The Study Of Chromosomal Instability In Primary Breast Cancer

[Background]Breast cancer is a leading cause of cancer-related morbidity and mortality among women worldwide. As cancers evolve, their genomes undergo many alterations, including point mutations, rearrangements, deletions, and amplifications (often including growth control genes, anti-apoptotic genes and cell cycle checkpoint genes). An understanding of these changes will allow the design of more rational therapies and, by providing precise diagnostic criteria, allow fitting the correct therapy to each patient according to need.Chromosomal instability refers to the rate with which whole chromosomes or large portions thereof are gained or lost in cancers. The accumulation of chromosomal instability is characteristic of all carcinomas, including breast cancer. Chromosomal instability has been proposed to play an important role in cancer by accelerating the accumulation of genetic changes responsible for cancer cell evolution.More recently, molecular profiling methods have been used to identify clinically-relevant tumor features. Expression profiling has been very effective at revealing phenotypic subtypes of breast cancer and clinically useful diagnostic patterns of gene expression in tumors. But microarray studies have not had sufficient resolution to detect copy number. Recent technological advances have provided platforms that allow genomic amplifications and deletions to be analyzed in association studies. The combination of the two newly developed techniques ROMA (representational oligonucleotide microarray analysis) and QM-FISH(quantitative multi-gene fluorescent in situ hybridization) allow high resolution genomic analysis of specific deletions and amplifications. ROMA is a high sensitive and specific microarray technique and measures copy number values across the genome averaged over the population of a tumor. QM-FISH is a cytological technique that measures copy number values in individual cells. It has recently been shown that the QM-FISH technique works on formaldehyde fixed tumour cells in paraffin blocks. QM-FISH is therefore a very suitable technique for large-scale retrospective clinical studies. In summary ROMA is the technique to identify interesting chromosomal regions and genes, but QM-FISH is the technique of choice to quantify copy number changes of these genes or chromosomal regions in clinical tumour samples. These two powerful tools open the possibility for high resolution genome wide analysis and a detailed study of chromosomal instabilities.The p53 gene is often referred as the gene most related to tumours. The p53 gene is mutated in many, but not all human malignancies.MDM2 and MDM4 are the key negative regulators of p53 function in vivo. MDM2 and MDMx overexpression have been observed in a subset of human tumors, some of which retain wild-type p53.The thesis composed of two parts. The specific aim of the first study was scanning a panel of specific molecular markers for identifying the genetic aberration of individual patients, defining the distinct genetic subtypes of breast cancer and comparison of patterns of chromosomal alterations in DCIS(ductal carcinoma in situ) and their concurrent invasive breast cancer. High resolution genome analysis in primary breast cancer was performed by QM-FISH based on the previous ROMA analysis. Furthermore, we explored associations between CNAs and clinicopathological parameters in breast cancer. The second study was focused on evaluation of abnormalities of p53, MDMx and MDM2 genes and the relationships between each other. Here we assessed the genetic instability at p53, MDMx and MDM2 using FISH protocol and detected the expression status of P53 and MDM2 proteins by immunohistochemistry in 34 archived primary breast cancers. This study may provide a basis for improved patient prognostication, as well as a starting point to define important genes contributing to breast cancer development and progression.[Methods]The first part was based on thirty-four primary invasive breast carcinoma samples with foci DCIS diagnosed at the Department of Pathology, Qilu Hospital of Shandong University. The part of the tissue that contains normal ducts or lobules, DCIS and invasive carcinoma was cored and placed in the recipient block forming the array. Frequently amplified and deleted loci were chosen based on the previous ROMA data and CGH data. Hybridization probes for FISH were created from bacterial artificial chromosomes (BAC) selected using the UCSC Genome Browser. QM-FISH for 30 genes were performed on 4μm slides. Evaluation of signals was carried out in an epifluorescence microscope. Selected cells were photographed in a Zeiss Axioplan 2 microscope equipped with an Axio Cam MRM CCD camera and Axio Vision software. The difference of patterns of chromosomal alterations in DCIS and invasive breast cancer and the correlation between FISH data and clinicopathological parameters were evaluated.In the second study, to evaluate the abnormalities of p53, MDMx and MDM2 genes and the relationship between each other, we assessed the genetic instability at p53, MDMx and MDM2 loci using FISH protocol and detected the expression status of p53 and MDM2 by immunohistochemistry in 34 archived primary breast cancers. The interaction of three genes and their correlation with clinicopathological parameters were also assessed.[Results]1. Overall, among the 34 breast cancers,11 were D-tumors(32%) and 23 were A-tumors (68%). 2. All cases had at least two chromosomal aberrations out of 30 loci. The mean number of chromosomal alterations was 6.5.3. Gains of CCNE2, C-erbB2, IGF1R,CKS1a,c-myc,CCND1 and loss of chkl,p53, Rbl,CDHl,chk2, Nek9 were observed in more than 25% of the analyzed cases .Gain of MDMx is the most frequently affected chromosomal region in breast cancer.4. The invasive component of individuals breast carcinoma totally presented 222 CNAs by QM-FISH, however the DCIS component presented 194 CNAs (P>0.05). All the pairs showed a strong similarity between the DCIS and IDC with few differences.5. There are significant association between CCND1 amplification and chk1 deletion (P <0.05), c-myc amplification and LZST1 deletion (P<0.01), MAPK3 amplification and JARID2 deletion (P<0.001), WTAP amplification and TPTE deletion(P <0.001), c-myc amplification and CCNE2 amplification(P <0.001).Inverse relationship between LOH (loss of heterozygosity) of p53 and MDMx amplification was found(P <0.05).6. The frequencies of occurrence of the gene copy number gains/losses in larger tumors(>2cm), A-tumors, and node positive tumors were higher than in smaller tumors, D-tumors and node negative tumors (P<0.05) .7. Allelic loss of p53 was detected in 47% (16/34) of the cases and amplification of MDMx was found in 20 cases (59%), while MDM2 amplification was identified only in one of these samples. But 27 out of 34 cases (79%) showed MDM2 overexpression indicating MDM2 amplification is an uncommon event in primary breast cancer.8. Most tumors contained either p53 dysfunction or MDM2 alteration, but not both. This distribution was significant (P < 0.01). Significant inverse correlation between MDM4 amplification and p53 expression was also observed (P < 0.05).[Conclusions]1. Breast cancer develops multiple chromosomal alteration (including gene amplifications and deletions) as they evolve. 2. Frequently affected gene aberrations include loss of chk1, chk2, Rb1, Nek9, p53 and CDH1, and gains of MDMx,CKS1a,CCNE2, IGF1R,C-erbB2,c-myc and CCND1. The identified CNAs may provide a basis for improved patient prognostication, as well as a starting point to define important genes to further our understanding of the pathobiology of breast cancer.3. Levels of genomic instability are equivalent in DCIS lesions and advanced invasive rumors. DCIS lesions have the same extent of genomic instability as the synchronous invasive carcinomas; thus supporting the notion that invasive carcinomas evolve from or in parallel with DCIS.4. The frequencies of occurrence of the gene copy number gains/losses in larger tumors(>2cm), aneuploid tumors, and node positive tumors were higher than in smaller tumors,diploid tumors and node negative tumors (p<0.05) . Distinct spectra of CNAs underlie the different clinicopathological subtypes of breast cancer.5. Several genes undergo amplifications or deletions in tumorgenensis of breast cancer. Uncontrlled cell cycle is crucial for tumorigenesis6. Human solid tumors commonly develop multiple genetic abnormalities. Single gene alteration can not explain tumorgenensis. Study on interaction of multigenes involved in tumor development and tumor progression would be helpful to understand the mechanism of tumorgenesis.7. Overexpression of MDM2 or MDMx and p53 mutations in primary breast cancer are mutually exclusive events and combined use of MDM2 and MDM4 antagonists should be considered in the treatment of breast cancer expressing wild-type p53.