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markers in breast cancer. J ClinOncol. 2007;25:5287-5312.

2. Gerlinger M, Rowan AJ, Horswell S,et al. Intratumor heterogeneity andbranched evolution revealed bymultiregion sequencing. N Engl JMed. 2012;366:883-892.

3. Alix-Panabieres C, Muller V,Pantel K. Current status in humanbreast cancer micrometastasis. CurrOpin Oncol. 2007;19:558-563.

4. Paoletti C, Smerage J, Hayes DF.Circulating tumor cells as a markerof prognosis. PPO Updates. 2012;26:1-8.

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5. Papadopoulou E, Davilas E,Sotiriou V, et al. Cell-free DNA andRNA in plasma as a new molecularmarker for prostate and breast cancer.Ann N Y Acad Sci. 2006;1075:235-243.

6. Teutsch SM, Bradley LA,Palomaki GE, et al; EGAPP WorkingGroup. The Evaluation of GenomicApplications in Practice andPrevention (EGAPP) Initiative:methods of the EGAPP WorkingGroup. Genet Med. 2009;11:3-14.

7. Moore HM, Kelly A, McShane LM,Vaught J. Biospecimen reporting for

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improved study quality (BRISQ).Clin Chim Acta. 2012;413:1305.

8. Simon RM, Paik S, Hayes DF. Use ofarchived specimens in evaluation ofprognostic and predictive biomarkers.J Natl Cancer Inst. 2009;101:1446-1452.

9. Hayes DF, Paoletti C. Circulatingtumour cells: insights into tumourheterogeneity. J Intern Med. 2013;274:137-143.

Identification of InheritedGenetic Variations InfluencingPrognosis in Early-Onset BreastCancer

Rafiq S, Tapper W, Collins A, et al (Univ ofSouthampton School of Medicine, Hants,UK; et al)

Cancer Res 73:1883-1891, 2013

Genome-Wide Association Studies(GWAS) have begun to investigate asso-ciations between inherited genetic vari-ations and breast cancer prognosis.Here, we report our findings froma GWAS conducted in 536 patientswith early-onset breast cancer aged40 or less at diagnosis and with amean follow-up period of 4.1 years(SD¼ 1.96). Patients were selectedfrom the Prospective Study of Out-comes in Sporadic versus Hereditarybreast cancer. A Bonferroni correctionfor multiple testing determined that aP value of 1.0� 10�7 was a statisticallysignificant association signal. Fol-lowing quality control, we identified487,496 single nucleotide polymor-phisms (SNP) for association tests instage 1. In stage 2, 35 SNPs with the

most significant associations weregenotyped in 1,516 independent casesfrom the same early-onset cohort. Instage 2, 11 SNPs remained associatedin the same direction (P# 0.05). Fixedeffects meta-analysis models identifiedone SNP associated at close to genomewide level of significance 556 kbupstream of the ARRDC3 locus[HR¼ 1.61; 95% confidence interval(CI), 1.33e1.96; P¼ 9.5� 10�7].Four further associations at or close tothe PBX1, RORa, NTN1, and SYT6loci also came close to genome-widesignificance levels (P¼ 10�6). In thefirst ever GWAS for the identificationof SNPs associated with prognosis inpatients with early-onset breast cancer,we report a SNP upstream of theARRDC3 locus as potentially associatedwith prognosis (median follow-uptime for genotypes: CC¼ 4 years,CT¼ 3 years, and TT¼ 2.7 years; Wil-coxon rank-sum test CC vs. CT,P¼ 4� 10�4 and CT vs. TT,P¼ 0.76). Four further loci may alsobe associated with prognosis.

GWASs assess SNPs at the whole-genome level and have shown greatpromise in identifying common, low-

penetrance genetic variants associatedwith the risk of developing complexdiseases. GWASs have been success-fully employed to identify genetic riskvariants associated with the develop-ment of all major cancers. To date,multiple large-scale GWASs haveidentified nearly 70 common breastcancer susceptibility loci, 41 of whichwere recently identified by the Collab-orative Oncological Gene-EnvironmentStudy consortium.1 In contrast, geneticvariants associated with the outcomesof patients after cancer diagnosis haveseldom been reported.2-5 We found 3prior studies3,5,6 that applied the GWASapproach to searching for commongenetic variants associated with clin-ical outcomes of breast cancer. In onestudy, Azzato and colleagues6 failed toidentify any prognostic SNPs. Inanother study, Azzato and colleagues5

queried a much smaller set of SNPs(w10 000) compared to the regularGWAS and identified a SNP(rs4778137) significantly associatedwith overall survival in estrogenreceptor (ER)-negative breast cancerpatients but not in ER-positive patients.Shu and colleagues3 conducted aGWAS in a Chinese population and

identified 2 loci associated with totalmortality, one of which (rs9934948)was replicated in an independentpopulation of Caucasian breast cancerpatients.

The limited success demonstratedthe challenges faced by these studies.The most notable challenge is the largesample size and multiple patient groupstypically required for replication inGWASs because of the considerablenumber of SNPs being analyzed and thegenerally modest effects of the variantalleles. In addition, detailed clinicaldata are often lacking in GWASs ofcancer outcomes because informationsuch as tumor characteristics, treat-ment regimen, toxicity, and follow-upfor tumor progression and cancer-specific survival, would require exten-sive review of medical records andcould be fiscally and logisticallyprohibitive. A well-characterizedpatient population is necessary tominimize the effects of heterogeneityand enhance the discovery of outcomedeterminants in subgroups of homoge-nous patient populations. For example,we successfully conducted a GWAS inadvanced-stage non-small-cell lungcancer patients who received primaryplatinum-based chemotherapy, leadingto the discovery of a SNP (rs1878022)in the chemokine-like receptor 1statistically significantly associatedwith poor overall survival.2

In the current article, Rafiq andcolleagues performed a GWAS inwomenwhowere diagnosedwith breastcancer at age 40 years or younger toidentify SNPs associated with breastcancer-specific survival. The subgroupof patients with early-onset breastcancers has been suggested to have agreater genetic predisposition to theircancer as well as poorer prognosisfollowing their cancer diagnosis. Rafiqand colleagues applied a multistagedesign with a discovery phase involving536 patients, followed by a validationphase of 1516 patients from the same

cohort. The discovery phase, which hasenough power to identify SNPs ofmoderate effect (HR $1.5), wasenriched with patients with either veryshort (<2 years) or relatively long(>4 years) of breast cancer-specificsurvival. The most notable associationwas an SNP (rs421379) located 556 kbupstream of the ARRDC3 gene that issignificant at close to the genome-widelevel, with a 1.61-fold increased risk ofbreast cancer-specific mortality in thecombined discovery and validationdata. Four additional SNPs were alsosuggestively associated with prognosis.Of note, the same associations werereplicated in a second cohort of youngbreast cancer patients (average age,35.7 years) but not in a third cohort ofmostly older breast cancer patients(average age, 56.8 years), furtherdemonstrating the importance of iden-tifying subgroups of patient popula-tions with homogenous characteristicsfor studies of clinical outcomes,including GWASs.

As mentioned by the authors,additional external cohorts of well-characterized early-onset breastcancer patients are necessary tofurther validate the findings. Since thestudy cohort included only symptom-atic breast cancer patients, it wouldbe of particular interest to assesswhether the findings can be general-ized to screening-detected breastcancer patients. In addition, a largersample size would be needed to iden-tify common genetic variants withweaker associations (HRs <1.5), asthe majority of the HRs for the SNPsreplicated in stage II and the meta-analysis were <1.50. Rafiq andcolleagues adjusted for the estrogenreceptor status, nodal involvement,and metastasis in their analyses andfound them to have a slight effect onthe strength of associations with someSNPs. It would be interesting to alsoexamine the effects of other clinicalvariables, such as tumor size, number

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of positive nodes, and tumor grade,that were included in several widelyaccepted online breast canceroutcome prediction tools, such asAdjuvant! Online7 and PREDICT.8 Ifthe effect of these SNPs remainssignificant after adjusting for theseknown clinical variables in furtherstudies, they may be considered forincorporation in these online tools toimprove outcome prediction in early-onset breast cancer patients. Onestudy9 has shown that Adjuvant!Online overestimated 10-year overallsurvival and breast cancer-specificsurvival in breast cancer patientsunder 40 years of age. Another study10

found that both Adjuvant! Online andPREDICT underestimated 10-yearmortality by 17.1% in women aged20-35 years. Therefore, there is aclear need to identify novelbiomarkers that can better predict theoutcome for early-onset breast cancerpatients. Rafiq and colleagues madethe first effort to tackle this problem byidentifying the germline genetic vari-ants associated with the outcome ofearly-onset breast cancer patients.

Y. Ye, PhDW. H. Chow, PhDX. Wu, MD, PhD

References1. Michailidou K, Hall P, Gonzalez-

Neira A, et al. Large-scalegenotyping identifies 41 new lociassociated with breast cancer risk.Nat Genet. 2013;45:353-361.

2. Wu X, Ye Y, Rosell R, et al.Genome-wide association study ofsurvival in non-small cell lungcancer patients receivingplatinum-based chemotherapy.J Natl Cancer Inst. 2011;103:817-825.

3. Shu XO, Long JR, Lu W, et al.Novel genetic markers of breastcancer survival identified by a

iseases: A Year Book� Quarterly 353Vol 24 No 4 2014

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genome-wide association study.Cancer Res. 2012;72:1182-1189.

4. Wu XF, Wang L, Ye Y, et al.Genome-wide association study ofgenetic predictors of overall survivalfor non-small cell lung cancer innever smokers. Cancer Res. 2013;73:4028-4038.

5. Azzato EM, Tyrer J, Fasching PA,et al. Association between a germlineOCA2polymorphism at chromosome15q13.1 and estrogen receptor-negative breast cancer survival. J NatlCancer Inst. 2010;102:650-662.

6. Azzato EM, Pharoah PD,Harrington P, et al. A genome-wide

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association study of prognosis inbreast cancer. Cancer EpidemiolBiomarkers Prev. 2010;19:1140-1143.

7. Ravdin PM, Siminoff LA, Davis GJ,et al. Computer program to assist inmaking decisions about adjuvanttherapy for women with early breastcancer. J Clin Oncol. 2001;19:980-991.

8. Wishart GC, Azzato EM,Greenberg DC, et al. PREDICT:a new UK prognostic model thatpredicts survival following surgeryfor invasive breast cancer. BreastCancer Res. 2010;12:R1.

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9. Mook S, Schmidt MK, Rutgers EJ,et al. Calibration and discriminatoryaccuracy of prognosis calculation forbreast cancer with the onlineAdjuvant! Program: a hospital-basedretrospective cohort study. LancetOncol. 2009;10:1070-1076.

10. Wishart GC, Bajdik CD,Azzato EM, et al. A population-based validation of the prognosticmodel PREDICT for early breastcancer. Eur J Surg Oncol. 2011;37:411-417.

NON-INVASIVE CANCER

Role of radiotherapy boost inwomen with ductal carcinomain situ: A single-centerexperience in a series of 389patients

Meattini I, Livi L, Franceschini D, et al(Univ of Florence, Italy; et al)

Eur J Surg Oncol 39:613-618, 2013

Background.dThe use of adjuvantradiotherapy in ductal carcinoma in situis accepted by most radiation oncolo-gists worldwide; the role of a booston the tumor bed is however morecontroversial.

Materials and Methods.dWereviewed our Institute experience inDCIS treatment, focusing on mainprognostic factors and impact of radia-tion boost on local relapse. A total of389 patients treated between 1990 and2007 were retrospectively analyzed.All patients received adjuvant radio-

therapy after breast-conserving surgeryfor a median dose of 50 Gy; 190patients (48.8%) received and addi-tional radiation boost on the tumor bed.

Results.dAt a mean follow up of7.7 years, we recorded 26 local recur-rence (6.7%). Concerning localrelapse-free survival, at Cox regressionunivariate analyses <1 mm surgicalmargins (p < 0.0001) and young age(p¼ 0.01) emerged as significant unfa-vorable prognostic factors.

At multivariate analysis Coxregression model, surgical margins(p < 0.001) and radiation boost(p¼ 0.014) resulted as the significantindependent predictors of recurrence.

Conclusions.dOur experienceshowed the negative prognostic impactof surgical margins <1 mm and theprotective role of radiation boost onLR rate. Anyway, results from ongoingprospective Phase III studies arestrongly necessary to better identifyhigh-risk DCIS patients.

The incidence of ductal carcinomain situ (DCIS) has increased signifi-cantly since the introduction ofscreening mammography, and treat-ment is focused primarily on prevent-ing local recurrence (LR). Currently,the standard treatment for DCIS isbreast-conserving therapy (BCT) fol-lowed by adjuvant radiotherapy (RT).The benefit of adjuvant whole-breastirradiation (WBI) in reducing LR inpatients undergoing BCT has beendemonstrated in 4 large prospectiverandomized controlled trials: NationalAdjuvant Breast and Bowel Project(NSABP) B-17; European Organisa-tion for Research and Treatment ofCancer 10853; United Kingdom,Australia, and New Zealand DCIStrial; and SweDCIS.1-4 However, therole of a RT boost is still controversial.

In this study, Meattini andcolleagues reviewed their experience inDCIS treatment, focusing on prog-nostic factors and the effect of a boost