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Leukemia Research 35 (2011) 1020– 1026

Contents lists available at ScienceDirect

Leukemia Research

jou rna l h omepa g e: www.elsev ier .com/ locate / leukres

AD51 and XRCC3 polymorphisms: Impact on the risk and treatment outcomes ofe novo inv(16) or t(16;16)/CBFˇ–MYH11(+) acute myeloid leukemia

iang Liua, Lin Yanga, Yingchang Mib, Jianxiang Wanga,b, Jianyong Li c, Yue Zhangd, Xiaotang Maa,iejun Qind, Zefeng Xud, Zhijian Xiaoa,d,∗

State Key Laboratory of Experimental Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China2th Department of Clinical Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, ChinaDepartment of Hematology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, China6th Department of Clinical Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China

r t i c l e i n f o

rticle history:eceived 10 October 2010eceived in revised form 15 January 2011ccepted 18 January 2011vailable online 5 February 2011

eywords:cute myeloid leukemia

a b s t r a c t

DNA double-strand break repair via homologous recombination (HR) is essential in maintaining geneticintegrity, and may modulate susceptibility to the development of acute myeloid leukemia (AML) andinfluence outcomes of AML. This study was designed to evaluate the effects of polymorphisms in HR repairgenes RAD51 and XRCC3 on the risk and treatment outcomes of inv(16)/t(16;16)/CBFˇ–MYH11(+) AML.The distribution of polymorphisms in RAD51-G135C and XRCC3-Thr241Met were studied by PCR–RFLPanalysis in 625 cases of de novo AML, including 105 cases with inv(16)/t(16;16)/CBFˇ–MYH11, 806 fam-ily controls and 704 volunteer controls. It was found that the XRCC3-241Met variant significantly increased

usceptibilityutcomeNA repairolymorphism

the risk of the development of the AML with inv(16)/t(16;16) as compared with both the volunteer con-trol (OR = 7.22; 95% CI, 4.37–11.91) and the family control (OR = 7.99; 95% CI, 5.03–12.69). A retrospectivestudy conducted in 103 inv(16)/t(16;16) AML patients. In multivariate analysis for the potential prog-nostic factors, the XRCC3-241Met variant significantly reduced disease-free survival (DFS) in completeremission (CR) achieved patients (HR = 2.34, 95% CI, 1.32–4.16). These data indicate that the XRCC3-241Met variant may not be only a susceptibility factor to the AML with inv(16)/t(16;16), but also an

ostic

independent poor-progn

. Introduction

The inv(16)(p13q22)/t(16;16)(p13;q22) fuses the CBF ̌ geneocated in 16q22 to the MYH11 gene located in 16p13, whichesults in CBF�–MYH11 fusion protein which hinders the differ-ntiation process of leukemic cells through sequestration of CBFA2n the cytoplasm [1]. However, expression of the chimeric proteinlone is not sufficient for leukemogenesis, additional mutationsay be needed for the development of acute myeloid leukemia

AML) [1]. The inv(16)/t(16;16) coding CBFˇ–MYH11 fusion gene

ccurs in approximately 4–7% de novo AML and 3–4% therapy-elated AML patients, respectively [2,3]. The inv(16)/t(16;16)atients are frequently associated with specific characteristics.orphologically, this AML subtype is more often diagnosedith French-American-British subtype M4eo with an abnormal

∗ Corresponding author at: 6th Department of Clinical Hematology, Institute ofematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences, 288anjing Road, Tianjin 300020, China. Tel.: +86 22 23909184; fax: +86 22 27219070.

E-mail addresses: zjxiao@hotmail.com, zjxiao@medmail.com.cn (Z. Xiao).

145-2126/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.leukres.2011.01.014

factor for this AML subtype.© 2011 Elsevier Ltd. All rights reserved.

eosinophilic differentiation. Cytogenetically, the specific aberrationmay be associated with trisomy 22, 8, and 21 and less frequentlywith deletion of the long arm of the chromosome 7 [3–5]. Clinically,this AML subtype trends to be associated with a higher completeremission (CR) rate and favorable outcome as compared with oth-ers, and prolonged CR can be achieved with intensive postremissionchemotherapy including high-dose cytarabine (HDAC) [2,3,5,6].

Up to now, the precise molecular mechanism of origin of theinv(16)/t(16;16) and CBFˇ–MYH11 is still unclear. However, thesequence analysis of genomic breakpoints [7–11] has indicatedthat the t(8;21)/AML1–ETO and t(15;17)/PML–RARA result fromnonhomologous recombination at double-strand DNA (dsDNA)breaks due to a dysfunctional DNA damage-repair mechanism.The inv(16)/t(16;16)/CBFˇ–MYH11, however, occurs by homolo-gous recombination (HR). HR is the main pathway to repair thebreaks of dsDNA. The major eukaryotic homologous recombinaseRAD51 protein is crucial for HR in repairing dsDNA breaks and

maintaining genomic diversity and stability [12–14]. The RAD51gene knocked-out mice are embryonically lethal [15,16]. Cells lack-ing RAD51 are characterized by an accumulation of chromosomalbreaks before cell death [17]. A paralog of RAD51, XRCC3 proteincan directly interact with and stabilize RAD51C to promote strand

L. Liu et al. / Leukemia Research 35 (2011) 1020– 1026 1021

Table 1Distributions of sex and age in case group and control groups.

All Male (%) Female (%) Median age, years (range)

Volunteer control 704 247 (35.1) 457 (64.9) 42 (15–90)Family control 806 388 (48.1) 418 (51.9) 39 (14–72)Total AML 625 361 (57.8) 264 (42.2) 33 (5–75)

Cytogenetics normal 155 82 (52.6) 73 (47.4) 35 (14–71)Cytogenetics abnormala 91 59 (64.8) 32 (35.2) 35 (14–73)inv(16)/t(16;16)/CBFˇ–MYH11 105 58 (55.2) 47 (44.8) 33 (5–69)t(15;17)/PML–RARA 111 63 (56.8) 48 (43.2) 35 (14–66)t(8;21)/AML1–ETO 151 93 (61.6) 58 (38.4) 31 (14–72)

6 (50

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a Exclude below four AML subtypes.

nvasion and/or pairing during HR [12–14]. The RAD51-G135C poly-orphism at position −135 in the 5′ UTR region may be relatedith RAD51 protein overexpression and DNA repair increase

18–20]. A polymorphism at codon 241(C → T) in the XRCC3 geneesults in a Thr-to-Met amino acid substitution [21]. The XRCC3-41Met variant resulted in reduced DNA repair activity with theossible involvement with dysfunctional XRCC3 protein [22–24].he RAD51-135C (G/C + C/C) and XRCC3-241Met (Thr/Met + Met/Met)ariants have already been included among the risk factors forhe development of AML and treatment associated AML (t-AML)25–29], and among the prognostic factors for the AML patients27,30]. Our previous study showed that the frequency of theRCC3-241Met variant in 17 AML patients with inv(16)/t(16;16)as been 41.2% as compared with 12.0% in 458 controls [oddsatio (OR), 6.13; 95% confidence interval (CI), 2.23–16.89; P < .001]29]. In the previous report, RAD51-G135C, XRCC3-Thr241Met,STT1 and GSTM1 genotypes were analyzed in 372 patientsith AML [including 54 inv(16)/t(16;16)/CBFˇ–MYH11(+) AMLatients], and we found that the relapse-free survival (RFS) ofhe inv(16)/t(16;16)/CBFˇ–MYH11(+) AML patients with XRCC3-hr241Met and GSTT1 double-wild genotypes(48.3 months) wereignificantly longer than those of patients with double-variants28.8 months) [30]. However, the RAD51-135C variant has hado significant effect on the risk and prognosis of this AML sub-ype. We subsequently have expanded the number of casesnd controls to 105 and 704, respectively; and the resultshich we will report are still consistent with our previous

tudy.The aims of this survey were especially (1) to examine whether

he polymorphisms of XRCC3 and RAD51 might modulate suscepti-ility to the development of the AML with inv(16)/t(16;16); and (2)o investigate the effects of polymorphisms of XRCC3 and RAD51 onrognosis of patients with this disease.

. Materials and methods

.1. Patients and controls selection

This study was retrospective investigate and approved by the Ethical Commit-ee of Institute of Hematology, CAMS and PUMC according to the guidelines of theeclaration of Helsinki. Informed consent was obtained from all subjects or theirarents. The patients were noncontinuity recruited from November 1, 1999 to July 1,009, who were diagnosed according to the WHO criteria. Karyotyping and substan-ial gene analyses were performed for each patient. The case group consisted of 625aive and primary AML patients, including 105 with inv(16)/t(16;16)/CBFˇ–MYH11,11 t(15;17)/PML–RARA, 151 t(8;21)/AML1–ETO, 12 11q23, 91 other cytogeneticbnormality, and 155 cytogenetic normal. Controls were divided into two groups.he first group consisted of 704 healthy volunteers from Han Chinese employeef two hospitals in north China; and the second group consisted of 806 healthy

rst-grade families of these AML patients, such as their parents, children, sisters orrothers. The distributions of sex and age in the case group and control groups arehown in Table 1.

In the treatment outcome study of the 105 AML patients with inv(16)/t(16;16),wo of whom lost follow-up and were excluded. The long-term follow-up wastarted from November 1, 1999 and ended on January 31, 2010. The median follow-

.0) 6 (50.0) 28 (18–60)

ups of all and survival patients were 28 (range, 1–106) months and 46 (range, 7–106)months, respectively.

2.2. Preparation of DNA

DNA was extracted from peripheral blood by the standard phenol/chloroformmethod, then dissolved in TE (10 mM Tris–HCl–1 mM EDTA) (pH 8.0), quantitatedwith spectrophotometer at 260 nm, and stored at 4 ◦C.

2.3. Genotyping

XRCC3-Thr241Met (rs861539) and RAD51-G135C (rs1801320) genotypes for SNPwere performed by polymerase chain reaction (PCR)–restriction fragment lengthpolymorphism (RFLP) analysis as described previously [26]. Briefly, the PCR systemwas identical for RAD51 and XRCC3. The 50 �l PCR mixture contained 0.2 �g DNA,300 �M primers, 0.2 mM dNTPs, and 2 U Taq polymerase. Amplification cycles forXRCC3 were: 95 ◦C for 5 min, then 35 cycles at 95 ◦C for 1 min, 60 ◦C for 1 min, 72 ◦Cfor 1 min, followed by 72 ◦C for 10 min. XRCC3-Thr241Met was amplified in a 415-bpproduct. The PCR products were digested at 37 ◦C overnight with 1 U of NlaIII (NewEngland Biolabs). All XRCC3 PCR products contain an internal NlaIII site, and thepresence of the Met polymorphism also generates a NlaIII site, which produces 104-bp, 141-bp, and 170-bp products for the polymorphic allele and 141-bp and 274-bpproducts for the wild-type threonine allele. The XRCC3-Thr241Thr, Thr241Met andMet241Met controls confirmed by sequencing included in each reaction to check theefficiency of the restriction enzyme. Amplification cycles for RAD51 were: 95 ◦C for5 min, then 35 cycles at 95 ◦C for 1 min, 53 ◦C for 1 min, 72 ◦C for 1 min, followed by72 ◦C for 10 min. The PCR products were digested with 1 U of MvaI (TaKaRa). TheRAD51-G135C polymorphism was amplified in a 157-bp fragment, the polymorphicC allele eliminates the MvaI restriction site, and, therefore, the digestion of poly-morphic samples resulted in a single band of 157-bp, whereas the wild-type alleleresulted in two bands of 71-bp and 87-bp. The RAD51-G135G, G135C and C135Ccontrols confirmed by sequencing included in each reaction to check the efficiencyof the restriction enzyme. Digestion products of XRCC3 and RAD51 were analyzedby electrophoresis in 1% agarose with 2% Synergel (Diversified Biotech, Italy) andviewed by ethidium bromide staining/UV trans-illumination.

Two reviewers independently scored all of the genotypes, there was essentiallyno discrepancy in genotype scores between the two reviewers. All the samples thatcould not be scored and a 10% random sample population of controls and AMLPCR products were sequenced, which had confirmed the RFLP findings. Sequenc-ing reactions were set up with 5–20 ng of purified PCR product (40 �l) and 10 pmolprimer using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).The primers used for sequencing were the same as those used for the PCR amplifi-cations. The reactions were capillary electrophoresed using an Applied Biosystems3730/3730xl DNA Analyzers (Applied Biosystems).

2.4. Antileukemic therapy

All the patients were treated with HAD regimen as induction therapy [31]. HADregimen consisted of homoharringtonine (HHT) 2.5 mg/m2 intravenously on days1–7, cytosine arabinoside (Ara-c) 150 mg/m2/d continuous infusion on days 1–7,and daunorubicin (DNR) 45 mg/m2 intravenously on days 1–3. The second courseof HAD induction therapy was applied to patients who did not obtain CR after theinitial course of induction therapy. Postremission therapy consisted of HA regimen(HHT and Ara-c) (course 1 and 4), DA regimen (DNR and Ara-c) (course 2 and 5), andMA [mitoxantrone (MTZ) and Ara-c] or HAM regimen (course 3 and 6) (Ara-c 1 g/m2,q12 h, intravenously on days 1–4, combined with MTZ 8 mg/m2, intravenously ondays 5–7). The number of courses of HDAC treatment was from 1 to 3 according topatient’s age and general condition.

2.5. Statistical methods

The observed genotype frequencies of the XRCC3-Thr241Met and RAD51-G135Cpolymorphisms in the control cohorts were compared with those calculated by theHardy–Weinberg equilibrium (HWE) (p2 + q2 + 2pq = 1, where p is the variant allele

1022 L. Liu et al. / Leukemia Research 35 (2011) 1020– 1026

Table 2Frequency of RAD51-G135C, XRCC3-Thr241Met polymorphisms in AML with inv(16)/t(16;16)/CBFˇ–MYH11 and control populations and the relative risk for this AML subtypeassociated with these genotypes.

Genotype Volunteer controls (n) Family Controls (n) Cases (n) Volunteer controls versus. cases Family controls versus. cases

OR (95% CI) P OR (95% CI) P

RAD51-G135C 704 806 105G/G 511 574 72 1.0 (Ref) 1.0 (Ref)G/C 175 218 25 1.03 (0.61–1.74) .906 0.94 (0.58–1.54) .812C/C 18 14 8 1.87 (1.16–3.01) .010 2.15 (1.35–3.41) .001G/C + C/C 193 232 33 1.26 (0.78–2.04) .342 1.17 (0.75–1.83) .487XRCC3-Thr241Met 704 806 105Thr/Thr 627 716 55 1 (Ref) 1 (Ref)Thr/Met 73 89 39 5.75 (3.39–9.76) <.001 6.36 (3.91–10.37) <.001

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(2%) with primary drug resistant AML died 5 and 6 months afterenrollment, respectively. Overall, the estimated 5-year DFS ratewas 26.4%, and the estimated 5-year survival rate was 43.6%. Themedian OS and median DFS were 53 and 27 months, respectively.

Table 3Patient characteristics and overall efficacy in AML with inv(16)/t(16;16)/CBFˇ–MYH11.

Patients no. 103

Sex, M/F 58 (56.3%)/45 (43.7%)Median age, y (range) 32 (5–69)Aged 15 years or younger 18 (17.5%)Aged older than 50 years 5 (4.9%)Median marrow blast percentage (range) 51 (26–91)Median WBC count × 109/L (range) 38.2 (1.6–325.0)Median hemoglobin count, g/L (range) 80 (28–137)Median platelet count × 109/L (range) 26 (4–221)CR rate 95/103 (92.2%)DFS of CR patients5-year DFS (95% CI) 26.4% (21.1%–31.7%)Median DFS, months (95% CI) 27.0 (22.9–31.1)Survival of all patients

Met/Met 4 1Thr/Met + Met/Met 77 90

R, odds ratio; CI, confidence interval; Ref, reference group.

requency). The distribution of genotypes in AML populations compared with theontrol populations were tested for significance using odds ratios (OR) and their5% confidence intervals (CI) calculated by logistic regression analysis and adjustedor the effects of sex and age using the SPSS 15.0 software. A P value less than .05ndicates statistical significance.

In the treatment outcome analysis of 103 patients with inv(16)/t(16;16) AML,he definitions of CR, relapse, overall survival (OS) or disease-free survival (DFS)ere referred to “Revised recommendation of the International Working Group foriagnosis, standardization of response criteria, treatment outcomes, an reporting

tandards for therapeutic trials in acute myeloid leukemia” [32]. OS was calculatedrom the time of diagnosis until death, and patients alive were censored at the timef last contact. DFS was calculated from the date of CR achievement until first relapser death in CR, and patients alive in CR were censored at the time of last contact. Aultivariable analysis was carried out. Variables for model inclusion were sex, age,BC count, platelet count, hemoglobin (Hb) level, cytogenetics, and polymorphisms

f XRCC3 and RAD51. The �2 test and Fisher exact test were used for binary variableomparisons. The Mann–Whitney test was used for median comparisons. Impactsf continuous variables on CR rate and multivariate analysis for CR achievementere tested using the maximum likelihood model. Data on treatment failure were

stimated by the Kaplan–Meier method and compared using the log-rank test. Innivariate evaluations of the prognostic impact of continuous variables (age, bloodounts), optimal cutpoints were determined using a corrected minimum P valueethod based on an approximation to the improved Bonferroni inequality [33]. Inultivariate analyses, outcome comparisons were adjusted with the Cox model and

ested by the likelihood ratio test [5]. Risks of relapse in CR and death were comparedsing the standard Kaplan–Meier method and the log-rank test. A P value less than

05 indicates statistical significance. Hazard ratios (HR) were calculated with 95% CI.ll calculations were performed using the SPSS 15.0 software.

. Results

.1. RAD51-G135C and XRCC3-Thr241Met polymorphisms

We have examined the frequency of two polymorphisms in25 cases of de novo AML, 704 volunteer controls and 806 fam-

ly controls. The variant allele frequencies for volunteer controlsere as follows: RAD51-135C, 0.15; and XRCC3-241Met, 0.06; for

amily controls: RAD51-135C, 0.15; and XRCC3-241Met, 0.06. TheAD51-G135C and XRCC3-Thr241Met genotype frequencies in theolunteer control population were consistent with those expectedrom the HWE: RAD51-G135C, �2 = .42, P = .52; XRCC3-Thr241Met,2 = 1.35, P = .25; which also the case in the family control popula-

ion: RAD51-G135C, �2 = 1.69, P = .20; XRCC3-Thr241Met, �2 = 1.08, = .30.

The ORs and their 95% CIs for each genotype were adjusted forex and age. The XRCC3-241Met variant increased the risk of devel-ping AML in total 625 AML patients, both to volunteer controlsOR = 1.67) and family controls (OR = 1.50). Stratified analysis ofML subtypes revealed that the XRCC3-241Met variant significantly

ncreased the risk for the inv(16)/t(16;16) AML; but not for otherML subtypes [including AML with t(15;17), AML with t(8;21), andML with 11q23]. There were 7.22-fold and 7.99-fold increase ofisk for developing inv(16)/t(16;16) AML in the XRCC3-241Met vari-nt as compared with that in the volunteer and family controls,

9.50 (4.23–21.33) <.001 14.21 (4.77–42.28) <.0017.22 (4.37–11.91) <.001 7.99 (5.03–12.69) <.001

respectively (Table 2). However, the RAD51-135C variant had noinfluence on the risk for developing AML including inv(16)/t(16;16)AML (Supplemental Tables 1–4).

3.2. Treatment outcomes of 103 AML patients withinv(16)/t(16;16)

3.2.1. Patient populationOutcomes and prognostic factors for CR achievement, DFS, and

survival were evaluated in 103, 144 and 108 AML patients withinv(16)/t(16;16), t(8;21) and t(15;17), respectively. Results of uni-variate and multivariate analysis in patients with t(8;21) andt(15;17) were shown in Supplemental Tables 6 and 7, respectively,and for patients with inv(16)/t(16;16) were as follows.

All 103 inv(16)/t(16;16) patients did not receive hematopoieticstem cell transplantation (HSCT). Clinical characteristics of thesepatients are presented in Table 3. There were 58 (56.3%) males and45 (43.7%) females with a median age of 32 years (range, 5–69years). There was no difference for median WBC count, plateletcount, and Hb level in patients aged 15 years or younger as com-pared with older patients (P = .321, .458 and .406, respectively). Theoverall CR rate was 92.2% (95/103). Six patients (5.8%) died fromtreatment related multiorgan failure including acute tumor lysissyndrome, coagulopathy, and pulmonary infection. Two patients

5-year survival (95% CI) 43.6% (37.7%–49.5%)Overall survival (95% CI) 36.1% (28.9%–43.3%)Median OS, months (95% CI) 53.0 (33.4–72.7)

WBC indicates white blood cell count.CR, complete remission; DFS, disease-free survival; OS, overall survival.

L. Liu et al. / Leukemia Research 35 (2011) 1020– 1026 1023

Table 4Distribution of RAD51-G135C, XRCC3-Thr241Met polymorphisms in sex and age in AML with inv(16)/t(16;16)/CBFˇ–MYH11.

Genotype All (n) Male (n) Female (n) P Aged 15 years or younger (n) Aged older than 15 years (n) P

RAD51-G135C 103 58 45 – 18 85 –G/G 70 39 31 0.859 12 58 .897G/C + C/C 33 19 14 6 27XRCC3-Thr241Met 103 58 45 – 18 85 –

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Thr/Met + Met/Met 50 27 23

not applicable.

As shown in Table 4, proportions of the RAD51-G135C wild-ype and variant were 68% and 32%, respectively. Proportions ofhe XRCC3-Thr241Met wild-type and variant were 51.5% and 48.5%,espectively. The proportion of the RAD51-135C variant had no sta-istical difference, either between patients aged 15 years or youngernd those older than 15 years (P = .897) or between male and femaleatients (P = .859); the same profiles were observed for the XRCC3-41Met variant as well (P = .155 and .646, respectively).

The standard of valid results of chromosomal karyotype requireshat there are at least 15 analyzed metaphases. A clone is defined byt least two bone marrow cells showing the same gain of chromoso-al material or the same structural aberration, or by at least three

one marrow cells showing loss of the same chromosome [34].here are at least 5 normal metaphases in abnormality/normalityAN) status. As shown in Table 5, in the 101 patients with validesults of chromosomal karyotype in the 103 patients, 100 werenv(16) and 1 t(16;16) translocation. There was no difference forrequent persistent normal metaphases (AN status) in patients aged5 years or younger as compared with older patients (P = .82). Asso-iated chromosome abnormalities were observed in 39 patients,ncluding 12 of trisomy 22, 10 trisomy 8, 4 trisomy 21, 2 mono-omy 18, and 5 del(7q). Ten patients presented only trisomy 22,

patients only trisomy 8, 4 patients only trisomy 21, and 5atients only del(7q) as additional anomaly. Other abnormalitiesere del(16q) in 2 patients and +9 +13, del(11q), t(6;10)(q23;q25),

(16;18)(q22;q12) in 1 patient each. Complex karyotype (defineds 3 or more unrelated abnormalities) was observed in 2 patientsith only limited chromosome number anomalies in addition to

he inv(16) (+9 +14 +22 in 1 patients; +8 +11 +13 in 1 patient). Over-ll, there was no difference for frequent additional chromosomebnormalities in patients aged 15 years or younger as comparedith older patients (P = .18).

.2.2. Prognostic factors for CR in all patients

.2.2.1. Univariate analysis. As a continuous variable, advanced agead no impact on CR rate (P = .240); but as a categoric variable with

able 5ytogenetic features in AML patients with inv(16)/t(16;16)/CBFˇ–MYH11.

All Aged 15

Patients no.a 101 18

inv(16)/t(16;16), no. 100/1 18/0

AN/AA status, no. 48/53 9/9

Complex karyotype, no. 2 0

Associated abnormalities, no. 39 4

Number abnormalities only, no. 29 4

Including +8 10 1

Including +21 4 1

Including +22 12 2

Including −18 2 0

Other 1 0

Including structure abnormalities, no. 10 0

del(7q) 5 0

Other 5 0

not applicable.a In 101 patients with valid results of chromosomal karyotype.

12 41 .1556 44

an age cutpoint at 30 years, the CR rate for patients older than30 years (87.0%) was lower than that for those under 30 (98.0%)(P = .060). Higher WBC was a significantly poor-prognosis factorfor CR rate when viewed as a continuous variable (P = .001). Themedian WBC count of patients not achieved CR was 114.0 × 109/Lversus that of 34.5 × 109/L in those achieved CR (P = .002). The opti-mal WBC cutpoint was 70 × 109/L. The CR rate in patients withWBC above 70 × 109/L (77.4%) was significantly lower than that inthose with WBC below 70 × 109/L (98.6%) (P = .001). The XRCC3 andRAD51 genotypes had no significant effect on CR rate: the CR rate inthe 50 patients with XRCC3-241Met variant was 96.0% versus 88.8%in the 53 patients with XRCC3-241Thr/Thr wild-type (P = .271), andin the 33 patients with RAD51-135C variant 97.0% versus 90.0% inthe 70 patients with RAD51-135GG wild-type (P = .137) (Table 6).The sex, platelet count and Hb level also had no significant impacton CR rate. The presence of additional chromosome structure ornumber abnormalities or persistence of normal metaphases hadno significant impact on CR rate.

3.2.2.2. Multivariate analysis. In the multivariate analysis, higherWBC count (P = .004, when considered as a continuous variable;P = .005, when cutpoint at 70 × 109/L) and older age (P = .076, whenconsidered as a continuous variable; P = .035, when cutpoint at 30years) were 2 independent poor-prognostic factors for CR achieve-ment (Tables 6 and 7).

3.2.3. Prognostic factors for DFS in CR patients3.2.3.1. Univariate analysis. Advanced age was not a poor-prognostic factor for DFS of CR patients, either considered as acontinuous variable (P = .165) or as a categoric variable (Table 6).Conversely, higher WBC count as a continuous variable was sig-

nificantly associated with worsened DFS of CR patients (P = .004).When the optimal WBC cutpoint set at 70 × 109/L, the estimated 5-year DFS rate was 6.5% (95% CI, 0.3–12.7%) for the above 70 × 109/Lpatients versus that of 33.1% (95% CI, 26.7–39.5%) for those below70 × 109/L (P = .006) (Fig. 1A). The XRCC3-241Met variant signif-

years or younger Aged older than 15 years P

83 –82/1 –39/44 .82

2 1.0035 .1825 .58

9 –3 –

10 –21 –

10 .205 –5 –

1024 L. Liu et al. / Leukemia Research 35 (2011) 1020– 1026

Table 6Univariate and multivariate analysis for CR in all patients, DFS in CR patients and OS in all patients with inv(16)/t(16;16)/CBFˇ–MYH11 when age and WBC as continuousvariables: P values.

Univariate analysis Multivariate analysis

P OR/HR (95% CI) P OR/HR (95% CI)

CR rateAge (continuous variable) .240 – .076 –WBC (continuous variable) .001 – .004 –XRCC3-241Met variant .271 0.33 (0.15–1.60) – –RAD51-135C variant .137 0.30 (0.12–1.98) – –5-year DFSAge (continuous variable) .165 – .179 –WBC (continuous variable) .004 – .009 –XRCC3-241Met variant .001 – .007 2.34 (1.32–4.16)RAD51-135C variant .340 – – –Cytogenetics with associated +22 .080 – .399 0.67 (0.26–1.71)5-year OSAge (continuous variable) .102 – .086 –WBC (continuous variable) <.001 – .002 –XRCC3-241Met variant .235 – – –RAD51-135C variant .128 – – –Cytogenetics with associated +8 .008 – .035 2.31 (1.08–4.83)

OR, odds ratio; HR, hazard ratio; CI, confidence interval; –, not applicable.CR, complete remission; DFS, disease-free survival; OS, overall survival.WBC, white blood count.

Table 7Univariate and multivariate analysis for CR in all patients, DFS in CR patients and OS in all patients with inv(16)/t(16;16)/CBFˇ–MYH11 when age and WBC as cutpointvariables: P values.

Univariate analysis Multivariate analysis

P OR/HR (95% CI) P OR/HR (95% CI)

CR rateAge (cutpoint at 30 years) .060 1.77 (1.27–2.46) .035 3.55 (1.93–6.57)WBC (cutpoint at 70 × 109/L) .001 3.46 (2.24–5.35) .005 4.82 (2.24–8.84)XRCC3-241Met variant .271 0.33 (0.15–1.60) – –RAD51-135C variant .137 0.30 (0.12–1.98) – –5-year DFSAge (cutpoint at 15 years) .186 – – –WBC (cutpoint at 70 × 109/L) .006 – .011 2.16 (1.22–3.82)XRCC3-241Met variant .001 – .006 2.37 (1.31–4.17)RAD51-135C variant .340 – – –Cytogenetics with associated +22 .080 – .400 0.65 (0.25–1.72)5-year OSAge (cutpoint at 15 years) .034 – .175 2.21 (0.60–6.91)WBC (cutpoint at 70 × 109/L) <.001 – .003 2.53 (1.39–4.61)XRCC3-241Met variant .235 – – –RAD51-135C variant .128 – – –Cytogenetics with associated +8 .008 – .033 2.29 (1.06–4.85)

OR, odds ratio; HR, hazard ratio; CI, confidence interval; –, not applicable.CR, complete remission; DFS, disease-free survival; OS, overall survival.WBC, white blood count.

Fig. 1. Prognostic impact of WBC count and XRCC3-Thr241Met genotype on DFS of CR patients with inv(16)/t(16;16)/CBFˇ–MYH11. (A) Prognostic impact of WBC count belowor above 70 × 109/L on DFS. (B) Prognostic impact of XRCC3-Thr241Thr wild-type or 241Met variant (Thr241Met + Met241Met) on DFS.

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cantly worsened DFS in CR patients. The estimated 5-year DFSate was 14.1% (95% CI, 8.8–19.4%) for the patients with XRCC3-41Met variant versus that of 44.2% (95% CI, 34.9–53.5%) in theemaining patients (P = .001) (Fig. 1B). However, the RAD51-135Cariant, sex, platelet count and Hb level had no significant impactn DFS in CR patients. Finally, the presence of an additional tri-omy 22 was associated with a trend to a higher DFS, the estimated-year DFS rate in patients with an associated trisomy 22 was2.1% (95% CI, 36.3–67.9%) versus 21.8% (95% CI, 16.4–27.2%) in theemaining patients (P = 080). Other additional numeral or struc-ural chromosome abnormalities had no significant impact onFS.

.2.3.2. Multivariate analysis. In the multivariate analysis, theRCC3-241Met variant (P = .007) and higher WBC count (P = .009,hen considered as a continuous variable; P = .011, when cutpoint

t 70 × 109/L) were two independent poor-prognostic factors for-year DFS (Tables 6 and 7).

.2.4. Prognostic factors for overall survival in all patients

.2.4.1. Univariate analysis. As a continuous variable, advanced agead no impact on survival for all patients (P = .102); but as a cat-goric variable with an age cutpoint at 15 years, the estimated-year survival rate for the older than 15 years group (38.4%) wasarkedly lower than that for the aged 15 years or younger group

78.9%) (P = .034). The higher WBC count as a continuous variableignificantly worsened survival for all patients (P < .001). When theptimal WBC cutpoint set at 70 × 109/L, as shown in Fig. 2A, thestimated 5-year survival rate was lower (19.1%) for the above0 × 109/L group than that (54.8%) for the below 70 × 109/L groupP < .001). As to genotype variants, the estimated 5-year survivalate for the XRCC3-241Met variant patients was 36.8% versus that of2.8% in the remaining patients (P = .235), and for the RAD51-135Cariant 51.6% versus 39.8% for the remaining patients (P = .128)Table 6). The sex, platelet count and Hb level had no significantmpact on survival. Among the additional numeral or structuralhromosome abnormalities, only trisomy 8 showed impact on sur-ival (Fig. 2B).

.2.4.2. Multivariate analysis. In the multivariate analysis, higher

BC count (P = .002, when considered as a continuous vari-

ble; P = .003, when cutpoint at 70 × 109/L) and cytogeneticbnormality with an associated trisomy 8 (P = .035) werewo independent poor-prognostic factors for 5-year survivalTables 6 and 7).

n OS of all patients with inv(16)/t(16;16)/CBFˇ–MYH11. (A) Prognostic impact ofr without an associated trisomy 8 on OS.

4. Discussion

Previous studies [25–29] have demonstrated the XRCC3-241Metvariant has a higher risk of the development of AML, which isconsistent with our finding. However, the stratified analysis hadnot been performed among the AML subtypes in earlier stud-ies, perhaps due to limited patients with special subtype AML.Our pilot study [29] showed that the frequency of the XRCC3-241Met variant in 17 AML patients with inv(16)/t(16;16) has been41.2% as compared with 12.0% in 458 controls (OR = 6.13; 95% CI,2.23–16.89; P < .001). In the present study, there are 105 cases withinv(16)/t(16;16), 111 t(15;17), 151 t(8;21), and 12 11q23 in 625AML patients. After stratified analysis and adjusting for the effectsof sex and age in these AML subtypes, we have shown for the firsttime that the XRCC3-241Met variant significantly increases the riskof the developing of the AML with inv(16)/t(16;16), which mayactually be a susceptibility factor to the development of this AMLsubtype. However, the XRCC3-241Met variant has no effect, eitheron the risk of the AML with t(15;17) or on the risk of the AMLwith t(8;21). These results indicate that the molecular mechanismof the occurring of inv(16)/t(16;16)/CBFˇ–MYH11 is not identi-cal with that of t(15;17)/PML–RARA and t(8;21)/AML1–ETO. Thevariants in DNA HR repair genes may be related with the occur-ring of inv(16)/t(16;16)/CBFˇ–MYH11 but not with the occurringof t(15;17)/PML–RARA and t(8;21)/AML1–ETO.

Some studies [5,35–37] have evaluated impact of age, sex,WBC count, platelet count, Hb level, karyotype, induction ther-apy and postremission therapy on the prognosis of patients withinv(16)/t(16;16) AML, but few similar studies have been carried outfor XRCC3 and RAD51 polymorphisms. Kuptsova et al. [38] indicatedthat XRCC3-Thr241Met polymorphisms had no impact on the out-comes in 372 AML patients. But their study had suffered from fewcases with inv(16)/t(16;16) AML. In the present study, at least to ourknowledge, we have shown for the first time that, after adjusting forpotential confounding variables, the XRCC3-241Met variant signif-icantly reduces the DFS of the AML patients with inv(16)/t(16;16)and may be an independent poor-prognostic factor for this AMLsubtype.

Higher WBC count was commonly associated with worsenedoutcome of patients with AML. In this study, higher WBC countrepresents an independent poor-prognostic factor for overall CR

rate, DFS and survival, both in the univariate and in the multivari-ate analysis; which is consistent with the results on the publishedliteratures [5,35,36]. Schlenk et al. [36] showed that cytogeneticabnormality with additional trisomy 22 represented a favorableprognostic factor in inv(16)/t(16;16) patients. However, we find

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hat the cytogenetic abnormal with an additional trisomy 8 isssociated with a lower survival rate, and an independent poor-rognostic factor for this AML subtype. Yet, this finding needs to beonfirmed further in clinical studies with more patients because ofhe fewer patients in our study.

In conclusion, our study yielded some new data. For the firstime, we have revealed the relationship between the polymor-hism of DNA HR gene XRCC3 and the risk and therapeuticutcomes of de novo inv(16) or t(16;16)/CBFˇ–MYH11(+) AML, andave suggested that the occurring of inv(16)/t(16;16)/CBFˇ–MYH11ay represent a new dysfunctional DNA HR repair mechanism. This

otential novel biological risk factor may be useful in prognosisrediction for such patients, and the molecular mechanisms of theccurring of AML with inv(16)/t(16;16)/CBFˇ–MYH11 needs to bestablished.

onflict of interest statement

All authors declare no conflict of interest.

cknowledgements

This work is supported in part by National Natural Science FundsNo. 81070403, No. 30670899), Tianjin Key Natural Science FundsNo. 08JCZDJC19200, No. 10JCYBJC12400), New Century Excellentalents in University (NCET-05-0173), High Tech Research andevelopment (863) Programme (2006AA02A405) and Tianjin Keyechnology R&D Program (09ZCZDSF03800).

Contributions. L.L. and L.Y. contributed equally to this study; Z.X.rovided conception and design of the study, drafting the article,nd final approval of the version; L.L. and L.Y. provided acquisitionf data, analysis and interpretation of data; Z.X. and L.L. drafted therticle and J.W., J.L., Y.M., Y.Z., T.Q., Z.X. and Z.X supplied acquisitionf data.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.leukres.2011.01.014.

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