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Pathophysiology of Hematologic Malignancies

Pathophysiology of Hematologic Malignancies. Mehtap Kaçar Koçak M.D., PhD Yeditepe University, Faculty of Medicine. WHO Classification of Hematopoietic and Lymphoid Tumors: B-cell Neoplasms. Aggressive Prolymphocytic leukemia Plasmacytoma/ multiple myeloma Mantle cell

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Pathophysiology of Hematologic Malignancies

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  1. Pathophysiology of Hematologic Malignancies Mehtap Kaçar Koçak M.D., PhD Yeditepe University, Faculty of Medicine

  2. WHO Classification of Hematopoietic and Lymphoid Tumors: B-cell Neoplasms Aggressive • Prolymphocytic leukemia • Plasmacytoma/multiple myeloma • Mantle cell • Follicle center lymphoma, follicular, grade III • Diffuse large B-cell lymphoma (DLBCL) • Primary mediastinal large B-cell lymphoma Very Aggressive • Precursor B-lymphoblastic lymphoma/leukemia • Burkitt lymphoma/ B-cell acute leukemia • Plasma cell leukemia Indolent • Chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma • Lymphoplasmacytic/Waldenstrom’s macroglobulinemia (WM) • Hairy cell leukemia • Marginal zone lymphoma • Extranodal mucosa-associated lymphoid tissue (MALT) • Nodal • Splenic • Follicle center lymphoma, follicular, grade I-II Jaffe E, et al. IARC Press, World Health Organization, 2001.

  3. Pathophysiology of Hematologic Malignancies: Relationship to Normal Hematopoiesis Basic mechanisms of oncogenesis + extraordinary features of hematologic cells: • Differentiation • Diverse and dynamic mechanisms of mobilization, localization, activation, targeting • Biology of immune system/response • Stromal interactions • Interface with infectious diseases

  4. Normal Immune System • Innate • Acquired B cells – antibody production T cells - detection of foreign peptide in setting of MHC

  5. How do the lymphocytes that can recognize (and attack) the infectious/foreign material become active?

  6. Innate Immunity CpG fmet LPS Teichoic acid mannan • Innate immunity: immediately identifies the infectious or “dangerous”agent and attacks it; focuses on crude and general features of the agent that identify it as foreign or “dangerous” Advances in Immunology: New England Journal of MedicineDelves, Roitt, Medzhitov, Janeway

  7. MHC MHC – window to the inside of the cell Virus Natural Killer Cells Virus

  8. Acquired Immunity requires diversity of B and T cells to allow interaction with an estimated 1014 epitopes

  9. Diversity: B cells • The body makes new lymphocytes by jumbling the recognition genes so that no 2 new lymphocytes that are made are identical: Gene rearrangements & mutations result in billions of new lymphocytes that recognize unique objects Each lymphocyte recognizes something different

  10. Diversity: T Cells (a spectrum of T cells, each with different receptors that can bind to different peptides)

  11. How do the T cells get smart enough to distinguish “self” from “foreign”?

  12. Early steps in T-cell selection: Get rid of the bad ones • T-cells leave BM • Undergo TCR gene rearrangement & selection in thymus • T-cells that have high level autoreactivity die • T-cells that are not capable of reacting with foreign peptides + MHC also die Bone marrow thymus RIP Can’t react with self peptide & can react with foreign peptide

  13. Immune Reaction Germinal Center

  14. Acquired Immune System: How to generate a specific immune response

  15. Events Leading to GC Reaction • Once stimulated by foreign organism or cell, resident DC take up ag and migrate to draining LN • If pathogens enter blood – filtered by DC in spleen • DCs directed towards T cell zones of secondary lymphoid tissue by CCR7 & chemokine ligands CCL19, CCL21 • DCs mature during migration – CD80, CD86 allowing presentation in MHC II to CD4+ cells (TCR [signal 1] & CD28 [signal 2]) with further maturation of T cells into Th

  16. Keeping the good T cells around: T-cells from thymus skin RIP Lymph node lymphatic T-cells that can react with antigen on cells survive; others die antigen

  17. Th cells result in activation of ag-specific B cell responses Initial Th-B cell interactions occur in T cell zones adjacent to follicles B cell receptor binding to ag [signal 1] and CD40 ligation [signal 2] required for B cell activation In addition to CD154, T cells also provide ICOS, a CD28-related protein that binds to B7-H2, a B7-like molecule constitutively expressed on B cells

  18. Functional Characteristics of GC B Cells • GC - extensive B cell proliferation and differentiation • Doubling times of 6-10 hours • Non-dividing GC B cells apparent towards the end of the first week • Humans proliferating and nonproliferating B cells separate into distinct zones in the GC • Centroblasts continue to expand in dark zone • Centrocytes falling out of cycle segregate in light zones • Molecular events of selection in GC: isotype switching, somatic hypermutation, affinity selection & apoptosis

  19. Cells Involved in the GC Reaction • GC founder B cells • CD4+ Th • FDC networks support GC-B cell differentiation by displaying immune complexed ag for extended periods of time and providing co-stimulation • Distinct set of macrophages becomes prominent and ingest apoptotic B cells resulting from failed selection – “tingible bodies”

  20. Kosco-Vilbois. Nature Immunol 3:764.2003 • Green – Ki67 • Red - IgM • Green – FDC M1 • Red – PNA (B cells) • Green – FDC M1 • Red - IgM

  21. Somatic hypermutation (SHM) localized to GC • point mutations in variable regions of L and H chains • requires transcription; AID; uracil DNA glycosylase (UNG); error prone DNA polymerases (h,i,z); MSH2 • most active in second and third week post-immunization • mutations first random [low replacement:silent ratio (R:S)] • change over time to selection of high affinity • immune complexes bound to FDC drive selection • low affinity (poor signaling through BCR) & autoreactive (signaling by soluble ag) GC clones eliminated (similar to selection events in BM) • GC B cells express high levels of Fas & Bax and low levels of bcl-2 • 10-5 - 10 -3/base pair/division • base pair substitutions beginning -150-200 and extending 1.5 kb to intronic enhancer, sparing C region • Sequence based “hot spots” – preferential targeting to W(A/T)R(A/G)CY(T/C) • Transitions > transversions

  22. Aberrant SHM • Targets 5’ regions of BCL-6, FAS/CD95, C-MYC, PAX5, RhoH/TTF • ? others

  23. Isotype switching • class switch recombination (CSR) – replacement of m with a, g or e constant regions • cutting, looping/excision and joining process • after challenge with T-dependent (TD) ag isotype switching is found in follicular and extra-follicular locations • 3-4 d after immunization transcripts localized to areas of extrafollicular B cell activation resulting in “switched” short-lived AFC early in response • isotype switching continues in GC

  24. Immune cells that can bind to foreign material travel to lymph nodes If there is anything to bind in the lymph node an editing process starts; changing a few letters at a time to enhance binding No/poor binding Lymphocyte death High level binding SURVIVAL (e.g after influenza vaccine only a lymphocyte that binds to flu lives) Editor

  25. Follicular Dendritic Cells • FDC processes form strings of immune complex coated bodies (iccosomes) • Trap and retain unprocessed ag • FcgRIIb (CD32) • FcRII (CD23) • Immune complexed ag + DC + c’- >> stimulation of B cells than soluble ag • Synapse + (GM1, BCR, phosphotyrosine containing proteins, PLCg2, actin) – (CD45, CD22, SHP1) • Interactions with FDC necessary to avert apoptosis – induction of FLIP; support ex vivo growth of NHL & H-RS cells • CD32 inhibition – prevention of autoimmunity

  26. Patient Question: How Did My Lymphoma Develop?

  27. Mutations:the foundation of oncogenesis Nucleus DNA- 6,000,000,000 nucleotides (letters) Spontaneous mutation rate:1/1,000,000,000 nucleotide/cell division 6 mistakes/cell division >10,000,000,000 bone marrow and lymphocyte cell divisions/day

  28. + lymphoma Editor Normal rearrangement “It was the best of dines,…” 6 mutations 6 mutations “It was the best of diners,…” 6 mutations 6 mutations 6 mutations “It was the best of dining,…”

  29. Patient Question: What Did I Do Wrong?

  30. Nothing: Many Mutations • 100,000,000,000,000,000 MUTATIONS IN EACH OF US FROM CONCEPTION-75 YEARS OLD • ~ 94,608,000,000,000,000 SECONDS in 3 BILLION YEARS What prevents the early uniform development of cancer ? • Mutations are random and most don’t significantly impact on the behavior of the cell; • Cancer occurs as a consequence of a series of very • specific mutations in very specific cells at critical times in • their development; • Protective mechanisms: any cell can cause trouble; • no single cell is criticalprogrammed cell death.

  31. The Hallmarks of Cancer D. Hanahan & R.A. Weinberg. Cell (2000) 100:57-70

  32. B Cell Receptor • Survival of normal B cells • Selection for for expression of BCR • Selection of pre-BCR in BM • Selection of functional non-autoreactive BCR in BM • Affinity selection in GC • Mature resting B cells must express BCR to avert apoptosis • BCR dependency of B-cell lymphomas • BCR expressed on nearly all B cell lymphomas • Translocations into non-productively rearranged allele • Ongoing V region gene mutations during clonal selection

  33. BCR • Rare B cell lymphomas do not express BCR • cHL (40% with EBV – ? LMP2A may replace BCR signaling – but entire complex downregulated); downregulation of transcription factors, inactive chromatin • PTLD • PEL • PMBCL

  34. BCR • Antigen stimulation of BCR in NHL • B-CLL • BCR binds autoantigens • HTLV-1 • subgroups with similar VH and VL gene rearrangement sequences • PCNS lymphoma • 80% carry somatic V region mutations; same VH region in ~50% • FL • ongoing somatic mutation • ~ 80% FL & BL carry somatic mutations that result in generation of carbohydrate linking motifs (<10% of nl B cells)

  35. Transformation Events • Reciprocal translocations involving Ig loci • Bcl2-IgH associated with FL have breakpoints that are directly adjacent to JH or DJH and loss of nucleotides at end of JH or DH - characteristic of VDJ recombination in BM • Breakpoints within rearranged VDJ with somatically mutated V regions - translocation as a byproduct of SHM • Breakpoints in IgH constant region switch point region – byproduct of class switch recombination • Strand breaks in non-Ig genes: aberrant SHM • Bcl2 gene altered DNA structure recognized by RAG nucleases

  36. Transformation Events • SHM • Chromosomal translocations • Targeting of non-Ig genes • Bcl-6 in normal and malignant GC B cells • Inactivating mutations of CD95 • Myc • Non reciprocal genetic alterations • P53 mutations, IkBa mutations, genomic amplification of Rel, API2-MALT1 • Infections • EBV, HHV-8

  37. Kwong Br J Haem 137:273.2007

  38. Microenvironment • In vitro dependence upon stroma and cytokines • Role of cellular microenvironment in cHL

  39. Role of the Microenvironment in cHL • Reactive cellular infiltrate • CD4+ lymphocytes – regulatory phenotype CTLA-4+ (CD4+CD10+ & CD4+CD25+) • Few CD4+Th1 & CD8+ CTL – production of IL-4, IL-13, IL-10, TGF-b by H-RS cells • Eosinophils attracted by IL-5 • Cytokines, chemokines & TNF R family members • Cytokines: favor Treg cell formation; VEGF • Chemokines: TARC (CCL17) & MDC (CCL22) bind to CCR4 on Th2 cells; eotaxin (CCL11); CCL28 – eosinophils & plasma cells • TNF receptor family: CD30 & CD30L expression but CD30 induction of NFkB is CD30L independent; CD40 & RANK (autocrine RANK & RANKL)

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