Establishment And Evaluation Of The Gene Diagnostic Methods For The Incidence And Metastases Risk Of Breast Cancer

Background:The risk of breast cancer and aggressive biological behavior are associated with human genetic background and the cancer-related genetic variation. Single nucleotide polymorphism (SNP) is the most common form of human genetic variation. Some studies showed that SNPs in many genes are the established risk factor for breast cancer. Identifying SNPs of tumor related genes and genotyping these SNPs may contribute to screening the population with high risk of breast cancer and aggressive biological behavior, thus providing evidence for prevention and individualized treatment for breast cancer patients. Multicenter and large sample sizes studies suggested that the polymorphisms of TGFβ1rs1982073(LeulOPro) and FGFR2rs1219648were strongly associated with breast cancer in western population, while researches for Chinese population haven’t been reported yet before.Breast cancer is generally viewed as a systemic disease; metastasis is the leading cause of death for patients with breast cancer. Micrometastases in lymph nodes and peripheral blood couldn’t be found sensitively by regular pathological and imaging diagnostic methods. Quantitative detection of circulating tumor cells in peripheral blood and the occult metastasis of histologically negative lymph nodes could contribute to distinguish patients with high metastases risk earlier, and to evaluate clinical stage of breast cancer patients more exactly, thus to guide clinical patient-tailored therapy effectively and improve survival rate of the breast cancer patients.Objective:To study the potential influences of TGFβ1rs1982073and FGFR2rs1219648on the incidence and development of breast cancer, and to evaluate clinical significance of identifing the people with high risk of breast cancer and aggressive biological behavior. To establish a real-time quantitative reverse transcription-PCR (QRT-PCR) method with CK19and CEA mRNA as the gene markers for detecting the circulating tumor cells in peripheral blood and the occult metastasis in axillary lymph nodes of breast cancer patients, and evaluate the clinical potential of monitoring the amount of the circulating tumor cells in peripheral blood and micrometastasis status of axillary lymph nodes. Methods:TaqMan allelic discrimination assay was performed to genotyping SNPs of rs1982073and rs1219648in435breast cancer cases and410healthy controls. QRT-PCR assay was established for detecting the level of CK19and CEA mRNA in peripheral blood nucleated cells of102healthy controls,189breast cancer patients and129histologically negative axillary lymph nodes of57breast cancer patients. SPSS13.0software was used for statistical analysis. The genotype frequencies in cases and controls were compared by χ2test, genotype-specific risks were estimated as odds ratios (ORs) and95%confidence intervals (CIs) by unconditional logistic regression; the difference of CK19and CEA mRNA in different groups were assessed by nonparametric test, and their positive rates were compared by x2test or Fisher exact test, and Spearman rank correlation analysis was used to measure the relations between CK19and CEA mRNA.Results:1. The results of SNP analysis:The genotype frequencies of rs1982073and rs1219648were not significantly different between breast cancer patients and healthy controls, but for the SNP rs1982073, C-carriers (T/C and C/C genotype) were more likely to be tumors with high malignancy (grade III) relative to patients with T/T genotype [OR=4.520(95%CI:1.785～11.449, P=0.001) and3.137(95%CI:1.090～9.057, P=0.034)]. For the SNP rs1219648, G-carriers (A/G and G/G genotype) were more likely to be lymph nodes positive relative to patients with A/A genotype (OR=1.606,95%CI:1.053～2.448, P=0.028).2. The results of micrometastases detection:Median CK19mRNA copy numbers in the control group and breast cancer patients were67and593(copies/ml blood) respectively (P<0.05), and median CEA mRNA copy numbers was7and14respectively (P<0.05). Taking the highest value (497and19copies/ml blood) in the control group as the cut-off,54.0%breast cancer patients were considered positive for CK19mRNA and29.6%for CEA mRNA, both significantly higher than control group (P<0.05).The positive rate of CK19mRNA and CEA mRNA enhanced with the increasing of clinical stage respectively (P=0.037,0.015), and positive rates for Ⅳ breast cancer patients were much higher than breast cancer patients with stage Ⅰ～Ⅱ (P=0.035,0.003); the positive rate of CEA mRNA was significantly associated with the status of PR (P=0.016), but there were not significantly difference between the CK19mRNA or CEA mRNA and other clinical pathological factors. The co-positive rate of CK19mRNA and CEA mRNA were significantly associated with the age, tumor size, clinical stage and status of LN (P<0.05). Either CK19mRNA or CEA mRNA positive is associated with clinical stage and the status of PR (P=0.021,0.017).For patients with negative lymph nodes by regular pathological method, the detection rate for breast cancer patients with CK19-positive was49.1%, CEA-positive was35.1%; The detection rate for LNs with CK19-positive was38.7%, CEA-positive was26.3%, the results of CK19detection were correlated well with CEA (r=0.482, P=0.000). In addition, either CK19mRNA or CEA mRNA positive associated with histological grade (P<0.05).Conclusion:The polymorphisms of TGFβ1rs1982073and FGFR2rs1219648were not associated with breast cancer risk in Chinese population, but the individuals carried C allele in rs1982073and G allele in rs1219648were more likely to be highly malignant tumor. So detecting of the genotypes of these two SNPs might contribute to identifying patients with poor prognosis.Circulating tumor cells in peripheral blood and the occult metastasis in axillary lymph nodes could be monitored by quantification of CK19and CEA mRNA. The progression of breast cancer could be evaluated more exactly by combining CK19and CEA, which might contribute to the decision on individual therapy. Thus, it might be a useful gene diagnostic method for predicting prognosis and monitoring the therapeutic effect of breast cancer patients.