Lymphoma Pathogenesis

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Lymphoma Pathogenesis

• Jares et al. Br J Haem 142149.2008
• Klein, Dalla-Favera Nat Immunol 822.2008
• Gleissner et al. Br J Haem 142162.2008
• Jung et al. Annual Rev Immunol 24541.2006
• Bende et al. Leukemia 2118.2007
• Kuppers. Nature Reviews Cancer 5251.2005
• Barreto et al. TRENDS in Immunology 2690. 2005
• deJong. J Clin Oncol. 236358.2005
• Li. et al. Genes Dev 18111.2004
• Wolniak KL, Shinall SM, Waldscmidt TJ. Crit Rev
  Immunol 2439-66.2004
• Kosco-Vilbors. Nat Rev Immunol. 3764.2003

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• Tissue infiltration
• Nodal
• Extranodal
• Abnormal cell products
• Monoclonal proteins hyperviscosity,
  neuropathy, autoimmune
• amyloid (nephrosis, malabsorption, neuropathy,
  CHF
• macroglossia)
• Cytokines fevers, chills, fatigue, asthenia,
  POEMS
• Mediators (e.g. histamine) pruritis, diarrhea,
  ulcers
• Coagulation stimuli DIC

www.methodsofhealing.com
www.urticare.com

• Metabolic
• Hypercalcemia
• Hyponatremia
• Real and pseudo - abnormalities (O2, K)

content.nejm.org
www.doctorspiller.com/macroglossia.htm
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Lymph Node Biopsies
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WHO Classification of Hematopoietic and Lymphoid
Tumors B-cell Neoplasms

• Indolent
• Chronic lymphocytic leukemia (CLL)/small
  lymphocytic lymphoma
• Lymphoplasmacytic/Waldenstroms
  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.
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Staging-Prognosis

• Extent of measurable disease and clinical setting
  may correlate with clinical course dictate
  therapy
• Prognostic Indices

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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

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Genetic Alterations

• Chromosomal translocations
• Regulatory
• Fusion transcripts
• Point mutations
• Deletions
• Amplifications
• Gene conversion/UPD

Physiology
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EBV
HTLV-1
H. pylori
HIV
HHV-8
Hepatitis C
borrelia
chlamydia
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Normal Immune System

• Innate
• -Rapid detection of foreign/dangerous material
  preparation for acquired immunity
• Acquired/Adaptive

-Diversity-genetic reorganization, DNA
breakage, random insertion of nucleotides,
mutation/repair, non-homologous recombination
suppression of detection of DNA
breaks -Selection of rare ag-reactive
clones -Rapid expansion of highly selected clones
(receptor signaling survival and
proliferative pathways) Efficiency in generating
diversity high metabolic, cellular, mutational
load
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Innate Immunity
CpG fmet LPS Teichoic acid mannan
Complement

• Innate immunity immediately identifies the
  infectious or dangerousagent 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
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MHC
MHC window to the inside of the cell
Virus
Natural Killer Cells
Virus
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Acquired Immune System How to generate a
specific immune response
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• Acquired Immunity requires diversity of B and T
  cells to allow interaction with an estimated 1014
  epitopes

7 different loci (IgH, k, l b,d and
a,g) Combinatorial recombination Nearly infinite
diversity

• 6 noses
• 6 ears
• 10 eyes
• 10 mouths
• 5 hats
• 18,000 Mr. Potato Heads (immune cells)
• 37 parts (gene)

http//neatorama.cachefly.net/images/2008-01/potat
o-head-presidential-candidates.jpg
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Diversity B cells
DS DNA breaks introduced by RAG at RSS
(recombination signal sequence) followed by
excision of intervening gene sequence and joining
by DNA repair machinery

• Site-specific recombination tightly controlled
• Cell type (developing lymphocytes)
• Order of locus
• Allele
• Recombination activating genes 1 2 (RAG)
• RAG endonuclease
• DNA ds breaks at borders between coding seq and
  flanking recomb signals (RSs)
• RSs heptamer/nonamer separated by 12 or 23 bp
  spacer
• RAG most efficient with one 12 and one 23 bp
  spacer
• ds breaks resolved by non-homologous end joining
  proteins with non-templated nucleotide additions
  by tdT (many non-productive)

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Diversity T Cells (a spectrum of T cells, each
with different receptors that can bind to
different peptide-MHC complexes)
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Immune Reaction

• Germinal Center
• Affinity maturation
• -somatic hypermutation
• -class switch recombination

-Rapid proliferation -High DNA mutation,
breakage -Suppression of DNA damage sensing
repair -Gross DNA rearrangement -High rate of
apoptosis
Foundation of Lymphomagenesis
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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

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• 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

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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

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Centroblasts (Dark zone)

• Generate a large of diverse binding sites
• Proliferation rate 6-12h (gene expression arrays)
• High mutation rate in immunoglobulin gene
  variable regions (10-3/nucleotide)
• Suppression of sensing and response to DNA damage
  (bcl6-p53)

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Centrocyte Differentiation

• Light zone reaction
• Selection of B cells with high affinity receptors
• Class switch recombination
• Initiation of differentiation into plasma cells
  (initiated by high affinity) or memory cells
  (stochastic)
• Testing of affinity via BCR (FDCs) and
  co-stimulatory molecules (T cells)

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• Somatic hypermutation (SHM) localized to GC
• point mutations in variable regions of L and H
  chains
• requires transcription activation-induced
  cytidine deaminase (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 replacementsilent
  ratio (RS)
• 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)

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SHM

• Can also target 5 regions of BCL-6, FAS/CD95,
  C-MYC, PAX5, RhoH/TTF
• ? others

Peled et al. Ann Rev Immunol. 26481.2008
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AID

• VDJ diverse primary repertoire
• AID dependent diversification
• SHM untemplated point mutations
• CSR
• Gene conversions using pseudogenes as template
• AID
• Deaminates ss DNA
• dC dU conversion
• High level expression in mature B cells
  responding to ag
• Overexpression increases SHM
• Transcription of gene required

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Barreto et al. TRENDS in Immunology 2690.2005
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• 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

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Class Switch Recombinatioin

• Requires AID
• Non-homologous end joining

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Role of bcl-6 in the GC reaction
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Affinity Maturation Requires B Cell Receptor
Signaling

• 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

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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

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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 (lt10 of nl B cells)

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• Role of the BCR complex
• Depending upon the stage of development BCR
  signaling has different outcomes
• Immature BM B cells - BCR signaling results in
  IgL deletion, anergy or clonal deletion
• Mature B cell - BCR signaling results in
  proliferation differentiation
• Tonic BCR signaling facilitates survival
• IgVH IgVL
• 4 framework lesions FR1-FR4
• 3 CDR - contact sites for ag
• IgVH CD3 is VDJ junction and most hypervariable
  region 15-85 bp length variation among B cells
• Low R/S in FR, high R/S CDR (codon compositions
  favor S and R respectively)

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Mantle Cell Lymphoma
t(1114) cyclin D1
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• t(1114)(q13q32) in early pre-B cell cyclin D1
• related to cells of primary follicle or mantle
  cells of secondary follicle
• CD5 (naïve B cells present in fetal blood and
  decrease with age)
• Produce low affinity polyreactive ab
• SHM in 15-40, biased IgHV subtypes
• Cyclin D1 dysregulation additional molecular
  features

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Burkitts Lymphoma
t (814) c-myc
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• Burkitts Lymphoma t(814) c-myc-IgH
• endemic-equatorial Africa age 4-7 malaria belt,
  EBV, exhaustion of EBV T cells and TLR9
  activation of B cells arboviruses jaws facial
  bones in 50
• sporadic-more common in children, median age in
  adults 30, EBV 30, extranodal, ileocecal,
  breast in pregnancy/lactation, mediastinum rare
• immunodeficiency associated-HIV with higher CD4,
  polyclonal activation of B cells
  post-transplant.

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Large Cell Lymphoma
spectrum of genetic abnormalities
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Diffuse Large B-cell Lymphoma

• 30-40 of NHL
• WHO classification Clinical Advisory Committee
• De novo vs transformation (CLL, follicular
  lymphoma, marginal zone lymphoma, nodular
  lymphocyte predominant Hodgkins disease)
• CD19, CD20, CD22, CD79a, CD45, sIgM
• Morphologic variants centroblastic,
  immunoblastic, T-cell/histiocytic rich,
  lymphomatoid granulomatosis-like, anaplastic
  large B-cell, plasmablastic
• Subtypes mediastinal large B-cell lymphoma,
  primary effusion lymphoma (HHV-8, CD30, CD38,
  CD138), intravascular large cell lymphoma
• IPI - P,L,E,A,S

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• Recurring cytogenetic abnormalities
• t(1418) with overexpression of bcl-2 (30)
  independent prognostic factor
• 3q27 rearrangements with overexpression of bcl-6
  (30-40)
• t(814) with overexpression of c-myc
• Bcl-2
• Bcl-6
• bcl-6 zinc finger DNA binding protein
• represses transcription of target genes
• required for GC formation
• Suppression of damaged DNA response
• Suppression of differentiation

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Lossos IS. Molecular Pathogenesis of Diffuse
Large B-Cell Lymphoma. J Clin Oncol.
23.6351.2005
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International Prognostic Index (IPI)

• Risk factors
• gt60 years
• 2-4
• Elevated
• gt1 site
• III-IV
• 2-4
• Elevated
• III-IV

• Patients of all ages
• Age
• Performance status
• Lactate dehydrogenase (LDH) level
• Extranodal involvement
• Stage (Ann Arbor)
• Patients 60 years (age-adjusted)
• Performance status
• LDH
• Stage

Shipp. N Engl J Med. 1993329987.
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International Prognostic Index - LCL
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Classification of B-Cell Large Cell Lymphoma -
Alizadeh et al. Nature 403503.2000
cDNA patterns determined in DLBCL, CLL, FL,
normal B-cells, activated B-cells, B-cell lines.
Distinct patterns for each. Subtypes of DLBCL
(i) normal germinal center B-cells (e.g. CD10,
CD38 bcl-6, OGG1, bcl-7A, LMO2) (ii) activated
B-cells (bcl-2)
Diffuse Large Cell Lymphoma
Gene expression profile similar to normal
germinal center B-cells GC
Gene expression profile similar to circulating
B-cells treated with cytokines ABC
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Rosenwald et al NEJM 3461937.2002
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Prognostic Impact of Gene Expression Cell of
Origin
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Lossos IS. Molecular Pathogenesis of Diffuse
Large B-Cell Lymphoma. J Clin Oncol. 23.6351.2005
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Gene expression profiling LCL-Survival

• Leukemia/Lymphoma Molecular Profiling Project
• Diversity of gene expression in DLBCL reflects
  variation in proliferative rate, host response
  differentiation state
• GC vs ABC prognostic significance
• 3 GC genes, 4 MHC II genes, 6 lymph node
  (reactive non-malignant) genes, 3 proliferation
  genes (poor prognosis), BMP6 (poor prognosis)
• Shipp et al
• B-cell receptor signaling, signaling pathways,
  apoptosis correlations with survival

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Rosenwald et al NEJM 3461937.2002
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Classification Based upon Clinical Outcome
Shipp et al Nature Medicine 868 (2002)
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Transcription Profiles in LCL
OxPhos Host response BCR
Monti et al. Blood. 20051051851-1861
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LCL6 gene expression prognostic score
Lossos et al. NEJM 3501828.2004
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HodgkinsDisease
EBV association 30-50 Orderly spread of
disease Constitutional complaints Staging
Stein, R. Hodgkin Disease in Wintrobess Clinical
Hematology
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Hodgkins LymphomaKuppers Nat Rev cancer
915.2009

• GC B cell origin (rearranged and somatic
  mutations IgV)
• 25 have mutation that prevent expression of BCR
  usually causes apoptosis of GC cells remainder
  may have low affinity mutations
• Downregulated transcription factor networks OCT2,
  BOB1
• Jak-stat and NF-kB pathway alterations

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Role of the Microenvironment in cHL

• Reactive cellular infiltrate
• CD4 lymphocytes regulatory phenotype CTLA-4
  (CD4CD10 CD4CD25)
• Few CD4Th1 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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EBV - Hodgkins Lymphoma

• EBV in 40
• Latent viral pattern
• EBNA1 maintains genome ORI
• LMP1 CD40 signaling
• LMP2 BCR like signaling ITAMs
• BARTs - microRNAs
• EBERS

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Environmental Networking of H-RS Cells
Re, Kuppers, Diehl. Molecular pathogenesis of HL.
JCO 236379.2005
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Follicular Lymphoma
t(1418) bcl-2
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• BCR binding
• BCR intact in FL and may bind ag, presumably on
  surface of FDC
• Immunohistochemical staining using soluble
  recombinant BCR did not bind GC structures or
  autoantigens in one study but 8/31 reacted with
  autoag in another study
• FL IgVH repertoire similar to normal B cell
  normal size CD3 (long CD3 associated with
  auto-ag)
• In contrast CLL MALT have restricted and
  recurrent repertoire with auto-ag specificity
• 55/79 FL R mutations in IgVH resulted in
  generation of aa motifs being N-glycosylation
  sites - enhanced signaling or microenvironment
  interaction

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• Secondary Genetic Alterations
• 5 recurrent chromosomal alterations each gt 20 of
  cases
• -1p32-36
• -6q11-27
• 7
• 12
• X
• X, 1p-, 1q, 12, 19p-, 17q- poorer prognosis
  6q- shorter survival time
• Gene Expression Profiling in FL Transformation
• Whole FL tissue samples (Dave)
• IR1 better survival - T cell specific genes
• IR2 shorter survival - macrophages DCs
• Loss of full FDC phenotype and Treg with
  progression

Dave S, et al. Prediction of Survival in
Follicular Lymphoma Based on Molecular Features
of Tumor-Infiltrating Immune Cells. NEJM
3512159.2004.
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Dave S, et al. Prediction of Survival in
Follicular Lymphoma Based on Molecular Features
of Tumor-Infiltrating Immune Cells. NEJM
3512159.2004.
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Pedro Farinha, Hamid Masoudi, Brian F. Skinnider,
Karey Shumansky, John J. Spinelli, Karamjit Gill,
Richard Klasa, Nicolas Voss, Joseph M. Connors,
and Randy D. Gascoyne Analysis of multiple
biomarkers shows that lymphoma-associated
macrophage (LAM) content is an independent
predictor of survival in follicular lymphoma
(FL). Blood 1062169.2005.
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MALT
3, t(1118), t(114)
H. pylori
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Bcl-10-IgH
MALT Lymphoma
API-MALT1

• Unsorted cells from gasrtric MALT die in lt5d
• Heat killed strain specific H.pylori survival
  proliferation of lymphoma cells
• T cell activation (IL-2 R)
• Autoreactive BCR, ongoing selection

MALT1-IgH
Farinha Gascoyne. JCO. 236370.2005 Isaacson.
Nat Rev Cancer 4644.2004
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• TREATMENT

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LYMPHOMA or LEUKEMIA
Signals cytokines hormones stromal
interactions cell-cell interactions
NORMAL
key regulatory pathways p53, pRb, cyclins, cdks,
mitochondrial regulation etc
BRAKES
--------
Mutations in key regulatory pathways (e.g. flt3,
ras, kit, Rb, cdk, p53), make cells independent
of some signals
Intact regulatory pathways that must be
physiologically regulated by signals to allow
cell division
Take the brakes off
energy generation cell cycle progression cell
preparation for DNA replication, chromosome
condensation, mitosis CELL DIVISION
The brakes are broken
Normally the brakes are on!
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NORMAL
LEUKEMIA or LYMPHOMA
CHEMOTHERAPY
CELL DAMAGE
CELL DAMAGE
Key regulatory pathways that detect DNA, protein
and membrane damage
Some key regulatory pathways that detect DNA,
protein and membrane damage are mutated during
oncogenesis
Cell cycle arrest
Less cell cycle arrest
REPAIR
Enhanced damage --- apoptosis, mitotic catastrophe
, prolonged arrest
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Normal
Broken Brakes
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Dont randomly damage the car and hope it crashes
into the wall (or auto-destructs) ! Identify
whats broken and target that defect
Evolving concepts in cancer biology
Preclinical studies Clinical trials New
therapies
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Monoclonal Antibody Therapy

• Biotherapy targeted
• treatment
• Effective, low toxicity
• Targets tumor cells
• Two types
• Unconjugated
• Conjugated

CD20 CD22 CD52 CD80 CD40
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Radioimmunotherapy

• Targets tumor cell
• Monoclonal antibody and radioisotope conjugate

Naked
Radiolabeled
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Rituximab Coiffier. Oncogene 263603.2007

• CD20 transmembrane protein expressed by gt95
  B-NHL
• First and most successful mab in lymphoma therapy
• CD20 binding homotypic aggregation
  translocation to rafts induction of apoptosis
• Consumption of complement (complement dependent
  cytotoxicity) in some cell types
• ADCC also may play a role FcgRIIIa alleles

• Resistance mechanisms

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New Therapies for the Future

• Antisense therapy
• Proteasone inhibitors
• Genetics
• Gene expression profiling
• Cytokines
• Vaccines
• Other monoclonals
• BCR signaling