1. Epstein-Barr Virus: Cancer and Immunosuppression

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
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 apoptosis Upregulates 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. Lane 1: EBV- B cell Lane 2: EBV+ B cell Lane 3: EBV- LPD
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
Type EBER EBNA-1 EBNA-2 EBNA-3 LMP-1 LMP-2 Disease 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. EBV+: 60-70% of cases in developing countries
Hodgkin Disease
EBV+: % 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. with Lymphoproliferative Disease
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 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

20. 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

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 )

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. Methotrexate, but not other Immunosuppressants,
Methotrexate, but not other Immunosuppressants, Induces EBV Lytic Replication
BMRF1
CY (100 g/ml)
Prednisone (10 m) _
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)
MPA (100 g/ml)
MTX (50 g/ml)
DRUG:
-actin
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
Age Transplant AT/LG Sites of Lymphoma Ref
kidney horse buttock, nodes
kidney horse buttock, nodes, liver
heart rabbit thigh, brain, lung, nodes 3
heart rabbit thigh, chest wall,
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, 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