How I Diagnose EBV-Positive B- and T-Cell Lymphoproliferative Disorders

Stefan D. Dojcinov, MD; Leticia Quintanilla-Martinez, MD

Am J Clin Pathol. 2023;159(1):14-33.

Abstract and Introduction

Abstract

Objectives: Epstein-Barr virus (EBV)–associated lymphoproliferative disorders (LPDs) encompass a group of well-defined entities of B-, T-, and natural killer (NK)–cell derivation. The diagnosis of these disorders is challenging because of clinical and morphologic features that may overlap with other benign and malignant EBV+ lymphoproliferations. This review describes our approach to the diagnosis of EBV-associated LPDs.

Methods: Two cases are presented that illustrate how we diagnose EBV-associated LPDs. The first case represents a systemic EBV+ T-cell lymphoma of childhood and the second case an EBV+ mucocutaneous ulcer. The clinicopathologic features that help distinguish these entities from biological and morphologic mimickers are emphasized.

Results: The accurate diagnosis of EBV-associated LPDs requires the incorporation of histologic and immunophenotypic features, the assessment of the EBV latency program, and, most important, complete clinical findings. Clonality analysis is not helpful in distinguishing benign from malignant EBV+ LPDs.

Conclusions: The better understanding of EBV-associated LPDs has resulted in the recognition of well-defined entities of B-, T-, and NK-cell derivation and consequently improvement of their treatment with curative intent. It is critical to distinguish benign from malignant EBV+ LPDs to avoid overtreatment.

Introduction

Epstein-Barr virus (EBV)–associated B-, T-, and natural killer (NK)–cell lymphoproliferations have a broad clinical spectrum ranging from reactive self-limited disorders to malignant proliferations with a fulminant clinical course.^[1–3] EBV is the most common viral infection in humans, with 95% of the population worldwide showing an asymptomatic lifelong carrier stage state.^[4,5] Most EBV infections occur asymptomatically in children; however, a small subset of patients develops infectious mononucleosis (IM), a self-limited disease usually occurring in the second decade of life.^[6,7] During EBV primary infection, the virus undergoes lytic replication in the epithelial cells of the oropharynx subsequently infecting circulating naive B cells.^[8] The latently EBV-infected B cells trigger a strong, specific primary cytotoxic response that controls the EBV infection. The quantity and the quality of the CD8+ T-cell response to EBV are critical to control the infection.^[9,10] Patients with low numbers of EBV-specific CD8+ cells are prone to develop chronic active EBV infection (CAEBV).^[11] Furthermore, it has been shown that patients with CAEBV have a decrease in the T-cell receptor (TCR)–β repertoire and expanded T-cell clones in the peripheral blood.^[12] To escape immunosurveillance, EBV downregulates antigen expression, establishing a stable reservoir of memory B cells that carry the viral genome, but viral antigen expression is maximally suppressed, so-called latency 0.^[13,14] There are three EBV latency patterns recognized.^[4,5] Latency III involves the unrestricted expression of all nine latent genes (six EBV-encoded nuclear antigens [EBNAs] and three latent membrane proteins [LMPs]) observed during acute EBV infection or in severely immunodeficient individuals. Latency II is an intermediate pattern with expression of many latent proteins except for EBNA2. This pattern is demonstrated in most T-cell LPDs. Latency I is restricted to the expression of EBNA1, characteristic of Burkitt lymphoma. The transcription of nonpolyadenylated RNAs EBER1 and EBER2 is a constant feature of all latent EBV infection patterns and, therefore, is the best marker to demonstrate EBV infection in routine diagnosis.^[1,2,15,16]

The exact mechanisms by which EBV promotes lymphomagenesis remain elusive; however, they involve impaired immune surveillance. The disruption of the finely regulated balance between virus and host immune system can result in EBV-associated lymphoproliferations of B-, T-, and NK-cell derivation, with disease manifestations generally depending on the type of EBV-infected cell and the state of host immunity Table 1^[17] and Figure 1.^[14] The diagnosis of EBV-associated lymphoproliferative disorders (LPDs) may be challenging because it requires detailed clinical information and correlation of morphologic findings. In general, clonality analysis is not helpful in distinguishing benign from malignant EBV+ lymphoproliferations. In the assessment of EBV-associated LPDs, the investigation of EBV latency in tissue biopsy specimens (LMP1 and EBNA2) and peripheral blood (PB) EBV DNA copy numbers provides valuable information regarding the extent of host immunosuppression. In this review, we discuss the clinicopathologic features of EBV-associated LPDs and emphasize the differential diagnosis by describing two cases: (1) systemic EBV+ T-cell lymphoma of childhood and (2) EBV+ mucocutaneous ulcer (EBV+ MCU).

(Enlarge Image)

Figure 1.

Differential diagnosis of EBER+ cells in lymph nodes and other tissues. The initial approach to the diagnosis of EBV+ lymphoproliferations is the identification of the cell type infected by EBV. The major criteria to identify the different disorders are depicted. EBV in T and NK cells is per default latency II. BCR, B-cell receptor; DLBCL, diffuse large B-cell lymphoma; EBV, Epstein-Barr virus; HLH, hemophagocytic lymphohistiocytosis; IM, infectious mononucleosis; LPD, lymphoproliferative disorder; LyG, lymphomatoid granulomatosis; NK, natural killer; NOS, not otherwise specified; RS-like, Reed-Sternberg–like cells; TCR, T-cell receptor.

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Abstract and Introduction
Cases
Systemic EBV+ T-cell Lymphoma of Childhood
EBV+ Mucocutaneous Ulcer
Diagnostic Challenges and Questions for the Experts
Conclusions
References
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Table 1. EBV-Associated Lymphoproliferative Disorders ^a
B-cell lymphoproliferations EBV-associated
Infectious mononucleosis
EBV+ mucocutaneous ulcer
EBV+ diffuse large B-cell lymphoma, NOS
Diffuse large B-cell lymphoma associated with chronic inflammation
Fibrin-associated diffuse large B-cell lymphoma
Lymphomatoid granulomatosis
T- and NK-cell lymphoproliferations EBV-associated
EBV+ hemophagocytic lymphohistiocytosis
EBV+ T- and NK-cell lymphoproliferative diseases of childhood
Hydroa-vacciniforme lymphoproliferative disorder
Severe mosquito bite allergy
Chronic active EBV disease, systemic
Systemic EBV+ T-cell lymphoma of childhood
Extranodal NK/T-cell lymphoma, nasal type
Aggressive NK-cell leukemia
Primary nodal EBV+ T- or NK-cell lymphoma

EBV, Epstein-Barr virus; NK, natural killer; NOS, not otherwise specified.
^aProvisional entities in the 2017 World Health Organization classification are italicized. In the recent 2022 international consensus classification, EBV+ mucocutaneous ulcer is recognized as a definitive entity, whereas primary nodal EBV+ T- or NK-cell lymphoma remains a provisional entity.¹⁷

Table 2. Differential Diagnosis of Systemic EBV+ T-Cell Lymphoma of Childhood
Disease	Clinical Features	Morphology	Immunophenotype	Lineage and Clonality	HLH Association
Nonfamilial EBV-associated HLH, benign disorder	More often in children and young adults Diagnostic criteria for HLH should be fulfilled (>5 criteria)^a 1. Temperature >38.5°C 2. Splenomegaly 3. Cytopenias 4. Hypertriglyceridemia 5. Hemophagocytosis 6. Low/absent NK-cell activity 7. Elevated ferritin 8. Elevated CD25	Hemophagocytosis in BM, spleen, or LNs Relatively small numbers of EBV+ T cells, rarely NK cells Bland cytology but a mild degree of atypia may be seen	Predominantly cytotoxic CD8+ T cells CD56+ phenotype raises the differential diagnosis with ANKL and CAEBV	T cells (80%) NK cells (20%) Monoclonal TCR in 50%-60% Cytogenetic analysis should be normal	Always
CAEBV, systemic	More often in children and young adults IM-like illness persistent for more than 3 months Fever, lymphadenopathy, hepatosplenomegaly, anemia, skin rash, diarrhea, and uveitis Rare manifestations: myocarditis, coronary aneurism, interstitial pneumonia, digestive tract ulcer/perforation, CNS involvement Protracted clinical course Risk of progression to EBV+ T- or NK-cell lymphoma	Nonspecific inflammatory changes with no evidence of malignant lymphoproliferations LNs often with follicular hyperplasia and expanded paracortical areas Bland cytology	CD4 >> CD8 > γ δ T cells CD56+ 40%	T cells (60%) NK cells (40%) Monoclonal TCR (40%-63%) Monoclonal EBV (84%) Somatic mutations in DDX3D and KMT2D have been demonstrated EBV genome harbors intragenic deletions	Common during disease progression Rare as initial symptom, obscures the diagnosis of CAEBV HLH as initial diagnosis associated with poor prognosis
Systemic EBV+ T-cell lymphoma of childhood	More often in children and young adults Occurs shortly after acute primary EBV infection and rare in the setting of CAEBV disease Abnormal serology against EBV with lack of anti-VCA IgM High fever, hepatosplenomegaly, pancytopenia, coagulopathy, abnormal liver function Lymphadenopathy might occur and be the dominant feature Fulminant clinical course that results in death within days to weeks	Cytologic atypia ranges from minimal to severe Histiocytic hyperplasia and striking hemophagocytosis in BM, spleen, and liver LNs look depleted with paracortical EBV+ CD8+ T cells	Predominantly cytotoxic CD8+ T cells Rare CD4 or double positive CD4 and CD8	T cells (100%) Monoclonal TCR (100%) Cytogenetic abnormalities and somatic genetic alterations favor this diagnosis over nonneoplastic HLH	Virtually always Overlapping clinical and morphologic features with HLH
ANKL	Characteristically in adults, might present in young adults High fever, general malaise, hepatosplenomegaly, liver failure, and pancytopenia Neoplastic NK cells in PB and BM Fulminant clinical course Rare cases EBV negative (<10%)	Varying degrees of leukemic infiltrate in BM, liver, LNs, and spleen LNs with diffuse infiltration by neoplastic cells and striking hemophagocytosis Broad cytologic spectrum from subtle to large pleomorphic cells Bland cytology raises the differential diagnosis with CAEBV and EBV-HLH of NK-cell type	CD3ε+, CD56+, CD2+, FASL+, CD16+ (75%) Surface CD3–, CD5–, CD57–	NK cells (100%) Polyclonal TCR	Almost always

ANKL, aggressive NK-cell leukemia; BM, bone marrow; CAEBV, Chronic active EBV infection; CNS, central nervous system; EBV, Epstein-Barr virus; HLH, hemophagocytic lymphohistiocytosis; IgM, immunoglobulin M; IM, infectious mononucleosis; LN, lymph node; NK, natural killer; PB, peripheral blood; TCR, T-cell receptor; VCA, viral capsid antigen.

Table 3. Differential Diagnosis of EBV+ B-Cell Lymphoproliferations With Hodgkin-Like Features
Disease	Clinical Features	Morphology	Immunophenotype	Lineage, Clonality, and Molecular Features
EBV+ mucocutaneous ulcer	Mostly elderly with well-circumscribed ulcerating lesions of skin and mucosas No breach of compartmental boundaries No lymphadenopathy or hepatosplenomegaly No LDH elevation Full resolution upon reduction/withdrawal of IS Waxing-wanning course in elderly with immunosenescence No progression to lymphoma	Good circumscription with a band-like infiltrate following contour of the overlying ulcerated surface Hodgkin-like features with HRS-like cells in a mixed inflammatory background Wide range of lesional cell sizes (highlighted by EBER) Angioinvasion Rim of lymphocytes at base	Retained B-cell phenotype (post–germinal center) CD45+/– CD20+/– CD79a+ PAX5+ (strong) OCT2+ (strong) BOB1+ (strong) MUM1+ BCL6–/+ CD10– CD30+ CD15+ (up to 50%) EBV latency III (50%) PD-L1–	B-cell clonal 50% T-cell (background lymphocytes) oligoclonal and "restricted" TCR rearrangement patterns Genetic features not studied fully No immune evasion features
EBV+ diffuse large B-cell lymphoma, NOS	Elderly with high-stage nodal and extranodal disease B-symptoms and elevated LDH Isolated lesions and bulky tumor masses breaching compartmental boundaries Aggressive lymphoma with poor outcomes	Diffuse, destructive, and infiltrative proliferations Range of morphologies with common Hodgkin-like features and polymorphism—wide range of lesional cell sizes (highlighted by EBER) Angioinvasion Copious necrosis Mucosal/cutaneous lesions show no distinctive architecture	Retained B-cell phenotype (post–germinal center) CD45+/– CD20+/– Cd79a+ PAX5+ (strong) OCT2+ (strong) BOB1+ (strong) MUM1+ BCL6–/+ CD10– CD30+ CD15+ (up to 50%) EBV latency III (9%-12%) PD-L1+/– (40%-60%) (extranodal)	B-cell clonal T-cell (background lymphocytes) oligoclonal and "restricted" TCR rearrangement patterns "Host response" lymphoma partly characterized by immune evasion A proportion with CIITA and PD-L1/L2 gene loci abnormalities
EBV+ classic Hodgkin lymphoma	Young and elderly with mediastinal and nodal presentation B-symptoms and elevated LDH Extremely rare primary extranodal presentation in skin and mucosas Extranodal involvement usually as part of widespread disease Excellent outcomes with appropriate combined therapies	Diffuse/nodular destructive and infiltrative proliferations HRS cells in a mixed inflammatory background Only large HRS cells with no intermediate or small cell component (highlighted by EBER) No angioinvasion Exceptionally rare mucosal/cutaneous lesions show no distinctive architecture	Shut off B-cell expression program CD45– CD20– Cd79a–/+ PAX5+ (weak) OCT2–/+ (weak) BOB1–/+ (weak) MUM1+ BCL6–/+ CD10– CD30+ CD15+ (variable) EBV latency II PD-L1+/– (>80%)	B-cell clonal T-cell (background lymphocytes) polyclonal; oligoclonal/"restricted" patterns in elderly "Host response" lymphoma characterized by immune evasion Higher prevalence of CIITA and PD-L1/L2 gene loci abnormalities

EBV, Epstein-Barr virus; HRS, Hodgkin and Reed-Sternberg; IS, immunosuppression; LDH, lactate dehydrogenase; NOS, not otherwise specified; TCR, T-cell receptor.

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How I Diagnose EBV-Positive B- and T-Cell Lymphoproliferative Disorders

Abstract and Introduction

Abstract

Introduction

Tables

Tables

Tables

References

Authors and Disclosures

Authors and Disclosures

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Key Points

Figure 1.

Figure 1.

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