Overview of Hodgkin Lymphoma pathogenesis

1. Hodgkin’s lymphoma:
Pathogensis and targets for
treatment
JG Nel
August 2014

2. Hodgkin’s Lymphoma
• Prior to the middle of the last
century HL was fatal for the
majority of cases.
• Introduction of radiotherapy
and development of very
effective chemotherapy
regimens led to great progress in
the management of the
condition.
• The success of these therapeutic
interventions are not without a
price tag
• the incidence of late toxicities
related to HL treatment is
directly associated with an
increasing cure rate of mostly
young patients.

3. History
Marcello Malphigi
• Macello Malphigi published
the first recorded description
of HD in De viscerum
structuru exercitatio
anatomica in the year 1666

4. History
Thomas Hodgkin
• 1832 publishes his paper on
lymphatic diseases: “On Some
Morbid Appearances of the
Absorbent Glands and Spleen”

5. History
Reed-Sternberg cells
• 1898 Carl Sternberg first
detailed description of giant
cells

6. History
Dorothy Reed
• 1902 Dorothy Reed
independently describes Reed-
Sternberg cells whilst working
in the Johns Hopkins Hospital

7. History
Ann Arbour, Michigan state
• 1971 Ann Arbor meeting:
Staging broken down into
▫ CS : xray, lymphangiogram
and Physical Examination
▫ PS: staging laparotomy,
splenectomy, liver biopsy
open marrow biopsy and
additional node biopsy

8. History
• 1994 Kuppers et al. definitive proof that Reed-
Sternberg and Hodgkin cells in Hodgkin’s
disease are clonally related B-cell-derived
malignant cells
• 2005 Mathas et al. determine the mechanism
that causes normal B lymphocytes to mutate into
cancer cells
• 2005 Robertson, Knight and Sharma determine
the link between EBV and several cancers via
molecular elimination of the Rb protein

9. Electron microscope picture of a RS
cell

10. Outline of this lecture
• Hodgkin’s in a nutshell
▫ Clinical presentation
▫ Haematological and biochemical features
▫ Diagnosis
▫ Histopathological classification
• Pathogenesis
▫ Biology of Hodgkin and Reed-Sternberg cells
▫ Cytokines and receptors in cHL
▫ Constitutive NF-KB activation
▫ Resistance to apoptosis
▫ EBV and Hodgkin Lymphoma
▫ HIV and HL
• Targeting the Lymphoma

11. In a nutshell

12. Hodgkin’s in a nutshell
▫ Clinical presentation
▫ Haematological and biochemical features
▫ Diagnosis
▫ Histopathological classification

13. Hodkgkin’s in a nutshell
• Bimodal age distribution
▫ 2nd-3rd decade
▫ 5th-6th decade
• Overall incidence stable, ~30% of lymphomas
• One of the more frequent malignancies in young
people

14. Hodkgkin’s in a nutshell
• Characteristics of Hodgkin lymphomas
▫ Usually arise in lymphnodes
▫ Neoplastic tissues contain a small number of
scattered HRS cells, abundant admixture of non-
neoplastic inflammatory and accessory cells
▫ Tumour cells are often ringed by T-lymphocytes in
a rosette like manner

15. Hodkgkin’s in a nutshell
• Clinical presentation
▫ enlarged but asymptomatic lumps of lower neck or
supraclavicular region(60-70%)
▫ Splenomegaly(50%)
▫ Mediastinal involvement(10%)

16. Clinical presentation
Cervical lump in HL Mediastinal involvement

17. Hodkgkin’s in a nutshell
• Clinical presentation, continued
▫ Cutaneous HD
▫ Constitutional symptoms
 Prominent in Pt with widespread disease
 Fever –continous or cyclic
 Pruritus
 Alcohol induced pain in areas where disease is present
 “B” symptoms

18. Hodkgkin’s in a nutshell
• Haematological and biochemical findings
▫ Normochromic, normocytic anaemia
▫ 1/3 patients Neutrophillia, Eosinophillia frequent
▫ ESR and CRP raised
▫ LDH raised initially
▫ Advanced disease:
 Lymphopaenia and loss of cell mediated immunity
 Platelet count reduced

19. Hodgkin’s Lymphoma
-getting to the diagnosis
Cells seen in HL
• Histological exmination of an
excised lymphnode
▫ Finding of HRS cells in an
appropriate cellular
background
 Reactive T-cells
 Eosinophils
 Varying degrees of fibrosis

20. Histological classification Hodgkin
Lymphomas (WHO)
• Two disease entities
▫ Classical
▫ Nodular lymphocyte predominant
• Entities differ
▫ Clinical features
▫ Behaviour
▫ Cellular background
▫ Morphology, immunophenotype,
preservation/extinction of B cell gene expression
program

21. Histological classification of Hodgkin
Lymphomas (WHO)
• Classical HL
▫ Nodular sclerosis
▫ Mixed cellularity
▫ Lymphocyte rich
▫ Lymphocyte depleted
Differ by:
 Sites of involvement
 Clinical features
 Growth pattern
 Fibrosis
 Atypia of tumour cells
 Presence of EBV
Similar immuno phenotype of
cells

22. Histologic classification

23. It in a nutshell

24. Pathogenesis
NOT FULLY UNDERTSOOD AT PRESENT
• Biology of Hodgkin and Reed-Sternberg cells
• Cytokines and receptors in cHL
• Constitutive NF-KB activation
• Resistance to apoptosis
• EBV and Hodgkin Lymphoma

25. Biology of HRS cells
• Not found in normal lymphoid tissues
• Rare in HL tissues –less than 1% of cellular
infiltrate

26. normal B-cell differentiation and
relationship to major B-cell neoplasms

27. HRS cells
• Post germinal center B cell
 presence of rearranged heavy and light chains
▫ IgV chain genes
 identical for a particlular tumour –monoclonality
 Heavily somatic mutated
• Lost typical markers such as CD20 and CD79a
• Express CD15 and CD30

28. Biology of HRS cells
• Expression of functional Ig prevented by
crippling mutations in rearranged genes
• Disturbed B-cell transcription factors ~absence
of classic B-cell phenotype
• Transforming events leading to the development
of HL not known yet.

29. Cytokines and receptors in cHL
• 99% of infiltrate made up out of immune cells
• HRS secrete mostly Th2 cytokines and
chemokines -leading to a favourable
environment for HRS cell survival
▫ TARC –rosetting of Th2 cells around HRS
▫ TNF
 fibroblasts-eotaxin
 Recruitment of more Th2

30. Cytokines and receptors in cHL
Cytokines secreted by immune cells:
• Th2 cells
▫ Il13 Stim autocrine rec Upregulation STAT6
• Treg cells
▫ Il10 inhibit cytotoxic T-cell function
▫ TGFB

31. Cytokines and receptors in cHL
Cell surface receptors and
their respective Icel mediators
• Attracted cells influence the
HRS phenotype favourably by
providing survival signals
• Soluble factors secreted by
HRS cells and bystanding
stromal cells induce
reorganization of stromal
micro environment

32. Other cells in cHL
IL13
TGFB
IL10
TNF
eotaxin
VEGF
TARC
CD30/CD40/RANK
JAGGED

34. NF-KB

35. NF-KB
• Protein complex that acts as a transcription
factor
• Key role in the immune response to infection
• Rapid acting primary transcription factor
▫ Present in cell in an inactive state and does not
require new protein synthesis to be activated
• Activation in response to a number of stimuli
▫ TLR, TNF-R,

36. NF-KB
• Inhibition
▫ Unstimulated cells-sequestered in the cytoplasm
by a family of inhibitors, IkB
▫ IkB Mask the nuclear localisation signals of NF-
KB
• Activation of NF-KB initiated by the signal-
induced degradation of IkB, via activation of IkB
kinase
▫ Phosphorylation of serine residues of IkB leads to
ubiquination and subsequent proteasomal
degradation

37. NF-KB in HL
• Constitutive NF-KB leads to inappropriate cell growth
• HRS constitutive NF-KB activation essential for tumour-
cell survival
▫ Via various mech
 JunB overexpression CD30, high levels leads to ligand
independent activation
 CD40
 RANK assoc with TRAF
 Inactivating mutations in IkB genes
• NF-KB target genes in HRS
▫ Chemokines
▫ Cytokines –TNFa,Il-13,Il-6,
▫ Anti-apoptotic molecules –c-FLIP,Bcl-XL,IAP2
▫ Transcription factors

38. Resistance to Apoptosis

39. Apoptosis
• Programmed cell death
• Important regulatory mechanism whereby
unwanted cells are eliminated
• Two distinct signalling pathways
▫ Triggering of death domain cell surface receptors
▫ Release of proapoptotic factors from
mitochondrria

40. Apoptosis mediated by cell surface
receptors:
CD95/FAS DISC Oligomerization
Caspase 8
Autoproteolytic
cleavage and
activation
Effector
caspases
apoptosis

41. Resistance to Apoptosis
• c-FLIP can also be recruited by DISC(death
inducing signaling complex)
• Recruitment of c-FLIP inhibits the
recruitment of caspase 8
• High levels of c-FLIP can inhibit apoptosis
• C-FLIP over expressed in HRS cells

42. Apoptosis mediated by the release of
proapoptotic factors from
mitochondria
• CytochromeC+procaspase9+Apaf-
1=Apoptosome
Activation of caspase9 by dimerization
Effector caspases
Apoptosis

43. Resistance to Apoptosis
• Inhibitors of apoptosis
▫ X-linked inhibitor of apoptosis (XIAP)
▫ Binds effector caspase3, blocks activation
• HRS cells constitutively over express XIAP

44. Tumor suppressor genes
• RASSF1A
▫ Tumour suppressor gene
▫ Inactivated epigenetically in classical HL

45. Anti-apoptotic mechanisms in HL

46. Resistance to Apoptosis
• c-FLIP over expression
• XIAP over expression
• inactivation of tumour suppressor gene
RASSF1A

47. A quick detour

48. EBV

49. EBV
• HHV 4
• One of the most common viruses in humans
• infection usually asymptomatic
• dsDNA virus
• On infecting the B-lymphocyte, the linear viral
genome circularises and the virus subsequently
persists as an epsiome

50. EBV
Lytic cycle
• Results in production of infectious virions
• Virions produced by budding from infected cell
Latent cycle
• Does not result in the production of virions
• Distinct set of viral proteins produced
• nuclear antigen (EBNA-1,2,3A,3B,3C)
• Leader protein
• Latent membrane proteins(LMP-1,2A,2B)
• Viral RNAs
• EBER
• 20 micro RNAs

51. EBV proteins
• EBNA-1
• EBNA-1 protein binds to a replication origin (oriP) within the viral genome
and mediates replication and partitioning of the episome during division of
the host cell. It is the only viral protein expressed during group I latency.
• EBNA-2
• EBNA-2 is the main viral transactivator.
• EBNA-3
• These genes also bind the host RBP-Jκ protein.
• LMP-1
• LMP-1 is a six-span transmembrane protein that is also essential for EBV-
mediated growth transformation.
• LMP-2
• LMP-2A/LMP-2B are transmembrane proteins that act to block tyrosine
kinase signaling.

52. EBV
Transformation
• EBV infection of B-cells in vitro leads to the formation of
lymphoblastoid cells capable of indefinite growth
• The retinoblastoma protein
• a major regulator of the cell cycle
• normally binds to E2F, turning off genes involved with
cell proliferation
• EBNA3C recruits a group of molecules called the SCF
complex, which attaches ubiquitin to Rb
• Rb taged for degradation by the proteosome machinery

53. Retinoblastoma protein
• Pocket proteins
• Sequester E2F transcription proteins
• Release of E2F dependant upon the
phosphorylation state of Rb protein
 E2F dissiociates from Rb –free to transcribe responder
genes
 Cyclin E –required for progression through restriction point

54. Regulation of the cell cycle by Rb
protein

55. The detour ends…

56. Hodgkins and EBV
Presence of EBV in HRS cells varies according to the
histological subtype and epidemiological factors
• Highest in Mixed cellularity cHL (75%)
• Lowest NSCHL
• EBV infection more prevalent in
• resource poor settings
• HIV infection
Recognition of the association between IM and HL
predates the discovery of EBV

57. Infectious Mononucleosis and HL
Lymphocytes from a pt with
IM
• Hx of IM linked to HL (1950s)
• Assoc between IM and HL
▫ Strongest in young adults
▫ Virus in tumour cells less
frequently detected in
tumours in young adults
• ?relationship
▫ Primary inf per se
▫ SYMPTOMATIC inf and HL

58. EBV and HL: The role of the virus
• Gene expressed in HL
▫ LMP2A
 Membrane protein
 Carries an ITAM
 When expressed at cell membrane tonic signal
that prevents apoptosis

59. HIV and HL
• Nearly uniformly associated with EBV
• Present at an advanced stage with B sx
• Most often mixed cellularity disease
• HL usually contiguous spread, HIV skip lesions

60. HIV and HL
• HIV specifically predisposed to
EBV+ tumours
• The risk for HL is lower with
lower CD4 counts
▫ ?inability of tumour cells to
recruit lymphocytes
required for tumour
survival in a
lymphodepleted host

61. Pathogenisis:what we know about HRS
cells
• Constitutive NF-KB activity
• Resistant to Apoptosis
• Sometimes carry EBV

62. Pathogenesis –the unanswered
questions
• ?Relationship between HRS cells and their
environment,? are the immune cells just
innocent bystanders
• Why a relationship with IM, but EBV not found
in HRS
• Initiating event?

64. Targeting the Lymphoma

65. Biologically based strategies for HL
• Receptor specific antibodies
• Protein specific small molecules
• Gene specific antisense oligonucleotides
• Antigen specific adoptive T-cell transfer

66. Biologically based strategies for HL
Receptor specific antibodies
• ADCC
▫ CD30 antibody
▫ CD20 antibody
▫ IL13 antibody
▫ CD40 antibody
▫ RANKL antibody
• Proapoptotic
▫ TRAIL-R1 antibody

67. Biologically based strategies for HL
• Intracellular level, novel compounds that target
the IKK-IkBa-NFkB cascade
▫ IKK inhibitors
▫ Proteasome inhibitors
▫ Direct NF-KB inhibitors

68. Targeted strategies for modulation of
NF-KB

69. Biologically based strategies for HL
• IKK inhibitors
▫ Preclinical phase of testing
▫ Median inhibitory [] in the nanomolar range
▫ Efficacy of IKK inhibitors in HL counteracted by
mutations rendering IkBa nonfunctional in 1/3
cases

70. Biologically based strategies for HL
• Proteasome inhibitors : Bortezomib
▫ Reversible inhibitor of 26S proteasome
 Interferes with the degradation of a number of
proteins including IkBa
 Has a strong apoptosis inducing activity even in
IKBA-mutated HRS cell lines
 IKK-independent effects of Bortezomib
▫ Increase P21 and Bax levels
▫ Downregulation of bcl2

71. Biologically based strategies for HL
• Direct NF-kB inhibition
▫ Expression of NF-kB targets under the influence
of coavtivators and corepressors
 Depsipeptide
 Inhibit HDAC enzymes interfere with NF-kB
transactivating potential mediate apoptosis by p21 up
regulation, inh c-FLIP, generation of reactive oxygen
species
 Favour the assembly of NF-kB with it’s suppresor IkB
and the nuclear export of NF-kB to the cytoplasm

72. Biologically based strategies for HL
• Under investigation
▫ XIAP inhibitors
▫ STAT3 inhibition
▫ TRAIL-R1 antibodies
▫ Bcl2 inhibition

73. Biologically based strategies for HL
• Immunological approaches
▫ cellular strategies against EBV encoded proteins
 In vitro generated LMP2a-specific autologous CD8
cytotoxic T-cells
 Limited by
 EBV positive indiv
 Each batch of autologous cells for each indicidual
needs to be tested before it is used
 Engineering sufficient cells of sufficient quality takes
months

74. Sources
• Daniel Re,Roman K Thomas,Behringer et al. From Hodgkin diseasse to Hodgkin lymphoma:biologic
insights and therapeutic potential.Blood.2005;105:4553-4560
• Richard F.Ambinder.Epstein-Barr Virus and Hodgkin Lymphoma.ASH 2007
• Felderbaum R. The Molecular Mechanisms of Classic Hodgkin’s Lymphoma.Yale Journal of Biology and
Medicine;78(2005)pp201-207
• NF-KB, EBV Wikipedia
• Knight, Sharma, Robertson.Epstein-Barr virus latent antigen 3C can mediate the degradation of the
retinoblastoma protein through an SCF cellular ubiquitin ligase.PNAS December 20,2005 vol 102 no 51
p18562-18566
• Ralf Kuppers, Martin-Leo Hansen. The Hodgkin and Reed-Sternberg cell .The International Journal of
Biochemistry and Cell Biology;37(2005) 511-517
• Brauninger, Schmitz, Bechtel et al. Molecular biology of Reed/Sternberg cells in Hodgkin’s lymphoma.
Int.J.Cancer:118,1853-1861(2006)
• Janz, Mathas.The pathogenesis of classical Hodgkin’s lymphoma:what we can learn from analyses of
genomic alterations in Hodgkin and Reed-Sternberg cells? Haematologica;2008;93(9)
• Raemaekers,vander Maazen.Hodgkin’s lymphoma news from an old disease.Netherlands journal of
medicine. December 2008, vol 66 , No11
• Essentail Haematology
• Postgraduate Haematology
• WHO classification of Tumours of the haematopoietic and lymphoid tissues 2008

75. The end