Epstein-Barr Virus: Cancer and Immunosuppression

Jeffrey I. Cohen Head, Medical Virology Section Laboratory of Clinical Infectious Diseases NIH

Pathogenesis of EBV Infection

Cohen NEJM 2000

Cellular Immune Responses Are Critical For Control of EBV

Early IM: NK cells non-HLA specific CTLs Late IM: HLA-restricted CTLs (CD8 and CD4): Lytic epitopes - up to 40% of CD8 cells Latent epitopes - up to 2% of CD8 cells

Healthy EBV seropositive persons: Latent epitopes- 4% of CD8 cells Lytic epitopes- 0.1 to 5% of CD8 cells

EBV Transforms B Cells In Vitro and the Cells Express Limited Viral and Cellular Proteins

EBV LCLs

EBV Latency Proteins

Cell Genes Induced

Rickinson and Kieff, Fields Virology

EBV Latency Proteins

Cohen NEJM 2000

LMP-1 is the EBV Oncogene Oncogene

Expression in transgenic mice leads to B cell lymphoma; expression in fibroblasts leads to tumors in nude mice

B Cell Proliferation
Upregulates adhesion molecules, CD23, CD40, IL-6, IL10, etc. Activates NF-B

H&E

Inhibits apoptosis
Upregulates Bcl-2, A20, Mcl-1
(Kulwichit et al PNAS 1998)

LMP-1

LMP-1 Mimics constitutively form of CD40 in B cells

Thorley-Lawson, Nature Rev Immunol, 2001

Activation of NF-B in Tumor from Patient with Post-Transplant EBV Lymphoproliferative Disease

Lane 1: EBV- B cell Lane 2: EBV+ B cell Lane 3: EBV- LPD Lane 4: EBV+ LPD

Liebowitz NEJM 1998

Diseases Associated with EBV

EBV in B Cell Infectious mononucleosis X-Linked Lymphoproliferative Disease Chronic active EBV Hodgkin Disease Burkitt Lymphoma Lymphoproliferative disease EBV in Other Cells Nasopharyngeal carcinoma Gastric carcinoma Nasal T/NK cell lymphomas Peripheral T cell lymphomas Oral hairy leukoplakia Smooth muscle tumors in transplant patients

Diseases Driven by Epstein-Barr Virus

Infectious mononucleosis Chronic Active EBV X-linked lymphoproliferative disease Lymphoproliferative disease Oral hairy leukoplakia Hodgkin disease Nasopharyngeal carcinoma T cell lymphoma Burkitt lymphoma EBV Gene Expression EBV-Driven Cell Proliferation

Patterns of EBV Latent Infection

Latency Type 1 2 3 Other EBER EBNA-1 EBNA-2 EBNA-3 LMP-1 LMP-2 + + + + + + + +/+ + + + + + +/Disease BL NPC, HD IM, LPD Carrier

Burkitt Lymphoma
EBV+: 90% of cases in developing countries jaw tumors 20% cases in US children with abdominal tumors AIDS patients tumors in lymph nodes EBV may be one hit but all tumors have c-myc translocations Dysregulation of c-myc oncogene Only EBV EBNA-1 expressed Therapy: Chemotherapy

Hodgkin Disease

EBV+: 60-70% of cases in developing countries 35-50% cases in US EBV in Reed-Sternberg cells Therapy: Chemotherapy, radiation Anti-EBV CTLs effective in some cases
LMP-1 expression

EBV-Associated Smooth Muscle Tumors

Occur in transplant recipients, AIDS patients, congenitial immunodeficiency Pathology: leiomyosarcomas and leiomyomas in various organs (especially transplant) and lymph nodes Some tumors regress with reduced immunosuppression

EBV Lymphoproliferative Disease

Occurs with immunodeficiency (AIDS, congenital) or after transplantation, RA and MTX Symptoms: Infectious Mononucleosis Mass lesions in organs (less often lymph nodes) Risk Factors: Primary infection GVHD with increased immune suppression T cell depleted bone marrow CMV

Cohen NEJM 2000

Risk for EBV PTLD

Primary infection- higher viral loads, no memory T cells to EBV CMV infection Polymorphisms corresponding to low production of IFN-, TNF-; high levels of IL-10 Level of intensity of T cell immunosuppression

EBV Viral Load is Increased in Patients with Lymphoproliferative Disease

Riddler, Blood 1994

Viral Load Used to Monitor Transplant Patients: Increased EBV load at onset of LPD Used to initiate preemptive therapy

Treatment of EBV Lymphoproliferative Disease

Reduce immunosuppressionEarly, polymorphic lesions often responsive Later monomorphic lesions can have chromosomal changes Excise localized lesions Radiation therapy (for CNS lesions) or chemotherapy Anti-CD20 monoclonal antibody (rituximab) Interferon- For stem cell transplant recipients: donor lymphocyte infusions or donor EBV-specific cytotoxic T cell infusions For solid organ transplant recipients: autologous or HLAmatched, EBV-specific, cytotoxic T cell infusions

Cutaneous Lymphomas Associated with EBV-infected T cells Non-immunosuppressed Patients

More often in Asians Hydroa vacciniforme: vesciulopapular lesions on face and hands, fever, can progress to T cell lymphoma Angiocentric NK/T cell lymphomas:ulcers, vesicles, nodules, papules on nose, checks, lips, extremities, trunk EBV subcutaneous T cell lymphoma: plaques, fever, hepatosplenomegaly, pancytopenia, panniculitis, hemophagocytosis

Cutaneous Lymphomas Associated with EBV-infected B cells Immunosuppressed Patients

Cutaneous ulcerated nodules- B cell lymphomas after transplant or in patients with AIDS Cutaneous B cell lymphomas in patients with rheumatoid arthritis or polymyositis receiving methotrexate- resolution in some after drug stopped

EBV LPD More Common at Sites with Chronic Inflammation

Disease more frequent in transplanted organ Higher frequency of EBV+ cells Antigenic stimulation with B cell proliferation Cytokine activation in organ Reports of EBV+ pyothorax-associated pleural lymphomas at site of pleural inflammation after tuberculosis (Arch Pathol Lab Med. 1996) Report of 3 cases of EBV+ large B cell lymphomas in patients with chronic inflammation (osteomyelitistumor at site of bone, chronic venous ulcers- tumor at site of ulcer) (J Pathol. 1997 )

Immunosuppressive Agents Associated with EBV LPD

Steroids and Azathioprine Methotrexate: Patients with RA, Polymyositits Antibodies: ATG: anti-thymocyte globulin ALG: anti-lymphocyte globulin OKT3: anti-CD3 Calcineurin inhibitors: cyclosporine, tacrolimus Sirolimus

AZA (1 g/ml) CsA (1 g/ml) CY (10 g/ml) MPA (10 g/ml) Prednisone (1 m) MTX (5 g/ml) AZA (10 g/ml) CsA (10 g/ml) CY (100 g/ml) MPA (100 g/ml) Prednisone (10 m) MTX (50 g/ml)

DRUG: _

Methotrexate, but not other Immunosuppressants, Induces EBV Lytic Replication

Feng et al JNCI 2004

-actin BMRF1

Calcineurin Inhibitors and PTLD: Cyclosporine, Tacrolimus

Inhibit generation of cytotoxic activity Induce expression of IL-6 and TGF- that supports B cell activation and proliferation Enhance survival of EBV-transformed cells in vitro by protecting from Fas-mediated apoptosis Lower doses of cyclosporine allow T cell responses to EBV in vitro and are associated with lower rates of lymphoma than higher doses In children tacrolimus is associated with a higher risk of LPD than cyclosporine in some, but not all studies.

Risk of PTLD in Pediatric Liver Transplant Recipients for Primary Tacrolimus Therapy

Cacciarelli et at Pediatric Transplantation 2001

Kaposis Sarcoma at the Site of Topical Tacrolimus

28 yo AIDS patient on HAART (CD 143) with psoriasis and seborrheic dermatitis treated with topical tacrolimus 0.1% ointment to axilla, groin, head for 1 month Developed KS at these sites and in lungs while on tacrolimus

Cho et al. J. Am Acad Dermatol. 50:149-50, 2004

Lymphoma at Site of ATG or ALG Injections

Age 32 33 32 18 Transplant kidney kidney heart heart AT/LG Sites of Lymphoma Ref horse buttock, nodes 1 horse buttock, nodes, liver 2 rabbit thigh, brain, lung, nodes 3 rabbit thigh, chest wall, 3 abdominal nodes

1. Deodhar et al N Engl J Med 280:1104-6, 1969 2. Cotton et al. Transplantation 16:154-7, 1973; follow-up Herrera et al. Mil Med. 146:652-4, 1981 3. Weintraub and Warnke Transplantation 33:347, 1982

Lymphoma at Site of ALG

(Cotton et al 1973; Herrera et al 1981)
47 y.o. renal transplant recipient thoracic duct canulation before and 3 wks after transplant to deplete lymphocytes; prednisone, azothioprine Horse ALG i.m. in buttocks post transplant on x 14 d, 3 x/wk x 1 yr 6 months after last ALG nodule at site >reticulum cell sarcoma (no EBV studies), immunosuppression reduced, radiation to site; One year later draining lymph nodes had histiocytic lymphoma, radiation (no EBV studies) 2 years later died of bacteremia-lymphoma in liver

Lymphoma at Site of ALG (Deodhar et al 1969)

32 y.o. renal transplant recipient on azathioprine and prednisone Rejection 7 months after transplant: treated with actinomycin C and graft irradiation Horse ALG i.m. in buttocks: 6 weeks later nodule at site, enlarge over 10 months; excisedreticulum cell sarcoma with lymph node involvement (no EBV studies); died of OI

Lymphoma at Site of ALG

(Weintraub and Warnke 1982)
7 patients with NHL/182 heart transplants, 2 developed lymphoma at site of ATG 32 yo cardiac transplant recipient underwent two allogeneic heart transplants Developed high grade immunoblastic lymphoma in thigh at site of rabbit ATG, later in brain and lung 18 yo cardiac transplant recipient underwent two allogeneic heart transplants Developed large noncleaved cell lymphoma in thigh at site of rabbit ATG, later chest wall and abdomen

Summary: EBV LPD in Persons Receiving Immunosuppressants

Most early, polymorphic lesions are EBV driven, and may respond to reduction in immunosuppression Later monomorphic lesions may have chromosomal changes and often require chemotherapy More common with primary EBV infection May have genetic component (cytokine polymorphisms) More common at site of chronic inflammation Some occur at sites of local immunosuppression: ATG or ALG injection sites all patients on other immunosuppression