The Study Of Cell Proliferation, Chromosomal Instability And Gene Copy Number Changes Of CCND1 And Chk1 In Breast Cancer

【Background】Breast cance is one of the most common malignant tumor in women.It is the number one cancer in Europoen countries such as Sweden. Its incidence is increasing in China recently.Numerous studies published during the last decades have clearly shown that genomic instability in terms of degree of aneuploidy and chromosomal rearrangements is closely related to tumor development and tumor progression.Breast tumors of the D-type generally progressed more slowly,and were clinically much less aggressive than their highly aneuploid,genomically unstable counterparts of the A-type.Chromosomal rearrangements in terms of deletions,duplications and amplifications,as studied by comparative genomic hybridization(CGH),were found to be much more frequent in the highly aneuploid breast tumors than in the diploid ones.In addition to genomic instability proliferative activity is a general property to be considered in the progression of tumors.Tumor cell proliferation has been widely investigated in breast cancer for its association with neoplastic growth,progression,metastatic potential,survival and response to chemotherapy.Proliferative activity could be assessed through immunohistochemical procedures detecting proliferation associated antigens,such as Ki67,or cell cycle specific proteins like cyclin A. Various studies have shown that a high expression of Ki67 or cyclin A is correlated with a worse prognosis in breast cancer.Ki67 is expressed during G1,S,G2 and M phases,but not during G0 phase,Cyclin A is known as an S- and G2-M phase regulatory protein.The ratio between Cyclin A and Ki67reflects the frequency of proliferating cells in S,and G2/M phases.High ratio implies more premature tumor cells going through Glcheck point,synthesis and mitosis,thus increases chromosome instability in breast cancer.Chromosomal band 11q13 is frequently amplified in breast carcinoma and assumed to be critically involved in tumor initiation and progression by proto-oncogene activation.It is well documented that the cyclin D1 is involved in the regulation of the G1-S-phase transition of the cell cycle. Overexpression of cyclin D1 has been shown to shorten the G1 phase of the cell cycle and lead to tumor formation.Cyclin D1 overexpression is reported to be more prevalent than CCND1 amplification,with the reported frequency ranging from 40 to 90%of cases of invasive breast cancer in the presence or absence of CCND1 gene amplification,which is observed in about 5-20%of tumors.The Chk1 kinase located on 11q24 is a key regulator of both S phase progression and mitotic entry,and has been proposed to be critical for genomic stability,apoptosis,and drug resistance.Alterations in Chk1 may play a critical role in the etiology of human cancer.To investigate the role of CCND1 and Chk1 in breast cancer and to explore their role in tumorigenesis,we used high resolution fluorescent in situ hybridization(FISH) to detect the copy number changes of CCND1 and chk1 in paraffin sections.The thesis composed of two parts.The specific aim of the first study was to investigate and compare in the same individual tumors,the relative influence of genomic instability and proliferative activity as risk factors for development of distant metastases in breast cancer.As one aspect of genomic instability,the degree of aneuploidy was quantified, and the tumors were separated in the two groups(A or D) with respect to ploidy level.The proliferative activity was analyzed by immunohistochemistry using antibodies against Ki67 and cyclin A.An important methodological aspect of the paper was the direct quantitative comparison performed between the Ki67 analysis and the cyclin A analysis in the same tumor areas.By combining ploidy type(D or A),proliferative activity(low or high),node-status(NO or N+) and tumor size(T1 or T2) breast cancers could be divided into two risk groups with respect to development of distant metastases.A detailed survival curve and Cox multiple factor regression analysis were performed in a large series of patients(n=378) with a long term follow up study(median,114 months). The prognostic values of proliferating markers(cyclin A and Ki67),ploidy, histological grade,tumor size,and axillary lymph node involvement were assessed for the relapse-free survival and tumor-specific survival.We also analysed the correlation between high ratio of cyclin A/Ki67 and the malignant behavior of breast cancer.Cytoplasmic cyclin A expression together with its significance in tumor maglignace was assessed.The second study was focus molecular analysis and clinico-pathological correlation in a set of 33 tumor samples obtained from Shandong Qilu hospital with breast cancer.Fluorescence in situ hybridization(FISH) with two probes(CCND1 and chk1) was performed on the 33 cases of invasive ductal carcinoma with foci of ductal carcimoa in situ(DCIS).The subcellular distribution of cyclin D1 and correlation with CCND1 amplification were also assessed.【Methods】The first part was based on the data of 428 patients with breast cancer analyzed at the department of Oncology-Pathology, Karolinska University Hospital,Solna at the time of diagnosis (1997-1998).All histological specimens were routinely Ki67 and cyclin A stained.In the 428 cases,378 patients available with clinical data were followed up from diagnosis until death or survivors for at least 9 years.By comparison with the H&E-stained sections,images of the same morphology areas expressing Ki67 and cyclin A were taken by digital camera in at least 5 to 14 high power fields(10×40 magnification).The percentage of positive cells was measured by two experienced pathologists blinded to each other.A minimum of 1000 tumor cells were counted.Only distinct nuclear staining was accepted as a positive reaction for both markers,whereas all cells with simultaneous nuclear and cytoplasmic cyclin A staining were regarded as positive for cyclin A.Nuclear DNA was measured by image cytometry on Feulgen stained imprints.The histograms were divided into two groups.Cases with a major peak near the 2c region (1.8c-2.2c) and＜10%cells exceeding 2.2c were denoted diploid.DNA profiles with a stemline outside the diploid and tetraploid region and distinctly scattered DNA values exceeding the tetraploid region (3.8c-4.2c) were classified as aneuploid.Furthermore the S-phase fraction(SPF) was measured based on the DNA distribution patterns. Statistical analyses were performed using the SPSS for Windows version 15.0.In the second study,A total of 33 invasive ductal carcinoma of the breast with foci of DCIS diagnosed at the department of Pathology,Shandong University Qilu Hospital were selected.High resolution two-color FISH was performed to detect the gene copy number changes of CCND1and chk1. Immunohistochemistry was used to assess the expression of ER,PR,C-erbB2, Ki67,P53,and cyclin D1.【Results】1.Aprogressive increase was found for the frequency of Ki67 and cyclin A positive cells from low grade tumor to high grade tumor with statistical significance observed between each group(p＜0.0001).The same trend was observed between breast carcinomas with tumor size＜2cm and tumor size≥2cm(p＜0.0001).No significant difference was found for the frequency of Ki67 and cyclin A positive cells between axillary lymph node positive and negative groups.Statistic difference for SPF was only found between A-tumors and D-tumors.2.The percentage of Ki67 positive cells was highly correlated (correlation coefficient 0.90) to the percentage of cyclin A positive cells.A lower,but still relatively good correlation was found between Ki67 and S-phase fraction(correlation coefficient 0.61) and cyclin A and S-phase fraction(correlation coefficient 0.65).3.Among the tumors with low proliferative activity,using a cut off value of 15%for Ki67 positive cells and 8%for cyclin A positive cells between 6.8 and 7.2%of all tumors had developed distant metastases during the 9-year follow up time.Among the tumors with high proliferative activity between 24.9 and 27.9%of all tumors bad developed distant metastases during the same time period.A significant diference was found between low and high proliferation tumors.4.An increased risk for distant metastases was also seen in the highly aneuploid A-tumors as compared to the near-diploid D-tumors.However, this increased risk could only be demonstrated in small(T1), node-negative(N0) tumors,among which the D-tumors exhibited a risk as low as 2 to 3%and the A-tumors as high as 20 to 25%to develop distant metastases.For the remaining tumor groups T2N0,T1N+ and T2N+ no significant difference between D- and A-tumors could be demonstrated.5.In the group T1N0 all D-tumors,independently of proliferative activity, showed a very low risk(less than 4%) to develop distant metastases.For the A-tumors in the group T1N0 the situation was different.A-tumors with low proliferative activity,however,showed the same low risk to develop distant metastases as the D-tumors,but for the A-tumors with high proliferative activity the risk to develop distant metastases was found to be high(close to 30%) and significantly increased(p＜0.001) over that of the D-tumors with high proliferative activity. 6.For large(T2) node-negative(N0) tumors,or small(T1) node-positive (N+) tumors the risk for distant metastases was only moderately increased for tumors with low proliferative activity(＜20%),and for high proliferative tumors the risk was dramatically increased(＞21%).7.Large(T2) node-positive(N+) tumors were at high risk for metastasis relatively independent of ploidy type or proliferative activity.8.Multivariate analysis showed that proliferation(both Ki67 and cyclin A) was the most critical factor in N+ tumors(p＜0.05) and ploidy was the most critical one in NO tumors(p＜0.05).9.High ratio between cyclin A and Ki67 was related with distant metastases in breast cancer(P＜0.05),especially in patients with axillary lymph node metastases(P＜0.001).10.When evaluated in the entire series of patients,increased expression of both Ki67 and cyclin A were significantly associated with shorter relapse-free survival(P＜0.0001) and tumor-specific survival(P＜0.0001). Only 14.9%(16.9%) patients relapse and 14.3%(15.4%) died with low expression of Ki67 or cyclin A,while as many as 48.4%(39.3%) relapse and 34.6%(37.2%) died if they had tumors with high expression of Ki67 or cyclin A.11.When patients were stratified by tumor size and axillary lymph node involvement,a high expression of both Ki67or cyclin A was associated with both shorter relapse-free survival and tumor-specific survival regardless of tumor size and axillary lymph node status.Increased expression of Ki67 or cyclin A was significantly associated with shorter relapse-free survival and tumor-specific survival observed in intermediate/ high grade tumors,but in low grade tumors,no such correlations were found12.Multivariate Cox regression analysis was performed in 182 invasive ductal carcinoma,including histology grade,nodal status,tumor size, ploidy and expression of Ki67 or cyclin A.The results revealed that high proliferation was an independent factor associated with a shorter survival(Ki67,P=0.003;cyclin A,P=0.001).13.In 428 breast cancer with cyclin A expression,20%cases were found positive cells with strong cytoplasmic staining,cyclin A positive cells with a cytoplasmic staining were related with high tumor grade,DNA aneuploid,high proliferation and high ration between cyclin A/ Ki67.14.Nuclear Cyclin D1 expression was detected in 53(86.9%) of 61 cases of breast cancer with Cytoplasmic cyclin D1 overexpression in 11(18%) of the 53 nuclear positive cases.Cytoplasmic cyclin D1 overexpression showed a strong correlation with strong nuclear Cyclin D1 staining (P＜0.0001),high tumor grade(P＜0.0001)15.CCND1 amplification was observed in 9 of 33 breast carcinomas with foci of DCIS component.Cytoplasmic cyclin D1 staining was found in 7out of 9 tumors with CCND1 amplification.Only one case showed cytoplasmic cyclin D1 staining for the 24 cases without CCND1 amplification.CCND1 amplification showed a strong direct correlation with cytoplasmic cyclin Dlexpression(P＜0.0001).16.Among the 33 infiltrating ductal carcinoma withcases with foci of DCIS, CCND1 amplification and cytoplasmic cyclin D1 expression were observed in both the infiltrating ductal areas and DCIS areas in 7 cases.In two cases with high grade infiltrating ductal areas and low grade DCIS,CCND1 amplification and cytoplasmic cyclin D1 overexpression were only found in the infiltrating ductal areas,but not in the DCIS areas.17.10 cases out of 33 breast carcinoma showed loss of Chkl copy number. 8 cases showed both CCND1 amplification and Chkl deletion.Allele lose of chkl was correlated with CCND1 amplifiction(P＜0.0001).[Conclusions]1.Ki67 and cyclin A correlate strongly to each other. Both of the two markers are good reliable proliferative indicators for estimating the relapse or overall survival in breast cancer. 2.For the low risk group(40%),consisting of all D-tumors in the T1N0 group together with the A-tumors with low proliferative activity in the same group plus all tumors with low proliferative activity in the T2NO and T1N+ groups,the risk to form distant metastases was less than 6%.3.For the high-risk group,consisting of all large(T2) node-positive (N+) tumors and all tumors with high proliferative activity,except the D-tumors in the TINO group,the risk for distant metastases was found to be in the range 30 to 60%,and around 37%in average.4.Cyclin A positive cells with a cytoplasmic staining cyclin A and high ratio between cyclin A/ Ki67 are related with malignant behaviour of breast cancer.5.Strong nuclear overexpression accompanied with cytoplasmic cyclin Dlexpression might predict CCND1 gene amplification in breast carcinoma.6.cyclin Dloverexpression is a early event in the tumorigenesis of breast cancer,CCND1 amplification is related to tumor invasion.7.CCND1 amplification and chkl allele loss often co-exist in breast cancer.