In non-tumorous cells, cyclin D1, together with the cyclins D2 and D3, is involved in regulating progression from G 1 to the S phase of the cell cycle (Barnes and Gillett, 1998;Steeg and Zhou, 1998). These cyclins form complexes with cdk4 or cdk6, which can then phosphorylate the retinoblastoma (Rb) protein, resulting in the release of E2F transcription factors and allowing cells to progress into the S phase. Cyclin D1 has other regulatory effects. Specifically, it has been implicated in the replication and repair of DNA, as it can bind to PCNA (proliferating cell nuclear antigen) (Xiong et al., 1992), a protein involved in DNA synthesis, and cells that overexpress cyclin D1 are unable to repair DNA damage induced by ultraviolet radiation (Pagano et al., 1994). Experimentally, a transfected rat liver cell line that overexpresses cyclin D1 showed an increased number of cells with CAD amplification, suggesting that under specific conditions cyclin D1 may enhance gene amplification and contribute to genomic instability (Zhou et al., 1996)
Given the multifarious effects of cyclin D1 on cell function and genomic integrity, it might be postulated that perturbations of normal cyclin D1 levels are related to altered cancer risk (Zhou et al., 2000). Indeed, since cyclin D1 amplification and/or protein overexpression have been observed not only in breast cancer (Buckley et al., 1993;Zhang et al., 1994;Zukerberg et al., 1995;Frierson et al., 1996;Barbareschi et al., 1997) but also in the putative early stages of breast neoplasia (Weinstat-Saslow et al., 1995;Simpson et al., 1997;Alle et al., 1998;Gillett et al., 1998;Zhu et al., 1998), it might be postulated that such changes contribute to breast cancer development. Therefore, in the cohort study reported here, we investigated whether the occurrence of cyclin D1 gene amplification and/or protein overexpression in benign breast tissue might identify women at increased risk of subsequent breast cancer development.
Materials and methods
Subjects and methods
The study methods have been described in detail elsewhere (Rohan et al., 1998). In brief, the investigation was undertaken as a case–control study nested within the cohort of 4888 women in the National Breast Screening Study (NBSS) who received a histopathological diagnosis of benign breast disease during the active follow-up phase of the NBSS. The NBSS is a multicentre randomized controlled trial of screening for breast cancer in 89 835 Canadian women who were recruited between 1980 and 1985, and who were followed actively until 1988 and passively thereafter (Miller et al., 1981, 1992). Women were eligible to participate if they were 40–59 years old and had no previous history of breast cancer (in situ or invasive).
Diagnosis of breast disease in the NBSS. In the NBSS, patients with clinical or radiological evidence of a lesion underwent either needle aspirates or biopsies. For those subjects who had biopsies, the histological sections were reviewed for study purposes by a reference pathologist. The study reported here was restricted to subjects who had no evidence of breast cancer (in situ or invasive) in their initial surgical biopsy as determined on review by an NBSS reference pathologist. Women with a history of previous benign breast disease were not excluded from participation. The characteristics of the cohort have been described previously (Rohan et al., 1998).
Ascertaining outcome. Incident cases of breast cancer were ascertained by record linkage with the provincial cancer registries, and a death clearance was performed by linkage to the Canadian National Mortality Database (Miller et al., 1992). The dates of the linkages varied by province, ranging from the end of 1988 to early 1991.
Definition of cases. Cases were the 92 women with a histological diagnosis of benign breast disease made by a reference pathologist during the active follow-up phase of the NBSS and who subsequently developed breast cancer. In this study, cancer was defined as any form of breast carcinoma; there were 16 cases with ductal carcinoma in situ (DCIS) and 76 cases with invasive carcinoma.
Definition and selection of controls. Controls were women with benign breast disease who had not developed breast cancer by (but were alive at) the date of diagnosis of the corresponding case. Five controls were selected randomly for each case from those non-cases available within strata defined by screening centre: NBSS study arm, year of birth and age at diagnosis of benign breast disease.
Questionnaires. At the time of their enrolment in the NBSS, all participants completed a questionnaire that sought data on potential breast cancer risk factors, including demographic characteristics, family history of breast cancer, and menstrual and reproductive history.
Acquisition of paraffin-embedded blocks of breast tissue. For the present study, hospitals and clinics storing the paraffin-embedded blocks of benign and malignant tissue were asked to send one representative block per lesion and to indicate the fixative type and whether the tissue had been frozen prior to fixation. Blocks or sections of paraffin-embedded benign tissue were obtained for 74 (80.4%) of the 92 cases and for 349 (75.9%) of the 460 controls; blocks or sections of malignant tissue were obtained for 62 (83.8%) of the 74 cases (Rohan et al., 1998).
Histopathology review. Sections from the blocks received were reviewed and classified according to the criteria developed by Page and Anderson (1987) and the recent consensus conference (Schwartz et al., 1997), without knowledge of the case–control status of the study subjects.
Breast cancer cell lines
The human breast carcinoma-derived cell lines ZR-75-1, which has two- to fivefold amplification of cyclin D1 (Bartkova et al., 1994), MDA-MB-231, which shows no cyclin D1 gene amplification (Frierson et al., 1996), and T47D, which shows cyclin D1 overexpression immunohistochemically (Bartkova et al., 1994), were obtained from the American Type Culture Collection (ATCC). The cells were grown in culture, harvested using trypsin–EDTA (Sigma Chemical Co, St Louis, MO, USA), and centrifuged to form pellets. The cell pellets were placed in 3% bacto-agar (Difco Laboratories, Detroit, MI, USA), fixed in 10% buffered formalin and then embedded in paraffin. Sections (5 μm) were cut and used as controls for the polymerase chain reaction (PCR) and/or immunostaining.
Cyclin D1 immunostaining. Immunostaining was performed as described previously (Zhu et al., 1998). Briefly, tissue sections that had been stored for up to 3 years underwent antigen retrieval (microwave pretreatment in 10 mmol/l citrate buffer, pH 6.0, for 15 minutes at a medium-high setting) and were incubated overnight at 4°C with antibody reactive with cyclin D1 protein (monoclonal, dilution 1:2000; Upstate Biotechnology, Lake Placid, NY, USA). After washing, the sections were incubated with biotinylated anti-mouse immunoglobulin G (dilution 1:200; Vector Laboratories, Burlingame, California, USA) for 30 minutes at room temperature, followed by avidin–biotin–peroxidase complex (Vectastain Elite ABC Kit; Vector Laboratories). Immunoreactivity was visualized with 3,3′-diaminobenzidine (Vector Laboratories), and the sections were counterstained briefly with haematoxylin. T47D cells embedded in paraffin served as the positive control. The negative control consisted of replacing the primary antibody with Tris-buffered saline or non-immune mouse serum (Dako, Carpinteria, CA, USA). Distinct nuclear staining in more than 1% of epithelial cells indicated a positive reaction and cytoplasmic staining was considered non-specific and interpreted as negative.
Determination of cyclin D1 gene amplification. Tissue microdissection. Sections (5 μm) were stained briefly with haematoxylin to visualize the epithelium. The sections were matched to the corresponding immunostained sections. The epithelium in the area of the tissue corresponding to that which had shown cyclin D1 immunoreactivity was microdissected out and placed in a microfuge tube. If the tissue had shown no immunostaining for cyclin D1 the corresponding section underwent random microdissection of epithelium. DNA was extracted by incubating the microdissected tissue in buffer (50 mmol/l Tris–HCl, pH 8.5, 10 mmol/l EDTA, 0.5% Tween 20) containing 0.5 mg/ml of proteinase K (Sigma Chemical Co) at 55°C for 48 h. The proteinase K was then inactivated by heating at 95°C for 15 minutes.
Differential polymerase chain reaction. Semiquantitative differential PCR was used to determine the presence of cyclin D1 gene amplification (Zhu et al., 1998). As fragmented genomic DNA (<200 bp) may influence the results of differential PCR, interferon ? (IFN?), which is a single copy gene, was analysed in a multiplex PCR reaction in order to indirectly assess DNA quality first, as described previously (Frye et al., 1989;Neubauer et al., 1992;Zhu et al., 1998). If the IFN?82/IFN?150 ratio of the PCR products was 3 or less, the tissue was considered suitable for further analysis. For such cases, aliquots of the proteinase K-digested tissue were then examined for cyclin D1 amplification using PCR. If chromosomal aneuploidy is present within the tissue it might simulate amplification, resulting in a false positive result. To prevent this we selected the dopamine receptor (DR) for co-amplification, as it is present on the same chromosome as cyclin D1 (Grandy et al., 1989;Gramlich et al., 1994), yet of sufficient distance from cyclin D1 that it is unlikely to be part of an amplified amplicon. Each run included DNA extracted from paraffin-embedded cell lines, MDA-MB-231 (negative control) and ZR-75-1 (positive control).
Briefly, 1 μl of the digest was mixed with 14 μl of PCR working solution containing 50 mmol/l Tris–HCl, pH 8.3, 50 mmol/l KCl, 1.5 mmol/l MgCl 2 , 0.01% gelatin, 100 μmol/l of each dNTP, 1 U of AmpliTaq DNA polymerase (Roche Diagnostic Systems Inc., Branchburg, New Jersey, USA) and 0.4 μmol/l of each primer. The primers and PCR conditions are shown in Table 1
. The PCR products were separated on a 12% polyacrylamide gel at 200 V for 2 hours and visualized following ethidium bromide staining. DNA from each sample was analysed at least twice in separate polymerase chain reactions. Samples showing reproducible amplification of cyclin D1 then underwent a third PCR that included samples of DNA that had been extracted from the patient's breast stromal tissue. As cyclin D1 is not amplified in this tissue, it served as an internal control for the presence of cyclin D1 amplification in the breast epithelium. Direct sequencing of selected PCR products using the sense primer and the Thermo Sequenase radiolabelled terminator cycle sequencing kit (Amersham Life Sciences, Cleveland, Ohio, USA) confirmed that the product was cyclin D1 as described previously (Zhu et al., 1998).
Quantification of cyclin D1 amplification. To determine whether there was cyclin D1 gene amplification, the ratio of the cyclin D1 PCR product to the DR PCR product was derived from photographic negatives of ethidium bromide-stained gels. The bands were quantified by laser densitometry (Computing Densitometer Model 300A, Molecular Dynamics, Sunnyvale, CA, USA). There were at least two gels per PCR product and each gel was scanned two times. A mean ratio of cyclin D1 to DR of greater than 0.88 was considered indicative of gene amplification. This value was determined by identifying the point two standard deviations above the average of the ratios (n = 93) obtained from the control cell line, MDA-MB-231, that had no gene amplification.
c-erbB-2 protein overexpression and p53 protein accumulation. Immunostaining for c-erbB-2 and p53 was performed as described previously (Rohan et al., 1998).
Odds ratios (OR) and 95% confidence intervals (CI) for the associations between cyclin D1 protein overexpression and gene amplification and risk of breast cancer were obtained from conditional logistic regression models (Breslow and Day, 1980). Adjusted odds ratio estimates were obtained by including terms representing the following potential confounders in the regression models: history of breast cancer in a first-degree relative, age at menarche, age at first live birth, menopausal status (pre-, peri- and post-menopausal), body mass index [weight (kg)/height (m) 2 ], and hyperplasia (ductal or lobular, with or without atypia). For categorical variables, tests for trend (on one degree of freedom) in associations were performed by fitting the categorized variables as continuous variables in conditional logistic regression models. Further analyses included within individual comparisons of cyclin D1 in benign breast disease and breast cancer. All statistical tests were two-sided.
As described previously (Rohan et al., 1998), blocks of benign tissue were obtained for 74 (80.4%) of the 92 cases and for 349 (75.9%) of the 460 controls; however, blocks were stained for 309 of the controls only, since for 40 controls benign tissue was not obtained for the corresponding case. For three cases and 15 controls, the benign tissue was inadequate for immunohistochemical analyses, and therefore the statistical analyses were based on 71 cases and 293 controls. DNA was extracted from paraffin blocks and yielded sufficient DNA suitable for analysis in a total of 356 subjects (69 cases, 287 controls).
As shown previously (Rohan et al., 1998) in this study population, risk of breast cancer was altered little in association with a family history of breast cancer, age at menarche, age at first live birth, menopausal status, Quetelet's index, and the presence of hyperplasia in benign tissue. However, the patterns of risk were mostly in accord with expectation. Also, there were few differences between those subjects for whom benign tissue was and was not obtained with respect to their distributions by breast cancer risk factors.
Of the subjects whose benign tissue was suitable for immunohistochemical analysis, 15 cases and 60 controls showed evidence of cyclin D1 immunostaining (Fig. 1
and Table 2
). There was essentially no association between cyclin D1 protein overexpression in benign breast tissue and risk of subsequent breast cancer, and there was little variation in risk by the percentage of cells showing immunostaining. Twelve cases and 29 controls showed cyclin D1 gene amplification (Fig. 2
and Table 2). After adjustment for potential confounding, there was a statistically non-significant 40% increase in risk of breast cancer in association with cyclin D1 gene amplification. For those with hyperplasia and cyclin D1 immunostaining, the adjusted OR was 1.66 (95% CI 0.75–3.71); for those with hyperplasia and cyclin D1 gene amplification, the adjusted OR was 1.55 (95% CI 0.52–4.69). Compared to those with neither cyclin D1 immunostaining nor cyclin D1 gene amplification, the adjusted OR (95% CI) for those with either or both of these changes was 1.47 (0.71–3.03) and 0.99 (0.34–2.90), respectively. As oestrogens regulate cyclin D1 expression (Prall et al., 1998) cyclin D1 levels might vary during the menstrual cycle. However, additional adjustment for days since last menstrual period had little effect on the odds ratio reported in Table 2 (data not shown).
After exclusion of the 19 cases (and their matched controls) whose diagnosis of breast cancer occurred within one year of their diagnosis of benign breast disease, the adjusted OR (95% CI) for the associations between cyclin D1 protein overexpression and gene amplification and risk of breast cancer were 1.15 (0.53–2.50) and 2.27 (0.90–5.71), respectively. When the analyses were restricted to the matched case–control sets containing cases with invasive breast cancer (that is, after exclusion of the 14 cases with DCIS and their matched controls), the adjusted OR for cyclin D1 immunopositivity was 1.29 (95% CI 0.58–2.87), while the adjusted OR for cyclin D1 gene amplification was 1.79 (95% CI 0.66–4.86). Also, when the 23 cases whose benign and malignant lesions occurred in opposite breasts were excluded, the adjusted ORs for cyclin D1 immunopositivity and gene amplification were 1.08 (95% CI 0.47–2.48) and 1.55 (95% CI 0.53–4.52), respectively. Also the results for cyclin D1 immunostaining and gene amplification did not differ between strata defined by age, menopausal status, NBSS study arm, history of previous breast disease, and whether the benign breast disease was screen-detected or interval-detected.
As described elsewhere (Rohan et al., 1998), risk of breast cancer was increased in those with positive immunostaining for p53 in their benign breast tissue but not in those with immunostaining for c-erbB-2. When risk was examined according to the number of markers for which positive immunostaining was observed (relative to the risk in those with negative immunostaining for all three markers), the adjusted OR (95% CI) associated with positive immunostaining for one only and for two or more markers (only one case and one control had positive immunostaining for all three markers) were 1.19 (0.63–2.23) and 0.96 (0.33–2.81), respectively.
shows the concordance between the immunohistochemical and gene amplification findings for the benign and malignant tissue for the cases. Of the 39 subjects who were negative for cyclin D1 protein overexpression in their benign tissue, about 31% (12/39) showed evidence of overexpression in their malignant tissue; four (28.6%) of the 14 subjects with immunostaining in their benign tissue had cancers which did not show immunostaining. For gene amplification, the corresponding values were 21.2% (7/33) and 77.8% (7/9).
Cyclin D1 gene amplification and/or protein expression has been detected in non-cancerous breast tissue. Breast epithelium that is either normal or has changes of benign breast disease, including breast papillomas, can show cyclin D1 protein overexpression immunohistochemically (Alle et al., 1998;Gillett et al., 1998;Zhu et al., 1998;Saddik et al., 1999). The frequency of cyclin D1 protein overexpression is greater in proliferative disease with atypia than in normal epithelium or in the presence of proliferative disease without atypia, as demonstrated in two studies (Alle et al., 1998;Zhu et al., 1998). In one of those studies (Zhu et al., 1998), cyclin D1 gene amplification was also examined and was detected in normal and benign breast tissue. In an in situ hybridization study, 18% of benign breast lesions showed cyclin D1 mRNA overexpression (Weinstat-Saslow et al., 1995). Cyclin D1 gene amplification and overexpression, as well as protein accumulation, can also occur in ductal carcinoma in situ (DCIS) (Weinstat-Saslow et al., 1995;Simpson et al., 1997;Alle et al., 1998;Gillett et al., 1998;Zhu et al., 1998) and breast cancer (Buckley et al., 1993;Zhang et al., 1994;Zukerberg et al., 1995;Frierson et al., 1996;Barbareschi et al., 1997). In the latter, cyclin D1 accumulation, as detected immunohistochemically, has been observed in up to 81% of cases (Zukerberg et al., 1995) and gene amplification has been observed in between 11 and 23% of cases (Lammie et al., 1991;Zhang et al., 1994;Courjal et al., 1996;Frierson et al., 1996;Worsley et al., 1996).
In the present study cyclin D1 gene amplification and protein overexpression were detected in breast tissues at similar frequencies to those reported by others. However, we did not observe associations between the presence of either or both of these cyclin D1 alterations and breast cancer risk. Furthermore, the presence of either cyclin D1 amplification and/or protein overexpression in combination with epithelial hyperplasia was not associated with altered risk. There were too few cases of epithelial hyperplasia with atypia to permit a statistically meaningful evaluation of risk in association with this histological abnormality and cyclin D1 changes.
The benign breast tissue of 75 subjects showed positive immunostaining whereas the benign tissue of only 41 subjects showed gene amplification. The lack of correlation between cyclin D1 gene amplification and protein overexpression has been described previously (Frierson et al., 1996;Pelosio et al., 1996;Worsley et al., 1996;Simpson et al., 1997;Zhu et al., 1998). It has been suggested that mechanisms other than gene amplification, such as post-transcriptional or post-translational mechanisms, could cause increased levels of cyclin D1 protein (Worsley et al., 1996;Simpson et al., 1997). For example, accumulation of cyclin D1 protein can occur because of transactivation of the cyclin D1 promoter by β-catenin (Lin et al., 2000), or increased stability of the protein, as has been demonstrated in human uterine sarcomas (Welcker et al., 1996), or decreased proteolysis, as has been shown to occur in the MCF-7 breast cancer cell line (Russell et al., 1999). The mechanism(s) causing cyclin D1 protein overexpression in the absence of gene amplification in these breast tissues is unknown.
It is possible that the methodology used in this study influenced the results. The differential PCR assay used to determine whether gene amplification was present was assessed previously for sensitivity and reproducibility (Zhu et al., 1998). Although a different housekeeping gene was used in that study, the results suggested that differential PCR is appropriate for determining whether the cyclin D1 gene is amplified and that it is sufficiently sensitive to detect twofold gene amplification. In addition, other studies have utilized this approach for semiquantitation of gene amplification (Nakagawa et al., 1995;Schneeberger et al., 1998;Suzuki et al., 1998). In terms of the immunostaining, the methodology that was used may have resulted in an underestimation of the number of subjects overexpressing cyclin D1. It has been shown that the type and duration of tissue fixation, the sensitivity of the antibody, and the extent of tissue sampling may influence the sensitivity of immunohistochemistry (Elias, 1996;Rohan et al., 1998). Also, given that the controls were women with biopsy-proven benign breast disease and that their risk for subsequent breast cancer is higher than that for women without benign breast disease (Page and Anderson, 1987) it is possible that we underestimated the magnitude of the association between cyclin D1 gene overexpression or protein accumulation and breast cancer risk.
Other than methodological limitations, there are several possible reasons why cyclin D1 changes were not associated with altered breast cancer risk. First, recent experimental data suggest that cyclin D1 is not a dominant oncogene but requires the presence of other oncogenes to form tumours (Barnes and Gillett, 1998). For example, transformation of BRK cells occurred when cyclin D1 was transfected together with the adenovirus E1A oncogene (Hinds et al., 1994) and transformation of rat fibroblasts by cyclin D1 required the presence of Ha-ras (Lovec et al., 1994). Also, transgenic mice containing cyclin D1 linked to an immunoglobulin enhancer rarely developed lymphoma until the mice were crossed with mice expressing the myc transgene (Bodrug et al., 1994). Secondly, the quantification of cyclin D1 gene amplification suggested that the level of amplification in the benign tissue was in the range of that observed in the ZR-75-1 cell line, which shows two- to fivefold amplification. This low level of amplification may be insufficient to affect cell proliferation. Thirdly, the effect of cyclin D1 on the cell cycle is controversial, as stable transfection of cyclin D1 into HBL-100, a mammary epithelial cell line, resulted in longer doubling time, an increased percentage of cells in S phase, and decreased tumorigenesis (Han et al., 1995). This is different from the effect observed for rat fibroblasts, suggesting that the effect of cyclin D1 overexpression may be dependent on the cell type and which other genes are expressed (Jiang et al., 1993). Fourthly, the presence of increased cyclin D1 mRNA levels (Utsumi et al., 2000) or moderate to strong staining for cyclin D1 (Gillett et al., 1996) in breast cancer has been associated with a better prognosis, an observation that raises the possibility that cyclin D1 may not be involved in the pathogenesis of breast cancer.
In conclusion, in this study cyclin D1 amplification and/or protein overexpression in normal or benign breast tissue were not associated with increased risk of developing breast cancer. As experimental evidence suggests that cyclin D1 requires other oncogenes to induce tumorigenesis, assessment of cyclin D1 alterations alone may not be sufficient to identify women at increased risk of breast cancer. Instead, it may not be until the cascade of molecular alterations leading to breast cancer development (Beckmann et al., 1997;Ingvarsson, 1999) are known that the putative role of cyclin D1 in this process can be identified.
We thank Lori Cutler and Isabelle Schell for their secretarial assistance. This work was supported by the US Army Medical Research and Materiel Command and was presented in part at the American Association for Cancer Research, Philadelphia, PA, 1–5 April 2000.
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