Epstein-Barr Virus: Cancer and Immunosuppression

1. Epstein-Barr Virus: Cancer and Immunosuppression Jeffrey I. Cohen Head, Medical Virology Section Laboratory of Clinical Infectious Diseases NIH

2. Pathogenesis of EBV Infection Cohen NEJM 2000

3. 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

4. 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

5. EBV Latency Proteins Cohen NEJM 2000

6. 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, IL-10, etc. Activates NF-B Inhibits apoptosisUpregulates Bcl-2, A20, Mcl-1 H & E LMP-1 (Kulwichit et al PNAS 1998)

7. LMP-1 Mimics constitutively form of CD40 in B cells Thorley-Lawson, Nature Rev Immunol, 2001

8. 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

9. 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

10. Diseases Driven by Epstein-Barr Virus Infectious mononucleosis Chronic Active EBV X-linked lymphoproliferative disease Lymphoproliferative disease Oral hairy leukoplakia Hodgkin disease EBV EBV-Driven Nasopharyngeal carcinoma Gene Cell T cell lymphoma Expression Proliferation Burkitt lymphoma

11. Patterns of EBV Latent Infection • Latency • TypeEBEREBNA-1EBNA-2EBNA-3LMP-1LMP-2Disease • 1 + + - - - - BL • + + - - + + NPC, HD • + + + + + + IM, LPD • Other + +/- - - - +/- Carrier

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. Treatment of EBV Lymphoproliferative Disease • Reduce immunosuppression- Early, 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 HLA-matched, EBV-specific, cytotoxic T cell infusions

19. Cutaneous Lymphomas Associated with EBV-infected T cellsNon-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

20. Cutaneous Lymphomas Associated with EBV-infected B cellsImmunosuppressed 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

21. 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 (osteomyelitis- tumor at site of bone, chronic venous ulcers- tumor at site of ulcer) (J Pathol. 1997 )

22. 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

23. CY (100 g/ml) Prednisone (10 m) Prednisone (1 m) CY (10 g/ml) MPA (100 g/ml) MPA (10 g/ml) MTX (50 g/ml) AZA (10 g/ml) AZA (1 g/ml) CsA (10 g/ml) CsA (1 g/ml) MTX (5 g/ml) _ DRUG: BMRF1 -actin Methotrexate, but not other Immunosuppressants, Induces EBV Lytic Replication Feng et al JNCI 2004

24. 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.

25. Risk of PTLD in Pediatric Liver Transplant Recipients for Primary Tacrolimus Therapy Cacciarelli et at Pediatric Transplantation 2001

26. Kaposi’s 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

27. Lymphoma at Site of ATG or ALG Injections AgeTransplantAT/LGSites of LymphomaRef • kidney horse buttock, nodes 1 • kidney horse buttock, nodes, liver 2 32 heart rabbit thigh, brain, lung, nodes 3 • heart 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

28. 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

29. 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; excised-reticulum cell sarcoma with lymph node involvement (no EBV studies); died of OI

30. 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

31. 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