S

a

crqadadaipr(dpe

Clinical Therapeutics/Volume 33, Number 3, 2011

Evaluating the Effect of 3 Glucocorticoid Receptor GenePolymorphisms on Risk of Relapse in 100 Iranian ChildrenWith Acute Lymphoblastic Leukemia: A Case-Control Study

Soha Namazi, PharmD, PhD1; Soheila Zareifar, MD2; Ahmad Monabati, MD3;hahla Ansari, MD4; and Iman Karimzadeh, PharmD1

1Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran; 2Hematology Research Center,Shiraz University of Medical Sciences, Shiraz, Iran; 3Hematology Research Center and Department ofPathology, Shiraz University of Medical Sciences, Shiraz, Iran; and 4Department of Pediatric Hematologynd Oncology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran

ospsel

ABSTRACTBackground: Glucocorticoids are an important

component of treatment for childhood acute lympho-blastic leukemia (ALL). To induce antileukemic effects,glucocorticoids have to bind their intracellular recep-tors. Little is known about probable mechanisms ofglucocorticoid resistance in ALL.

Objective: The aim of this study was to evaluate thepossible association between 3 prominent glucocorticoidreceptor gene polymorphisms—BclI, N363S, and ER22/23EK—and the risk of relapse in children with ALL.

Methods: We conducted a case-control study on 100hildren with ALL, aged 0 to 15 years, including 50 non-elapsed (control) and 50 relapsed (case) subjects. Re-uired patient information such as demographic char-cteristics; relevant clinical and paraclinical findings atiagnosis; chemotherapy protocols used at diagnosis;nd relapse properties, including time interval fromate of initial diagnosis to relapse and number, type,nd site of relapse, were gathered from patients’ med-cal files. Genotyping of BclI, N363S, and ER22/23EKolymorphisms was carried out by polymerase chaineaction restriction fragment–length polymorphismPCR–RFLP). Statistical analysis was performed. Theistribution of BclI, N363S, and ER22/23EK polymor-hism genotypes in our population and in populationsxamined in similar studies was compared using the �2

test or Fischer exact test.Results: One hundred children with ALL, consisting

of 65 males and 35 females, were recruited into this study.Their mean (SD) age was 5.39 (3.02) years. No relapsedor nonrelapsed individuals were homozygous for N363Sand ER22/23EK polymorphisms. The allelic frequenciesof mutant alleles of BclI, N363S, and ER22/23EK poly-

morphisms in all patients were 0.195, 0.02, and 0.005,

280

respectively. No statistically significant association be-tween BclI, N363S, and ER22/23EK polymorphisms andrisk of relapse in children with ALL was observed (P �0.104 [BclI], not calculated for the last 2 polymorphisms[N363S and ER22/23EK]). The incidence of BclI poly-morphism in our population (35/100) differed signifi-cantly from that in a Canadian population with Euro-pean descent (135/219) and a Dutch population (29/53)(P � 0.001 and P � 0.007, respectively).

Conclusion: Our data suggest that there did not ap-pear to be any prognostic value of BclI, ER22/23EK,and N363S polymorphisms for predicting relapse inthis population of 100 Iranian children with ALL.(Clin Ther. 2011;33:280–290) © 2011 Elsevier HSJournals, Inc. All rights reserved.

Key words: acute lymphoblastic leukemia, child-hood, glucocorticoid receptor gene, Iranian popula-tion, polymorphism, relapse.

INTRODUCTIONAcute lymphoblastic leukemia (ALL) is the most fre-quent childhood malignancy. It accounts for nearlyone third (30%) of all pediatric cancers.1 Optimal usef antileukemic medications and implementation oftringent risk-adapted strategies have resulted in im-rovement of treatment outcome and disease progno-is.2 Ninety-five to 97% of patients with ALL experi-nce a first complete remission3; the results of aiterature review on published articles regarding child-

Accepted for publication March 16, 2011.doi:10.1016/j.clinthera.2011.03.0040149-2918/$ - see front matter

© 2011 Elsevier HS Journals, Inc. All rights reserved.

Volume 33 Number 3

y8

gsgagf

otbTng

s(ansaTac

pc

t

2ccaEcvalc

S. Namazi et al.

hood cancers in Iran indicated that the rate of achiev-ing complete remission in Iranian children with ALLwas 79%.4 The 5-year survival of children aged 1 to 14ears increased from 58%, between 1975 and 1977, to8%, between 1996 and 2004 in the United States.5

Despite these successes, 25% to 30% of children withALL still relapse,6 and at least 15% of patients will dieof their disease. Leukemic relapse is the most commoncause of treatment failure in childhood ALL.7 The ma-jor cause of relapse and treatment failure is the devel-opment of resistance to antileukemic agents.8

Glucocorticoids are important antileukemic medi-cations, having been used in the multidrug chemother-apy protocols of childhood ALL for more than 50years.9 The Berlin–Frankfurt–Münster (BFM) studyroup trials reported that in vivo initial treatment re-ponse to prednisone monotherapy is one of the stron-est prognostic factors for treatment outcome in pedi-tric ALL.10,11 Leukemic cell in vitro resistance tolucocorticoids at diagnosis is also a strong risk factoror poor prognosis.12–14 Glucocorticoids exert their

antileukemic effects through reduction of cell prolifer-ation, promotion of G1 cell cycle arrest, and inductionf apoptosis. To induce antileukemic effects, glucocor-icoids have to bind their intracellular receptor, whichelongs to the nuclear hormone receptor superfamily.he ligand-receptor complex then translocates to theucleus and transactivates or transrepresses, or both,lucocorticoid responsive genes.9

Despite the clinical importance of glucocorticoids inthe treatment of ALL, exact mechanisms involved inglucocorticoid sensitivity and development of resis-tance are still poorly understood.15 Previously, someomatic mutations of the glucocorticoid receptor geneNR3C1) were described that were linked with gener-lized cortisol resistance syndromes.16,17 Besides ge-etic mutations, several polymorphisms have been de-cribed within the NR3C1. Among these, some weressociated with altered sensitivity to glucocorticoids.he BclI polymorphism (rs41423247) that consists ofC to G substitution (TGATCA to TGATGA) is lo-

ated 646 bp downstream from exon 2.18 It has beenassociated with an increased sensitivity to glucocortico-ids, resulting in high systolic blood pressure, hyperinsu-linemia, elevated body mass index, high abdominal fatdistribution,19,20 and increased cortisol suppression af-ter low-dose dexamethasone administration.18 Anotherolymorphism resulting in increased sensitivity for gluco-

orticoids is N363S (rs6195) that results in an asparagine

March 2011

o serine substitution at codon 363 of exon 2.21 N363Spolymorphism has been related to an increased bodymass index and low bone mineral density.22 The ER22/3EK polymorphism (also known as rs6189 and rs6190)onsisting of 2 linked single-nucleotide mutations inodons 22 and 23 in exon 2 of NR3C1 caused an aminocid change from arginine (R) to lysine (K).21 TheR22/23EK polymorphism has been correlated withortisol resistance, and ER22/23EK carriers have a fa-orable metabolic health profile, including lower totalnd low-density lipoprotein cholesterol levels andower tendency to develop type 2 diabetes mellitus andardiovascular disease.23

In spite of clinical significance, the number of stud-ies that have analyzed the effect of these polymor-phisms on treatment response in childhood ALL arefew. A literature search resulted in the identification ofonly 4 published articles regarding the association be-tween NR3C1 polymorphisms and treatment responseor relapse in childhood ALL. In the present study, weinvestigated the possible association between 3NR3C1 variants (BclI, N363S, and ER22/23EK) andrisk of relapse in children with ALL. To our knowl-edge, this study is the first survey of its kind in Asia.

PATIENTS AND METHODSStudy Population

The patients included in the present case-controlstudy were children up to 15 years of age diagnosedwith ALL between 1997 and 2007 and subsequentlytreated at 2 pediatric leukemia referral centers in Iran(Hazrat Ali-Asghar Children’s Hospital, affiliated withTehran University of Medical Sciences, in Tehran, andMotahari Clinic and Faghihi Hospital, both affiliatedwith Shiraz University of Medical Sciences, in Shiraz).The study was approved by the Medical Ethics Com-mittee of both centers. Written informed consent wasobtained from the patients or their parents or guard-ians before blood sample collection. Required data,including demographic characteristics, relevant clini-cal and paraclinical findings at diagnosis (eg, completeblood count, immunophenotype, and cytogenetics),chemotherapy protocol used at diagnosis, and relapseproperties (time interval from date of initial diagnosisto relapse, type and site of relapse), were recorded ret-rospectively from patients’ medical files.

The study population was divided into 2 groups:nonrelapsed (control) and relapsed (case). The dura-

tion of remission in the nonrelapsed group was �18

281

((3edwa12

Clinical Therapeutics

months. Relapses were divided into early (first completeremission duration �18 months) and late relapse (firstcomplete remission duration � 18 months).6 No inclu-sion-exclusion criteria regarding number (first, second, ormore), site (bone marrow, central nervous system, testis,or others), type (isolated or combined), and time of re-lapses (early or late) were applied for selection of relapsedpatients. Children who died during remission inductionbefore their early response status was known were ex-cluded from this study. The parents and all 4 grandpar-ents of each subject were of Iranian origin.

Blood SamplingTo avoid confusing somatic mutations with poly-

morphisms, peripheral blood samples were collectedfrom all patients at complete remission. A 10-mL ve-nous blood sample was drawn into a EDTA-containingtube. The mononuclear cell (buffy-coat) fraction wasisolated by Lymphoflot density gradient centrifugation(Biotest, Dreieich, Germany) to increase the yield ofDNA extraction.

Genetic AnalysisPolymerase Chain Reaction Amplification

Genomic DNA was extracted from mononuclearcell fraction by use of QIAamp DNA Mini Kit (Qiagen,Hilden, Germany) according to the protocol suppliedby the manufacturer. Polymerase chain reaction (PCR)amplification of DNA segments containing the poly-morphic sites was carried out using primer sequencesand the PCR reaction mixture described previ-ously.21,24 Forward and reverse primer sequences usedfor each polymorphism were as follows: 5=-TGCT-GCCTTATTTGTAAATTCGT-3= (forward) and 5=-AAGCTTAACAATTTTGGCCATC-3= (reverse) forBclI polymorphism, 5=-GATTCGGAGTTAACTA-AAAG-3= (forward) and 5=-ATCCCAGGTCATTTC-CCATC-3= (reverse) for ER22/23EK polymorphism,and 5=-AGTACCTCTGGAGGACAGAT-3= (forwardexon 2–5) and 5=-GTCCATTCTTAAGAAACAGG-3=(reverse exon 2-5) for N363S polymorphism. The PCRprotocol included 1 cycle of denaturation at 95°C for 5minutes followed by 30 cycles of denaturation at 94°Cfor 1 minute, annealing at 55°C for 1.5 minutes (exceptfor N363S polymorphism [exon 2–5] at 51°C), exten-sion at 72°C for 1.5 minutes, and a final cycle at 72°C

for 5 minutes.

282

GenotypingRestriction fragment-length polymorphism (RFLP)

analysis was performed to determine NR3C1 geno-types as described previously.23–25 To determine BclIpolymorphism genotypes, 20 �L of relevant PCR prod-uct (335 bp) was digested by 20 units of BclI restrictionendonuclease (Roche Applied Science, Mannheim, Ger-many) for 1.5 hours at 50°C. The DNA fragments wereseparated on 2% agarose gel electrophoresis (FermentasUAB, Vilnius, Lithuania) and visualized by ethidium bro-mide staining. The CC genotype produced 2 bands (117and 222 bp), the CG variant gave 3 fragments (117, 222,and 335 bp), and the GG genotype remained undigested(335 bp).

The ER22/23EK polymorphism genotypes were de-termined by digesting 20 �L of relevant PCR product482 bp) with 1.25 units of MnlI restriction enzymeNew England Biolabs, Beverly, Massachusetts) at7°C for 1.5 hours. Fragments were visualized withthidium bromide on a 4% agarose gel. Endonucleaseigestion yielded fragments of 142 and 163 bp for theild-type allele (ER22/23ER genotype); heterozygousllele (ER22/23EK genotype) appeared as 3 bands of42, 163, and 177 bp; and homozygous allele (EK22/3EK genotype) gave 163 and 177 bp fragments.

To determine N363S polymorphism genotypes, 20�L of relevant PCR product (248 bp) were digestedwith 1 unit of Tsp509I restriction endonuclease (NewEngland Biolabs, Beverly, Massachusetts) for 1.5hours at 65°C. Fragments were visualized by ethidiumbromide staining after electrophoresis on agarose gel3%. Digestion produced 3 fragments of 19, 95, and134 bp (wild type allele, N363N) or 2 bands of 95 and153 bp (heterozygous mutant allele, N363S).

To confirm the accuracy of the genotyping per-formed by PCR-RFLP, 1 randomly selected samplefrom each BclI polymorphism genotype (CC, CG, andGG), ER22/23EK (ER22/23ER and ER22/23EK), andN363S (N363N and N363S) was genotyped by directDNA sequencing. A random number generator pro-vided by Excel 2007 (Microsoft, Redmond, Washing-ton) was used to select random samples. Each samplewas sequenced once. ABI 310 Genetic Analyzer (Ap-plied Biosystems, Carlsbad, California) was used fordirect DNA sequencing. No discrepancies were found.

Statistical AnalysisCategorical data were expressed as a percentage.

Continuous variables were reported as mean (SD). The

Volume 33 Number 3

sopbvdp(OHt

tcib(mat

tcsLAM

fp

Istnwttdum

psitrpnm

tt

irlwbwcnfi3i3

mjr

S. Namazi et al.

association between prognostic factors and occurrenceof relapse was examined with logistic regression anal-ysis as described.26 To test for any possible associationbetween the genotypes of the relevant glucocorticoidreceptor gene polymorphisms and prognostic factors,as well as the risk of relapse, �2 test or Fischer exact testwas used. The �2 test or Fischer exact test was also usedto compare the incidence of different BclI, N363S, andER22/23EK polymorphism genotypes in our popula-tion and those recorded in 4 similar studies in otherpopulations (Canadian with European descent [2 stud-ies],27,28 Dutch,29 and Italian30) identified by literatureearch in the MEDLINE database (http://www.pubmed.rg/) using relevant search terms without any language orublication date limitations. P � 0.05 was considered toe statistically significant. For statistical analysis, SPSSersion 13.0 (SPSS Inc., Chicago, Illinois) was used. Har-y-Weinberg equilibrium was calculated for BclIolymorphism genotypes with Arlequin version 3.11Ecology and Evolution Inc., Bern, Switzerland).wing to the low frequency of mutant genotypes,ardy-Weinberg equilibrium was not calculated for

he N363S and ER22/23EK polymorphisms.

RESULTSIn this study, 100 pediatric patients with ALL wereenrolled, including 50 nonrelapsed and 50 relapsedsubjects. Their mean (SD) age was 5.39 (3.02) years.Demographic characteristics and laboratory findingsrelated to treatment efficacy in relapsed and nonre-lapsed subjects are shown in Table I. The percentage ofmales was higher than that of females in both groups.Seventy nine (79%) subjects had initial leukocytecounts �50,000/�L. Early pre–B-cell immunopheno-ype was identified in 34 (34%) of the patients, pre–B-ell in 28 (28%). No statistically significant differencesn demographic characteristics and laboratory findingsetween relapsed and nonrelapsed subjects were foundP � 0.05). As immunophenotype, ploidy, and bonearrow status at day 14 of remission induction were only

vailable for 68%, 33%, and 35% of patients, respec-ively, statistical analysis of these items was impossible.

Treatment protocols of studied populations at ini-ial diagnosis are summarized in Table II. The mostommon chemotherapy protocol was Medical Re-earch Council United Kingdom Acute Lymphoblasticeukemia XI (MRC UK ALL XI) (38%),31 followed bycute Lymphoblastic Leukemia Berlin–Frankfurt–

ünster (ALL IC-BFM) 2002 (30%),32 Berlin–Frank- l

March 2011

urt–Münster (BFM) 1997 (14%),33 and standard riskre–B-cell ALL (5%).34 Suggested glucocorticoid in all

phases of the MRC UK ALL XI regimen is prednisone(60 mg/m2/d PO). As prednisone is not available inran, and as prednisone and prednisolone have theame dose equivalency, prednisolone is used instead. Inhe remission-induction phase, patients received pred-isolone from days 0 to 28. After day 28, prednisoloneas tapered over 2 weeks. Prednisolone treatment in

he consolidation phase continued for 7 days. In main-enance therapy, prednisolone was given in the first 5ays of each month, and its administration was contin-ed until completion of 32 (for boys) or 20 (for girls)onths of maintenance therapy.Intrathecal administration of hydrocortisone was

art of triple intrathecal prophylaxis of central nervousystem leukemia in the MRC UK ALL XI protocol, andts dose was age dependent (9–15 mg). According tohe modified ALL IC-BFM 2002 protocol for standardisk patients, after a 7-day prephase monotherapy withrednisolone (60 mg/m2/d PO)—along with the begin-ing of L-asparaginase (5000 IU/m2), vincristine (1.5g/m2), and daunorubicin (30 mg/m2) for remission

induction—prednisolone was given orally at doses of60 mg/m2/d from days 8 through 28. After day 28,prednisolone was tapered over 9 days. In the reinduc-tion-reintensification phase, prednisolone was re-placed by oral or intravenous dexamethasone at dosesof 10 mg/m2/d. Dexamethasone was given from days 8hrough 28 of reinduction-reintensification phase andapered over 9 days.

Table III provides information on relapse character-stics from this study’s relapsed subjects. A total of 85elapses were recorded from the 50 patients in the re-apsed group. Sixty-nine out of 85 relapses (81.18%)ere considered to be isolated, 13 (15.29%) were com-ined, and the type of the remaining 3 relapses (3.53%)as unknown. Bone marrow (52.94%) was the most

ommon site of leukemic relapse, followed by centralervous system (47.06%) and testis (8.24%). Of 49rst relapses, 13 (26.53%) were classified as early and3 (67.35%) as late; the time interval from the date ofnitial diagnosis to relapse occurrence was unknown infirst relapses (6.12%).The distribution of glucocorticoid receptor poly-

orphism genotypes in relapsed and nonrelapsed sub-ects and the association of these variants with risk ofelapse occurrence are displayed in Table IV. No re-

apsed or nonrelapsed individuals were homozygous

283

Clinical Therapeutics

Table I. Demographic and clinical characteristics of total patients (N � 100). Data are number (%) ofpatients.

CharacteristicNonrelapsed

(n � 50)Relapsed(n � 50) P*

SexMale 32 (64) 33 (66) 0.726Female 18 (36) 17 (34)

Age at diagnosis (y)�5 26 (52) 24 (48) 0.525–10 17 (34) 22 (44)�10 7 (14) 4 (8)

Initial leukocyte count (�L)�10,000 27 (54) 24 (48) 0.85310,000–49,000 15 (30) 13 (26)�50,000 8 (16) 5 (10)Unknown — 8 (16)

Initial peripheral lymphoblast count (�L)�1000 30 (60) 27 (54) 0.763�1000 19 (38) 15 (30)Unknown 1 (2) 8 (16)

Platelet count (�L)�20,000 4 (8) 6 (12) 0.59520,000–100,000 31 (62) 23 (46)�100,000 15 (30) 12 (24)Unknown — 9 (18)

Morphology (FAB classification system)L1 20 (40) 26 (52) 0.457L2 26 (52) 20 (40)L3 1 (2) 1 (2)Unknown 3 (6) 3 (6)

ImmunophenotypeEarly pre–B-cell 18 (36) 16 (32) NDPre–B-cell 10 (20) 18 (36)Mature B-cell 1 (2) 1 (2)T-cell 1 (2) 3 (6)Unknown 20 (40) 12 (24)

PloidyNormal diploidy 12 (24) 8 (16) NDHyperdiploidy 6 (12) 7 (14)Unknown 32 (64) 35 (70)

BM status at day 14 of remission inductionRapid early response 20 (40) 8 (16) NDSlow early response 2 (4) 5 (10)Unknown 28 (56) 37 (74)

FAB � French–American–British; ND � not done (owing to limited number of available variables); BM � bone marrow.*�2 or Fisher’s exact test.

284 Volume 33 Number 3

psaps

d

D

salo

pkt

dNt

S. Namazi et al.

for ER22/23EK and N363S polymorphisms. The al-lelic frequencies of the mutant allele of BclI, N363S,and ER22/23EK polymorphisms in all patients were0.195, 0.02, and 0.005, respectively. The distributionof different BclI polymorphism genotypes was in ac-cordance with Hardy-Weinberg equilibrium. No sta-tistically significant association between different BclIolymorphism genotypes and risk of relapse was ob-erved (P � 0.104). The number of the heterozygousnd homozygous carriers of N363S and ER22/23EKolymorphisms was too small to test for statisticalignificance.

Apart from peripheral blood lymphoblast count atiagnosis for BclI polymorphism (P � 0.027), no par-

ticular association between studied prognostic factors(eg, age, leukocyte count, and immunophenotype atdiagnosis) and different BclI, N363S, and ER22/23EKpolymorphism genotypes were found (P � 0.05, datanot shown). The possible interrelationship among dif-ferent variants of studied polymorphisms could not bedetermined statistically because the allelic frequenciesof mutant alleles of N363S and ER22/23EK polymor-phisms were very low.

Table V provides a comparison between incidenceof studied polymorphisms in our population and otherpopulations in childhood ALL. Except for BclI poly-morphism in Canadian with European descent27 and

Table II. Treatment protocol of study populationat initial diagnosis (N � 100).

Treatment Protocol No. (%) Reference

MRC UK ALL XI 38 (38) 31ALL IC-BFM 2002 30 (30) 32BFM 1997 14 (14) 33Standard risk pre–B-cell ALL 5 (5) 34ALL high risk for rapid earlyresponders

3 (3) 34

T cell 3 (3) 34LMB 89 1 (1) 34Unknown 6 (6) —

ALL IC-BFM � Acute Lymphoblastic Leukemia Intercon-tinental Berlin–Frankfurt–Münster; LMB � lymphomamalignancy B; MRC UK ALL XI � Medical Research Coun-cil United Kingdom Acute Lymphoblastic Leukemia XI.

utch29 populations (P � 0.001 and P � 0.007, re-

March 2011

pectively), the mutant allele distribution of N363Snd ER22/23EK polymorphisms in the Iranian popu-ation was in accordance with the incidence reported inther populations (P � 0.05).

DISCUSSIONAccording to a general model of ALL, interactionsamong host, disease, and treatment factors determinetreatment efficacy.35 Host pharmacogenetics affectharmacokinetics and pharmacodynamics of antileu-emic drugs including absorption, metabolism, excre-ion, cellular transport, targets, and target pathways.36

Glucocorticoids are an important component of child-hood ALL treatment.37 The in vivo prednisone re-sponse and the in vitro glucocorticoid sensitivity oflymphoblasts at initial diagnosis have been reported tobe predictors of ALL outcome in children.10–14 As glu-cocorticoids exert antileukemic actions in leukemiccells through their intracellular receptors, glucocorti-coid resistance might be the result of genetic variationswithin the NR3C1.9 Among several polymorphismsescribed in the NR3C1, the clinical relevance of BclI,363S, and ER22/23EK with altered responsiveness

o glucocorticoids has been studied.29

According to our literature search, the effect ofNR3C1 polymorphisms on the efficacy and resistanceto glucocorticoids in childhood ALL has been consid-ered in only a few clinical studies. Fleury et al con-ducted a cohort study on 222 children with ALL who

Table III. Classification of recorded relapses in 50relapsed subjects (total relapses � 85).

Type/Site of Relapse No. (%)

IsolatedBM 33 (38.82)CNS 31 (36.47)Testis 4 (4.71)Ovary 1 (1.18)

CombinedBM and CNS 9 (10.59)BM and testis 3 (3.53)Kidneys and peritoneal lymph nodes 1 (1.18)

Unknown 3 (3.53)

BM � bone marrow; CNS � central nervous system.

285

hmta

t

Clinical Therapeutics

were of European descent and living in the province ofQuebec, Canada.27 They investigated the effect of cy-tochrome P450 3A4 -290A/G substitution and threeNR3C1 polymorphisms (BclI, N363S, and ER22/23EK) on the outcome of pediatric ALL treatment.Statistical analysis showed that among studied polymor-phisms, there was only a significant reduction in overallsurvival probability (but not disease-free survival andevent-free survival) in the homozygous carriers of BclIpolymorphism (GG genotype) compared with those withother BclI genotypes (CC and CG).27 Their results con-tradicted findings about BclI polymorphism associationwith increased glucocorticoid sensitivity.18,38,39 The au-thors explained this discrepancy by citing a study thatreported that lysozymes of cultured leukocytes of GGindividuals tend to have a lower sensitivity to dexameth-asone inhibitory effects in vitro.40

To better understand the reasons for the observedassociation, the same study group analyzed the possi-ble association of BclI and 3 other functional polymor-phisms of NR3C1, including –627A/G, –3807A/G,and 9bT/C, with pediatric ALL outcome in a largersample size (n � 310) from the same topographicalregion. In accordance with previous results, BclI GGgenotype was associated with reduced overall survival.

Table IV. Distribution of different BclI, N363S, and Ewith risk of relapse (N � 100). Data are nu

Polymorphism

BclIWild-type (TCA/TCA)Heterozygous carriers (TCA/TGA)Homozygous carriers (TGA/TGA)

N363SWild-type (ATT/ATT)Heterozygous carriers (ATT/GTT)Homozygous carriers (GTT/GTT)

ER22/23EKWild-type (GAGAGG/GAGAGG)Heterozygous carriers (GAGAGG/GAAAAG)Homozygous carriers (GAAAAG/GAAAAG)

ND � not done (because of too small number of heterozyg

In the more recent study, quantitative real-time–PCR

286

(RT-PCR) analysis showed a higher �/� ratio in theeterozygous and homozygous carriers of BclI poly-orphism (CG and GG genotypes).28 It was reported

hat the transactivation capacity of the � isoform isbout 50% less than that of the � isoform, the func-

tional splice variant of the glucocorticoid receptor.9

Preliminary evidence suggests a possible associationbetween high levels of � isoform and in vitro glucocor-icoid resistance41 as well as in vivo prednisone poor

response in childhood ALL.9

The Dutch Childhood Oncology Group (DCOG)analyzed the incidence of genetic variations in theNR3C1 and their correlation with in vitro and in vivoglucocorticoid resistance in 57 Dutch children withALL.29 The different genotypes of the detected poly-morphisms such as BclI, N363S, and ER22/23EK werenot correlated with prednisone resistance. Further-more, no association was found between the presenceof NR3C1 polymorphisms and ALL outcomes. Ma-rino et al30 investigated the role of polymorphisms ofsome genes involved in the pharmacokinetics and phar-macodynamics of glucocorticoids such as NR3C1 in glu-cocorticoid response and toxicity in 36 Italian pediatricpatients with ALL. Patients were stratified into standard,medium, and high-risk groups according to a number of

23EK polymorphism genotypes and their associationr (%) of patients.

Nonrelapsed(n � 50)

Relapsed(n � 50) P

33 (66) 32 (64) 0.10413 (26) 18 (36)

4 (8) 0

49 (98) 47 (94) ND1 (2) 3 (6)

0 0

49 (98) 50 (100) ND1 (2) 0

0 0

d homozygous carriers).

R22/mbe

ous an

criteria (eg, response to 7-day prephase monotherapy

Volume 33 Number 3

m

sasN

bwnBN

S. Namazi et al.

with prednisone). Differences in allelic frequencies ofBclI, N363S, and ER22/23EK polymorphisms among thedifferent risk groups were not significant.

Our case-control study analyzed the possible asso-ciation between typical polymorphisms of NR3C1 andrisk of relapse in children with ALL. Along with theDCOG and Marino et al studies, we found no statisti-cally significant association between different BclI,N363S, and ER22/23EK polymorphism genotypes andrisk of relapse in pediatric ALL. These results could bedue to the small sample sizes in the present and afore-mentioned studies. The low frequency of N363S (0.02)and ER22/23EK (0.005) polymorphisms makes thesample size issue more important. The incidence ofN363S and ER22/23EK polymorphisms reported inthe literature varies substantially among different pop-ulations. For example, allelic frequency of the mutantallele of N363S was found to be 3.1% in the elderlyhealthy Dutch Caucasians21 and 7.4% in healthy nor-

otensive adults of an Australian population.42 The

Table V. Comparison among incidence of BclI,N363S, and ER22/23EK polymorphismsin our population (Iranian) and otherpopulations in childhood ALL.

PolymorphismPopulation(Reference) Incidence P*

BclI (G allele) Canadian (27) 135/219 �0.001Iranian 35/100Dutch (29) 29/53 0.007Iranian 35/100Italian (30) 13/36 0.68Iranian 35/100

N363S Canadian (27) 7/220 0.744Iranian 4/100Dutch (29) 7/57 0.1Iranian 4/100Italian (30) 1/36 1.0Iranian 4/100

ER22/23EK Canadian (27) 11/222 0.114Iranian 1/100Dutch (29) 2/57 0.554Iranian 1/100Italian (30) 0/36 1.0Iranian 1/100

ALL � acute lymphoblastic leukemia.*�2 or Fisher’s exact test.

frequency of N363S reported in a heterogeneous pop-

March 2011

ulation of South Asian origin (Indian, Pakistani, andBangladeshi) was 0.3%.43 The N363S variant has notbeen detected in Japanese or Chinese populationsyet.44

Apart from sample size, a combination of allelesfrom several polymorphisms in 1 gene (haplotypes) ora combination of a polymorphism in 1 gene with ge-netic variations from other relevant genes might lead toclinically detectable phenotypic changes.44 In this re-gard, TthIII1 polymorphism, which causes the C to Tnucleotide change in the promoter region of NR3C1,has been reported to have linkage with the ER22/23EKallele. Carriers of both polymorphisms (TthIII1 CT orTT and ER22/23EK) showed a relative resistance toglucocorticoids measured by a dexamethasone sup-pression test, whereas no differences in responsivenessto glucocorticoids were found between the group ofnoncarriers of both variant alleles (TthIII1 CC andER22/23ER) and the group of carriers of only theTthIII1 T allele (TthIII1 CT or TT and ER22/23ER).45

As TthIII1 polymorphism was not determined in ourstudy, analyzing the impact of combined mutant allelesof TthIII1 and ER22/23EK polymorphisms on risk ofrelapse was not feasible.

Results of a study conducted on patients with severeobesity suggested that the BclI and N363S variantscould act in synergy.46 Fleury et al27 observed no syn-ergistic effects of these 2 polymorphisms in their firststudy. The simultaneous presence of N363S and BclIpolymorphisms in an individual was not correlatedwith in vivo or in vitro glucocorticoid resistance in theDCOG survey.29 This issue was not considered in thetudy by Marino et al.30 The frequency of the mutantllele of the N363S variant was too low in the presenttudy (0.02) to analyze the possible synergistic effect of363S and BclI polymorphisms on risk of relapse.Among different prognostic factors, only peripheral

lood lymphoblast count at diagnosis was associatedith BclI polymorphism. Other prognostic factors didot have any statistically significant association withclI and 2 other polymorphisms (ER22/23EK and363S). The study by Fleury et al27 observed that

among different prognostic factors assessed, only pa-tient’s sex was associated with BclI genotypes. Thepossible association between NR3C1 polymorphismsand demographic and clinical characteristics that haveprognostic value was not analyzed in either of the other

studies.29,30

287

pt

1

1

1

1

1

1

1

Clinical Therapeutics

The present study had 2 main limitations. First, oursample size was relatively small. Given the low fre-quency of N363S and ER22/23EK polymorphisms, theinability to detect a statistically significant associationbetween the studied polymorphisms and the risk ofrelapse in pediatric ALL might be due to the limitedstatistical power of our survey. Second, determinationof in vivo prednisone response was impossible becausechemotherapy protocols rather than ALL-BFM werealso used in our study population. Furthermore, lym-phoblasts of patients at initial diagnosis were not avail-able for in vitro glucocorticoid sensitivity assessmentby 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide assay. As a consequence, evaluating theassociation of BclI, N363S, and ER22/23EK polymor-phisms with in vivo prednisone response as well as invitro glucocorticoid sensitivity was not feasible.

CONCLUSIONOur data suggest that there did not appear to be anyprognostic value of BclI, ER22/23EK, and N363Solymorphisms for predicting relapse in this popula-ion of 100 Iranian children with ALL.

ACKNOWLEDGMENTSThe authors would like to thank the children and theirfamilies who participated in this study. We also ac-knowledge all the staff of both pediatric leukemia referralcenters (Hazrat Ali-Asghar Children’s Hospital and Mo-tahari Clinic and Faghihi Hospital) for blood collectionand the personnel and chairman of Payvand MedicalLaboratory, Tehran, Iran, Dr. Behzad Poopak, forbuffy-coat isolation. We are especially grateful to Beh-naz Valibeigi for the excellent assistance with geneticanalysis of patients’ samples. The authors have indi-cated that they have no conflicts of interest regardingthe content of this article.

Dr. Namazi contributed to the study design, datainterpretation, and manuscript review. Dr. Zareifarcontributed to the study design, patient selection, datainterpretation, and manuscript review. Dr. Monabaticontributed to the study design, genetic analysis, andmanuscript review. Dr. Ansari contributed to the pa-tient selection, data interpretation, and manuscript re-view. Dr. Karimzadeh contributed to blood sampling,data collection, genetic analysis, and the drafting of the

manuscript.

288

REFERENCES1. Margolin JF, Steuber SC, Poplack DG. Acute lymphoblastic

leukemia. In: Pizzo PA, Poplack DG, eds. Principles andPractice of Pediatric Oncology.Philadelphia, Pa: LippincottWilliams & Wilkins; 2005:538–590.

2. Pui CH, Evans WE. Treatment of acute lymphoblasticleukemia. N Engl J Med. 2006;354:166–178.

3. Mahoney DH. Acute lymphoblastic leukemia. In: McMillanUA, DeAngelis CD, Feigin RD, Warshaw JB, eds. Oski’sPediatrics.Philadelphia, Pa: Lippincott Williams & Wilkins;2006: 1750–1758.

4. Mousavi SM, Pourfeizi A, Dastgiri S. Childhood cancer inIran. J Pediatr Hematol Oncol. 2010;32:376–382.

5. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2009. CACancer J Clin. 2009;59:225–249.

6. Chessells JM. Relapsed lymphoblastic leukaemia in children: acontinuing challenge. Br J Haematol. 1998;102:423–438.

7. Gaynon PS. Childhood acute lymphoblastic leukaemia andrelapse. Br J Haematol. 2005;131:579–587.

8. Verrills NM, Liem NL, Liaw TY, et al. Proteomic analysisreveals a novel role for the actin cytoskeleton in vincristineresistant childhood leukemia—an in vivo study. Proteomics.2006;6:1681–1694.

9. Tissing WJ, Meijerink JP, den Boer ML, Pieters R. Moleculardeterminants of glucocorticoid sensitivity and resistance inacute lymphoblastic leukemia. Leukemia. 2003;17:17–25.

0. Schrappe M, Reiter A, Zimmermann M, et al. Long-termresults of four consecutive trials in childhood ALL per-formed by the ALL-BFM study group from 1981 to 1995.Berlin-Frankfurt-Münster. Leukemia. 2000;14:2205–2222.

1. Lauten M, Stanulla M, Zimmermann M, et al. Clinicaloutcome of patients with childhood acute lymphoblasticleukaemia and an initial leukaemic blood blast count of lessthan 1000 per microliter. Klin Padiatr. 2001;213:169–174.

2. Pieters R, Huismans DR, Loonen AH, et al. Relation ofcellular drug resistance to long-term clinical outcome inchildhood acute lymphoblastic leukaemia. Lancet. 1991;338:399–403.

3. Hongo T, Yajima S, Sakurai M, et al. In vitro drug sensitivitytesting can predict induction failure and early relapse ofchildhood acute lymphoblastic leukemia. Blood. 1997;89:2959–2965.

4. Kaspers GJ, Pieters R, Van Zantwijk CH, et al. Prednisoloneresistance in childhood acute lymphoblastic leukemia:vitro-vivo correlations and cross-resistance to other drugs.Blood. 1998;92:259–266.

5. Kofler R, Schmidt S, Kofler A, Ausserlechner MJ. Resistanceto glucocorticoid-induced apoptosis in lymphoblastic leu-kemia. J Endocrinol. 2003;178:19–27.

6. Hurley DM, Accili D, Stratakis CA, et al. Point mutationcausing a single amino acid substitution in the hormone

binding domain of the glucocorticoid receptor in famil-

Volume 33 Number 3

2

2

2

2

2

3

3

3

3

3

3

3

3

3

3

4

4

4

4

4

S. Namazi et al.

ial glucocorticoid resistance. J ClinInvest. 1991;87:680 – 686.

17. Ruiz M, Lind U, Gafvels M, et al.Characterization of two novel muta-tions in the glucocorticoid receptorgene in patients with primary corti-sol resistance. Clin Endocrinol. 2001;55:363–371.

18. van Rossum EF, Koper JW, van denBeld AW, et al. Identification of theBclI polymorphism in the glucocorti-coid receptor gene: association withsensitivity to glucocorticoids in vivoand body mass index. Clinl Endocri-nol. 2003;59:585–592.

19. Weaver JU, Hitman GA, KopelmanPG. An association between a Bc1Irestriction fragment length polymor-phism of the glucocorticoid recep-tor locus and hyperinsulinaemia inobese women. J Mol Endocrinol. 1992;9:295–300.

20. Bueman B, Vohl MC, Chagnon M, etal. Abdominal visceral fat is associ-ated with a BclI restriction fragmentlength polymorphism at the gluco-corticoid receptor gene locus. ObesRes. 1997;5:186–192.

21. Koper JW, Stolk RP, de Lange P, etal. Lack of association between fivepolymorphisms in the human gluco-corticoid receptor gene and gluco-corticoid resistance. Hum Genet.1997;99:663–668.

22. Huizenga NA, Koper JW, De LangeP, et al. A polymorphism in theglucocorticoid receptor gene may beassociated with an increased sensi-tivity to glucocorticoids in vivo. J ClinEndocrinol Metab. 1998;83:144–151.

23. van Rossum EF, Koper JW, HuizengaNA, et al. A polymorphism in theglucocorticoid receptor gene, whichdecreases sensitivity to glucocortico-ids in vivo, is associated with lowinsulin and cholesterol levels. Diabe-tes. 2002;51:3128–3134.

24. Bachmann AW, Sedgley TL, JacksonRV, et al. Glucocorticoid receptorpolymorphisms and post-traumaticstress disorder. Psychoneuroendocri-

nology. 2005;30:297–306.

March 2011

5. Lin RC, Wang WY, Morris BJ. Asso-ciation and linkage analyses of glu-cocorticoid receptor gene markersin essential hypertension. Hyperten-sion. 1999;34:1186–1192.

6. Dawson B, Trapp R. Basic and ClinicalBiostatistics. New York: McGraw-HillMedical; 2004.

7. Fleury I, Primeau M, Doreau A, et al.Polymorphisms in genes involved inthe corticosteroid response and theoutcome of childhood acute lym-phoblastic leukemia. Am J Pharmacog-enomics. 2004;4:331–341.

8. Labuda M, Gahier A, Gagné V, et al.Polymorphisms in glucocorticoid re-ceptor gene and the outcome of child-hood acute lymphoblastic leukemia(ALL). Leuk Res. 2010;34:492–497.

9. Tissing WJ, Meijerink JP, den BoerML, et al. Genetic variations in theglucocorticoid receptor gene are notrelated to glucocorticoid resistancein childhood acute lymphoblasticleukemia. Clin Cancer Res. 2005;11:6050–6056.

0. Marino S, Verzegnassi F, Tamaro P,et al. Response to glucocorticoidsand toxicity in childhood acute lym-phoblastic leukemia: role of poly-morphisms of genes involved in glu-cocorticoid response. Pediatr BloodCancer. 2009;53:984–991.

1. Hann IM, Richards SM, Eden OB,Hill FG. Analysis of the immunophe-notype of children treated on theMedical Research Council UnitedKingdom Acute Lymphoblastic Leu-kaemia Trial XI (MRC UKALLXI).Medical Research Council Child-hood Leukaemia Working Party. Leu-kemia. 1998;12:1249–1255.

2. ALL IC-BFM 2002. A randomizedtrial of the I-BFM-SG for the manage-ment of childhood non-B acute lym-phoblastic leukemia. http://www.sld.cu/galerias/pdf/sitios/hematologia/all_ic-bfm_2002_-_bp.pdf. AccessedDecember 17, 2010.

3. Lanzkowsky P. Manual of PediatricHematology and Oncology. 3rd ed. San

Diego, Calif: Academic Press; 2000.

4. Lanzkowsky P. Manual of PediatricHematology and Oncology. 4th ed. Bur-lington, Mass: Elsevier; 2005.

5. Aplenc R, Lange B. Pharmacoge-netic determinants of outcome inacute lymphoblastic leukaemia. Br JHaematol. 2004;125:421–434.

6. Davidsen ML, Dalhoff K, SchmiegelowK. Pharmacogenetics influence treat-ment efficacy in childhood acute lym-phoblastic leukemia. J Pediatr HematolOncol. 2008;30:831–849.

7. Haarman EG, Kaspers GJ, VeermanAJ. Glucocorticoid resistance inchildhood leukaemia: mechanismsand modulation. Br J Haematol.2003;120:19–29.

8. Stevens A, Ray DW, Zeggini E, et al.Glucocorticoid sensitivity is deter-mined by a specific glucocorticoidreceptor haplotype. J Clin EndocrinolMetab. 2004;89:892–897.

9. van Rossum EF, Lamberts SW. Poly-morphisms in the glucocorticoid re-ceptor gene and their associationswith metabolic parameters and bodycomposition. Recent Prog Horm Res.2004;59:333–357.

0. Panarelli M, Holloway CD, Fraser R,et al. Glucocorticoid receptor poly-morphism, skin vasoconstriction,and other metabolic intermediatephenotypes in normal human sub-jects. J Clin Endocrinol Metab. 1998;83:1846–1852.

1. Haarman EG, Kaspers GJ, Pieters R,et al. Glucocorticoid receptor alpha,beta and gamma expression vs invitro glucocorticod resistance inchildhood leukemia. Leukemia. 2004;18:530–537.

2. Lin RC, Wang WY, Morris BJ. Highpenetrance,overweight,andglucocor-ticoid receptor variant: case-controlstudy. BMJ. 1999;319:1337–1338.

3. Syed AA, Irving JA, Redfern CP, et al.Low prevalence of the N363S poly-morphism of the glucocorticoid re-ceptor in South Asians living in theUnited Kingdom. J Clin EndocrinolMetab. 2004;89:232–235.

4. van Rossum EF, Russcher H, Lam-

berts SW. Genetic polymorphisms

289

Clinical Therapeutics

and multifactorial diseases: factsand fallacies revealed by the glu-cocorticoid receptor gene. TrendsEndocrinol Metab. 2005;16:445–450.

45. van Rossum EF, Roks PH, de JongFH, et al. Characterization of a pro-moter polymorphism in the glucocor-ticoid receptor gene and its relation-ship to three other polymorphisms.Clin Endocrinol. 2004;61:573–581.

46. Di Blasio AM, van Rossum EF, Mae-strini S, et al. The relation betweentwo polymorphisms in the glucocor-ticoid receptor gene and body massindex, blood pressure and choles-terol in obese patients. Clin Endocri-nol (Oxf). 2003;59:68–74.

290

Address correspondence to: Iman Karimzadeh, PharmD, Faculty of Phar-macy, Shiraz University of Medical Sciences, Shiraz, Iran. E-mail:

karimzadehiman@yahoo.com

Volume 33 Number 3