Subscribe Now Subscribe Today
Abstract
Fulltext PDF
References
Research Article
 

Significance of Cyclin D1 Overexpression and Amplification in Ductal Hyperplasia, Carcinoma in situ and Invasive Carcinoma in Egyptian Female Breast



Nehad MR Abd El-Maqsoud and Moatasem M. Aly
 
ABSTRACT

Cyclin D1 is involved in regulating the transition of G1 to S-phase in the cell cycle. Amplification and overexpression of the cyclin D1 gene have been reported to be implicated in breast carcinoma and are suggested to play important roles in breast carcinogenesis. In the present study, we tried to evaluate the correlation between cyclin D1 expression and gene amplification and analyze the correlations between cyclin D1overexpression with clinicopathological features in different breast lesions. Cyclin D1 gene amplification and protein overexpression were assessed in 20 cases of ductal hyperplasia without atypia, 20 cases of atypical ductal hyperplasia, 24 cases ductal carcinoma in situ and 114 cases of invasive carcinoma. Cyclin D1 overexpression was found in 0, 30, 58.3 and 63.2%, respectively. While gene amplification was detected in 0, 0, 16.7 and 15.8%, respectively. In ductal carcinoma in situ, no-significant correlations between either cyclin D1 overexpression or amplification and any of clinicpathological features. In cases of invasive carcinoma, cyclin D1 overexpression and amplification showed a strong direct correlation with expression in both hormonal receptors. There was a significant correlation between cyclin D1 expression and good prognostic parameters, including low histological grade (p = 0.04) and small tumor size (p = 0.003). There was a strong correlation between cyclin Dl overexpression and histological tumor type (p = 0.008). In conclusion, amplification and overexpression of cyclin D1 in atypical ductal hyperplasia, ductal carcinoma in situ and invasive carcinoma suggests its role in early and late stages of breast cancer.

Services
Related Articles in ASCI
Similar Articles in this Journal
Search in Google Scholar
View Citation
Report Citation

 
  How to cite this article:

Nehad MR Abd El-Maqsoud and Moatasem M. Aly, 2010. Significance of Cyclin D1 Overexpression and Amplification in Ductal Hyperplasia, Carcinoma in situ and Invasive Carcinoma in Egyptian Female Breast. International Journal of Cancer Research, 6: 202-219.

DOI: 10.3923/ijcr.2010.202.219

URL: https://scialert.net/abstract/?doi=ijcr.2010.202.219
 
Received: June 24, 2010; Accepted: August 16, 2010; Published: November 04, 2010

INTRODUCTION

Breast cancer is the most common malignancy among Egyptian females accounting for 37.6% of all malignancies in women (Parkin et al., 2002). Breast cancer in Egyptian patients is biologically more aggressive than that encountered in the West. This is explained partly by the predominance of premenopausal patients and partly by the late presentation of patients at an advanced stage (El-Bolkainy et al., 2005).

It was found that genetic alterations play an important role in the development of invasive carcinoma. Preinvasive lesions indicate the presence of intermediate stages in the development of invasive carcinoma in some cases but we still have little understanding of what genetic/epigenetic events are likely to be associated with the earliest phases of the disease (Heffelfinger et al., 2000). Cyclin D1 is claimed to be one of the genes that are known to be involved in preinvasive breast lesions (Naidu et al., 2002).

Gene amplification is a well-defined cause of oncogene activation during tumor development (Luo et al., 2006). Amplification of chromosome locus 11q13 has been reported at high frequencies in a wide variety of human cancers, such as bladder (Zaharieva et al., 2003), lung (Shibata et al., 2005), breast (Elsheikh et al., 2008) and ovarian (Brown et al., 2006) carcinomas.

The cyclins are a family of key regulatory proteins that control the progression of human cells through critical transition points in the cell cycle. Several classes of cyclins have been identified, displaying sequential expression in different phases of the cell cycle. The D-type of cyclins control the transition through the G1 phase enabling the entry into S phase playing a pivotal role in the regulation of progression from the G1 to S phase through the formation of active enzyme complexes with cyclin dependent kinases Cdk4 and Cdk6 (Esteva and Hortobagyi, 2004).

Cyclin D1 overexpression has been reported in invasive breast cancer with expression rates between 35 to 90% of cases. Overexpression may occur with or without CCND1 gene amplification, which is observed in about 5-20% of tumors (Gillett et al., 1994; Barnes, 1997; Naidu et al., 2002; Bieche et al., 2002; Molland et al., 2004; Stendahl et al., 2004; Jirstrom et al., 2005; Ahnstrom et al., 2005; Butt et al., 2005; Reis-Filho et al., 2006) and therefore qualifies as one of the most commonly overexpressed proteins in breast cancer (Ormandy et al., 2003; Butt et al., 2005). Though CCND1 amplification correlates well with the overexpression of the protein (Mrhalova et al., 2002; Jirstrom et al., 2005), high expression of cyclin D1 is not always secondary to gene amplification implying that other mechanisms contribute to maintain cyclin D1 overexpression.

CCND1 amplification has been investigated in breast cancer by Southern blotting (Zukerberg et al., 1995; Cuny et al., 2000), fluorescent in situ hybridization (FISH) (Gillett et al., 1994; Stendahl et al., 2004; Jirstrom et al., 2005) and real-time polymerase chain reaction-based methods (Bieche et al., 2002).

Recently, cyclin D1 overexpression and CCND1 amplification, have received great attention due to data from clinical trials implicating cyclin D1 overexpression in resistance to tamoxifen treatment in postmenopausal (Jirstrom et al., 2005) and in premenopausal breast cancer patients (Stendahl et al., 2004).

Estrogen receptor status has been used in predicting the response to adjuvant tamoxifen therapy for more than 20 years and has remained the most powerful molecular marker for treatment decision. ER-positive invasive ductal carcinoma (IDC) have a 40-70% response rate in tamoxifen treatment (Fitzgibbons et al., 2000). It is not known what makes the difference between responders and non-responders towards therapeutic agents such as Trastuzumab or tamoxifen. It has been suggested that cyclin D1 can bind directly to and activate estrogen receptors independently from estrogen (Neuman et al., 1997).

Until now, the prognostic value of cyclin D1 protein has been controversial, with studies reporting both a positive and negative role in breast cancer, whereas amplification of the CCND1 gene is predominantly related with worse outcome in ER positive patients (Sutherland and Musgrove, 2004).

To the best of our knowledge, this is the first report of simultaneous evaluation of cyclin D1 protein and gene amplification status in both of preinvasive and invasive breast cancer using immunohistochemistry (IHC) and FISH in Egypt, together with studying the correlations between both of them and clinicopathological features with hormone receptor status.

MATERIALS AND METHODS

Patient's Selection
For ductal hyperlasia (DH), atyical ductal hyperlasia (ADH) and ductal carinoma in situ (DCIS) cases, excisional biopsies were taken with full history of the patients from Minia University Hospital and private clinic of the co-author, in the period between 2006 and 2009. Paraffin embedded formalin fixed blocks were prepared for the present study. Twenty cases of DH, 20 cases of ADH without invasive or in situ carcinoma that were accidentally found beside benign breast lesions. ADH was diagnosed using the criteria previously described (Page and Rogers, 1992). Twenty-four cases of DCIS without any associated invasive carcinoma were investigated. No lobular carcinoma in situ (LCIS) cases were found. The histological subtypes were divided into comedo and non-comedo types. The histologic grade was also classified into well, intermediately and poorly differentiated as the previously classified (Holland et al., 1994).

One hundred fourteen cases with invasive carcinoma (IC) were selected for this study. All cases undergone modified radical mastectomy. We were found adjacent DCIS in 42 cases. Tumors were graded according to a modified Bloom-Richardson scoring system (Elston and Ellis, 1991). Tumor staging was performed according to the TNM system of the International Union against Cancer (Hermanek and Sobin, 1992). Tumor size and lymph node status were categorized according to Carter et al. (1989) and Recht et al. (2001).

Regarding DH cases, the mean age of the patient was 42.5±7.47 years (range 30-55 years) and the median of the cases was 42 years. In cases of ADH, the mean age of the patient was 46.7±5.57 years (range 39-54 years) and the median of the cases was 46.5 years. Regarding DCIS cases, the mean age of the patient was 45.75±7.64 years (range 35-60 years) and the median of the cases was 47.5 years. In invasive cases, the mean age was 51.43±8.59 years (range 35-69 years) and the median of the cases was 51 years.

Immunohistochemical Staining
It has been shown in previous studies that for cyclin D1, the use of the rabbit monoclonal antibody SP4, would give reproducible results (Cheuk et al., 2004; Reis-Filho et al., 2006; Elsheikh et al., 2008). The SP4 antibody is a rabbit monoclonal antibody raised against a synthetic peptide from C-terminus of human cyclin D1, which was deemed specific to cyclin D1, identifying a single 36 kDa band on Western blot analysis (Reis-Filho et al., 2006). In addition, the SP4 clone was reported to be at least as specific as other monoclonal antibodies against cyclin D1, but is reported to be more sensitive than other clones (Loden et al., 2002; Cheuk et al., 2004; Reis-Filho et al., 2006). In contrast to other antibodies, the rabbit anticyclin D1 monoclonal antibody shows a strong correlation with CCND1 gene amplification. Therefore, to determine cyclin D1 protein expression we chose this antibody for employing the protocol described previously (Cheuk et al., 2004).

Immunohistochemistry for Cyclin D1 was Performed as follows
First, the sections were dewaxed and hydrated through graded alcohol then the slides are lowered into boiling EDTA buffer (pH 8.0) in a pressure cooker and boil for 3 min under full pressure and cooling down slides in a sink of cold running tap water.

The slides then rinsed in Tris-buffer saline for 3 min and the endogenous peroxidase was blocked with 3% hydrogen peroxide for 3 minutes and incubated with 1:10 dilution of cyclin D1 rabbit monoclonal antibody (clone SP4, LabVision) for 1.5 h and washed with distilled water and incubated with EnVisionanti-rabbit labeled polymer (Dako, Denmark) for 1 h.

Lastly the slides were rinsed with three changes of Tris-buffer saline, 5 min each, incubated with diaminobenzidine solution (Dako, Denmark) for 4 min and checked with microscope for optimal staining then washed thoroughly with distilled water and incubate with 0.5% copper sulphate solution in saline for 6 min.

At last, washing the slides in running tap water and counterstaining nuclei with hematoxylin, dehydrating, clear and mount.

For Hormone Receptors Status

ER and PR expression were evaluated on formalin-fixed and paraffin embedded sections after enhanced microwave antigen retrieval using an anti-estrogen receptor antibody (ER ID5, DAKO) and an anti-progesterone receptor antibody (PR 1A6, DAKO). Staining was performed by a standard streptavidin-biotin-peroxidase technique using DAB for visualization and hematoxylin for nuclear counterstaining.

Evaluation of Staining Results
Scoring of the Cyclin D1 in DH, ADH and DCIS
Due to the restricted number of these lesions, variations in cyclin D1 expression were not seen. Therefore, cases were stratified into positive or negative. Overexpression of cyclin D1 was defined as cells greater than 10% with moderate/strong nuclear staining. This cut-off value was chosen in accordance with the available literature (Fiche et al., 2000; Oh et al., 2001; Lebeau et al., 2003). Faint nuclear staining or cytoplasmic staining was not considered significant.

Scoring of the Cyclin D1 in IC
Scoring of the cyclin D1 reactivity was performed using the Allred method (Harvey et al., 1999; Reis-Filho et al., 2006). With this method, the intensity of the immunohistochemical reaction was recorded as 0, negative (no staining of any nuclei even at high magnification); 1, weak (only visible at high magnification); 2, moderate (readily visible at low magnification) or 3, strong (strikingly positive even at low power magnification). The proportion of tumor nuclei showing positive staining was also recorded as either: 0, no staining; 1, <1% nuclei staining; 2, 1-10%; 3, 11-33%; 4, 34-66% and 5, 67-100% nuclei staining. The proportion and intensity scores were subsequently added to obtain a total score, which ranged from 0 to 8. Tumors were categorized into four groups: negative 0, weak (total scores 1–2), moderate (total scores 3-5) and strong (total scores 6-8). Only nuclear staining was considered specific.

Scoring of the Hormone Receptors
The slides in which more than 10% of tumor cells were stained were scored as positive. Staining intensity was not evaluated (Nishimura et al., 2007).

FISH Study
FISH was performed on 4 μm paraffin sections according to previously reported protocol (Lebeau et al., 2001, 2003). In brief, the slide-mounted tissue sections were air-dried and baked overnight at 56°C. Slides were dewaxed in xylene for 10 min x3, followed by immersion in 100% ethanol for 5 min x2. Air-dried tissue sections were subsequently covered with 1 M NaSCN (Sodium Isothiocyanate) and placed in an oven for 30 min at 80°C. Afterwards slides were washed in Aqua bidest and treated in a pepsin solution (8 mg/mL H2O; pH 2.0) for 30 min at 37°C. Slides were washed in Aqua bidest and air dried. CCND1 gene copies were determined by using a spectrum orange-labelled probe purchased from Vysis (Vysis SA, Maurens Scopont, France). Cyclin D1 probe was prepared as follows (1 μL probe, 2 μL purified water, 7 μL LSI Hybridization Buffer). Prepared probes were centrifuged after preparation. Slides were incubated at 80°C for 20 min. Twenty microliter of the probe in appropriate hybridisation buffer (Vysis SA) were applied to the sections and incubated 10 min at 80°. Hybridisation was carried out overnight at 37°C in a moist chamber. Washes were performed at 42°C three times in 0.1x SSC (sodium chloride/sodium citrate) and Post Hybridization Wash Buffer (PHWB) for 1 min each. Tissue sections were counterstained with DAPI/Vectashield.

Scoring Criteria
Slides were evaluated for CCND1 gene amplification according to previously published criteria (Lebeau et al., 2001) using a Bausch and Lomb fluorescence microscope (Balplan research illuminator) equipped with appropriate filters (Vysis, Inc., Downers Grove, IL). The cyclin D1 gene to chromosome/centromere ratio was measured in at least 60 nuclei from the tumor cells and an average score was taken. More than two copies of cyclin D1 for each chromosome was considered to be a positive sign for cyclin D1 gene amplification

Statistical Analysis
Statistical analyses were performed using the SPSS Version 17 for Windows (SPSS Inc., Chicago, IL) program package. The two-sided Chi-squared (χ2) test was used to compare categorical variables, if the sample size was large. Fischer’s exact test was used when the sample size was small. Student t test was used to compare means of patients ages. The significance level was considered at 0.05.

RESULTS

Cases of DH and ADH
In cases of DH, the expression of cyclin D1 was not found. Among ADH cases, overexpression of cyclin D1 was found in six cases (30%), While CCND1 gene amplification was not identified (Table 1; Fig. 1a, b). We found no significant correlation between cyclin D1 and ER expression in spite that all cases that were positive with cyclin D1 also were positive for ER (p = 0.29).

Cases of DCIS
In the DCIS cases, cyclin D1 overexpression was seen in 14 cases (58.3%) and no staining in ten cases (41.7%). while, CCND1 gene status was amplified in four patients (16.7%) and non-amplified in 20 (83.3%) patients. We found that the four amplified cases showed strong cyclin D1 protein expression, but p-value was not statically significant (p = 0.31) (Table 1; Fig. 1c, d, e).

Correlation of Cyclin D1 Overexpression and CCND1 Gene Amplification with Clinicpathological Features of DCIS cases
Table 2 shows the association between cyclin D1 expression and clinicpathological features of DCIS. Non-significant correlations between cyclin D1 expression and amplification with any of clinicpathological features could be detected.

Fig. 1a: Cyclin D1 overexpression in ADH

Fig. 1b: Fluorescent in situ hybridization signals in ADH showing non-amplified cyclin D1 gene. (Cyclin D1, Spectrum Orange, Centromere 11 Spectrum Green)

Fig. 1c: Cyclin D1 overexpression in comedo DCIS

Fig. 1d: Cyclin D1 overexpression in non- comedo DCIS

Fig. 1e: Fluorescent in situ hybridization cyclin D1 signals in DCIS showing Cyclin D1 gene amplification clusters (Cyclin D1 gene/CEP 11 ratio = 2)

Fig. 1f:: Cyclin D1 overexpression in IDC grade I, strong expression

Fig. 1g: Cyclin D1 overexpression in IDC grade III, weak expression

Fig. 1h: Cyclin D1 overexpression in IDC with DCIS component, showing same expression pattern

Fig. 1i: Cyclin D1 overexpression in IL, strong expression

Fig. 1j: Fluorescent in situ hybridization cyclin D1 signals in IC, showing cyclin D1 gene amplification clusters and multiple scattered amplification signals


Table 1:

Cyclin D1 overexpression and CCND1 gene amplification in different breast lesions

Cases of IC
Cyclin D1 expression as detected by immunohistochemistry was negative in 42 (36.8%) cases and positive in 72 (63.2%) of cases (Table 1; Fig. 1f-i,). Positive cases were categorized into three groups; weak 24 (21.1%), moderate 28 (24.6%) and strong 20 (17.5%). We noticed that positive cases for cyclin D1 maintain the same expression score in in situ component as well as invasive component.

CCND1 amplification as detected by FISH was amplified in 18 patients (15.8%) from these cases we found four cases with in situ component and non-amplified in 96 patients (84.2%) of invasive breast cancer (Table 1; Fig. 1j).

Correlation between CCND1 Gene Amplification and Cyclin D1 Protein Expression in IC
An excellent correlation between CCND1 amplification and cyclin D1 overexpression was found (p = 0.01). Most tumors with CCND1 amplification showed moderate or strong cyclin D1 protein expression. Ten out of 18 tumors with CCND1 amplification showed strong cyclin D1 expression, whereas 6/18 showed moderate expression (Table 3).

Correlation of Cyclin D1 Expression with Clinicopathologicl Parameters in IC
Table 4 shows the association between cyclin D1 expression and different clinicopathologic parameters. Cyclin D1 expression showed a strong direct correlation with expression of ER (p = 0.03) and PR (p = 0.01). There was a significant correlation between moderate/strong cyclin D1 expression and good prognostic parameters, including low histological grade (p = 0.04) and small tumor size (p = 0.003). There was a strong correlation between cyclin Dl overexpression and histological type (p = 0.008).


Table 2:

Correlation of cyclin D1 overexpression and CCND1 gene amplification with DCIS clinicpathological features

Test of significance: Fisher’s exact test; p-value ≤0.05 are considered significant


Table 3:

Correlation between CCND1 gene amplification and cyclin D1 protein expression in IC

p = 0.01 with χ2-test; *Allred scoring system

Positive cyclin Dl staining was seen in 12/14 of lobular carcinomas (85%), most of the cases show strong expression 8/12 cases (Fig. 1i) while all medullary carcinomas were completely negative.

Correlation of CCND1 Gene Amplification with Clinicopathological Parameters in IC
CCND1 gene amplification showed a significant correlation with expression of ER (p = 0.04) and PR (p = 0.01). Fourteen out of eighteen tumors showing CCND1 amplification also showed positive ER and PR expression. Non-significant correlations between CCND1 gene amplification and any of clinicpathological features were found as shown in Table 5.

DISCUSSION

Cyclin D1 gene amplification and overexpression has been reported in breast cancers. In situ hybridisation studies suggest that cyclin D1 overexpression occurs at the transition from in situ to invasive cancer (Fiche et al., 2000), while immunohistochemical studies indicate that cyclin D1 overexpression increases progressively from ADH, to DCIS and to IC (Heffelfinger et al., 2000; Lebeau et al., 2003). Cyclin D1 overexpression was present in 64% of cases of DCIS and in only 14% of cases of ADH (Gillett et al., 1998). Gradual increase in cyclin D1 overexpression from DH to ADH to DCIS and finally to IC with significant differences not always being seen between the lesions (Alle et al., 1998; Mommers et al., 1998; Shoker et al., 2001).


Table 4:

Correlation of cyclin D1 expression with different clinicopathological parameters in IC

Test of significance: chi-square test; p-value ≤0.05 are considered significant

Our own findings agree with these studies.

We examined preinvasive breast lesions for the expression of cyclin D1 to determine the beginning of invasion alterations in the expression levels and to correlate these changes with the clinicopathological feature. Our study demonstrated high expression of cyclin D1 in 58.3% in DCIS cases. This is in line with other studies (Alle et al., 1998; Gillett et al., 1998; Oh et al., 2001; Lebeau et al., 2003), who reported that cyclin D1 expression in DCIS range from 48 to 64%. The frequency of CCND1 amplifications as defined by FISH in the present study was 16.8% in DICS. It was reported by others that CCND1 was amplified in 33% and 18% of DICS respectively (Simpson et al., 1997; Fiche et al., 2000). In accordance with previous studies, we did not find a correlation between cyclin D1 overexpression or CCND1 amplification and any of clinicopathological features in DCIS cases (Vos et al., 1999; Umekita and Yoshida, 2000; Oh et al., 2001; Lebeau et al., 2003).

In the present study, the rate of cyclin D1 overexpression in cases of IC is 63.2% that was correspond to reported results that were 64% cyclin D1 overexpression in IC (Lee et al., 2007). However, there is a wide range of cyclin D1 expression in breast cancer, varying between 35-90% (Hwang et al., 2003; Sutherland and Musgrove, 2004; Bilalovic et al., 2005; Cho et al., 2008; Elsheikh et al., 2008). These might have resulted from different criteria for defining cyclin D1 overexpression and immunostaining techniques.

In this study, we found that, expression of cyclin D1 did not increase with the progression from DCIS to IC. Similarly, there was no change in expression from low-grade to high-grade DCIS.


Table 5:

Correlation of CCND1 gene amplification with different clinicopathological parameters in IC

Test of significance: chi-square test and Fisher’s exact test; p-value ≤ 0.05 are considered significant; ILC: Invasive lobular carcinoma; MC: Medullary carcinoma

Cyclin D1 overexpression is elevated in both DCIS and IC to similar levels and is increased above normal levels in preneoplastic hyperproliferative lesions implying that molecular alterations leading to cyclin D1 overexpression occur relatively early during breast carcinogenesis (Alle et al., 1998).

In our study, CCND1 amplifications was 15.8% in IC, which was similar to that reported by Worsley et al. (1996), Loden et al. (2002) and Jirstrom et al. (2005) using FISH, or using chromogenic in situ hybridization (CISH) (Reis-Filho et al., 2006). We have identified a strong significant correlation between cyclin D1 protein expression and CCND1 amplification (p = 0.01). Interestingly, all cases with CCND1 gene amplification showed either moderate or strong cyclin D1 expression. Similar results were reported before in a series of 485 cases of IC, an excellent correlation between cyclin D1 overexpression and CCND1 amplification (p = 0.001). All cases with CCND1 gene amplification showed either moderate (42%) or strong (58%) cyclin D1 expression. In our study, we found non-significant correlations between CCND1 gene amplification and any of clinicpathological features (Elsheikh et al., 2008). The same finding was reported by Worsley et al. (1996) using FISH technique and using CISH Reis-Filho et al. (2006).

Other reported discordance between the amplification of the cyclin D1 gene and the overexpression of cyclin D1 protein in breast cancers which may be due to several different pathways, including the ER, c-myc and fibroblast growth factor receptor pathways (Fu et al., 2004; Butt et al., 2005; Arnold and Papanikolaou, 2005).

As with previous immunohistochemical studies, the localization of the cyclin D1 protein was predominantly confined to the nucleus of the breast cancer cells (Hwang et al., 2003; Bilalovic et al., 2005) and the intensity and percentage of staining varied within the individual tumor and from cell to cell within the same tumor signifying that cyclin D1 overexpression is regulated in a cyclical manner. These observations support the original suggestion that cyclin D1 protein is not constantly expressed and must be degraded or expelled from the nucleus before progression into the S-phase (Baldin et al., 1993).

In this study, we found that, cyclin D1 overexpression and CCND1 amplification are associated with positive hormone receptor status. Similar correlation was reported by other studies (Hwang et al., 2003; Al-Kuraya et al., 2004; Jirstrom et al., 2005; Reis-Filho et al., 2006; Elsheikh et al., 2008). In DCIS and ADH, this relationship is apparently not of the same significance. Two studies reported a significant association (Vos et al., 1999; Oh et al., 2001), whereas others (Gillett et al., 1998; Lebeau et al., 2003) like the present study failed to find a statistically significant association. In the present study, despite those cases with positive cyclin D1 expression showed hormone receptor positivity there was no significant correlation between both of them.

A potential functional link between ER expression and cyclin D1 expression is supported by evidence that cyclin D1 is a major downstream target of estrogen action and plays a pivotal role in estrogen-induced mitogenesis in breast cancer cells (Planas-Silva and Weinberg, 1997). This supports the finding that cyclin D1 is an ER-regulated gene (Sutherland et al., 1995). The disparity between the amplification of the cyclin D1 gene and the overexpression of cyclin D1 protein in breast cancers (Barnes, 1997) may therefore be due to the dysregulation of ER that occurs in the majority of these lesions (Shoker et al., 2000, 2001). These data along with our result support the concept that high ER expression may help in the maintenance of high levels of cyclin D1 protein expression. Given the high prevalence of ER expression in breast cancers, it is not surprising that cyclin D1 overexpression is frequently due to upregulation by ER than as a result of CCND1 gene amplification and that anti-oestrogen therapy may offset some of these upregulating effects (Elsheikh et al., 2008).

In this study, a significant correlation was observed between cyclin D1 overexpression and smaller size of the tumor (p = 0.003), well-differentiated carcinomas (p = 0.04), positive ER (p = 0.009) and PR (p = 0.01) all this features known to be associated with a good prognosis.

Until now, the prognostic value of cyclin D1 expression on breast cancer outcome has been controversial. Some studies have reported that cyclin D1 overexpression indicates a better prognosis in breast cancers (Hwang et al., 2003; Bilalovic et al., 2005; Cho et al., 2008) while others reported it to be of no prognostic value (Worsley et al., 1996; Lee et al., 2007), at the same time, Ahnstrom et al. (2005) reported that cyclin D1 overexpression is associated with a poor prognosis in breast cancer. This difference may be due to the use of different antibodies, different thresholds for cyclin D1 positivity and different methods for the analysis of cyclin D1 expression by Western blotting, IHC and FISH. This is also in agreement with studies in which cyclin D1 was predominantly expressed in well differentiated, low-grade and slow growing breast cancers (Naidu et al., 2002; Hwang et al., 2003; Stendahl et al., 2004). In other tumors, like lung cancer and colorectal cancer, expression of cyclin D1 correlated with a worse outcome and a positive correlation with proliferative markers. This indicates that cyclin D1 activities might be not only diverse but also tissue specific (Fu et al., 2004).

In this study, there was a strong correlation between cyclin Dl overexpression and histological type (p = 0.008). Positive cyclin Dl staining was seen in 12 of 14 lobular carcinomas (85%) most of cases show strong expression 8/12 cases. This finding was in line with other studies (Van Diest et al., 1997; Soslow et al., 2000; Naidu et al., 2002; Reis-Filho et al., 2006) who reported that, overexpression was found in 73 to 90% of lobular cancer, which also usually show low nuclear atypicality, low proliferation and positive steroid receptor status and are associated with an intermediate prognosis (Ellis et al., 1992).

As regard medullary carcinoma, all five cases were completely negative for cyclin D1 expression or CNND1 amplification. Similar result by IHC and CISH were reported (Van Diest et al., 1997; Elsheikh et al., 2008). This may be due to its high nuclear atypicality, high proliferation and poor prognosis (Ellis et al., 1992). On contrary to our results, a study reported 27% (4/15) of the medullary carcinomas was positive (Naidu et al., 2002). So, overexpression of cyclin D1 is confined to specific phenotypes, implying different roles in different subtypes of the disease.

In conclusion, Overexpression and amplification of cyclin D1 in preinvasive alone or with adjacent invasive lesions and invasive carcinomas suggest that the gene may play an important role in early and late stages of breast carcinogenesis. Our results demonstrate a strong correlation between CCND1 amplification and its protein expression in invasive breast cancer. Our data are in agreement with the results of previous studies showing that cyclin D1 is not associated with poor prognosis. However, this study has a limitation due to lack of follow up data and treatment information. Therefore survival analysis could not be conducted. Further studies are needed to clarify the function of cyclin D1 as a prognostic factor.

ABBREVIATIONS

ADH : Atypical Ductal Hyperplasia
CISH : Chromogenic in situ hybridization
DH : Ductal Hyperplasia without atypia
FISH :

Fluorescence in situ hybridization

IDC : Invasive duct carcinoma
ILC : Invasive lobular carcinoma
MC : Medullary carcinoma
DCIS : Ductal carcinoma in situ
ER : Estrogen receptor
IC : Invasive carcinoma
IHC : Immunohistochemistry
LCIS : Lobular carcinoma in situ
PR : Progesterone receptor
REFERENCES
Ahnstrom, M., B. Nordenskjold, L.E. Rutqvist, L. Skoog and O. Stal, 2005. Role of cyclin D1 in ErbB2-positive breast cancer and tamoxifen resistance. Breast Cancer Res. Treat, 91: 145-151.
CrossRef  |  

Al-Kuraya, K., P. Schraml, J. Torhorst, C. Tapia and B. Zaharieva et al., 2004. Prognostic relevance of gene amplifications and coamplifications in breast cancer. Cancer Res., 64: 8534-8540.
CrossRef  |  

Alle, K.M., S.M. Henshall, A.S. Field and R.L. Sutherland, 1998. Cyclin D1 protein is overexpressed in hyperplasia and intraductal carcinoma of the breast. Clin Cancer Res., 4: 847-854.
PubMed  |  

Arnold, A. and A. Papanikolaou, 2005. Cyclin D1 in breast cancer pathogenesis. J. Clin. Oncol., 23: 4215-4224.
CrossRef  |  

Baldin, V., J. Lukas, M.J. Marcote, M. Pagano and G. Draetta, 1993. Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev., 7: 812-821.
PubMed  |  

Barnes, D.M., 1997. Cyclin D1 in mammary carcinoma. J. Pathol., 181: 267-269.
PubMed  |  

Bieche, I., M. Olivi, C. Nogues, M. Vidaud and R. Lidereau, 2002. Prognostic value of CCND1 gene status in sporadic breast tumours, as determined by real-time quantitative PCR assays. Br. J. Cancer, 86: 580-586.
PubMed  |  

Bilalovic, N., S. Vranic, H. Basic, A. Tatarevic and I. Selak, 2005. Immunohistochemical evaluation of cyclin D1 in breast cancer. Croat Med. J., 46: 382-388.
PubMed  |  

Brown, L.A., J. Irving, R. Parker, H. Kim and J.Z. Press et al., 2006. Amplification of EMSY, a novel oncogene on 11q13, in high grade ovarian surface epithelial carcinomas. Gynecol. Oncol., 100: 264-270.
PubMed  |  

Butt, A.J., C.M. McNeil, E.A. Musgrove and R.L. Sutherland, 2005. Downstream targets of growth factor and oestrogen signalling and endocrine resistance: The potential roles of c-Myc, cyclin D1 and cyclin E. Endocr Relat Cancer, 12: S47-S59.
CrossRef  |  Direct Link  |  

Carter, C.L., C. Allen and D.E. Henson, 1989. Relation of tumor size, lymph node status and survival in 24, 740 breast cancer cases. Cancer, 63: 181-187.
CrossRef  |  PubMed  |  Direct Link  |  

Cheuk, W., K.O. Wong, C.S. Wong and J.K. Chan, 2004. Consistent immunostaining for cyclin D1 can be achieved on a routine basis using a newly available rabbit monoclonal antibody. Am. J. Surg. Pathol., 28: 801-807.
PubMed  |  

Cho, E.Y., Y.L. Choi, J.J. Han, K.M. Kim and Y.L. Oh, 2008. Expression and amplification of Her2, EGFR and cyclin D1 in breast cancer: Immunohistochemistry and chromogenic in situ hybridization. Pathol. Int., 58: 17-25.
PubMed  |  

Cuny, M., A. Kramar, F. Courjal, V. Johannsdottir and B. Iacopetta et al., 2000. Relating genotype and phenotype in breast cancer: An analysis of the prognostic significance of amplification at eight different genes or loci and of p53 mutations. Cancer Res., 60: 1077-1083.
PubMed  |  

El-Bolkainy, M.N., M.A. Nouh and T.K. El-Bolkainy, 2005. Topgraphic Pathology of Cancer. 3rd Edn., Chap. 7. Cairo University, Egypt, pp: 57-64.

Ellis, I.O., M. Galea, N. Broughton, A. Locker and R.W. Blamey et al., 1992. Pathological prognostic factors in breast cancer: II. Histological type: Relationship with survival in a large study with long term follow up. Histopathology, 20: 479-489.
PubMed  |  

Elsheikh, S., A.R. Green, M.A. Aleskandarany, M. Grainge and C.E. Paish et al., 2008. CCND1 amplification and cyclin D1 expression in breast cancer and their relation with proteomic subgroups and patient outcome. Breast Cancer Res. Treat, 109: 325-335.
CrossRef  |  

Elston, C.W. and I.O. Ellis, 1991. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: Experience from a large study with long-term follow-up. Histopathology, 19: 403-410.
PubMed  |  

Esteva, F.J. and G.N. Hortobagyi, 2004. Prognostic molecular markers in early breast cancer. Breast Cancer Res., 6: 109-118.
Direct Link  |  

Fiche, M., H. Avet-Loiseau, C.M. Maugard, C. Sagan and M.F. Heymann et al., 2000. Gene amplifications detected by fluorescence in situ hybridization in pure intraductal breast carcinomas: Relation to morphology, cell proliferation and expression of breast cancer-related genes. Int. J. Cancer, 89: 403-410.
PubMed  |  

Fitzgibbons, P.L., D.L. Page, D. Weaver, A.D. Thor and D.C. Allred et al., 2000. Prognostic factors in breast cancer. College of American Pathologists Consensus Statement 1999. Arch. Pathol. Lab. Med., 124: 966-978.
PubMed  |  

Fu, M., C. Wang, Z. Li, T. Sakamaki and R.G. Pestell, 2004. Minireview: Cyclin D1: Normal and abnormal functions. Endocrinology, 145: 5439-5447.
CrossRef  |  

Gillett, C., V. Fantl and R. Smith, 1994. Amplification and overexpression of cyclin D1 in breast cancer detected by immunohistochemical staining. Cancer Res., 54: 1812-1817.
Direct Link  |  

Gillett, C.E., A.H. Lee, R.R. Millis and D.M. Barnes, 1998. Cyclin D1 and associated proteins in mammary ductal carcinoma in situ and atypical ductal hyperplasia. J. Pathol., 184: 396-400.
PubMed  |  

Harvey, J.M., G.M. Clark, C.K. Osborne and D.C. Allred, 1999. Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer. J. Clin. Oncol., 17: 1474-1481.
PubMed  |  

Heffelfinger, S.C., R. Yassin, M.A. Miller and E.E. Lower, 2000. Cyclin D1, retinoblastoma, p53 and Her2/neu protein expression in preinvasive breast pathologies: Correlation with vascularity. Pathobiology, 68: 129-136.
PubMed  |  

Hermanek, P. and L.H. Sobin, 1992. TNM Classification of International Union Against Cancer: TNM Atlas. 4th Edn., 3rd Revision, Springer-Verlag, Berlin.

Holland, R., J.L. Peterse, R.R. Millis, V. Eusebi and D. Faverly et al., 1994. Ductal carcinoma in situ: A proposal for a new classification. Semin. Diagn. Pathol., 11: 167-180.
PubMed  |  

Hwang, T.S., H.S. Han, Y.C. Hong, H.J. Lee and N.S. Paik, 2003. Prognostic value of combined analysis of cyclin D1 and estrogen receptor status in breast cancer patients. Pathol. Int., 53: 74-80.
PubMed  |  

Jirstrom, K., M. Stendahl and L. Ryden, 2005. Adverse effect of adjuvant tamoxifen in premenopausal breast cancer with cyclin D1 gene amplification. Cancer Res., 65: 8009-8016.
Direct Link  |  

Lebeau, A., A. Unholzer, G. Amann, M. Kronawitter and I. Bauerfeind et al., 2003. EGFR, HER-2/neu, cyclin D1, p21 and p53 in correlation to cell proliferation and steroid hormone receptor status in ductal carcinoma in situ of the breast. Breast Cancer Res. Treat, 79: 187-198.
CrossRef  |  PubMed  |  

Lebeau, A., D. Deimling, C. Kaltz, A. Sendelhofert and A. Iff et al., 2001. Her-2/neu analysis in archival tissue samples of human breast cancer: Comparison of immunohistochemistry and fluorescence in situ hybridization. J. Clin. Oncol., 19: 354-363.
PubMed  |  

Lee, A., W.C. Park, H.W. Yim, M.A. Lee and G. Park et al., 2007. Expression of c-erbB2, cyclin D1 and estrogen receptor and their clinical implications in the invasive ductal carcinoma of the breast. Jap. J. Clin. Oncol., 37: 708-714.
CrossRef  |  

Loden, M., M. Stighall, N.H. Nielsen, G. Roos and S.O. Emdin et al., 2002. The cyclin D1 high and cyclin E high subgroups of breast cancer: Separate pathways in tumorogenesis based on pattern of genetic aberrations and inactivation of the pRb node. Oncogene, 21: 4680-4690.
PubMed  |  

Luo, M.L., X.M. Shen, Y. Zhang, F. Wei and X. Xu et al., 2006. Amplification and overexpression of CTTN (EMS1) contribute to the metastasis of esophageal squamous cell carcinoma by promoting cell migration and anoikis resistance. Cancer Res., 66: 11690-11699.
CrossRef  |  

Molland, J.G., M. Donnellan, N.C. Janu, H.L. Carmalt and C.W, Kennedy et al., 2004. Infiltrating lobular carcinoma: A comparison of diagnosis, management and outcome with infiltrating duct carcinoma. Breast, 13: 389-396.
CrossRef  |  

Mommers, E.C.M, P.J. van Diest, A.M. Leonhart, C.J.L.M. Meijer and J.P.A. Baak, 1998. Expression of proliferation and apoptosis-related proteins in usual ductalhyperplasia of the breast. Human Pathol., 29: 1539-1545.
PubMed  |  Direct Link  |  

Mrhalova, M., R. Kodet and P. Strnad, 2002. Invasive ductal carcinoma of the breast: Study of the number of copies of the CCND1 gene and chromosome 11 using fluorescence in situ hybridization (FISH) in comparison with expression of cyclin D1 protein and estrogen receptors (ER alpha) with immunohistochemical detection. Cas Lek Cesk, 141: 708-714.
PubMed  |  

Naidu, R., N.A. Wahab, M.M. Yadav and M.K. Kutty, 2002. Expression and amplification of cyclin D1 in primary breast carcinomas: Relationship with histopathological types and clinico-pathological parameters. Oncol. Rep., 9: 409-416.
PubMed  |  

Neuman, E., M.H. Ladha, N. Lin, T.M. Upton and S.J. Miller et al., 1997. Cyclin D1 stimulation of estrogen receptor transcriptional activity independent of cdk4. Mol. Cell. Biol., 17: 5338-5347.

Nishimura, R., T. Saeki, S. Ohsumi, Y. Tani and S. Takashima, 2007. Immunohistochemical expression of hormone receptors and the histological characteristics of biochemically hormone receptor negative breast cancers. Breast Cancer, 14: 100-104.
PubMed  |  

Oh, Y.L., J.S. Choi, S.Y. Song, Y.H. Ko and B.K. Han et al., 2001. Expression of p21Waf1, p27Kip1 and cyclin D1 proteins in breast ductal carcinoma in situ: Relation with clinicopathologic characteristics and with p53 expression and estrogen receptor status. Pathol. Int., 51: 94-99.
PubMed  |  Direct Link  |  

Ormandy, C.J., E.A. Musgrove, R. Hui, R.J. Daly and R.L. Sutherland, 2003. Cyclin D1, EMS1and 11q13 amplification in breast cancer. Breast Cancer Res. Treat, 78: 323-335.
CrossRef  |  

Page, D.L. and L.W. Rogers, 1992. Combined histologic and cytologic criteria for the diagnosis of mammary atypical ductal hyperplasia. Human Pathol., 23: 1095-1097.
PubMed  |  

Parkin, D.M., S.L. Whelan, J. Ferlay, L. Teppo and D.B. Thomas, 2002. Cancer Incidence in Five Continents. Vol. 8. IARC Scientific Publications No. 155. International Agency for Research on Cancer, Lyon, France, ISBN-13: 9789283221555, pp: 838.

Planas-Silva, M.D. and R.A. Weinberg, 1997. Estrogen dependent cyclin E-cdk2 activation through p21 redistribution. Mol. Cell Biol., 17: 4059-4069.
PubMed  |  Direct Link  |  

Recht, A., S.B. Edge, L.J. Solin, D.S. Robinson and A. Estabrook et al., 2001. Postmastectomy radiotherapy: Clinical practice guidelines of the American Society of Clinical Oncology. J. Clin. Oncol., 19: 1539-1569.
Direct Link  |  

Reis-Filho, J.S., K. Savage, M.B. Lambros, M. James and D. Steele et al., 2006. Cyclin D1 protein overexpression and CCND1 amplification in breast carcinomas: An immunohistochemical and chromogenic in situ hybridisation analysis. Mod. Pathol., 19: 999-1009.
CrossRef  |  

Shibata, T., S. Uryu, A. Kokubu, F. Hosoda and M. Ohki et al., 2005. Genetic classification of lung adenocarcinoma based on array-based comparative genomic hybridization analysis: Its association with clinicopathologic features. Clin. Cancer Res., 11: 6177-6185.
CrossRef  |  

Shoker, B.S., C. Jarvis, M.P.A. Davies, M. Iqbal and D.R. Sibson et al., 2001. Sloane JP. Immunodetectable cyclin D1 is associated with oestrogen receptor but not Ki67 in normal, cancerous and precancerous breast lesions. Br. J. Cancer, 84: 1064-1069.
CrossRef  |  PubMed  |  

Shoker, B.S., C. Jarvis, R.B. Clarke, E. Anderson and C. Munro et al., 2000. Abnormal regulation of oestrogen receptor in benign breast lesions. J. Clin. Pathol., 53: 778-783.
CrossRef  |  

Simpson, J.F., D.E. Quan, F. O'Malley, T. Odom-Maryon and P.E. Clarke, 1997. Amplification of CCND1 and expression of its protein product, cyclin D1, in ductal carcinoma in situ of the breast. Am. J. Pathol., 151: 161-168.
PubMed  |  

Soslow, R.A., D.L. Carlson, M.G. Horenstein and M.P. Osborne, 2000. A comparison of cell cycle markers in well-differentiated lobular and ductal carcinomas. Breast Cancer Res. Treat, 61: 161-170.
PubMed  |  

Stendahl, M., A. Kronblad, L. Ryden, S. Emdin, N.O. Bengtsson and G. Landberg, 2004. Cyclin D1overexpression is a negative predictive factor for tamoxifen response in postmenopausal breast cancer patients. Br. J. Cancer, 90: 1942-1948.
CrossRef  |  PubMed  |  

Sutherland, R.L. and E.A. Musgrove, 2004. Cyclins and breast cancer. J. Mammary Gland Biol. Neoplasia, 9: 95-104.

Sutherland, R.L., J.A. Hamilton, K.J.E. Sweeney, C.K.W. Watts and E.A. Musgrove, 1995. Expression and regulation of cyclin genes in breast cancer. Acta Oncol., 34: 651-656.
CrossRef  |  

Umekita, Y. and H. Yoshida, 2000. Cyclin D1 expression in ductal carcinoma in situ, atypical ductal hyperplasia and usual ductal hyperplasia: An immunohistochemical study. Pathol. Int., 50: 527-530.
PubMed  |  

Van Diest, P.J., R.J. Michalides, L. Jannink, P. van der Valk and H.L. Peterse et al., 1997. Cyclin D1 expression in invasive breast cancer. Correlations and prognostic value. Am. J. Pathol., 150: 705-711.
PubMed  |  Direct Link  |  

Vos, C.B., N.T. Ter Haar, J.L. Peterse, C.J. Cornelisse and M J. van de Vijver, 1999. Cyclin D1 gene amplification and overexpression are present in ductal carcinoma in situ of the breast. J. Pathol., 187: 279-284.
PubMed  |  

Worsley, S.D., B.A. Jennings, K.H. Khalil, M. Mole and A.C. Girling, 1996. Cyclin D1 amplification and expression in human breast carcinoma: Correlation with histological prognostic markers and oestrogen receptor expression. Clin. Mol. Pathol., 49: M46-M50.
Direct Link  |  

Zaharieva, B.M., R. Simon, P.A. Diener, D. Ackermann and R. Maurer et al., 2003. High-throughput tissue microarray analysis of 11q13 gene amplification (CCND1, FGF3, FGF4, EMS1) in urinary bladder cancer. J. Pathol., 201: 603-608.
PubMed  |  

Zukerberg, L.R., W.I. Yang, M. Gadd, A.D. Thor and F.C. Koerner et al., 1995. Cyclin D1 (PRAD1) protein expression in breast cancer: Approximately one-third of infiltrating mammary carcinomas show overexpression of the cyclin D1 oncogene. Mod. Pathol., 8: 560-567.
PubMed  |  

©  2019 Science Alert. All Rights Reserved
Fulltext PDF References Abstract