| ObjectiveGenomic integrity is critical to organism survival. DNA damage response promotesfaithful transmission of genomes in dividing cells by reversing the extrinsic and intrinsicDNA damage, and is required for cell survival during replication. Based on therelationship between chromosomal instability and cancer, it is postulated that thedeifciency in DNA damage response is a major driving force for breast cancer’soccurrence and development. DNA topoisomerase II&-binding protein1(TopBPl) andataxia telangiectasia mutated and Rad3related protein (ATR) are two important membersin DNA damage response network and, in particular, TopBPl is required for the fullactivation of ATR. Previous studies have shown that mutations or polymorphisms ofTopBPl and ATR are associated with human cancer risk. This study was performed todetect the expression of these two proteins in breast cancer, especially in triple-negativebreast cancer (TNBC), by tissue microarray (TMA) and immunohistochemistry (IHC)methods, and to explore their correlations with clinicopathological factors. We sought toelucidate whether TopBPl and ATR play an important role in the pathogenesis andprogression of breast cancer, and to provide a theoretical basis for the selection of TNBCclinical treatment strategies.MethodsPart1:272cases of breast cancer tissue and118cases of adjacent non-canceroustissue from Shanghai Changhai Hospital and Huangpu District Central Hospital werecollected to construct TMAs. IHC of Envision method was used to detect proteinexpression of TopBPl and ATR with rabbit anti-human polyclonal antibodies (Abeam,UK). Each case was evaluated as negative or positive staining by multiplying the scorefor staining intensity and score for percentage of positively stained cells. Relevantclinicopathological information, such as age of patient, menopausal status, pathologicaltype, histological grade, tumor size，lymph node metastasis, TNM stage and ER，PR,HER2, p53status, was collected to perform correlation analysis. The SPSS19.0softwarewas used for statistical analysis. The difference of protein expression between cancertissue and adjacent non-cancerous tissue as well as the relationship between proteinexpression and clinicopathological parameter was examined with Chi-square test. Analysis of the relationship between TopBPl and ATR expression levels was performedusing Spearman rank correlation. Multivariate analysis was evaluated by logisticregression. All statistical tests were two sided and尸<0.05was considered signiifcant.Part2:78cases of TNBC tissue and53cases of matched adjacent non-canceroustissue from Shanghai Xinhua Hospital were collected to construct TMAs. Expression ofTopBPl and ATR was detected by IHC as Part1described. All these78patients as wellas46patients of TNBC from Part1cohort were followed up for survival data. The SPSS19.0software was used for statistical analysis. Difference of protein expression betweenTNBC tissue and matched adjacent non-cancerous tissue was examined with McNemar’stest. Survival analysis of overall survival (OS) was assessed by Kaplan-Meier methodwith log-rank test for difference between expression levels of each protein. Coxregression analysis was used to estimate the relationship between prognostic variables.ResultsPart1:236cases of invasive breast cancer tissue and113cases of adjacentnon-cancerous tissue obtained the experimental results of both TopBPl and ATR. IHCstaining demonstrated that TopBPl was expressed almost exclusively in the nucleus ofthe normal breast epithelium while in breast cancer tissue TopBPl was aberrantlyexpressed in both cytoplasm and nucleus. ATR was expressed in the nucleus of bothbreast cancer tissue and adjacent non-cancerous tissue. Compared with adjacentnon-cancerous tissue, the expressions of both TopBPl and ATR were signiifcantlyincreased in breast cancer tissue (尸<0.001). High expression of TopBPl was signiifcantlyassociated with positive PR and negative p53status, while the expression of ATR wassigniifcantly lower in the cases with tumor size Tl，stage I，negative HER2status andTNBC subgroup (尸<0.05). No association was found between the expression levels ofTopBPl and ATR. In multivariate logistic regression analysis, TopBPl demonstratedsigniifcant association with PR while ATR was signiifcantly correlated with tumor size.Part2: In this part74cases of TNBC and53cases of matched adjacentnon-cancerous tissue obtained the IHC staining results of both TopBPl and ATR.Including46cases from Part1cohort, a total of120TNBC cases entered the statisticalanalysis. Expression of TopBPl was signiifcantly higher in TNBC compared withmatched adjacent non-cancerous tissue (尸<0.001)，while no signiifcant difference ofATR expression was detected between these two groups. Low expression of TopBPl wassigniifcantly associated with lymph node metastasis (尸<0.01) but no relationship between ATR expression level and any clinicopathological parameter of TNBC wasfound. No association was detected between the expression of TopBPl and ATR inTNBC. No signiifcant difference was found in OS of TNBC according to differentTopBPl, ATR, or TopBPl/ATR expression levels. Cox regression analysis revealed thatTNM stage was an independent prognostic factor for OS in TNBC patients.ConclusionExpression of TopBP1and ATR was signiifcantly higher in breast cancer tissue butno correlation was found between the two proteins. TopBPl was aberrantly expressed inboth cytoplasm and nucleus of breast cancer cells，the expression of TopBPl wassigniifcantly associated with PR and p53status in breast cancer and with lymph nodemetastasis in TNBC subgroup, all of these demonstrated that TopBPl might play animportant role in the pathogenesis and progression of breast cancer. The expression ofATR was associated with tumor size，TNM stage, HER2status and triple-negativephenotype in breast cancer consisting with its function in DNA damage response, but norelationship between ATR expression level and clinicopathological parameters of TNBCwas found. The association between ATR and the development of breast cancer needfurther investigation. In TNBC patients TNM stage was an independent prognostic factorfor OS. Chemotherapy with DNA cross-linking agents, such as cisplatin, may be areasonable choice for TNBC patients. |
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