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
Moatasem M. Aly
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.
June 24, 2010; Accepted: August 16, 2010;
Published: November 04, 2010
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
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
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.
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
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
12), 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 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
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 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.
Fischers 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.
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
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.
||Cyclin D1 overexpression in ADH
||Fluorescent in situ hybridization signals in ADH showing non-amplified
cyclin D1 gene. (Cyclin D1, Spectrum Orange, Centromere 11 Spectrum Green)
||Cyclin D1 overexpression in comedo DCIS
||Cyclin D1 overexpression in non- comedo DCIS
||Fluorescent in situ hybridization cyclin D1 signals
in DCIS showing Cyclin D1 gene amplification clusters (Cyclin D1 gene/CEP
11 ratio = 2)
||Cyclin D1 overexpression in IDC grade I, strong expression
||Cyclin D1 overexpression in IDC grade III, weak expression
||Cyclin D1 overexpression in IDC with DCIS component, showing
same expression pattern
||Cyclin D1 overexpression in IL, strong expression
||Fluorescent in situ hybridization cyclin D1 signals
in IC, showing cyclin D1 gene amplification clusters and multiple scattered
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;
Correlation between CCND1 Gene Amplification and Cyclin D1 Protein Expression
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
Correlation of Cyclin D1 Expression with Clinicopathologicl Parameters in
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).
of cyclin D1 overexpression and CCND1 gene amplification with DCIS clinicpathological
of significance: Fishers exact test; p-value ≤0.05 are considered
between CCND1 gene amplification and cyclin D1 protein expression in IC
= 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
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.
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).
of cyclin D1 expression with different clinicopathological parameters
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.
of CCND1 gene amplification with different clinicopathological parameters
of significance: chi-square test and Fishers 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
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.
||Atypical Ductal Hyperplasia
||Chromogenic in situ hybridization
||Ductal Hyperplasia without atypia
Fluorescence in situ hybridization
||Invasive duct carcinoma
||Invasive lobular carcinoma
||Ductal carcinoma in situ
||Lobular carcinoma in situ
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