Microsatellite Instability And Mismatch Repair Genes Expression In Sporadic Breast Cancer And Precancerous Lesion

Breast cancer is the most common malignancy among females affecting approximately one out of ten women. Worldwide incidence of breast cancer continues to rise in women of all nationalities, with disproportionate increases now occurring in countries historically characterized by a low prevalence of breast cancer. Commonly, the occurrence of breast cancer is related to the modern, western lifestyle. In the USA, breast cancer has become the leading cause of death in women aged 35-54 years. In China, the incidence of this malignancy is increasing by speed of 3% per year along with the change of lifestyle. However, early diagnosis and treatment are helpful to enhance curability and survival. Studies indicate that the curability of breast cancer in the early stage is high to 80%-90%, while that in late stage is only about 40%. Therefore, it is necessary to understand the mechanism of breast carcinogenesis deeply and seek the useful genetic markers in the early stage.So far, many breast cancer predisposing factors have been identified includingadvanced age, low parity, delayed age at first delivery, short duration of breastfeeding and so on;however, all these diverse risks can be assigned to either of two major categories: excessive exposure to estrogens and deficiency in maintenance of genomic integrity. It is suggested since recently, that some of estrogen metabolites were shown to cause DNA damage directly (i.e. contribute in the breast cancer initiation). Generally, breast carcinomas arise through a well-defined, but non-obligatory, sequence of histological changes from normal epithelium through hyperplasia, atypical hyplasia, in situ carcinoma, and invasive malignant disease. This sequence is a multigenetic and multistep progression. Recent knowledge in molecular biology indicates that breast cancer is a genetic disease. Like other human cancers, it is thought to occur as the result of progressive accumulation genetic aberrations. Several changes at the cellular level usually take place sequentially, but genetic changes accumulate stochastically, with some genetic alterations linked to specific stages of differentiation and other changes apparently unlinked to tumor development. Instability of the genome in human breast cells is an important contributor to heritable and somatic genetic changes that drive tumorigenic processes. Genomic instability is highly complex, because breast carcinomas develop with a range of genetic alterations, and even subtle genetic differences can have a striking effect on clinical outcome. It has been proposed that genomic instability in human cancers can be divided into two types: one is microsatellite instability (MSI), which is usually equated with DNA polymerase error, and the other is chromosomal instability (CIN) or loss of heterozygosity (LOH), which can result from errors in chromosome partitioning. In this study, we investigated the MSI status in breast cancer and precancerous lesion as well as its correlation with the clinicopathologic features of breast cancer.Microsatellites (MS) are those short, tandem, repeated nucleotide sequences thatrepeat unit is less than 10 bp interspersed throughout the genome and are useful markers for genetic mapping. In recent years, more and more microsatellite markers have been cloned and located. Meanwhile, results of human genome project research have directly provided people probe used for sequence analysis and primer sequence. Analyses of microsatellite combining with their character of abundant polymorphism and low mutation have made an important contribution to our understanding and widespread application of tumor genetic. Microsatellite instability (MSI) indicates the changes in number of repeat unit. Addition to mainly reported in HNPCC, MSI is also present in significant subsets of nonhereditary forms of cancer of the gastric, the endometrium, the lung, the ovarian and the soft-tissue. The status and relevance of MSI in sporadic breast cancer is currently under debate. However, it is difficult to compare various outcomes of different groups for lack of uniform standard. The occurrence of MSI is related to mismatch repair genes (MMR). Many recent studies have implicated that MMR deficiency, especially genetic or epigenetic alteration of two key MMR genes hMSH2 and hMLHl were associated with some cancers displaying MSI. Nevertheless, some other researchers did not agree that MSI was all caused by MMR genes deficiency. To reveal the relationship between MSI and expression of MMR genes, we evaluated the expression of mismatch repair proteins hMSH2 and hMLHl by immunohistochemistry as well.In this study, 41 breast cancers and corresponding-tissues were collected from patients underwent operation at the Second Affiliated Hospital of ZheJiang University College of Medicine between 2002 and 2003. All the specimens were freshly taken during the operation and frozen in -80 °C immediately. Besides, 13 precancerous lesions which were archival paraffin-embedded samples and 7 couples of benign hyperplasia were included for comparion. A panel of 12microsatellite markers were analyzed by polymerase chain reaction (PCR)-single strand length polymorphism (SSLP)-silver stain. The expression of proteins of hMSH2 and hMLHl was detected by immunohistochemistry. Results were statistically analyzed by SPSS 12.0 software. The X2-test or Fisher's exact test was employed for statistical analysis of the status of MSI and MMR protein expression and clinicopathological parameters. Spearman's-test was used for correlation analysis. Ap value less than 0.05 was considered significant.MSI analysis results revealed that 15 out of 41 (36.6 %) of the carcinomas were observed MSI in more than 2 microsatellite markers, almost all belonging to low or intermediate differentiation. MSI was highly associated with poor differentiation (p = 0.033) in breast cancers. There was no significant association seen between MSI status and other clinicopathological variables. 9 of the 13 cases of precancerous lesion showed instabelity, amongst which 2 cases had more than 2 MSI sites. There was no MSI in benign hyperplasia. MSI was targeted predominately at D3S1766, D2S2739 in both of carcinomas and precancerous lesions.There were 40 effective cases in immunohistochemical analyses. The positive expression of hMLHl and hMSH2 protein were detected in breast carcinomas in scattered form and their positive rate were 45 % and 40 %, respectively. In precancerous lesions, hMLHl and hMSH2 protein showed diffuse expression and positive rate were 61.54 % and 76.92 % showing significant less from the control tissues in which positive rate of the two protein were 92.9% and 100% (P > 0.05). The positive expression of hMLHl and hMSH2 protein in grade of high differentiation were much more than that in low-intermediate differentiation. Attenuated expression of hMSH2 in breast cancers were much more than in precancerous lesions, whereas there was no statistical difference between those two diseases for hMLHl. All MSI-positive tumors showed abnormal expression ofeither hMLHl (66.7%) or hMSH2 (77.3%), absent expression rate of the MMR protein was 100% while only 52% of MSI-L and MSS tumors showed absent expression of the two proteins. There was a strong negative correlation between the expression of the two proteins and MSI-positive (y - -0.507, p = 0.001).In conclusion, defective expression of MMR genes is closely associated with the development of breast cancer. Genomic instability might play a role in an early stage during the multi-step breast-carcinogenesis. MSI indicates poor histological differentiation in breast carcinoma. D3S1766 and D2S2739 might be the sensitive sites to detect MSI in breast carcinoma transformation.