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RAS, KIT, FLT3 and JAK2 gene mutations in Acute Myeloid Leukemia (AML) with inv(16) and t(8;21) [Elektronische Ressource] : incidence and relevance on clinical outcome / presented by Juan Du

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98 pages
UniversitätsklinikumUlmZentrumfürInnereMedizinKlinikfürInnereMedizinIIIHämatologie,Onkologie,RheumatologieundInfektionskrankheitenÄrztlicherDirektor:Prof.Dr.med.HartmutDöhner RAS,KIT,FLT3andJAK2 GeneMutationsinAcuteMyeloidLeukemia(AML)withinv(16)andt(8;21):IncidenceandRelevanceonClinicalOutcomeDissertation fortheattainmentoftheDoctoralDegreeofMedicine(Dr.med.)attheFacultyofMedicine,UniversityofUlm,Ulm,GermanyPresentedbyJuanDuborninMudanjiang,HeilongjiangProvince,P.R.China2007 AmtierenderDekan:1.Berichterstatter:2.Berichterstatter:TagderPromotion: ContentContent Abbreviations………………………………………………………………III 1. Introduction........................................................................................... 1 1.1. Acutemyeloidleukemia.......................................................................... 1 1.2. Prognosticsubgroupsbasedoncytogeneticfindings............................. 3 1.2.1. Corebindingfactor(CBF)acutemyeloidleukemia................................. 5 1.3. MutationclassesinAML......................................................................... 9 1.3.1. ClassImutations .................................................................................. 10 1.3.2. ClassIImutations ..............................................................
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UniversitätsklinikumUlm
ZentrumfürInnereMedizin
KlinikfürInnereMedizinIII
Hämatologie,Onkologie,Rheumatologieund
Infektionskrankheiten
ÄrztlicherDirektor:Prof.Dr.med.HartmutDöhner


RAS,KIT,FLT3andJAK2 GeneMutationsin
AcuteMyeloidLeukemia(AML)withinv(16)
andt(8;21):IncidenceandRelevanceon
ClinicalOutcome



Dissertation
fortheattainmentofthe
DoctoralDegreeofMedicine(Dr.med.)
attheFacultyofMedicine,UniversityofUlm,Ulm,Germany



PresentedbyJuanDu
borninMudanjiang,HeilongjiangProvince,P.R.China


2007









AmtierenderDekan:
1.Berichterstatter:
2.Berichterstatter:



TagderPromotion:


Content
Content
Abbreviations………………………………………………………………III
1. Introduction........................................................................................... 1
1.1. Acutemyeloidleukemia.......................................................................... 1
1.2. Prognosticsubgroupsbasedoncytogeneticfindings............................. 3
1.2.1. Corebindingfactor(CBF)acutemyeloidleukemia................................. 5
1.3. MutationclassesinAML......................................................................... 9
1.3.1. ClassImutations .................................................................................. 10
1.3.2. ClassIImutations ................................................................................. 15
1.4. Theaimofthestudy ............................................................................. 16
2. MaterialandMethods ......................................................................... 18
2.1. Material................................................................................................. 18
2.1.1. DNAextraction...................................................................................... 18
2.1.2. Polymerasechainreaction(PCR)......................................................... 18
2.1.3. Agarosegelelectrophoresis ................................................................. 20
2.1.4. Denaturinghighperformanceliquidchromatography(DHPLC)............ 20
2.1.5. PurificationofPCRproducts................................................................. 21
2.1.6. Cyclesequencingreaction(CSR)......................................................... 21
2.1.7. Dyeremoval.......................................................................................... 21
2.1.8. DirectDNAsequencing ........................................................................ 21
2.2. Patients................................................................................................. 21
2.3. Treatment ............................................................................................. 23
2.4. Cytogeneticandmoleculargeneticanalysis......................................... 25
2.5. AnalysisofRAS,KIT,FLT3andJAK2genemutations ........................ 25
2.5.1. DNAextraction...................................................................................... 25
2.5.2. Polymerasechainreaction(PCR)......................................................... 26
2.5.3. PCRwithrestrictionfragmentlengthpolymorphism(PCRCRFLP)........ 26
2.5.4. DetectionofJAK2mutationsusingalleleCspecificPCR....................... 27
2.5.5. Agarosegelelectrophoresis ................................................................. 28
2.5.6. IsolationoftheDNAfragmentsfromagarosegels ............................... 28
2.5.7. Denaturinghighperformanceliquidchromatography(DHPLC)............ 28
2.5.8. PurificationofPCRproducts................................................................. 30
2.5.9. Cyclesequencingreaction(CSR)......................................................... 31
IContent
2.5.10. DyeremovalusingDyeExspinkit ........................................................ 31
2.5.11. DirectDNAsequencing ........................................................................ 31
2.5.12. SensitivityofDHPLCandsequencingassay........................................ 31
2.6. Statisticalanalyses ............................................................................... 32
3. Results................................................................................................. 33
3.1. IncidenceofRAS,KIT,FLT3andJAK2genemutations ...................... 33
3.1.1. RASmutations...................................................................................... 34
3.1.2. KITmutations........................................................................................ 35
3.1.3. FLT3mutations ..................................................................................... 36
3.1.4. JAK2mutations..................................................................................... 36
3.2. Associatedgenemutations................................................................... 36
3.2.1. Associatedgenemutationsininv(16)Cpositivepatients ........................ 36
3.2.2. Associatedgenemutationsint(8;21)Cpositivepatients......................... 37
3.3. Patientscharacteristics......................................................................... 38
3.3.1. Pretreatmentcharacteristicsforinv(16)Cpositivepatients ..................... 38
3.3.2. Pretreatmentcharacteristicsfort(8;21)Cpositivepatients...................... 43
3.4. Responsetoinductiontherapy ............................................................. 48
3.4.1. Responsetoinductiontherapyforinv(16)Cpositivep atients ................. 48
3.4.2. Responsetoinductiontherapyfort(8;21)Cpositivep atients.................. 48
3.5. Survivalanalysis................................................................................... 51
3.5.1. Survivalanalysisforinv(16)Cpositivepatients ...................................... 51
3.5.2. Survivalanalysisfort(8;21)Cpositivepatients........................................ 56
4. Discussion .......................................................................................... 60
4.1. RASanalysis ........................................................................................ 60
4.2. KITanalysis .......................................................................................... 62
4.3. FLT3analysis ....................................................................................... 64
4.4. JAK2analysis ....................................................................................... 66
4.5. Conclusion............................................................................................ 67
5. Summary ............................................................................................. 69
6. References .......................................................................................... 71
7. Curriculumvitae ................................................................................. 87
8. Acknowledgements............................................................................ 90
IIAbbreviations
Abbreviations
ACN Acetonitrile
Allo6SCT AllogeneicstemCcelltransplantation
AML Acutemyeloidleukemia
AMLSG AMLStudyGroup
APL Acutepromyelocyticleukemia
ATRA alltransretinoicacid
Auto6SCT AutologousstemCcelltransplantation
BM Bonemarrow
bp Basepair
CBF CoreCbindingfactor
CBFB CoreCbindingfactorβ
CBFs CoreCbindingfactors
CEP AntiCcancercompound
CI Confidenceinterval
CIR Cumulativeincidenceofrelapse
CALGB CancerandLeukemiaassociatedGroupB
CMPD Chronicmyeloproliferativediseases
CNS Centralnervoussystem
CR Completeremission
CRi Completeremissionwithincompletehematologicalrecovery
CSR Cyclesequencingreaction
DFS DiseaseCfreesurvival
DHPLC Denaturinghighperformanceliquidchromatography
DNA Deoxyribonucleicacid
dNTP Deoxynucleotidetriphosphate
ED Earlydeath
EDTA Ethylenediaminetetraaceticacid
EFS EventCfreesurvival
EML Extramedullaryinvolvement
ETO EighttwentyCone
EUN NCethylCNCnitorsourea
FAB FrenchCAmericanCBritish
IIIAbbreviations
Fluorescenceinsituhybridization FISH
FTIs Farnesyltransferaseinhibitors
GISTs Gastrointestinalstromaltumors
HAM HighCdosecytarabineandmitoxantrone
HiDAC HighCdosecytarabine
HOVON HematoCOncologievoorVolwassenenNederland
HPV Humanpapillomavirus
HR Hazardratio
HSC Hematopoieticstemcell
HSCs Hematopoieticstemcells
HSPCs Hematopoieticstem/progenitorcells
HUGO HumanGenomeOrganization
IC Idarubicin,cytarabine
ICE Idarubicin,cytarabine,etoposide
IDAC IntermediateCdosecytatabine
IE Idarubicin,etoposide
ins/del Insertion/deletionmutations
inv(16) Inversioninv(16)
ITD Internaltandemduplication
JAK Januskinase
JM Juxtamembrane
KI Kinaseinsert
LDH Lactatedehydrogense
LSCs Leukemiastemcells
MCD Mastcelldisease
MDS Myelodysplasticsyndrome
MgCl Magnesiumchloride2
MgSO Magnesiumsulfate4
MPD Myeloproliferativedisorders
MRCAML10 MedicalResearchCouncil's10thAMLtrial
MYH11 Myosinheavychain11
NCAM Neuralcelladhesionmolecule
OR Oddsratio
OS Overallsurvival
IVAbbreviations
PB PeripheralBlood
PCR Polymerasechainreaction
PCR6RFLP PCRwithrestrictionfragmentlengthpolymorphism
PDGFRa PlateletCderivedgrowthfactorCreceptorCα
PDGFRB PlateletCderivedgrowthfactorCreceptorC β
PKC ProteinKinaseC
PML Promyelocyticleukaemia
RARA RetinoidCacidreceptorCα
RD Resistantdisease
RFS RelapseCfreesurvival
rpm RotationperMinute
RT6PCR ReversetranscriptionCpolymerasechainreaction
RTKs Receptortyrosinekinases
RUNX1 RuntCrelatedtranscriptionfactor1
S6AML SecondaryAML
SCF Stemcellfactor
SCT Stemcelltransplantation
SMMHC SmoothCmusclemyosinheavychain
SNL Sinonasallymphomas
STAT Signaltransducerandactivatoroftranscription
STI571 Signaltransductioninhibitornumber571
t(8;21) Translocationt(8;21)
t6AML TherapyCrelatedAML
TE TrisCEDTA
TEAA Triethylammoniumacetate
TK Tyrosinekinase
TKD Tyrosinekinasedomain
TKIs Tyrosinekinaseinhibitors
TM Transmembranedomain
Tris Tris(hydroxymethyl)aminomethane
UVlight Ultravioletlight
WBC Whitebloodcell
WHO WorldHealthOrganization
VIntroduction
1. Introduction
1.1. Acutemyeloidleukemia
Acutemyeloid leukemia (AML) is a heterogeneous malignant hematopoietic
disordercharacterizedbyclonalexpansionofimmaturemyeloidcellsinthebone
marrow, blood or other organs. The affected cells underly an uncontrolled
proliferationandimpaireddifferentiationprogram.Typically,thecellsareblockedat
variousmaturationstepsandareresistanttocelldeath(Martellietal.2006).AML
accountsforapproximately80%ofalladultleukemias.Completeremission(CR)
canbeachievedinapproximately60%to70%ofadultAMLfollowingappropriate
inductiontherapyandmorethan15%oftheAMLpatients(about25%ofthosewho
attainCR)canbeexpectedtosurvive3ormoreyearsandmaybecured(Tallman
etal.2005).Increasedmorbidityandmortalityduringinductiontherapyappearto
be directly related to age, as well as other adverse prognostic factors including
centralnervoussystem(CNS)involvement,severeinfectionatdiagnosis,elevated
white blood cell (WBC) count, treatmentCinduced AML, or history of
myelodysplasticsyndrome(MDS)(Camposetal.1992;Lowenbergetal.1999).
Standard in the diagnosis of AML is the cytologic examination of
WrightCGiemsaorMayCGrünwaldGiemsastainedbloodandbonemarrowsmears
by light microscopy allowing reproducible classification by experienced
morphologists in most of the cases. Until 1999, the most widely used AML
classificationwastheFrenchCAmericanCBritish(FA)Bclassificationthatdescribed
thedegreeofdifferentiationandthelineageofleukemiabasedonpredominantly
morphologicalcriteria(Table1)(Bennettetal.1985).TheFABclassificationalso
included cytochemical stains. With the intention to link previous, predominantly
morphologicclassificationsystemswithnewlyemergingscientificdata,thecurrent
WorldHealthOrganization(WHO)classificationofhematopoieticneoplasmswas
recentlyimplemented.Thisnewclassificationcomprisesfourmajorcategoriesof
AML(Table2)(Harrisetal.1999).



1Introduction
Table1.FABclassificationofAML(Bennettetal.1985)
Category Morphology Incidence(%)
M0 AMLwithdifferentiation 3
M1 AMLwithoutmaturation 15C20
M2 AMLwithmaturation 25C30
M3 Acutepromyelocyticleukemia(APL) 5C10
M4 Acutemyelomonocyticleukemia 25C30
M5 Acutemonocytic/monoblasticleukemia 2C10
M6 Acuteerythroleukemia 3C5
M7 Acutemegakaryoblasticleukemia 3C12
FAB:FrenchCAmericanCBritish;AML:Acutemyeloidelukemia.
Table2.WHOclassificationofAML(Harrisetal.1999)
Category Morphology Incidence(%)
AML with recurrent AMLwitht(8;21)(q22;q22),AML1/ETO 5C12
cytogenetic 10C15AMLwith(15;17)(q22;q11C12),P( ML/RARα)
translocations andvariants,
5AMLinv(16)(p13q22)ort(16;16)(p13;q11),
3C5(CBFb/MYH11)
AMLwith11q23(MLL)abnormalities
AML with multilineage Withpriormyelodysplasticsyndrome 10C15
dysplasia Withoutpriormyelodysplasticsyndrome
AMLand Alkylatingagent–related 5C10
mylodysplastic EpipodophyllotoxinCrelated(somemaybe
syndromes(MDS), lymphoid)
therapy6related Othertypes
AMLnototherwise AMLminimallydifferentiated 40C50
categorized AMLwithoutmaturation
AMLwithmaturation
Acutemyelomonocyticleukemia
Acutemonocyticleukemia
Acuteerythroidleukemia
Acutemegakaryocyticleukemia
Acutebasophilicleukemia
Acutepanmyelosiswithmyelofibrosis
WHO: World Health Organization; AML: Acute myeloid leukemia; ETO: Eight twentyCone;
CBFb:CoreCbindingfactorβ;M YH11:Myosinheavychain11.
2Introduction
1.2. Prognosticsubgroupsbasedoncytogeneticfindings
Intheninetiesanumberofprospectiveandretrospectivestudieshaveclearly
demonstrated,thatcytogeneticaberrationsdetectableintheleukemiccellsatthe
time of diagnosis are the most important prognostic factors for clinical outcome
(Grimwadeetal.1998).BasedonthesefindingsAMLisnowcategorizedinthree
major cytogenetic groups: lowCrisk group, intermedai teCrisk group and highCrisk
group (Table 3). Using conventional cytogenteic analysis acquired clonal
chromosome aberrations (ie, reciprocal translocations, inversions, insertions,
deletions,trisomies,andmonosomies)canbedetectedinthepretreatmentmarrow
of50%to60%ofadultswithdenovoAML(Figure1).
Table3.RiskgroupassignmentsofAMLwithfrequentcytogeneticfindings(Grimwadeet
al.1998)
Favorable6RiskGroup
Balancedstructuralrearrangements t(15;17)(q22;q12C21)
t(8;21)(q22;q22)
inv(16)(p13q22)/t(16;16)(p13;q22)
Intermediate6RiskGroup
Normalkaryotype
Balancedstructuralrearrangement t(9;11)(p22;q23)
Unbalancedstructuralrearrangements del(7q)
del(9q)
Numericalaberrations –Y
+8
+21
+22
Unfavorable6RiskGroup
Complexkaryotype
Balancedstructuralrearrangements inv(3)(q21q26)/t(3;3)(q21;q26)
Unbalancedstructuralrearrangement del(5q)
Numericalaberrations –5
–7
AML:Acutemyeloidleukemia;del:deletion;inv:inversiton;t:translocation.

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