Hodgkins lymphoma pathogenesis and targets for therapy

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. H istory 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 2 nd -3 rd decade 5 th -6 th 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

33. NF-KB

34. 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,

35. 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

36. 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

37. Resistance to Apoptosis

38. 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

39. Apoptosis mediated by cell surface receptors: apoptosis

40. 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

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

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

43. Tumor suppressor genes RASSF1A Tumour suppressor gene Inactivated epigenetically in classical HL

44. Anti-apoptotic mechanisms in HL

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

46. A quick detour

47. EBV

48. 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

49. EBV

50. 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.

51. EBV

52. 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

53. Regulation of the cell cycle by Rb protein

54. The detour ends…

55. Hodgkins and EBV

56. 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

57. 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

58. 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

59. 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

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

61. 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?

62. Targeting the Lymphoma

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

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

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

66. Targeted strategies for modulation of NF-KB

67. 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

68. 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

69. 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

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

71. 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

72. 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

73. The end