Hindawi Publishing CorporationDisease MarkersVolume 35 (2013) Issue 6 Pages 701ndash710httpdxdoiorg1011552013762685

Research ArticleInvestigation of Genetic Polymorphisms Related to the Outcomeof Radiotherapy for Prostate Cancer Patients

Hellen Silva Cintra12 Juliana Castro Dourado Pinezi34

Graziella Dias Pinheiro Machado24 Gustavo Moura de Carvalho23

Ana Terra Silva Carvalho12 Thalles Eduardo Dias dos Santos23

Ricardo Duarte Marciano23 and Renata de Bastos Ascenccedilo Soares123

1 Programa de Mestrado em Genetica Pontifıcia Universidade Catolica de Goias Avenida Universitaria 1440 Setor Universitario74605-010 Goiania GO Brazil

2 Laboratorio de Oncogenetica e Radiobiologia Associacao de Combate ao Cancer em Goias Rua 239 N52 Lt29Setor Universitario 74605-070 Goiania GO Brazil

3 Departamento de Medicina Pontifıcia Universidade Catolica de Goias Avenida Universitaria 1440 Setor Universitario74605-010 Goiania GO Brazil

4 Servico de Radioterapia Hospital Araujo Jorge Associacao de Combate ao Cancer em Goias Rua 239 N52 Lt181Setor Universitario 74605-070 Goiania GO Brazil

Correspondence should be addressed to Renata de Bastos Ascenco Soares renatasoarespucgoiasedubr

Received 30 June 2013 Accepted 8 October 2013

Academic Editor Esperanza Ortega

Copyright copy 2013 Hellen Silva Cintra et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The purpose of this study was to evaluate the association between ATM TP53 and MDM2 polymorphisms in prostate cancerpatients and morbidity after radiotherapy The presence of ATM (rs1801516) TP53 (rs1042522 rs1800371 rs17878362 rs17883323and rs35117667) andMDM2 (rs2279744) polymorphismswas assessed by direct sequencing of PCR fragments from48 patientswithhistologically proven prostate adenocarcinoma and treated with external beam radiation The side effects were classified accordingto the Radiation Therapy Oncology Group (RTOG) score The results showed no association between clinical characteristics andthe development of radiation toxicities (P gt 005) The CgtT transition in the position 16273 (intron 3) of TP53 (rs35117667) wassignificantly associated with the risk of acute skin toxicity (OR 00072 95 CI 00002ndash0227 P = 0003) The intronic TP53polymorphism at position 16250 (rs17883323) was associated with chronic urinary toxicity (OR 0071 95CI 0006ndash0784 P= 0032) No significant associations were found for the remaining polymorphisms (P gt 005) The results show that clinicalcharacteristics were not determinant on the developing of radiation sensitivity in prostate cancer patients and intronic TP53polymorphisms would be associated with increased acute and chronic radiation toxicities These observations corroborate theimportance of investigating the genetic profile to predict adverse side effects in patients undergoing radiotherapy

1 IntroductionRadiotherapy is the most important nonsurgical modality forthe curative treatment of cancer The success of radiotherapyin eradicating a tumor depends mainly on the total radiationdose given but the tolerance of the normal tissues surround-ing the tumor limits this dose There is a significant variationbetween patients regarding the severity of toxicity followinga given dose of radiotherapy As a consequence the dose issubmaximal in the majority of the radiotherapy patients [1]

Chronic side effects from radiotherapy are often irre-versible and can decrease health-related quality of life aswell as limit treatment intensity in radical radiotherapy regi-mensQuantification of acute and chronic toxicity is thereforecrucial in the assessment of the therapeutic benefit of radio-therapyVarious studiesworldwide have attempted to identifycommon genetic variations associated with the developmentof radiation toxicity as a step in the process of identifying sucha subset of toxicity prone patients [2 3]

702 Disease Markers

Researchers have long recognized that genetic variationcontributes to individual differences in radiotherapy toxicityStudies among prostate cancer patients have identified singlenucleotide polymorphisms (SNP) in candidate genes mostlyinvolved in the DNA damage response including ataxia-telangiectasia mutated (ATM) transforming growth factorbeta 1 gene (TGFB1) X-ray repair cross-complementingprotein 1 gene (XRCC1) X-ray repair cross-complementingprotein 3 gene (XRCC3) and superoxide dismutase 2 gene(SOD2) among others that are associated with the devel-opment of toxicity in response to radiation therapy [4ndash7]One study using a genome-wide approach has also identifiedvariants associated with the development of long term sideeffects of radiation therapy [8] Polymorphisms in genesresponsible for DNA damage signaling and repair mightmodulate DNA repair capacity and therefore affect cell andtissue response to radiation and influence individual radio-sensitivity [9 10]

The tumor suppressor protein p53 encoded by the tumorprotein 53 gene (TP53) a key regulator of cellular responsesto genotoxic stress is stabilized and activated after DNAdamage [11] The rapid activation of p53 by ionizing radia-tion is largely dependent on the ATM kinase The proteinp53 is phosphorylated by ATM shortly after DNA damageresulting in enhanced stability and activity of p53 [12 13]Themurine double minute 2 (MDM2) oncoprotein is a pivotalnegative regulator of p53 In response to ionizing radiationMDM2 undergoes rapid ATM-dependent phosphorylationprior to p53 accumulationThe increase inMDM2basal levelsinduces degradation and blockade of transcriptional activityof p53 protein [14 15]

The functional p53 polymorphism characterized by CgtGchange at the second position of codon 72 (rs1042522) resultsin ArggtPro amino acid substitution It has been shown that72Arg allele may induce apoptosis with faster kinetics thanPro72 allele [16] On the other hand the Pro72 variant seemsto be more competent in inducing cell cycle arrest andDNA repair [17] Therefore Pro72 could be associated witha lower incidence of side effects to radiotherapy by beingmore efficient in cell cycle arrest allowing repair of radi-ation-induced damage The p53 polymorphism at codon 47(rs1800371) is also functionally significant since it has beendemonstrated that the Ser47 variant has a decreased abilityto induce apoptosis compared with the Pro47 p53 allele [18]The TP53 PIN3 insertion (rs17878362) was chosen based ona study by Tan et al 2006 [19] which reported no associationbetween this insertion and the development of radiationtoxicity however we found that it was important to confirmthe lack of association in our study populationThe remainingintronic polymorphisms (rs35117667 and rs17883323) wereselected because they are located in nearby regions to thestudied polymorphisms and also because they have not beenevaluated by any study so far

The MDM2 single-nucleotide polymorphism T309G(rs2279744) is located in the promoter region of MDM2 Asdemonstrated in a different study it increases the promoteraffinity for the stimulatory protein (Sp) 1 intensifyingMDM2expression and subsequent attenuation of the p53 pathway[20] We hypothesized that the increased degradation of

p53 by MDM2 could lead to adverse effects of radiationtherapy

Mutations and polymorphisms of the ATM gene havebeen investigated as possible causes of normal tissue radia-tion sensitivity in breast cancer patients [21] Breast cancerpatients undergoing radiotherapy presented with a higherincidence of adverse chronic effects (fibrosis) when the exon39 polymorphism D1853N (rs1801516) were present [21]ATM protein presents kinase activity induced by ionizingradiation and it is involved in DNA damage detection andin the control of cell cycle progression [22]

The purpose of this study was to examine the hypothesisthat the presence of ATMcodonAsp1853Asn (5557GgtA) pol-ymorphism (rs1801516) TP53 Pro72Arg (rs1042522) TP53Pro47Ser (rs1800371) 3 intronic TP53 polymorphisms (rs1-7878362 rs17883323 and rs35117667) or MDM2 309TgtGpolymorphism (rs2279744) (Table 1) is predictive for the dev-elopment of adverse radiotherapy responses among prostatecancer patients It was hypothesized that polymorphisms inthese geneswould influenceDNArepair capacitymodulatingresponses to radiation and the risk of acute or chronic radi-ation-induced side effects We explored any possible asso-ciation of acute and chronic skin urinary and rectal func-tional outcomes with ATM and TP53 polymorphisms usingthe Radiation Therapy Oncology Group (RTOG) morbiditycriteria [23]

2 Materials and Methods

21 Study Population and Case Definition The present studywas performed with patients from the Associacao de Com-bate ao Cancer em Goias (ACCG) who were recruitedbetween January 2009 and December 2010 The 48 eligiblepatients had histologically confirmed prostate cancer andunderwent conventional external beam radiation therapywith curative intent at the Department of Radiotherapy ofAraujo Jorge Hospital in Goiania Goias Brazil The trea-tment was performed using a daily dose of two Grays andhigh-energy photons (15MV) to obtain deeper penetrationand better dose uniformity

Patients with metastatic carcinoma were excluded fromthis study Patients whose followup was less than 24 monthswere not included on chronic toxicity evaluations The studywas approved by the internal ethical committee and allpatients agreed to participate in the study by signing an info-rmed consent before the biological sample was collected

The clinical information of the patients such as treatmenttype radiation dose pretreatment symptoms age and comor-bidities such as diabetes and vascular disease and radiation-induced injury were obtained retrospectively from the med-ical record of each patient Acute and chronic radiation-related toxicities were graded using the European Organiza-tion for Research and Treatment of CancerRadiation Ther-apy Oncology Group (EORTCRTOG) morbidity criteria[23] Patients who developed RTOG grading level 2 or morewere classified as having a high grade (HG) adverse responseand those with RTOG grading level 1 and level 0 responseswere classified as low grade (LG) responses

Disease Markers 703

Table1Descriptio

nof

thea

nalysedpo

lymorph

ismsinAT

MM

DM2andTP

53genes

Genen

ame

Genom

iclocatio

nEx

onin

tron

Genom

icdescrip

tion

Cod

ingdescrip

tion

Proteindescrip

tion

refSNP

Effect

TP53

17p131

Intro

n3

g16225delGinsA

CCTG

GAG

GGCT

GGGG

c97-54delGinsA

CCTG

GAG

GGCT

GGGG

prs17878362

Intro

nic

Intro

n3

g16250CgtA

c97-29Cgt

Ap

rs17883323

Intro

nic

Intro

n3

g16273CgtT

c97-6CgtT

prs35117

667

Intro

nic

Exon

4g16321CgtT

c139Cgt

TpP4

7Srs1800371

Miss

ense

Exon

4g16397CgtG

c215Cgt

GpP7

2Rrs1042522

Miss

ense

ATM

11q22-q23

Exon

39g86904GgtA

c5557GgtA

pD1853N

rs1801516

Miss

ense

MDM2

12q143-q15

Intro

n1

g5610Tgt

G309TgtG

prs2279744

Intro

nic

Abbreviatio

nsrefSN

PreferenceS

NPclu

sterreport

704 Disease Markers

Table 2 Primer sequences and optimal annealing

Gene name Primer orientation Primer sequences (51015840-31015840) Annealing temperature (∘C)

TP53 Forward ATGGAGGAGCCGCAGTCAGA 613Reverse TCTGGGAAGGGACAGAAGA

ATM Forward AGCAGTATGTTGAGTTTATGGC 543Reverse TGAATCCAAGTTTGCAGG

MDM2 Forward CGGGAGTTCAGGGTAAAGGT 603Reverse AGCAAGTCGGTGCTTACCTG

This study used a candidate gene approach to search forassociations between genetic variants such as single nucle-otide polymorphisms and radiation treatment toxicity

22 Genotyping For genetic analysis each patientwas appro-ached at the time of follow-up evaluation A consent formwaspresented to the patient prior to the collection of their clinicaldata and blood sample and all patients included agreed tosign the consent form for this research study This study wasapproved by the Ethicrsquos Committee from ACCG

DNA was isolated from peripheral blood through theiPrep Purification system developed by Invitrogen Corpo-ration CA according to the manufacturerrsquos protocol Poly-merase chain reaction (PCR) was used to amplify eachfragment using specific primers (Table 2) designed from thegenomic sequence obtained from the National Center forBiotechnology Information (httpwwwncbinlmnihgov)The optimal annealing temperature for each primer wasascertained experimentally on a gradient PCR machine

In brief PCR was performed at a 50120583L reaction mixturecontaining 100 ng of DNA 10 120583M of each primer 02120583L ofAccuprime Taq High Fidelity and a reaction buffer contain-ing 600mM Tris-SO

4 180mM (NH

4)2SO4 20mMMgSO

4

and 2mM of each deoxynucleoside triphosphate (InvitrogenCA) The PCR profile consisted of an initial melting step of10 minutes at 95∘C followed by 40 cycles of 30 seconds at95∘C 30 seconds at 543∘C for ATM at 613∘C for TP53 or at603∘C for MDM2 amplification 1 minute at 72∘C and a finalelongation step of 7 minutes at 72∘C

The PCR reactions were first subjected to purificationusing the PureLink Quick Gel Extraction and PCR Purifi-cation Combo Kit (Invitrogen CA) treated with Exo SAP-IT (USB Corporation Cleveland Ohio USA) to removedNTPs and primers and then underwent nucleotide primerextension reaction The sequencing reactions (BigDye Ter-minator v 31 Ready Reaction Cycle Sequencing Kit AppliedBiosystems Foster City CA) were subjected to purificationusing BigDye Xterminator (Applied Biosystems Foster CityCA) for a complete removal of ddNTPs and underwentcapillary electrophoresis on an Applied Biosystems 3130xlDNA Analyzer The data were analysed by the SeqscapeSoftware v26 (Applied Biosystems Foster City CA)

23 Statistical Analysis Each polymorphism was tested fordeviation from Hardy-Weinberg equilibrium by compar-ing the observed and expected genotype frequencies usingthe chi-square test with one degree of freedom Analyseswere performed using the SigmaStat version 150 statistical

software package (SPSS Inc Chicago IL) The associationbetween ATMTP53 andMDM2 polymorphisms and adverseresponse to radiotherapy was estimated by chi-square test orFisherrsquos exact test with one degree of freedom All analyseswere performed with 120572 = 5 and confidence interval of 95

3 Results

31 Patientrsquos Characteristics Patientrsquos characteristics are des-cribed in Table 3Themean age was 673 years Of 48 patients292 had family history of cancer 479 had hypertension125 were diabetic and 542 had a history of smokingduring some time in their lives

A total of 45 patients (937) had tumor Gleason scorehigher than five 25 patients (521) had mean prostatespecific antigen (PSA) higher than 10 ngmL before treatmentand in 771 of the patients PSAdecreased after radiotherapyTwelve patients (25) underwent concomitant hormonaltherapy and seven (146) had undergone prior prostatec-tomy The average radiation dose in the 50 patients was 701Grays (Gy) and the treatment lasted for an average of 615days The followup ranged from 2 to 92 months

Thepatientswere classified as having an adverse responseRTOG high grade or not having an adverse response (RTOGlow grade) Tables 4 and 5 show the number of patients havingacute or chronic adverse effects of radiotherapy respectively

There were no significant differences between patientswho developed severe acute or chronic radiation toxicitiesand those who did not in relation to age smoking status Gle-ason score family history of cancer hypertension or diabetesand radiotherapy dose and patients who underwent con-comitant hormonal therapy or had prior prostatectomy (119875 gt005)

32 Polymorphisms and Risk for Development of RadiationToxicity From the initial group of 48 patients eligible forthe present study we analysed polymorphisms in the genesATM TP53 and MDM2 For the ATM and MDM2 SNPsall patients were successfully genotyped For TP53 gene weanalysed four polymorphisms in the PCRamplified fragmentComplete data was obtained for the TP53 polymorphism atcodon 72 It was possible to verify the presence or absenceof this polymorphism in all 48 patients while data regardingTP53 polymorphisms at codon 47 and intronic regions 16250and 16225 were analysed in 47 patients and region 16273of intron three was analysed in 46 patients The frequenciesof each polymorphism are shown in Table 6 For Chronicanalysis three patients were excluded because their followup

Disease Markers 705

Table 3 Patients clinical characteristics

Characteristic No of patients ()Age (y) median (range) 673 (52ndash82)Family history of cancer 14 (292)Hypertension 19 (396)Diabetes mellitus 2 (42)Hypertension + Diabetes mellitus 4 (83)Other Comorbidities 6 (125)Any history of smoking 26 (542)Gleason scorele5 3 (63)6 25 (521)7 16 (333)8-9 4 (84)

PSA (ngmL) before treatment no of patients (PSA range)le4 9 (063ndash380)5ndash10 14 (413ndash1000)10ndash20 16 (1060ndash1950)gt20 9 (2138ndash151)

PSA outcome after radiotherapyPSA decreased 37 (771)PSA did not decrease 11 (229)

Concomitant hormonal therapy 12 (25)Prior surgery (prostatectomy) 7 (146)Radiotherapy interruption 5 (104)Total radiation dose (Gy) median (range) 701 (64ndash74)Treatment duration (months) median (range) 615 (49ndash86)Follow up (months) median (range) 48 (2ndash92)Abbreviations PSA prostate specific antigen Gy Grays

Table 4 RTOG acute radiation morbidity (toxicity grading)

Skin RTOG Lower GI tract RTOG Urinary RTOGn () n () n ()

High grade 2 (42) 7 (146) 23 (479)Low grade 46 (958) 41 (854) 25 (521)Abbreviations RTOG radiation therapy oncology group GI tract gastrointestinal tractHigh grade RTOG ge2 e Low grade RTOG lt2

Table 5 RTOG chronic radiation morbidity (toxicity grading)lowast

Skin RTOG Lower GI tract RTOG Urinary tract RTOGn () n () n ()

High grade 1 (22) 2 (44) 10 (222)Low grade 44 (978) 43 (956) 35 (778)Abbreviations RTOG radiation therapy oncology group GI tract gastrointestinal tractHigh grade RTOG ge2 e Low grade RTOG lt2lowastFor chronic analyses it was considered patients with followup higher than 24 months

was less than 24 months The frequencies of polymorphismsfor chronic associations are shown in Table 7

In univariate cross-table analysis the ATM Asp1853Asnpolymorphism was not associated with occurrence of eitheracute or chronic toxicity after radiotherapy (119875 gt 005)The intronic polymorphism located on region 16273 with a

homozygous change of CgtT was significantly associated tothe development of acute skin toxicity RTOG grade two orhigher (OR = 00072 95 CI 00002ndash0227 119875 = 0003) asshown in Table 6The heterozygous change of AgtC on region16250 was strongly associated to the developing of chronicurinary toxicity RTOG grade two or higher (OR = 0071

706 Disease Markers

Table6As

sociationof

thep

olym

orph

ismsa

nalysedwith

acutes

kin

gastrointestinaland

urinaryradiationtoxicity

Acutes

kinRT

OG

AcuteG

astro

intestinalRT

OG

AcuteU

rinaryTractR

TOG

Gene

(refSN

P)Genotype

n(

)HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

ATM

GG

36(75)

135

531

1719

(rs1801516)

CAlowast

12(25)

111

0314

0018ndash5452

044

12

100806

0135ndash4821

16

60895

0242ndash3307

1TP

53GG

25(52)

223

421

1213

(rs104

2522)

GC

17(35)

017

3723

0168ndash82615

0506

215

1429

0231ndash8840

19

80820

0239ndash

2819

1CC

6(13)

06

1383

0059ndash

32563

11

50952

0086ndash

10489

12

418

460284ndash

11983

066

4TP

53CC

45(96)

243

639

2223

(rs1800371)

CTdagger

2(4)

02

0287

0011ndash7744

11

16500

0357ndash11837

0278

02

4787

0217ndash10539

0491

TP53

CC43

(91)

241

538

1924

(rs17883323)

CAlowast

4(9)

04

0542

0022ndash1315

71

22

0132

0015ndash1153

0100

31

0264

0025ndash2745

0328

TP53

CC41

(89)

041

635

1823

(rs35117667)

CT2(4)

02

mdashmdash

Dagger0

20915

0039ndash

21368

11

10783

004

6ndash1339

91

TT3(7)

21

0007

000

02ndash0

227

000

31

22917

0227ndash37425

0413

21

2556

0214ndash

30469

0583

TP53

A1

40(87)

238

634

2020

(rs17878362)

A2

6(13)

06

0844

0036ndash

19676

11

51133

0112ndash11484

12

40500

0082ndash304

60667

MDM2

TT18

(38)

117

117

99

(rs2279744

)GT

27(56)

126

1529

0089ndash

2613

61

621

0206

0023ndash18

800215

1215

1250

0378ndash

4133

0767

GG

3(6)

03

060

0002ndash179

961

03

060

00020ndash

17996

12

10500

0038ndash6547

1Ab

breviatio

nsrefSN

PreferenceS

NPclu

sterreportRT

OGradiatio

ntherapyo

ncolog

ygroup

HGhighgradeR

TOGLGlow

gradeR

TOGO

Rod

dsratio

CIconfi

denceintervalT

omakec

alculations

ofFishers

exacttestp

ossib

lewhenon

evalue

iszero05was

addedto

thev

alue

lowastNocarriersof

anAAho

mozygou

sgenotypew

erefou

nd

daggerNocarriersof

aTTho

mozygou

sgenotypew

erefou

nd

DaggerNopatient

high

gradefor

theg

enotypes

analysed

Disease Markers 707

Table7As

sociationof

thep

olym

orph

ismsa

nalysedwith

chronics

kin

gastrointestinaland

urinaryradiotoxicitylowast

Chronics

kinRT

OG

Chronicg

astro

intestinalRT

OG

Chronicu

rinarytractR

TOG

Gene

(refSN

P)Genotype

n(

)HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

ATM

GG

35(78)

035

233

827

(rs1801516)

GAlowastlowast

10(22)

19

0089

0003ndash2372

0222

010

1567

0069ndash

3532

31

28

1185

0208ndash6740

1TP

53GG

23(51)

023

221

518

(rs104

2522)

GC

16(36)

115

0220

0008ndash5756

0410

016

3837

0172ndash8553

30503

214

1944

0327ndash11563

0678

CC6(13)

06

mdashmdash

Dagger0

615

12006

4ndash35678

13

30278

004

2ndash18

250305

TP53

CC42 (955)

141

240

933

(rs1800371)

CTdagger

2(45)

02

0181

0058ndash5673

10

20307

0011ndash8325

11

13667

0208ndash64

54

040

7TP

53CC

40(91)

040

238

733

(rs17883323)

CAlowastlowast

4(9)

13

0029

0001ndash0848

0091

04

0584

0024ndash

1419

91

31

0071

000

6ndash0784

0032

TP53

CC38

(88)

137

137

830

(rs35117667)

CT2(5)

02

0200

000

6ndash6288

11

10027

0001ndash0819

0099

02

1393

0061ndash31901

1TT

3(7)

03

0280

000

9ndash8254

10

30280

000

9ndash8254

11

218

750150ndash

2339

60535

TP53

A1

37(86)

136

235

829

(rs17878362)

A2

6(14

)0

60534

0019ndash

14607

10

60915

0039ndash

21368

12

418

130280ndash

11750

0611

MDM2

TT17

(38)

017

116

413

(rs2279744

)GT

25(55)

124

046

70018ndash1215

21

124

1500

0087ndash25753

16

190974

0229ndash

4148

1GG

3(7)

03

mdashmdash

Dagger0

30636

0212ndash1912

41

03

2333

0100ndash

54460

1Ab

breviatio

nsrefSN

PreferenceS

NPclu

sterreportRT

OGradiatio

ntherapyo

ncolog

ygroup

HGhighgradeR

TOGLGlow

gradeR

TOGO

Rod

dsratio

CIconfi

denceintervalT

omakec

alculations

ofFishers

exacttestp

ossib

lewhenon

evalue

iszero05was

addedto

thev

alue

lowastFo

rchron

icanalysesitw

asconsidered

patientsw

ithfollo

wup

high

erthan

24mon

ths

lowastlowastNocarriersof

anAAho

mozygou

sgenotypew

erefou

nd

daggerNocarriersof

aTTho

mozygou

sgenotypew

erefou

nd

DaggerNopatient

high

gradefor

theg

enotypes

analysed

708 Disease Markers

95CI 0006ndash0784119875 = 0032) None of the remaining TP53polymorphisms including the one located on codon 72 weresignificantly associated with the presence of acute or chronicskin gastrointestinal and urinary toxicities (Tables 6 and 7)

4 Discussion

41 Clinical Data Associated with Radiation Toxicity Ouranalysis showed as expected that clinical features were notdeterminants of acute or chronic toxicities of the prostateadjacent tissues when submitted to radiotherapy (119875 gt 005)These results confirm the importance of investigating thegenetic profile of patients undergoing radiotherapy in orderto find if genetic differences are involved or not involvedin the development of radiation toxicities during and aftertreatment

42 Polymorphisms and Radiation Toxicity There is a beliefthat the genomic revolution triggered by full sequencingof the human genome and technological development oflarge-scale methodologies announces the future of person-alized medicine In oncology this progress should increasethe possibility to predict individual patient responses toradiotherapy and chemotherapy Andreassen et al (2002)[24] hypothesized that the radiation sensitivity of normaltissues should be associated with a so-called ldquotrait-dependentcomplexrdquo which adds the effect ofmany genetic determinantsand single nucleotide polymorphisms that could correspondin part to the frequency and severity of these components

Some suggestive evidence was obtained from the cor-relation between the ATM codon Asp1853Asn (5557GgtA)SNP and the high risk of the development of radiotherapy-induced acute skin complications in breast cancer patients[21 25 26] For prostate cancer data are controversial ATMsequence alterations including codon 1853 polymorphismwere associated with the development of radiation-inducedproctitis [5] although no relation was shown between thismarker and bladder or rectal toxicities arising from theradiation therapy in Canadian prostate cancer patients [27]Our findings showed that Asp1853Asn polymorphism wasnot associated with the development of any acute or chronicadverse effects in patients with prostate cancer treated withradiotherapy (119875 gt 005) ATM is a large gene with many vari-ants documented Further investigations will be necessary toverify the association of other ATM SNPs in more repre-sentative sample groups in order to establish whether singlenucleotide polymorphisms or gene haplotypes may actuallycontribute to the toxicity of normal tissue

The single base polymorphism in the promoter ofMDM2gene (SNP309 rs2279744) increases gene expression and it isshown to associate with accelerated tumor formation in bothhereditary and sporadic cancers [20] This SNP creates animproved site of action that regulates basal levels of MDM2The increase of MDM2 basal levels will also increase thedegradation of p53 tumor suppressor protein and may stopits transcriptional activity leading to increased apoptosis [1415 28 29] The hypothesis that increased apoptosis couldcause necrosis of normal tissue leading to radiotherapy sideeffects was not confirmed by our results Among the prostate

cancer patients of this study no association of the investigatedMDM2 polymorphism (rs2279744) and the incidence ofradiotherapy-induced acute and chronic effects were foundthus indicating no major contribution in this cohort ofprostate cancer Similar lack of association was reported byPopanda et al 2009 [30] Another study also showed that theMDM2 SNP309 polymorphism was not found to be involvedwith clinical pathologic variables recurrence risk and overallsurvival outcome in prostate cancer [31]

Many studies have showed the association between theTP53 Pro72Arg polymorphism and cancer susceptibility [32ndash34] An association between the risk of acute skin toxicityand TP53 Pro72 carriers in those with the CDKN1A 31Sergenotype in a subset of normal weight patients treatedwith radiotherapy for breast cancer has been shown [19]Another study found evidence that genetic polymorphismsin the ATM-P53 pathway can influence susceptibility todeveloping radiation-induced pneumonitis in lung cancerpatients after radiotherapy [35] Despite the strong hypothesisfor involvement of this gene the association between thePro72Arg polymorphism and the development of normaltissue reaction to radiotherapy in prostate cancer patientshas not been fully investigated previously This was the firststudy to evaluate the acute and chronic radiation toxicitiesin patients with prostate cancer and TP53 Pro72Arg poly-morphism No association was found in the studied groupeven though 27 (54) patients presented the combinationArgArg which has been described as a more predisposed toapoptosis genotype [16 36]

Another polymorphism in TP53 gene at codon 47(rs1800371) is also functionally significant Codon 47encodes proline in wild type p53 but in a small subset ofindividuals it can encode a serine It was showed that theserine 47 variant has up to 5-fold decreased ability to induceapoptosis compared with wild type p53 [18] However onour study the percentage of patients with this polymorphismwas not statistically sufficient to find a relationship with theadverse effects of radiotherapy even though it is a variantthat induces apoptosis There is no other literature data ofassociation between this polymorphism and toxicity inducedby radiation therapy

Our findings showed that a polymorphism located in theregion 16273 of intron 3 of TP53 (rs35117667) was associatedwith high risk of developing acute skin adverse effects (OR00072 95CI 00002ndash0227119875 = 0003)This polymorphismhas not been investigated by any study so far The presentstudy is the first to observe the frequencies of this polymor-phism in a specific population and also the first to associatethis polymorphism with radiotherapy There is also evidenceof association between the intronic polymorphism at position16250 (rs17883323) and chronic high grade toxicity of urinarytract (OR 0071 95 CI 0006ndash0784 119875 = 0032) Thispolymorphism has not been previously studied in a clinicalpopulation according to searches on scientific websites Bothintronic polymorphisms (rs35117667 and rs17883323) werevalidated by genotype data showing minor alleles in at leasttwo chromosomes [37] It is now necessary to evaluate themolecular basis of the association between these intronicpolymorphisms and the risk for radiotherapy side effects

Disease Markers 709

An intronic polymorphism is not implicated directly in analtered protein however it may lead to alternative splicing orit can be related to MicroRNAs [38ndash40] A protein modifiedby these processesmay be associated for example an increaseof apoptosis

Gemignani and colleagues [41] in 2004 reported that aninsertionduplication of 16 base pairs in intron 3 of TP53 genethe so called TP53 PIN3 (rs17878362) described by Lazar et al[42] (1993) is associated with an increased risk of developingcolorectal cancer (OR 155 95 CI 1 10 to 2 18 119875 = 0012)Tan et al [19] (2006) reported no association between theTP53 PIN3 (rs17878362) insertion and the development ofadverse effects of radiotherapy (119875 gt 005) Our data corro-borate the results of Tan et al [19] (2006) which also foundno association between this polymorphism and the risk oftoxicity after radiotherapy Our study showed that 42 patients(84) had this polymorphism The frequency of the SNPsuggest the importance of a case-control study to assess theassociation between this polymorphism and the developmentof prostate cancer and thus at this stage the functionalsignificance of TP53 PIN3 polymorphism remains poorlyexplored

5 Conclusions

Until now most of studies in this field have been carriedout using candidate genes as was done in the present studyThese studies can provide valuable information howeverthey are limited and have not yet succeeded in providinga predictive test to identify radiosensitivity in patients Inresponse to lack of success in candidate gene studies and thesubsequent recognition that this approach is too limited inscope radiogenomic investigators have now begun a muchbroader search to identify genes and SNPs associated withradiation response Several large-scale genome wide asso-ciation (GWA) studies have been initiated in which radio-therapy patients are being genotyped for large numbers ofcommon SNPs [8 43]

The results of this study hint to a few candidate geneshowever our study was limited by the small sample size andtherefore low statistical power to detect associations Thisstudy shows the importance of genetic investigations as auseful tool for individualization of radiotherapy strategies Itis anticipated that over the next few years SNPs correlatedwith susceptibility for the development of adverse effectsresulting from radiotherapy will be identified from GWAstudies The data provided by studies in radiogenomics willopen new perspectives for interpretation of the results ofcandidate gene studies such as ours and can be the basis forthe development of a predictive assay that may be useful topersonalize and optimize cancer treatment

Acknowledgments

This work was supported by grants from Financiadora deEstudos e Projetos FINEP MCT Brazil and Fundacao deAmparo a Pesquisa do Estado de Goias FAPEG BrazilHellen Cintra had a fellowship from CAPES MEC Brazil

The author would like to thank Dr Alan George Smulian forthe paper revising

References

[1] G C Barnett C M L West A M Dunning et al ldquoNormaltissue reactions to radiotherapy towards tailoring treatmentdose by genotyperdquoNature Reviews Cancer vol 9 no 2 pp 134ndash142 2009

[2] N G Burnet R M Elliott A Dunning and C M L WestldquoRadiosensitivity Radiogenomics and RAPPERrdquo Clinical Onc-ology vol 18 no 7 pp 525ndash528 2006

[3] C M L West A M Dunning and B S Rosenstein ldquoGenome-wide association studies and prediction of normal tissue toxi-cityrdquo Seminars in Radiation Oncology vol 22 no 2 pp 91ndash992012

[4] J A Cesaretti R G Stock S Lehrer et al ldquoATM sequencevariants are predictive of adverse radiotherapy response amongpatients treated for prostate cancerrdquo International Journal ofRadiation Oncology Biology Physics vol 61 no 1 pp 196ndash2022005

[5] J A Cesaretti R G Stock D P Atencio et al ldquoA geneticallydetermined dose-volume histogram predicts for rectal bleedingamong patients treated with prostate brachytherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 68 no5 pp 1410ndash1416 2007

[6] C A Peters R G Stock J A Cesaretti et al ldquoTGFB1 singlenucleotide polymorphisms are associated with adverse qualityof life in prostate cancer patients treated with radiotherapyrdquoInternational Journal of Radiation Oncology Biology Physics vol70 no 3 pp 752ndash759 2008

[7] R J Burri R G Stock J A Cesaretti et al ldquoAssociation of singlenucleotide polymorphisms in SOD2 XRCC1 and XRCC3 withsusceptibility for the development of adverse effects resultingfrom radiotherapy for prostate cancerrdquo Radiation Research vol170 no 1 pp 49ndash59 2008

[8] S L Kerns H Ostrer R Stock et al ldquoGenome-wide associationstudy to identify single nucleotide polymorphisms (SNPs)associated with the development of erectile dysfunction inAfrican-American men after radiotherapy for prostate cancerrdquoInternational Journal of Radiation Oncology Biology Physics vol78 no 5 pp 1292ndash1300 2010

[9] G Iliakis ldquoThe role of DNA double strand breaks in ionizingradiation-induced killing of eukaryotic cellsrdquo BioEssays vol 13no 12 pp 641ndash648 1991

[10] F Altieri C Grillo M Maceroni and S Chichiarelli ldquoDNAdamage and repair from molecular mechanisms to healthimplicationsrdquo Antioxidants and Redox Signaling vol 10 no 5pp 891ndash937 2008

[11] A J Levine and M Oren ldquoThe first 30 years of p53 growingever more complexrdquo Nature Reviews Cancer vol 9 no 10 pp749ndash758 2009

[12] M B Kastan andD-S Lim ldquoThemany substrates and functionsof ATMrdquoNature ReviewsMolecular Cell Biology vol 1 no 3 pp179ndash186 2000

[13] A Tichy J Vavrova J Pejchal and M Rezacova ldquoAtaxia-telangiectasia mutated kinase (ATM) as a central regulator ofradiation-inducedDNAdamage responserdquoActaMedica vol 53no 1 pp 13ndash17 2010

[14] T Soussi and K G Wiman ldquoShaping genetic alterations inhuman cancer the p53 mutation paradigmrdquo Cancer Cell vol12 no 4 pp 303ndash312 2007

710 Disease Markers

[15] Y Levav-Cohen S Haupt and Y Haupt ldquoMdm2 in growthsignaling and cancerrdquoGrowth Factors vol 23 no 3 pp 183ndash1922005

[16] P Dumont J I-J Leu A C Della Pietra III D L George andM Murphy ldquoThe codon 72 polymorphic variants of p53 havemarkedly different apoptotic potentialrdquoNature Genetics vol 33no 3 pp 357ndash365 2003

[17] D D Oslashrsted S E Bojesen A Tybjaeligrg-Hansen and B G Nor-destgaard ldquoTumor suppressor p53 Arg72Pro polymorphismand longevity cancer survival and risk of cancer in the generalpopulationrdquo Journal of Experimental Medicine vol 204 no 6pp 1295ndash1301 2007

[18] X Li P Dumont A Della Pietra G Shetler andM E MurphyldquoThe codon 47 polymorphism in p53 is functionally significantrdquoJournal of Biological Chemistry vol 280 no 25 pp 24245ndash24251 2005

[19] X-L Tan O Popanda C B Ambrosone et al ldquoAssociationbetween TP53 and p21 genetic polymorphisms and acute sideeffects of radiotherapy in breast cancer patientsrdquo Breast CancerResearch and Treatment vol 97 no 3 pp 255ndash262 2006

[20] G L Bond W Hu E E Bond et al ldquoA single nucleotide poly-morphism in the MDM2 promoter attenuates the p53 tumorsuppressor pathway and accelerates tumor formation inhumansrdquo Cell vol 119 no 5 pp 591ndash602 2004

[21] A Y Ho G Fan D P Atencio et al ldquoPossession of ATMsequence variants as predictor for late normal tissue responsesin breast cancer patients treated with radiotherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 69 no3 pp 677ndash684 2007

[22] C E Canman D-S Lim K A Cimprich et al ldquoActivation ofthe ATM kinase by ionizing radiation and phosphorylation ofp53rdquo Science vol 281 no 5383 pp 1677ndash1679 1998

[23] J D Cox J Stetz and T F Pajak ldquoToxicity criteria of the Radi-ation Therapy Oncology Group (RTOG) and the EuropeanOrganization for Research and Treatment of Cancer (EORTC)rdquoInternational Journal of Radiation Oncology Biology Physics vol31 no 5 pp 1341ndash1346 1995

[24] C N Andreassen J Alsner and J Overgaard ldquoDoes variabilityin normal tissue reactions after radiotherapy have a geneticbasismdashwhere and how to look for itrdquo Radiotherapy and Onc-ology vol 64 no 2 pp 131ndash140 2002

[25] S Angele P Romestaing N Moullan et al ldquoATM haplotypesand cellular response to DNA damage association with breastcancer risk and clinical radiosensitivityrdquo Cancer Research vol63 no 24 pp 8717ndash8725 2003

[26] C N Andreassen J Overgaard J Alsner et al ldquoATM sequencevariants and risk of radiation-induced subcutaneous fibrosisafter postmastectomy radiotherapyrdquo International Journal ofRadiation Oncology Biology Physics vol 64 no 3 pp 776ndash7832006

[27] S Damaraju D Murray J Dufour et al ldquoAssociation of DNArepair and steroid metabolism gene polymorphisms with clini-cal late toxicity in patients treated with conformal radiotherapyfor prostate cancerrdquo Clinical Cancer Research vol 12 no 8 pp2545ndash2554 2006

[28] G L Bond W Hu and A Levine ldquoA single nucleotide poly-morphism in the MDM2 gene from a molecular and cellularexplanation to clinical effectrdquo Cancer Research vol 65 no 13pp 5481ndash5484 2005

[29] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[30] O Popanda J UMarquardt J Chang-Claude and P SchmezerldquoGenetic variation in normal tissue toxicity induced by ionizingradiationrdquoMutation Research vol 667 no 1-2 pp 58ndash69 2009

[31] J J Jaboin M Hwang C A Perez et al ldquoNo evidence forassociation of the MDM2-309 TG promoter polymorphismwith prostate cancer outcomesrdquo Urologic Oncology vol 29 no3 pp 319ndash323 2011

[32] L Sreeja V Syamala P B Raveendran S Santhi J Madha-van and R Ankathil ldquop53 Arg72Pro polymorphism predictssurvival outcome in lung cancer patients in Indian populationrdquoCancer Investigation vol 26 no 1 pp 41ndash46 2008

[33] M B Parliament and D Murray ldquoSingle nucleotide polymor-phisms of DNA repair genes as predictors of radioresponserdquoSeminars in Radiation Oncology vol 20 no 4 pp 232ndash2402010

[34] T Pugh M Keyes L Barclay et al ldquoSequence variant discoveryin DNA repair genes from radiosensitive and radiotolerantprostate brachytherapy patientsrdquo Clinical Cancer Research vol15 no 15 pp 5008ndash5016 2009

[35] M Yang L Zhang N Bi et al ldquoAssociation of P53 and ATMpolymorphisms with risk of radiation-induced pneumonitis inlung cancer patients treated with radiotherapyrdquo InternationalJournal of Radiation Oncology Biology Physics vol 79 no 5 pp1402ndash1407 2011

[36] G Alsbeih N Al-Harbi M Al-Buhairi K Al-Hadyan and MAl-Hamed ldquoAssociation between TP53 codon 72 single-nuc-leotide polymorphism and radiation sensitivity of human fibro-blastsrdquo Radiation Research vol 167 no 5 pp 535ndash540 2007

[37] M C Zody M Garber D J Adams et al ldquoDNA sequence ofhuman chromosome 17 and analysis of rearrangement in thehuman lineagerdquoNature vol 440 no 7087 pp 1045ndash1049 2006

[38] H I Suzuki K Yamagata K Sugimoto T Iwamoto S Kato andK Miyazono ldquoModulation of microRNA processing by p53rdquoNature vol 460 no 7254 pp 529ndash533 2009

[39] A K L Leung and P A Sharp ldquomicroRNAs a safeguard againstturmoilrdquo Cell vol 130 no 4 pp 581ndash585 2007

[40] Z Peng Y Cheng B C-M Tan et al ldquoComprehensive analysisof RNA-Seq data reveals extensive RNA editing in a humantranscriptomerdquo Nature Biotechnology vol 30 no 3 pp 253ndash260 2012

[41] F Gemignani VMoreno S Landi et al ldquoATP53 polymorphismis associated with increased risk of colorectal cancer and withreduced levels of TP53 mRNArdquo Oncogene vol 23 no 10 pp1954ndash1956 2004

[42] V Lazar F Hazard F Bertin N Janin D Bellet and B BressacldquoSimple sequence repeat polymorphism within the p53 generdquoOncogene vol 8 no 6 pp 1703ndash1705 1993

[43] CWest B S Rosenstein and J Alsner ldquoEstablishment of a radi-ogenomics consortiumrdquo International Journal of RadiationOnc-ology Biology Physics vol 76 no 5 pp 1295ndash1296 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

702 Disease Markers

Researchers have long recognized that genetic variationcontributes to individual differences in radiotherapy toxicityStudies among prostate cancer patients have identified singlenucleotide polymorphisms (SNP) in candidate genes mostlyinvolved in the DNA damage response including ataxia-telangiectasia mutated (ATM) transforming growth factorbeta 1 gene (TGFB1) X-ray repair cross-complementingprotein 1 gene (XRCC1) X-ray repair cross-complementingprotein 3 gene (XRCC3) and superoxide dismutase 2 gene(SOD2) among others that are associated with the devel-opment of toxicity in response to radiation therapy [4ndash7]One study using a genome-wide approach has also identifiedvariants associated with the development of long term sideeffects of radiation therapy [8] Polymorphisms in genesresponsible for DNA damage signaling and repair mightmodulate DNA repair capacity and therefore affect cell andtissue response to radiation and influence individual radio-sensitivity [9 10]

The tumor suppressor protein p53 encoded by the tumorprotein 53 gene (TP53) a key regulator of cellular responsesto genotoxic stress is stabilized and activated after DNAdamage [11] The rapid activation of p53 by ionizing radia-tion is largely dependent on the ATM kinase The proteinp53 is phosphorylated by ATM shortly after DNA damageresulting in enhanced stability and activity of p53 [12 13]Themurine double minute 2 (MDM2) oncoprotein is a pivotalnegative regulator of p53 In response to ionizing radiationMDM2 undergoes rapid ATM-dependent phosphorylationprior to p53 accumulationThe increase inMDM2basal levelsinduces degradation and blockade of transcriptional activityof p53 protein [14 15]

The functional p53 polymorphism characterized by CgtGchange at the second position of codon 72 (rs1042522) resultsin ArggtPro amino acid substitution It has been shown that72Arg allele may induce apoptosis with faster kinetics thanPro72 allele [16] On the other hand the Pro72 variant seemsto be more competent in inducing cell cycle arrest andDNA repair [17] Therefore Pro72 could be associated witha lower incidence of side effects to radiotherapy by beingmore efficient in cell cycle arrest allowing repair of radi-ation-induced damage The p53 polymorphism at codon 47(rs1800371) is also functionally significant since it has beendemonstrated that the Ser47 variant has a decreased abilityto induce apoptosis compared with the Pro47 p53 allele [18]The TP53 PIN3 insertion (rs17878362) was chosen based ona study by Tan et al 2006 [19] which reported no associationbetween this insertion and the development of radiationtoxicity however we found that it was important to confirmthe lack of association in our study populationThe remainingintronic polymorphisms (rs35117667 and rs17883323) wereselected because they are located in nearby regions to thestudied polymorphisms and also because they have not beenevaluated by any study so far

The MDM2 single-nucleotide polymorphism T309G(rs2279744) is located in the promoter region of MDM2 Asdemonstrated in a different study it increases the promoteraffinity for the stimulatory protein (Sp) 1 intensifyingMDM2expression and subsequent attenuation of the p53 pathway[20] We hypothesized that the increased degradation of

p53 by MDM2 could lead to adverse effects of radiationtherapy

Mutations and polymorphisms of the ATM gene havebeen investigated as possible causes of normal tissue radia-tion sensitivity in breast cancer patients [21] Breast cancerpatients undergoing radiotherapy presented with a higherincidence of adverse chronic effects (fibrosis) when the exon39 polymorphism D1853N (rs1801516) were present [21]ATM protein presents kinase activity induced by ionizingradiation and it is involved in DNA damage detection andin the control of cell cycle progression [22]

The purpose of this study was to examine the hypothesisthat the presence of ATMcodonAsp1853Asn (5557GgtA) pol-ymorphism (rs1801516) TP53 Pro72Arg (rs1042522) TP53Pro47Ser (rs1800371) 3 intronic TP53 polymorphisms (rs1-7878362 rs17883323 and rs35117667) or MDM2 309TgtGpolymorphism (rs2279744) (Table 1) is predictive for the dev-elopment of adverse radiotherapy responses among prostatecancer patients It was hypothesized that polymorphisms inthese geneswould influenceDNArepair capacitymodulatingresponses to radiation and the risk of acute or chronic radi-ation-induced side effects We explored any possible asso-ciation of acute and chronic skin urinary and rectal func-tional outcomes with ATM and TP53 polymorphisms usingthe Radiation Therapy Oncology Group (RTOG) morbiditycriteria [23]

2 Materials and Methods

21 Study Population and Case Definition The present studywas performed with patients from the Associacao de Com-bate ao Cancer em Goias (ACCG) who were recruitedbetween January 2009 and December 2010 The 48 eligiblepatients had histologically confirmed prostate cancer andunderwent conventional external beam radiation therapywith curative intent at the Department of Radiotherapy ofAraujo Jorge Hospital in Goiania Goias Brazil The trea-tment was performed using a daily dose of two Grays andhigh-energy photons (15MV) to obtain deeper penetrationand better dose uniformity

Patients with metastatic carcinoma were excluded fromthis study Patients whose followup was less than 24 monthswere not included on chronic toxicity evaluations The studywas approved by the internal ethical committee and allpatients agreed to participate in the study by signing an info-rmed consent before the biological sample was collected

The clinical information of the patients such as treatmenttype radiation dose pretreatment symptoms age and comor-bidities such as diabetes and vascular disease and radiation-induced injury were obtained retrospectively from the med-ical record of each patient Acute and chronic radiation-related toxicities were graded using the European Organiza-tion for Research and Treatment of CancerRadiation Ther-apy Oncology Group (EORTCRTOG) morbidity criteria[23] Patients who developed RTOG grading level 2 or morewere classified as having a high grade (HG) adverse responseand those with RTOG grading level 1 and level 0 responseswere classified as low grade (LG) responses

Disease Markers 703

Table1Descriptio

nof

thea

nalysedpo

lymorph

ismsinAT

MM

DM2andTP

53genes

Genen

ame

Genom

iclocatio

nEx

onin

tron

Genom

icdescrip

tion

Cod

ingdescrip

tion

Proteindescrip

tion

refSNP

Effect

TP53

17p131

Intro

n3

g16225delGinsA

CCTG

GAG

GGCT

GGGG

c97-54delGinsA

CCTG

GAG

GGCT

GGGG

prs17878362

Intro

nic

Intro

n3

g16250CgtA

c97-29Cgt

Ap

rs17883323

Intro

nic

Intro

n3

g16273CgtT

c97-6CgtT

prs35117

667

Intro

nic

Exon

4g16321CgtT

c139Cgt

TpP4

7Srs1800371

Miss

ense

Exon

4g16397CgtG

c215Cgt

GpP7

2Rrs1042522

Miss

ense

ATM

11q22-q23

Exon

39g86904GgtA

c5557GgtA

pD1853N

rs1801516

Miss

ense

MDM2

12q143-q15

Intro

n1

g5610Tgt

G309TgtG

prs2279744

Intro

nic

Abbreviatio

nsrefSN

PreferenceS

NPclu

sterreport

704 Disease Markers

Table 2 Primer sequences and optimal annealing

Gene name Primer orientation Primer sequences (51015840-31015840) Annealing temperature (∘C)

TP53 Forward ATGGAGGAGCCGCAGTCAGA 613Reverse TCTGGGAAGGGACAGAAGA

ATM Forward AGCAGTATGTTGAGTTTATGGC 543Reverse TGAATCCAAGTTTGCAGG

MDM2 Forward CGGGAGTTCAGGGTAAAGGT 603Reverse AGCAAGTCGGTGCTTACCTG

This study used a candidate gene approach to search forassociations between genetic variants such as single nucle-otide polymorphisms and radiation treatment toxicity

22 Genotyping For genetic analysis each patientwas appro-ached at the time of follow-up evaluation A consent formwaspresented to the patient prior to the collection of their clinicaldata and blood sample and all patients included agreed tosign the consent form for this research study This study wasapproved by the Ethicrsquos Committee from ACCG

DNA was isolated from peripheral blood through theiPrep Purification system developed by Invitrogen Corpo-ration CA according to the manufacturerrsquos protocol Poly-merase chain reaction (PCR) was used to amplify eachfragment using specific primers (Table 2) designed from thegenomic sequence obtained from the National Center forBiotechnology Information (httpwwwncbinlmnihgov)The optimal annealing temperature for each primer wasascertained experimentally on a gradient PCR machine

In brief PCR was performed at a 50120583L reaction mixturecontaining 100 ng of DNA 10 120583M of each primer 02120583L ofAccuprime Taq High Fidelity and a reaction buffer contain-ing 600mM Tris-SO

4 180mM (NH

4)2SO4 20mMMgSO

4

and 2mM of each deoxynucleoside triphosphate (InvitrogenCA) The PCR profile consisted of an initial melting step of10 minutes at 95∘C followed by 40 cycles of 30 seconds at95∘C 30 seconds at 543∘C for ATM at 613∘C for TP53 or at603∘C for MDM2 amplification 1 minute at 72∘C and a finalelongation step of 7 minutes at 72∘C

The PCR reactions were first subjected to purificationusing the PureLink Quick Gel Extraction and PCR Purifi-cation Combo Kit (Invitrogen CA) treated with Exo SAP-IT (USB Corporation Cleveland Ohio USA) to removedNTPs and primers and then underwent nucleotide primerextension reaction The sequencing reactions (BigDye Ter-minator v 31 Ready Reaction Cycle Sequencing Kit AppliedBiosystems Foster City CA) were subjected to purificationusing BigDye Xterminator (Applied Biosystems Foster CityCA) for a complete removal of ddNTPs and underwentcapillary electrophoresis on an Applied Biosystems 3130xlDNA Analyzer The data were analysed by the SeqscapeSoftware v26 (Applied Biosystems Foster City CA)

23 Statistical Analysis Each polymorphism was tested fordeviation from Hardy-Weinberg equilibrium by compar-ing the observed and expected genotype frequencies usingthe chi-square test with one degree of freedom Analyseswere performed using the SigmaStat version 150 statistical

software package (SPSS Inc Chicago IL) The associationbetween ATMTP53 andMDM2 polymorphisms and adverseresponse to radiotherapy was estimated by chi-square test orFisherrsquos exact test with one degree of freedom All analyseswere performed with 120572 = 5 and confidence interval of 95

3 Results

31 Patientrsquos Characteristics Patientrsquos characteristics are des-cribed in Table 3Themean age was 673 years Of 48 patients292 had family history of cancer 479 had hypertension125 were diabetic and 542 had a history of smokingduring some time in their lives

A total of 45 patients (937) had tumor Gleason scorehigher than five 25 patients (521) had mean prostatespecific antigen (PSA) higher than 10 ngmL before treatmentand in 771 of the patients PSAdecreased after radiotherapyTwelve patients (25) underwent concomitant hormonaltherapy and seven (146) had undergone prior prostatec-tomy The average radiation dose in the 50 patients was 701Grays (Gy) and the treatment lasted for an average of 615days The followup ranged from 2 to 92 months

Thepatientswere classified as having an adverse responseRTOG high grade or not having an adverse response (RTOGlow grade) Tables 4 and 5 show the number of patients havingacute or chronic adverse effects of radiotherapy respectively

There were no significant differences between patientswho developed severe acute or chronic radiation toxicitiesand those who did not in relation to age smoking status Gle-ason score family history of cancer hypertension or diabetesand radiotherapy dose and patients who underwent con-comitant hormonal therapy or had prior prostatectomy (119875 gt005)

32 Polymorphisms and Risk for Development of RadiationToxicity From the initial group of 48 patients eligible forthe present study we analysed polymorphisms in the genesATM TP53 and MDM2 For the ATM and MDM2 SNPsall patients were successfully genotyped For TP53 gene weanalysed four polymorphisms in the PCRamplified fragmentComplete data was obtained for the TP53 polymorphism atcodon 72 It was possible to verify the presence or absenceof this polymorphism in all 48 patients while data regardingTP53 polymorphisms at codon 47 and intronic regions 16250and 16225 were analysed in 47 patients and region 16273of intron three was analysed in 46 patients The frequenciesof each polymorphism are shown in Table 6 For Chronicanalysis three patients were excluded because their followup

Disease Markers 705

Table 3 Patients clinical characteristics

Characteristic No of patients ()Age (y) median (range) 673 (52ndash82)Family history of cancer 14 (292)Hypertension 19 (396)Diabetes mellitus 2 (42)Hypertension + Diabetes mellitus 4 (83)Other Comorbidities 6 (125)Any history of smoking 26 (542)Gleason scorele5 3 (63)6 25 (521)7 16 (333)8-9 4 (84)

PSA (ngmL) before treatment no of patients (PSA range)le4 9 (063ndash380)5ndash10 14 (413ndash1000)10ndash20 16 (1060ndash1950)gt20 9 (2138ndash151)

PSA outcome after radiotherapyPSA decreased 37 (771)PSA did not decrease 11 (229)

Concomitant hormonal therapy 12 (25)Prior surgery (prostatectomy) 7 (146)Radiotherapy interruption 5 (104)Total radiation dose (Gy) median (range) 701 (64ndash74)Treatment duration (months) median (range) 615 (49ndash86)Follow up (months) median (range) 48 (2ndash92)Abbreviations PSA prostate specific antigen Gy Grays

Table 4 RTOG acute radiation morbidity (toxicity grading)

Skin RTOG Lower GI tract RTOG Urinary RTOGn () n () n ()

High grade 2 (42) 7 (146) 23 (479)Low grade 46 (958) 41 (854) 25 (521)Abbreviations RTOG radiation therapy oncology group GI tract gastrointestinal tractHigh grade RTOG ge2 e Low grade RTOG lt2

Table 5 RTOG chronic radiation morbidity (toxicity grading)lowast

Skin RTOG Lower GI tract RTOG Urinary tract RTOGn () n () n ()

High grade 1 (22) 2 (44) 10 (222)Low grade 44 (978) 43 (956) 35 (778)Abbreviations RTOG radiation therapy oncology group GI tract gastrointestinal tractHigh grade RTOG ge2 e Low grade RTOG lt2lowastFor chronic analyses it was considered patients with followup higher than 24 months

was less than 24 months The frequencies of polymorphismsfor chronic associations are shown in Table 7

In univariate cross-table analysis the ATM Asp1853Asnpolymorphism was not associated with occurrence of eitheracute or chronic toxicity after radiotherapy (119875 gt 005)The intronic polymorphism located on region 16273 with a

homozygous change of CgtT was significantly associated tothe development of acute skin toxicity RTOG grade two orhigher (OR = 00072 95 CI 00002ndash0227 119875 = 0003) asshown in Table 6The heterozygous change of AgtC on region16250 was strongly associated to the developing of chronicurinary toxicity RTOG grade two or higher (OR = 0071

706 Disease Markers

Table6As

sociationof

thep

olym

orph

ismsa

nalysedwith

acutes

kin

gastrointestinaland

urinaryradiationtoxicity

Acutes

kinRT

OG

AcuteG

astro

intestinalRT

OG

AcuteU

rinaryTractR

TOG

Gene

(refSN

P)Genotype

n(

)HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

ATM

GG

36(75)

135

531

1719

(rs1801516)

CAlowast

12(25)

111

0314

0018ndash5452

044

12

100806

0135ndash4821

16

60895

0242ndash3307

1TP

53GG

25(52)

223

421

1213

(rs104

2522)

GC

17(35)

017

3723

0168ndash82615

0506

215

1429

0231ndash8840

19

80820

0239ndash

2819

1CC

6(13)

06

1383

0059ndash

32563

11

50952

0086ndash

10489

12

418

460284ndash

11983

066

4TP

53CC

45(96)

243

639

2223

(rs1800371)

CTdagger

2(4)

02

0287

0011ndash7744

11

16500

0357ndash11837

0278

02

4787

0217ndash10539

0491

TP53

CC43

(91)

241

538

1924

(rs17883323)

CAlowast

4(9)

04

0542

0022ndash1315

71

22

0132

0015ndash1153

0100

31

0264

0025ndash2745

0328

TP53

CC41

(89)

041

635

1823

(rs35117667)

CT2(4)

02

mdashmdash

Dagger0

20915

0039ndash

21368

11

10783

004

6ndash1339

91

TT3(7)

21

0007

000

02ndash0

227

000

31

22917

0227ndash37425

0413

21

2556

0214ndash

30469

0583

TP53

A1

40(87)

238

634

2020

(rs17878362)

A2

6(13)

06

0844

0036ndash

19676

11

51133

0112ndash11484

12

40500

0082ndash304

60667

MDM2

TT18

(38)

117

117

99

(rs2279744

)GT

27(56)

126

1529

0089ndash

2613

61

621

0206

0023ndash18

800215

1215

1250

0378ndash

4133

0767

GG

3(6)

03

060

0002ndash179

961

03

060

00020ndash

17996

12

10500

0038ndash6547

1Ab

breviatio

nsrefSN

PreferenceS

NPclu

sterreportRT

OGradiatio

ntherapyo

ncolog

ygroup

HGhighgradeR

TOGLGlow

gradeR

TOGO

Rod

dsratio

CIconfi

denceintervalT

omakec

alculations

ofFishers

exacttestp

ossib

lewhenon

evalue

iszero05was

addedto

thev

alue

lowastNocarriersof

anAAho

mozygou

sgenotypew

erefou

nd

daggerNocarriersof

aTTho

mozygou

sgenotypew

erefou

nd

DaggerNopatient

high

gradefor

theg

enotypes

analysed

Disease Markers 707

Table7As

sociationof

thep

olym

orph

ismsa

nalysedwith

chronics

kin

gastrointestinaland

urinaryradiotoxicitylowast

Chronics

kinRT

OG

Chronicg

astro

intestinalRT

OG

Chronicu

rinarytractR

TOG

Gene

(refSN

P)Genotype

n(

)HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

ATM

GG

35(78)

035

233

827

(rs1801516)

GAlowastlowast

10(22)

19

0089

0003ndash2372

0222

010

1567

0069ndash

3532

31

28

1185

0208ndash6740

1TP

53GG

23(51)

023

221

518

(rs104

2522)

GC

16(36)

115

0220

0008ndash5756

0410

016

3837

0172ndash8553

30503

214

1944

0327ndash11563

0678

CC6(13)

06

mdashmdash

Dagger0

615

12006

4ndash35678

13

30278

004

2ndash18

250305

TP53

CC42 (955)

141

240

933

(rs1800371)

CTdagger

2(45)

02

0181

0058ndash5673

10

20307

0011ndash8325

11

13667

0208ndash64

54

040

7TP

53CC

40(91)

040

238

733

(rs17883323)

CAlowastlowast

4(9)

13

0029

0001ndash0848

0091

04

0584

0024ndash

1419

91

31

0071

000

6ndash0784

0032

TP53

CC38

(88)

137

137

830

(rs35117667)

CT2(5)

02

0200

000

6ndash6288

11

10027

0001ndash0819

0099

02

1393

0061ndash31901

1TT

3(7)

03

0280

000

9ndash8254

10

30280

000

9ndash8254

11

218

750150ndash

2339

60535

TP53

A1

37(86)

136

235

829

(rs17878362)

A2

6(14

)0

60534

0019ndash

14607

10

60915

0039ndash

21368

12

418

130280ndash

11750

0611

MDM2

TT17

(38)

017

116

413

(rs2279744

)GT

25(55)

124

046

70018ndash1215

21

124

1500

0087ndash25753

16

190974

0229ndash

4148

1GG

3(7)

03

mdashmdash

Dagger0

30636

0212ndash1912

41

03

2333

0100ndash

54460

1Ab

breviatio

nsrefSN

PreferenceS

NPclu

sterreportRT

OGradiatio

ntherapyo

ncolog

ygroup

HGhighgradeR

TOGLGlow

gradeR

TOGO

Rod

dsratio

CIconfi

denceintervalT

omakec

alculations

ofFishers

exacttestp

ossib

lewhenon

evalue

iszero05was

addedto

thev

alue

lowastFo

rchron

icanalysesitw

asconsidered

patientsw

ithfollo

wup

high

erthan

24mon

ths

lowastlowastNocarriersof

anAAho

mozygou

sgenotypew

erefou

nd

daggerNocarriersof

aTTho

mozygou

sgenotypew

erefou

nd

DaggerNopatient

high

gradefor

theg

enotypes

analysed

708 Disease Markers

95CI 0006ndash0784119875 = 0032) None of the remaining TP53polymorphisms including the one located on codon 72 weresignificantly associated with the presence of acute or chronicskin gastrointestinal and urinary toxicities (Tables 6 and 7)

4 Discussion

41 Clinical Data Associated with Radiation Toxicity Ouranalysis showed as expected that clinical features were notdeterminants of acute or chronic toxicities of the prostateadjacent tissues when submitted to radiotherapy (119875 gt 005)These results confirm the importance of investigating thegenetic profile of patients undergoing radiotherapy in orderto find if genetic differences are involved or not involvedin the development of radiation toxicities during and aftertreatment

42 Polymorphisms and Radiation Toxicity There is a beliefthat the genomic revolution triggered by full sequencingof the human genome and technological development oflarge-scale methodologies announces the future of person-alized medicine In oncology this progress should increasethe possibility to predict individual patient responses toradiotherapy and chemotherapy Andreassen et al (2002)[24] hypothesized that the radiation sensitivity of normaltissues should be associated with a so-called ldquotrait-dependentcomplexrdquo which adds the effect ofmany genetic determinantsand single nucleotide polymorphisms that could correspondin part to the frequency and severity of these components

Some suggestive evidence was obtained from the cor-relation between the ATM codon Asp1853Asn (5557GgtA)SNP and the high risk of the development of radiotherapy-induced acute skin complications in breast cancer patients[21 25 26] For prostate cancer data are controversial ATMsequence alterations including codon 1853 polymorphismwere associated with the development of radiation-inducedproctitis [5] although no relation was shown between thismarker and bladder or rectal toxicities arising from theradiation therapy in Canadian prostate cancer patients [27]Our findings showed that Asp1853Asn polymorphism wasnot associated with the development of any acute or chronicadverse effects in patients with prostate cancer treated withradiotherapy (119875 gt 005) ATM is a large gene with many vari-ants documented Further investigations will be necessary toverify the association of other ATM SNPs in more repre-sentative sample groups in order to establish whether singlenucleotide polymorphisms or gene haplotypes may actuallycontribute to the toxicity of normal tissue

The single base polymorphism in the promoter ofMDM2gene (SNP309 rs2279744) increases gene expression and it isshown to associate with accelerated tumor formation in bothhereditary and sporadic cancers [20] This SNP creates animproved site of action that regulates basal levels of MDM2The increase of MDM2 basal levels will also increase thedegradation of p53 tumor suppressor protein and may stopits transcriptional activity leading to increased apoptosis [1415 28 29] The hypothesis that increased apoptosis couldcause necrosis of normal tissue leading to radiotherapy sideeffects was not confirmed by our results Among the prostate

cancer patients of this study no association of the investigatedMDM2 polymorphism (rs2279744) and the incidence ofradiotherapy-induced acute and chronic effects were foundthus indicating no major contribution in this cohort ofprostate cancer Similar lack of association was reported byPopanda et al 2009 [30] Another study also showed that theMDM2 SNP309 polymorphism was not found to be involvedwith clinical pathologic variables recurrence risk and overallsurvival outcome in prostate cancer [31]

Many studies have showed the association between theTP53 Pro72Arg polymorphism and cancer susceptibility [32ndash34] An association between the risk of acute skin toxicityand TP53 Pro72 carriers in those with the CDKN1A 31Sergenotype in a subset of normal weight patients treatedwith radiotherapy for breast cancer has been shown [19]Another study found evidence that genetic polymorphismsin the ATM-P53 pathway can influence susceptibility todeveloping radiation-induced pneumonitis in lung cancerpatients after radiotherapy [35] Despite the strong hypothesisfor involvement of this gene the association between thePro72Arg polymorphism and the development of normaltissue reaction to radiotherapy in prostate cancer patientshas not been fully investigated previously This was the firststudy to evaluate the acute and chronic radiation toxicitiesin patients with prostate cancer and TP53 Pro72Arg poly-morphism No association was found in the studied groupeven though 27 (54) patients presented the combinationArgArg which has been described as a more predisposed toapoptosis genotype [16 36]

Another polymorphism in TP53 gene at codon 47(rs1800371) is also functionally significant Codon 47encodes proline in wild type p53 but in a small subset ofindividuals it can encode a serine It was showed that theserine 47 variant has up to 5-fold decreased ability to induceapoptosis compared with wild type p53 [18] However onour study the percentage of patients with this polymorphismwas not statistically sufficient to find a relationship with theadverse effects of radiotherapy even though it is a variantthat induces apoptosis There is no other literature data ofassociation between this polymorphism and toxicity inducedby radiation therapy

Our findings showed that a polymorphism located in theregion 16273 of intron 3 of TP53 (rs35117667) was associatedwith high risk of developing acute skin adverse effects (OR00072 95CI 00002ndash0227119875 = 0003)This polymorphismhas not been investigated by any study so far The presentstudy is the first to observe the frequencies of this polymor-phism in a specific population and also the first to associatethis polymorphism with radiotherapy There is also evidenceof association between the intronic polymorphism at position16250 (rs17883323) and chronic high grade toxicity of urinarytract (OR 0071 95 CI 0006ndash0784 119875 = 0032) Thispolymorphism has not been previously studied in a clinicalpopulation according to searches on scientific websites Bothintronic polymorphisms (rs35117667 and rs17883323) werevalidated by genotype data showing minor alleles in at leasttwo chromosomes [37] It is now necessary to evaluate themolecular basis of the association between these intronicpolymorphisms and the risk for radiotherapy side effects

Disease Markers 709

An intronic polymorphism is not implicated directly in analtered protein however it may lead to alternative splicing orit can be related to MicroRNAs [38ndash40] A protein modifiedby these processesmay be associated for example an increaseof apoptosis

Gemignani and colleagues [41] in 2004 reported that aninsertionduplication of 16 base pairs in intron 3 of TP53 genethe so called TP53 PIN3 (rs17878362) described by Lazar et al[42] (1993) is associated with an increased risk of developingcolorectal cancer (OR 155 95 CI 1 10 to 2 18 119875 = 0012)Tan et al [19] (2006) reported no association between theTP53 PIN3 (rs17878362) insertion and the development ofadverse effects of radiotherapy (119875 gt 005) Our data corro-borate the results of Tan et al [19] (2006) which also foundno association between this polymorphism and the risk oftoxicity after radiotherapy Our study showed that 42 patients(84) had this polymorphism The frequency of the SNPsuggest the importance of a case-control study to assess theassociation between this polymorphism and the developmentof prostate cancer and thus at this stage the functionalsignificance of TP53 PIN3 polymorphism remains poorlyexplored

5 Conclusions

Until now most of studies in this field have been carriedout using candidate genes as was done in the present studyThese studies can provide valuable information howeverthey are limited and have not yet succeeded in providinga predictive test to identify radiosensitivity in patients Inresponse to lack of success in candidate gene studies and thesubsequent recognition that this approach is too limited inscope radiogenomic investigators have now begun a muchbroader search to identify genes and SNPs associated withradiation response Several large-scale genome wide asso-ciation (GWA) studies have been initiated in which radio-therapy patients are being genotyped for large numbers ofcommon SNPs [8 43]

The results of this study hint to a few candidate geneshowever our study was limited by the small sample size andtherefore low statistical power to detect associations Thisstudy shows the importance of genetic investigations as auseful tool for individualization of radiotherapy strategies Itis anticipated that over the next few years SNPs correlatedwith susceptibility for the development of adverse effectsresulting from radiotherapy will be identified from GWAstudies The data provided by studies in radiogenomics willopen new perspectives for interpretation of the results ofcandidate gene studies such as ours and can be the basis forthe development of a predictive assay that may be useful topersonalize and optimize cancer treatment

Acknowledgments

This work was supported by grants from Financiadora deEstudos e Projetos FINEP MCT Brazil and Fundacao deAmparo a Pesquisa do Estado de Goias FAPEG BrazilHellen Cintra had a fellowship from CAPES MEC Brazil

The author would like to thank Dr Alan George Smulian forthe paper revising

References

[1] G C Barnett C M L West A M Dunning et al ldquoNormaltissue reactions to radiotherapy towards tailoring treatmentdose by genotyperdquoNature Reviews Cancer vol 9 no 2 pp 134ndash142 2009

[2] N G Burnet R M Elliott A Dunning and C M L WestldquoRadiosensitivity Radiogenomics and RAPPERrdquo Clinical Onc-ology vol 18 no 7 pp 525ndash528 2006

[3] C M L West A M Dunning and B S Rosenstein ldquoGenome-wide association studies and prediction of normal tissue toxi-cityrdquo Seminars in Radiation Oncology vol 22 no 2 pp 91ndash992012

[4] J A Cesaretti R G Stock S Lehrer et al ldquoATM sequencevariants are predictive of adverse radiotherapy response amongpatients treated for prostate cancerrdquo International Journal ofRadiation Oncology Biology Physics vol 61 no 1 pp 196ndash2022005

[5] J A Cesaretti R G Stock D P Atencio et al ldquoA geneticallydetermined dose-volume histogram predicts for rectal bleedingamong patients treated with prostate brachytherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 68 no5 pp 1410ndash1416 2007

[6] C A Peters R G Stock J A Cesaretti et al ldquoTGFB1 singlenucleotide polymorphisms are associated with adverse qualityof life in prostate cancer patients treated with radiotherapyrdquoInternational Journal of Radiation Oncology Biology Physics vol70 no 3 pp 752ndash759 2008

[7] R J Burri R G Stock J A Cesaretti et al ldquoAssociation of singlenucleotide polymorphisms in SOD2 XRCC1 and XRCC3 withsusceptibility for the development of adverse effects resultingfrom radiotherapy for prostate cancerrdquo Radiation Research vol170 no 1 pp 49ndash59 2008

[8] S L Kerns H Ostrer R Stock et al ldquoGenome-wide associationstudy to identify single nucleotide polymorphisms (SNPs)associated with the development of erectile dysfunction inAfrican-American men after radiotherapy for prostate cancerrdquoInternational Journal of Radiation Oncology Biology Physics vol78 no 5 pp 1292ndash1300 2010

[9] G Iliakis ldquoThe role of DNA double strand breaks in ionizingradiation-induced killing of eukaryotic cellsrdquo BioEssays vol 13no 12 pp 641ndash648 1991

[10] F Altieri C Grillo M Maceroni and S Chichiarelli ldquoDNAdamage and repair from molecular mechanisms to healthimplicationsrdquo Antioxidants and Redox Signaling vol 10 no 5pp 891ndash937 2008

[11] A J Levine and M Oren ldquoThe first 30 years of p53 growingever more complexrdquo Nature Reviews Cancer vol 9 no 10 pp749ndash758 2009

[12] M B Kastan andD-S Lim ldquoThemany substrates and functionsof ATMrdquoNature ReviewsMolecular Cell Biology vol 1 no 3 pp179ndash186 2000

[13] A Tichy J Vavrova J Pejchal and M Rezacova ldquoAtaxia-telangiectasia mutated kinase (ATM) as a central regulator ofradiation-inducedDNAdamage responserdquoActaMedica vol 53no 1 pp 13ndash17 2010

[14] T Soussi and K G Wiman ldquoShaping genetic alterations inhuman cancer the p53 mutation paradigmrdquo Cancer Cell vol12 no 4 pp 303ndash312 2007

710 Disease Markers

[15] Y Levav-Cohen S Haupt and Y Haupt ldquoMdm2 in growthsignaling and cancerrdquoGrowth Factors vol 23 no 3 pp 183ndash1922005

[16] P Dumont J I-J Leu A C Della Pietra III D L George andM Murphy ldquoThe codon 72 polymorphic variants of p53 havemarkedly different apoptotic potentialrdquoNature Genetics vol 33no 3 pp 357ndash365 2003

[17] D D Oslashrsted S E Bojesen A Tybjaeligrg-Hansen and B G Nor-destgaard ldquoTumor suppressor p53 Arg72Pro polymorphismand longevity cancer survival and risk of cancer in the generalpopulationrdquo Journal of Experimental Medicine vol 204 no 6pp 1295ndash1301 2007

[18] X Li P Dumont A Della Pietra G Shetler andM E MurphyldquoThe codon 47 polymorphism in p53 is functionally significantrdquoJournal of Biological Chemistry vol 280 no 25 pp 24245ndash24251 2005

[19] X-L Tan O Popanda C B Ambrosone et al ldquoAssociationbetween TP53 and p21 genetic polymorphisms and acute sideeffects of radiotherapy in breast cancer patientsrdquo Breast CancerResearch and Treatment vol 97 no 3 pp 255ndash262 2006

[20] G L Bond W Hu E E Bond et al ldquoA single nucleotide poly-morphism in the MDM2 promoter attenuates the p53 tumorsuppressor pathway and accelerates tumor formation inhumansrdquo Cell vol 119 no 5 pp 591ndash602 2004

[21] A Y Ho G Fan D P Atencio et al ldquoPossession of ATMsequence variants as predictor for late normal tissue responsesin breast cancer patients treated with radiotherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 69 no3 pp 677ndash684 2007

[22] C E Canman D-S Lim K A Cimprich et al ldquoActivation ofthe ATM kinase by ionizing radiation and phosphorylation ofp53rdquo Science vol 281 no 5383 pp 1677ndash1679 1998

[23] J D Cox J Stetz and T F Pajak ldquoToxicity criteria of the Radi-ation Therapy Oncology Group (RTOG) and the EuropeanOrganization for Research and Treatment of Cancer (EORTC)rdquoInternational Journal of Radiation Oncology Biology Physics vol31 no 5 pp 1341ndash1346 1995

[24] C N Andreassen J Alsner and J Overgaard ldquoDoes variabilityin normal tissue reactions after radiotherapy have a geneticbasismdashwhere and how to look for itrdquo Radiotherapy and Onc-ology vol 64 no 2 pp 131ndash140 2002

[25] S Angele P Romestaing N Moullan et al ldquoATM haplotypesand cellular response to DNA damage association with breastcancer risk and clinical radiosensitivityrdquo Cancer Research vol63 no 24 pp 8717ndash8725 2003

[26] C N Andreassen J Overgaard J Alsner et al ldquoATM sequencevariants and risk of radiation-induced subcutaneous fibrosisafter postmastectomy radiotherapyrdquo International Journal ofRadiation Oncology Biology Physics vol 64 no 3 pp 776ndash7832006

[27] S Damaraju D Murray J Dufour et al ldquoAssociation of DNArepair and steroid metabolism gene polymorphisms with clini-cal late toxicity in patients treated with conformal radiotherapyfor prostate cancerrdquo Clinical Cancer Research vol 12 no 8 pp2545ndash2554 2006

[28] G L Bond W Hu and A Levine ldquoA single nucleotide poly-morphism in the MDM2 gene from a molecular and cellularexplanation to clinical effectrdquo Cancer Research vol 65 no 13pp 5481ndash5484 2005

[29] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[30] O Popanda J UMarquardt J Chang-Claude and P SchmezerldquoGenetic variation in normal tissue toxicity induced by ionizingradiationrdquoMutation Research vol 667 no 1-2 pp 58ndash69 2009

[31] J J Jaboin M Hwang C A Perez et al ldquoNo evidence forassociation of the MDM2-309 TG promoter polymorphismwith prostate cancer outcomesrdquo Urologic Oncology vol 29 no3 pp 319ndash323 2011

[32] L Sreeja V Syamala P B Raveendran S Santhi J Madha-van and R Ankathil ldquop53 Arg72Pro polymorphism predictssurvival outcome in lung cancer patients in Indian populationrdquoCancer Investigation vol 26 no 1 pp 41ndash46 2008

[33] M B Parliament and D Murray ldquoSingle nucleotide polymor-phisms of DNA repair genes as predictors of radioresponserdquoSeminars in Radiation Oncology vol 20 no 4 pp 232ndash2402010

[34] T Pugh M Keyes L Barclay et al ldquoSequence variant discoveryin DNA repair genes from radiosensitive and radiotolerantprostate brachytherapy patientsrdquo Clinical Cancer Research vol15 no 15 pp 5008ndash5016 2009

[35] M Yang L Zhang N Bi et al ldquoAssociation of P53 and ATMpolymorphisms with risk of radiation-induced pneumonitis inlung cancer patients treated with radiotherapyrdquo InternationalJournal of Radiation Oncology Biology Physics vol 79 no 5 pp1402ndash1407 2011

[36] G Alsbeih N Al-Harbi M Al-Buhairi K Al-Hadyan and MAl-Hamed ldquoAssociation between TP53 codon 72 single-nuc-leotide polymorphism and radiation sensitivity of human fibro-blastsrdquo Radiation Research vol 167 no 5 pp 535ndash540 2007

[37] M C Zody M Garber D J Adams et al ldquoDNA sequence ofhuman chromosome 17 and analysis of rearrangement in thehuman lineagerdquoNature vol 440 no 7087 pp 1045ndash1049 2006

[38] H I Suzuki K Yamagata K Sugimoto T Iwamoto S Kato andK Miyazono ldquoModulation of microRNA processing by p53rdquoNature vol 460 no 7254 pp 529ndash533 2009

[39] A K L Leung and P A Sharp ldquomicroRNAs a safeguard againstturmoilrdquo Cell vol 130 no 4 pp 581ndash585 2007

[40] Z Peng Y Cheng B C-M Tan et al ldquoComprehensive analysisof RNA-Seq data reveals extensive RNA editing in a humantranscriptomerdquo Nature Biotechnology vol 30 no 3 pp 253ndash260 2012

[41] F Gemignani VMoreno S Landi et al ldquoATP53 polymorphismis associated with increased risk of colorectal cancer and withreduced levels of TP53 mRNArdquo Oncogene vol 23 no 10 pp1954ndash1956 2004

[42] V Lazar F Hazard F Bertin N Janin D Bellet and B BressacldquoSimple sequence repeat polymorphism within the p53 generdquoOncogene vol 8 no 6 pp 1703ndash1705 1993

[43] CWest B S Rosenstein and J Alsner ldquoEstablishment of a radi-ogenomics consortiumrdquo International Journal of RadiationOnc-ology Biology Physics vol 76 no 5 pp 1295ndash1296 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Research and TreatmentAIDS

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 703

Table1Descriptio

nof

thea

nalysedpo

lymorph

ismsinAT

MM

DM2andTP

53genes

Genen

ame

Genom

iclocatio

nEx

onin

tron

Genom

icdescrip

tion

Cod

ingdescrip

tion

Proteindescrip

tion

refSNP

Effect

TP53

17p131

Intro

n3

g16225delGinsA

CCTG

GAG

GGCT

GGGG

c97-54delGinsA

CCTG

GAG

GGCT

GGGG

prs17878362

Intro

nic

Intro

n3

g16250CgtA

c97-29Cgt

Ap

rs17883323

Intro

nic

Intro

n3

g16273CgtT

c97-6CgtT

prs35117

667

Intro

nic

Exon

4g16321CgtT

c139Cgt

TpP4

7Srs1800371

Miss

ense

Exon

4g16397CgtG

c215Cgt

GpP7

2Rrs1042522

Miss

ense

ATM

11q22-q23

Exon

39g86904GgtA

c5557GgtA

pD1853N

rs1801516

Miss

ense

MDM2

12q143-q15

Intro

n1

g5610Tgt

G309TgtG

prs2279744

Intro

nic

Abbreviatio

nsrefSN

PreferenceS

NPclu

sterreport

704 Disease Markers

Table 2 Primer sequences and optimal annealing

Gene name Primer orientation Primer sequences (51015840-31015840) Annealing temperature (∘C)

TP53 Forward ATGGAGGAGCCGCAGTCAGA 613Reverse TCTGGGAAGGGACAGAAGA

ATM Forward AGCAGTATGTTGAGTTTATGGC 543Reverse TGAATCCAAGTTTGCAGG

MDM2 Forward CGGGAGTTCAGGGTAAAGGT 603Reverse AGCAAGTCGGTGCTTACCTG

This study used a candidate gene approach to search forassociations between genetic variants such as single nucle-otide polymorphisms and radiation treatment toxicity

22 Genotyping For genetic analysis each patientwas appro-ached at the time of follow-up evaluation A consent formwaspresented to the patient prior to the collection of their clinicaldata and blood sample and all patients included agreed tosign the consent form for this research study This study wasapproved by the Ethicrsquos Committee from ACCG

DNA was isolated from peripheral blood through theiPrep Purification system developed by Invitrogen Corpo-ration CA according to the manufacturerrsquos protocol Poly-merase chain reaction (PCR) was used to amplify eachfragment using specific primers (Table 2) designed from thegenomic sequence obtained from the National Center forBiotechnology Information (httpwwwncbinlmnihgov)The optimal annealing temperature for each primer wasascertained experimentally on a gradient PCR machine

In brief PCR was performed at a 50120583L reaction mixturecontaining 100 ng of DNA 10 120583M of each primer 02120583L ofAccuprime Taq High Fidelity and a reaction buffer contain-ing 600mM Tris-SO

4 180mM (NH

4)2SO4 20mMMgSO

4

and 2mM of each deoxynucleoside triphosphate (InvitrogenCA) The PCR profile consisted of an initial melting step of10 minutes at 95∘C followed by 40 cycles of 30 seconds at95∘C 30 seconds at 543∘C for ATM at 613∘C for TP53 or at603∘C for MDM2 amplification 1 minute at 72∘C and a finalelongation step of 7 minutes at 72∘C

The PCR reactions were first subjected to purificationusing the PureLink Quick Gel Extraction and PCR Purifi-cation Combo Kit (Invitrogen CA) treated with Exo SAP-IT (USB Corporation Cleveland Ohio USA) to removedNTPs and primers and then underwent nucleotide primerextension reaction The sequencing reactions (BigDye Ter-minator v 31 Ready Reaction Cycle Sequencing Kit AppliedBiosystems Foster City CA) were subjected to purificationusing BigDye Xterminator (Applied Biosystems Foster CityCA) for a complete removal of ddNTPs and underwentcapillary electrophoresis on an Applied Biosystems 3130xlDNA Analyzer The data were analysed by the SeqscapeSoftware v26 (Applied Biosystems Foster City CA)

23 Statistical Analysis Each polymorphism was tested fordeviation from Hardy-Weinberg equilibrium by compar-ing the observed and expected genotype frequencies usingthe chi-square test with one degree of freedom Analyseswere performed using the SigmaStat version 150 statistical

software package (SPSS Inc Chicago IL) The associationbetween ATMTP53 andMDM2 polymorphisms and adverseresponse to radiotherapy was estimated by chi-square test orFisherrsquos exact test with one degree of freedom All analyseswere performed with 120572 = 5 and confidence interval of 95

3 Results

31 Patientrsquos Characteristics Patientrsquos characteristics are des-cribed in Table 3Themean age was 673 years Of 48 patients292 had family history of cancer 479 had hypertension125 were diabetic and 542 had a history of smokingduring some time in their lives

A total of 45 patients (937) had tumor Gleason scorehigher than five 25 patients (521) had mean prostatespecific antigen (PSA) higher than 10 ngmL before treatmentand in 771 of the patients PSAdecreased after radiotherapyTwelve patients (25) underwent concomitant hormonaltherapy and seven (146) had undergone prior prostatec-tomy The average radiation dose in the 50 patients was 701Grays (Gy) and the treatment lasted for an average of 615days The followup ranged from 2 to 92 months

Thepatientswere classified as having an adverse responseRTOG high grade or not having an adverse response (RTOGlow grade) Tables 4 and 5 show the number of patients havingacute or chronic adverse effects of radiotherapy respectively

There were no significant differences between patientswho developed severe acute or chronic radiation toxicitiesand those who did not in relation to age smoking status Gle-ason score family history of cancer hypertension or diabetesand radiotherapy dose and patients who underwent con-comitant hormonal therapy or had prior prostatectomy (119875 gt005)

32 Polymorphisms and Risk for Development of RadiationToxicity From the initial group of 48 patients eligible forthe present study we analysed polymorphisms in the genesATM TP53 and MDM2 For the ATM and MDM2 SNPsall patients were successfully genotyped For TP53 gene weanalysed four polymorphisms in the PCRamplified fragmentComplete data was obtained for the TP53 polymorphism atcodon 72 It was possible to verify the presence or absenceof this polymorphism in all 48 patients while data regardingTP53 polymorphisms at codon 47 and intronic regions 16250and 16225 were analysed in 47 patients and region 16273of intron three was analysed in 46 patients The frequenciesof each polymorphism are shown in Table 6 For Chronicanalysis three patients were excluded because their followup

Disease Markers 705

Table 3 Patients clinical characteristics

Characteristic No of patients ()Age (y) median (range) 673 (52ndash82)Family history of cancer 14 (292)Hypertension 19 (396)Diabetes mellitus 2 (42)Hypertension + Diabetes mellitus 4 (83)Other Comorbidities 6 (125)Any history of smoking 26 (542)Gleason scorele5 3 (63)6 25 (521)7 16 (333)8-9 4 (84)

PSA (ngmL) before treatment no of patients (PSA range)le4 9 (063ndash380)5ndash10 14 (413ndash1000)10ndash20 16 (1060ndash1950)gt20 9 (2138ndash151)

PSA outcome after radiotherapyPSA decreased 37 (771)PSA did not decrease 11 (229)

Concomitant hormonal therapy 12 (25)Prior surgery (prostatectomy) 7 (146)Radiotherapy interruption 5 (104)Total radiation dose (Gy) median (range) 701 (64ndash74)Treatment duration (months) median (range) 615 (49ndash86)Follow up (months) median (range) 48 (2ndash92)Abbreviations PSA prostate specific antigen Gy Grays

Table 4 RTOG acute radiation morbidity (toxicity grading)

Skin RTOG Lower GI tract RTOG Urinary RTOGn () n () n ()

High grade 2 (42) 7 (146) 23 (479)Low grade 46 (958) 41 (854) 25 (521)Abbreviations RTOG radiation therapy oncology group GI tract gastrointestinal tractHigh grade RTOG ge2 e Low grade RTOG lt2

Table 5 RTOG chronic radiation morbidity (toxicity grading)lowast

Skin RTOG Lower GI tract RTOG Urinary tract RTOGn () n () n ()

High grade 1 (22) 2 (44) 10 (222)Low grade 44 (978) 43 (956) 35 (778)Abbreviations RTOG radiation therapy oncology group GI tract gastrointestinal tractHigh grade RTOG ge2 e Low grade RTOG lt2lowastFor chronic analyses it was considered patients with followup higher than 24 months

was less than 24 months The frequencies of polymorphismsfor chronic associations are shown in Table 7

In univariate cross-table analysis the ATM Asp1853Asnpolymorphism was not associated with occurrence of eitheracute or chronic toxicity after radiotherapy (119875 gt 005)The intronic polymorphism located on region 16273 with a

homozygous change of CgtT was significantly associated tothe development of acute skin toxicity RTOG grade two orhigher (OR = 00072 95 CI 00002ndash0227 119875 = 0003) asshown in Table 6The heterozygous change of AgtC on region16250 was strongly associated to the developing of chronicurinary toxicity RTOG grade two or higher (OR = 0071

706 Disease Markers

Table6As

sociationof

thep

olym

orph

ismsa

nalysedwith

acutes

kin

gastrointestinaland

urinaryradiationtoxicity

Acutes

kinRT

OG

AcuteG

astro

intestinalRT

OG

AcuteU

rinaryTractR

TOG

Gene

(refSN

P)Genotype

n(

)HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

ATM

GG

36(75)

135

531

1719

(rs1801516)

CAlowast

12(25)

111

0314

0018ndash5452

044

12

100806

0135ndash4821

16

60895

0242ndash3307

1TP

53GG

25(52)

223

421

1213

(rs104

2522)

GC

17(35)

017

3723

0168ndash82615

0506

215

1429

0231ndash8840

19

80820

0239ndash

2819

1CC

6(13)

06

1383

0059ndash

32563

11

50952

0086ndash

10489

12

418

460284ndash

11983

066

4TP

53CC

45(96)

243

639

2223

(rs1800371)

CTdagger

2(4)

02

0287

0011ndash7744

11

16500

0357ndash11837

0278

02

4787

0217ndash10539

0491

TP53

CC43

(91)

241

538

1924

(rs17883323)

CAlowast

4(9)

04

0542

0022ndash1315

71

22

0132

0015ndash1153

0100

31

0264

0025ndash2745

0328

TP53

CC41

(89)

041

635

1823

(rs35117667)

CT2(4)

02

mdashmdash

Dagger0

20915

0039ndash

21368

11

10783

004

6ndash1339

91

TT3(7)

21

0007

000

02ndash0

227

000

31

22917

0227ndash37425

0413

21

2556

0214ndash

30469

0583

TP53

A1

40(87)

238

634

2020

(rs17878362)

A2

6(13)

06

0844

0036ndash

19676

11

51133

0112ndash11484

12

40500

0082ndash304

60667

MDM2

TT18

(38)

117

117

99

(rs2279744

)GT

27(56)

126

1529

0089ndash

2613

61

621

0206

0023ndash18

800215

1215

1250

0378ndash

4133

0767

GG

3(6)

03

060

0002ndash179

961

03

060

00020ndash

17996

12

10500

0038ndash6547

1Ab

breviatio

nsrefSN

PreferenceS

NPclu

sterreportRT

OGradiatio

ntherapyo

ncolog

ygroup

HGhighgradeR

TOGLGlow

gradeR

TOGO

Rod

dsratio

CIconfi

denceintervalT

omakec

alculations

ofFishers

exacttestp

ossib

lewhenon

evalue

iszero05was

addedto

thev

alue

lowastNocarriersof

anAAho

mozygou

sgenotypew

erefou

nd

daggerNocarriersof

aTTho

mozygou

sgenotypew

erefou

nd

DaggerNopatient

high

gradefor

theg

enotypes

analysed

Disease Markers 707

Table7As

sociationof

thep

olym

orph

ismsa

nalysedwith

chronics

kin

gastrointestinaland

urinaryradiotoxicitylowast

Chronics

kinRT

OG

Chronicg

astro

intestinalRT

OG

Chronicu

rinarytractR

TOG

Gene

(refSN

P)Genotype

n(

)HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

ATM

GG

35(78)

035

233

827

(rs1801516)

GAlowastlowast

10(22)

19

0089

0003ndash2372

0222

010

1567

0069ndash

3532

31

28

1185

0208ndash6740

1TP

53GG

23(51)

023

221

518

(rs104

2522)

GC

16(36)

115

0220

0008ndash5756

0410

016

3837

0172ndash8553

30503

214

1944

0327ndash11563

0678

CC6(13)

06

mdashmdash

Dagger0

615

12006

4ndash35678

13

30278

004

2ndash18

250305

TP53

CC42 (955)

141

240

933

(rs1800371)

CTdagger

2(45)

02

0181

0058ndash5673

10

20307

0011ndash8325

11

13667

0208ndash64

54

040

7TP

53CC

40(91)

040

238

733

(rs17883323)

CAlowastlowast

4(9)

13

0029

0001ndash0848

0091

04

0584

0024ndash

1419

91

31

0071

000

6ndash0784

0032

TP53

CC38

(88)

137

137

830

(rs35117667)

CT2(5)

02

0200

000

6ndash6288

11

10027

0001ndash0819

0099

02

1393

0061ndash31901

1TT

3(7)

03

0280

000

9ndash8254

10

30280

000

9ndash8254

11

218

750150ndash

2339

60535

TP53

A1

37(86)

136

235

829

(rs17878362)

A2

6(14

)0

60534

0019ndash

14607

10

60915

0039ndash

21368

12

418

130280ndash

11750

0611

MDM2

TT17

(38)

017

116

413

(rs2279744

)GT

25(55)

124

046

70018ndash1215

21

124

1500

0087ndash25753

16

190974

0229ndash

4148

1GG

3(7)

03

mdashmdash

Dagger0

30636

0212ndash1912

41

03

2333

0100ndash

54460

1Ab

breviatio

nsrefSN

PreferenceS

NPclu

sterreportRT

OGradiatio

ntherapyo

ncolog

ygroup

HGhighgradeR

TOGLGlow

gradeR

TOGO

Rod

dsratio

CIconfi

denceintervalT

omakec

alculations

ofFishers

exacttestp

ossib

lewhenon

evalue

iszero05was

addedto

thev

alue

lowastFo

rchron

icanalysesitw

asconsidered

patientsw

ithfollo

wup

high

erthan

24mon

ths

lowastlowastNocarriersof

anAAho

mozygou

sgenotypew

erefou

nd

daggerNocarriersof

aTTho

mozygou

sgenotypew

erefou

nd

DaggerNopatient

high

gradefor

theg

enotypes

analysed

708 Disease Markers

95CI 0006ndash0784119875 = 0032) None of the remaining TP53polymorphisms including the one located on codon 72 weresignificantly associated with the presence of acute or chronicskin gastrointestinal and urinary toxicities (Tables 6 and 7)

4 Discussion

41 Clinical Data Associated with Radiation Toxicity Ouranalysis showed as expected that clinical features were notdeterminants of acute or chronic toxicities of the prostateadjacent tissues when submitted to radiotherapy (119875 gt 005)These results confirm the importance of investigating thegenetic profile of patients undergoing radiotherapy in orderto find if genetic differences are involved or not involvedin the development of radiation toxicities during and aftertreatment

42 Polymorphisms and Radiation Toxicity There is a beliefthat the genomic revolution triggered by full sequencingof the human genome and technological development oflarge-scale methodologies announces the future of person-alized medicine In oncology this progress should increasethe possibility to predict individual patient responses toradiotherapy and chemotherapy Andreassen et al (2002)[24] hypothesized that the radiation sensitivity of normaltissues should be associated with a so-called ldquotrait-dependentcomplexrdquo which adds the effect ofmany genetic determinantsand single nucleotide polymorphisms that could correspondin part to the frequency and severity of these components

Some suggestive evidence was obtained from the cor-relation between the ATM codon Asp1853Asn (5557GgtA)SNP and the high risk of the development of radiotherapy-induced acute skin complications in breast cancer patients[21 25 26] For prostate cancer data are controversial ATMsequence alterations including codon 1853 polymorphismwere associated with the development of radiation-inducedproctitis [5] although no relation was shown between thismarker and bladder or rectal toxicities arising from theradiation therapy in Canadian prostate cancer patients [27]Our findings showed that Asp1853Asn polymorphism wasnot associated with the development of any acute or chronicadverse effects in patients with prostate cancer treated withradiotherapy (119875 gt 005) ATM is a large gene with many vari-ants documented Further investigations will be necessary toverify the association of other ATM SNPs in more repre-sentative sample groups in order to establish whether singlenucleotide polymorphisms or gene haplotypes may actuallycontribute to the toxicity of normal tissue

The single base polymorphism in the promoter ofMDM2gene (SNP309 rs2279744) increases gene expression and it isshown to associate with accelerated tumor formation in bothhereditary and sporadic cancers [20] This SNP creates animproved site of action that regulates basal levels of MDM2The increase of MDM2 basal levels will also increase thedegradation of p53 tumor suppressor protein and may stopits transcriptional activity leading to increased apoptosis [1415 28 29] The hypothesis that increased apoptosis couldcause necrosis of normal tissue leading to radiotherapy sideeffects was not confirmed by our results Among the prostate

cancer patients of this study no association of the investigatedMDM2 polymorphism (rs2279744) and the incidence ofradiotherapy-induced acute and chronic effects were foundthus indicating no major contribution in this cohort ofprostate cancer Similar lack of association was reported byPopanda et al 2009 [30] Another study also showed that theMDM2 SNP309 polymorphism was not found to be involvedwith clinical pathologic variables recurrence risk and overallsurvival outcome in prostate cancer [31]

Many studies have showed the association between theTP53 Pro72Arg polymorphism and cancer susceptibility [32ndash34] An association between the risk of acute skin toxicityand TP53 Pro72 carriers in those with the CDKN1A 31Sergenotype in a subset of normal weight patients treatedwith radiotherapy for breast cancer has been shown [19]Another study found evidence that genetic polymorphismsin the ATM-P53 pathway can influence susceptibility todeveloping radiation-induced pneumonitis in lung cancerpatients after radiotherapy [35] Despite the strong hypothesisfor involvement of this gene the association between thePro72Arg polymorphism and the development of normaltissue reaction to radiotherapy in prostate cancer patientshas not been fully investigated previously This was the firststudy to evaluate the acute and chronic radiation toxicitiesin patients with prostate cancer and TP53 Pro72Arg poly-morphism No association was found in the studied groupeven though 27 (54) patients presented the combinationArgArg which has been described as a more predisposed toapoptosis genotype [16 36]

Another polymorphism in TP53 gene at codon 47(rs1800371) is also functionally significant Codon 47encodes proline in wild type p53 but in a small subset ofindividuals it can encode a serine It was showed that theserine 47 variant has up to 5-fold decreased ability to induceapoptosis compared with wild type p53 [18] However onour study the percentage of patients with this polymorphismwas not statistically sufficient to find a relationship with theadverse effects of radiotherapy even though it is a variantthat induces apoptosis There is no other literature data ofassociation between this polymorphism and toxicity inducedby radiation therapy

Our findings showed that a polymorphism located in theregion 16273 of intron 3 of TP53 (rs35117667) was associatedwith high risk of developing acute skin adverse effects (OR00072 95CI 00002ndash0227119875 = 0003)This polymorphismhas not been investigated by any study so far The presentstudy is the first to observe the frequencies of this polymor-phism in a specific population and also the first to associatethis polymorphism with radiotherapy There is also evidenceof association between the intronic polymorphism at position16250 (rs17883323) and chronic high grade toxicity of urinarytract (OR 0071 95 CI 0006ndash0784 119875 = 0032) Thispolymorphism has not been previously studied in a clinicalpopulation according to searches on scientific websites Bothintronic polymorphisms (rs35117667 and rs17883323) werevalidated by genotype data showing minor alleles in at leasttwo chromosomes [37] It is now necessary to evaluate themolecular basis of the association between these intronicpolymorphisms and the risk for radiotherapy side effects

Disease Markers 709

An intronic polymorphism is not implicated directly in analtered protein however it may lead to alternative splicing orit can be related to MicroRNAs [38ndash40] A protein modifiedby these processesmay be associated for example an increaseof apoptosis

Gemignani and colleagues [41] in 2004 reported that aninsertionduplication of 16 base pairs in intron 3 of TP53 genethe so called TP53 PIN3 (rs17878362) described by Lazar et al[42] (1993) is associated with an increased risk of developingcolorectal cancer (OR 155 95 CI 1 10 to 2 18 119875 = 0012)Tan et al [19] (2006) reported no association between theTP53 PIN3 (rs17878362) insertion and the development ofadverse effects of radiotherapy (119875 gt 005) Our data corro-borate the results of Tan et al [19] (2006) which also foundno association between this polymorphism and the risk oftoxicity after radiotherapy Our study showed that 42 patients(84) had this polymorphism The frequency of the SNPsuggest the importance of a case-control study to assess theassociation between this polymorphism and the developmentof prostate cancer and thus at this stage the functionalsignificance of TP53 PIN3 polymorphism remains poorlyexplored

5 Conclusions

Until now most of studies in this field have been carriedout using candidate genes as was done in the present studyThese studies can provide valuable information howeverthey are limited and have not yet succeeded in providinga predictive test to identify radiosensitivity in patients Inresponse to lack of success in candidate gene studies and thesubsequent recognition that this approach is too limited inscope radiogenomic investigators have now begun a muchbroader search to identify genes and SNPs associated withradiation response Several large-scale genome wide asso-ciation (GWA) studies have been initiated in which radio-therapy patients are being genotyped for large numbers ofcommon SNPs [8 43]

The results of this study hint to a few candidate geneshowever our study was limited by the small sample size andtherefore low statistical power to detect associations Thisstudy shows the importance of genetic investigations as auseful tool for individualization of radiotherapy strategies Itis anticipated that over the next few years SNPs correlatedwith susceptibility for the development of adverse effectsresulting from radiotherapy will be identified from GWAstudies The data provided by studies in radiogenomics willopen new perspectives for interpretation of the results ofcandidate gene studies such as ours and can be the basis forthe development of a predictive assay that may be useful topersonalize and optimize cancer treatment

Acknowledgments

This work was supported by grants from Financiadora deEstudos e Projetos FINEP MCT Brazil and Fundacao deAmparo a Pesquisa do Estado de Goias FAPEG BrazilHellen Cintra had a fellowship from CAPES MEC Brazil

The author would like to thank Dr Alan George Smulian forthe paper revising

References

[1] G C Barnett C M L West A M Dunning et al ldquoNormaltissue reactions to radiotherapy towards tailoring treatmentdose by genotyperdquoNature Reviews Cancer vol 9 no 2 pp 134ndash142 2009

[2] N G Burnet R M Elliott A Dunning and C M L WestldquoRadiosensitivity Radiogenomics and RAPPERrdquo Clinical Onc-ology vol 18 no 7 pp 525ndash528 2006

[3] C M L West A M Dunning and B S Rosenstein ldquoGenome-wide association studies and prediction of normal tissue toxi-cityrdquo Seminars in Radiation Oncology vol 22 no 2 pp 91ndash992012

[4] J A Cesaretti R G Stock S Lehrer et al ldquoATM sequencevariants are predictive of adverse radiotherapy response amongpatients treated for prostate cancerrdquo International Journal ofRadiation Oncology Biology Physics vol 61 no 1 pp 196ndash2022005

[5] J A Cesaretti R G Stock D P Atencio et al ldquoA geneticallydetermined dose-volume histogram predicts for rectal bleedingamong patients treated with prostate brachytherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 68 no5 pp 1410ndash1416 2007

[6] C A Peters R G Stock J A Cesaretti et al ldquoTGFB1 singlenucleotide polymorphisms are associated with adverse qualityof life in prostate cancer patients treated with radiotherapyrdquoInternational Journal of Radiation Oncology Biology Physics vol70 no 3 pp 752ndash759 2008

[7] R J Burri R G Stock J A Cesaretti et al ldquoAssociation of singlenucleotide polymorphisms in SOD2 XRCC1 and XRCC3 withsusceptibility for the development of adverse effects resultingfrom radiotherapy for prostate cancerrdquo Radiation Research vol170 no 1 pp 49ndash59 2008

[8] S L Kerns H Ostrer R Stock et al ldquoGenome-wide associationstudy to identify single nucleotide polymorphisms (SNPs)associated with the development of erectile dysfunction inAfrican-American men after radiotherapy for prostate cancerrdquoInternational Journal of Radiation Oncology Biology Physics vol78 no 5 pp 1292ndash1300 2010

[9] G Iliakis ldquoThe role of DNA double strand breaks in ionizingradiation-induced killing of eukaryotic cellsrdquo BioEssays vol 13no 12 pp 641ndash648 1991

[10] F Altieri C Grillo M Maceroni and S Chichiarelli ldquoDNAdamage and repair from molecular mechanisms to healthimplicationsrdquo Antioxidants and Redox Signaling vol 10 no 5pp 891ndash937 2008

[11] A J Levine and M Oren ldquoThe first 30 years of p53 growingever more complexrdquo Nature Reviews Cancer vol 9 no 10 pp749ndash758 2009

[12] M B Kastan andD-S Lim ldquoThemany substrates and functionsof ATMrdquoNature ReviewsMolecular Cell Biology vol 1 no 3 pp179ndash186 2000

[13] A Tichy J Vavrova J Pejchal and M Rezacova ldquoAtaxia-telangiectasia mutated kinase (ATM) as a central regulator ofradiation-inducedDNAdamage responserdquoActaMedica vol 53no 1 pp 13ndash17 2010

[14] T Soussi and K G Wiman ldquoShaping genetic alterations inhuman cancer the p53 mutation paradigmrdquo Cancer Cell vol12 no 4 pp 303ndash312 2007

710 Disease Markers

[15] Y Levav-Cohen S Haupt and Y Haupt ldquoMdm2 in growthsignaling and cancerrdquoGrowth Factors vol 23 no 3 pp 183ndash1922005

[16] P Dumont J I-J Leu A C Della Pietra III D L George andM Murphy ldquoThe codon 72 polymorphic variants of p53 havemarkedly different apoptotic potentialrdquoNature Genetics vol 33no 3 pp 357ndash365 2003

[17] D D Oslashrsted S E Bojesen A Tybjaeligrg-Hansen and B G Nor-destgaard ldquoTumor suppressor p53 Arg72Pro polymorphismand longevity cancer survival and risk of cancer in the generalpopulationrdquo Journal of Experimental Medicine vol 204 no 6pp 1295ndash1301 2007

[18] X Li P Dumont A Della Pietra G Shetler andM E MurphyldquoThe codon 47 polymorphism in p53 is functionally significantrdquoJournal of Biological Chemistry vol 280 no 25 pp 24245ndash24251 2005

[19] X-L Tan O Popanda C B Ambrosone et al ldquoAssociationbetween TP53 and p21 genetic polymorphisms and acute sideeffects of radiotherapy in breast cancer patientsrdquo Breast CancerResearch and Treatment vol 97 no 3 pp 255ndash262 2006

[20] G L Bond W Hu E E Bond et al ldquoA single nucleotide poly-morphism in the MDM2 promoter attenuates the p53 tumorsuppressor pathway and accelerates tumor formation inhumansrdquo Cell vol 119 no 5 pp 591ndash602 2004

[21] A Y Ho G Fan D P Atencio et al ldquoPossession of ATMsequence variants as predictor for late normal tissue responsesin breast cancer patients treated with radiotherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 69 no3 pp 677ndash684 2007

[22] C E Canman D-S Lim K A Cimprich et al ldquoActivation ofthe ATM kinase by ionizing radiation and phosphorylation ofp53rdquo Science vol 281 no 5383 pp 1677ndash1679 1998

[23] J D Cox J Stetz and T F Pajak ldquoToxicity criteria of the Radi-ation Therapy Oncology Group (RTOG) and the EuropeanOrganization for Research and Treatment of Cancer (EORTC)rdquoInternational Journal of Radiation Oncology Biology Physics vol31 no 5 pp 1341ndash1346 1995

[24] C N Andreassen J Alsner and J Overgaard ldquoDoes variabilityin normal tissue reactions after radiotherapy have a geneticbasismdashwhere and how to look for itrdquo Radiotherapy and Onc-ology vol 64 no 2 pp 131ndash140 2002

[25] S Angele P Romestaing N Moullan et al ldquoATM haplotypesand cellular response to DNA damage association with breastcancer risk and clinical radiosensitivityrdquo Cancer Research vol63 no 24 pp 8717ndash8725 2003

[26] C N Andreassen J Overgaard J Alsner et al ldquoATM sequencevariants and risk of radiation-induced subcutaneous fibrosisafter postmastectomy radiotherapyrdquo International Journal ofRadiation Oncology Biology Physics vol 64 no 3 pp 776ndash7832006

[27] S Damaraju D Murray J Dufour et al ldquoAssociation of DNArepair and steroid metabolism gene polymorphisms with clini-cal late toxicity in patients treated with conformal radiotherapyfor prostate cancerrdquo Clinical Cancer Research vol 12 no 8 pp2545ndash2554 2006

[28] G L Bond W Hu and A Levine ldquoA single nucleotide poly-morphism in the MDM2 gene from a molecular and cellularexplanation to clinical effectrdquo Cancer Research vol 65 no 13pp 5481ndash5484 2005

[29] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[30] O Popanda J UMarquardt J Chang-Claude and P SchmezerldquoGenetic variation in normal tissue toxicity induced by ionizingradiationrdquoMutation Research vol 667 no 1-2 pp 58ndash69 2009

[31] J J Jaboin M Hwang C A Perez et al ldquoNo evidence forassociation of the MDM2-309 TG promoter polymorphismwith prostate cancer outcomesrdquo Urologic Oncology vol 29 no3 pp 319ndash323 2011

[32] L Sreeja V Syamala P B Raveendran S Santhi J Madha-van and R Ankathil ldquop53 Arg72Pro polymorphism predictssurvival outcome in lung cancer patients in Indian populationrdquoCancer Investigation vol 26 no 1 pp 41ndash46 2008

[33] M B Parliament and D Murray ldquoSingle nucleotide polymor-phisms of DNA repair genes as predictors of radioresponserdquoSeminars in Radiation Oncology vol 20 no 4 pp 232ndash2402010

[34] T Pugh M Keyes L Barclay et al ldquoSequence variant discoveryin DNA repair genes from radiosensitive and radiotolerantprostate brachytherapy patientsrdquo Clinical Cancer Research vol15 no 15 pp 5008ndash5016 2009

[35] M Yang L Zhang N Bi et al ldquoAssociation of P53 and ATMpolymorphisms with risk of radiation-induced pneumonitis inlung cancer patients treated with radiotherapyrdquo InternationalJournal of Radiation Oncology Biology Physics vol 79 no 5 pp1402ndash1407 2011

[36] G Alsbeih N Al-Harbi M Al-Buhairi K Al-Hadyan and MAl-Hamed ldquoAssociation between TP53 codon 72 single-nuc-leotide polymorphism and radiation sensitivity of human fibro-blastsrdquo Radiation Research vol 167 no 5 pp 535ndash540 2007

[37] M C Zody M Garber D J Adams et al ldquoDNA sequence ofhuman chromosome 17 and analysis of rearrangement in thehuman lineagerdquoNature vol 440 no 7087 pp 1045ndash1049 2006

[38] H I Suzuki K Yamagata K Sugimoto T Iwamoto S Kato andK Miyazono ldquoModulation of microRNA processing by p53rdquoNature vol 460 no 7254 pp 529ndash533 2009

[39] A K L Leung and P A Sharp ldquomicroRNAs a safeguard againstturmoilrdquo Cell vol 130 no 4 pp 581ndash585 2007

[40] Z Peng Y Cheng B C-M Tan et al ldquoComprehensive analysisof RNA-Seq data reveals extensive RNA editing in a humantranscriptomerdquo Nature Biotechnology vol 30 no 3 pp 253ndash260 2012

[41] F Gemignani VMoreno S Landi et al ldquoATP53 polymorphismis associated with increased risk of colorectal cancer and withreduced levels of TP53 mRNArdquo Oncogene vol 23 no 10 pp1954ndash1956 2004

[42] V Lazar F Hazard F Bertin N Janin D Bellet and B BressacldquoSimple sequence repeat polymorphism within the p53 generdquoOncogene vol 8 no 6 pp 1703ndash1705 1993

[43] CWest B S Rosenstein and J Alsner ldquoEstablishment of a radi-ogenomics consortiumrdquo International Journal of RadiationOnc-ology Biology Physics vol 76 no 5 pp 1295ndash1296 2010

Submit your manuscripts athttpwwwhindawicom

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Disease Markers

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704 Disease Markers

Table 2 Primer sequences and optimal annealing

Gene name Primer orientation Primer sequences (51015840-31015840) Annealing temperature (∘C)

TP53 Forward ATGGAGGAGCCGCAGTCAGA 613Reverse TCTGGGAAGGGACAGAAGA

ATM Forward AGCAGTATGTTGAGTTTATGGC 543Reverse TGAATCCAAGTTTGCAGG

MDM2 Forward CGGGAGTTCAGGGTAAAGGT 603Reverse AGCAAGTCGGTGCTTACCTG

This study used a candidate gene approach to search forassociations between genetic variants such as single nucle-otide polymorphisms and radiation treatment toxicity

22 Genotyping For genetic analysis each patientwas appro-ached at the time of follow-up evaluation A consent formwaspresented to the patient prior to the collection of their clinicaldata and blood sample and all patients included agreed tosign the consent form for this research study This study wasapproved by the Ethicrsquos Committee from ACCG

DNA was isolated from peripheral blood through theiPrep Purification system developed by Invitrogen Corpo-ration CA according to the manufacturerrsquos protocol Poly-merase chain reaction (PCR) was used to amplify eachfragment using specific primers (Table 2) designed from thegenomic sequence obtained from the National Center forBiotechnology Information (httpwwwncbinlmnihgov)The optimal annealing temperature for each primer wasascertained experimentally on a gradient PCR machine

In brief PCR was performed at a 50120583L reaction mixturecontaining 100 ng of DNA 10 120583M of each primer 02120583L ofAccuprime Taq High Fidelity and a reaction buffer contain-ing 600mM Tris-SO

4 180mM (NH

4)2SO4 20mMMgSO

4

and 2mM of each deoxynucleoside triphosphate (InvitrogenCA) The PCR profile consisted of an initial melting step of10 minutes at 95∘C followed by 40 cycles of 30 seconds at95∘C 30 seconds at 543∘C for ATM at 613∘C for TP53 or at603∘C for MDM2 amplification 1 minute at 72∘C and a finalelongation step of 7 minutes at 72∘C

The PCR reactions were first subjected to purificationusing the PureLink Quick Gel Extraction and PCR Purifi-cation Combo Kit (Invitrogen CA) treated with Exo SAP-IT (USB Corporation Cleveland Ohio USA) to removedNTPs and primers and then underwent nucleotide primerextension reaction The sequencing reactions (BigDye Ter-minator v 31 Ready Reaction Cycle Sequencing Kit AppliedBiosystems Foster City CA) were subjected to purificationusing BigDye Xterminator (Applied Biosystems Foster CityCA) for a complete removal of ddNTPs and underwentcapillary electrophoresis on an Applied Biosystems 3130xlDNA Analyzer The data were analysed by the SeqscapeSoftware v26 (Applied Biosystems Foster City CA)

23 Statistical Analysis Each polymorphism was tested fordeviation from Hardy-Weinberg equilibrium by compar-ing the observed and expected genotype frequencies usingthe chi-square test with one degree of freedom Analyseswere performed using the SigmaStat version 150 statistical

software package (SPSS Inc Chicago IL) The associationbetween ATMTP53 andMDM2 polymorphisms and adverseresponse to radiotherapy was estimated by chi-square test orFisherrsquos exact test with one degree of freedom All analyseswere performed with 120572 = 5 and confidence interval of 95

3 Results

31 Patientrsquos Characteristics Patientrsquos characteristics are des-cribed in Table 3Themean age was 673 years Of 48 patients292 had family history of cancer 479 had hypertension125 were diabetic and 542 had a history of smokingduring some time in their lives

A total of 45 patients (937) had tumor Gleason scorehigher than five 25 patients (521) had mean prostatespecific antigen (PSA) higher than 10 ngmL before treatmentand in 771 of the patients PSAdecreased after radiotherapyTwelve patients (25) underwent concomitant hormonaltherapy and seven (146) had undergone prior prostatec-tomy The average radiation dose in the 50 patients was 701Grays (Gy) and the treatment lasted for an average of 615days The followup ranged from 2 to 92 months

Thepatientswere classified as having an adverse responseRTOG high grade or not having an adverse response (RTOGlow grade) Tables 4 and 5 show the number of patients havingacute or chronic adverse effects of radiotherapy respectively

There were no significant differences between patientswho developed severe acute or chronic radiation toxicitiesand those who did not in relation to age smoking status Gle-ason score family history of cancer hypertension or diabetesand radiotherapy dose and patients who underwent con-comitant hormonal therapy or had prior prostatectomy (119875 gt005)

32 Polymorphisms and Risk for Development of RadiationToxicity From the initial group of 48 patients eligible forthe present study we analysed polymorphisms in the genesATM TP53 and MDM2 For the ATM and MDM2 SNPsall patients were successfully genotyped For TP53 gene weanalysed four polymorphisms in the PCRamplified fragmentComplete data was obtained for the TP53 polymorphism atcodon 72 It was possible to verify the presence or absenceof this polymorphism in all 48 patients while data regardingTP53 polymorphisms at codon 47 and intronic regions 16250and 16225 were analysed in 47 patients and region 16273of intron three was analysed in 46 patients The frequenciesof each polymorphism are shown in Table 6 For Chronicanalysis three patients were excluded because their followup

Disease Markers 705

Table 3 Patients clinical characteristics

Characteristic No of patients ()Age (y) median (range) 673 (52ndash82)Family history of cancer 14 (292)Hypertension 19 (396)Diabetes mellitus 2 (42)Hypertension + Diabetes mellitus 4 (83)Other Comorbidities 6 (125)Any history of smoking 26 (542)Gleason scorele5 3 (63)6 25 (521)7 16 (333)8-9 4 (84)

PSA (ngmL) before treatment no of patients (PSA range)le4 9 (063ndash380)5ndash10 14 (413ndash1000)10ndash20 16 (1060ndash1950)gt20 9 (2138ndash151)

PSA outcome after radiotherapyPSA decreased 37 (771)PSA did not decrease 11 (229)

Concomitant hormonal therapy 12 (25)Prior surgery (prostatectomy) 7 (146)Radiotherapy interruption 5 (104)Total radiation dose (Gy) median (range) 701 (64ndash74)Treatment duration (months) median (range) 615 (49ndash86)Follow up (months) median (range) 48 (2ndash92)Abbreviations PSA prostate specific antigen Gy Grays

Table 4 RTOG acute radiation morbidity (toxicity grading)

Skin RTOG Lower GI tract RTOG Urinary RTOGn () n () n ()

High grade 2 (42) 7 (146) 23 (479)Low grade 46 (958) 41 (854) 25 (521)Abbreviations RTOG radiation therapy oncology group GI tract gastrointestinal tractHigh grade RTOG ge2 e Low grade RTOG lt2

Table 5 RTOG chronic radiation morbidity (toxicity grading)lowast

Skin RTOG Lower GI tract RTOG Urinary tract RTOGn () n () n ()

High grade 1 (22) 2 (44) 10 (222)Low grade 44 (978) 43 (956) 35 (778)Abbreviations RTOG radiation therapy oncology group GI tract gastrointestinal tractHigh grade RTOG ge2 e Low grade RTOG lt2lowastFor chronic analyses it was considered patients with followup higher than 24 months

was less than 24 months The frequencies of polymorphismsfor chronic associations are shown in Table 7

In univariate cross-table analysis the ATM Asp1853Asnpolymorphism was not associated with occurrence of eitheracute or chronic toxicity after radiotherapy (119875 gt 005)The intronic polymorphism located on region 16273 with a

homozygous change of CgtT was significantly associated tothe development of acute skin toxicity RTOG grade two orhigher (OR = 00072 95 CI 00002ndash0227 119875 = 0003) asshown in Table 6The heterozygous change of AgtC on region16250 was strongly associated to the developing of chronicurinary toxicity RTOG grade two or higher (OR = 0071

706 Disease Markers

Table6As

sociationof

thep

olym

orph

ismsa

nalysedwith

acutes

kin

gastrointestinaland

urinaryradiationtoxicity

Acutes

kinRT

OG

AcuteG

astro

intestinalRT

OG

AcuteU

rinaryTractR

TOG

Gene

(refSN

P)Genotype

n(

)HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

ATM

GG

36(75)

135

531

1719

(rs1801516)

CAlowast

12(25)

111

0314

0018ndash5452

044

12

100806

0135ndash4821

16

60895

0242ndash3307

1TP

53GG

25(52)

223

421

1213

(rs104

2522)

GC

17(35)

017

3723

0168ndash82615

0506

215

1429

0231ndash8840

19

80820

0239ndash

2819

1CC

6(13)

06

1383

0059ndash

32563

11

50952

0086ndash

10489

12

418

460284ndash

11983

066

4TP

53CC

45(96)

243

639

2223

(rs1800371)

CTdagger

2(4)

02

0287

0011ndash7744

11

16500

0357ndash11837

0278

02

4787

0217ndash10539

0491

TP53

CC43

(91)

241

538

1924

(rs17883323)

CAlowast

4(9)

04

0542

0022ndash1315

71

22

0132

0015ndash1153

0100

31

0264

0025ndash2745

0328

TP53

CC41

(89)

041

635

1823

(rs35117667)

CT2(4)

02

mdashmdash

Dagger0

20915

0039ndash

21368

11

10783

004

6ndash1339

91

TT3(7)

21

0007

000

02ndash0

227

000

31

22917

0227ndash37425

0413

21

2556

0214ndash

30469

0583

TP53

A1

40(87)

238

634

2020

(rs17878362)

A2

6(13)

06

0844

0036ndash

19676

11

51133

0112ndash11484

12

40500

0082ndash304

60667

MDM2

TT18

(38)

117

117

99

(rs2279744

)GT

27(56)

126

1529

0089ndash

2613

61

621

0206

0023ndash18

800215

1215

1250

0378ndash

4133

0767

GG

3(6)

03

060

0002ndash179

961

03

060

00020ndash

17996

12

10500

0038ndash6547

1Ab

breviatio

nsrefSN

PreferenceS

NPclu

sterreportRT

OGradiatio

ntherapyo

ncolog

ygroup

HGhighgradeR

TOGLGlow

gradeR

TOGO

Rod

dsratio

CIconfi

denceintervalT

omakec

alculations

ofFishers

exacttestp

ossib

lewhenon

evalue

iszero05was

addedto

thev

alue

lowastNocarriersof

anAAho

mozygou

sgenotypew

erefou

nd

daggerNocarriersof

aTTho

mozygou

sgenotypew

erefou

nd

DaggerNopatient

high

gradefor

theg

enotypes

analysed

Disease Markers 707

Table7As

sociationof

thep

olym

orph

ismsa

nalysedwith

chronics

kin

gastrointestinaland

urinaryradiotoxicitylowast

Chronics

kinRT

OG

Chronicg

astro

intestinalRT

OG

Chronicu

rinarytractR

TOG

Gene

(refSN

P)Genotype

n(

)HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

ATM

GG

35(78)

035

233

827

(rs1801516)

GAlowastlowast

10(22)

19

0089

0003ndash2372

0222

010

1567

0069ndash

3532

31

28

1185

0208ndash6740

1TP

53GG

23(51)

023

221

518

(rs104

2522)

GC

16(36)

115

0220

0008ndash5756

0410

016

3837

0172ndash8553

30503

214

1944

0327ndash11563

0678

CC6(13)

06

mdashmdash

Dagger0

615

12006

4ndash35678

13

30278

004

2ndash18

250305

TP53

CC42 (955)

141

240

933

(rs1800371)

CTdagger

2(45)

02

0181

0058ndash5673

10

20307

0011ndash8325

11

13667

0208ndash64

54

040

7TP

53CC

40(91)

040

238

733

(rs17883323)

CAlowastlowast

4(9)

13

0029

0001ndash0848

0091

04

0584

0024ndash

1419

91

31

0071

000

6ndash0784

0032

TP53

CC38

(88)

137

137

830

(rs35117667)

CT2(5)

02

0200

000

6ndash6288

11

10027

0001ndash0819

0099

02

1393

0061ndash31901

1TT

3(7)

03

0280

000

9ndash8254

10

30280

000

9ndash8254

11

218

750150ndash

2339

60535

TP53

A1

37(86)

136

235

829

(rs17878362)

A2

6(14

)0

60534

0019ndash

14607

10

60915

0039ndash

21368

12

418

130280ndash

11750

0611

MDM2

TT17

(38)

017

116

413

(rs2279744

)GT

25(55)

124

046

70018ndash1215

21

124

1500

0087ndash25753

16

190974

0229ndash

4148

1GG

3(7)

03

mdashmdash

Dagger0

30636

0212ndash1912

41

03

2333

0100ndash

54460

1Ab

breviatio

nsrefSN

PreferenceS

NPclu

sterreportRT

OGradiatio

ntherapyo

ncolog

ygroup

HGhighgradeR

TOGLGlow

gradeR

TOGO

Rod

dsratio

CIconfi

denceintervalT

omakec

alculations

ofFishers

exacttestp

ossib

lewhenon

evalue

iszero05was

addedto

thev

alue

lowastFo

rchron

icanalysesitw

asconsidered

patientsw

ithfollo

wup

high

erthan

24mon

ths

lowastlowastNocarriersof

anAAho

mozygou

sgenotypew

erefou

nd

daggerNocarriersof

aTTho

mozygou

sgenotypew

erefou

nd

DaggerNopatient

high

gradefor

theg

enotypes

analysed

708 Disease Markers

95CI 0006ndash0784119875 = 0032) None of the remaining TP53polymorphisms including the one located on codon 72 weresignificantly associated with the presence of acute or chronicskin gastrointestinal and urinary toxicities (Tables 6 and 7)

4 Discussion

41 Clinical Data Associated with Radiation Toxicity Ouranalysis showed as expected that clinical features were notdeterminants of acute or chronic toxicities of the prostateadjacent tissues when submitted to radiotherapy (119875 gt 005)These results confirm the importance of investigating thegenetic profile of patients undergoing radiotherapy in orderto find if genetic differences are involved or not involvedin the development of radiation toxicities during and aftertreatment

42 Polymorphisms and Radiation Toxicity There is a beliefthat the genomic revolution triggered by full sequencingof the human genome and technological development oflarge-scale methodologies announces the future of person-alized medicine In oncology this progress should increasethe possibility to predict individual patient responses toradiotherapy and chemotherapy Andreassen et al (2002)[24] hypothesized that the radiation sensitivity of normaltissues should be associated with a so-called ldquotrait-dependentcomplexrdquo which adds the effect ofmany genetic determinantsand single nucleotide polymorphisms that could correspondin part to the frequency and severity of these components

Some suggestive evidence was obtained from the cor-relation between the ATM codon Asp1853Asn (5557GgtA)SNP and the high risk of the development of radiotherapy-induced acute skin complications in breast cancer patients[21 25 26] For prostate cancer data are controversial ATMsequence alterations including codon 1853 polymorphismwere associated with the development of radiation-inducedproctitis [5] although no relation was shown between thismarker and bladder or rectal toxicities arising from theradiation therapy in Canadian prostate cancer patients [27]Our findings showed that Asp1853Asn polymorphism wasnot associated with the development of any acute or chronicadverse effects in patients with prostate cancer treated withradiotherapy (119875 gt 005) ATM is a large gene with many vari-ants documented Further investigations will be necessary toverify the association of other ATM SNPs in more repre-sentative sample groups in order to establish whether singlenucleotide polymorphisms or gene haplotypes may actuallycontribute to the toxicity of normal tissue

The single base polymorphism in the promoter ofMDM2gene (SNP309 rs2279744) increases gene expression and it isshown to associate with accelerated tumor formation in bothhereditary and sporadic cancers [20] This SNP creates animproved site of action that regulates basal levels of MDM2The increase of MDM2 basal levels will also increase thedegradation of p53 tumor suppressor protein and may stopits transcriptional activity leading to increased apoptosis [1415 28 29] The hypothesis that increased apoptosis couldcause necrosis of normal tissue leading to radiotherapy sideeffects was not confirmed by our results Among the prostate

cancer patients of this study no association of the investigatedMDM2 polymorphism (rs2279744) and the incidence ofradiotherapy-induced acute and chronic effects were foundthus indicating no major contribution in this cohort ofprostate cancer Similar lack of association was reported byPopanda et al 2009 [30] Another study also showed that theMDM2 SNP309 polymorphism was not found to be involvedwith clinical pathologic variables recurrence risk and overallsurvival outcome in prostate cancer [31]

Many studies have showed the association between theTP53 Pro72Arg polymorphism and cancer susceptibility [32ndash34] An association between the risk of acute skin toxicityand TP53 Pro72 carriers in those with the CDKN1A 31Sergenotype in a subset of normal weight patients treatedwith radiotherapy for breast cancer has been shown [19]Another study found evidence that genetic polymorphismsin the ATM-P53 pathway can influence susceptibility todeveloping radiation-induced pneumonitis in lung cancerpatients after radiotherapy [35] Despite the strong hypothesisfor involvement of this gene the association between thePro72Arg polymorphism and the development of normaltissue reaction to radiotherapy in prostate cancer patientshas not been fully investigated previously This was the firststudy to evaluate the acute and chronic radiation toxicitiesin patients with prostate cancer and TP53 Pro72Arg poly-morphism No association was found in the studied groupeven though 27 (54) patients presented the combinationArgArg which has been described as a more predisposed toapoptosis genotype [16 36]

Another polymorphism in TP53 gene at codon 47(rs1800371) is also functionally significant Codon 47encodes proline in wild type p53 but in a small subset ofindividuals it can encode a serine It was showed that theserine 47 variant has up to 5-fold decreased ability to induceapoptosis compared with wild type p53 [18] However onour study the percentage of patients with this polymorphismwas not statistically sufficient to find a relationship with theadverse effects of radiotherapy even though it is a variantthat induces apoptosis There is no other literature data ofassociation between this polymorphism and toxicity inducedby radiation therapy

Our findings showed that a polymorphism located in theregion 16273 of intron 3 of TP53 (rs35117667) was associatedwith high risk of developing acute skin adverse effects (OR00072 95CI 00002ndash0227119875 = 0003)This polymorphismhas not been investigated by any study so far The presentstudy is the first to observe the frequencies of this polymor-phism in a specific population and also the first to associatethis polymorphism with radiotherapy There is also evidenceof association between the intronic polymorphism at position16250 (rs17883323) and chronic high grade toxicity of urinarytract (OR 0071 95 CI 0006ndash0784 119875 = 0032) Thispolymorphism has not been previously studied in a clinicalpopulation according to searches on scientific websites Bothintronic polymorphisms (rs35117667 and rs17883323) werevalidated by genotype data showing minor alleles in at leasttwo chromosomes [37] It is now necessary to evaluate themolecular basis of the association between these intronicpolymorphisms and the risk for radiotherapy side effects

Disease Markers 709

An intronic polymorphism is not implicated directly in analtered protein however it may lead to alternative splicing orit can be related to MicroRNAs [38ndash40] A protein modifiedby these processesmay be associated for example an increaseof apoptosis

Gemignani and colleagues [41] in 2004 reported that aninsertionduplication of 16 base pairs in intron 3 of TP53 genethe so called TP53 PIN3 (rs17878362) described by Lazar et al[42] (1993) is associated with an increased risk of developingcolorectal cancer (OR 155 95 CI 1 10 to 2 18 119875 = 0012)Tan et al [19] (2006) reported no association between theTP53 PIN3 (rs17878362) insertion and the development ofadverse effects of radiotherapy (119875 gt 005) Our data corro-borate the results of Tan et al [19] (2006) which also foundno association between this polymorphism and the risk oftoxicity after radiotherapy Our study showed that 42 patients(84) had this polymorphism The frequency of the SNPsuggest the importance of a case-control study to assess theassociation between this polymorphism and the developmentof prostate cancer and thus at this stage the functionalsignificance of TP53 PIN3 polymorphism remains poorlyexplored

5 Conclusions

Until now most of studies in this field have been carriedout using candidate genes as was done in the present studyThese studies can provide valuable information howeverthey are limited and have not yet succeeded in providinga predictive test to identify radiosensitivity in patients Inresponse to lack of success in candidate gene studies and thesubsequent recognition that this approach is too limited inscope radiogenomic investigators have now begun a muchbroader search to identify genes and SNPs associated withradiation response Several large-scale genome wide asso-ciation (GWA) studies have been initiated in which radio-therapy patients are being genotyped for large numbers ofcommon SNPs [8 43]

The results of this study hint to a few candidate geneshowever our study was limited by the small sample size andtherefore low statistical power to detect associations Thisstudy shows the importance of genetic investigations as auseful tool for individualization of radiotherapy strategies Itis anticipated that over the next few years SNPs correlatedwith susceptibility for the development of adverse effectsresulting from radiotherapy will be identified from GWAstudies The data provided by studies in radiogenomics willopen new perspectives for interpretation of the results ofcandidate gene studies such as ours and can be the basis forthe development of a predictive assay that may be useful topersonalize and optimize cancer treatment

Acknowledgments

This work was supported by grants from Financiadora deEstudos e Projetos FINEP MCT Brazil and Fundacao deAmparo a Pesquisa do Estado de Goias FAPEG BrazilHellen Cintra had a fellowship from CAPES MEC Brazil

The author would like to thank Dr Alan George Smulian forthe paper revising

References

[1] G C Barnett C M L West A M Dunning et al ldquoNormaltissue reactions to radiotherapy towards tailoring treatmentdose by genotyperdquoNature Reviews Cancer vol 9 no 2 pp 134ndash142 2009

[2] N G Burnet R M Elliott A Dunning and C M L WestldquoRadiosensitivity Radiogenomics and RAPPERrdquo Clinical Onc-ology vol 18 no 7 pp 525ndash528 2006

[3] C M L West A M Dunning and B S Rosenstein ldquoGenome-wide association studies and prediction of normal tissue toxi-cityrdquo Seminars in Radiation Oncology vol 22 no 2 pp 91ndash992012

[4] J A Cesaretti R G Stock S Lehrer et al ldquoATM sequencevariants are predictive of adverse radiotherapy response amongpatients treated for prostate cancerrdquo International Journal ofRadiation Oncology Biology Physics vol 61 no 1 pp 196ndash2022005

[5] J A Cesaretti R G Stock D P Atencio et al ldquoA geneticallydetermined dose-volume histogram predicts for rectal bleedingamong patients treated with prostate brachytherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 68 no5 pp 1410ndash1416 2007

[6] C A Peters R G Stock J A Cesaretti et al ldquoTGFB1 singlenucleotide polymorphisms are associated with adverse qualityof life in prostate cancer patients treated with radiotherapyrdquoInternational Journal of Radiation Oncology Biology Physics vol70 no 3 pp 752ndash759 2008

[7] R J Burri R G Stock J A Cesaretti et al ldquoAssociation of singlenucleotide polymorphisms in SOD2 XRCC1 and XRCC3 withsusceptibility for the development of adverse effects resultingfrom radiotherapy for prostate cancerrdquo Radiation Research vol170 no 1 pp 49ndash59 2008

[8] S L Kerns H Ostrer R Stock et al ldquoGenome-wide associationstudy to identify single nucleotide polymorphisms (SNPs)associated with the development of erectile dysfunction inAfrican-American men after radiotherapy for prostate cancerrdquoInternational Journal of Radiation Oncology Biology Physics vol78 no 5 pp 1292ndash1300 2010

[9] G Iliakis ldquoThe role of DNA double strand breaks in ionizingradiation-induced killing of eukaryotic cellsrdquo BioEssays vol 13no 12 pp 641ndash648 1991

[10] F Altieri C Grillo M Maceroni and S Chichiarelli ldquoDNAdamage and repair from molecular mechanisms to healthimplicationsrdquo Antioxidants and Redox Signaling vol 10 no 5pp 891ndash937 2008

[11] A J Levine and M Oren ldquoThe first 30 years of p53 growingever more complexrdquo Nature Reviews Cancer vol 9 no 10 pp749ndash758 2009

[12] M B Kastan andD-S Lim ldquoThemany substrates and functionsof ATMrdquoNature ReviewsMolecular Cell Biology vol 1 no 3 pp179ndash186 2000

[13] A Tichy J Vavrova J Pejchal and M Rezacova ldquoAtaxia-telangiectasia mutated kinase (ATM) as a central regulator ofradiation-inducedDNAdamage responserdquoActaMedica vol 53no 1 pp 13ndash17 2010

[14] T Soussi and K G Wiman ldquoShaping genetic alterations inhuman cancer the p53 mutation paradigmrdquo Cancer Cell vol12 no 4 pp 303ndash312 2007

710 Disease Markers

[15] Y Levav-Cohen S Haupt and Y Haupt ldquoMdm2 in growthsignaling and cancerrdquoGrowth Factors vol 23 no 3 pp 183ndash1922005

[16] P Dumont J I-J Leu A C Della Pietra III D L George andM Murphy ldquoThe codon 72 polymorphic variants of p53 havemarkedly different apoptotic potentialrdquoNature Genetics vol 33no 3 pp 357ndash365 2003

[17] D D Oslashrsted S E Bojesen A Tybjaeligrg-Hansen and B G Nor-destgaard ldquoTumor suppressor p53 Arg72Pro polymorphismand longevity cancer survival and risk of cancer in the generalpopulationrdquo Journal of Experimental Medicine vol 204 no 6pp 1295ndash1301 2007

[18] X Li P Dumont A Della Pietra G Shetler andM E MurphyldquoThe codon 47 polymorphism in p53 is functionally significantrdquoJournal of Biological Chemistry vol 280 no 25 pp 24245ndash24251 2005

[19] X-L Tan O Popanda C B Ambrosone et al ldquoAssociationbetween TP53 and p21 genetic polymorphisms and acute sideeffects of radiotherapy in breast cancer patientsrdquo Breast CancerResearch and Treatment vol 97 no 3 pp 255ndash262 2006

[20] G L Bond W Hu E E Bond et al ldquoA single nucleotide poly-morphism in the MDM2 promoter attenuates the p53 tumorsuppressor pathway and accelerates tumor formation inhumansrdquo Cell vol 119 no 5 pp 591ndash602 2004

[21] A Y Ho G Fan D P Atencio et al ldquoPossession of ATMsequence variants as predictor for late normal tissue responsesin breast cancer patients treated with radiotherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 69 no3 pp 677ndash684 2007

[22] C E Canman D-S Lim K A Cimprich et al ldquoActivation ofthe ATM kinase by ionizing radiation and phosphorylation ofp53rdquo Science vol 281 no 5383 pp 1677ndash1679 1998

[23] J D Cox J Stetz and T F Pajak ldquoToxicity criteria of the Radi-ation Therapy Oncology Group (RTOG) and the EuropeanOrganization for Research and Treatment of Cancer (EORTC)rdquoInternational Journal of Radiation Oncology Biology Physics vol31 no 5 pp 1341ndash1346 1995

[24] C N Andreassen J Alsner and J Overgaard ldquoDoes variabilityin normal tissue reactions after radiotherapy have a geneticbasismdashwhere and how to look for itrdquo Radiotherapy and Onc-ology vol 64 no 2 pp 131ndash140 2002

[25] S Angele P Romestaing N Moullan et al ldquoATM haplotypesand cellular response to DNA damage association with breastcancer risk and clinical radiosensitivityrdquo Cancer Research vol63 no 24 pp 8717ndash8725 2003

[26] C N Andreassen J Overgaard J Alsner et al ldquoATM sequencevariants and risk of radiation-induced subcutaneous fibrosisafter postmastectomy radiotherapyrdquo International Journal ofRadiation Oncology Biology Physics vol 64 no 3 pp 776ndash7832006

[27] S Damaraju D Murray J Dufour et al ldquoAssociation of DNArepair and steroid metabolism gene polymorphisms with clini-cal late toxicity in patients treated with conformal radiotherapyfor prostate cancerrdquo Clinical Cancer Research vol 12 no 8 pp2545ndash2554 2006

[28] G L Bond W Hu and A Levine ldquoA single nucleotide poly-morphism in the MDM2 gene from a molecular and cellularexplanation to clinical effectrdquo Cancer Research vol 65 no 13pp 5481ndash5484 2005

[29] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[30] O Popanda J UMarquardt J Chang-Claude and P SchmezerldquoGenetic variation in normal tissue toxicity induced by ionizingradiationrdquoMutation Research vol 667 no 1-2 pp 58ndash69 2009

[31] J J Jaboin M Hwang C A Perez et al ldquoNo evidence forassociation of the MDM2-309 TG promoter polymorphismwith prostate cancer outcomesrdquo Urologic Oncology vol 29 no3 pp 319ndash323 2011

[32] L Sreeja V Syamala P B Raveendran S Santhi J Madha-van and R Ankathil ldquop53 Arg72Pro polymorphism predictssurvival outcome in lung cancer patients in Indian populationrdquoCancer Investigation vol 26 no 1 pp 41ndash46 2008

[33] M B Parliament and D Murray ldquoSingle nucleotide polymor-phisms of DNA repair genes as predictors of radioresponserdquoSeminars in Radiation Oncology vol 20 no 4 pp 232ndash2402010

[34] T Pugh M Keyes L Barclay et al ldquoSequence variant discoveryin DNA repair genes from radiosensitive and radiotolerantprostate brachytherapy patientsrdquo Clinical Cancer Research vol15 no 15 pp 5008ndash5016 2009

[35] M Yang L Zhang N Bi et al ldquoAssociation of P53 and ATMpolymorphisms with risk of radiation-induced pneumonitis inlung cancer patients treated with radiotherapyrdquo InternationalJournal of Radiation Oncology Biology Physics vol 79 no 5 pp1402ndash1407 2011

[36] G Alsbeih N Al-Harbi M Al-Buhairi K Al-Hadyan and MAl-Hamed ldquoAssociation between TP53 codon 72 single-nuc-leotide polymorphism and radiation sensitivity of human fibro-blastsrdquo Radiation Research vol 167 no 5 pp 535ndash540 2007

[37] M C Zody M Garber D J Adams et al ldquoDNA sequence ofhuman chromosome 17 and analysis of rearrangement in thehuman lineagerdquoNature vol 440 no 7087 pp 1045ndash1049 2006

[38] H I Suzuki K Yamagata K Sugimoto T Iwamoto S Kato andK Miyazono ldquoModulation of microRNA processing by p53rdquoNature vol 460 no 7254 pp 529ndash533 2009

[39] A K L Leung and P A Sharp ldquomicroRNAs a safeguard againstturmoilrdquo Cell vol 130 no 4 pp 581ndash585 2007

[40] Z Peng Y Cheng B C-M Tan et al ldquoComprehensive analysisof RNA-Seq data reveals extensive RNA editing in a humantranscriptomerdquo Nature Biotechnology vol 30 no 3 pp 253ndash260 2012

[41] F Gemignani VMoreno S Landi et al ldquoATP53 polymorphismis associated with increased risk of colorectal cancer and withreduced levels of TP53 mRNArdquo Oncogene vol 23 no 10 pp1954ndash1956 2004

[42] V Lazar F Hazard F Bertin N Janin D Bellet and B BressacldquoSimple sequence repeat polymorphism within the p53 generdquoOncogene vol 8 no 6 pp 1703ndash1705 1993

[43] CWest B S Rosenstein and J Alsner ldquoEstablishment of a radi-ogenomics consortiumrdquo International Journal of RadiationOnc-ology Biology Physics vol 76 no 5 pp 1295ndash1296 2010

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 705

Table 3 Patients clinical characteristics

Characteristic No of patients ()Age (y) median (range) 673 (52ndash82)Family history of cancer 14 (292)Hypertension 19 (396)Diabetes mellitus 2 (42)Hypertension + Diabetes mellitus 4 (83)Other Comorbidities 6 (125)Any history of smoking 26 (542)Gleason scorele5 3 (63)6 25 (521)7 16 (333)8-9 4 (84)

PSA (ngmL) before treatment no of patients (PSA range)le4 9 (063ndash380)5ndash10 14 (413ndash1000)10ndash20 16 (1060ndash1950)gt20 9 (2138ndash151)

PSA outcome after radiotherapyPSA decreased 37 (771)PSA did not decrease 11 (229)

Concomitant hormonal therapy 12 (25)Prior surgery (prostatectomy) 7 (146)Radiotherapy interruption 5 (104)Total radiation dose (Gy) median (range) 701 (64ndash74)Treatment duration (months) median (range) 615 (49ndash86)Follow up (months) median (range) 48 (2ndash92)Abbreviations PSA prostate specific antigen Gy Grays

Table 4 RTOG acute radiation morbidity (toxicity grading)

Skin RTOG Lower GI tract RTOG Urinary RTOGn () n () n ()

High grade 2 (42) 7 (146) 23 (479)Low grade 46 (958) 41 (854) 25 (521)Abbreviations RTOG radiation therapy oncology group GI tract gastrointestinal tractHigh grade RTOG ge2 e Low grade RTOG lt2

Table 5 RTOG chronic radiation morbidity (toxicity grading)lowast

Skin RTOG Lower GI tract RTOG Urinary tract RTOGn () n () n ()

High grade 1 (22) 2 (44) 10 (222)Low grade 44 (978) 43 (956) 35 (778)Abbreviations RTOG radiation therapy oncology group GI tract gastrointestinal tractHigh grade RTOG ge2 e Low grade RTOG lt2lowastFor chronic analyses it was considered patients with followup higher than 24 months

was less than 24 months The frequencies of polymorphismsfor chronic associations are shown in Table 7

In univariate cross-table analysis the ATM Asp1853Asnpolymorphism was not associated with occurrence of eitheracute or chronic toxicity after radiotherapy (119875 gt 005)The intronic polymorphism located on region 16273 with a

homozygous change of CgtT was significantly associated tothe development of acute skin toxicity RTOG grade two orhigher (OR = 00072 95 CI 00002ndash0227 119875 = 0003) asshown in Table 6The heterozygous change of AgtC on region16250 was strongly associated to the developing of chronicurinary toxicity RTOG grade two or higher (OR = 0071

706 Disease Markers

Table6As

sociationof

thep

olym

orph

ismsa

nalysedwith

acutes

kin

gastrointestinaland

urinaryradiationtoxicity

Acutes

kinRT

OG

AcuteG

astro

intestinalRT

OG

AcuteU

rinaryTractR

TOG

Gene

(refSN

P)Genotype

n(

)HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

ATM

GG

36(75)

135

531

1719

(rs1801516)

CAlowast

12(25)

111

0314

0018ndash5452

044

12

100806

0135ndash4821

16

60895

0242ndash3307

1TP

53GG

25(52)

223

421

1213

(rs104

2522)

GC

17(35)

017

3723

0168ndash82615

0506

215

1429

0231ndash8840

19

80820

0239ndash

2819

1CC

6(13)

06

1383

0059ndash

32563

11

50952

0086ndash

10489

12

418

460284ndash

11983

066

4TP

53CC

45(96)

243

639

2223

(rs1800371)

CTdagger

2(4)

02

0287

0011ndash7744

11

16500

0357ndash11837

0278

02

4787

0217ndash10539

0491

TP53

CC43

(91)

241

538

1924

(rs17883323)

CAlowast

4(9)

04

0542

0022ndash1315

71

22

0132

0015ndash1153

0100

31

0264

0025ndash2745

0328

TP53

CC41

(89)

041

635

1823

(rs35117667)

CT2(4)

02

mdashmdash

Dagger0

20915

0039ndash

21368

11

10783

004

6ndash1339

91

TT3(7)

21

0007

000

02ndash0

227

000

31

22917

0227ndash37425

0413

21

2556

0214ndash

30469

0583

TP53

A1

40(87)

238

634

2020

(rs17878362)

A2

6(13)

06

0844

0036ndash

19676

11

51133

0112ndash11484

12

40500

0082ndash304

60667

MDM2

TT18

(38)

117

117

99

(rs2279744

)GT

27(56)

126

1529

0089ndash

2613

61

621

0206

0023ndash18

800215

1215

1250

0378ndash

4133

0767

GG

3(6)

03

060

0002ndash179

961

03

060

00020ndash

17996

12

10500

0038ndash6547

1Ab

breviatio

nsrefSN

PreferenceS

NPclu

sterreportRT

OGradiatio

ntherapyo

ncolog

ygroup

HGhighgradeR

TOGLGlow

gradeR

TOGO

Rod

dsratio

CIconfi

denceintervalT

omakec

alculations

ofFishers

exacttestp

ossib

lewhenon

evalue

iszero05was

addedto

thev

alue

lowastNocarriersof

anAAho

mozygou

sgenotypew

erefou

nd

daggerNocarriersof

aTTho

mozygou

sgenotypew

erefou

nd

DaggerNopatient

high

gradefor

theg

enotypes

analysed

Disease Markers 707

Table7As

sociationof

thep

olym

orph

ismsa

nalysedwith

chronics

kin

gastrointestinaland

urinaryradiotoxicitylowast

Chronics

kinRT

OG

Chronicg

astro

intestinalRT

OG

Chronicu

rinarytractR

TOG

Gene

(refSN

P)Genotype

n(

)HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

ATM

GG

35(78)

035

233

827

(rs1801516)

GAlowastlowast

10(22)

19

0089

0003ndash2372

0222

010

1567

0069ndash

3532

31

28

1185

0208ndash6740

1TP

53GG

23(51)

023

221

518

(rs104

2522)

GC

16(36)

115

0220

0008ndash5756

0410

016

3837

0172ndash8553

30503

214

1944

0327ndash11563

0678

CC6(13)

06

mdashmdash

Dagger0

615

12006

4ndash35678

13

30278

004

2ndash18

250305

TP53

CC42 (955)

141

240

933

(rs1800371)

CTdagger

2(45)

02

0181

0058ndash5673

10

20307

0011ndash8325

11

13667

0208ndash64

54

040

7TP

53CC

40(91)

040

238

733

(rs17883323)

CAlowastlowast

4(9)

13

0029

0001ndash0848

0091

04

0584

0024ndash

1419

91

31

0071

000

6ndash0784

0032

TP53

CC38

(88)

137

137

830

(rs35117667)

CT2(5)

02

0200

000

6ndash6288

11

10027

0001ndash0819

0099

02

1393

0061ndash31901

1TT

3(7)

03

0280

000

9ndash8254

10

30280

000

9ndash8254

11

218

750150ndash

2339

60535

TP53

A1

37(86)

136

235

829

(rs17878362)

A2

6(14

)0

60534

0019ndash

14607

10

60915

0039ndash

21368

12

418

130280ndash

11750

0611

MDM2

TT17

(38)

017

116

413

(rs2279744

)GT

25(55)

124

046

70018ndash1215

21

124

1500

0087ndash25753

16

190974

0229ndash

4148

1GG

3(7)

03

mdashmdash

Dagger0

30636

0212ndash1912

41

03

2333

0100ndash

54460

1Ab

breviatio

nsrefSN

PreferenceS

NPclu

sterreportRT

OGradiatio

ntherapyo

ncolog

ygroup

HGhighgradeR

TOGLGlow

gradeR

TOGO

Rod

dsratio

CIconfi

denceintervalT

omakec

alculations

ofFishers

exacttestp

ossib

lewhenon

evalue

iszero05was

addedto

thev

alue

lowastFo

rchron

icanalysesitw

asconsidered

patientsw

ithfollo

wup

high

erthan

24mon

ths

lowastlowastNocarriersof

anAAho

mozygou

sgenotypew

erefou

nd

daggerNocarriersof

aTTho

mozygou

sgenotypew

erefou

nd

DaggerNopatient

high

gradefor

theg

enotypes

analysed

708 Disease Markers

95CI 0006ndash0784119875 = 0032) None of the remaining TP53polymorphisms including the one located on codon 72 weresignificantly associated with the presence of acute or chronicskin gastrointestinal and urinary toxicities (Tables 6 and 7)

4 Discussion

41 Clinical Data Associated with Radiation Toxicity Ouranalysis showed as expected that clinical features were notdeterminants of acute or chronic toxicities of the prostateadjacent tissues when submitted to radiotherapy (119875 gt 005)These results confirm the importance of investigating thegenetic profile of patients undergoing radiotherapy in orderto find if genetic differences are involved or not involvedin the development of radiation toxicities during and aftertreatment

42 Polymorphisms and Radiation Toxicity There is a beliefthat the genomic revolution triggered by full sequencingof the human genome and technological development oflarge-scale methodologies announces the future of person-alized medicine In oncology this progress should increasethe possibility to predict individual patient responses toradiotherapy and chemotherapy Andreassen et al (2002)[24] hypothesized that the radiation sensitivity of normaltissues should be associated with a so-called ldquotrait-dependentcomplexrdquo which adds the effect ofmany genetic determinantsand single nucleotide polymorphisms that could correspondin part to the frequency and severity of these components

Some suggestive evidence was obtained from the cor-relation between the ATM codon Asp1853Asn (5557GgtA)SNP and the high risk of the development of radiotherapy-induced acute skin complications in breast cancer patients[21 25 26] For prostate cancer data are controversial ATMsequence alterations including codon 1853 polymorphismwere associated with the development of radiation-inducedproctitis [5] although no relation was shown between thismarker and bladder or rectal toxicities arising from theradiation therapy in Canadian prostate cancer patients [27]Our findings showed that Asp1853Asn polymorphism wasnot associated with the development of any acute or chronicadverse effects in patients with prostate cancer treated withradiotherapy (119875 gt 005) ATM is a large gene with many vari-ants documented Further investigations will be necessary toverify the association of other ATM SNPs in more repre-sentative sample groups in order to establish whether singlenucleotide polymorphisms or gene haplotypes may actuallycontribute to the toxicity of normal tissue

The single base polymorphism in the promoter ofMDM2gene (SNP309 rs2279744) increases gene expression and it isshown to associate with accelerated tumor formation in bothhereditary and sporadic cancers [20] This SNP creates animproved site of action that regulates basal levels of MDM2The increase of MDM2 basal levels will also increase thedegradation of p53 tumor suppressor protein and may stopits transcriptional activity leading to increased apoptosis [1415 28 29] The hypothesis that increased apoptosis couldcause necrosis of normal tissue leading to radiotherapy sideeffects was not confirmed by our results Among the prostate

cancer patients of this study no association of the investigatedMDM2 polymorphism (rs2279744) and the incidence ofradiotherapy-induced acute and chronic effects were foundthus indicating no major contribution in this cohort ofprostate cancer Similar lack of association was reported byPopanda et al 2009 [30] Another study also showed that theMDM2 SNP309 polymorphism was not found to be involvedwith clinical pathologic variables recurrence risk and overallsurvival outcome in prostate cancer [31]

Many studies have showed the association between theTP53 Pro72Arg polymorphism and cancer susceptibility [32ndash34] An association between the risk of acute skin toxicityand TP53 Pro72 carriers in those with the CDKN1A 31Sergenotype in a subset of normal weight patients treatedwith radiotherapy for breast cancer has been shown [19]Another study found evidence that genetic polymorphismsin the ATM-P53 pathway can influence susceptibility todeveloping radiation-induced pneumonitis in lung cancerpatients after radiotherapy [35] Despite the strong hypothesisfor involvement of this gene the association between thePro72Arg polymorphism and the development of normaltissue reaction to radiotherapy in prostate cancer patientshas not been fully investigated previously This was the firststudy to evaluate the acute and chronic radiation toxicitiesin patients with prostate cancer and TP53 Pro72Arg poly-morphism No association was found in the studied groupeven though 27 (54) patients presented the combinationArgArg which has been described as a more predisposed toapoptosis genotype [16 36]

Another polymorphism in TP53 gene at codon 47(rs1800371) is also functionally significant Codon 47encodes proline in wild type p53 but in a small subset ofindividuals it can encode a serine It was showed that theserine 47 variant has up to 5-fold decreased ability to induceapoptosis compared with wild type p53 [18] However onour study the percentage of patients with this polymorphismwas not statistically sufficient to find a relationship with theadverse effects of radiotherapy even though it is a variantthat induces apoptosis There is no other literature data ofassociation between this polymorphism and toxicity inducedby radiation therapy

Our findings showed that a polymorphism located in theregion 16273 of intron 3 of TP53 (rs35117667) was associatedwith high risk of developing acute skin adverse effects (OR00072 95CI 00002ndash0227119875 = 0003)This polymorphismhas not been investigated by any study so far The presentstudy is the first to observe the frequencies of this polymor-phism in a specific population and also the first to associatethis polymorphism with radiotherapy There is also evidenceof association between the intronic polymorphism at position16250 (rs17883323) and chronic high grade toxicity of urinarytract (OR 0071 95 CI 0006ndash0784 119875 = 0032) Thispolymorphism has not been previously studied in a clinicalpopulation according to searches on scientific websites Bothintronic polymorphisms (rs35117667 and rs17883323) werevalidated by genotype data showing minor alleles in at leasttwo chromosomes [37] It is now necessary to evaluate themolecular basis of the association between these intronicpolymorphisms and the risk for radiotherapy side effects

Disease Markers 709

An intronic polymorphism is not implicated directly in analtered protein however it may lead to alternative splicing orit can be related to MicroRNAs [38ndash40] A protein modifiedby these processesmay be associated for example an increaseof apoptosis

Gemignani and colleagues [41] in 2004 reported that aninsertionduplication of 16 base pairs in intron 3 of TP53 genethe so called TP53 PIN3 (rs17878362) described by Lazar et al[42] (1993) is associated with an increased risk of developingcolorectal cancer (OR 155 95 CI 1 10 to 2 18 119875 = 0012)Tan et al [19] (2006) reported no association between theTP53 PIN3 (rs17878362) insertion and the development ofadverse effects of radiotherapy (119875 gt 005) Our data corro-borate the results of Tan et al [19] (2006) which also foundno association between this polymorphism and the risk oftoxicity after radiotherapy Our study showed that 42 patients(84) had this polymorphism The frequency of the SNPsuggest the importance of a case-control study to assess theassociation between this polymorphism and the developmentof prostate cancer and thus at this stage the functionalsignificance of TP53 PIN3 polymorphism remains poorlyexplored

5 Conclusions

Until now most of studies in this field have been carriedout using candidate genes as was done in the present studyThese studies can provide valuable information howeverthey are limited and have not yet succeeded in providinga predictive test to identify radiosensitivity in patients Inresponse to lack of success in candidate gene studies and thesubsequent recognition that this approach is too limited inscope radiogenomic investigators have now begun a muchbroader search to identify genes and SNPs associated withradiation response Several large-scale genome wide asso-ciation (GWA) studies have been initiated in which radio-therapy patients are being genotyped for large numbers ofcommon SNPs [8 43]

The results of this study hint to a few candidate geneshowever our study was limited by the small sample size andtherefore low statistical power to detect associations Thisstudy shows the importance of genetic investigations as auseful tool for individualization of radiotherapy strategies Itis anticipated that over the next few years SNPs correlatedwith susceptibility for the development of adverse effectsresulting from radiotherapy will be identified from GWAstudies The data provided by studies in radiogenomics willopen new perspectives for interpretation of the results ofcandidate gene studies such as ours and can be the basis forthe development of a predictive assay that may be useful topersonalize and optimize cancer treatment

Acknowledgments

This work was supported by grants from Financiadora deEstudos e Projetos FINEP MCT Brazil and Fundacao deAmparo a Pesquisa do Estado de Goias FAPEG BrazilHellen Cintra had a fellowship from CAPES MEC Brazil

The author would like to thank Dr Alan George Smulian forthe paper revising

References

[1] G C Barnett C M L West A M Dunning et al ldquoNormaltissue reactions to radiotherapy towards tailoring treatmentdose by genotyperdquoNature Reviews Cancer vol 9 no 2 pp 134ndash142 2009

[2] N G Burnet R M Elliott A Dunning and C M L WestldquoRadiosensitivity Radiogenomics and RAPPERrdquo Clinical Onc-ology vol 18 no 7 pp 525ndash528 2006

[3] C M L West A M Dunning and B S Rosenstein ldquoGenome-wide association studies and prediction of normal tissue toxi-cityrdquo Seminars in Radiation Oncology vol 22 no 2 pp 91ndash992012

[4] J A Cesaretti R G Stock S Lehrer et al ldquoATM sequencevariants are predictive of adverse radiotherapy response amongpatients treated for prostate cancerrdquo International Journal ofRadiation Oncology Biology Physics vol 61 no 1 pp 196ndash2022005

[5] J A Cesaretti R G Stock D P Atencio et al ldquoA geneticallydetermined dose-volume histogram predicts for rectal bleedingamong patients treated with prostate brachytherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 68 no5 pp 1410ndash1416 2007

[6] C A Peters R G Stock J A Cesaretti et al ldquoTGFB1 singlenucleotide polymorphisms are associated with adverse qualityof life in prostate cancer patients treated with radiotherapyrdquoInternational Journal of Radiation Oncology Biology Physics vol70 no 3 pp 752ndash759 2008

[7] R J Burri R G Stock J A Cesaretti et al ldquoAssociation of singlenucleotide polymorphisms in SOD2 XRCC1 and XRCC3 withsusceptibility for the development of adverse effects resultingfrom radiotherapy for prostate cancerrdquo Radiation Research vol170 no 1 pp 49ndash59 2008

[8] S L Kerns H Ostrer R Stock et al ldquoGenome-wide associationstudy to identify single nucleotide polymorphisms (SNPs)associated with the development of erectile dysfunction inAfrican-American men after radiotherapy for prostate cancerrdquoInternational Journal of Radiation Oncology Biology Physics vol78 no 5 pp 1292ndash1300 2010

[9] G Iliakis ldquoThe role of DNA double strand breaks in ionizingradiation-induced killing of eukaryotic cellsrdquo BioEssays vol 13no 12 pp 641ndash648 1991

[10] F Altieri C Grillo M Maceroni and S Chichiarelli ldquoDNAdamage and repair from molecular mechanisms to healthimplicationsrdquo Antioxidants and Redox Signaling vol 10 no 5pp 891ndash937 2008

[11] A J Levine and M Oren ldquoThe first 30 years of p53 growingever more complexrdquo Nature Reviews Cancer vol 9 no 10 pp749ndash758 2009

[12] M B Kastan andD-S Lim ldquoThemany substrates and functionsof ATMrdquoNature ReviewsMolecular Cell Biology vol 1 no 3 pp179ndash186 2000

[13] A Tichy J Vavrova J Pejchal and M Rezacova ldquoAtaxia-telangiectasia mutated kinase (ATM) as a central regulator ofradiation-inducedDNAdamage responserdquoActaMedica vol 53no 1 pp 13ndash17 2010

[14] T Soussi and K G Wiman ldquoShaping genetic alterations inhuman cancer the p53 mutation paradigmrdquo Cancer Cell vol12 no 4 pp 303ndash312 2007

710 Disease Markers

[15] Y Levav-Cohen S Haupt and Y Haupt ldquoMdm2 in growthsignaling and cancerrdquoGrowth Factors vol 23 no 3 pp 183ndash1922005

[16] P Dumont J I-J Leu A C Della Pietra III D L George andM Murphy ldquoThe codon 72 polymorphic variants of p53 havemarkedly different apoptotic potentialrdquoNature Genetics vol 33no 3 pp 357ndash365 2003

[17] D D Oslashrsted S E Bojesen A Tybjaeligrg-Hansen and B G Nor-destgaard ldquoTumor suppressor p53 Arg72Pro polymorphismand longevity cancer survival and risk of cancer in the generalpopulationrdquo Journal of Experimental Medicine vol 204 no 6pp 1295ndash1301 2007

[18] X Li P Dumont A Della Pietra G Shetler andM E MurphyldquoThe codon 47 polymorphism in p53 is functionally significantrdquoJournal of Biological Chemistry vol 280 no 25 pp 24245ndash24251 2005

[19] X-L Tan O Popanda C B Ambrosone et al ldquoAssociationbetween TP53 and p21 genetic polymorphisms and acute sideeffects of radiotherapy in breast cancer patientsrdquo Breast CancerResearch and Treatment vol 97 no 3 pp 255ndash262 2006

[20] G L Bond W Hu E E Bond et al ldquoA single nucleotide poly-morphism in the MDM2 promoter attenuates the p53 tumorsuppressor pathway and accelerates tumor formation inhumansrdquo Cell vol 119 no 5 pp 591ndash602 2004

[21] A Y Ho G Fan D P Atencio et al ldquoPossession of ATMsequence variants as predictor for late normal tissue responsesin breast cancer patients treated with radiotherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 69 no3 pp 677ndash684 2007

[22] C E Canman D-S Lim K A Cimprich et al ldquoActivation ofthe ATM kinase by ionizing radiation and phosphorylation ofp53rdquo Science vol 281 no 5383 pp 1677ndash1679 1998

[23] J D Cox J Stetz and T F Pajak ldquoToxicity criteria of the Radi-ation Therapy Oncology Group (RTOG) and the EuropeanOrganization for Research and Treatment of Cancer (EORTC)rdquoInternational Journal of Radiation Oncology Biology Physics vol31 no 5 pp 1341ndash1346 1995

[24] C N Andreassen J Alsner and J Overgaard ldquoDoes variabilityin normal tissue reactions after radiotherapy have a geneticbasismdashwhere and how to look for itrdquo Radiotherapy and Onc-ology vol 64 no 2 pp 131ndash140 2002

[25] S Angele P Romestaing N Moullan et al ldquoATM haplotypesand cellular response to DNA damage association with breastcancer risk and clinical radiosensitivityrdquo Cancer Research vol63 no 24 pp 8717ndash8725 2003

[26] C N Andreassen J Overgaard J Alsner et al ldquoATM sequencevariants and risk of radiation-induced subcutaneous fibrosisafter postmastectomy radiotherapyrdquo International Journal ofRadiation Oncology Biology Physics vol 64 no 3 pp 776ndash7832006

[27] S Damaraju D Murray J Dufour et al ldquoAssociation of DNArepair and steroid metabolism gene polymorphisms with clini-cal late toxicity in patients treated with conformal radiotherapyfor prostate cancerrdquo Clinical Cancer Research vol 12 no 8 pp2545ndash2554 2006

[28] G L Bond W Hu and A Levine ldquoA single nucleotide poly-morphism in the MDM2 gene from a molecular and cellularexplanation to clinical effectrdquo Cancer Research vol 65 no 13pp 5481ndash5484 2005

[29] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[30] O Popanda J UMarquardt J Chang-Claude and P SchmezerldquoGenetic variation in normal tissue toxicity induced by ionizingradiationrdquoMutation Research vol 667 no 1-2 pp 58ndash69 2009

[31] J J Jaboin M Hwang C A Perez et al ldquoNo evidence forassociation of the MDM2-309 TG promoter polymorphismwith prostate cancer outcomesrdquo Urologic Oncology vol 29 no3 pp 319ndash323 2011

[32] L Sreeja V Syamala P B Raveendran S Santhi J Madha-van and R Ankathil ldquop53 Arg72Pro polymorphism predictssurvival outcome in lung cancer patients in Indian populationrdquoCancer Investigation vol 26 no 1 pp 41ndash46 2008

[33] M B Parliament and D Murray ldquoSingle nucleotide polymor-phisms of DNA repair genes as predictors of radioresponserdquoSeminars in Radiation Oncology vol 20 no 4 pp 232ndash2402010

[34] T Pugh M Keyes L Barclay et al ldquoSequence variant discoveryin DNA repair genes from radiosensitive and radiotolerantprostate brachytherapy patientsrdquo Clinical Cancer Research vol15 no 15 pp 5008ndash5016 2009

[35] M Yang L Zhang N Bi et al ldquoAssociation of P53 and ATMpolymorphisms with risk of radiation-induced pneumonitis inlung cancer patients treated with radiotherapyrdquo InternationalJournal of Radiation Oncology Biology Physics vol 79 no 5 pp1402ndash1407 2011

[36] G Alsbeih N Al-Harbi M Al-Buhairi K Al-Hadyan and MAl-Hamed ldquoAssociation between TP53 codon 72 single-nuc-leotide polymorphism and radiation sensitivity of human fibro-blastsrdquo Radiation Research vol 167 no 5 pp 535ndash540 2007

[37] M C Zody M Garber D J Adams et al ldquoDNA sequence ofhuman chromosome 17 and analysis of rearrangement in thehuman lineagerdquoNature vol 440 no 7087 pp 1045ndash1049 2006

[38] H I Suzuki K Yamagata K Sugimoto T Iwamoto S Kato andK Miyazono ldquoModulation of microRNA processing by p53rdquoNature vol 460 no 7254 pp 529ndash533 2009

[39] A K L Leung and P A Sharp ldquomicroRNAs a safeguard againstturmoilrdquo Cell vol 130 no 4 pp 581ndash585 2007

[40] Z Peng Y Cheng B C-M Tan et al ldquoComprehensive analysisof RNA-Seq data reveals extensive RNA editing in a humantranscriptomerdquo Nature Biotechnology vol 30 no 3 pp 253ndash260 2012

[41] F Gemignani VMoreno S Landi et al ldquoATP53 polymorphismis associated with increased risk of colorectal cancer and withreduced levels of TP53 mRNArdquo Oncogene vol 23 no 10 pp1954ndash1956 2004

[42] V Lazar F Hazard F Bertin N Janin D Bellet and B BressacldquoSimple sequence repeat polymorphism within the p53 generdquoOncogene vol 8 no 6 pp 1703ndash1705 1993

[43] CWest B S Rosenstein and J Alsner ldquoEstablishment of a radi-ogenomics consortiumrdquo International Journal of RadiationOnc-ology Biology Physics vol 76 no 5 pp 1295ndash1296 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

706 Disease Markers

Table6As

sociationof

thep

olym

orph

ismsa

nalysedwith

acutes

kin

gastrointestinaland

urinaryradiationtoxicity

Acutes

kinRT

OG

AcuteG

astro

intestinalRT

OG

AcuteU

rinaryTractR

TOG

Gene

(refSN

P)Genotype

n(

)HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

ATM

GG

36(75)

135

531

1719

(rs1801516)

CAlowast

12(25)

111

0314

0018ndash5452

044

12

100806

0135ndash4821

16

60895

0242ndash3307

1TP

53GG

25(52)

223

421

1213

(rs104

2522)

GC

17(35)

017

3723

0168ndash82615

0506

215

1429

0231ndash8840

19

80820

0239ndash

2819

1CC

6(13)

06

1383

0059ndash

32563

11

50952

0086ndash

10489

12

418

460284ndash

11983

066

4TP

53CC

45(96)

243

639

2223

(rs1800371)

CTdagger

2(4)

02

0287

0011ndash7744

11

16500

0357ndash11837

0278

02

4787

0217ndash10539

0491

TP53

CC43

(91)

241

538

1924

(rs17883323)

CAlowast

4(9)

04

0542

0022ndash1315

71

22

0132

0015ndash1153

0100

31

0264

0025ndash2745

0328

TP53

CC41

(89)

041

635

1823

(rs35117667)

CT2(4)

02

mdashmdash

Dagger0

20915

0039ndash

21368

11

10783

004

6ndash1339

91

TT3(7)

21

0007

000

02ndash0

227

000

31

22917

0227ndash37425

0413

21

2556

0214ndash

30469

0583

TP53

A1

40(87)

238

634

2020

(rs17878362)

A2

6(13)

06

0844

0036ndash

19676

11

51133

0112ndash11484

12

40500

0082ndash304

60667

MDM2

TT18

(38)

117

117

99

(rs2279744

)GT

27(56)

126

1529

0089ndash

2613

61

621

0206

0023ndash18

800215

1215

1250

0378ndash

4133

0767

GG

3(6)

03

060

0002ndash179

961

03

060

00020ndash

17996

12

10500

0038ndash6547

1Ab

breviatio

nsrefSN

PreferenceS

NPclu

sterreportRT

OGradiatio

ntherapyo

ncolog

ygroup

HGhighgradeR

TOGLGlow

gradeR

TOGO

Rod

dsratio

CIconfi

denceintervalT

omakec

alculations

ofFishers

exacttestp

ossib

lewhenon

evalue

iszero05was

addedto

thev

alue

lowastNocarriersof

anAAho

mozygou

sgenotypew

erefou

nd

daggerNocarriersof

aTTho

mozygou

sgenotypew

erefou

nd

DaggerNopatient

high

gradefor

theg

enotypes

analysed

Disease Markers 707

Table7As

sociationof

thep

olym

orph

ismsa

nalysedwith

chronics

kin

gastrointestinaland

urinaryradiotoxicitylowast

Chronics

kinRT

OG

Chronicg

astro

intestinalRT

OG

Chronicu

rinarytractR

TOG

Gene

(refSN

P)Genotype

n(

)HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

ATM

GG

35(78)

035

233

827

(rs1801516)

GAlowastlowast

10(22)

19

0089

0003ndash2372

0222

010

1567

0069ndash

3532

31

28

1185

0208ndash6740

1TP

53GG

23(51)

023

221

518

(rs104

2522)

GC

16(36)

115

0220

0008ndash5756

0410

016

3837

0172ndash8553

30503

214

1944

0327ndash11563

0678

CC6(13)

06

mdashmdash

Dagger0

615

12006

4ndash35678

13

30278

004

2ndash18

250305

TP53

CC42 (955)

141

240

933

(rs1800371)

CTdagger

2(45)

02

0181

0058ndash5673

10

20307

0011ndash8325

11

13667

0208ndash64

54

040

7TP

53CC

40(91)

040

238

733

(rs17883323)

CAlowastlowast

4(9)

13

0029

0001ndash0848

0091

04

0584

0024ndash

1419

91

31

0071

000

6ndash0784

0032

TP53

CC38

(88)

137

137

830

(rs35117667)

CT2(5)

02

0200

000

6ndash6288

11

10027

0001ndash0819

0099

02

1393

0061ndash31901

1TT

3(7)

03

0280

000

9ndash8254

10

30280

000

9ndash8254

11

218

750150ndash

2339

60535

TP53

A1

37(86)

136

235

829

(rs17878362)

A2

6(14

)0

60534

0019ndash

14607

10

60915

0039ndash

21368

12

418

130280ndash

11750

0611

MDM2

TT17

(38)

017

116

413

(rs2279744

)GT

25(55)

124

046

70018ndash1215

21

124

1500

0087ndash25753

16

190974

0229ndash

4148

1GG

3(7)

03

mdashmdash

Dagger0

30636

0212ndash1912

41

03

2333

0100ndash

54460

1Ab

breviatio

nsrefSN

PreferenceS

NPclu

sterreportRT

OGradiatio

ntherapyo

ncolog

ygroup

HGhighgradeR

TOGLGlow

gradeR

TOGO

Rod

dsratio

CIconfi

denceintervalT

omakec

alculations

ofFishers

exacttestp

ossib

lewhenon

evalue

iszero05was

addedto

thev

alue

lowastFo

rchron

icanalysesitw

asconsidered

patientsw

ithfollo

wup

high

erthan

24mon

ths

lowastlowastNocarriersof

anAAho

mozygou

sgenotypew

erefou

nd

daggerNocarriersof

aTTho

mozygou

sgenotypew

erefou

nd

DaggerNopatient

high

gradefor

theg

enotypes

analysed

708 Disease Markers

95CI 0006ndash0784119875 = 0032) None of the remaining TP53polymorphisms including the one located on codon 72 weresignificantly associated with the presence of acute or chronicskin gastrointestinal and urinary toxicities (Tables 6 and 7)

4 Discussion

41 Clinical Data Associated with Radiation Toxicity Ouranalysis showed as expected that clinical features were notdeterminants of acute or chronic toxicities of the prostateadjacent tissues when submitted to radiotherapy (119875 gt 005)These results confirm the importance of investigating thegenetic profile of patients undergoing radiotherapy in orderto find if genetic differences are involved or not involvedin the development of radiation toxicities during and aftertreatment

42 Polymorphisms and Radiation Toxicity There is a beliefthat the genomic revolution triggered by full sequencingof the human genome and technological development oflarge-scale methodologies announces the future of person-alized medicine In oncology this progress should increasethe possibility to predict individual patient responses toradiotherapy and chemotherapy Andreassen et al (2002)[24] hypothesized that the radiation sensitivity of normaltissues should be associated with a so-called ldquotrait-dependentcomplexrdquo which adds the effect ofmany genetic determinantsand single nucleotide polymorphisms that could correspondin part to the frequency and severity of these components

Some suggestive evidence was obtained from the cor-relation between the ATM codon Asp1853Asn (5557GgtA)SNP and the high risk of the development of radiotherapy-induced acute skin complications in breast cancer patients[21 25 26] For prostate cancer data are controversial ATMsequence alterations including codon 1853 polymorphismwere associated with the development of radiation-inducedproctitis [5] although no relation was shown between thismarker and bladder or rectal toxicities arising from theradiation therapy in Canadian prostate cancer patients [27]Our findings showed that Asp1853Asn polymorphism wasnot associated with the development of any acute or chronicadverse effects in patients with prostate cancer treated withradiotherapy (119875 gt 005) ATM is a large gene with many vari-ants documented Further investigations will be necessary toverify the association of other ATM SNPs in more repre-sentative sample groups in order to establish whether singlenucleotide polymorphisms or gene haplotypes may actuallycontribute to the toxicity of normal tissue

The single base polymorphism in the promoter ofMDM2gene (SNP309 rs2279744) increases gene expression and it isshown to associate with accelerated tumor formation in bothhereditary and sporadic cancers [20] This SNP creates animproved site of action that regulates basal levels of MDM2The increase of MDM2 basal levels will also increase thedegradation of p53 tumor suppressor protein and may stopits transcriptional activity leading to increased apoptosis [1415 28 29] The hypothesis that increased apoptosis couldcause necrosis of normal tissue leading to radiotherapy sideeffects was not confirmed by our results Among the prostate

cancer patients of this study no association of the investigatedMDM2 polymorphism (rs2279744) and the incidence ofradiotherapy-induced acute and chronic effects were foundthus indicating no major contribution in this cohort ofprostate cancer Similar lack of association was reported byPopanda et al 2009 [30] Another study also showed that theMDM2 SNP309 polymorphism was not found to be involvedwith clinical pathologic variables recurrence risk and overallsurvival outcome in prostate cancer [31]

Many studies have showed the association between theTP53 Pro72Arg polymorphism and cancer susceptibility [32ndash34] An association between the risk of acute skin toxicityand TP53 Pro72 carriers in those with the CDKN1A 31Sergenotype in a subset of normal weight patients treatedwith radiotherapy for breast cancer has been shown [19]Another study found evidence that genetic polymorphismsin the ATM-P53 pathway can influence susceptibility todeveloping radiation-induced pneumonitis in lung cancerpatients after radiotherapy [35] Despite the strong hypothesisfor involvement of this gene the association between thePro72Arg polymorphism and the development of normaltissue reaction to radiotherapy in prostate cancer patientshas not been fully investigated previously This was the firststudy to evaluate the acute and chronic radiation toxicitiesin patients with prostate cancer and TP53 Pro72Arg poly-morphism No association was found in the studied groupeven though 27 (54) patients presented the combinationArgArg which has been described as a more predisposed toapoptosis genotype [16 36]

Another polymorphism in TP53 gene at codon 47(rs1800371) is also functionally significant Codon 47encodes proline in wild type p53 but in a small subset ofindividuals it can encode a serine It was showed that theserine 47 variant has up to 5-fold decreased ability to induceapoptosis compared with wild type p53 [18] However onour study the percentage of patients with this polymorphismwas not statistically sufficient to find a relationship with theadverse effects of radiotherapy even though it is a variantthat induces apoptosis There is no other literature data ofassociation between this polymorphism and toxicity inducedby radiation therapy

Our findings showed that a polymorphism located in theregion 16273 of intron 3 of TP53 (rs35117667) was associatedwith high risk of developing acute skin adverse effects (OR00072 95CI 00002ndash0227119875 = 0003)This polymorphismhas not been investigated by any study so far The presentstudy is the first to observe the frequencies of this polymor-phism in a specific population and also the first to associatethis polymorphism with radiotherapy There is also evidenceof association between the intronic polymorphism at position16250 (rs17883323) and chronic high grade toxicity of urinarytract (OR 0071 95 CI 0006ndash0784 119875 = 0032) Thispolymorphism has not been previously studied in a clinicalpopulation according to searches on scientific websites Bothintronic polymorphisms (rs35117667 and rs17883323) werevalidated by genotype data showing minor alleles in at leasttwo chromosomes [37] It is now necessary to evaluate themolecular basis of the association between these intronicpolymorphisms and the risk for radiotherapy side effects

Disease Markers 709

An intronic polymorphism is not implicated directly in analtered protein however it may lead to alternative splicing orit can be related to MicroRNAs [38ndash40] A protein modifiedby these processesmay be associated for example an increaseof apoptosis

Gemignani and colleagues [41] in 2004 reported that aninsertionduplication of 16 base pairs in intron 3 of TP53 genethe so called TP53 PIN3 (rs17878362) described by Lazar et al[42] (1993) is associated with an increased risk of developingcolorectal cancer (OR 155 95 CI 1 10 to 2 18 119875 = 0012)Tan et al [19] (2006) reported no association between theTP53 PIN3 (rs17878362) insertion and the development ofadverse effects of radiotherapy (119875 gt 005) Our data corro-borate the results of Tan et al [19] (2006) which also foundno association between this polymorphism and the risk oftoxicity after radiotherapy Our study showed that 42 patients(84) had this polymorphism The frequency of the SNPsuggest the importance of a case-control study to assess theassociation between this polymorphism and the developmentof prostate cancer and thus at this stage the functionalsignificance of TP53 PIN3 polymorphism remains poorlyexplored

5 Conclusions

Until now most of studies in this field have been carriedout using candidate genes as was done in the present studyThese studies can provide valuable information howeverthey are limited and have not yet succeeded in providinga predictive test to identify radiosensitivity in patients Inresponse to lack of success in candidate gene studies and thesubsequent recognition that this approach is too limited inscope radiogenomic investigators have now begun a muchbroader search to identify genes and SNPs associated withradiation response Several large-scale genome wide asso-ciation (GWA) studies have been initiated in which radio-therapy patients are being genotyped for large numbers ofcommon SNPs [8 43]

The results of this study hint to a few candidate geneshowever our study was limited by the small sample size andtherefore low statistical power to detect associations Thisstudy shows the importance of genetic investigations as auseful tool for individualization of radiotherapy strategies Itis anticipated that over the next few years SNPs correlatedwith susceptibility for the development of adverse effectsresulting from radiotherapy will be identified from GWAstudies The data provided by studies in radiogenomics willopen new perspectives for interpretation of the results ofcandidate gene studies such as ours and can be the basis forthe development of a predictive assay that may be useful topersonalize and optimize cancer treatment

Acknowledgments

This work was supported by grants from Financiadora deEstudos e Projetos FINEP MCT Brazil and Fundacao deAmparo a Pesquisa do Estado de Goias FAPEG BrazilHellen Cintra had a fellowship from CAPES MEC Brazil

The author would like to thank Dr Alan George Smulian forthe paper revising

References

[1] G C Barnett C M L West A M Dunning et al ldquoNormaltissue reactions to radiotherapy towards tailoring treatmentdose by genotyperdquoNature Reviews Cancer vol 9 no 2 pp 134ndash142 2009

[2] N G Burnet R M Elliott A Dunning and C M L WestldquoRadiosensitivity Radiogenomics and RAPPERrdquo Clinical Onc-ology vol 18 no 7 pp 525ndash528 2006

[3] C M L West A M Dunning and B S Rosenstein ldquoGenome-wide association studies and prediction of normal tissue toxi-cityrdquo Seminars in Radiation Oncology vol 22 no 2 pp 91ndash992012

[4] J A Cesaretti R G Stock S Lehrer et al ldquoATM sequencevariants are predictive of adverse radiotherapy response amongpatients treated for prostate cancerrdquo International Journal ofRadiation Oncology Biology Physics vol 61 no 1 pp 196ndash2022005

[5] J A Cesaretti R G Stock D P Atencio et al ldquoA geneticallydetermined dose-volume histogram predicts for rectal bleedingamong patients treated with prostate brachytherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 68 no5 pp 1410ndash1416 2007

[6] C A Peters R G Stock J A Cesaretti et al ldquoTGFB1 singlenucleotide polymorphisms are associated with adverse qualityof life in prostate cancer patients treated with radiotherapyrdquoInternational Journal of Radiation Oncology Biology Physics vol70 no 3 pp 752ndash759 2008

[7] R J Burri R G Stock J A Cesaretti et al ldquoAssociation of singlenucleotide polymorphisms in SOD2 XRCC1 and XRCC3 withsusceptibility for the development of adverse effects resultingfrom radiotherapy for prostate cancerrdquo Radiation Research vol170 no 1 pp 49ndash59 2008

[8] S L Kerns H Ostrer R Stock et al ldquoGenome-wide associationstudy to identify single nucleotide polymorphisms (SNPs)associated with the development of erectile dysfunction inAfrican-American men after radiotherapy for prostate cancerrdquoInternational Journal of Radiation Oncology Biology Physics vol78 no 5 pp 1292ndash1300 2010

[9] G Iliakis ldquoThe role of DNA double strand breaks in ionizingradiation-induced killing of eukaryotic cellsrdquo BioEssays vol 13no 12 pp 641ndash648 1991

[10] F Altieri C Grillo M Maceroni and S Chichiarelli ldquoDNAdamage and repair from molecular mechanisms to healthimplicationsrdquo Antioxidants and Redox Signaling vol 10 no 5pp 891ndash937 2008

[11] A J Levine and M Oren ldquoThe first 30 years of p53 growingever more complexrdquo Nature Reviews Cancer vol 9 no 10 pp749ndash758 2009

[12] M B Kastan andD-S Lim ldquoThemany substrates and functionsof ATMrdquoNature ReviewsMolecular Cell Biology vol 1 no 3 pp179ndash186 2000

[13] A Tichy J Vavrova J Pejchal and M Rezacova ldquoAtaxia-telangiectasia mutated kinase (ATM) as a central regulator ofradiation-inducedDNAdamage responserdquoActaMedica vol 53no 1 pp 13ndash17 2010

[14] T Soussi and K G Wiman ldquoShaping genetic alterations inhuman cancer the p53 mutation paradigmrdquo Cancer Cell vol12 no 4 pp 303ndash312 2007

710 Disease Markers

[15] Y Levav-Cohen S Haupt and Y Haupt ldquoMdm2 in growthsignaling and cancerrdquoGrowth Factors vol 23 no 3 pp 183ndash1922005

[16] P Dumont J I-J Leu A C Della Pietra III D L George andM Murphy ldquoThe codon 72 polymorphic variants of p53 havemarkedly different apoptotic potentialrdquoNature Genetics vol 33no 3 pp 357ndash365 2003

[17] D D Oslashrsted S E Bojesen A Tybjaeligrg-Hansen and B G Nor-destgaard ldquoTumor suppressor p53 Arg72Pro polymorphismand longevity cancer survival and risk of cancer in the generalpopulationrdquo Journal of Experimental Medicine vol 204 no 6pp 1295ndash1301 2007

[18] X Li P Dumont A Della Pietra G Shetler andM E MurphyldquoThe codon 47 polymorphism in p53 is functionally significantrdquoJournal of Biological Chemistry vol 280 no 25 pp 24245ndash24251 2005

[19] X-L Tan O Popanda C B Ambrosone et al ldquoAssociationbetween TP53 and p21 genetic polymorphisms and acute sideeffects of radiotherapy in breast cancer patientsrdquo Breast CancerResearch and Treatment vol 97 no 3 pp 255ndash262 2006

[20] G L Bond W Hu E E Bond et al ldquoA single nucleotide poly-morphism in the MDM2 promoter attenuates the p53 tumorsuppressor pathway and accelerates tumor formation inhumansrdquo Cell vol 119 no 5 pp 591ndash602 2004

[21] A Y Ho G Fan D P Atencio et al ldquoPossession of ATMsequence variants as predictor for late normal tissue responsesin breast cancer patients treated with radiotherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 69 no3 pp 677ndash684 2007

[22] C E Canman D-S Lim K A Cimprich et al ldquoActivation ofthe ATM kinase by ionizing radiation and phosphorylation ofp53rdquo Science vol 281 no 5383 pp 1677ndash1679 1998

[23] J D Cox J Stetz and T F Pajak ldquoToxicity criteria of the Radi-ation Therapy Oncology Group (RTOG) and the EuropeanOrganization for Research and Treatment of Cancer (EORTC)rdquoInternational Journal of Radiation Oncology Biology Physics vol31 no 5 pp 1341ndash1346 1995

[24] C N Andreassen J Alsner and J Overgaard ldquoDoes variabilityin normal tissue reactions after radiotherapy have a geneticbasismdashwhere and how to look for itrdquo Radiotherapy and Onc-ology vol 64 no 2 pp 131ndash140 2002

[25] S Angele P Romestaing N Moullan et al ldquoATM haplotypesand cellular response to DNA damage association with breastcancer risk and clinical radiosensitivityrdquo Cancer Research vol63 no 24 pp 8717ndash8725 2003

[26] C N Andreassen J Overgaard J Alsner et al ldquoATM sequencevariants and risk of radiation-induced subcutaneous fibrosisafter postmastectomy radiotherapyrdquo International Journal ofRadiation Oncology Biology Physics vol 64 no 3 pp 776ndash7832006

[27] S Damaraju D Murray J Dufour et al ldquoAssociation of DNArepair and steroid metabolism gene polymorphisms with clini-cal late toxicity in patients treated with conformal radiotherapyfor prostate cancerrdquo Clinical Cancer Research vol 12 no 8 pp2545ndash2554 2006

[28] G L Bond W Hu and A Levine ldquoA single nucleotide poly-morphism in the MDM2 gene from a molecular and cellularexplanation to clinical effectrdquo Cancer Research vol 65 no 13pp 5481ndash5484 2005

[29] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[30] O Popanda J UMarquardt J Chang-Claude and P SchmezerldquoGenetic variation in normal tissue toxicity induced by ionizingradiationrdquoMutation Research vol 667 no 1-2 pp 58ndash69 2009

[31] J J Jaboin M Hwang C A Perez et al ldquoNo evidence forassociation of the MDM2-309 TG promoter polymorphismwith prostate cancer outcomesrdquo Urologic Oncology vol 29 no3 pp 319ndash323 2011

[32] L Sreeja V Syamala P B Raveendran S Santhi J Madha-van and R Ankathil ldquop53 Arg72Pro polymorphism predictssurvival outcome in lung cancer patients in Indian populationrdquoCancer Investigation vol 26 no 1 pp 41ndash46 2008

[33] M B Parliament and D Murray ldquoSingle nucleotide polymor-phisms of DNA repair genes as predictors of radioresponserdquoSeminars in Radiation Oncology vol 20 no 4 pp 232ndash2402010

[34] T Pugh M Keyes L Barclay et al ldquoSequence variant discoveryin DNA repair genes from radiosensitive and radiotolerantprostate brachytherapy patientsrdquo Clinical Cancer Research vol15 no 15 pp 5008ndash5016 2009

[35] M Yang L Zhang N Bi et al ldquoAssociation of P53 and ATMpolymorphisms with risk of radiation-induced pneumonitis inlung cancer patients treated with radiotherapyrdquo InternationalJournal of Radiation Oncology Biology Physics vol 79 no 5 pp1402ndash1407 2011

[36] G Alsbeih N Al-Harbi M Al-Buhairi K Al-Hadyan and MAl-Hamed ldquoAssociation between TP53 codon 72 single-nuc-leotide polymorphism and radiation sensitivity of human fibro-blastsrdquo Radiation Research vol 167 no 5 pp 535ndash540 2007

[37] M C Zody M Garber D J Adams et al ldquoDNA sequence ofhuman chromosome 17 and analysis of rearrangement in thehuman lineagerdquoNature vol 440 no 7087 pp 1045ndash1049 2006

[38] H I Suzuki K Yamagata K Sugimoto T Iwamoto S Kato andK Miyazono ldquoModulation of microRNA processing by p53rdquoNature vol 460 no 7254 pp 529ndash533 2009

[39] A K L Leung and P A Sharp ldquomicroRNAs a safeguard againstturmoilrdquo Cell vol 130 no 4 pp 581ndash585 2007

[40] Z Peng Y Cheng B C-M Tan et al ldquoComprehensive analysisof RNA-Seq data reveals extensive RNA editing in a humantranscriptomerdquo Nature Biotechnology vol 30 no 3 pp 253ndash260 2012

[41] F Gemignani VMoreno S Landi et al ldquoATP53 polymorphismis associated with increased risk of colorectal cancer and withreduced levels of TP53 mRNArdquo Oncogene vol 23 no 10 pp1954ndash1956 2004

[42] V Lazar F Hazard F Bertin N Janin D Bellet and B BressacldquoSimple sequence repeat polymorphism within the p53 generdquoOncogene vol 8 no 6 pp 1703ndash1705 1993

[43] CWest B S Rosenstein and J Alsner ldquoEstablishment of a radi-ogenomics consortiumrdquo International Journal of RadiationOnc-ology Biology Physics vol 76 no 5 pp 1295ndash1296 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 707

Table7As

sociationof

thep

olym

orph

ismsa

nalysedwith

chronics

kin

gastrointestinaland

urinaryradiotoxicitylowast

Chronics

kinRT

OG

Chronicg

astro

intestinalRT

OG

Chronicu

rinarytractR

TOG

Gene

(refSN

P)Genotype

n(

)HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

HG

LGOR

95CI

Pvalue

ATM

GG

35(78)

035

233

827

(rs1801516)

GAlowastlowast

10(22)

19

0089

0003ndash2372

0222

010

1567

0069ndash

3532

31

28

1185

0208ndash6740

1TP

53GG

23(51)

023

221

518

(rs104

2522)

GC

16(36)

115

0220

0008ndash5756

0410

016

3837

0172ndash8553

30503

214

1944

0327ndash11563

0678

CC6(13)

06

mdashmdash

Dagger0

615

12006

4ndash35678

13

30278

004

2ndash18

250305

TP53

CC42 (955)

141

240

933

(rs1800371)

CTdagger

2(45)

02

0181

0058ndash5673

10

20307

0011ndash8325

11

13667

0208ndash64

54

040

7TP

53CC

40(91)

040

238

733

(rs17883323)

CAlowastlowast

4(9)

13

0029

0001ndash0848

0091

04

0584

0024ndash

1419

91

31

0071

000

6ndash0784

0032

TP53

CC38

(88)

137

137

830

(rs35117667)

CT2(5)

02

0200

000

6ndash6288

11

10027

0001ndash0819

0099

02

1393

0061ndash31901

1TT

3(7)

03

0280

000

9ndash8254

10

30280

000

9ndash8254

11

218

750150ndash

2339

60535

TP53

A1

37(86)

136

235

829

(rs17878362)

A2

6(14

)0

60534

0019ndash

14607

10

60915

0039ndash

21368

12

418

130280ndash

11750

0611

MDM2

TT17

(38)

017

116

413

(rs2279744

)GT

25(55)

124

046

70018ndash1215

21

124

1500

0087ndash25753

16

190974

0229ndash

4148

1GG

3(7)

03

mdashmdash

Dagger0

30636

0212ndash1912

41

03

2333

0100ndash

54460

1Ab

breviatio

nsrefSN

PreferenceS

NPclu

sterreportRT

OGradiatio

ntherapyo

ncolog

ygroup

HGhighgradeR

TOGLGlow

gradeR

TOGO

Rod

dsratio

CIconfi

denceintervalT

omakec

alculations

ofFishers

exacttestp

ossib

lewhenon

evalue

iszero05was

addedto

thev

alue

lowastFo

rchron

icanalysesitw

asconsidered

patientsw

ithfollo

wup

high

erthan

24mon

ths

lowastlowastNocarriersof

anAAho

mozygou

sgenotypew

erefou

nd

daggerNocarriersof

aTTho

mozygou

sgenotypew

erefou

nd

DaggerNopatient

high

gradefor

theg

enotypes

analysed

708 Disease Markers

95CI 0006ndash0784119875 = 0032) None of the remaining TP53polymorphisms including the one located on codon 72 weresignificantly associated with the presence of acute or chronicskin gastrointestinal and urinary toxicities (Tables 6 and 7)

4 Discussion

41 Clinical Data Associated with Radiation Toxicity Ouranalysis showed as expected that clinical features were notdeterminants of acute or chronic toxicities of the prostateadjacent tissues when submitted to radiotherapy (119875 gt 005)These results confirm the importance of investigating thegenetic profile of patients undergoing radiotherapy in orderto find if genetic differences are involved or not involvedin the development of radiation toxicities during and aftertreatment

42 Polymorphisms and Radiation Toxicity There is a beliefthat the genomic revolution triggered by full sequencingof the human genome and technological development oflarge-scale methodologies announces the future of person-alized medicine In oncology this progress should increasethe possibility to predict individual patient responses toradiotherapy and chemotherapy Andreassen et al (2002)[24] hypothesized that the radiation sensitivity of normaltissues should be associated with a so-called ldquotrait-dependentcomplexrdquo which adds the effect ofmany genetic determinantsand single nucleotide polymorphisms that could correspondin part to the frequency and severity of these components

Some suggestive evidence was obtained from the cor-relation between the ATM codon Asp1853Asn (5557GgtA)SNP and the high risk of the development of radiotherapy-induced acute skin complications in breast cancer patients[21 25 26] For prostate cancer data are controversial ATMsequence alterations including codon 1853 polymorphismwere associated with the development of radiation-inducedproctitis [5] although no relation was shown between thismarker and bladder or rectal toxicities arising from theradiation therapy in Canadian prostate cancer patients [27]Our findings showed that Asp1853Asn polymorphism wasnot associated with the development of any acute or chronicadverse effects in patients with prostate cancer treated withradiotherapy (119875 gt 005) ATM is a large gene with many vari-ants documented Further investigations will be necessary toverify the association of other ATM SNPs in more repre-sentative sample groups in order to establish whether singlenucleotide polymorphisms or gene haplotypes may actuallycontribute to the toxicity of normal tissue

The single base polymorphism in the promoter ofMDM2gene (SNP309 rs2279744) increases gene expression and it isshown to associate with accelerated tumor formation in bothhereditary and sporadic cancers [20] This SNP creates animproved site of action that regulates basal levels of MDM2The increase of MDM2 basal levels will also increase thedegradation of p53 tumor suppressor protein and may stopits transcriptional activity leading to increased apoptosis [1415 28 29] The hypothesis that increased apoptosis couldcause necrosis of normal tissue leading to radiotherapy sideeffects was not confirmed by our results Among the prostate

cancer patients of this study no association of the investigatedMDM2 polymorphism (rs2279744) and the incidence ofradiotherapy-induced acute and chronic effects were foundthus indicating no major contribution in this cohort ofprostate cancer Similar lack of association was reported byPopanda et al 2009 [30] Another study also showed that theMDM2 SNP309 polymorphism was not found to be involvedwith clinical pathologic variables recurrence risk and overallsurvival outcome in prostate cancer [31]

Many studies have showed the association between theTP53 Pro72Arg polymorphism and cancer susceptibility [32ndash34] An association between the risk of acute skin toxicityand TP53 Pro72 carriers in those with the CDKN1A 31Sergenotype in a subset of normal weight patients treatedwith radiotherapy for breast cancer has been shown [19]Another study found evidence that genetic polymorphismsin the ATM-P53 pathway can influence susceptibility todeveloping radiation-induced pneumonitis in lung cancerpatients after radiotherapy [35] Despite the strong hypothesisfor involvement of this gene the association between thePro72Arg polymorphism and the development of normaltissue reaction to radiotherapy in prostate cancer patientshas not been fully investigated previously This was the firststudy to evaluate the acute and chronic radiation toxicitiesin patients with prostate cancer and TP53 Pro72Arg poly-morphism No association was found in the studied groupeven though 27 (54) patients presented the combinationArgArg which has been described as a more predisposed toapoptosis genotype [16 36]

Another polymorphism in TP53 gene at codon 47(rs1800371) is also functionally significant Codon 47encodes proline in wild type p53 but in a small subset ofindividuals it can encode a serine It was showed that theserine 47 variant has up to 5-fold decreased ability to induceapoptosis compared with wild type p53 [18] However onour study the percentage of patients with this polymorphismwas not statistically sufficient to find a relationship with theadverse effects of radiotherapy even though it is a variantthat induces apoptosis There is no other literature data ofassociation between this polymorphism and toxicity inducedby radiation therapy

Our findings showed that a polymorphism located in theregion 16273 of intron 3 of TP53 (rs35117667) was associatedwith high risk of developing acute skin adverse effects (OR00072 95CI 00002ndash0227119875 = 0003)This polymorphismhas not been investigated by any study so far The presentstudy is the first to observe the frequencies of this polymor-phism in a specific population and also the first to associatethis polymorphism with radiotherapy There is also evidenceof association between the intronic polymorphism at position16250 (rs17883323) and chronic high grade toxicity of urinarytract (OR 0071 95 CI 0006ndash0784 119875 = 0032) Thispolymorphism has not been previously studied in a clinicalpopulation according to searches on scientific websites Bothintronic polymorphisms (rs35117667 and rs17883323) werevalidated by genotype data showing minor alleles in at leasttwo chromosomes [37] It is now necessary to evaluate themolecular basis of the association between these intronicpolymorphisms and the risk for radiotherapy side effects

Disease Markers 709

An intronic polymorphism is not implicated directly in analtered protein however it may lead to alternative splicing orit can be related to MicroRNAs [38ndash40] A protein modifiedby these processesmay be associated for example an increaseof apoptosis

Gemignani and colleagues [41] in 2004 reported that aninsertionduplication of 16 base pairs in intron 3 of TP53 genethe so called TP53 PIN3 (rs17878362) described by Lazar et al[42] (1993) is associated with an increased risk of developingcolorectal cancer (OR 155 95 CI 1 10 to 2 18 119875 = 0012)Tan et al [19] (2006) reported no association between theTP53 PIN3 (rs17878362) insertion and the development ofadverse effects of radiotherapy (119875 gt 005) Our data corro-borate the results of Tan et al [19] (2006) which also foundno association between this polymorphism and the risk oftoxicity after radiotherapy Our study showed that 42 patients(84) had this polymorphism The frequency of the SNPsuggest the importance of a case-control study to assess theassociation between this polymorphism and the developmentof prostate cancer and thus at this stage the functionalsignificance of TP53 PIN3 polymorphism remains poorlyexplored

5 Conclusions

Until now most of studies in this field have been carriedout using candidate genes as was done in the present studyThese studies can provide valuable information howeverthey are limited and have not yet succeeded in providinga predictive test to identify radiosensitivity in patients Inresponse to lack of success in candidate gene studies and thesubsequent recognition that this approach is too limited inscope radiogenomic investigators have now begun a muchbroader search to identify genes and SNPs associated withradiation response Several large-scale genome wide asso-ciation (GWA) studies have been initiated in which radio-therapy patients are being genotyped for large numbers ofcommon SNPs [8 43]

The results of this study hint to a few candidate geneshowever our study was limited by the small sample size andtherefore low statistical power to detect associations Thisstudy shows the importance of genetic investigations as auseful tool for individualization of radiotherapy strategies Itis anticipated that over the next few years SNPs correlatedwith susceptibility for the development of adverse effectsresulting from radiotherapy will be identified from GWAstudies The data provided by studies in radiogenomics willopen new perspectives for interpretation of the results ofcandidate gene studies such as ours and can be the basis forthe development of a predictive assay that may be useful topersonalize and optimize cancer treatment

Acknowledgments

This work was supported by grants from Financiadora deEstudos e Projetos FINEP MCT Brazil and Fundacao deAmparo a Pesquisa do Estado de Goias FAPEG BrazilHellen Cintra had a fellowship from CAPES MEC Brazil

The author would like to thank Dr Alan George Smulian forthe paper revising

References

[1] G C Barnett C M L West A M Dunning et al ldquoNormaltissue reactions to radiotherapy towards tailoring treatmentdose by genotyperdquoNature Reviews Cancer vol 9 no 2 pp 134ndash142 2009

[2] N G Burnet R M Elliott A Dunning and C M L WestldquoRadiosensitivity Radiogenomics and RAPPERrdquo Clinical Onc-ology vol 18 no 7 pp 525ndash528 2006

[3] C M L West A M Dunning and B S Rosenstein ldquoGenome-wide association studies and prediction of normal tissue toxi-cityrdquo Seminars in Radiation Oncology vol 22 no 2 pp 91ndash992012

[4] J A Cesaretti R G Stock S Lehrer et al ldquoATM sequencevariants are predictive of adverse radiotherapy response amongpatients treated for prostate cancerrdquo International Journal ofRadiation Oncology Biology Physics vol 61 no 1 pp 196ndash2022005

[5] J A Cesaretti R G Stock D P Atencio et al ldquoA geneticallydetermined dose-volume histogram predicts for rectal bleedingamong patients treated with prostate brachytherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 68 no5 pp 1410ndash1416 2007

[6] C A Peters R G Stock J A Cesaretti et al ldquoTGFB1 singlenucleotide polymorphisms are associated with adverse qualityof life in prostate cancer patients treated with radiotherapyrdquoInternational Journal of Radiation Oncology Biology Physics vol70 no 3 pp 752ndash759 2008

[7] R J Burri R G Stock J A Cesaretti et al ldquoAssociation of singlenucleotide polymorphisms in SOD2 XRCC1 and XRCC3 withsusceptibility for the development of adverse effects resultingfrom radiotherapy for prostate cancerrdquo Radiation Research vol170 no 1 pp 49ndash59 2008

[8] S L Kerns H Ostrer R Stock et al ldquoGenome-wide associationstudy to identify single nucleotide polymorphisms (SNPs)associated with the development of erectile dysfunction inAfrican-American men after radiotherapy for prostate cancerrdquoInternational Journal of Radiation Oncology Biology Physics vol78 no 5 pp 1292ndash1300 2010

[9] G Iliakis ldquoThe role of DNA double strand breaks in ionizingradiation-induced killing of eukaryotic cellsrdquo BioEssays vol 13no 12 pp 641ndash648 1991

[10] F Altieri C Grillo M Maceroni and S Chichiarelli ldquoDNAdamage and repair from molecular mechanisms to healthimplicationsrdquo Antioxidants and Redox Signaling vol 10 no 5pp 891ndash937 2008

[11] A J Levine and M Oren ldquoThe first 30 years of p53 growingever more complexrdquo Nature Reviews Cancer vol 9 no 10 pp749ndash758 2009

[12] M B Kastan andD-S Lim ldquoThemany substrates and functionsof ATMrdquoNature ReviewsMolecular Cell Biology vol 1 no 3 pp179ndash186 2000

[13] A Tichy J Vavrova J Pejchal and M Rezacova ldquoAtaxia-telangiectasia mutated kinase (ATM) as a central regulator ofradiation-inducedDNAdamage responserdquoActaMedica vol 53no 1 pp 13ndash17 2010

[14] T Soussi and K G Wiman ldquoShaping genetic alterations inhuman cancer the p53 mutation paradigmrdquo Cancer Cell vol12 no 4 pp 303ndash312 2007

710 Disease Markers

[15] Y Levav-Cohen S Haupt and Y Haupt ldquoMdm2 in growthsignaling and cancerrdquoGrowth Factors vol 23 no 3 pp 183ndash1922005

[16] P Dumont J I-J Leu A C Della Pietra III D L George andM Murphy ldquoThe codon 72 polymorphic variants of p53 havemarkedly different apoptotic potentialrdquoNature Genetics vol 33no 3 pp 357ndash365 2003

[17] D D Oslashrsted S E Bojesen A Tybjaeligrg-Hansen and B G Nor-destgaard ldquoTumor suppressor p53 Arg72Pro polymorphismand longevity cancer survival and risk of cancer in the generalpopulationrdquo Journal of Experimental Medicine vol 204 no 6pp 1295ndash1301 2007

[18] X Li P Dumont A Della Pietra G Shetler andM E MurphyldquoThe codon 47 polymorphism in p53 is functionally significantrdquoJournal of Biological Chemistry vol 280 no 25 pp 24245ndash24251 2005

[19] X-L Tan O Popanda C B Ambrosone et al ldquoAssociationbetween TP53 and p21 genetic polymorphisms and acute sideeffects of radiotherapy in breast cancer patientsrdquo Breast CancerResearch and Treatment vol 97 no 3 pp 255ndash262 2006

[20] G L Bond W Hu E E Bond et al ldquoA single nucleotide poly-morphism in the MDM2 promoter attenuates the p53 tumorsuppressor pathway and accelerates tumor formation inhumansrdquo Cell vol 119 no 5 pp 591ndash602 2004

[21] A Y Ho G Fan D P Atencio et al ldquoPossession of ATMsequence variants as predictor for late normal tissue responsesin breast cancer patients treated with radiotherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 69 no3 pp 677ndash684 2007

[22] C E Canman D-S Lim K A Cimprich et al ldquoActivation ofthe ATM kinase by ionizing radiation and phosphorylation ofp53rdquo Science vol 281 no 5383 pp 1677ndash1679 1998

[23] J D Cox J Stetz and T F Pajak ldquoToxicity criteria of the Radi-ation Therapy Oncology Group (RTOG) and the EuropeanOrganization for Research and Treatment of Cancer (EORTC)rdquoInternational Journal of Radiation Oncology Biology Physics vol31 no 5 pp 1341ndash1346 1995

[24] C N Andreassen J Alsner and J Overgaard ldquoDoes variabilityin normal tissue reactions after radiotherapy have a geneticbasismdashwhere and how to look for itrdquo Radiotherapy and Onc-ology vol 64 no 2 pp 131ndash140 2002

[25] S Angele P Romestaing N Moullan et al ldquoATM haplotypesand cellular response to DNA damage association with breastcancer risk and clinical radiosensitivityrdquo Cancer Research vol63 no 24 pp 8717ndash8725 2003

[26] C N Andreassen J Overgaard J Alsner et al ldquoATM sequencevariants and risk of radiation-induced subcutaneous fibrosisafter postmastectomy radiotherapyrdquo International Journal ofRadiation Oncology Biology Physics vol 64 no 3 pp 776ndash7832006

[27] S Damaraju D Murray J Dufour et al ldquoAssociation of DNArepair and steroid metabolism gene polymorphisms with clini-cal late toxicity in patients treated with conformal radiotherapyfor prostate cancerrdquo Clinical Cancer Research vol 12 no 8 pp2545ndash2554 2006

[28] G L Bond W Hu and A Levine ldquoA single nucleotide poly-morphism in the MDM2 gene from a molecular and cellularexplanation to clinical effectrdquo Cancer Research vol 65 no 13pp 5481ndash5484 2005

[29] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[30] O Popanda J UMarquardt J Chang-Claude and P SchmezerldquoGenetic variation in normal tissue toxicity induced by ionizingradiationrdquoMutation Research vol 667 no 1-2 pp 58ndash69 2009

[31] J J Jaboin M Hwang C A Perez et al ldquoNo evidence forassociation of the MDM2-309 TG promoter polymorphismwith prostate cancer outcomesrdquo Urologic Oncology vol 29 no3 pp 319ndash323 2011

[32] L Sreeja V Syamala P B Raveendran S Santhi J Madha-van and R Ankathil ldquop53 Arg72Pro polymorphism predictssurvival outcome in lung cancer patients in Indian populationrdquoCancer Investigation vol 26 no 1 pp 41ndash46 2008

[33] M B Parliament and D Murray ldquoSingle nucleotide polymor-phisms of DNA repair genes as predictors of radioresponserdquoSeminars in Radiation Oncology vol 20 no 4 pp 232ndash2402010

[34] T Pugh M Keyes L Barclay et al ldquoSequence variant discoveryin DNA repair genes from radiosensitive and radiotolerantprostate brachytherapy patientsrdquo Clinical Cancer Research vol15 no 15 pp 5008ndash5016 2009

[35] M Yang L Zhang N Bi et al ldquoAssociation of P53 and ATMpolymorphisms with risk of radiation-induced pneumonitis inlung cancer patients treated with radiotherapyrdquo InternationalJournal of Radiation Oncology Biology Physics vol 79 no 5 pp1402ndash1407 2011

[36] G Alsbeih N Al-Harbi M Al-Buhairi K Al-Hadyan and MAl-Hamed ldquoAssociation between TP53 codon 72 single-nuc-leotide polymorphism and radiation sensitivity of human fibro-blastsrdquo Radiation Research vol 167 no 5 pp 535ndash540 2007

[37] M C Zody M Garber D J Adams et al ldquoDNA sequence ofhuman chromosome 17 and analysis of rearrangement in thehuman lineagerdquoNature vol 440 no 7087 pp 1045ndash1049 2006

[38] H I Suzuki K Yamagata K Sugimoto T Iwamoto S Kato andK Miyazono ldquoModulation of microRNA processing by p53rdquoNature vol 460 no 7254 pp 529ndash533 2009

[39] A K L Leung and P A Sharp ldquomicroRNAs a safeguard againstturmoilrdquo Cell vol 130 no 4 pp 581ndash585 2007

[40] Z Peng Y Cheng B C-M Tan et al ldquoComprehensive analysisof RNA-Seq data reveals extensive RNA editing in a humantranscriptomerdquo Nature Biotechnology vol 30 no 3 pp 253ndash260 2012

[41] F Gemignani VMoreno S Landi et al ldquoATP53 polymorphismis associated with increased risk of colorectal cancer and withreduced levels of TP53 mRNArdquo Oncogene vol 23 no 10 pp1954ndash1956 2004

[42] V Lazar F Hazard F Bertin N Janin D Bellet and B BressacldquoSimple sequence repeat polymorphism within the p53 generdquoOncogene vol 8 no 6 pp 1703ndash1705 1993

[43] CWest B S Rosenstein and J Alsner ldquoEstablishment of a radi-ogenomics consortiumrdquo International Journal of RadiationOnc-ology Biology Physics vol 76 no 5 pp 1295ndash1296 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

708 Disease Markers

95CI 0006ndash0784119875 = 0032) None of the remaining TP53polymorphisms including the one located on codon 72 weresignificantly associated with the presence of acute or chronicskin gastrointestinal and urinary toxicities (Tables 6 and 7)

4 Discussion

41 Clinical Data Associated with Radiation Toxicity Ouranalysis showed as expected that clinical features were notdeterminants of acute or chronic toxicities of the prostateadjacent tissues when submitted to radiotherapy (119875 gt 005)These results confirm the importance of investigating thegenetic profile of patients undergoing radiotherapy in orderto find if genetic differences are involved or not involvedin the development of radiation toxicities during and aftertreatment

42 Polymorphisms and Radiation Toxicity There is a beliefthat the genomic revolution triggered by full sequencingof the human genome and technological development oflarge-scale methodologies announces the future of person-alized medicine In oncology this progress should increasethe possibility to predict individual patient responses toradiotherapy and chemotherapy Andreassen et al (2002)[24] hypothesized that the radiation sensitivity of normaltissues should be associated with a so-called ldquotrait-dependentcomplexrdquo which adds the effect ofmany genetic determinantsand single nucleotide polymorphisms that could correspondin part to the frequency and severity of these components

Some suggestive evidence was obtained from the cor-relation between the ATM codon Asp1853Asn (5557GgtA)SNP and the high risk of the development of radiotherapy-induced acute skin complications in breast cancer patients[21 25 26] For prostate cancer data are controversial ATMsequence alterations including codon 1853 polymorphismwere associated with the development of radiation-inducedproctitis [5] although no relation was shown between thismarker and bladder or rectal toxicities arising from theradiation therapy in Canadian prostate cancer patients [27]Our findings showed that Asp1853Asn polymorphism wasnot associated with the development of any acute or chronicadverse effects in patients with prostate cancer treated withradiotherapy (119875 gt 005) ATM is a large gene with many vari-ants documented Further investigations will be necessary toverify the association of other ATM SNPs in more repre-sentative sample groups in order to establish whether singlenucleotide polymorphisms or gene haplotypes may actuallycontribute to the toxicity of normal tissue

The single base polymorphism in the promoter ofMDM2gene (SNP309 rs2279744) increases gene expression and it isshown to associate with accelerated tumor formation in bothhereditary and sporadic cancers [20] This SNP creates animproved site of action that regulates basal levels of MDM2The increase of MDM2 basal levels will also increase thedegradation of p53 tumor suppressor protein and may stopits transcriptional activity leading to increased apoptosis [1415 28 29] The hypothesis that increased apoptosis couldcause necrosis of normal tissue leading to radiotherapy sideeffects was not confirmed by our results Among the prostate

cancer patients of this study no association of the investigatedMDM2 polymorphism (rs2279744) and the incidence ofradiotherapy-induced acute and chronic effects were foundthus indicating no major contribution in this cohort ofprostate cancer Similar lack of association was reported byPopanda et al 2009 [30] Another study also showed that theMDM2 SNP309 polymorphism was not found to be involvedwith clinical pathologic variables recurrence risk and overallsurvival outcome in prostate cancer [31]

Many studies have showed the association between theTP53 Pro72Arg polymorphism and cancer susceptibility [32ndash34] An association between the risk of acute skin toxicityand TP53 Pro72 carriers in those with the CDKN1A 31Sergenotype in a subset of normal weight patients treatedwith radiotherapy for breast cancer has been shown [19]Another study found evidence that genetic polymorphismsin the ATM-P53 pathway can influence susceptibility todeveloping radiation-induced pneumonitis in lung cancerpatients after radiotherapy [35] Despite the strong hypothesisfor involvement of this gene the association between thePro72Arg polymorphism and the development of normaltissue reaction to radiotherapy in prostate cancer patientshas not been fully investigated previously This was the firststudy to evaluate the acute and chronic radiation toxicitiesin patients with prostate cancer and TP53 Pro72Arg poly-morphism No association was found in the studied groupeven though 27 (54) patients presented the combinationArgArg which has been described as a more predisposed toapoptosis genotype [16 36]

Another polymorphism in TP53 gene at codon 47(rs1800371) is also functionally significant Codon 47encodes proline in wild type p53 but in a small subset ofindividuals it can encode a serine It was showed that theserine 47 variant has up to 5-fold decreased ability to induceapoptosis compared with wild type p53 [18] However onour study the percentage of patients with this polymorphismwas not statistically sufficient to find a relationship with theadverse effects of radiotherapy even though it is a variantthat induces apoptosis There is no other literature data ofassociation between this polymorphism and toxicity inducedby radiation therapy

Our findings showed that a polymorphism located in theregion 16273 of intron 3 of TP53 (rs35117667) was associatedwith high risk of developing acute skin adverse effects (OR00072 95CI 00002ndash0227119875 = 0003)This polymorphismhas not been investigated by any study so far The presentstudy is the first to observe the frequencies of this polymor-phism in a specific population and also the first to associatethis polymorphism with radiotherapy There is also evidenceof association between the intronic polymorphism at position16250 (rs17883323) and chronic high grade toxicity of urinarytract (OR 0071 95 CI 0006ndash0784 119875 = 0032) Thispolymorphism has not been previously studied in a clinicalpopulation according to searches on scientific websites Bothintronic polymorphisms (rs35117667 and rs17883323) werevalidated by genotype data showing minor alleles in at leasttwo chromosomes [37] It is now necessary to evaluate themolecular basis of the association between these intronicpolymorphisms and the risk for radiotherapy side effects

Disease Markers 709

An intronic polymorphism is not implicated directly in analtered protein however it may lead to alternative splicing orit can be related to MicroRNAs [38ndash40] A protein modifiedby these processesmay be associated for example an increaseof apoptosis

Gemignani and colleagues [41] in 2004 reported that aninsertionduplication of 16 base pairs in intron 3 of TP53 genethe so called TP53 PIN3 (rs17878362) described by Lazar et al[42] (1993) is associated with an increased risk of developingcolorectal cancer (OR 155 95 CI 1 10 to 2 18 119875 = 0012)Tan et al [19] (2006) reported no association between theTP53 PIN3 (rs17878362) insertion and the development ofadverse effects of radiotherapy (119875 gt 005) Our data corro-borate the results of Tan et al [19] (2006) which also foundno association between this polymorphism and the risk oftoxicity after radiotherapy Our study showed that 42 patients(84) had this polymorphism The frequency of the SNPsuggest the importance of a case-control study to assess theassociation between this polymorphism and the developmentof prostate cancer and thus at this stage the functionalsignificance of TP53 PIN3 polymorphism remains poorlyexplored

5 Conclusions

Until now most of studies in this field have been carriedout using candidate genes as was done in the present studyThese studies can provide valuable information howeverthey are limited and have not yet succeeded in providinga predictive test to identify radiosensitivity in patients Inresponse to lack of success in candidate gene studies and thesubsequent recognition that this approach is too limited inscope radiogenomic investigators have now begun a muchbroader search to identify genes and SNPs associated withradiation response Several large-scale genome wide asso-ciation (GWA) studies have been initiated in which radio-therapy patients are being genotyped for large numbers ofcommon SNPs [8 43]

The results of this study hint to a few candidate geneshowever our study was limited by the small sample size andtherefore low statistical power to detect associations Thisstudy shows the importance of genetic investigations as auseful tool for individualization of radiotherapy strategies Itis anticipated that over the next few years SNPs correlatedwith susceptibility for the development of adverse effectsresulting from radiotherapy will be identified from GWAstudies The data provided by studies in radiogenomics willopen new perspectives for interpretation of the results ofcandidate gene studies such as ours and can be the basis forthe development of a predictive assay that may be useful topersonalize and optimize cancer treatment

Acknowledgments

This work was supported by grants from Financiadora deEstudos e Projetos FINEP MCT Brazil and Fundacao deAmparo a Pesquisa do Estado de Goias FAPEG BrazilHellen Cintra had a fellowship from CAPES MEC Brazil

The author would like to thank Dr Alan George Smulian forthe paper revising

References

[1] G C Barnett C M L West A M Dunning et al ldquoNormaltissue reactions to radiotherapy towards tailoring treatmentdose by genotyperdquoNature Reviews Cancer vol 9 no 2 pp 134ndash142 2009

[2] N G Burnet R M Elliott A Dunning and C M L WestldquoRadiosensitivity Radiogenomics and RAPPERrdquo Clinical Onc-ology vol 18 no 7 pp 525ndash528 2006

[3] C M L West A M Dunning and B S Rosenstein ldquoGenome-wide association studies and prediction of normal tissue toxi-cityrdquo Seminars in Radiation Oncology vol 22 no 2 pp 91ndash992012

[4] J A Cesaretti R G Stock S Lehrer et al ldquoATM sequencevariants are predictive of adverse radiotherapy response amongpatients treated for prostate cancerrdquo International Journal ofRadiation Oncology Biology Physics vol 61 no 1 pp 196ndash2022005

[5] J A Cesaretti R G Stock D P Atencio et al ldquoA geneticallydetermined dose-volume histogram predicts for rectal bleedingamong patients treated with prostate brachytherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 68 no5 pp 1410ndash1416 2007

[6] C A Peters R G Stock J A Cesaretti et al ldquoTGFB1 singlenucleotide polymorphisms are associated with adverse qualityof life in prostate cancer patients treated with radiotherapyrdquoInternational Journal of Radiation Oncology Biology Physics vol70 no 3 pp 752ndash759 2008

[7] R J Burri R G Stock J A Cesaretti et al ldquoAssociation of singlenucleotide polymorphisms in SOD2 XRCC1 and XRCC3 withsusceptibility for the development of adverse effects resultingfrom radiotherapy for prostate cancerrdquo Radiation Research vol170 no 1 pp 49ndash59 2008

[8] S L Kerns H Ostrer R Stock et al ldquoGenome-wide associationstudy to identify single nucleotide polymorphisms (SNPs)associated with the development of erectile dysfunction inAfrican-American men after radiotherapy for prostate cancerrdquoInternational Journal of Radiation Oncology Biology Physics vol78 no 5 pp 1292ndash1300 2010

[9] G Iliakis ldquoThe role of DNA double strand breaks in ionizingradiation-induced killing of eukaryotic cellsrdquo BioEssays vol 13no 12 pp 641ndash648 1991

[10] F Altieri C Grillo M Maceroni and S Chichiarelli ldquoDNAdamage and repair from molecular mechanisms to healthimplicationsrdquo Antioxidants and Redox Signaling vol 10 no 5pp 891ndash937 2008

[11] A J Levine and M Oren ldquoThe first 30 years of p53 growingever more complexrdquo Nature Reviews Cancer vol 9 no 10 pp749ndash758 2009

[12] M B Kastan andD-S Lim ldquoThemany substrates and functionsof ATMrdquoNature ReviewsMolecular Cell Biology vol 1 no 3 pp179ndash186 2000

[13] A Tichy J Vavrova J Pejchal and M Rezacova ldquoAtaxia-telangiectasia mutated kinase (ATM) as a central regulator ofradiation-inducedDNAdamage responserdquoActaMedica vol 53no 1 pp 13ndash17 2010

[14] T Soussi and K G Wiman ldquoShaping genetic alterations inhuman cancer the p53 mutation paradigmrdquo Cancer Cell vol12 no 4 pp 303ndash312 2007

710 Disease Markers

[15] Y Levav-Cohen S Haupt and Y Haupt ldquoMdm2 in growthsignaling and cancerrdquoGrowth Factors vol 23 no 3 pp 183ndash1922005

[16] P Dumont J I-J Leu A C Della Pietra III D L George andM Murphy ldquoThe codon 72 polymorphic variants of p53 havemarkedly different apoptotic potentialrdquoNature Genetics vol 33no 3 pp 357ndash365 2003

[17] D D Oslashrsted S E Bojesen A Tybjaeligrg-Hansen and B G Nor-destgaard ldquoTumor suppressor p53 Arg72Pro polymorphismand longevity cancer survival and risk of cancer in the generalpopulationrdquo Journal of Experimental Medicine vol 204 no 6pp 1295ndash1301 2007

[18] X Li P Dumont A Della Pietra G Shetler andM E MurphyldquoThe codon 47 polymorphism in p53 is functionally significantrdquoJournal of Biological Chemistry vol 280 no 25 pp 24245ndash24251 2005

[19] X-L Tan O Popanda C B Ambrosone et al ldquoAssociationbetween TP53 and p21 genetic polymorphisms and acute sideeffects of radiotherapy in breast cancer patientsrdquo Breast CancerResearch and Treatment vol 97 no 3 pp 255ndash262 2006

[20] G L Bond W Hu E E Bond et al ldquoA single nucleotide poly-morphism in the MDM2 promoter attenuates the p53 tumorsuppressor pathway and accelerates tumor formation inhumansrdquo Cell vol 119 no 5 pp 591ndash602 2004

[21] A Y Ho G Fan D P Atencio et al ldquoPossession of ATMsequence variants as predictor for late normal tissue responsesin breast cancer patients treated with radiotherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 69 no3 pp 677ndash684 2007

[22] C E Canman D-S Lim K A Cimprich et al ldquoActivation ofthe ATM kinase by ionizing radiation and phosphorylation ofp53rdquo Science vol 281 no 5383 pp 1677ndash1679 1998

[23] J D Cox J Stetz and T F Pajak ldquoToxicity criteria of the Radi-ation Therapy Oncology Group (RTOG) and the EuropeanOrganization for Research and Treatment of Cancer (EORTC)rdquoInternational Journal of Radiation Oncology Biology Physics vol31 no 5 pp 1341ndash1346 1995

[24] C N Andreassen J Alsner and J Overgaard ldquoDoes variabilityin normal tissue reactions after radiotherapy have a geneticbasismdashwhere and how to look for itrdquo Radiotherapy and Onc-ology vol 64 no 2 pp 131ndash140 2002

[25] S Angele P Romestaing N Moullan et al ldquoATM haplotypesand cellular response to DNA damage association with breastcancer risk and clinical radiosensitivityrdquo Cancer Research vol63 no 24 pp 8717ndash8725 2003

[26] C N Andreassen J Overgaard J Alsner et al ldquoATM sequencevariants and risk of radiation-induced subcutaneous fibrosisafter postmastectomy radiotherapyrdquo International Journal ofRadiation Oncology Biology Physics vol 64 no 3 pp 776ndash7832006

[27] S Damaraju D Murray J Dufour et al ldquoAssociation of DNArepair and steroid metabolism gene polymorphisms with clini-cal late toxicity in patients treated with conformal radiotherapyfor prostate cancerrdquo Clinical Cancer Research vol 12 no 8 pp2545ndash2554 2006

[28] G L Bond W Hu and A Levine ldquoA single nucleotide poly-morphism in the MDM2 gene from a molecular and cellularexplanation to clinical effectrdquo Cancer Research vol 65 no 13pp 5481ndash5484 2005

[29] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[30] O Popanda J UMarquardt J Chang-Claude and P SchmezerldquoGenetic variation in normal tissue toxicity induced by ionizingradiationrdquoMutation Research vol 667 no 1-2 pp 58ndash69 2009

[31] J J Jaboin M Hwang C A Perez et al ldquoNo evidence forassociation of the MDM2-309 TG promoter polymorphismwith prostate cancer outcomesrdquo Urologic Oncology vol 29 no3 pp 319ndash323 2011

[32] L Sreeja V Syamala P B Raveendran S Santhi J Madha-van and R Ankathil ldquop53 Arg72Pro polymorphism predictssurvival outcome in lung cancer patients in Indian populationrdquoCancer Investigation vol 26 no 1 pp 41ndash46 2008

[33] M B Parliament and D Murray ldquoSingle nucleotide polymor-phisms of DNA repair genes as predictors of radioresponserdquoSeminars in Radiation Oncology vol 20 no 4 pp 232ndash2402010

[34] T Pugh M Keyes L Barclay et al ldquoSequence variant discoveryin DNA repair genes from radiosensitive and radiotolerantprostate brachytherapy patientsrdquo Clinical Cancer Research vol15 no 15 pp 5008ndash5016 2009

[35] M Yang L Zhang N Bi et al ldquoAssociation of P53 and ATMpolymorphisms with risk of radiation-induced pneumonitis inlung cancer patients treated with radiotherapyrdquo InternationalJournal of Radiation Oncology Biology Physics vol 79 no 5 pp1402ndash1407 2011

[36] G Alsbeih N Al-Harbi M Al-Buhairi K Al-Hadyan and MAl-Hamed ldquoAssociation between TP53 codon 72 single-nuc-leotide polymorphism and radiation sensitivity of human fibro-blastsrdquo Radiation Research vol 167 no 5 pp 535ndash540 2007

[37] M C Zody M Garber D J Adams et al ldquoDNA sequence ofhuman chromosome 17 and analysis of rearrangement in thehuman lineagerdquoNature vol 440 no 7087 pp 1045ndash1049 2006

[38] H I Suzuki K Yamagata K Sugimoto T Iwamoto S Kato andK Miyazono ldquoModulation of microRNA processing by p53rdquoNature vol 460 no 7254 pp 529ndash533 2009

[39] A K L Leung and P A Sharp ldquomicroRNAs a safeguard againstturmoilrdquo Cell vol 130 no 4 pp 581ndash585 2007

[40] Z Peng Y Cheng B C-M Tan et al ldquoComprehensive analysisof RNA-Seq data reveals extensive RNA editing in a humantranscriptomerdquo Nature Biotechnology vol 30 no 3 pp 253ndash260 2012

[41] F Gemignani VMoreno S Landi et al ldquoATP53 polymorphismis associated with increased risk of colorectal cancer and withreduced levels of TP53 mRNArdquo Oncogene vol 23 no 10 pp1954ndash1956 2004

[42] V Lazar F Hazard F Bertin N Janin D Bellet and B BressacldquoSimple sequence repeat polymorphism within the p53 generdquoOncogene vol 8 no 6 pp 1703ndash1705 1993

[43] CWest B S Rosenstein and J Alsner ldquoEstablishment of a radi-ogenomics consortiumrdquo International Journal of RadiationOnc-ology Biology Physics vol 76 no 5 pp 1295ndash1296 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Disease Markers 709

An intronic polymorphism is not implicated directly in analtered protein however it may lead to alternative splicing orit can be related to MicroRNAs [38ndash40] A protein modifiedby these processesmay be associated for example an increaseof apoptosis

Gemignani and colleagues [41] in 2004 reported that aninsertionduplication of 16 base pairs in intron 3 of TP53 genethe so called TP53 PIN3 (rs17878362) described by Lazar et al[42] (1993) is associated with an increased risk of developingcolorectal cancer (OR 155 95 CI 1 10 to 2 18 119875 = 0012)Tan et al [19] (2006) reported no association between theTP53 PIN3 (rs17878362) insertion and the development ofadverse effects of radiotherapy (119875 gt 005) Our data corro-borate the results of Tan et al [19] (2006) which also foundno association between this polymorphism and the risk oftoxicity after radiotherapy Our study showed that 42 patients(84) had this polymorphism The frequency of the SNPsuggest the importance of a case-control study to assess theassociation between this polymorphism and the developmentof prostate cancer and thus at this stage the functionalsignificance of TP53 PIN3 polymorphism remains poorlyexplored

5 Conclusions

Until now most of studies in this field have been carriedout using candidate genes as was done in the present studyThese studies can provide valuable information howeverthey are limited and have not yet succeeded in providinga predictive test to identify radiosensitivity in patients Inresponse to lack of success in candidate gene studies and thesubsequent recognition that this approach is too limited inscope radiogenomic investigators have now begun a muchbroader search to identify genes and SNPs associated withradiation response Several large-scale genome wide asso-ciation (GWA) studies have been initiated in which radio-therapy patients are being genotyped for large numbers ofcommon SNPs [8 43]

The results of this study hint to a few candidate geneshowever our study was limited by the small sample size andtherefore low statistical power to detect associations Thisstudy shows the importance of genetic investigations as auseful tool for individualization of radiotherapy strategies Itis anticipated that over the next few years SNPs correlatedwith susceptibility for the development of adverse effectsresulting from radiotherapy will be identified from GWAstudies The data provided by studies in radiogenomics willopen new perspectives for interpretation of the results ofcandidate gene studies such as ours and can be the basis forthe development of a predictive assay that may be useful topersonalize and optimize cancer treatment

Acknowledgments

This work was supported by grants from Financiadora deEstudos e Projetos FINEP MCT Brazil and Fundacao deAmparo a Pesquisa do Estado de Goias FAPEG BrazilHellen Cintra had a fellowship from CAPES MEC Brazil

The author would like to thank Dr Alan George Smulian forthe paper revising

References

[1] G C Barnett C M L West A M Dunning et al ldquoNormaltissue reactions to radiotherapy towards tailoring treatmentdose by genotyperdquoNature Reviews Cancer vol 9 no 2 pp 134ndash142 2009

[2] N G Burnet R M Elliott A Dunning and C M L WestldquoRadiosensitivity Radiogenomics and RAPPERrdquo Clinical Onc-ology vol 18 no 7 pp 525ndash528 2006

[3] C M L West A M Dunning and B S Rosenstein ldquoGenome-wide association studies and prediction of normal tissue toxi-cityrdquo Seminars in Radiation Oncology vol 22 no 2 pp 91ndash992012

[4] J A Cesaretti R G Stock S Lehrer et al ldquoATM sequencevariants are predictive of adverse radiotherapy response amongpatients treated for prostate cancerrdquo International Journal ofRadiation Oncology Biology Physics vol 61 no 1 pp 196ndash2022005

[5] J A Cesaretti R G Stock D P Atencio et al ldquoA geneticallydetermined dose-volume histogram predicts for rectal bleedingamong patients treated with prostate brachytherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 68 no5 pp 1410ndash1416 2007

[6] C A Peters R G Stock J A Cesaretti et al ldquoTGFB1 singlenucleotide polymorphisms are associated with adverse qualityof life in prostate cancer patients treated with radiotherapyrdquoInternational Journal of Radiation Oncology Biology Physics vol70 no 3 pp 752ndash759 2008

[7] R J Burri R G Stock J A Cesaretti et al ldquoAssociation of singlenucleotide polymorphisms in SOD2 XRCC1 and XRCC3 withsusceptibility for the development of adverse effects resultingfrom radiotherapy for prostate cancerrdquo Radiation Research vol170 no 1 pp 49ndash59 2008

[8] S L Kerns H Ostrer R Stock et al ldquoGenome-wide associationstudy to identify single nucleotide polymorphisms (SNPs)associated with the development of erectile dysfunction inAfrican-American men after radiotherapy for prostate cancerrdquoInternational Journal of Radiation Oncology Biology Physics vol78 no 5 pp 1292ndash1300 2010

[9] G Iliakis ldquoThe role of DNA double strand breaks in ionizingradiation-induced killing of eukaryotic cellsrdquo BioEssays vol 13no 12 pp 641ndash648 1991

[10] F Altieri C Grillo M Maceroni and S Chichiarelli ldquoDNAdamage and repair from molecular mechanisms to healthimplicationsrdquo Antioxidants and Redox Signaling vol 10 no 5pp 891ndash937 2008

[11] A J Levine and M Oren ldquoThe first 30 years of p53 growingever more complexrdquo Nature Reviews Cancer vol 9 no 10 pp749ndash758 2009

[12] M B Kastan andD-S Lim ldquoThemany substrates and functionsof ATMrdquoNature ReviewsMolecular Cell Biology vol 1 no 3 pp179ndash186 2000

[13] A Tichy J Vavrova J Pejchal and M Rezacova ldquoAtaxia-telangiectasia mutated kinase (ATM) as a central regulator ofradiation-inducedDNAdamage responserdquoActaMedica vol 53no 1 pp 13ndash17 2010

[14] T Soussi and K G Wiman ldquoShaping genetic alterations inhuman cancer the p53 mutation paradigmrdquo Cancer Cell vol12 no 4 pp 303ndash312 2007

710 Disease Markers

[15] Y Levav-Cohen S Haupt and Y Haupt ldquoMdm2 in growthsignaling and cancerrdquoGrowth Factors vol 23 no 3 pp 183ndash1922005

[16] P Dumont J I-J Leu A C Della Pietra III D L George andM Murphy ldquoThe codon 72 polymorphic variants of p53 havemarkedly different apoptotic potentialrdquoNature Genetics vol 33no 3 pp 357ndash365 2003

[17] D D Oslashrsted S E Bojesen A Tybjaeligrg-Hansen and B G Nor-destgaard ldquoTumor suppressor p53 Arg72Pro polymorphismand longevity cancer survival and risk of cancer in the generalpopulationrdquo Journal of Experimental Medicine vol 204 no 6pp 1295ndash1301 2007

[18] X Li P Dumont A Della Pietra G Shetler andM E MurphyldquoThe codon 47 polymorphism in p53 is functionally significantrdquoJournal of Biological Chemistry vol 280 no 25 pp 24245ndash24251 2005

[19] X-L Tan O Popanda C B Ambrosone et al ldquoAssociationbetween TP53 and p21 genetic polymorphisms and acute sideeffects of radiotherapy in breast cancer patientsrdquo Breast CancerResearch and Treatment vol 97 no 3 pp 255ndash262 2006

[20] G L Bond W Hu E E Bond et al ldquoA single nucleotide poly-morphism in the MDM2 promoter attenuates the p53 tumorsuppressor pathway and accelerates tumor formation inhumansrdquo Cell vol 119 no 5 pp 591ndash602 2004

[21] A Y Ho G Fan D P Atencio et al ldquoPossession of ATMsequence variants as predictor for late normal tissue responsesin breast cancer patients treated with radiotherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 69 no3 pp 677ndash684 2007

[22] C E Canman D-S Lim K A Cimprich et al ldquoActivation ofthe ATM kinase by ionizing radiation and phosphorylation ofp53rdquo Science vol 281 no 5383 pp 1677ndash1679 1998

[23] J D Cox J Stetz and T F Pajak ldquoToxicity criteria of the Radi-ation Therapy Oncology Group (RTOG) and the EuropeanOrganization for Research and Treatment of Cancer (EORTC)rdquoInternational Journal of Radiation Oncology Biology Physics vol31 no 5 pp 1341ndash1346 1995

[24] C N Andreassen J Alsner and J Overgaard ldquoDoes variabilityin normal tissue reactions after radiotherapy have a geneticbasismdashwhere and how to look for itrdquo Radiotherapy and Onc-ology vol 64 no 2 pp 131ndash140 2002

[25] S Angele P Romestaing N Moullan et al ldquoATM haplotypesand cellular response to DNA damage association with breastcancer risk and clinical radiosensitivityrdquo Cancer Research vol63 no 24 pp 8717ndash8725 2003

[26] C N Andreassen J Overgaard J Alsner et al ldquoATM sequencevariants and risk of radiation-induced subcutaneous fibrosisafter postmastectomy radiotherapyrdquo International Journal ofRadiation Oncology Biology Physics vol 64 no 3 pp 776ndash7832006

[27] S Damaraju D Murray J Dufour et al ldquoAssociation of DNArepair and steroid metabolism gene polymorphisms with clini-cal late toxicity in patients treated with conformal radiotherapyfor prostate cancerrdquo Clinical Cancer Research vol 12 no 8 pp2545ndash2554 2006

[28] G L Bond W Hu and A Levine ldquoA single nucleotide poly-morphism in the MDM2 gene from a molecular and cellularexplanation to clinical effectrdquo Cancer Research vol 65 no 13pp 5481ndash5484 2005

[29] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[30] O Popanda J UMarquardt J Chang-Claude and P SchmezerldquoGenetic variation in normal tissue toxicity induced by ionizingradiationrdquoMutation Research vol 667 no 1-2 pp 58ndash69 2009

[31] J J Jaboin M Hwang C A Perez et al ldquoNo evidence forassociation of the MDM2-309 TG promoter polymorphismwith prostate cancer outcomesrdquo Urologic Oncology vol 29 no3 pp 319ndash323 2011

[32] L Sreeja V Syamala P B Raveendran S Santhi J Madha-van and R Ankathil ldquop53 Arg72Pro polymorphism predictssurvival outcome in lung cancer patients in Indian populationrdquoCancer Investigation vol 26 no 1 pp 41ndash46 2008

[33] M B Parliament and D Murray ldquoSingle nucleotide polymor-phisms of DNA repair genes as predictors of radioresponserdquoSeminars in Radiation Oncology vol 20 no 4 pp 232ndash2402010

[34] T Pugh M Keyes L Barclay et al ldquoSequence variant discoveryin DNA repair genes from radiosensitive and radiotolerantprostate brachytherapy patientsrdquo Clinical Cancer Research vol15 no 15 pp 5008ndash5016 2009

[35] M Yang L Zhang N Bi et al ldquoAssociation of P53 and ATMpolymorphisms with risk of radiation-induced pneumonitis inlung cancer patients treated with radiotherapyrdquo InternationalJournal of Radiation Oncology Biology Physics vol 79 no 5 pp1402ndash1407 2011

[36] G Alsbeih N Al-Harbi M Al-Buhairi K Al-Hadyan and MAl-Hamed ldquoAssociation between TP53 codon 72 single-nuc-leotide polymorphism and radiation sensitivity of human fibro-blastsrdquo Radiation Research vol 167 no 5 pp 535ndash540 2007

[37] M C Zody M Garber D J Adams et al ldquoDNA sequence ofhuman chromosome 17 and analysis of rearrangement in thehuman lineagerdquoNature vol 440 no 7087 pp 1045ndash1049 2006

[38] H I Suzuki K Yamagata K Sugimoto T Iwamoto S Kato andK Miyazono ldquoModulation of microRNA processing by p53rdquoNature vol 460 no 7254 pp 529ndash533 2009

[39] A K L Leung and P A Sharp ldquomicroRNAs a safeguard againstturmoilrdquo Cell vol 130 no 4 pp 581ndash585 2007

[40] Z Peng Y Cheng B C-M Tan et al ldquoComprehensive analysisof RNA-Seq data reveals extensive RNA editing in a humantranscriptomerdquo Nature Biotechnology vol 30 no 3 pp 253ndash260 2012

[41] F Gemignani VMoreno S Landi et al ldquoATP53 polymorphismis associated with increased risk of colorectal cancer and withreduced levels of TP53 mRNArdquo Oncogene vol 23 no 10 pp1954ndash1956 2004

[42] V Lazar F Hazard F Bertin N Janin D Bellet and B BressacldquoSimple sequence repeat polymorphism within the p53 generdquoOncogene vol 8 no 6 pp 1703ndash1705 1993

[43] CWest B S Rosenstein and J Alsner ldquoEstablishment of a radi-ogenomics consortiumrdquo International Journal of RadiationOnc-ology Biology Physics vol 76 no 5 pp 1295ndash1296 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

710 Disease Markers

[15] Y Levav-Cohen S Haupt and Y Haupt ldquoMdm2 in growthsignaling and cancerrdquoGrowth Factors vol 23 no 3 pp 183ndash1922005

[16] P Dumont J I-J Leu A C Della Pietra III D L George andM Murphy ldquoThe codon 72 polymorphic variants of p53 havemarkedly different apoptotic potentialrdquoNature Genetics vol 33no 3 pp 357ndash365 2003

[17] D D Oslashrsted S E Bojesen A Tybjaeligrg-Hansen and B G Nor-destgaard ldquoTumor suppressor p53 Arg72Pro polymorphismand longevity cancer survival and risk of cancer in the generalpopulationrdquo Journal of Experimental Medicine vol 204 no 6pp 1295ndash1301 2007

[18] X Li P Dumont A Della Pietra G Shetler andM E MurphyldquoThe codon 47 polymorphism in p53 is functionally significantrdquoJournal of Biological Chemistry vol 280 no 25 pp 24245ndash24251 2005

[19] X-L Tan O Popanda C B Ambrosone et al ldquoAssociationbetween TP53 and p21 genetic polymorphisms and acute sideeffects of radiotherapy in breast cancer patientsrdquo Breast CancerResearch and Treatment vol 97 no 3 pp 255ndash262 2006

[20] G L Bond W Hu E E Bond et al ldquoA single nucleotide poly-morphism in the MDM2 promoter attenuates the p53 tumorsuppressor pathway and accelerates tumor formation inhumansrdquo Cell vol 119 no 5 pp 591ndash602 2004

[21] A Y Ho G Fan D P Atencio et al ldquoPossession of ATMsequence variants as predictor for late normal tissue responsesin breast cancer patients treated with radiotherapyrdquo Interna-tional Journal of Radiation Oncology Biology Physics vol 69 no3 pp 677ndash684 2007

[22] C E Canman D-S Lim K A Cimprich et al ldquoActivation ofthe ATM kinase by ionizing radiation and phosphorylation ofp53rdquo Science vol 281 no 5383 pp 1677ndash1679 1998

[23] J D Cox J Stetz and T F Pajak ldquoToxicity criteria of the Radi-ation Therapy Oncology Group (RTOG) and the EuropeanOrganization for Research and Treatment of Cancer (EORTC)rdquoInternational Journal of Radiation Oncology Biology Physics vol31 no 5 pp 1341ndash1346 1995

[24] C N Andreassen J Alsner and J Overgaard ldquoDoes variabilityin normal tissue reactions after radiotherapy have a geneticbasismdashwhere and how to look for itrdquo Radiotherapy and Onc-ology vol 64 no 2 pp 131ndash140 2002

[25] S Angele P Romestaing N Moullan et al ldquoATM haplotypesand cellular response to DNA damage association with breastcancer risk and clinical radiosensitivityrdquo Cancer Research vol63 no 24 pp 8717ndash8725 2003

[26] C N Andreassen J Overgaard J Alsner et al ldquoATM sequencevariants and risk of radiation-induced subcutaneous fibrosisafter postmastectomy radiotherapyrdquo International Journal ofRadiation Oncology Biology Physics vol 64 no 3 pp 776ndash7832006

[27] S Damaraju D Murray J Dufour et al ldquoAssociation of DNArepair and steroid metabolism gene polymorphisms with clini-cal late toxicity in patients treated with conformal radiotherapyfor prostate cancerrdquo Clinical Cancer Research vol 12 no 8 pp2545ndash2554 2006

[28] G L Bond W Hu and A Levine ldquoA single nucleotide poly-morphism in the MDM2 gene from a molecular and cellularexplanation to clinical effectrdquo Cancer Research vol 65 no 13pp 5481ndash5484 2005

[29] S L Harris G Gil H Robins et al ldquoDetection of functionalsingle-nucleotide polymorphisms that affect apoptosisrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 102 no 45 pp 16297ndash16302 2005

[30] O Popanda J UMarquardt J Chang-Claude and P SchmezerldquoGenetic variation in normal tissue toxicity induced by ionizingradiationrdquoMutation Research vol 667 no 1-2 pp 58ndash69 2009

[31] J J Jaboin M Hwang C A Perez et al ldquoNo evidence forassociation of the MDM2-309 TG promoter polymorphismwith prostate cancer outcomesrdquo Urologic Oncology vol 29 no3 pp 319ndash323 2011

[32] L Sreeja V Syamala P B Raveendran S Santhi J Madha-van and R Ankathil ldquop53 Arg72Pro polymorphism predictssurvival outcome in lung cancer patients in Indian populationrdquoCancer Investigation vol 26 no 1 pp 41ndash46 2008

[33] M B Parliament and D Murray ldquoSingle nucleotide polymor-phisms of DNA repair genes as predictors of radioresponserdquoSeminars in Radiation Oncology vol 20 no 4 pp 232ndash2402010

[34] T Pugh M Keyes L Barclay et al ldquoSequence variant discoveryin DNA repair genes from radiosensitive and radiotolerantprostate brachytherapy patientsrdquo Clinical Cancer Research vol15 no 15 pp 5008ndash5016 2009

[35] M Yang L Zhang N Bi et al ldquoAssociation of P53 and ATMpolymorphisms with risk of radiation-induced pneumonitis inlung cancer patients treated with radiotherapyrdquo InternationalJournal of Radiation Oncology Biology Physics vol 79 no 5 pp1402ndash1407 2011

[36] G Alsbeih N Al-Harbi M Al-Buhairi K Al-Hadyan and MAl-Hamed ldquoAssociation between TP53 codon 72 single-nuc-leotide polymorphism and radiation sensitivity of human fibro-blastsrdquo Radiation Research vol 167 no 5 pp 535ndash540 2007

[37] M C Zody M Garber D J Adams et al ldquoDNA sequence ofhuman chromosome 17 and analysis of rearrangement in thehuman lineagerdquoNature vol 440 no 7087 pp 1045ndash1049 2006

[38] H I Suzuki K Yamagata K Sugimoto T Iwamoto S Kato andK Miyazono ldquoModulation of microRNA processing by p53rdquoNature vol 460 no 7254 pp 529ndash533 2009

[39] A K L Leung and P A Sharp ldquomicroRNAs a safeguard againstturmoilrdquo Cell vol 130 no 4 pp 581ndash585 2007

[40] Z Peng Y Cheng B C-M Tan et al ldquoComprehensive analysisof RNA-Seq data reveals extensive RNA editing in a humantranscriptomerdquo Nature Biotechnology vol 30 no 3 pp 253ndash260 2012

[41] F Gemignani VMoreno S Landi et al ldquoATP53 polymorphismis associated with increased risk of colorectal cancer and withreduced levels of TP53 mRNArdquo Oncogene vol 23 no 10 pp1954ndash1956 2004

[42] V Lazar F Hazard F Bertin N Janin D Bellet and B BressacldquoSimple sequence repeat polymorphism within the p53 generdquoOncogene vol 8 no 6 pp 1703ndash1705 1993

[43] CWest B S Rosenstein and J Alsner ldquoEstablishment of a radi-ogenomics consortiumrdquo International Journal of RadiationOnc-ology Biology Physics vol 76 no 5 pp 1295ndash1296 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom