Ior DFS in cases with gene gain or amplification and with

Ior DFS in cases with gene gain or amplification and with inferior DFS in the absence of ESR1 gene gain. Prognostic factors with independent Title Loaded From File significance for superior OS were small tumor size, less than four involved axillary nodes, Ki67,14 , and the interaction of the Gene Functional profile with HER2 tumor status (p = 0.029). Irrespective of gene ratio status, patients with ESR1 functional tumors fared better than those with nonfunctional tumors only in the absence of HER2 amplification/ESR1 Gene Amplification in Early Breast CancerFigure 5. Multivariate analysis for DFS (a) and OS (b) presented by forest plots. doi:10.1371/journal.pone.0070634.goverexpression. In the presence of HER2 amplification/overexpression, the prognostic impact of functional ESR1 was lost.DiscussionER is encoded by the ESR1 gene localized on chromosome 6q25.1, and copy number changes of ESR1 have only recently become the focus of interest. Holst et al reported a FISH ESR1 amplification rate of 20.6 in 2000 breast carcinomas loaded in tissue microarrays, the majority showing a clustered arrangement of tight signals and corresponding to 12?6 gene copies per nucleus by qPCR [6]. However, other groups soon refuted these findings, reporting amplification rates as low as 0.9 [8?1]. Differences in patient populations, tumor characteristics and methodologies and definitions used (qPCR, MLPA, aCGH, FISH) only partly explain such discrepancies. We used strict protocolquality guidelines for data capture and central FISH/IHC assessment in .1000 tumors in order to report an amplification rate of 4.2 , mostly low-level (five or more gene copies per nucleus in only 3 of cases) and a deletion rate of 15.7 . Our reported incidence of ESR1 amplification is intermediary between that reported by Brown (FISH, 1 ) [8], Vincent-Salomon (aCGH, 0.9 ) [10], Moelans (MLPA, 2 ) [19], Horlings (aCGH and FISH, 2.3 ) [9], Reis-Filho (FISH, 4 ) [11] and that reported by Ooi (RNAse FISH, 5.9 ) [18], Ejlertsen (FISH, 13.6 ) [19], Nielsen (FISH, 14 ) [20], Tomita (FISH, 22.6 )[7]. In contrast to Holst et al, we used a manual scoring algorithm in order to count the number of gene signals and assess the ESR1/ CEP6 ratio, rather than consider all cases with tight clusters as amplification Title Loaded From File events. Cases with gene clusters were seen in 9.5 of cases (almost all scored as gain and amplification events). Despite varying incidence, some of our findings confirm those reported by other groups. ESR1 gene amplification 23727046 was low-level and correlated with high histological grade, in keeping with data reported by Ejlertsen et al [19] and Moelans et al [22]. The correlation of ESR1 gene gain or amplification with protein expression was rather weak, , in agreement with data from other groups. We report deleted ESR1 cases in 15.7 , an incidence which is higher than the one reported by Ejlertsen (4.2 ) [19], though in agreement with preclinical observations showing gene deletion in four out of six breast cancer cell line [21]. Moreover, some of the deleted cases were due to a high number of CEP6 copies in the presence of normal ESR1 gene copy number. . We did observe a favorable prognostic significance of ER mRNA and protein expression, but failed to find any for ESR1 gene ratio, despite the numerical association of copy number with increased risk of relapse and death. Even when we ommitted the CEP6 gene copy number as a possible confounder and studied only ESR1 gene copies, we failed to demonstrate an unequivo.Ior DFS in cases with gene gain or amplification and with inferior DFS in the absence of ESR1 gene gain. Prognostic factors with independent significance for superior OS were small tumor size, less than four involved axillary nodes, Ki67,14 , and the interaction of the Gene Functional profile with HER2 tumor status (p = 0.029). Irrespective of gene ratio status, patients with ESR1 functional tumors fared better than those with nonfunctional tumors only in the absence of HER2 amplification/ESR1 Gene Amplification in Early Breast CancerFigure 5. Multivariate analysis for DFS (a) and OS (b) presented by forest plots. doi:10.1371/journal.pone.0070634.goverexpression. In the presence of HER2 amplification/overexpression, the prognostic impact of functional ESR1 was lost.DiscussionER is encoded by the ESR1 gene localized on chromosome 6q25.1, and copy number changes of ESR1 have only recently become the focus of interest. Holst et al reported a FISH ESR1 amplification rate of 20.6 in 2000 breast carcinomas loaded in tissue microarrays, the majority showing a clustered arrangement of tight signals and corresponding to 12?6 gene copies per nucleus by qPCR [6]. However, other groups soon refuted these findings, reporting amplification rates as low as 0.9 [8?1]. Differences in patient populations, tumor characteristics and methodologies and definitions used (qPCR, MLPA, aCGH, FISH) only partly explain such discrepancies. We used strict protocolquality guidelines for data capture and central FISH/IHC assessment in .1000 tumors in order to report an amplification rate of 4.2 , mostly low-level (five or more gene copies per nucleus in only 3 of cases) and a deletion rate of 15.7 . Our reported incidence of ESR1 amplification is intermediary between that reported by Brown (FISH, 1 ) [8], Vincent-Salomon (aCGH, 0.9 ) [10], Moelans (MLPA, 2 ) [19], Horlings (aCGH and FISH, 2.3 ) [9], Reis-Filho (FISH, 4 ) [11] and that reported by Ooi (RNAse FISH, 5.9 ) [18], Ejlertsen (FISH, 13.6 ) [19], Nielsen (FISH, 14 ) [20], Tomita (FISH, 22.6 )[7]. In contrast to Holst et al, we used a manual scoring algorithm in order to count the number of gene signals and assess the ESR1/ CEP6 ratio, rather than consider all cases with tight clusters as amplification events. Cases with gene clusters were seen in 9.5 of cases (almost all scored as gain and amplification events). Despite varying incidence, some of our findings confirm those reported by other groups. ESR1 gene amplification 23727046 was low-level and correlated with high histological grade, in keeping with data reported by Ejlertsen et al [19] and Moelans et al [22]. The correlation of ESR1 gene gain or amplification with protein expression was rather weak, , in agreement with data from other groups. We report deleted ESR1 cases in 15.7 , an incidence which is higher than the one reported by Ejlertsen (4.2 ) [19], though in agreement with preclinical observations showing gene deletion in four out of six breast cancer cell line [21]. Moreover, some of the deleted cases were due to a high number of CEP6 copies in the presence of normal ESR1 gene copy number. . We did observe a favorable prognostic significance of ER mRNA and protein expression, but failed to find any for ESR1 gene ratio, despite the numerical association of copy number with increased risk of relapse and death. Even when we ommitted the CEP6 gene copy number as a possible confounder and studied only ESR1 gene copies, we failed to demonstrate an unequivo.