incidence and prognostic impact of dnmt3a mutations in korean … · 2018. 5. 11. · in the first...

Research ArticleIncidence and Prognostic Impact of DNMT3A Mutations inKorean Normal Karyotype Acute Myeloid Leukemia Patients

Sang Hyuk Park12 Jae-Cheol Choi3 Shine Young Kim12 Jongyoun Yi12

Seung Hwan Oh4 In-Suk Kim5 Hyung-Hoi Kim12 Chulhun Ludgerus Chang5

Eun Yup Lee12 Moo-Kon Song6 Ho-Jin Shin6 and Joo Seop Chung6

1Department of Laboratory Medicine Pusan National University School of Medicine Pusan National University Hospital179 Gudeok-ro Seo-gu Busan 602-739 Republic of Korea2Biomedical Research Institute Pusan National University Hospital 179 Gudeok-ro Seo-gu Busan 602-739 Republic of Korea3Department of Laboratory Medicine Hanmaeum Hospital 21 Woni-daero 682 Beon-gil Seongsan-guChangwon-si Gyeongsangnam-do 642-832 Republic of Korea4Department of Laboratory Medicine Inje University College of Medicine 75 Bokji-ro Busanjin-gu Busan 614-735 Republic of Korea5Department of Laboratory Medicine Pusan National University School of Medicine Pusan National University Yangsan Hospital20 Geumo-ro Mulgeum-eup Yangsan-si Gyeongsangnam-do 626-770 Republic of Korea6Division of Hematology-Oncology Department of Internal Medicine Pusan National University School of MedicinePusan National University Hospital 179 Gudeok-ro Seo-gu Busan 602-739 Republic of Korea

Correspondence should be addressed to Eun Yup Lee eyleepusanackr

Received 10 October 2014 Revised 22 December 2014 Accepted 22 December 2014

Academic Editor Martin Bornhaeuser

Copyright copy 2015 Sang Hyuk Park et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

BackgroundDNAmethyltransferase 3A (DNMT3A)mutationwas recently introduced as a prognostic indicator in normal karyotype(NK) AML and we evaluated the incidence and prognostic impact of DNMT3Amutations in Korean NK AML patientsMethodsTotal 67 NK AML patients diagnosed during the recent 10 years were enrolled DNMT3A mutations were analyzed by directsequencing and categorized into nonsynonymous variations (NSV) deleterious mutations (DM) and R882 mutation based onin silico analysis results Clinical features and prognosis were compared with respect to DNMT3A mutation status Results Threenovel (I158M K219V and E177V) and two known (R736H and R882H) NSVs were identified and the latter three were predicted asDMs DNMT3A NSVs DMs and R882 mutation were identified in 149ndash179 103ndash104 and 75 of patients respectivelyDNMT3A mutations were frequently detected in FLT3 ITD mutated patients (P = 0054 0071 and 0071 in NSV DMs and R882mutation resp) but did not affect clinical features and prognosis significantly Conclusions Incidences of DNMT3A NSVs DMsand R882 mutation are 149ndash179 103ndash104 and 75 respectively in Korean NK AML patients DNMT3A mutations areassociated with FLT3 ITD mutations but do not affect clinical outcome significantly in Korean NK AML patients

1 Introduction

Acute myeloid leukemia (AML) is a clonal hematologicdisorder exhibiting heterogeneous prognosis based on theresults of cytogenetic andmolecular aberration analysis [1 2]Among patients with AML approximately half of them arecategorized into the intermediate prognosis group definedin the recent National Comprehensive Cancer Network(NCCN) guidelines [3] In AML patients with intermediateprognosis group most of them show normal karyotype (NK)

and the prognosis of these patients generally depends onmolecular aberrations and fms-related tyrosine kinase 3 geneinternal tandem duplications (FLT3 ITD) which are detectedin 30 of AML patients and possess poor prognosis regard-less of mutation status in other genes [4ndash8] nucleophosmin(NPM1) and CCAATenhancer binding protein 120572 (CEBPA)mutations which are detected in 50 and 13ndash15 of NKAML respectively and exhibit good prognosis [9ndash15] havingbeen well established based on various previous studies Inaddition the mutations in isocitrate dehydrogenase (IDH)1

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 723682 11 pageshttpdxdoiorg1011552015723682

2 BioMed Research International

and 2 genes which are identified in 60ndash159 of AMLpatients were recently introduced as an unfavorable prognos-tic indicator in NPM1mutated AML patients [16ndash19]

Besides these mutations described above recent studiesbased on the whole-genome sequencing approach founda new prognostic marker of mutations in DNA methyl-transferase 3A (DNMT3A) gene involving DNA methyla-tion which is the most important epigenetic process inthe regulation of gene expression and chromatin structuralremodeling in the patients withNKAML [20ndash23]DNMT3Amutations have been reported in 18ndash22 of AML (29ndash34of CN-AML) [24ndash27] and previous studies showed that themutations of DNMT3A in AML are frequently found inthe patients with NPM1 mutations and have the mutationhotspot of arginine in 882th codon (R882) [20ndash27] Someprevious studies reported that the patients with DNMT3Amutations show inferior overall survival (OS) compared tothose without mutations [20 21 25] and another recent studyreported a trend of more frequent relapse and inferior OSin the patients who achieved complete remission (CR) whenthey possess DNMT3A mutations [27] However in contrasttoNPM1 andCEBPAmutations inwhich the prognostic valuewas confirmed and which were included as a provisionalentity in the 2008 WHO classifications the prognostic valueof DNMT3A mutations as well as IDH12 mutations has notbeen confirmed up to present time In addition although tworecent studies reported poor prognostic impact of DNMT3Amutations in Chinese and Taiwanese AML patients [28 29]studies which analyzed the clinical relevance of DNMT3Amutations in Asian population are outnumbered comparedto Western population and there have been no reports onthe clinical impact of DNMT3A mutations in Korean AMLpatients

In this study we performed direct sequencing of all 23exons in DNMT3A gene in 39 NK AML patients who werediagnosed at single tertiary hospital in Korea and analyzedthe mutation characteristics In addition we performed insilico analysis to predict the deleterious effect of detectednonsynonymous variations on the protein function andstructure and estimated the clinical relevance of DNMT3Amutations on the prediction of clinical course while includinganother 28 NK AML patients

2 Materials and Methods

21 Patients Selection A total of 60 NK AML patients whowere diagnosed from January 2004 to July 2010 and treatedin PusanNational University Hospital were initially recruitedfrom the retrospective review of electronic medical record(EMR) and a total of 39 patients in whom the quality ofextracted bonemarrow (BM)DNAat diagnosis was sufficientto be analyzed by direct sequencing were finally enrolled inthe first study cohort In these 39 patients direct sequencingof all 23 exons in DNMT3A gene was performed to analyzethe incidence and distribution characteristics of all detectedvariations in DNMT3A gene The median age and follow-upinterval of first study cohort was 390 years (range 110 yearsndash830 years) and 52 months (range 00 monthsndash720 months)

respectively and this cohort included 25 male (641) and 14female (359) patients

In addition total 28 NK AML patients who were diag-nosed from August 2010 to March 2014 at the same hospitalwere additionally enrolled as second study cohort to evaluatethe clinical and prognostic impact of mutation hotspot R882in DNMT3A gene In these patients direct sequencing ofDNMT3A exon 23 was performed The median age andfollow-up interval of second study cohort were 280 years(range 150 yearsndash800 years) and 64 months (range 01monthsndash426 months) respectively and this cohort included12 male (429) and 16 female (571) patients

22 Patients Treatment All these patients received inductionchemotherapy with cytarabine 100mgm2day for seven daysplus daunorubicin 45mgm2day for three days (the ADregimen) or cytarabine 100mgm2day for seven days plusidarubicin 12mgm2day for three days (the AI regimen)CR was defined by less than 5 of residual leukemic blastsin the BM aspirates and more than 20 of BM cellularitynormal maturation of all cellular components (erythrocyticgranulocytic andmegakaryocytic series) in the BMaspiratesnormal values for absolute neutrophil counts (gt1000120583L)and platelet counts (gt100000120583L) after the first or secondcourse of induction chemotherapy Relapsewas defined as thepresence of ge5 leukemic blasts in BM aspirates for patientswho had previously achieved CR Stem cell transplantation(SCT) after first CR achievement were considered as afinal treatment modality depending on the patientrsquos ageavailability of a suitable donor and FLT3 ITDmutation status

As a prognosis indicator CR relapse and death rateswere used and both OS and event-free survival (EFS) wereadditionally calculated OS was defined as the interval fromdiagnosis to death or last follow-up EFS was defined as theinterval from CR to relapse or death for patients who wererelapsed or expired during follow-up or the interval from CRto last follow-up for surviving patients who were not relapsedduring follow-up interval If the patient underwent SCT theCR date was replaced by the SCT date in the calculationof EFS This study was approved by the institutional reviewboard of authorrsquos institution

23 Mutation Analysis of DNMT3A Gene DNA sampleswere extracted from the unstained remnant smear slidesof BM aspirates or chromosomal cell pellets obtained atdiagnosis using DNA Blood Mini extraction kit (QiagenHilden Germany) as recommended by manufacturer All 23exons of DNMT3A gene were amplified with primers listedin Table 1 All forward and reverse primers hadM13F bindingtag (tgtaaaacgacggccagt) andM13Rbinding tag (caggaaacagc-tatgacc) on the 51015840 ends of each primer Polymerase chainreactions (PCR) were conducted with hot start procedureaccording to the manufacturerrsquos instructions (i-StarTaqINTRON Biotechnology Sungnam Korea) described belowan initial denaturation at 95∘C for 5 minutes and by 30 cyclesof following denaturation at 95∘C for 30 seconds annealingat 60∘C for 30 seconds and extension at 72∘C for 30 seconds

BioMed Research International 3

Table 1 The forward and reverse primer sequences used in the amplification of all 23 exons in DNMT3A gene

Primer Primer sequencegtDNMT02F tgtaaaacgacggccagtTCCAAAGACCACGATAATTCCTTCgtDNMT03F tgtaaaacgacggccagtCTGGAATGCTACACTGCTGGGgtDNMT04F tgtaaaacgacggccagtACTTGGAGAAGCGGAGTGAGCgtDNMT05F tgtaaaacgacggccagtGGATGTGTAAAGAAGGAGGAGGGgtDNMT06F tgtaaaacgacggccagtACATTGTGTTTGAGGCGAGTGCgtDNMT07F tgtaaaacgacggccagtCTAATTCCTGGAGAGGTCAAGGTGgtDNMT08F tgtaaaacgacggccagtTCTTGCCTCATTCAGATGGAGCgtDNMT09F tgtaaaacgacggccagtGTGCTTGCAAGTGTAAGCCTCGgtDNMT10F tgtaaaacgacggccagtCTTGAGCCTGACCCATCTGCgtDNMT11F tgtaaaacgacggccagtTTCCTGTCAGCCTGTAACTGACCgtDNMT12F tgtaaaacgacggccagtTTATTGATGAGCGCACAAGAGGgtDNMT13F tgtaaaacgacggccagtAGGGAGAGGCCCTTCGGTGGgtDNMT14F tgtaaaacgacggccagtGGTCATGTCTTCAGGGCTTAGGgtDNMT15F tgtaaaacgacggccagtTTTCCATTCCAGGTAGCACACCgtDNMT16F tgtaaaacgacggccagtAGGGTGTGTGGGTCTAGGAGCgtDNMT17F tgtaaaacgacggccagtGACTTGGGCCTACAGCTGACCgtDNMT18F tgtaaaacgacggccagtATAGGACAGTGGTGTGGCTCGgtDNMT19F tgtaaaacgacggccagtGACAGCTATTCCCGATGACCCgtDNMT20F tgtaaaacgacggccagtGCCGGCGCTGTTTCATGCgtDNMT21F tgtaaaacgacggccagtCCTTCCCGCTGTTATCCAGGgtDNMT22F tgtaaaacgacggccagtTGGCATATTTGGTAGACGCATGACgtDNMT23F tgtaaaacgacggccagtTCCCAGTCCACTATACTGACGTCTCgtDNMT91F tgtaaaacgacggccagtACAAAGAAAATGTTCCCTCCCTCCgtDNMT92F tgtaaaacgacggccagtAAATCTGGTATGGTGGAAATGGGgtDNMT02R caggaaacagctatgaccTCCCTCTCCCAGGCCAGAgtDNMT03R caggaaacagctatgaccATACATCACTGCCATCGACAGGgtDNMT04R caggaaacagctatgaccAAGCAGACCTTTAGCCACGACCgtDNMT05R caggaaacagctatgaccGAACAGCTAAACGGCCAGAGGgtDNMT06R caggaaacagctatgaccACTGAGGCCCATCACTTCTGGgtDNMT07R caggaaacagctatgaccAGATGGAGAGAGGAGAGCAGGACgtDNMT08R caggaaacagctatgaccCCTGGGATCAAGAACCTTCCCgtDNMT09R caggaaacagctatgaccCCTGCACTCCAACTTCCAGGgtDNMT10R caggaaacagctatgaccGCAGGTCATTCAAGTCCTGACCgtDNMT11R caggaaacagctatgaccATGCAGGCCTCCTGGTGCgtDNMT12R caggaaacagctatgaccTCCCATGTCATTCAAACCTTCCgtDNMT13R caggaaacagctatgaccACACAGTCAGCCAGAAGGCCGgtDNMT14R caggaaacagctatgaccTGCTACCTGGAATGGAAAGACCgtDNMT15R caggaaacagctatgaccAGGCTCCTAGACCCACACACCgtDNMT16R caggaaacagctatgaccGCTGTGAAGCTAACCATCATTTCGgtDNMT17R caggaaacagctatgaccAAATGAAAGGAGGCAAGGGCgtDNMT18R caggaaacagctatgaccTTCTTCCTGTCTGCCTCTGTCCgtDNMT19R caggaaacagctatgaccTGCAGATGAGACAGGATGAAGCgtDNMT20R caggaaacagctatgaccCCACTATGGGTCATCCCACCTGCgtDNMT21R caggaaacagctatgaccCATCCTGCCCTTCCTTCTCCgtDNMT22R caggaaacagctatgaccTGGGAAATGCTTGATAAAACCCACgtDNMT23R caggaaacagctatgaccTCTCTCCATCCTCATGTTCTTGGgtDNMT91R caggaaacagctatgaccCCAGCACTAAGTCAGCATCTCCAGgtDNMT92R caggaaacagctatgaccTGGAGTTCTTATGGATCACACCCDNMT3A DNA methyltransferase 3A F forward R reverse


A final extension was done at 72∘C at 5 minutes on Verity-96 well thermal cycler PCR system (Applied Biosystems IncFoster City CA USA)

PCR products were purified with AccuPrep kit (BioneerDaejeon Korea) and were used in the sequencing reactionusing BigDye Terminator v31 Cycle Sequencing Ready Reac-tion kit (Applied Biosystems Inc USA) Direct sequencing ofall 23 exons was performed with ABI 3130 Genetic Analyzer(Applied Biosystems Inc USA) The SeqScape softwareversion 26 (Applied Biosystems Inc USA) was used to alignthe obtained and reference sequences (NM 1756292) and alldetected nonsynonymous and synonymous variations weredescribed according to the guidelines of the Human GeneVariation Society (HGVS httpwwwhgvsorg)

24 In Silico Analysis to Predict the Deleterious Effect of AllDetected Nonsynonymous Variations on the Protein Functionand Structure To predict the deleterious effect of novel andknown sequence variations all detected nonsynonymousvariations were submitted to three web-based programsSorting Intolerant From Tolerent (SIFT httpsiftjcviorg)Polyphen-2 (version 222 httpgeneticsbwhharvardedupph) and MutationTaster (httpwwwmutationtasterorg)Theweb-based database dbSNP (httpwwwncbinlmnihgovprojectsSNP) was applied in the confirmation ofknown SNP and the novelty of detected nonsynonymousvariations was validated by web-based database Catalogueof Somatic Mutations in Cancer (COSMIC httpcancersangeracukcancergenomeprojectscosmic) Cn3D mac-romolecular structure viewer was used to annotate themutation sites in 3D structure [30]

25 Comparison of Clinical Characteristics and Prognosis inPatientswith respect toMutation Status ofDNMT3AGene Toassess the clinical relevance of DNMT3A mutation analysisthe comparison of clinical characteristics and prognosisin NK AML patients with respect to mutation status ofDNMT3A geneswas performedAll patientswere categorizedinto two subgroups with respect to nonsynonymous variationstatus of DNMT3A gene and both clinical features and prog-nosis were compared between two subgroups to evaluate theclinical relevance of nonsynonymous variations of DNMT3Agene Subsequently nonsynonymous variations which werepredicted as deleterious mutation in at least two of three insilico analyses were recruited and identical comparisons wereperformed again between patients with deleteriousmutationsand those without deleterious mutations to further evaluatethe clinical relevance of deleterious mutations in DNMT3Agene

Finally to evaluate the clinical relevance of DNMT3AR882mutation hotspot all patients were categorized into twosubgroups with respect to DNMT3A R882 mutation statusand identical comparisons were performed again Clinicaldata of patients were obtained from retrospective reviewof EMR and included age and gender complete blood cell(CBC) counts blasts () in BMaspirates FLT3 ITD andD835mutation status (at diagnosis) and date of CR relapse anddeath (during follow-up)

26 Multivariate Analysis of Overall Survival For the iden-tification of prognostic impact in DNMT3A mutations inassociation with other possible prognostic indicators suchas FLT3 ITD mutation status age BM blast () and SCTperformance status the multivariate analysis was performedwhen the clinical variablesmentioned above were included ascovariables

27 Statistical Analysis Pearson chi-square yest or Fisherrsquosexact test (for small numbers less than 5) were applied tocompare categorical variables (gender CR rates relapse ratesand death rates) between each two patient subgroups Mann-Whitney 119880 test was used to compare continuous variables(age CBC counts at diagnosis and interval from diagnosis toCR) between each two patient subgroups Estimates of OSand EFS were generated using Kaplan-Meier methods andwere compared using a log-rank test Multivariate analysesof OS were performed with a Coxrsquos proportional hazardsmodel For all analyses tests were two-tailed and 119875 values le005 and 005ndash010 were considered statistically significantand marginally significant respectively All calculations wereperformed using SPSS 1301 forWindows (SPSS Inc ChicagoIL USA)

3 Results

31 Summary of Detected Variations in DNMT3A GeneAmong 39 patients included in the first study cohort twoknown synonymous variations (c27CgtT (rs41284843 minorallele frequency = 00851) and c1266GgtA (rs2276598 minorallele frequency = 02337)) and one novel synonymousvariation (c2043CgtG) were detected in 5 19 (including 2homozygotes) and 6 patients (128 487 and 154)respectively

Total five different nonsynonymous variations whichare all missense mutations were detected in seven (179)patients and included two known mutations (c2207GgtA(pArg736His COSM133737 detected in one patient) andc2645GgtA (pArg882His COSM52946 detected in twopatients)) and three novelmutations (c474CgtG(pIle158Metdetected in two patients) c530AgtT (pGlu177Val detectedin one patient) and c656AgtG (pLys219Arg detected in twopatients)) All 7 patients with DNMT3A mutations harboredsingle mutation except for one patient who showed bothK219R and R736H mutations simultaneously All 2 knownmutations (R736HandR882H) induced protein change in thecatalytic domain of DNMT3A protein which has critical rolein methyltransferase activity and all three novel mutations(I158M E177V and K219R) were involved in protein changeat the regulatory domain of DNMT3A protein These resultsare summarized in Figure 1

Subsequently performed direct sequencing of DNMT3Aexon 23 in 28 NKAML patients who were included in secondstudy cohort detected additional three R882H mutationsWhen summarizing these results the incidence of DNMT3Anonsynonymous variations was calculated to be 739 (179)in the first study cohort and finally estimated to be 1067(149) when adding data from 28 patients in the second


S

A A ACC C CCCY CCC

S G

GG G GGGG

P D T

T

G

tccag gg gggga ac cccc c cc

E E

A A A A A A A AC C C C CS

SI

G G G G

MT

T T T

N

t ttc c c c c ca a aa a aaag g g g

E F

A A AC CC CC CC CRG GG TT T T T T

V R L L H

tt t t t tc cc cc cc ca a ag g g g

P E

AA A A ACC CC CC CCR

PL

GGG G G

PK

T T

G

t tcc cc cc ccaa a a aggg gg g

AA AA A ACC CC CC CCGGG G GT T


P E PL PK G

AC CC CC CGGG GGG G GTT T T T

tt t t tc cc cc ca aggg ggg g g

L S SE LG W

N AA A A AC CC C C CG G GG G GGT TT

t ttc cc c c caa a a ag g g g g ggg

N L AR RM S

R

Q M

A AA A A AC C CS

YI

GGGG G G G

VG

T T T T

K

t t t tc c c ca aa a a agggg g g g

K V

AA AAA A A ACC C CR

IK

G G GG G

AK

T TT

A

t ttcc caa aaa aa a cag g gg g

P9 = (c27CgtT) N = 5

L422 = (c1266GgtA) N = 17

HomozygoteL422 = (c1266GgtA) N = 2

I681= (c2043CgtG) N = 6

I158M= (c474CgtG) N = 2 R736H = (c2207GgtA) N = 1

E177V = (c530AgtT) N = 1 R882H = (c2645GgtA) N = 2

K219R= (c656AgtG) N = 2

Synonymous variationsNonsynonymous variations

Regulatory domain Catalytic domain

(a)

PWWP ZNF MeTFase

0 100 200 300 400 500 600 700 800 900

P9 =

I158M

E177V

K219R

L422 =

I681= R736HR882H

(b)

Figure 1 Electropherograms of all DNMT3A variations including synonymous and nonsynonymous variations observed in this study (a)and schematic summary representing locations and frequency of all detected DNMT3A variations (b) Note (a) shows electropherogram ofall detected DNMT3A variations in 39 patients included in the first study cohort (b) shows schematic summary of all detected DNMT3Avariation including location and frequency The number of circles indicates the frequency of detected variations Closed black and whitecircle indicates heterozygous nonsynonymous and synonymous variation case respectively and closed double-layered white circle indicateshomozygous synonymous variation case Abbreviations P proline I isoleucine M methionine R arginine H histidine L leucine Eglutamate V valine K lysine PWWP Pro-Trp-Trp-Pro motif ZNF zinc finger MeTFase methyltransferase

study cohort and the frequency ofDNMT3AR882mutationswas exactly calculated to be 567 (75)

32 In Silico Analysis Results to Predict the Deleterious Effectof Detected Nonsynonymous Variations on the Protein Func-tion and Structure Among five DNMT3A nonsynonymousvariations detected in this study R882H was predicted as

a deleterious mutation by all three web-based predictionprograms as reported in the previous study [21] and anotherknown variation (R736H) was also predicted as a deleteriousmutation by two programs In addition one novel variation(E177V) was also predicted as a deleterious mutation by allthree programs In contrast two novel variations (I158M andK219R) were predicted as a benign or tolerated variation in


Table 2 In silico analysis results of three web-based programs to predict the deleterious effect of detected nonsynonymous variations on theprotein function and structure

Mutations Sequence variationdatabase

SIFT Polyphen-2 MutationTasterScore Prediction Score Prediction Score Prediction

I158M Novel 021 Tolerated 0191 Benign 027 PolymorphismE177V Novel 002 Damaging 0981 Probably damaging 330 Disease causingK219R Novel 071 Tolerated 0002 Benign 071 Polymorphism

R736H rs139293773COSM133737 039 Tolerated 0997 Probably damaging 079 Disease causing

R882H rs147001633COSM52946 003 Damaging 0651 Possibly damaging 079 Disease causing

I isoleucine M methionine E glutamate V valine K lysine R arginine H histidine

all three programs These results are represented in Table 2With these results the frequency of deleterious mutation ofDNMT3A gene was calculated to be 439 (103) in the firststudy cohort and finally estimated to be 767 (104) whenadding data from 28 patients in the second study cohort

In the 3D structure model of methyltransferase domainsR882 residue was located in the region interfacing with twoDNMT3A molecules which is consistent with the results ofprevious literature [21] and R736 residue was located on theopposite side and toward DNMT3L molecule These resultsare illustrated in the Supplemental Figure 1 available onlineat httpdxdoiorg1011552015723682

33 Comparison of Clinical Characteristics and Prognosis inPatients with respect toMutation Status of DNMT3AGene Inthe comparison of patients with DNMT3A nonsynonymousvariations and those without nonsynonymous variationsboth clinical features and prognosis (OS and EFS) werenot significantly different between two patient subgroupsexcept for the trend of high FLT3 ITD mutation frequencyin patients with DNMT3A nonsynonymous variations thanthose who do not harbor (667 versus 244 119875 = 0054)Identical results were found in the comparison with respectto DNMT3A deleterious mutation status since the patientswith DNMT3A deleterious mutations also showed the trendof more frequent FLT3 ITD mutation than those withoutDNMT3A deleterious mutations (750 versus 255 119875 =0071) Additionally performed comparison between patientswith DNMT3A R882 mutation and those without R882mutation also showed identical results againThese results aresummarized in Table 3 and Figure 2

34 Multivariate Analysis of Overall Survival Themultivari-ate analysis of OS in total 67 NK AML patients demonstratedthat the presence of FLT3 ITD mutation is a significantlypoor prognostic indicator of OS (hazard ratio 2798 119875 =0041) and the performance of SCT during follow-up isa significantly good prognostic indicator of OS (hazard ratio0118 119875 = 0001) as expected However the presence ofDNMT3A nonsynonymous variations (hazard ratio 1229119875 = 0724) deleterious mutations and R882 mutations(hazard ratio 2285 119875 = 0313 in both) did not affect OSsignificantly as well as increasing age and BM blasts ()

although the trend was toward to adverse prognosis Theseresults are described in Table 4

4 Discussion

Previous studies that analyzed DNMT3A mutations showedthat the incidence of DNMT3A mutation is 18ndash22 of AMLand 29ndash34 of NK AML [24ndash27] including the largeststudy which analyzed 489 AML patients and reported thefrequency of DNMT3A mutation as 178 of AML patientsand 272 of NK AML patients [24] In Korean populationalthough the data of clinical outcome was not provideda recent study reported the incidence of DNMT3A R882mutation as 94 in adulthood AML [31] Our present studyshowed that the incidence of DNMT3A nonsynonymousvariations is 179 in first study cohort and 149 in extendedcohort which is similar to the results from previous studies[24ndash27] In addition our present study analyzed the inci-dence of DNMT3A R882 mutation as 75 which is alsosimilar to the previous data in Korean population [31] Allthese results demonstrate thatDNMT3Amutation can be alsoconsidered as a frequently occurring nonrandommutation inKorean NK AML patients

Our present study applied direct sequencing of all 23exons in DNMT3A gene to identify all possible variationsaffectingDNMT3A protein function and structure and coulddetect total 8 different sequence variations including 3synonymous and 5 nonsynonymous variations Among threesynonymous variations two known variations (c27CgtT andc1266GgtA) of which minor allele frequencies are 851 and2337 were found with the frequency of 128 and 487respectively and therefore these two variations can be con-sidered as constitutional single nucleotide polymorphisms(SNP) In addition our present study could detect one novelsynonymous variation (c2043CgtG) with the incidence of154 and this novel SNP can be listed in the current SNPdatabases

For the nonsynonymous variations we could find 3different novel variations (I158M E177V and K219V) in theregulatory domains of DNMT3A protein that have a rolein interaction with other molecules and protein localizationto the nuclei as well as 2 known variations (R736H andR882) located in the catalytic methyltransferase domain of


Table3Com

paris

onof

demograph

icandclinicalfeaturesinall67NKAMLpatie

ntsw

ithrespecttoDNMT3

Ano

nsyn

onym

ousv

ariatio

nsta

tus

Clinicalcharacteris

tics

DNMT3

Ano

nsyn

onym

ousv

ariatio

nsta

tus119875value

DNMT3

Adeleterio

usmutationsta

tus119875value

DNMT3

AR8

82mutationstatus

119875value

Novaria

tion(119873=57)

Varia

tion(119873=10)

Nomutation(119873=60)

Mutation(119873=7)


Mutation(119873=5)

Sex(M

F)lowast

3324

46

NS

3426

34

NS

3527

23

NS

Ageyearsm

edian(range)lowastlowast

560(110

ndash800)

620(180ndash

830)

NS

560(110

ndash800)

590(180ndash

830)

NS

560(110

ndash830)

590(180ndash

770)

NS

Labo

ratory

finding

sat

diagno

sism

edian(range)

WBC

(times10

9 L)lowastlowast

356(09ndash3689)

638(06ndash2822)

NS

363(06ndash3689)

555(09ndash2060)

NS

336(06ndash3689)

722(09ndash2060)

NS

Hb(gdL)lowastlowast

86(54ndash142)

77(56ndash107)

NS

86(54ndash142)

73(56ndash107)

NS

86(54ndash142)

73(56ndash107)

0097

Platele

ts(times10

9 L)lowastlowast

540(50ndash1720)

620(380ndash

2700)

NS

540(50ndash1720)

960(430ndash

2700)

NS

535(50ndash1720)

1130(510

ndash2700)

NS

BMblasts(

)lowastlowast

730(221ndash970)

743(295

ndash962)

NS

729(221ndash970)

750(295

ndash843)

NS

729(221ndash970)

750(295

ndash843)

NS

FLT3

ITDmutation(

)lowast1145(244)

46(667)

0054

1247(255)

34(750)

0071

1247(255)

34(750)

0071

FLT3

D835mutation(

)lowast245

(44)

06(00)

NS

247

(43)

04(00)

NS

247

(43)

04(00)

NS

CRrates()lowast

3357(579

)310

(300)

NS

3460(567)

27(286)

NS

3462(548)

25(400)

NS

Intervalfro

mDxto

CRdays

median(range)lowastlowast

340(210

ndash830)

530(250ndash

1640)

NS

350(210

ndash164

0)

390(250ndash

530)

NS

350(210

ndash164

0)

390(250ndash

530)

NS

Relap

serates()lowast

1633(485)

13(333)

NS

1734(500)

02(00)

NS

1734(500)

02(00)

NS

Death

rates()lowast

3157(544)

510

(500)

NS

3460(567)

27(286)

NS

3462(548)

25(400)

NS

SCTperfo

rmance

rates()lowast

1957(333)

110(100)

NS

1960(317

)17

(143)

NS

1962(306)

15(200)

NS

Note119875values

wereo

btainedfro

mCh

i-squ

aretestF

isherrsquosexacttestlowast

andMann-Whitney119880testlowastlowast

WBC

white

bloo

dcell

Hb

hemoglobin

BMb

onemarrowF

LT3

fms-related

tyrosin

ekinase

3ITDinternaltandem

duplicationsD

aspartateC

Rcompleteremission

Dx

diagno

sisS

CTstem

cell

transplantation

DNMT3

AD

NAmethyltransfe

rase

3AR

arginineNSno

tsignificant


25002000150010005000Overall survival date (days)

10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

Nonsynonymous variations

P = 0384

Yes (N = 10)No (N = 57)

(a)

Yes (N = 7)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

Deleterious mutations

P = 0833

No (N = 60)

(b)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

R882 mutation

P = 0726

Yes (N = 5)No (N = 62)

(c)


Yes (N = 10)

2000150010005000Event free survival date (days)

10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0771

No (N = 57)

(d)

Yes (N = 7)



10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0351

No (N = 60)

(e)

R882 mutation

Yes (N = 5)No (N = 62)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0351

(f)

Figure 2 Comparisons of overall survival and event-free survival in total 67 NK AML patients with respect to DNMT3A nonsynonymousvariations ((a) and (d)) deleterious mutations ((b) and (e)) and R882 mutation ((c) and (f)) respectively Abbreviation R arginine

DNMT3A protein In addition in silico analysis performedin our study predicted not only both R736H and R882H vari-ations in catalytic domain but also a novel E177V variation inregulatory domain to be deleterious to protein function andpreviously known mutation hotspot R882 was not frequentlydetected in our present study These results can be novelfindings of our present study since R882 residue ofDNMT3Agene has been regarded as a frequently mutated hotspot inprevious studies [20 21 24ndash27] which would have a gain-of-function activity or dominant negative effects [20] Allthese results may suggest the presence of ethnic variationin the characteristics of DNMT3A mutations and justify theapplication of detection method which covers all exons ofDNMT3A gene rather thanmutation hotspot R882 in Koreanpopulation

Our structural analysis using 3D model showed thatR736 residue is exposed to the DNMT3L molecules thatare a stimulating factor of DNMT3A and a mediator oftranscriptional repression through interaction with histonedeacetylase 1 [30] Therefore it may be considered thatthe alteration of R736 residue may affect the efficiency and

regulation of DNMT3A protein function as similarly withpreviously demonstrated R882 residue [21] In contrast inthe regulatory domain of DNMT3A protein only E177V wasidentified as deleterious mutation and we could not providethe possible explanation for this result in our present studyMore extensive functional analysis on the DNMT3A proteinshould be required for the clarification of this finding

DNMT3A mutations have been reported to occur inpatients with NPM1 and FLT3 mutations [24 25 27] butthe clinical and prognostic impact of DNMT3A mutationshas not been confirmed yet Some studies revealed thepoor prognostic effect of DNMT3A mutations on survivaloutcome both in overall [25] and in high risk (FLT3 ITDmutated but NPM1 nonmutated) NK AML patients [24]Two studies performed in Asian population also reportedpoor prognostic impact of DNMT3Amutations in NK AML[28 29] In contrast other studies also reported no significantprognostic effect of DNMT3A mutations in AML patientswith intermediate risk group [27] and in low risk (NPM1mutated but FLT3 ITD unmutated) NK AML patients [24]A recent study reported that the poor prognostic relevance


Table 4 Multivariate analysis of overall survival

Variables Overall survivalHR (95 CI) 119875 value Prognostic impact

All 67 patientsFLT3 ITD mutation (compared with wild type) 2798 (1041ndash7517) 0041 PoorDNMT3Amutation (compared with wild type)Nonsynonymous variations 1229 (0391ndash3864) 0724 NSlowast

Deleterious mutations 2285 (0459ndash11374) 0313 NSlowast

R882 mutation 2285 (0459ndash11374) 0313 NSlowast

Increasing age 1023 (0991ndash1055) 0157 NSIncrerasing BM blasts 1018 (0986ndash1051) 0285 NSSCT performed (compared to not performed) 0118 (0035ndash0399) 0001 Good

Note 119875 values were adjusted with FLT3 ITD mutation status age BM blast percentage and stem cell transplantation performance status (in all 67 patientslowast)FLT3 fms-related tyrosine kinase 3 ITD internal tandem duplications DNMT3A DNAmethyltransferase 3A R arginine BM bone marrow SCT stem celltransplantation HR hazard ratio CI confidence interval NS not significant

of DNMT3A mutation is dependent on age and its mutationtype (R882 versus non-R882) [26] Our present study couldalso demonstrate a trend of association between FLT3 ITDmutation and DNMT3A mutations in terms of mutationfrequency as reported in the previous literature [24 25 27]However our present study could not demonstrate a signif-icant effect of DNMT3A mutations on both clinical featuresand prognosis regardless of mutation type (nonsynonymousvariations deleterious mutations or R882 mutation hotspot)in Korean NK AML patients although the trend was towardadverse prognosis This result may support previously givenhypothesis in our present study referring the presence ofethnic variation in the characteristics ofDNMT3AmutationsIn addition from our present study result it can be speculatedthat the adverse prognostic effect ofDNMT3Amutationsmaynot exist in overall Korean NK AML patients and althoughsome variations are predicted to be deleterious to proteinfunction in our study the adverse prognostic impact mayreside in all non-synonymous variations of DNMT3A geneand therefore all 23 exons of DNMT3A gene should bescreened for the detection of clinically important DNMT3Amutations

Our study has some important limitations in terms ofsmall patient numbers in each subgroup and mutation anal-ysis of other genes with clinical significance First our studycohort included total 67 patients (39 in first and 28 in secondstudy cohort) who were diagnosed and treated in singletertiary hospital and statistical power in the comparisonof clinical features and prognosis with respect to DNMT3Amutation status especially in the analysis focused on FLT3ITD mutated patients is estimated to be significantly lowTherefore our present results should be interpreted withcaution and regarded to be preliminary data providing futureissues to be addressed Second although both NPM1 andCEBPA mutations have prognostic value in NK AML andsome previous study also suggested the association of NPM1and DNMT3A mutations [24 25 27] our present studydid not include the data of NPM1 and CEBPA mutationstatus and therefore comprehensive analysis in terms ofmutational relations among three genes (NPM1 CEBPA andDNMT3A) could not be performed More large scaled and

comprehensive study with these data should be required inthe future to address these points

5 Conclusions

Our present study identified three novel (I158M E177V andK219V in the regulatory domain) and two known (R736HandR882H in the methyltransferase domain) DNMT3A nonsyn-onymous variations and three (E177V R736H and R882H)of them were predicted to be deleterious to protein functionand structure The incidences of DNMT3A nonsynonymousvariations deleterious mutations and R882 mutation were149ndash179 103ndash104 and 75 respectively in KoreanNKAMLpatients without predominance of knownmutationhotspot R882 DNMT3Amutations were frequently detectedin FLT3 ITD mutated NK AML patients but did not signif-icantly affect clinical features and prognosis in Korean NKAML patients and this issue needs to be further evaluatedin the future

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by Biomedical Research Instituteand Pusan Cancer Center Grant (2011-30) Pusan NationalUniversity Hospital

References

[1] J C Byrd K Mrozek R K Dodge et al ldquoPretreatmentcytogenetic abnormalities are predictive of induction successcumulative incidence of relapse and overall survival in adultpatients with de novo acute myeloid leukemia results fromcancer and leukemia group B (CALGB 8461)rdquo Blood vol 100no 13 pp 4325ndash4336 2002

[2] D Grimwade H Walker F Oliver et al ldquoThe importanceof diagnostic cytogenetics on outcome in AML analysis of


1612 patients entered into the MRC AML 10 trial The MedicalResearch Council Adult and Childrenrsquos Leukaemia WorkingPartiesrdquo Blood vol 92 pp 2322ndash2333 1998

[3] M R OrsquoDonnell C N Abboud J Altman et al ldquoAcute myeloidleukemiardquo Journal of the National Comprehensive Cancer Net-work vol 10 no 8 pp 984ndash1021 2012

[4] S P Whitman K J Archer L Feng et al ldquoAbsence of thewild-type allele predicts poor prognosis in adult de novo acutemyeloid leukemia with normal cytogenetics and the internaltandem duplication of FLT3 a cancer and leukemia group Bstudyrdquo Cancer Research vol 61 no 19 pp 7233ndash7239 2001

[5] Y Yamamoto H Kiyoi Y Nakano et al ldquoActivatingmutation ofD835 within the activation loop of FLT3 in human hematologicmalignanciesrdquo Blood vol 97 no 8 pp 2434ndash2439 2001

[6] B Kainz D Heintel R Marculescu et al ldquoVariable prognosticvalue of FLT3 internal tandem duplications in patients with denovo AML and a normal karyotype t(1517) t(821) or inv(16)rdquoThe Hematology Journal vol 3 no 6 pp 283ndash289 2002

[7] S P Whitman K Maharry M D Radmacher et al ldquoFLT3internal tandem duplication associates with adverse outcomeand gene- and microRNA-expression signatures in patients 60years of age or older with primary cytogenetically normal acutemyeloid leukemia a cancer and leukemia group B studyrdquo Bloodvol 116 no 18 pp 3622ndash3626 2010

[8] S P Whitman A S Ruppert M D Radmacher et al ldquoFLT3D835I836 mutations are associated with poor disease-free sur-vival and a distinct gene-expression signature among youngeradults with de novo cytogenetically normal acute myeloidleukemia lacking FLT3 internal tandem duplicationsrdquo Bloodvol 111 no 3 pp 1552ndash1559 2008

[9] S Schnittger C Schoch W Kern et al ldquoNucleophosmingene mutations are predictors of favorable prognosis in acutemyelogenous leukemia with a normal karyotyperdquo Blood vol106 no 12 pp 3733ndash3739 2005

[10] K Dohner R F Schlenk M Habdank et al ldquoMutant nucle-ophosmin (NPM1) predicts favorable prognosis in youngeradults with acute myeloid leukemia and normal cytogeneticsinteraction with other gene mutationsrdquo Blood vol 106 no 12pp 3740ndash3746 2005

[11] R G W Verhaak C S Goudswaard W van Putten etal ldquoMutations in nucleophosmin (NPM1) in acute myeloidleukemia (AML) association with other gene abnormalitiesand previously established gene expression signatures and theirfavorable prognostic significancerdquo Blood vol 106 no 12 pp3747ndash3754 2005

[12] S Frohling R F Schlenk I Stolze et al ldquoCEBPA mutationsin younger adults with acute myeloid leukemia and normalcytogenetics prognostic relevance and analysis of cooperatingmutationsrdquo Journal of Clinical Oncology vol 22 no 4 pp 624ndash633 2004

[13] C L Green K K Koo R K Hills A K Burnett D CLinch and R E Gale ldquoPrognostic significance of CEBPAmutations in a large cohort of younger adult patients with acutemyeloid leukemia impact of double CEBPAmutations and theinteraction with FLT3 andNPM1mutationsrdquo Journal of ClinicalOncology vol 28 no 16 pp 2739ndash2747 2010

[14] C Preudhomme C Sagot N Boissel et al ldquoFavorable prog-nostic significance of CEBPA mutations in patients with denovo acutemyeloid leukemia a study from the Acute LeukemiaFrench Association (ALFA)rdquo Blood vol 100 no 8 pp 2717ndash2723 2002

[15] S H Park H S Chi Y U Cho S Jang and C J Park ldquoCEBPAsingle mutation can be a possible favorable prognostic indicatorin NPM1 and FLT3-ITD wild-type acute myeloid leukemiapatientswith intermediate cytogenetic riskrdquoLeukemiaResearchvol 37 no 11 pp 1488ndash1494 2013

[16] S Abbas S Lugthart F G Kavelaars et al ldquoAcquiredmutationsin the genes encoding IDH1 and IDH2 both are recurrent aber-rations in acute myeloid leukemia prevalence and prognosticvaluerdquo Blood vol 116 no 12 pp 2122ndash2126 2010

[17] P Paschka R F Schlenk V I Gaidzik et al ldquoIDH1 and IDH2mutations are frequent genetic alterations in acute myeloidleukemia and confer adverse prognosis in cytogenetically nor-mal acutemyeloid leukemiawithNPM1mutationwithout FLT3internal tandem duplicationrdquo Journal of Clinical Oncology vol28 no 22 pp 3636ndash3643 2010

[18] H-Y Chao Z-X Jia T Chen et al ldquoIDH2 mutations arefrequent in Chinese patients with acute myeloid leukemiaand associated with NPM1 mutations and FAB-M2 subtyperdquoInternational Journal of Laboratory Hematology vol 34 no 5pp 502ndash509 2012

[19] F Ravandi K Patel R Luthra et al ldquoPrognostic significanceof alterations in IDH enzyme isoforms in patients with AMLtreated with high-dose cytarabine and idarubicinrdquo Cancer vol118 no 10 pp 2665ndash2673 2012

[20] T J Ley L Ding M J Walter et al ldquoDNMT3A mutations inacute myeloid leukemiardquoTheNew England Journal of Medicinevol 363 no 25 pp 2424ndash2433 2010

[21] X-J Yan J Xu Z-H Gu et al ldquoExome sequencing identifiessomatic mutations of DNA methyltransferase gene DNMT3Ain acute monocytic leukemiardquo Nature Genetics vol 43 no 4pp 309ndash315 2011

[22] M A McDevitt ldquoClinical applications of epigenetic markersand epigenetic profiling in myeloid malignanciesrdquo Seminars inOncology vol 39 no 1 pp 109ndash122 2012

[23] J J Trowbridge J W Snow J Kim and S H Orkin ldquoDNAmethyltransferase 1 is essential for and uniquely regulateshematopoietic stem and progenitor cellsrdquo Cell Stem Cell vol 5no 4 pp 442ndash449 2009

[24] FThol F Damm A Ludeking et al ldquoIncidence and prognosticinfluence of DNMT3A mutations in acute myeloid leukemiardquoJournal of Clinical Oncology vol 29 no 21 pp 2889ndash2896 2011

[25] A Renneville N Boissel O Nibourel et al ldquoPrognosticsignificance of DNAmethyltransferase 3Amutations in cytoge-netically normal acute myeloid leukemia a study by the AcuteLeukemia French Associationrdquo Leukemia vol 26 no 6 pp1247ndash1254 2012

[26] G Marcucci K H Metzeler S Schwind et al ldquoAge-relatedprognostic impact of different types of DNMT3A mutationsin adults with primary cytogenetically normal acute myeloidleukemiardquo Journal of Clinical Oncology vol 30 no 7 pp 742ndash750 2012

[27] J Markova P Michkova K Burckova et al ldquoPrognostic impactof DNMT3A mutations in patients with intermediate cytoge-netic risk profile acute myeloid leukemiardquo European Journal ofHaematology vol 88 no 2 pp 128ndash135 2012

[28] Y Shen Y M Zhu X Fan et al ldquoGene mutation patterns andtheir prognostic impact in a cohort of 1185 patients with acutemyeloid leukemiardquo Blood vol 118 no 20 pp 5593ndash5603 2011

[29] H-A Hou Y-Y Kuo C-Y Liu et al ldquoDNMT3A mutations inacute myeloid leukemia stability during disease evolution andclinical implicationsrdquo Blood vol 119 no 2 pp 559ndash568 2012


[30] Y Wang L Y Geer C Chappey J A Kans and S H BryantldquoCn3D sequence and structure views for Entrezrdquo Trends inBiochemical Sciences vol 25 no 6 pp 300ndash302 2000

[31] M S Kim Y R Kim N J Yoo and S H Lee ldquoMutationalanalysis of DNMT3A gene in acute leukemias and commonsolid cancersrdquo APMIS vol 121 no 2 pp 85ndash94 2013


and 2 genes which are identified in 60ndash159 of AMLpatients were recently introduced as an unfavorable prognos-tic indicator in NPM1mutated AML patients [16ndash19]

Besides these mutations described above recent studiesbased on the whole-genome sequencing approach founda new prognostic marker of mutations in DNA methyl-transferase 3A (DNMT3A) gene involving DNA methyla-tion which is the most important epigenetic process inthe regulation of gene expression and chromatin structuralremodeling in the patients withNKAML [20ndash23]DNMT3Amutations have been reported in 18ndash22 of AML (29ndash34of CN-AML) [24ndash27] and previous studies showed that themutations of DNMT3A in AML are frequently found inthe patients with NPM1 mutations and have the mutationhotspot of arginine in 882th codon (R882) [20ndash27] Someprevious studies reported that the patients with DNMT3Amutations show inferior overall survival (OS) compared tothose without mutations [20 21 25] and another recent studyreported a trend of more frequent relapse and inferior OSin the patients who achieved complete remission (CR) whenthey possess DNMT3A mutations [27] However in contrasttoNPM1 andCEBPAmutations inwhich the prognostic valuewas confirmed and which were included as a provisionalentity in the 2008 WHO classifications the prognostic valueof DNMT3A mutations as well as IDH12 mutations has notbeen confirmed up to present time In addition although tworecent studies reported poor prognostic impact of DNMT3Amutations in Chinese and Taiwanese AML patients [28 29]studies which analyzed the clinical relevance of DNMT3Amutations in Asian population are outnumbered comparedto Western population and there have been no reports onthe clinical impact of DNMT3A mutations in Korean AMLpatients

In this study we performed direct sequencing of all 23exons in DNMT3A gene in 39 NK AML patients who werediagnosed at single tertiary hospital in Korea and analyzedthe mutation characteristics In addition we performed insilico analysis to predict the deleterious effect of detectednonsynonymous variations on the protein function andstructure and estimated the clinical relevance of DNMT3Amutations on the prediction of clinical course while includinganother 28 NK AML patients

2 Materials and Methods

21 Patients Selection A total of 60 NK AML patients whowere diagnosed from January 2004 to July 2010 and treatedin PusanNational University Hospital were initially recruitedfrom the retrospective review of electronic medical record(EMR) and a total of 39 patients in whom the quality ofextracted bonemarrow (BM)DNAat diagnosis was sufficientto be analyzed by direct sequencing were finally enrolled inthe first study cohort In these 39 patients direct sequencingof all 23 exons in DNMT3A gene was performed to analyzethe incidence and distribution characteristics of all detectedvariations in DNMT3A gene The median age and follow-upinterval of first study cohort was 390 years (range 110 yearsndash830 years) and 52 months (range 00 monthsndash720 months)

respectively and this cohort included 25 male (641) and 14female (359) patients

In addition total 28 NK AML patients who were diag-nosed from August 2010 to March 2014 at the same hospitalwere additionally enrolled as second study cohort to evaluatethe clinical and prognostic impact of mutation hotspot R882in DNMT3A gene In these patients direct sequencing ofDNMT3A exon 23 was performed The median age andfollow-up interval of second study cohort were 280 years(range 150 yearsndash800 years) and 64 months (range 01monthsndash426 months) respectively and this cohort included12 male (429) and 16 female (571) patients

22 Patients Treatment All these patients received inductionchemotherapy with cytarabine 100mgm2day for seven daysplus daunorubicin 45mgm2day for three days (the ADregimen) or cytarabine 100mgm2day for seven days plusidarubicin 12mgm2day for three days (the AI regimen)CR was defined by less than 5 of residual leukemic blastsin the BM aspirates and more than 20 of BM cellularitynormal maturation of all cellular components (erythrocyticgranulocytic andmegakaryocytic series) in the BMaspiratesnormal values for absolute neutrophil counts (gt1000120583L)and platelet counts (gt100000120583L) after the first or secondcourse of induction chemotherapy Relapsewas defined as thepresence of ge5 leukemic blasts in BM aspirates for patientswho had previously achieved CR Stem cell transplantation(SCT) after first CR achievement were considered as afinal treatment modality depending on the patientrsquos ageavailability of a suitable donor and FLT3 ITDmutation status

As a prognosis indicator CR relapse and death rateswere used and both OS and event-free survival (EFS) wereadditionally calculated OS was defined as the interval fromdiagnosis to death or last follow-up EFS was defined as theinterval from CR to relapse or death for patients who wererelapsed or expired during follow-up or the interval from CRto last follow-up for surviving patients who were not relapsedduring follow-up interval If the patient underwent SCT theCR date was replaced by the SCT date in the calculationof EFS This study was approved by the institutional reviewboard of authorrsquos institution

23 Mutation Analysis of DNMT3A Gene DNA sampleswere extracted from the unstained remnant smear slidesof BM aspirates or chromosomal cell pellets obtained atdiagnosis using DNA Blood Mini extraction kit (QiagenHilden Germany) as recommended by manufacturer All 23exons of DNMT3A gene were amplified with primers listedin Table 1 All forward and reverse primers hadM13F bindingtag (tgtaaaacgacggccagt) andM13Rbinding tag (caggaaacagc-tatgacc) on the 51015840 ends of each primer Polymerase chainreactions (PCR) were conducted with hot start procedureaccording to the manufacturerrsquos instructions (i-StarTaqINTRON Biotechnology Sungnam Korea) described belowan initial denaturation at 95∘C for 5 minutes and by 30 cyclesof following denaturation at 95∘C for 30 seconds annealingat 60∘C for 30 seconds and extension at 72∘C for 30 seconds












3 Results





S

A A ACC C CCCY CCC

S G

GG G GGGG

P D T

T

G


E E


SI

G G G G

MT

T T T

N


E F


V R L L H


P E


PL

GGG G G

PK

T T

G




P E PL PK G



L S SE LG W



N L AR RM S

R

Q M

A AA A A AC C CS

YI

GGGG G G G

VG

T T T T

K


K V

AA AAA A A ACC C CR

IK

G G GG G

AK

T TT

A


P9 = (c27CgtT) N = 5

L422 = (c1266GgtA) N = 17


I681= (c2043CgtG) N = 6



K219R= (c656AgtG) N = 2



(a)

PWWP ZNF MeTFase

0 100 200 300 400 500 600 700 800 900

P9 =

I158M

E177V

K219R

L422 =

I681= R736HR882H

(b)


















4 Discussion





Table3Com

paris

onof

demograph


ntsw

ithrespecttoDNMT3

Ano

nsyn

onym

ousv

ariatio

nsta

tus

Clinicalcharacteris

tics

DNMT3

Ano

nsyn

onym

ousv

ariatio

nsta

tus119875value

DNMT3

Adeleterio

usmutationsta

tus119875value

DNMT3

AR8

82mutationstatus

119875value

Novaria

tion(119873=57)

Varia

tion(119873=10)


Mutation(119873=7)


Mutation(119873=5)

Sex(M

F)lowast

3324

46

NS

3426

34

NS

3527

23

NS

Ageyearsm


560(110

ndash800)

620(180ndash

830)

NS

560(110

ndash800)

590(180ndash

830)

NS

560(110

ndash830)

590(180ndash

770)

NS

Labo

ratory

finding

sat

diagno

sism

edian(range)

WBC

(times10

9 L)lowastlowast

356(09ndash3689)

638(06ndash2822)

NS

363(06ndash3689)

555(09ndash2060)

NS

336(06ndash3689)

722(09ndash2060)

NS

Hb(gdL)lowastlowast

86(54ndash142)

77(56ndash107)

NS

86(54ndash142)

73(56ndash107)

NS

86(54ndash142)

73(56ndash107)

0097

Platele

ts(times10

9 L)lowastlowast

540(50ndash1720)

620(380ndash

2700)

NS

540(50ndash1720)

960(430ndash

2700)

NS

535(50ndash1720)

1130(510

ndash2700)

NS

BMblasts(

)lowastlowast

730(221ndash970)

743(295

ndash962)

NS

729(221ndash970)

750(295

ndash843)

NS

729(221ndash970)

750(295

ndash843)

NS

FLT3

ITDmutation(

)lowast1145(244)

46(667)

0054

1247(255)

34(750)

0071

1247(255)

34(750)

0071

FLT3

D835mutation(

)lowast245

(44)

06(00)

NS

247

(43)

04(00)

NS

247

(43)

04(00)

NS

CRrates()lowast

3357(579

)310

(300)

NS

3460(567)

27(286)

NS

3462(548)

25(400)

NS

Intervalfro

mDxto

CRdays


340(210

ndash830)

530(250ndash

1640)

NS

350(210

ndash164

0)

390(250ndash

530)

NS

350(210

ndash164

0)

390(250ndash

530)

NS

Relap

serates()lowast

1633(485)

13(333)

NS

1734(500)

02(00)

NS

1734(500)

02(00)

NS

Death

rates()lowast

3157(544)

510

(500)

NS

3460(567)

27(286)

NS

3462(548)

25(400)

NS

SCTperfo

rmance

rates()lowast

1957(333)

110(100)

NS

1960(317

)17

(143)

NS

1962(306)

15(200)

NS

Note119875values

wereo

btainedfro

mCh

i-squ

aretestF



WBC

white

bloo

dcell

Hb

hemoglobin

BMb

onemarrowF

LT3

fms-related

tyrosin

ekinase

3ITDinternaltandem

duplicationsD

aspartateC

Rcompleteremission

Dx

diagno

sisS

CTstem

cell

transplantation

DNMT3

AD

NAmethyltransfe

rase

3AR

arginineNSno

tsignificant



10

08

06

04

02

00

Cum

ulat

ive s

urvi

val


P = 0384

Yes (N = 10)No (N = 57)

(a)

Yes (N = 7)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val


P = 0833

No (N = 60)

(b)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

R882 mutation

P = 0726

Yes (N = 5)No (N = 62)

(c)


Yes (N = 10)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0771

No (N = 57)

(d)

Yes (N = 7)



10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0351

No (N = 60)

(e)

R882 mutation

Yes (N = 5)No (N = 62)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0351

(f)

















5 Conclusions




Acknowledgments


References














































3 Results





S

A A ACC C CCCY CCC

S G

GG G GGGG

P D T

T

G


E E


SI

G G G G

MT

T T T

N


E F


V R L L H


P E


PL

GGG G G

PK

T T

G




P E PL PK G



L S SE LG W



N L AR RM S

R

Q M

A AA A A AC C CS

YI

GGGG G G G

VG

T T T T

K


K V

AA AAA A A ACC C CR

IK

G G GG G

AK

T TT

A


P9 = (c27CgtT) N = 5

L422 = (c1266GgtA) N = 17


I681= (c2043CgtG) N = 6



K219R= (c656AgtG) N = 2



(a)

PWWP ZNF MeTFase

0 100 200 300 400 500 600 700 800 900

P9 =

I158M

E177V

K219R

L422 =

I681= R736HR882H

(b)


















4 Discussion





Table3Com

paris

onof

demograph


ntsw

ithrespecttoDNMT3

Ano

nsyn

onym

ousv

ariatio

nsta

tus

Clinicalcharacteris

tics

DNMT3

Ano

nsyn

onym

ousv

ariatio

nsta

tus119875value

DNMT3

Adeleterio

usmutationsta

tus119875value

DNMT3

AR8

82mutationstatus

119875value

Novaria

tion(119873=57)

Varia

tion(119873=10)


Mutation(119873=7)


Mutation(119873=5)

Sex(M

F)lowast

3324

46

NS

3426

34

NS

3527

23

NS

Ageyearsm


560(110

ndash800)

620(180ndash

830)

NS

560(110

ndash800)

590(180ndash

830)

NS

560(110

ndash830)

590(180ndash

770)

NS

Labo

ratory

finding

sat

diagno

sism

edian(range)

WBC

(times10

9 L)lowastlowast

356(09ndash3689)

638(06ndash2822)

NS

363(06ndash3689)

555(09ndash2060)

NS

336(06ndash3689)

722(09ndash2060)

NS

Hb(gdL)lowastlowast

86(54ndash142)

77(56ndash107)

NS

86(54ndash142)

73(56ndash107)

NS

86(54ndash142)

73(56ndash107)

0097

Platele

ts(times10

9 L)lowastlowast

540(50ndash1720)

620(380ndash

2700)

NS

540(50ndash1720)

960(430ndash

2700)

NS

535(50ndash1720)

1130(510

ndash2700)

NS

BMblasts(

)lowastlowast

730(221ndash970)

743(295

ndash962)

NS

729(221ndash970)

750(295

ndash843)

NS

729(221ndash970)

750(295

ndash843)

NS

FLT3

ITDmutation(

)lowast1145(244)

46(667)

0054

1247(255)

34(750)

0071

1247(255)

34(750)

0071

FLT3

D835mutation(

)lowast245

(44)

06(00)

NS

247

(43)

04(00)

NS

247

(43)

04(00)

NS

CRrates()lowast

3357(579

)310

(300)

NS

3460(567)

27(286)

NS

3462(548)

25(400)

NS

Intervalfro

mDxto

CRdays


340(210

ndash830)

530(250ndash

1640)

NS

350(210

ndash164

0)

390(250ndash

530)

NS

350(210

ndash164

0)

390(250ndash

530)

NS

Relap

serates()lowast

1633(485)

13(333)

NS

1734(500)

02(00)

NS

1734(500)

02(00)

NS

Death

rates()lowast

3157(544)

510

(500)

NS

3460(567)

27(286)

NS

3462(548)

25(400)

NS

SCTperfo

rmance

rates()lowast

1957(333)

110(100)

NS

1960(317

)17

(143)

NS

1962(306)

15(200)

NS

Note119875values

wereo

btainedfro

mCh

i-squ

aretestF



WBC

white

bloo

dcell

Hb

hemoglobin

BMb

onemarrowF

LT3

fms-related

tyrosin

ekinase

3ITDinternaltandem

duplicationsD

aspartateC

Rcompleteremission

Dx

diagno

sisS

CTstem

cell

transplantation

DNMT3

AD

NAmethyltransfe

rase

3AR

arginineNSno

tsignificant



10

08

06

04

02

00

Cum

ulat

ive s

urvi

val


P = 0384

Yes (N = 10)No (N = 57)

(a)

Yes (N = 7)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val


P = 0833

No (N = 60)

(b)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

R882 mutation

P = 0726

Yes (N = 5)No (N = 62)

(c)


Yes (N = 10)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0771

No (N = 57)

(d)

Yes (N = 7)



10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0351

No (N = 60)

(e)

R882 mutation

Yes (N = 5)No (N = 62)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0351

(f)

















5 Conclusions




Acknowledgments


References




































S

A A ACC C CCCY CCC

S G

GG G GGGG

P D T

T

G


E E


SI

G G G G

MT

T T T

N


E F


V R L L H


P E


PL

GGG G G

PK

T T

G




P E PL PK G



L S SE LG W



N L AR RM S

R

Q M

A AA A A AC C CS

YI

GGGG G G G

VG

T T T T

K


K V

AA AAA A A ACC C CR

IK

G G GG G

AK

T TT

A


P9 = (c27CgtT) N = 5

L422 = (c1266GgtA) N = 17


I681= (c2043CgtG) N = 6



K219R= (c656AgtG) N = 2



(a)

PWWP ZNF MeTFase

0 100 200 300 400 500 600 700 800 900

P9 =

I158M

E177V

K219R

L422 =

I681= R736HR882H

(b)


















4 Discussion





Table3Com

paris

onof

demograph


ntsw

ithrespecttoDNMT3

Ano

nsyn

onym

ousv

ariatio

nsta

tus

Clinicalcharacteris

tics

DNMT3

Ano

nsyn

onym

ousv

ariatio

nsta

tus119875value

DNMT3

Adeleterio

usmutationsta

tus119875value

DNMT3

AR8

82mutationstatus

119875value

Novaria

tion(119873=57)

Varia

tion(119873=10)


Mutation(119873=7)


Mutation(119873=5)

Sex(M

F)lowast

3324

46

NS

3426

34

NS

3527

23

NS

Ageyearsm


560(110

ndash800)

620(180ndash

830)

NS

560(110

ndash800)

590(180ndash

830)

NS

560(110

ndash830)

590(180ndash

770)

NS

Labo

ratory

finding

sat

diagno

sism

edian(range)

WBC

(times10

9 L)lowastlowast

356(09ndash3689)

638(06ndash2822)

NS

363(06ndash3689)

555(09ndash2060)

NS

336(06ndash3689)

722(09ndash2060)

NS

Hb(gdL)lowastlowast

86(54ndash142)

77(56ndash107)

NS

86(54ndash142)

73(56ndash107)

NS

86(54ndash142)

73(56ndash107)

0097

Platele

ts(times10

9 L)lowastlowast

540(50ndash1720)

620(380ndash

2700)

NS

540(50ndash1720)

960(430ndash

2700)

NS

535(50ndash1720)

1130(510

ndash2700)

NS

BMblasts(

)lowastlowast

730(221ndash970)

743(295

ndash962)

NS

729(221ndash970)

750(295

ndash843)

NS

729(221ndash970)

750(295

ndash843)

NS

FLT3

ITDmutation(

)lowast1145(244)

46(667)

0054

1247(255)

34(750)

0071

1247(255)

34(750)

0071

FLT3

D835mutation(

)lowast245

(44)

06(00)

NS

247

(43)

04(00)

NS

247

(43)

04(00)

NS

CRrates()lowast

3357(579

)310

(300)

NS

3460(567)

27(286)

NS

3462(548)

25(400)

NS

Intervalfro

mDxto

CRdays


340(210

ndash830)

530(250ndash

1640)

NS

350(210

ndash164

0)

390(250ndash

530)

NS

350(210

ndash164

0)

390(250ndash

530)

NS

Relap

serates()lowast

1633(485)

13(333)

NS

1734(500)

02(00)

NS

1734(500)

02(00)

NS

Death

rates()lowast

3157(544)

510

(500)

NS

3460(567)

27(286)

NS

3462(548)

25(400)

NS

SCTperfo

rmance

rates()lowast

1957(333)

110(100)

NS

1960(317

)17

(143)

NS

1962(306)

15(200)

NS

Note119875values

wereo

btainedfro

mCh

i-squ

aretestF



WBC

white

bloo

dcell

Hb

hemoglobin

BMb

onemarrowF

LT3

fms-related

tyrosin

ekinase

3ITDinternaltandem

duplicationsD

aspartateC

Rcompleteremission

Dx

diagno

sisS

CTstem

cell

transplantation

DNMT3

AD

NAmethyltransfe

rase

3AR

arginineNSno

tsignificant



10

08

06

04

02

00

Cum

ulat

ive s

urvi

val


P = 0384

Yes (N = 10)No (N = 57)

(a)

Yes (N = 7)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val


P = 0833

No (N = 60)

(b)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

R882 mutation

P = 0726

Yes (N = 5)No (N = 62)

(c)


Yes (N = 10)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0771

No (N = 57)

(d)

Yes (N = 7)



10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0351

No (N = 60)

(e)

R882 mutation

Yes (N = 5)No (N = 62)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0351

(f)

















5 Conclusions




Acknowledgments


References
















































4 Discussion





Table3Com

paris

onof

demograph


ntsw

ithrespecttoDNMT3

Ano

nsyn

onym

ousv

ariatio

nsta

tus

Clinicalcharacteris

tics

DNMT3

Ano

nsyn

onym

ousv

ariatio

nsta

tus119875value

DNMT3

Adeleterio

usmutationsta

tus119875value

DNMT3

AR8

82mutationstatus

119875value

Novaria

tion(119873=57)

Varia

tion(119873=10)


Mutation(119873=7)


Mutation(119873=5)

Sex(M

F)lowast

3324

46

NS

3426

34

NS

3527

23

NS

Ageyearsm


560(110

ndash800)

620(180ndash

830)

NS

560(110

ndash800)

590(180ndash

830)

NS

560(110

ndash830)

590(180ndash

770)

NS

Labo

ratory

finding

sat

diagno

sism

edian(range)

WBC

(times10

9 L)lowastlowast

356(09ndash3689)

638(06ndash2822)

NS

363(06ndash3689)

555(09ndash2060)

NS

336(06ndash3689)

722(09ndash2060)

NS

Hb(gdL)lowastlowast

86(54ndash142)

77(56ndash107)

NS

86(54ndash142)

73(56ndash107)

NS

86(54ndash142)

73(56ndash107)

0097

Platele

ts(times10

9 L)lowastlowast

540(50ndash1720)

620(380ndash

2700)

NS

540(50ndash1720)

960(430ndash

2700)

NS

535(50ndash1720)

1130(510

ndash2700)

NS

BMblasts(

)lowastlowast

730(221ndash970)

743(295

ndash962)

NS

729(221ndash970)

750(295

ndash843)

NS

729(221ndash970)

750(295

ndash843)

NS

FLT3

ITDmutation(

)lowast1145(244)

46(667)

0054

1247(255)

34(750)

0071

1247(255)

34(750)

0071

FLT3

D835mutation(

)lowast245

(44)

06(00)

NS

247

(43)

04(00)

NS

247

(43)

04(00)

NS

CRrates()lowast

3357(579

)310

(300)

NS

3460(567)

27(286)

NS

3462(548)

25(400)

NS

Intervalfro

mDxto

CRdays


340(210

ndash830)

530(250ndash

1640)

NS

350(210

ndash164

0)

390(250ndash

530)

NS

350(210

ndash164

0)

390(250ndash

530)

NS

Relap

serates()lowast

1633(485)

13(333)

NS

1734(500)

02(00)

NS

1734(500)

02(00)

NS

Death

rates()lowast

3157(544)

510

(500)

NS

3460(567)

27(286)

NS

3462(548)

25(400)

NS

SCTperfo

rmance

rates()lowast

1957(333)

110(100)

NS

1960(317

)17

(143)

NS

1962(306)

15(200)

NS

Note119875values

wereo

btainedfro

mCh

i-squ

aretestF



WBC

white

bloo

dcell

Hb

hemoglobin

BMb

onemarrowF

LT3

fms-related

tyrosin

ekinase

3ITDinternaltandem

duplicationsD

aspartateC

Rcompleteremission

Dx

diagno

sisS

CTstem

cell

transplantation

DNMT3

AD

NAmethyltransfe

rase

3AR

arginineNSno

tsignificant



10

08

06

04

02

00

Cum

ulat

ive s

urvi

val


P = 0384

Yes (N = 10)No (N = 57)

(a)

Yes (N = 7)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val


P = 0833

No (N = 60)

(b)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

R882 mutation

P = 0726

Yes (N = 5)No (N = 62)

(c)


Yes (N = 10)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0771

No (N = 57)

(d)

Yes (N = 7)



10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0351

No (N = 60)

(e)

R882 mutation

Yes (N = 5)No (N = 62)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0351

(f)

















5 Conclusions




Acknowledgments


References




































Table3Com

paris

onof

demograph


ntsw

ithrespecttoDNMT3

Ano

nsyn

onym

ousv

ariatio

nsta

tus

Clinicalcharacteris

tics

DNMT3

Ano

nsyn

onym

ousv

ariatio

nsta

tus119875value

DNMT3

Adeleterio

usmutationsta

tus119875value

DNMT3

AR8

82mutationstatus

119875value

Novaria

tion(119873=57)

Varia

tion(119873=10)


Mutation(119873=7)


Mutation(119873=5)

Sex(M

F)lowast

3324

46

NS

3426

34

NS

3527

23

NS

Ageyearsm


560(110

ndash800)

620(180ndash

830)

NS

560(110

ndash800)

590(180ndash

830)

NS

560(110

ndash830)

590(180ndash

770)

NS

Labo

ratory

finding

sat

diagno

sism

edian(range)

WBC

(times10

9 L)lowastlowast

356(09ndash3689)

638(06ndash2822)

NS

363(06ndash3689)

555(09ndash2060)

NS

336(06ndash3689)

722(09ndash2060)

NS

Hb(gdL)lowastlowast

86(54ndash142)

77(56ndash107)

NS

86(54ndash142)

73(56ndash107)

NS

86(54ndash142)

73(56ndash107)

0097

Platele

ts(times10

9 L)lowastlowast

540(50ndash1720)

620(380ndash

2700)

NS

540(50ndash1720)

960(430ndash

2700)

NS

535(50ndash1720)

1130(510

ndash2700)

NS

BMblasts(

)lowastlowast

730(221ndash970)

743(295

ndash962)

NS

729(221ndash970)

750(295

ndash843)

NS

729(221ndash970)

750(295

ndash843)

NS

FLT3

ITDmutation(

)lowast1145(244)

46(667)

0054

1247(255)

34(750)

0071

1247(255)

34(750)

0071

FLT3

D835mutation(

)lowast245

(44)

06(00)

NS

247

(43)

04(00)

NS

247

(43)

04(00)

NS

CRrates()lowast

3357(579

)310

(300)

NS

3460(567)

27(286)

NS

3462(548)

25(400)

NS

Intervalfro

mDxto

CRdays


340(210

ndash830)

530(250ndash

1640)

NS

350(210

ndash164

0)

390(250ndash

530)

NS

350(210

ndash164

0)

390(250ndash

530)

NS

Relap

serates()lowast

1633(485)

13(333)

NS

1734(500)

02(00)

NS

1734(500)

02(00)

NS

Death

rates()lowast

3157(544)

510

(500)

NS

3460(567)

27(286)

NS

3462(548)

25(400)

NS

SCTperfo

rmance

rates()lowast

1957(333)

110(100)

NS

1960(317

)17

(143)

NS

1962(306)

15(200)

NS

Note119875values

wereo

btainedfro

mCh

i-squ

aretestF



WBC

white

bloo

dcell

Hb

hemoglobin

BMb

onemarrowF

LT3

fms-related

tyrosin

ekinase

3ITDinternaltandem

duplicationsD

aspartateC

Rcompleteremission

Dx

diagno

sisS

CTstem

cell

transplantation

DNMT3

AD

NAmethyltransfe

rase

3AR

arginineNSno

tsignificant



10

08

06

04

02

00

Cum

ulat

ive s

urvi

val


P = 0384

Yes (N = 10)No (N = 57)

(a)

Yes (N = 7)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val


P = 0833

No (N = 60)

(b)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

R882 mutation

P = 0726

Yes (N = 5)No (N = 62)

(c)


Yes (N = 10)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0771

No (N = 57)

(d)

Yes (N = 7)



10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0351

No (N = 60)

(e)

R882 mutation

Yes (N = 5)No (N = 62)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0351

(f)

















5 Conclusions




Acknowledgments


References





































10

08

06

04

02

00

Cum

ulat

ive s

urvi

val


P = 0384

Yes (N = 10)No (N = 57)

(a)

Yes (N = 7)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val


P = 0833

No (N = 60)

(b)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

R882 mutation

P = 0726

Yes (N = 5)No (N = 62)

(c)


Yes (N = 10)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0771

No (N = 57)

(d)

Yes (N = 7)



10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0351

No (N = 60)

(e)

R882 mutation

Yes (N = 5)No (N = 62)


10

08

06

04

02

00

Cum

ulat

ive s

urvi

val

P = 0351

(f)

















5 Conclusions




Acknowledgments


References














































5 Conclusions




Acknowledgments


References



































incidence and prognostic impact of dnmt3a mutations in korean … · 2018. 5. 11. · in the first...

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