Burkitt lymphoma in Africa: 50 years on

1. Burkitt lymphoma in Africa: 50 years on Ian Magrath www.inctr.org

2. Denis Parsons Burkitt Born 1911, Enniskillen, Ireland; Died 1993 Lost his right eye at the age of 11 in an accident, Trained as a surgeon in Trinity College, Dublin Went to East Africa (Kenya, Somaliland, then Uganda) in World War II. Joined the colonial service in Uganda in 1946 as GP in Lira, then became a surgeon in Mulago Hospital, Kampala. Saw his first case of jaw tumor in a boy of 5 in the children’s ward in 1957, then a second a few weeks later. Surgery not possible: both children died soon after.

3. Recognition of Burkitt Lymphoma 1934-57 Descriptions of Jaw Tumors and High Frequency of Lymphomas in African Children 1910 Albert Cooke Describes Jaw Tumor in Mengo Hospital 1958 Denis Burkitt Describes a Clinical Syndrome O’Connor 1960-61 Lymphoma Burkitt 1962: Climatic distribution

4. First Definitive Papers D. P. Burkitt:A sarcoma involving the jaws in African children.British Journal of Surgery, 1958, 46: 218-223. Clinical description of 38 cases seen at Mulago Hospital over 7 years. 15 also tumor outside jaw O’Connor GT, Davies JNP. Malignant tumors in African children with special reference to malignant lymphoma. Tropical Pediatrics 1960;56:526-35. Experience of the Kampala Cancer Registry during its 7 years of existence. 57 of 125 tumors in children 0-14 years were “poorly differentiated lymphocytic lymphoma” or “lymphosarcoma.” 29 jaw; 20 abd; 2 neck node

5. A Tumor Paradigm • BL not amenable to surgery; radiation therapy was not available, but chemotherapy highly successful • encouraged pioneer chemotherapists and eventually led to 90% cure rates in pediatric BL • First human tumor shown to be associated with a virus – inspired search for others; prevention by vaccine (HBV, HPV): • EBV subsequently shown to be the cause of Infectious mononucleosis and associated with NPC, HD, T cell lymphomas and tumors in immunodeficient states • Chromosomal translocation identified; shown to juxtapose Ig sequences to MYC • leading to understanding molecular pathogenesis and providing a model for other lymphomas and leukemias

6. Key Discoveries in 1960s: Treatment • Burkitt in Uganda and Clifford in Nigeria with drugs sent from US and UK showed dramatic responses with some long term survivors with 1 or 2 doses of several drugs • NCI, Bethesda, supported early clinical trials eventually leading to COM that was used in the NCI to good effect and provided a foundation which led to today’s success (90% cure rate in childhood B cell tumors) • Still being used in Africa with survival in 60% of patients (recent INCTR study)

7. Key Discoveries in 1960s: Epstein-Barr Virus 1964 Epstein, Achong and Barr Discover EBV by EM in a BL Cell Line EB1 1966 Henle’s discover VCA in 2-5% of Cells in BL Lines and Show Ubiquity of Infection X 1967-68 Henle’s, Diehl and Pope show EBV transformation of B cells

8. Key Discoveries in 1970s • EBV infection can be latent (no virus production) • All cells in cell lines and tumors, even if not producing virus, contain EBV DNA (Zur Hausen, 1970) and EBV nuclear antigens (EBNA – Reedman and Klein 1973) • Some BLs lack EBV DNA • Nonoyma et al. 1975 show that American BL is usually EBV-negative • BL contains a specific chromosomal translocation • Zech et al. 1976 show (8:14) in BL biopsies and cell lines

9. EBV Latent and Lytic Cycles Zebra protein 6 Nuclear Proteins EBNA-1 EBNA-2 EBNA-3a,b,c EBNA-LP 3 Membrane Proteins LMP-1 LMP-2A LMP-2B Rp 2 EBERs; >20 mRNAs Latent Infection Lytic Infection

10. The 1970s Paradigm • EBV is able to induce proliferation in B cells via latent genes and probably drives proliferation of Burkitt lymphoma cells • Pathogenesis of EBV negative tumors unknown • Significance of 8;14 translocations unknown

11. Key Discoveries in 1980s • Genes participating in t8;14 translocation (Ig and MYC) identified: Dalla-Favera, Croce, Taub, Leder, Marcu, Bernard, Adams and others, 1982 • Variant translocations (2;8 and 8;22) identified • Bernheim et al. kappa or lambda Ig translated to MYC • Complete sequence of EBV genome (B95-8) published 172,282 bp • Baer et al. 1984 • Predisposition to NHL in HIV positive men shown • Ziegler et al. 1984 (90 homosexual men in San Francisco) • Differential expression of EBV latent antigens in BL cell lines (EBNA-1 only) and EBV-transformed B cells demonstrated • Rowe et al. 1987

12. Ig Translocations in BL 8;14 Myc P E IgH Telomere Der Chromosome 14 Variant (2;8, 8;22) IgL E Myc P Telomere Der Chromosome 8

13. EBV Latency Patterns • I – EBNA1 only (+microRNAs, EBERs) • Seen in occasional replicating memory cells, and Burkitt lymphoma • Cells not seen by CD8+ cytotoxic T cells • II – EBNA1, LMP1, LMP2a and LMP2b • Seen in germinal center cells, Hodgkin lymphoma and NPC • III – All 6 EBNAs, LMP1, 2a and 2b • Occurs in EBV transformed cells in vitro • Lymphomas in immunosuppressed hosts • Highly immunogenic to CD8+ T cells

14. Paradigm Shift • The EBV latent proteins that drive proliferation of EBV transformed B cells, particularly EBNA 2, which induces MYC expression and LMP1 and 2 are not expressed in BL and cannot drive proliferation • MYC is deregulated in BL cells, by juxtaposition to Ig sequences – whether heavy (chr 14) or light chain (chr 2 or 22) and is probably the main driver of proliferation • Role of EBV uncertain

15. Epidemiology and Pathogenesis

16. Malaria Association with BL • 1964: Dalldorf suggests climatic distribution could relate to holoendemic malaria • Geographic distribution of malaria and BL very similar (NB. Zanzibar – formerly free of both) • BL has higher incidence in regions of intense malarial infection (some exceptions) • De Thé: chloroquine prophylaxis in Mara Masai region: decrease (?) in incidence of BL • Sickle cell trait protects against malaria trend to protection against BL, but insufficient data

17. Malaria (and HIV) – Possible Pathogenetic Mechanisms • High parasitemia rates may cause earlier EBV infection (through increasing EBV production) • Influences immune responses to EBV • More EBV infected cells and greater chance of Myc translocation occurring in an EBV+ cell • May increase risk of Ig-Myc translocation either directly, or stochastically via B cell hyperplasia • Other B cell tumors (e.g., pre-B ALL and DLCL) decreased • Increases RAG expressing cells in periphery (mice) • HIV increases risk of BL 200-1000 fold: prior to major immunosuppression (but probably requires B cell hyperplasia) • Only 30-40% of HIV+ BL in the USA is EBV+

18. EBV Association with BL Caveat: may vary within countries – region, SE status, age

19. Does EBV Have a Causal Role? • BL in Africa almost invariably EBV associated: yet most B cells are not infected with EBV • There are a 1-50 per million EBV infected cells in the circulation; “chance” association in BL highly unlikely • Mechanism of EBV persistence involves protecting EBV-infected cells from apoptosis during passage through the germinal center to become memory cells • EBV may also prevent apoptosis in cells with a Myc translocation, although additional lesions must arise to prevent apoptosis once EBNAs (except EBNA1) are switched off • Germinal center passage may increase likelihood of genetic lesions (SHM, DNA recombination could predispose to mutations and Ig translocations)

20. Germinal Center Passage • Activation induced cytidine deaminase (AID), important to SHV mutations and class switching may have a role in translocations and mutations • EBV + cells appear to need to passage through the germinal center to become memory B cells – in which EBV persists throughout life. EBV protects against apoptosis in the absence of antigen • Bcl6 protects against mutations in germinal center cells as part of the process of affinity maturation of B cells (high affinity Ab via SHV mutations) Passage through the germinal center could both increase the risk of a translocation and protect against apoptosis that it would normally induce

21. MYC Growth (anaerobic) and immortality H-Ferritin Telomerase Transferrin receptor p27 LDH-A Cdk4 Myc Cyclin D1 Bim C/EBP Rb p21 ARF Apoptosis E2F p53 Inhibition of Differentiation Cell Cycle Progression

22. Evidence for Importance of Deregulated Myc • Myc not expressed in normal germinal follicle cells • Multiple types of translocation (8;14, 2;8 and 8;22 as well as (rarely) non-Ig translocations are associated with major structural changes or mutations (regulatory region or coding region) in Myc • Myc translocations also present in B cell neoplasms in mice and rats

23. Evidence for Importance of Deregulated Myc • Myc under the regulation of various Ig enhancers induces B cell tumors in transgenic mice (although no SHM) • Inhibition of Myc via anti-Ig Ab or antisense to Myc inhibit growth of BL cell lines • Inhibition of Eμ, in BL cell lines where Eμ juxtaposed to Myc, by peptide nucleic acids inhibits cell growth in vitro or in skid mice

24. Chromosome 8 Breakpoints Outside HindIII’ P1 P2 A Exon 1 Exon 2 Exon 3 H H P1 Imm 5’ P2 B Exon 1 Exon 2 Exon 3 H Exon 1/Intron “P3” C Exon 2 Exon 3 H

25. Translocations and Mutations Deregulation may occur at various levels, including transcription and protein interactions

26. Breakpoints in IgH Region

27. Inferences from Breakpoint Data • Mechanisms of Myc deregulation differ • The translocations may occur in B cells at different stages of differentiation – immature or GC • Environmental factors such as malaria, HIV, EBV or a combination may influence mechanism of translocation

28. Treatment

29. Chemotherapy in Africa • Early Studies (1960s - Burkitt, Clifford, Ngu, NCI, Sloane Kettering, Royal Marsden) • Demonstration of activity (CR) of various agents used alone (especially CTX, MTX, VCR, Ara-C) • Identified many key characteristics: • prolonged CR to even single doses of CTX and MTX • non-cross resistance of VCR/MTX with CTX • tumor burden correlates with prognosis • high risk of uricosemia • high risk of CNS disease • relapse period within a year of last therapy • salvage or patients who relapsed after minimal therapy especially with other drugs

30. Treatment Results Complete Remission

31. Prognosis: Tumor Burden Serum LDH and EA titre also correlate with survival

32. Survival From Last Relapse (Early 1970s, All Patients)

33. Multi-Center Study of the Treatment and Characterization of Burkitt Lymphoma in Africa A collaborative protocol of INCTR’s African Burkitt Lymphoma Strategy Group

34. Participating Institutions • Nigeria • University College Hospital, Ibadan • Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC), Ile-Ife • Kenya • Kenyatta National Hospital, Nairobi • Nyanza General Hospital, Kisumu (soon to participate) • Aga Khan University Hospital, Nairobi (probable) • Tanzania • Ocean Road Cancer Institute, Dar es Salaam • Uganda • Lacor Hospital, Gulu (soon to participate)

35. Patient Characteristics: 228 Patients

36. Response to First-Line Treatment

37. Need to Improve Access to Care for More Children 3.5 weeks Total cost of chemo ~ $200 INCTR African BL Treatment Project

38. Second Line Treatment - Outcome • Survival ranges to < 1 month to 28 months • 17 of 35 patients remain alive • Estimated survival is: • 40% at 6 months • 32% at 12 months

39. Survival in African BL; INCTR 03-06 192 patients entered on-study to July 2007

40. Conclusions • Central feature of BL is deregulated Myc, but protection against apoptosis required: EBV genes, lesions in proapoptotic genes, Myc mutations, Bcl6 • The high incidence of BL in equatorial Africa is probably a consequence of “cooperation” between Malaria and EBV – increase in fraction of EBV+ cells • HIV increases risk of BL – effect on CD 4+ T cells? This predisposition is not totally dependent on EBV (30% EBV+); mechanism not the same as malaria • In Africa, the combination of HIV, malaria and EBV, all of which predispose to BL could influence the pattern of NHLs occurring there • There are opportunities to cure patients with BL (HIV+/-with simple therapy while learning more about pathogenesis

41. Acknowledgements • UHC, Ibadan • Dr Yetunde Aken’Ova • Dr Biobele Brown • Mrs Tosin Olajedo • ORCI, Dar es Salaam • Dr Twalib Ngoma • Dr Jane Kaijage • Mr Seif Mkamba • KNH, Nairobi • Dr Jessie Githang’a • Mr Oliver Oruko • OAUTHC, Ile-Ife • Dr Muheez Durosinmi • Dr OO Adeodu • Dr L Salawu • Mr Lukman Bashir • NCI, Bethesda, MD USA • Dr David Venzon INCTR: Ian Magrath, Ama Rohatiner, Melissa Adde