QM-FISH Analysis Of The Genes Involved In The G1/S Checkpoint Signaling Pathway In Triple-negative Breast Cancer

Objective:1. To analyze copy number alterations(CNAs) of the genes involved in the Gl/S checkpoint signaling pathway of triple-negative breast cancer (TNBC) and non-triple-negative breast cancer(non-TNBC)2. To discover the relationship between copy number alterations (CNAs)of the related genes and clinical characteristics of TNBC and non-TNBC.3. To evaluate their clinical value in the prognosis of TNBC and non-TNBC and find specific CNAs associated with overall survive for TNBC.In all,to provide an effective target for future treatment.Methods:1. We selected the samples randomly form Tianjin Cancer Hospital between August,2006and August,2007,which contained one hundred TNBC cases and one hundred non-TNBC cases. Quantitative multi-gene fluorescence in situ hybridization (QM-FISH) was used to study CNAs of the genes involved in the G1/S checkpoint signaling pathway, including CCNDl,c-Myc,p21, CHEK2, p16, Rbl,Mdm2and p53.2. The samples were diagnosed with primary breast invasive ductal carcinoma confirmed by pathology. All of the patients were incipient breast cancer cases and had not been treated with chemotherapy, radiotherapy or endocrine therapy. The criteria for TNBC diagnosis included the following:the immuno-histochemical analysis results of ER and PR were negative and the FISH analysis resμlt of HER-2was negative. After collection, the specimens were fixed with10%formaldehyde, dehydrated and then embedded in paraffin. Paraffin sections were sliced at a4-μm thickness.3. SPSS17.0software (SPSS Inc., Chicago, IL, USA) was used to perform the statistical analyses. The basic conditions,clinical characteristics and CNAs of the patients were tested using the Chi-square test and the Fisher exact test. Patient survival was analyzed using the Kaplan-Meier method, and the survival rates were compared using the Log-rank test. The Cox proportional hazards model was used to analyze the mμltiple prognostic factors. Two-sided tests were performed,and p<0.05was considered to indicate significantly different resμlts in the statistical tests. Results:1. The TNBC were associated with lower percentage of T1stage(P=0.02) and breast-conserving surgery(P<0.001),but higher percentage of radiotherapy (P=0.03).2. CNAs of the8genes were analyzed individually:four out of eight genes showed significant differences between the TNBC group and the non-TNBC group, the amplification rates of c-Myc and the deletion rates of p53in TNBC tended to be higher than those in non-TNBC, whereas the amplification rates of CCND1and Mdm2in non-TNBC tended to be higher than those in TNBC.3. The amplification rates of CCND1in TNBC tended to be higher in the young group (50%,P=0.001), and were more common in patients with vessel carcinoma embolus (11%,P=0.025). The deletion rates of Rb1also tended to be higher in the lymph node positive group(50%,P=0.028).whereas the amplification rates of Mdm2(32.4%,P=0.01) in non-TNBC tended to be higher in the patients with lymph node positive metastasis. Otherwise,The deletion of p21(P=0.02), Rb1(P=0.02)and pl6(40.5%,P=0.03) were associated with histological grade, Pathological stage and lymph node positive metastasis respectively.4. The amplification of c-Myc and the deletion of p53in TNBC were associated with higher mortality risk (HR,2.87;95%CI,1.14-7.21[P=0.03]; HR,3.34;95%CI,1.22-9.09[P=0.02]), whereas the mortality risk tended to be higher in the patients with amplification of CCND1(HR,4.35;95%CI,1.23-15.39[P=0.02]) in non-TNBC.Conclusions:1. The amplification rates of c-Myc and deletion rates of p53in TNBC tended to be higher than those in non-TNBC, whereas the amplification rate of CCND1and Mdm2tended to be higher in non-TNBC.2. The CNAs was associated with different clinical pathology in TNBC and non-TNBC.3. c-Myc and p53were independent prognostic factors for TNBC, while CCND1was the independent prognostic factors for non-TNBC.