Burkitt lymphoma in Africa: 50 years on

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Burkitt lymphoma in Africa 50 years on
Ian Magrath
www.inctr.org
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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 childrens ward in 1957, then a
second a few weeks later. Surgery not possible
both children died soon after.
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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
OConnor 1960-61 Lymphoma
Burkitt 1962 Climatic distribution
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First Definitive Papers
D. P. BurkittA 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
OConnor GT, Davies JNP. Malignant tumors in
African children with special reference to
malignant lymphoma. Tropical Pediatrics
196056526-35.
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
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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

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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 todays success (90 cure rate in
  childhood B cell tumors)
• Still being used in Africa with survival in 60
  of patients (recent INCTR study)

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Key Discoveries in 1960s Epstein-Barr Virus
1964 Epstein, Achong and Barr Discover EBV by
EM in a BL Cell Line EB1
1966 Henles discover VCA in 2-5 of Cells in BL
Lines and Show Ubiquity of Infection
X
1967-68 Henles, Diehl and Pope show EBV
transformation of B cells
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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 (814) in BL biopsies and
  cell lines

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EBV Latent and Lytic Cycles
Zebra protein
6 Nuclear Proteins EBNA-1 EBNA-2 EBNA-3a,b,c EBN
A-LP
3 Membrane Proteins LMP-1 LMP-2A LMP-2B
Rp
2 EBERs gt20 mRNAs
Latent Infection
Lytic Infection
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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 814 translocations unknown

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Key Discoveries in 1980s

• Genes participating in t814 translocation (Ig
  and MYC) identified Dalla-Favera, Croce, Taub,
  Leder, Marcu, Bernard, Adams and others, 1982
• Variant translocations (28 and 822) 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

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Ig Translocations in BL
814
IgH
E
Myc
P
Telomere
Der Chromosome 14
Variant (28, 822)
Myc
IgL
E
P
Der Chromosome 8
Telomere
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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

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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

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Epidemiology and Pathogenesis
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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

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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

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EBV Association with BL
Caveat may vary within countries region, SE
status, age
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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)

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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
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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
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Evidence for Importance of Deregulated Myc

• Myc not expressed in normal germinal follicle
  cells
• Multiple types of translocation (814, 28 and
  822 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

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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

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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
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Translocations and Mutations
Deregulation may occur at various levels,
including transcription and protein interactions
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Breakpoints in IgH Region
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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

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Treatment
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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

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Treatment Results
Complete Remission
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Prognosis Tumor Burden
Serum LDH and EA titre also correlate with
survival
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Survival From Last Relapse (Early 1970s, All
Patients)
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Multi-Center Study of the Treatment and
Characterization of Burkitt Lymphoma in Africa

• A collaborative protocol of INCTRs African
  Burkitt Lymphoma Strategy Group

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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)

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Patient Characteristics 228 Patients
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Response to First-Line Treatment
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Need to Improve Access to Care for More Children
3.5 weeks
Total cost of chemo 200
INCTR African BL Treatment Project
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Second Line Treatment - Outcome

• Survival ranges to lt 1 month to 28 months
• 17 of 35 patients remain alive
• Estimated survival is
• 40 at 6 months
• 32 at 12 months

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

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Acknowledgements

• KNH, Nairobi
• Dr Jessie Githanga
• 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

• UHC, Ibadan
• Dr Yetunde AkenOva
• Dr Biobele Brown
• Mrs Tosin Olajedo
• ORCI, Dar es Salaam
• Dr Twalib Ngoma
• Dr Jane Kaijage
• Mr Seif Mkamba

INCTR Ian Magrath, Ama Rohatiner, Melissa Adde