosis or focal necrosis is common. More geographic necrosis is
associated with fulminant (fatal) EBV infections. Reticulin staining will often highlight the retained
architectural features, in particular irregular follicular structures.
Immunophenotype
Staining of tissues with EBV infection will show the immunoblastic proliferation in the pararcortical
areas to be a mixture of B and T-lymphocytes. Cells will often show positivity with CD30, reflecting its
recognition of an activation epitope. This may be a pitfall when Reed-Sternberg-like cells are present,
but unlike true Reed-Sternberg cell, they will not be positive with CD15 (LeuM1).
Stains for EBV (either immunoperoxidase staining with LMP or in-situ hybridization with EBER) will
show the presence of EBV in a spectrum of cell types.
Laboratory Testing
EBV-specific antibodies include viral capsid antigen (VCA)-specific IgM or VCA-IgG, IgG early antigen
(EA) and Epstein Barr nuclear antigen (EBNA) appear sequentially after the infection and represent
different phases of the immune response to infection. These are particularly helpful when there is an
atypical clinical presentation or when the agglutination screening tests (MonoSpot or heterophil test)
are negative.
Figure 1: Time course for appearance of various EBV antibodies
useful in diagnosis following EBV
infection.
Differential Diagnosis
The differential diagnosis includes:
| 1. | Other viral infections - CMV, other viral lymphadenitis. Many of the changes (paracortical immunoblastic proliferation and increased atypical lymphocytes in the blood) may be seen with a variety of viral infections. Serologies and MonoSpot test will be negative for EBV. |
| 2. | Toxoplasmosis - The prominent follicular hyperplasia and interfollicular expansion may be seen in both EBV infection and toxoplasmosis, however the increased numbers of epitheliod histiocytes that infiltrate germinal centers and monocytoid cells near vessels and sinuses that characterize toxoplasmosis are not seen in EBV infections. |
| 3. | Non-Hodgkin's Lymphoma: Diffuse large cell lymphoma is often the most difficult entity to exclude, particularly when large sheets of immunoblasts are present. The presence of a significant reactive component (follicular hyperplasia) and the usually polymorphous phenotype of the large cells (a mixture of T and B-cells) can be helpful, although occasionally a case will have a marked predominance of either T or B-immunoblasts. A reticulin stain may be useful to demonstrate retained architecture by highlighting follicle centers.
Anaplastic large cell lymphoma - The large cells and the occasionally strikingly positive CD30 staining may create a differential diagnosis of anaplastic large cell lymphoma. Usually ALCL is a T-cell process, whereas EBV infection often shows a mixture of cell types that are CD30 positive. ALK-1 stains to demonstrate an ALK rearrangement protein product will be negative in EBV. EMA may be positive in ALCL, but will be negative in EBV infection. |
| 4. | Hodgkin's lymphoma - The presence of large, multinucleated cells with CD30 positivity may create a differential of classical Hodgkin's lymphoma, In EBV infection the cells often lack the classic eosinophilic macronucloli seen in Reed-Sternberg cells and will be negative for CD15, while demonstrating staining with CD45 and T or B-cell markers. The surrounding infiltrate in Hodgkin's lymphoma is usually mixed, including plasma cells and eosinophils, which would not be seen in EBV infection. The presence of EBV staining may be seen in either entity, so is not useful in distinguishing between Hodgkin's lymphoma and EBV infection. |
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