Tumor Biology

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Tumor Biology

• Leukemia and lymphoma

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Leukemia

•   
• Leukemia is a malignant disease of
  hematopoietic tissue, characterized by
  replacement of normal bone marrow elements with
  abnormal blood cells. These leukemic cells are
  frequently present in the peripheral blood and
  may filtrate any organ of the body, particularly
  the spleen, liver and lymph nodes.

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Hematopoietic development
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FAB classification

• Acute leukemia
• acute lymphocytic leukemia (ALL)
• acute myelogenous leukemia (AML)
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• Chronic leukemia
• chronic lymphocytic leukemia (CLL)
• chronic myelogenous leukemia (CML)

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Lymphoma

• Lymphoma is a general term for a diverse group
  of proliferative malignant neoplasms that
  originate primarily in the T cells and B cells.
  These diseases generally arise in the lymph nodes
  but may develop in extranodal tissue as well.
  Lymphomas are currently separated into two major
  groups, Hodgkin's disease (HD) and non-Hodgkin's
  lymphoma (NHL), with different disease biology.

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Differentiation of B- and T-cell
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REAL-WHO classification

• B-cell lymphoma
• Burkitt's lymphoma
• Mantle cell lymphoma
• Follicular lymphoma
• Marginal zone B-cell lymphoma
• Diffuse large cell lymphoma
• Plasma cell myeloma
• Small lymphocytic lymphoma
• Precursor B-lymphoblastic lymphoma
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REAL-WHO classification

• T-cell lymphoma
• T-cell lymphoma
• HTLV-I() adult T-cell lymphoma/leukemia
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Overview of the molecular methods in the
diagnosis of leukemia and lymphoma
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• By 1956, in vitro cell culture and hypotonic
  treatment of cells resulted in adequate
  separation of chromosomes so that the correct
  chromosome number could be identified.
• From 1956 to 1969, all cytogenetic studies were
  performed using Giemsa staining without
  pretreatment of the chromosomes.

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• In 1960, Peter Nowell discovered that exposure of
  cells to phytohemaglutinin stimulated the T
  lymphocytes to divide within 48 to 72 hours.
  Simultaneously, Nowell and Hungerford discovered
  Philadelphia or Ph chromosome in patients with
  chronic myeloid leukemia (CML).

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• From 1969 to 1971, quinacrine staining (Q
  banding) was developed, and each pair of
  chromosomes had a unique pattern of bands.
  Pretreatment of chromosome with trypsin or with
  heat followed by Giemsa staining (G banding) also
  show a banding pattern.

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Procedure of karyotyping
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• Using of Q banding in 1972 led to the
  identification of the first consistent
  translocation of t(821) in acute myeloblastic
  leukemia. In 1970s also discovered a number of
  other chromosome translocations, including the
  t(814) in Burkitts lymphoma, the t(1517) in
  acute promyelocytic leukemia, and the t(1418) in
  follicular lymphoma.

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• In the mid 1980s, probe labeled with various
  fluorochromes were used to identify specific
  genes or chromosome regions. The technique
  fluorescence in situ hybridization (FISH) is used
  to determine whether particular genes or regions
  are involved in translocation.

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• The probes specific for each human chromosome
  were developed, this new technique is called
  spectral karyotyping (SKY) or multicolor FISH
  (M-FISH).

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• Chromosome deletion or amplification can be
  detected by comparative genomic hybridization
  (CGH).

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• Some of the genetic alterations can also be
  identified using Southern blot or reverse
  transcriptase-polymerase chain reaction (RT-PCR)
  analysis.

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• DNA chip technology can be used to detect
  specific RNA or DNA, which is resulted from
  either chromosomal translocation or point
  mutation.

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• Approximately 150 apparently balanced
  translocations in leukemia and lymphoma have been
  found, the presence of a specific translocation
  is often of diagnostic and prognostic importance.
  
• In addition to translocations, a number of
  recurring chromosome gains, losses, deletions,
  and point mutations have been identified in
  leukemic cells.

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General mechanisms for proto-oncogene activation
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AML
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ALL
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Lymphoma
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Molecular genetics of human leukemia
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•       Chromosomal translocations involving
  tyrosine kinases
• Translocations Fusion genes Disease
• t(922)(q34q22) BCR-ABL ALL, CML
• t(912)(q34p13) TEL-ABL ALL, AML
• t(512)(q33p13) TEL-PDGFbR CMML
• t(57)(q33q11.2) HIP1- PDGFbR
• t(912)(p24p13) TEL-JAK2 ALL, CML
•  

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• Oligomerization mediated by BCR, TEL and HIP1
  induces the intrinsic tyrosine kinase activity of
  PDGFb, ABL and JAK2.
• Activation of these three tyrosine kinases leads
  to activation of the RAS signal transduction
  pathway with subsequent phosphorylation of a
  family of latent cytoplasmic transcription
  factors known as signal transducers and
  activators of transcription (STATs).

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• STATs are important transcription factors
  associated with signal transduction mechanisms
  used by several cytokines and growth factors.
• Activation of JAK/STAT signaling pathway may lead
  to an up-regulation of hematopoietic growth and
  differentiation factor genes that contributes to
  leukemogenesis.

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• Chromosomal translocations involving
  transcription factor
• Core binding factor (CBF)
• Translocations Fusion genes Disease
• t(821)(q22q22) AML1-ETO AML-M2
  t(321)(q26q22) AML1-EVII MDS, AML
  t(1221)(p13q22) TEL-AML1 Pro-B-ALL
  inv(16)(p13q22) CBFb-MYH11 AML  

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• Translocations involving the core-binding factor
  (CBF) transcription factor genes are the most
  frequent chromosomal changes in human acute
  leukemias.
• CBF is a heterodimeric transcription factor,
  composed of AML1 and CBFb subunits, that play an
  essential role in hematopoietic cell development,
  function, and differentiation.
• AML1 contains a DNA-binding (runt) domain, a
  histone acetyltransferase (HAT)-interacting
  region and a transactivation domain.
• AML1 has been shown to be involved with 14 other
  translocation partners.

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• The TEL-AML1 fusion protein interacts with the
  CBFb, and bind to the enhancer motif TGTGGT,
  however, interferes its interaction with the
  transcription coactivator CBP/P300. Furthermore,
  the expression of TEL-AML1 is regulated by TEL
  promoter.
• ETO is a zinc-binding protein, which is
  associated with N-CoR-Sin-histone deacetylase
  (HDAC) complex. The AML1-ETO fusion protein acts
  as a dominant negative inhibitor of the wild-type
  AML1.
• The CBFb-MYH11 (smooth muscle myosin heavy chain)
  fusion protein dramatically reduces the binding
  of wild-type CBF to DNA.
• These fusion proteins play as dominant negative
  regulators of CBF.

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• Chromosomal translocations involving
  transcription factor
• Retinoic acid receptors
• Translocations Gene fusions Disease
• t(1517)(q22q11) PML-RARa AML-M3
  t(1117)(p13q11) PLZF-RARa atypical AML-M3
• t(517)(q31q11) NPM-RARa atypical AML-M3

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• Retinoic acid receptor-a (RARa) protein binds to
  retinoic acid ligand through a defined
  ligand-binding domain and to DNA through a
  separate zinc-finger region.
• RARa and RXR form a heterodimer that binds to the
  DNA with RAR responsive element.
• RARa regulates myeloid cell development at the
  promyeloid stage by altering the normal
  sequential pattern of HOX gene expression.
• PML is required for the hematopoietic
  differentiation, growth-suppressing activities of
  retinoic acid.

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• PML interacts with HDAC complex, which consists
  of the nuclear corepressor (N-CoR or SMRT), Sin3,
  and histone deacetylase. The whole complex is
  capable of repressing transcription of target
  genes by promoting histone acetylation.
• The aberrant PML-RARa fusion protein acts as a
  dominant negative inhibitor of both PML and RARa
  through the formation of heterodimers with PML
  and other retinoic acid-binding proteins, which
  induce differentiation arrest in the promyelocyte
  stage.

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RA
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• All-trans retinoic acid (ATRA) was routinely used
  to treat patients with t(1517).
• Binding of ATRA to the PML-RARa releases HDAC
  complex from PML-RARa and recruit transcriptional
  coactivators, including CBP/p300 and SRC1 which
  allows the normal expression of genes encoding
  hematopoietic cell differentiation factors.
• PLZF also contains an HDAC-binding site but the
  PLZF-RARa fusion protein is much less responsive
  to ARTA.
• NPM-RARa behaves similarly to PML-RARa in its
  reduced RA sensitivity in interaction with
  Co-repressors.

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RA
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Differentiation therapy

• Retinoic acid treatment
• Histone deacetylase inhibitor treatment

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• Chromosomal translocations involving
  transcription factor
• HOX genes.
• Translocations Fusion genes Disease
• t(711)(p15p15) NUP98-HOXA9 T cell ALL
• t(1014)(q24q11) TCRd-HOX11 T cell ALL
• t(710)(q35q24) TCRb-HOX11 T cell ALL
• t(119)(q23p13.3) E2A-PBX1 pre-B ALL 

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• Homeobox genes are categorized in two large
  groups. The class I genes are called HOX genes.
  Human 39 HOX genes are organized in four clusters
  (HOXA, -B, -C, -D). The class II, or diverged
  homeobox genes are grouped in several families,
  such as Pax family, POU domain family. PBX1 is
  also a diverged class II homeobox gene.
• Expression of HOX genes is closely associated
  with embryo development and hematopoiesis. HOXC
  cluster plays a role in regulation of lymphoid
  lineage, HOXA for the myeloid lineage, HOXB for
  the erythroid lineage, HOXD gene might also be
  involved in myeloid differentiation.

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• PBX1 form DNA-binding complexes with HOX
  proteins. E2A-PBX1 interferes both E2A and PBX1
  functions. Complex of E2A-PBX1 with HOX induces
  cell transformation.
• HOX11 could interact with the protein
  phosphatases PP2A and PP1 resulting in disruption
  of G2/M cell cycle checkpoint.

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• Chromosomal translocations involving
  transcription factorTAL1/E2A/LMO2 complex
• Translocations Fusion
  genes Disease
• t(119)(q23p13) E2A-PBX1 Pre-B-ALL
• t(1719)(q22p13) E2A-HLF Pro-B-ALL
• t(114)(p32q11) PTCRa/d-TAL1 T-cell ALL
• t(1114)(p13q11) PTCRa/d-LMO2 T-cell ALL
• t(711)(q35p13) PTCRb-LMO2 T-cell ALL
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• In most tissues, E2A heterodimerizes with
  tissue-specific bHLH family members to coordinate
  gene expression during development. E2A is able
  to bind E-box sequences (CANNTG) as a homodimer
  complex.
• TAL1 can dimerize with E2A protein through its
  bHLH domain to form DNA binding complexes, and
  with the LMO2 protein to activate specific sets
  of target genes that regulate hematopoiesis.
• A complex involving LMO2, TAL1, E2A, GATA-1, and
  Ldb1 could form in erythroid cells, and
  specifically activate transcription from the
  E-box-GATA sequence.

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• E2A-PBX1 and E2A-HLF fusion proteins remain two
  transcriptional activation domains of E2A, but
  not the bHLH DNA binding-protein interaction
  domain. Instead, PBX1 segment providing a
  homeodomain motif and HLF providing a leucine
  zipper domain.
• Chromosomal translocations of TAL1 and LMO2 near
  enhancers of TCR genes, can express genes that
  are normally quiescent in T-lineage progenitors
  or act by repressing E2A activity during T-cell
  development.

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• Chromosomal translocations involving
  transcription factor
• MYC
• Translocations Fusion genes Disease
• t(814)(q24q32) IgH-MYC B-ALL, Burkitt lymphoma
• t(28)(p12q24) Igk-MYC B-ALL, Burkitt lymphoma
• t(822)(q24q11) IgL-MYC B-ALL, Burkitt lymphoma
• t(814)(q24q11) TCRa/d-MYC T-ALL
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• MYC is a bHLH/leucine zipper transcription
  factor, whose regulation on a transcriptional
  network comprising other factors, such as MAX,
  and MAD. MAD is able to repress the transcription
  of down-stream target genes by recruiting HDAC
  complex.
• In Burkitts lymphoma and B-cell leukemia,
  rearrangement of MYC from chromosome 8 to a site
  near strong Ig enhancer elements leads to
  dysregulated expression of the MYC oncoprotein.

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• Chromosomal translocations involving
  transcription factor
• BCL6
• Translocations Fusion genes Disease
• t(314)(q27q32) IgH-BCL6 Diffuse B-cell
  lymphoma t(34)(q27p11) PTTF-BCL6 Diffuse B-cell
  lymphoma
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• BCL6 encodes a transcription factor containing
  six zinc-finger DNA-binding motifs near the
  carboxyl-terminus and a POZ regulatory domain
  near the carboxyl-terminus. BCL6 POZ domain binds
  SMRT and silence transcription by recruiting HDAC
  complex.

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• BCL6 is normally down-regulated before B cells
  exit from the germinal center. Constitutively
  expressing of BCL6 in the activated B
  lymphoblasts provides proliferative capacity,
  which leads to a large-cell lymphoma formation.
• In addition to gene rearrangement mediated by
  translocation, somatic point mutations of the 5
  regulatory region of the BCL6 gene have been
  identified at high frequency in both the diffuse
  large cell and the follicular lymphoma of B-cell
  origin.

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• Chromosomal translocations involving
  transcription factor
• TEL
• Translocations Fusion genes Disease
• t(1221)(p13q22) TEL-AML1 ALL
• t(512)(q33p13) TEL-PDGFRb CMML
• t(912)(q34p12) TEL-ABL ALL, AML
• t(912)(p24p12) TEL-JAK2 ALL, CML
• t(1222)(p13q11) MN1-TEL MDS, AML
• t(312)(q26p12) TEL-EVI1 MDS
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• TEL is a sequence-specific DNA binding
  transcriptional regulator. It contains a
  DNA-binding ETS domain on C-terminal, and a
  helix-loop-helix (HLH) domain on N-terminal. The
  HLH domain mediates homotypic oligomerization.

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• A total of 34 different chromosomal
  translocations involved with TEL have been
  identified. Abnormalities of TEL have been
  observed in a variety of hematologic
  malignancies, including about 5 of AML and MDS
  cases.
• HLH domain of TEL can mediated dimerization
  between chimeric protein and TEL, and alter the
  expression of TEL target genes. On the other
  hand, partner genes may provide new functions to
  the chimeric proteins.

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• Translocations translocation involving
  transcriptional modulatory factor
• MLL
• Translocations Fusion genes Disease
• t(911)(p22q23) MLL-AF9 AML
• t(1119)(q23p13.3) MLL-ENL AML, ALL
• t(411)(q21q23) MLL-AF4 ALL
• t(611)(q27q23) MLL-AF6 AML
• t(1011)(p12q23) MLL-AF10 AML
• t(1119)(q23p13.1) MLL-ELL AML
• MLL partial tandem duplication AML
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• MLL can induce or repress gene expression by
  directly modifying chromatin structure. It is a
  positive regulator of homeobox (HOX) genes, which
  are involved in embryonic development and
  hematopoiesis.

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• MLL is involved in more than 30 different
  translocations, they were identified in about 7
  of patients with AML, as well as in up to 10 of
  patients with ALL.
• MLL fusion proteins all contain the amino
  terminal sequences of MLL, including the AT hooks
  and the methyltransferase (MT) homology domain.
  It was speculated that the MLL AT hooks and MT
  domain bind to the minor groove of DNA at
  enhancer sites, whose interaction may facilitate
  the recruitment of transcription factors to the
  major groove or may alter local chromatin
  structure to regulate gene expression.

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• Translocations involving transcriptional
  coactivators/repressors CBP/p300
• Translocations Fusion genes Disease
• t(1116)(q23p13) MLL-CBP t-AML
• t(1122)(q23q13) MLL-p300 t-AML
• t(816)(p11p13) MOZ-CBP AML-M4, M5
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• CBP (cyclic-AMP response element binding protein
  (CREB)-binding protein) and p300 are
  transcriptional coactivators that serve as a
  bridge between the cellular transcriptional
  machinery and transcription factors. CBP express
  intrinsic histone acetyltransferase activity,
  which open chromatin and allow gene
  transcription.
• CBP and p300 do not bind directly to DNA but
  rather function to recruit and activate
  transcription factors, including RAR, STATs, BCL6
  and CBF. Through their ability to regulate gene
  expression, these proteins play important roles
  in cell differentiation, cell growth control, and
  signal transduction.

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CBP
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CBP
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• Translocations affecting apoptosis
• Translocations Fusion genes Disease
• t(1418)(q21q32) IgH-BCL2 Follicular lymphoma
• t(1118)(q21q21) API2-MLT MALT lymphoma
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• BCL2 inhibits cytochrome C release by modulating
  the proapoptotic family members such as Bax and
  Bak to facilitate opening of the VDAC pore. BCL2
  also binds antiapoptotic Bcl-XL to Apaf-1
  prevents the binding and activation of
  procaspase-9.
• IAP family can regulate the activation of
  procaspases. API2 (cIAP2) inhibit procaspase-9
  activation in the presence of cytochrome C and
  dATP.
• Constitutive expression of BCL2, as well as the
  presence of API2-MLT chimeric protein, inhibits
  apoptosis and confer a survival benefit to B-cell
  lymphomas.

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• Translocations affecting cell cycle control
• cyclin D1
• Translocation Fusion gene Disease
• t(1114)(q13q34) IgH-CCND1 Mantle cell lymphoma

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• Cyclin D1 (CCND1) act in concert with their
  catalytic partners, the cyclin-dependent kinases
  (CDK4 and CDK6), to initiate the phosphorylation
  of the retinoblastoma protein, pRB, thus coupling
  growth factor induced mitogenic signals to the
  biochemical machinery of the cell cycle.
• B cell normally do not express Cyclin D1.
  Aberrantly express of Cyclin D1 by translocation
  provide a constitutive proliferative stimulus and
  unable to exit the cell cycle.

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• Gene mutations in tumor suppressor genes
• Tumor Suppressor Gene Chromosome Location
• Rb 13q14
• p53 17q13
• p15/INK4B 9p21
• p16/INK4A 9p21
• p18/INKC 1p32
• p21/waf1/cip1 6p21
• p27/Kip1 12p13
• ATM 11q23
• Kai1/CD82 11p11.2
• Bax 19q13.3

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• Tumor suppressor genes encode for proteins that
  are involved in programmed cell death (apoptosis)
  and cell growth control, and many of these
  proteins block cell proliferation at the level of
  the cell cycle.
• In general, the effect of a tumor suppressor gene
  is recessive, both alleles must be inactivated
  before the cell inappropriately proliferates.
  Both tumor suppressor genes may be inactivated as
  acquired abnormalities. However, in familial
  cancer syndromes one defective allele is
  inherited, greatly increasing the statistical
  chance of cancers developing in the target
  tissues.

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• Gene mutations often result from missense or
  nonsense point mutations often results in
  transcription of non-functional mutant protein.
  Mutations also can result in the generation of
  stop codons that prevent protein synthesis.

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Mutations other than chromosomal translocation
AML1 M0 biallelic mutations GATA1
M7 amino-terminal mutations PU.1 M0,
M4, M5, M6 C/EBPa M1, M2,
M4 down-regulation in RNA level

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Hematopoietic development
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• Virus infection that associated with lymphoma
  and leukemia
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• Epstein-Barr Virus (EBV) infection was associated
  with Hodgkin lymphoma.
• Human T-cell lymphotropic virus 1-associated
  adult T-cell lymphoma/leukemia.

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Invariably, Burkitts lymphoma cells contain
Ig-MYC translocations
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• 3.        Rubnitz, J. E., and A. T. Look (1998)
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• 7.        Van Oostveen, J., et al. (1999) The
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