Modern Pathology (2012) 25, 1149–1159 & 2012 USCAP, Inc. All rights reserved 0893-3952/12 $32.00 1149

Infectious mononucleosis mimicking : distinguishing morphological and immunophenotypic features

Abner Louissaint Jr, Judith A Ferry, Chad P Soupir, Robert P Hasserjian, Nancy L Harris and Lawrence R Zukerberg

The James Homer Wright Pathology Laboratories, Massachusetts General Hospital, Boston, MA, USA

The diagnosis of (acute Epstein–Barr virus (EBV) infection) is usually made on the basis of clinical and laboratory findings. However, an atypical clinical presentation occasionally results in a or tonsillar . The morphological features of EBV-infected lymphoid tissue can easily mimic lymphoma. Furthermore, the immunophenotype of the immunoblasts has not been well characterized. To assess the morphological spectrum of acute EBV infection and the utility of immunohistochemistry in diagnosing difficult cases that resemble lymphoma, we reviewed 18 cases of acute EBV infection submitted in consultation to our institution with an initial diagnosis of/or suspicion for lymphoma. Patients included nine male and nine female individuals with a median age of 18 years (range 9–69). were obtained from lymph nodes (3/18) or Waldeyer’s ring (15/18). Infectious mononucleosis was confirmed by monospot or serological assays in 72% of cases (13/18). All cases featured architectural distortion by a polymorphous infiltrate with an immunoblastic proliferation, sometimes forming sheets. Reed–Sternberg-like cells were present in 8/18 (44%) of the cases. Infiltrates were often accompanied by necrosis (10/18) and mucosal ulceration (6/15). The majority of immunoblasts in all cases were CD20 þ B cells with a post-germinal center immunophenotype (strongly positive for MUM1/IRF4 (18/18), CD10À (18/18 negative) and BCL-6À (16/18 negative; 2/18 faint BCL-6 expression in o10% of immunoblasts)). Immunoblasts showed variable weak expression of BCL-2 and polyclonal expression of j and k immunoglobulin light chains in 81% cases. Reed– Sternberg-like cells in 8/8 cases were CD30 þ , CD15À, BOB.1 þ and OCT-2 þ . In conclusion, an atypical lymphoid infiltrate with numerous MUM1 þ , CD10À, BCL-6À immunoblasts should raise the suspicion of a reactive process, such as infectious mononucleosis, and warrants additional consideration before a diagnosis of lymphoma is made. Modern Pathology (2012) 25, 1149–1159; doi:10.1038/modpathol.2012.70; published online 25 May 2012

Keywords: Epstein–Barr virus; infectious mononucleosis; lymphoma

Epstein–Barr virus (EBV) infects more than 90% of acute infectious mononucleosis begins to rise in the human population and persists in most indivi- adolescence and declines through adulthood.2 Pri- duals as a lifelong, asymptomatic infection of B mary EBV infections in young children are common lymphocytes. Acute infectious mononucleosis, most and frequently subclinical. However, a small subset specifically characterized by the triad of tonsillitis/ of young infants and children with primary EBV pharyngitis, cervical and fever, is infection will also present with infectious mono- the most common acute clinical manifestation of nucleosis.3 For most cases, the diagnosis of acute primary EBV infection.1 Although primary EBV infectious mononucleosis is made on the basis of infection may occur at any age, the incidence of clinical presentation and laboratory findings, inclu- ding the presence of lymphocytosis with atypical lymphocytes and confirmatory serology/monospot Correspondence: Dr A Louissaint, MD, PhD, The James Homer test.4 However, acute EBV infection can occasionally Wright Pathology Laboratories, Massachusetts General Hospital, be associated with large or asymmetric lymph nodes 55 Fruit Street, Warren 2, Boston, MA 02114, USA. E-mail: [email protected] or tonsillar masses (frequently out of proportion to Received 25 October 2011; revised 22 December 2011; accepted pharyngitis/tonsillitis) that may be biopsied to rule 23 December 2011; published online 25 May 2012 out lymphoma. Unfortunately, these biopsies can www.modernpathology.org Infectious mononucleosis mimicking lymphoma 1150 A Louissaint Jr et al

lead to a misdiagnosis of lymphoma, as involvement Table 1 Antibodies and probes used for immunophenotypic of lymphoid tissue by acute EBV infection can analysis resemble both Hodgkin lymphoma and non-Hodgkin Antibody Source Dilution lymphoma.5–20 This relatively common pitfall can result in inappropriate treatment. Morphological CD20 Ventana Prediluted features mimicking non-Hodgkin lymphoma include CD10 Ventana Prediluted extensive proliferation of immunoblasts, often in BCL-2 Ventana Prediluted sheets, with marked cytological atypia.13,14,17,20 Simi- MUM1/IRF4 Dako 1:20 larly, Hodgkin lymphoma can be simulated, in part, BCL-6 Novocastra 1:100 CD3 Ventana Prediluted by the presence of atypical Reed–Sternberg-like CD4 Novocastra NEAT (undiluted) 9–11,20,21 cells. CD8 Ventana Prediluted Although the morphological features associated CD30 Ventana Prediluted with acute EBV infection of lymphoid tissue have CD15 Ventana Prediluted OCT-2 Santa Cruz 1:500 been described, the immunophenotypic features BOB.1 Santa Cruz 1:300 have not been fully characterized. In this report, we review 18 cases of acute EBV infection in which Immunoglobulin heavy and light chains the involved lymph node or Waldeyer’s ring tissue k Dako 1:2000 was biopsied or sampled. In most cases, the biopsy l Dako 1:1400 m Dako 1:3500 was prompted by a large or asymmetric mass and/or g Dako 1:3000 an unusual clinical presentation for infectious a Dako 1:1000 mononucleosis (ie older patient,22 the presence of d TFS NEAT symptomatic lymphadenopathy/airway obstruc- 12 In-situ probe tion ). The majority of cases were submitted to k, l (in-situ hybridization) Ventana Prediluted our department by pathologists who were concerned EBV-EBER (in situ Ventana Prediluted for lymphoma on the basis of a worrisome histo- hybridization) logical appearance. In this study, we describe the morphological and immunophenotypic features that Sources: Ventana, Tucson, AZ, USA; Dako Corporation, Carpinteria, CA, USA; Novocastra, Burlingame, CA, USA; Santa Cruz, Santa Cruz, are shared by these cases of infectious mononucleo- CA, USA; Thermo Fisher Scientific (TFS), Fremont, CA, USA. sis mimicking lymphoma and may be helpful in their recognition.

blasts), 1 þ (o10% immunoblasts positive), 2 þ Materials and methods (10–80% immunoblasts positive) and 3 þ (480% immunoblasts positive). In all, 18 cases of acute EBV infection involving Clinical follow-up information was obtained from or Waldeyer’s ring tissue were pathologists and clinicians at the referring institu- identified from the consultation files of two of the tion in all cases. This project was reviewed and authors. The cases were initially reviewed between approved by the Partners Institutional Review the years of 2001 and 2009. The original hema- Board. toxylin- and eosin-stained and immunohistochemi- cally stained slides were retrieved and reviewed in all cases. A panel including in-situ hybridization Results and immunohistochemical stains was performed Clinical Features and evaluated in all cases on formalin-fixed, paraffin-embedded sections. Immunohistochemical The 18 patients ranged from 9 to 69 years of age stains and in-situ hybridizations performed most (mean 24 years; median 18 years), with 3/18 (17%) recently were performed using the Ventana Bench- older than 25 years, and included nine female and mark Autostainer (Ventana Medical Systems, Tucson, nine male patients. Patients presented with neck AZ, USA) using the Ventana 3, 30-diaminobenzidine and tonsillar pain (7/18), pharyngitis (2/18), tonsil- tetrahydrochloride kit according to the manufacturer’s litis (4/18) and/or (4/18), but instructions. Primary antibodies included antibodies were occasionally asymptomatic (4/18). None of the to CD3, CD4, CD8, CD20, MUM1/IRF4, BCL-2, BCL-6, patients had a history of immunodeficiency or OCT-2, BOB.1, CD10, CD30, CD15, CD138, l and k immunosuppression. In all 18 patients, the mass immunoglobulin light chains, and IgD, IgM, IgG and/or lymphadenopathy raised a concern of lym- and IgA heavy chains (Table 1). For each case, the phoma and resulted in a biopsy. Eight (8/18; 44%) of immunophenotype of the cells comprising the im- the cases had at least one particularly unusual or munoblast population and the background infiltrate worrisome clinical feature, such as age 425 years was determined based on this panel of markers. To (3/18) and/or airway obstruction (4/18) (Table 2). assess the relative expression of particular antigens Biopsies were taken from cervical lymph nodes by immunoblasts, the positivity of each immunostain (3/18, 17%) or tonsil/adenoid/associated Waldeyer was scored as follows: 0 (absent staining in immuno- ring tissue (15/18, 83%). All cases were received in

Modern Pathology (2012) 25, 1149–1159 Table 2 Clinical features and follow-up

No. Age Sex Clinical presentation Extent of LAD Specimen Initial diagnosis Serology F/U (months)

1 9 M Asymptomatic LN (cervical) LN (cervical) Lymphoma (DLBCL ND 54 vs Hodgkin) 2 19 M Right neck mass LN (cervical) LN (cervical) (?) lymphoma Positive serology: VCA IgM (+); 19 VCA IgG (+); EA IgG (+); EBNA IgG (À) 3 69 M Asymptomatic lymphadenopathy LN (cervical, axillary) LN (cervical) (?) lymphoma Positive serology: VCA IgM (+); 101 VCA IgG (À); 4 11 F Nasopharyngeal mass Adenoids, Adenoids (?) Lymphoma Positive monospot 17 terminal ileum 5 17 F Peritonsillar abscess Tonsils (b) Tonsils (b) (?) Hodgkin lymphoma ND 53 6 17 F Asymptomatic Tonsils (b) Tonsils (b) (?) Hodgkin lymphoma ND 27 7 17 M Airway obstruction Tonsils (b) Tonsils (b) (?) Lymphoma Positive monospot 92 8 17 M Sore throat, fatigue, malaise, Tonsils (b), Tonsils (b) DLBCL Positive monospot 41 peritonsillar abscess, splenomegaly LN (mediastinal, cervical, axillary) 9 18 F Airway compromise, pharyngitis, Tonsils (b) and adenoids, Tonsils (b) (?) Lymphoma Positive monospot 29 cervical lymphadenopathy LN (cervical) 10 18 F Chronic tonsillitis Tonsils (b) Tonsils (b) Atypical lymphoid Positive monospot 55 infiltrate 11 18 F Sinusitis, tonsillitis, airway Tonsils (b) Tonsils (b) Hodgkin lymphoma ND 13 compromise adenoids 12 19 F Neck and tonsillar pain Tonsils (b) and adenoids, Tonsils (b) (?) Hodgkin lymphoma Positive monospot 70

LN (cervical) adenoids Jr Louissaint A lymphoma mimicking mononucleosis Infectious 13 20 M Severely hypertrophic tonsils Tonsils (b) Tonsils (l) Atypical lymphoid infiltrate Positive monospot 64 14 21 M Acute pharyngitis and peritonsillar Tonsils (l) Tonsils (l) (?) Lymphoma Positive monospot 29 abscess 15 22 F Pharyngeal obstruction, bilateral neck Tonsils (b); LN (cervical, Tonsil, Plasmablastic Positive monospot 39

and mediastinal lymphadenopathy, mediastinal) biopsy (l) lymphoma al et enlarged tonsils 16 23 M Acute tonsillar pharyngitis and Tonsils (b) Tonsil (b) (?) Lymphoma ND 51 peritonsillar abscess 17 39 M Neck and tonsillar pain Tonsils (b) Tonsils (b) DLBCL Positive serology: VCA IgM (+); 67 VCA IgG (+); EA IgG (+); EBNA IgG (À) 18 60 F Asymptomatic asymmetric Tonsil (r) Tonsil, biopsy (?) Lymphoma Positive serology: VCA IgM (+); 56 enlargement VCA IgG (À); of right tonsil

oenPathology Modern EA, early antigen; EBNA, Epstein–Barr virus nuclear antigen; EBV, Epstein–Barr virus; VCA, viral capsid antigen; DLBCL, diffuse large B-cell lymphoma; LN, lymph node; R, right; L, left; B, bilateral; ND, not done; M, male; F, female. 21)2,1149–1159 25, (2012) 1151 Infectious mononucleosis mimicking lymphoma 1152 A Louissaint Jr et al

consultation with a question of lymphoma (Table 2). of immunoblasts were CD20 þ B cells, although A diagnosis of lymphoma (Hodgkin lymphoma, occasional CD3 þ immunoblast-like cells were diffuse large B-cell lymphoma, plasmablastic lym- focally present as well in some cases (Figures 3a phoma) had already been made in 5 of 18 cases and b). In each case, the majority of immunoblasts (28%) before submission to our department. In the (450%) were positive for MUM1/IRF4 (18/18) and other cases, the diagnosis of lymphoma had been negative for germinal center markers CD10 (negative considered: lymphoma (8), Hodgkin lymphoma (3) in 18/18 cases) and BCL-6 (negative in 16/18 cases, and atypical lymphoproliferative (2). Testing of the o10% BCL-6 faintly positive immunoblasts in 2/18 patient for acute EBV infection was suggested at the cases), consistent with a post-germinal center time of consultation for confirmation of primary phenotype (Figures 3c and e and Table 4). Immuno- systemic infectious mononucleosis. In 13/18 cases, blasts were weakly positive for BCL-2 9/17 cases evidence of acute EBV infection was confirmed by (Table 4 and Figure 3f). In all cases examined, the EBV serology or positive monospot (Table 2). majority of immunoblasts expressed OCT-2, BOB.1 and CD30 (Figures 3g and i), but were negative for CD15 (Table 4). Reed–Sternberg-like cells had an Histopathological Features identical immunophenotype to the immunoblasts in all cases in which they were identified (8/8 cases). In all cases, there was expansion of the interfolli- In-situ hybridization for k and l light chains showed cular areas by a polymorphous infiltrate, resulting in numerous plasma cells with polyclonal expression; distortion, but not obliteration, of the normal tissue expression of light chains in immunoblasts was architecture (Figures 1a and b). The interfollicular often difficult to define using this technique infiltrate characteristically consisted of large round (Figures 4a and b). However, using immunohisto- to irregular cells with vesicular nuclei, prominent chemical stains for k and l light chains, there was nucleoli and scant basophilic cytoplasm, consistent polytypic expression in immunoblasts in 13/16 with immunoblasts, in a background of small- and cases (Figures 4c and d and Table 4). Immunoblasts intermediate-sized lymphoid cells, plasma cells, were found to express predominantly a mixture of histiocytes and admixed high endothelial venules IgG and IgM heavy chains. (Figure 1b). Immunoblasts were frequently present Immunoblasts were admixed within a polymor- in aggregates or occasionally sheets and were phous infiltrate, predominantly composed of small mitotically active (Figures 1c and d and Table 3). CD3 þ T cells with admixed small CD20 þ B cells Pleomorphic Reed–Sternberg-like cells were present and CD138 þ plasma cells. The number of CD3 þ T in a subset of cases (8/18) (Figure 1c, inset). More cells greatly outnumbered the CD20 þ B cells in the than half of the cases also contained areas of majority of cases. Moreover, immunohistochemical geographic necrosis (11/18), usually within ex- stains for CD4 and CD8 revealed a significantly panded interfollicular areas (Figure 1f and Table decreased or inverted CD4:CD8 ratio in most cases 3). Despite these worrisome features (distortion of evaluated (12/14). Plasma cells were polyclonal by the architecture, aggregates and sheets of large cells, in-situ hybridization (8/8 cases) and/or immunohis- Reed–Sternberg-like cells and necrosis), significant tochemistry for k and l light chains (10/10 cases) areas of normal lymph node or tonsillar architecture (Figures 4a and b). Plasma cells expressed IgG, IgM were retained in all cases. Areas of normal lymphoid and IgA heavy chains, with rare IgD þ plasma cells. architecture often merged with more abnormal areas. In Waldeyer ring tissue, crypts were pre- served, but focally associated with ulceration of the Clinical Follow-up tonsillar mucosa in 11/15 cases (73%) (Figure 1e). Peritonsillar abscesses were present in 5/15 cases Monospot testing (nine cases) or serological testing (33%). In lymph node tissue, reactive follicles as (four cases) was performed and the results were well as subcapsular, cortical and medullary sinuses available. Monospot testing was found to be positive were generally preserved; follicular hyperplasia was in 9/9 cases, and EBV-specific serologic tests were present in 4/18 cases (Table 3). consistent with recent infection in 4/4 cases (Table 2). In all patients, lymphadenopathy and symptoms subsided with treatment. There was no evidence of Immunohistochemistry and In-Situ Hybridization lymphoma or recurrence or progression of disease, with follow-up ranging from 13 to 102 months In all cases, in-situ hybridization for EBV-encoded (mean 49; median 52 months). RNA revealed a heterogeneous population of small to large EBV þ cells, predominantly within the interfollicular infiltrate, many of which were im- Discussion munoblasts (Figure 2a). Rare small EBV þ lymphoid cells were present in germinal centers, and occa- We report 18 cases of patients with lymphadeno- sional scattered EBV þ cells were identified within pathy or enlargement of Waldeyer’s ring tissue follicle mantles, but EBV þ immunoblasts were not associated with acute EBV infection, in which a found in germinal centers (Figure 2b). The majority biopsy was performed to evaluate for the possibility

Modern Pathology (2012) 25, 1149–1159 Infectious mononucleosis mimicking lymphoma A Louissaint Jr et al 1153

Figure 1 Characteristic morphological features of infectious mononucleosis cases. All cases showed interfollicular expansion with at least focal distortion of normal tissue architecture (a). The infiltrate was usually polymorphous, consisting of scattered immunoblasts in a background of numerous small mature lymphocytes, plasma cells and histiocytes (b). In areas, the immunoblastic proliferation was extensive with marked cytological atypia, numerous mitoses and focally forming sheets (c, d). Reed–Sternberg-like cells were present in 9/18 cases (d, inset). A significant number of tonsillar specimens were associated with ulceration of the tonsillar mucosa (e). More than half of the cases also featured focal geographic necrosis (11/18), usually within expanded interfollicular areas (f). of malignancy. All cases were considered worri- a florid immunoblastic proliferation, distortion of some for lymphoma by pathologists, on the basis of the underlying nodal or tonsillar architecture, the morphological features, including the presence of presence of necrosis and/or the presence of Reed–

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Sternberg-like or bizarre large cells within the infil- common extranodal site.24 Diffuse large B-cell lym- trate. The cases shared a number of morphological phoma characteristically shows a diffuse pattern of and immunophenotypic features that proved help- infiltration, commonly resulting in total architectur- ful in the distinction of acute EBV infection from al effacement of lymph or tonsillar architecture. lymphoma. However, in some cases, involvement by diffuse The most commonly considered diagnosis by large B-cell lymphoma may show partial architec- referring pathologists in the initial evaluation of tural effacement, and partial nodal involvement may the current infectious mononucleosis cases was occasionally be interfollicular, sometimes making diffuse large B-cell lymphoma. In fact, 20% of the distinction from infectious mononucleosis particu- cases in this series were submitted having had larly difficult.25 already been incorrectly been diagnosed as diffuse In all of the cases reviewed, certain morphological large B-cell lymphoma. While the distinction be- features raised the possibility of a reactive process, tween lymphoma and infectious mononucleosis is even in the presence of the worrisome features often not difficult, the overcalling of florid cases of described previously. One consistent morphological infectious mononucleosis continues to be a diag- feature in all cases was the predominantly poly- nostic pitfall for the practicing pathologist in many morphous nature of the infiltrate. Even when large cases.14,20 Diffuse large B-cell lymphoma is the most aggregates or sheets of immunoblasts were present, common type of non-Hodgkin lymphoma (31% of the majority of the infiltrate was almost always cases).23 The median age is 64 years, but patients of composed of reactive appearing small lymphoid any age can be affected.24 Patients typically present cells, plasma cells, histiocytes and high endothelial with rapidly enlarging lymph nodes or tumor venules. Moreover, despite the architectural distor- masses in extranodal sites.24 Waldeyer’s ring is a tion resulting from the frequently extensive inter- follicular expansion, sinuses and follicles frequently remained intact, although occasionally obscured. In Table 3 Summary of morphological features addition, significant areas of preserved lymph node or tonsillar architecture were identified in all cases Lymph node Waldeyer’s ring reviewed. (n ¼ 3) (n ¼ 15) We reviewed a panel of markers (including MUM1/IRF4, CD10, BCL-6 and BCL-2) that was Paracortical expansion 3/3 15/15 found to be particularly helpful in distinguishing Architectural distortion 3/3 15/15 the large activated cells of infectious mononucleosis Polymorphous infiltrate 3/3 15/15 Increased immunoblasts 3/3 15/15 from large neoplastic cells of diffuse large B-cell Preserved areas 3/3 15/15 lymphoma. A majority of stains in the panel were Reed–Sternberg-like cells 1/3 8/15 applied at the time of consultation, with any Follicular hyperplasia 0/3 4/15 remaining stains performed at the time of re-review Abscess 0/3 5/15 Ulcerated mucosa N/A 11/15 for the purposes of this study. In each case, we Necrosis 2/3 9/15 found that the immunoblasts consistently had a non-germinal center phenotype: MUM1 þ , BCL-6À

Figure 2 Detection of Epstein–Barr virus encoded RNA by in-situ hybridization. EBV-encoded RNA þ cells including immunoblasts and small- to medium-sized cells were present in all cases (a). Interestingly, EBV-encoded RNA þ immunoblasts were predominantly present within the interfollicular infiltrate, with scattered EBV-encoded RNA þ cells within follicle mantles and only rare small EBV-encoded RNA þ cells within germinal centers. EBV-encoded RNA þ immunoblasts were generally restricted from germinal centers (b).

Modern Pathology (2012) 25, 1149–1159 Infectious mononucleosis mimicking lymphoma A Louissaint Jr et al 1155

Figure 3 Characteristic immunophenotype of immunoblasts. Immunoblasts were characteristically CD20 þ B cells (a); while most CD3 þ cells corresponded to small mature lymphocytes, there were occasional larger CD3 þ cells with immunoblast-like morphology (b). Immunoblasts were positive for MUM1(c), negative for BCL-6 (d) and CD10 (e), and showed focal dim staining for BCL-2 (f). Immunoblasts were positive for CD30 (g), negative for CD15 (not shown) and retained expression of both BOB.1 (h) and OCT-2 (i). This was also true for Reed–Sternberg-like cells (insets, h and i), which helped distinguish them from neoplastic Reed–Sternberg cells.

and CD10À. The immunoblasts were also gene- ment in rheumatoid arthritis. The group of EBV þ rally positive for CD30 and EBV-encoded RNA, and diffuse large B-cell includes a recently frequently showed polyclonal expression of k and l defined subtype known as diffuse large B-cell light chains. The combination of EBV-encoded lymphoma of the elderly, in which immunological RNA þ ,CD30þ large activated cells with non- deterioration is additionally associated with the germinal center phenotype (MUM1/IRF4 þ ,CD10À, aging process.27–35 The polymorphous subtype of BCL-6À), in a polymorphous background with inver- diffuse large B-cell lymphoma of the elderly, in ted CD4:CD8, is relatively unusual in diffuse large particular, is additionally associated with a poly- B-cell lymphoma. Therefore, in an adolescent or morphous background consisting of a broad range young adult, an atypical lymphoid infiltrate with of B-cell maturation and mixture of reactive ele- numerous MUM1 þ ,CD10À,BCL-6À immunoblasts ments, including small lymphocytes, plasma cells should raise the suspicion of a reactive process, such and histiocytes, similar to that seen in acute EBV as infectious mononucleosis. infection. These cases can also be associated However, it is important to note that the non- with large areas of geographic necrosis and Reed– germinal center phenotype can be seen in approxi- Sternberg-like cells. Thus, in an older and/or mately 25% of diffuse large B-cell lymphoma immunocompromised patient, the distinction bet- cases,26 including a minority of EBV þ CD30 þ ween EBV þ diffuse large B-cell lymphoma and diffuse large B-cell lymphoma associated with acute EBV infection can be challenging. Of note, acquired immunodeficiency, including HIV infec- there have also been reports describing rare cases tion, organ transplantation and methotrexate treat- of EBV-positive diffuse large B-cell lymphoma

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Table 4 Immunoblast immunophenotype

Score 0+(absent) 1+ 2+ 3+

No. % No. % No. % No. %

CD20 0 0 0 0 7 39 11 61 EBERa,b 0 0 0 0 15 83 3 17 MUM1/IRF4 0 0 0 0 8 44 10 56 OCT-2 0 0 0 0 9 56 7 44 BOB.1 0 0 0 0 10 63 6 38 CD30 0 0 1 8 10 77 2 15 CD15 11 100 0 0 0000 BCL-2 8 47 3 18 6 35 0 0 CD312676330000 BCL-6 16 88 2 12 0000 CD10 18 100 0 0 0000

No staining Polyclonal Clonal

No. % No. % No. %

k/l IHC 3 19 13 72 0 0

To assess the relative expression of particular antigens by immunoblasts, the positivity of the each immunostain was scored as follows: 0+, absent staining in immunoblasts; 1+, o10% immunoblasts positive; 2+, 10–80% immunoblasts positive; 3+, 480% immunoblasts positive. aIn-situ hybridization for EBER (Epstein–Barr virus-encoded RNA); all other markers assessed by immunohistochemistry. bThe overall percentage of cells that were positive for EBER ranged from 10 to 70% in the most strongly positive areas (mean: 35%; median: 30%).

occurring in younger, apparently immunocompetent Hodgkin lymphoma and infectious mononucleosis patients.36 These cases are morphologically and are characterized by a similar polymorphous back- immunophenotypically similar to cases observed ground consisting of plasma cells, histiocytes and in patients 450 years old, and seem to have a small lymphocytes, but eosinophils are rare in similarly poor prognosis. Thus, EBV-positive diffuse infectious mononucleosis and usually numerous in large B-cell lymphoma cannot be ruled out solely on classical Hodgkin lymphoma. The presence of the basis of age and apparent immunocompetence. Reed–Sternberg-like cells in some cases of infec- In these rarer cases, the demonstration of polyclonal tious mononucleosis is notorious for causing confu- k and l immunoglobulin expression in EBV þ sion with Hodgkin’s disease. The Reed–Sternberg immunoblasts may be additionally helpful in dis- cell of classical Hodgkin lymphoma and the Reed– tinguishing acute EBV infection from EBV þ diffuse Sternberg-like cell of infectious mononucleosis are large B-cell lymphoma. Heterophile/serological as- not only similar cytologically, but are also similar in says for acute EBV infection and molecular genetic their expression of the activation antigen CD30, studies (eg for clonality of the immunoglobulin EBV-encoded RNA transcripts (although not com- genes) may also be important in making this mon in young adults with nodular sclerosing distinction. However, it is important to note that classical Hodgkin lymphoma) and their lack of PCR amplification of spurious or nonspecific clones CD45/LCA expression.5,10,11,20,21 One helpful differ- in the setting of restricted but benign reactive entiating feature is the expression of CD15 in Reed– populations of normal lymphocytes can sometimes Sternberg cells of classical Hodgkin lymphoma and lead to false positives.30,33,37 lack of expression of CD15 in Reed–Sternberg-like Classical Hodgkin lymphoma was considered or cells of infectious mononucleosis.21 However, clas- diagnosed by the submitting pathologist in four of sical Hodgkin lymphoma can infrequently be nega- the cases reviewed. Classical Hodgkin lymphoma tive for CD15,27,42–44 making this distinction more accounts for 490% of all Hodgkin lymphoma, and difficult. Moreover, Reed–Sternberg cells of classical shows a bimodal age distribution, with the first peak Hodgkin lymphoma have the same non-germinal in early adulthood (age 15–35 years) and second center immunophenotype that characterized the peak in those older than 55 years.38 Cervical lymph immunoblasts of the current infectious mononu- nodes are the most common site of involvement cleosis cases. Therefore, the retention of both OCT-2 (75% of cases). Notably, classical Hodgkin lympho- and BOB.1 in the current series of infectious ma occurs rarely in mucosa-associated lymphoid mononucleosis cases mimicking classical Hodgkin tissue, such as Waldeyer’s ring.38–41 Both classical lymphoma may particularly be helpful in difficult

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Figure 4 Immunoglobulin light-chain expression. In-situ hybridization for k and l immunoglobulin light chains revealed numerous polyclonal plasma cells within the interfollicular infiltrate (a, b)(a: k; b: l). It was often difficult to definitively identify light-chain expression on immunoblasts with this assay. Immunohistochemical stains for k and l light chains revealed polyclonal light-chain expression on occasional immunoblasts (arrows), in addition to scattered polyclonal plasma cells (arrowheads) (c, d)(c: k; d: l).

cases, as one of these markers is usually lost in made. Immunohistochemical stains for OCT-2/ Reed–Sternberg cells of classical Hodgkin lympho- BOB.1 and k/l light chains may be additionally ma. Of note, molecular genetic assays for B-cell helpful in particularly difficult cases mimicking clonality are not very useful in distinguishing diffuse large B-cell lymphoma or classical Hodgkin infectious mononucleosis from classical Hodgkin lymphoma. This immunophenotypic profile is an lymphoma, as such assays usually fail to detect the important one to keep in mind to avoid a mis- rare neoplastic Reed–Sternberg cells in classical diagnosis of lymphoma in challenging cases. Hodgkin lymphoma (thus appearing falsely nega- tive), as a result of the paucity of neoplastic cells in these lymphomas45,46 Disclosure/conflict of interest In summary, infectious mononucleosis should be The authors declare no conflict of interest. considered in any immunoblastic proliferation occurring in cervical lymph nodes and Waldeyer’s ring tissue. The distinction between infectious mononucleosis and lymphoma will often be made on morphological grounds; however, in difficult References cases, an atypical lymphoid infiltrate with numer- 1 Luzuriaga K, Sullivan JL. Infectious mononucleosis. ous MUM1/IRF4 þ , CD10À, BCL-6À immunoblasts N Engl J Med 2010;362:1993–2000. should raise the suspicion of infectious mononu- 2 Heath Jr CW, Brodsky AL, Potolsky AI. Infectious cleosis/acute EBV infection and warrants additional mononucleosis in a general population. Am J Epide- consideration before a diagnosis of lymphoma is miol 1972;95:46–52.

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