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

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

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

Jaffe E, et al. IARC Press, World Health Organization, 2001.

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

Normal Immune System

• Innate

• Acquired

B cells – antibody production

T cells - detection of foreign peptide in setting of MHC

How do the lymphocytes that can recognize (and attack) the infectious/foreign material

become active?

Innate Immunity

• 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

CpGfmetLPSTeichoic acidmannan

MHC – window to the inside of the cell

Natural Killer CellsVirus

Virus

MHC

Acquired Immunity requires diversity of B and T cells to allow interaction with an

estimated 1014 epitopes

Diversity: B cells• The body makes new lymphocytes by jumbling the

recognition genes so that no 2 new lymphocytes that are made are identical:

Each lymphocyte recognizes something different

Gene rearrangements & mutations result in billions of new lymphocytes that

recognize unique objects

Diversity: T Cells (a spectrum of T cells, each with

different receptors that can bind to different peptides)

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

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

thymus

Bone marrow

Can’t react with self peptide & canreact with foreignpeptide

RIP

Immune Reaction

Germinal Center

Acquired Immune System: How to generate a specific immune response

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

skin

antigen

Lymph node

T-cells that can react withantigen on cells survive;

others die

lymphatic

T-cells from thymus

RIP

Keeping the good T cells around:

• 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

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

• 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”

Cells Involved in the GC Reaction

a. Green – Ki67Red - IgM

b. Green – FDC M1Red – PNA (B cells)

c. Green – FDC M1Red - IgM

Kosco-Vilbois. Nature Immunol3:764.2003

• 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 (); 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

Aberrant SHM

• Targets 5’ regions of BCL-6, FAS/CD95, C-MYC, PAX5, RhoH/TTF

• ? others

• Isotype switching – class switch recombination (CSR) – replacement

of with or 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

Immune cells that canbind to foreign materialtravel to lymph nodes

If there is anything to bindin the lymph node an editing processstarts; changing a few letters at a time to enhance binding

No/poor binding Lymphocyte death

High level bindingSURVIVAL(e.g after influenza vaccine only a lymphocyte that binds to flu lives) Editor

Follicular Dendritic Cells• FDC processes form strings of immune complex

coated bodies (iccosomes)• Trap and retain unprocessed ag

– FcRIIb (CD32)– FcRII (CD23)

• Immune complexed ag + DC + c’- >> stimulation of B cells than soluble ag

• Synapse + (GM1, BCR, phosphotyrosine containing proteins, PLC2, 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

Patient Question: How Did My Lymphoma Develop?

Mutations:the foundation of oncogenesis

Spontaneous mutation rate:1/1,000,000,000nucleotide/cell division

6 mistakes/cell division

>10,000,000,000 bone marrow and lymphocyte cell divisions/day

Nucleus DNA- 6,000,000,000 nucleotides (letters)

6 mutations

6 mutations6 mutations

6 mutations

6 mutations

“It was the best of dines,…”

“It was the best of diners,…”

“It was the best of dining,…”

+lymphoma

Normal rearrangement

Editor

Patient Question: What Did I Do Wrong?

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

•Mutations are random and most don’t significantly impact on the behavior of the cell;

What prevents the early uniform development of cancer ?

•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 criticalprogrammed cell death.

D. Hanahan & R.A. Weinberg. Cell (2000)

100:57-70

The The Hallmarks of Hallmarks of CancerCancer

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

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

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)

• 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

Transformation Events

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, IkB mutations, genomic

amplification of Rel, API2-MALT1

• Infections– EBV, HHV-8

WHO SHM Ongoin

g SHMGCB

Mol Path Origin

SLL +/- - -

MCL - - - t(11;14) Pre GCB

BL + - + t(8;14); t(2;8), t(8;22)

GCB

DLCL GCB

+ + + bcl-2, bcl-6 GCB

DLCL ABC

+ - -

MZL + + - t(11;18); t(1;14); t(14;18)

Post GCB

Kwong Br J Haem 137:273.2007

Microenvironment• In vitro dependence upon stroma and

cytokines• Role of cellular microenvironment in cHL

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- 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)