RESEARCH Open Access
An analysis of Cyclin D1, Cytokeratin 5/6 and
Cytokeratin 8/18 expression in breast papillomas
and papillary carcinomas
Yu Wang, Jin-fu Zhu, Ying-ying Liu and Gui-ping Han*
Background: To evaluate the expression levels of Cyclin D1 in breast papillomas and papillary carcinomas, and to
analyze the types of cells that co-express Cyclin D1 with Cytokeratin 5/6 (CK 5/6) or with Cytokeratin 8/18(CK 8/18).
Methods: Fifty-nine cases of papillary lesions including 36 papillomas and 23 intracystic papillary carcinomas were
examined. Cyclin D1, CK 5/6 and CK 8/18 expression levels were evaluated by double immunostaining.
Results: Cyclin D1 is highly expressed in papillary carcinomas (27.54% ±15.43%) compared with papillomas (8.81%±
8.41%, p<0.01). Cyclin D1 is predominantly expressed in Cytokeratin 8/18- expressing cells, rather than in Cytokeratin
5/6-expressing cells, regardless of the type of lesion. In Papillomas, Cyclin D1 exhibited a mean 11.42% (11.42%±
10.17%) co-expression rate with Cytokeratin 8/18 compared with a mean 2.50% (2.50%±3.24%) co-expression rate
with Cytokeratin 5/6 (p<0.01). In papillary carcinomas, Cyclin D1 exhibited a mean 34.74% (34.74%±16.32%)
co-expression rate with Cytokeratin 8/18 compared with a co-expression rate of 0.70% (0.70%±0.93%) with Cytokeratin
Conclusions: The increase in Cyclin D1 suggests an association of Cyclin D1 staining with papillary carcinomas.
Although Cyclin D1 is an effective marker for the differential diagnosis of other papillary lesions, it cannot be used to
distinguish between papilloma and papillary carcinoma lesions because its expression occurs in both lesions. Our
results show that Cyclin D1 and CK 5/6 staining could be used in concert to distinguish between the diagnosis of
papilloma (Cyclin D1<4.20%, CK 5/6 positive) or papillary carcinoma (Cyclin D1>37.00%, CK 5/6 negative). In addition,
our data suggest that Cyclin D1 is expressed only in the cancer stem or progenitor cells that co-immunostained with
CK 8/18 in papillary carcinomas, and predominantly with CK 8/18 in the papillomas.
Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/
Keywords: Cyclin D1, Cytokeratin, Papillary carcinoma, Papilloma, Double immunostaining
Papillary breast tumors consist of proliferative mammary
epithelial cells that invade the ductal lumen and form
fibro-vascular stalks that may evolve into branching arbo-
rescent structures. Intraductal papillomas and papillary
ductal carcinomas in situ (DCIS) are examples of papillary
breast lesions. Despite the well-described histological fea-
tures of these two tumors, it is occasionally difficult to dis-
tinguish between them because of overlapping microscopic
characteristics . The key histological feature used
to delineate benign papilloma from papillary DCIS is
the presence of myoepithelial cells, which is preserved
in the former and scant or absent in the latter. CK 5/6,
SMA (smooth muscle actin) and p63 are generally accepted
markers for the differential diagnosis of papillomas and
papillary carcinomas .
In recent years, the elevated expression of Cyclin D1
in papillary carcinomas compared with papillomas has
attracted significant attention. Several studies have reported
that Cyclin D1 expression levels are sufficiently sensitive to
distinguish between these two types of lesions . Whether
* Correspondence: email@example.com.
Department of Pathology, The 2nd affiliated hospital of Harbin Medical
University, Xuefu Road 246, Harbin, Nanggang District, China
© 2013 Wang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Wang et al. Diagnostic Pathology 2013, 8:8
these two types of lesions can be distinguished by Cyclin
D1 expression levels alone requires further investigation.
Moreover, the mechanism underlying the elevated expres-
sion of Cyclin D1 in breast carcinomas remains unknown.
Cyclin D1 is a cell -cycle regulator that is essential for
progression through G1 phase and is a candidate proto-
oncogene. This protein has also been implicated in the
pathogenesis of several human tumor types including
breast carcinomas. To the best of our knowledge, there
are no available published studies regarding the expression
of Cyclin D1 in different breast cell types. Moreover, the
use of Cyclin D1 alone to make a differential diagnosis
between papilloma and papillary carcinoma remains a
controversial topic. In this study, we aimed to evaluate the
expression of Cyclin D1 in different cell types and to
assess its potential to distinguish between papillomas and
papillary carcinomas using a double immunostaining tech-
nique. Cells expressing CK 5/6 or CK 8/18 exhibited pale
red or red-stained cytoplasm, whereas cells expressing
Cyclin D1 exhibited a black nuclear staining pattern.
We examined 59 papillary breast lesions (Table 1) from
the database of the Department of Pathology of the 2nd
affilliated hospital of Harbin Medical University, inclu-
ding 36 cases of intraductal papillomas and 23 cases of
intracystic papillary carcinomas. All of the diagnoses
were made with Cytokeratin 5/6, SMA, p63, CD10
Double immunostaining analyses were performed. The
tissues were sectioned into sequential slices (two serial sec-
tions were cut from each block, specifically, one for Cyclin
D1 and CK 5/6 staining, and one for Cyclin D1 and CK 8/
18 staining). Initial immunostaining was performed using a
Cyclin D1 antibody purchased from QuanHui (Beijing,
China), and a Cytokeratin 5/6 (CK 5/6) or Cytokeratin 8/18
(CK 8/18) antibody, both of which were purchased from
Maxim Bio (Fuzhou, Fujian, China). Each primary antibody
was applied at a final dilution of 1:50. The Maxim DouMax
Visiontmkit was purchased from Maxim Bio, which
included Endogenous Peroxidase Blocking Solution,
Non-Immune Serum, Biotinylated Secondary Antibody,
Streptavidin Alkaline Phosphatase, BCIP/NBT, Amplifer,
Streptavidin Peroxidase, AEC Buffer, AEC Substrate, AEC
Chromogen, Hematoxylin, and ClearMount.
All tissues were fixed in a 10% formalin solution. Four
micron-thick serial sections were generated from paraffin-
embedded blocks. For epitope retrieval, the slides were
boiled in a cooker in EDTA buffer pH 9.0 for 20 minutes.
Next, the sections were incubated with the Cyclin D1
primary antibody over-night at 4 degrees centigrade. The
remaining steps of the procedure were performed accor-
ding to the manufacturer’s recommended protocol. The
sections were visualized by incubation in BCIP/NBT for
30 minutes at room temperature. The nuclei of cells that
expressed Cyclin D1 were stained black.
Next, the sections were treated with Amplifer for
10 minutes, incubated in 10% normal goat serum for
30 minutes at room temperature, and incubated with a
second primary antibody to either CK 5/6 or CK 8/18 for
1 hour at room temperature. The remaining steps of the
procedures were performed according to the manufac-
turer’s recommended protocol. The sections were visua-
lized using a cocktail of AEC Buffer, AEC Substrate and
AEC Chromogen for 10 minutes at room temperature.
The cytoplasm of CK 5/6 or CK 8/18-expressing cells
appeared pale red or red after staining. Hematoxylin was
used as a counter stain, and the nuclei were thus stained
blue-purple, unless the nuclei were positive for Cyclin D1:
in those cases, the nuclei were stained black.
Next, Cyclin D1, CK 5/6 or CK 8/18 staining was classi-
fied as either negative or positive for each antibody. Five
fields were selected randomly at magnification of 400×.
The number of Cyclin D1 expressing cells and, CK 5/6 or
CK 8/18 co-expressing cells was quantified – out of 200
cells for every selected field. The mean expression rate of
Cyclin D1 was calculated. Data were analyzed using the
SPSS 13.0 software package (IBM, Armonk, NY, USA) and
presented as?X±SD. The Mann–Whitney U test was used
to analyze discrepancies. P-values<0.01 were considered
to be statistically significant.
We detected negligible levels of Cyclin D1 expression
within papilloma sections (shown in Figure 1A, B) com-
pared with high levels of Cyclin D1 expression in papil-
lary carcinomas (shown in Figure 1C, D). In keeping
with these results, the mean rate of Cyclin D1 expression
in the papillomas was 8.81% (8.81%±8.41%), whereas
the mean expression rate of Cyclin D1 in the papillary
carcinomas exhibited a statistically significant increase
to 27.54% (27.54% ±15.43%, p<0.01, Figure 2).
In our study, the rate of Cyclin D1 expression in papil-
loma cells was 0-37%, whereas the rate of Cyclin D1
expression in papillary carcinoma cells was 4.20%-60.80%.
Although there was overlap in Cyclin D1 expression
between these two lesions, a small number of papilloma
cases exhibited higher Cyclin D1 expression than the
Table 1 The distribution of the different age groups of
patients with papillary lesions
Age (year) Papilloma Papillary carcinoma
16-39(mean 29.5) 112
40-59(mean 45.5) 19 16
Wang et al. Diagnostic Pathology 2013, 8:8
Page 2 of 7
average Cyclin D1 expression of the papillary carcinomas
and vice versa, the Cyclin D1 together with CK 5/6 staining
could be used to distinguish between papillary carcinoma
and papilloma samples. Statistical analysis indicated that
Cyclin D1 expression could distinguish papilloma from
papillary carcinoma (p < 0.01). If the percentage of the
Cyclin D1 expression was over 37.00% and negative for CK
5/6 expression, a diagnosis of papillary cacinoma was made.
Conversely, if the percentage of the Cyclin D1 expression
was below 4.20% and posirive for CK 5/6 expression, a
diagnosis of papilloma was made.
In the papillomas, Cyclin D1 was predominantly ex-
pressed in CK 8/18 positive cells with limited expression
CK 5/6 positive cells (shown in Figure 1a, b). The mean co-
expression rate of Cyclin D1 with CK 8/18 was 11.42%
(11.42%±10.17%), whereas the mean co-expression rate of
Cyclin D1 with CK 5/6 was 2.50% (2.50%±3.24%, p<0.01,
Figure 3). Thus, there is a statistically significant difference
in the co-expression rates of Cyclin D1 and CK 8/18 or CK
5/6 between these two lesion types. Cyclin D1 was almost
exclusively expressed in the cells that co-expressed CK 8/
18, whereas CK 5/6 is almost never expressed in the papil-
lary carcinomas (Figure 1c, d). The mean co-expression rate
of Cyclin D1 with CK 8/18 is 34.74%(34.74%±16.32%), and
the mean co-expression rate of Cyclin D1 with CK 5/6 is
0.70% (0.70%±0.93%, p<0.01, Figure 3), indicating that
the overexpression of Cyclin D1 is significantly correlated
with cells expressing CK 8/18.
Stem cells found within the breast tissue have the ability
to self-renew and generate daughter cells, including all
of the cell types found in mature breast tissues .
Multi-potent mammary stem cells (MaSCs) produce
committed progenitors, which subsequently differentiate
into myoepithelial and luminal epithelial cells .
During the differentiation process, the self-renewal
capacity of the cells is gradually lost. Of the MaSCs
and the committed progenitors, it is believed that at
least one is a bi-potent progenitor. These progenitors,
Figure 1 Immunohistochemical double staining for Cyclin D1 and CK 5/6 or CK 8/18 in papilloma (A B, original magnification×200; a
b, original magnification× 400) and in papillary carcinoma (C D, original magnification×200; c d, original magnification× 400). The
cytoplasm of the cells expressing CK 5/6 is stained pale red and CK 8/18 was stained red, whereas the cytoplasm of cells expressing CK 8/18 is
stained red. The nuclei of Cyclin D1 positive cells are stained black. (A, a) Immunohistochemical double staining for Cyclin D1 and CK 5/6 in a
papilloma. The Cyclin D1 positive cells were hardly detected in (A). (B, b) Immunohistochemical double staining for Cyclin D1 and CK 8/18 in a
papilloma. There were a few Cyclin D1 positive cells detected in (B). Upon comparing (a) and (b), Cyclin D1 expression is clear in the cells that
are positive for CK 8/18 in contrast to the cells that are positive for CK 5/6 in the papillomas. (C, c) Immunohistochemical double staining for
Cyclin D1 and CK 5/6 in papillary carcinoma. The cells are not immunoreactive for CK 5/6. (D,d) Immunohistochemical double staining for Cyclin
D1 and CK 8/18 in papillary carcinomas. Cyclin D1 (D) localizes in practically the same position compared with (C). The number of the Cyclin D1
positive cells (C, D) clearly increases, in contrast to (A) and (B). As in the papillomas, Cyclin D1 only appears in the cells that co-express CK 8/18.
Wang et al. Diagnostic Pathology 2013, 8:8
Page 3 of 7
which include bi-potent progenitors, all express basal
markers, such as CK5, CK6, and CK14 . In
addition to those progenitors previously mentioned,
the luminal progenitors also express such markers as
CK8 and CK18. Consequently, the existence of a lu-
minal progenitor population has been identified ,
but the myoepithelial progenitor could not be identified
because the progenitors also express basal markers, simi-
lar to MaSCs . Myoepithelial progenitors differentiate
into the myoepithelial cells, and luminal progenitors dif-
ferentiate into cells that are restricted to either ductal or
alveolar lineages. The terminal luminal epithelial cells or
alveolar cells lose the basal markers, expressing only CK8
and CK18, whereas the terminal myoepithelial cells main-
tain the expression of basal markers CK5, CK6, CK14 and
the new myoepithelial marker P63, SMA (smooth muscle
actin), and calponin.
These findings form the basis of the cancer stem cell
(CSC) hypothesis, which posits that certain tumors are
initiated by one or more self-renewing CSCs that diffe-
rentiate into large populations of non-self-renewing but
rapidly dividing, cells which are responsible for genera-
ting the tumor mass . The cancer stem cells could
potentially be derived from bi-potent stem cells or from
more differentiated cells that have acquired self-renewal
capabilities. In contrast to the normal stem cells, the
cancer stem cells lose their capacity for multi-directional
differentiation and only produce tumors with features of
a particular lineage, including luminal or basal like
lineages. Regardings to tumor heterogeneity, there still
remains controversy. Current explanations comprise two
predominant models: the cancer stem cell hypothesis
and the clonal evolution and selection hypothesis.
According to the clonal evolution hypothetical model,
tumor cell phenotypes are determined by the phenotype of
the original cell type of the tumor-initiating cell. In this
study, papillary carcinoma cells expressing CK 8/18 should
be differentiated from one cancer stem cell/progenitor of
luminal cell lineage, and accordingly, the luminal progeni-
tor or terminal cells should express the same markers. This
phenomenon would explain the clonal evolution of papil-
lary carcinomas. In our study, the papilloma masses con-
sisted of cells that were positive for CK 5/6 and CK 8/18,
clearly indicating that these cells originated from different
tumor stem/progenitor cells. Taken together, the two
models indicate that the cellular phenotypes are unstable
and can change as the tumor evolves. The heterogeneity
of the papilloma cells can be considered to characterize a
stage of the tumor progression, specifically, a competition
among tumor cells with different phenotypes. It is possible
that certain papillary carcinomas derived from the papillo-
mas might result from the CK 8/18-positive tumor cells
that replace the CK 5/6 positive cells. The two models are
complementary in explaining tumor heterogeneity and
Figure 2 The mean expression rate of Cyclin D1 in the papillomas and the papillary carcinomas. The expression rate of Cyclin D1 is
27.54%± 15.43% in papillary carcinomas and 8.81%± 8.41% in papillomas (p< 0.01). The values are expressed as?X±SD. *P<0.01 (Mann–Whitney U
test). The difference exhibited clear statistical significance.
Wang et al. Diagnostic Pathology 2013, 8:8
Page 4 of 7
progression. However, the hypothesis that the papillary
carcinoma cells could be derived from the papilloma
cells which have the same progenitor of the cancer
cells, requires further research. However, the validity
of the explanation above regarding tumor progression
remains to be confirmed.
In our results, Cyclin D1 staining was predominantly
detected in the cells that were also immunoreactive for CK
8/18 in either the papillary carcinomas or papillomas. Pre-
vious studies have demonstrated a correlation between Cyc-
lin D1 over-expression and breast cancers. The hypothesis
that papillary carcinoma cells could be derived from papi-
lloma cells which have the same progenitors as the cancer
cells might be explained by the expression pattern of Cyclin
D1 in cells co-expressing CK 8/18.
Cyclin D1, one of the protein mediators of the G1/ S
cell-cycle transition, is commonly altered in breast cancer
and contributes to tumorigenesis, presumably by increa-
sing proliferation . It is generally accepted that the ini-
tiation of most tumors is triggered by chromosomal
instability (CIN), which is characterized by chromosomal
abnormalities and an altered gene expression profile. Data
from Mathew et al.  suggest that Cyclin D1 contributes
to CIN and tumorigenesis by directly regulating a tran-
scriptional program that governs chromosomal stability.
During maturation of the breast cell, the differentiation
and proliferative behaviors are regulated by a series of
signaling pathways, such as the Notch , Hedgehog
, and Wnt  pathways. The CCND1 gene encodes a
subunit of the Cyclin D1 holoenzyme, which can phos-
phorylate and inactivate pRB and NRF1 to regulate
nuclear synthesis and mitochondrial biogenesis [13-17].
The biological effects of Cyclin D1 overexpression in the
process of tumorigenesis are exhibited through the path-
ways mentioned above. Several studies have demonstrated
that the disrupted regulation of these pathways can lead to
the development of breast cancer in mice [18-21] and in
humans [22-24]. One report indicated that deletion of the
CCND1 gene leads to failed mammary gland development
in mice . Another study demonstrated that overex-
pression of the Cyclin D1 oncogene can induce mammary
gland tumors in mice . These findings further suggest
that Cyclin D1 might directly or indirectly trigger the dif-
ferentiation of mammary glandular cells. Other studies
have suggested that Cyclin D1 might inhibit the differen-
tiation of adipocytes . In addition, many studies have
indicated that high Cyclin D1 expression levels correlate
with CIN, specifically in the luminal B subtype tumors .
This phenomenon provides a possible explanation for why
Cyclin D1 was mainly expressed in the cells that expressed
Figure 3 The mean co-expression rate of Cyclin D1with CK 5/6 or CK 8/18 in papilloma and papillary carcinoma cells. The mean
co-expression rate of Cyclin D1 with CK 8/18 was 11.42%±10.17%, and that with CK 5/6 was 2.50%± 3.24% (p<0.01); whereas in papillary
carcinomas, the mean co-expression rate of Cyclin D1 with CK 8/18 was 34.74%±16.32%, and that with CK 5/6 was 0.70%± 0.93% (p<0.01).
The values are expressed as?X±SD.*P<0.01 (Mann–Whitney U test). The differences exhibited statistical significance.
Wang et al. Diagnostic Pathology 2013, 8:8
Page 5 of 7
CK 8/18 but not in those that expressed CK 5/6. Cyclin
D1 over-expression leads to the sustained proliferation of
mammary epithelial cells, which is associated with a delay
in acinar development in vitro models  and a failure to
undergo terminal differention in mouse models . The
cells in either the papillomas or the papillary carcinomas
that expressed CK 8/18 could be derived from the same
cancer stem/progenitor cells, which exhibit the capacity of
self-renewal and strict luminal differentiation, and which
over-express Cyclin D1 proteins because of various
genetic or epigenetic events. The distinct expression
patterns of Cyclin D1 between the papillomas and papil-
lary carcinomas might be explained by their occurrence
during different stages of tumorigenesis.
In this study, we evaluated the levels of Cyclin D1 expres-
sion in CK 5/6- or CK 8/18-positive cells from breast papil-
loma and papillary carcinoma. Double immunostaining was
used to analyze the types of cells that co-express Cyclin D1
with CK 5/6 or CK 8/18. Increased levels of Cyclin D1 are
associated with inreased likehood of papillary carcinomas.
This study also demonstrated the usefullness of CK 5/6 in
distinguishing breast papilloma (Cyclin D1<4.20%) from
papillary carcinoma (Cyclin D1>37.00%). During the
differentiation of breast cells, various immunochemical
markers appeared or disappeared at different time-points.
In our results, Cyclin D1 was almost exclusively expressed
in the tumor cells stained by CK 8/18, which is a marker of
luminal cells in normal breast during development
into the final luminal format. We therefore conclude
that Cyclin D1 is expressed exclusively in the cancer
stem or progenitor cells that positively co-immunostained
for CK 8/18 in papillary carcinomas and predominantly
for CK 8/18 the papilloma lesions.
CK 5/6: Cytokeratin 5/6; CK 8/18: Cytokeratin 8/18; DCIS: Ductal carcinomas in
situ; MaSCs: Multipotent mammary stem cells; SMA: Smooth muscle actin;
CSC: Cancer stem cell; CIN: Chromosomal instability.
The Project Fund: the Scientific Research Foundation for the Returned
Overseas Chinese Scholars, State Education Ministry (2009–1001).
The authors declare that they have no competing interests.
YW and JZ carried out the experiments and analysis of results obtained. All
authors participated in the design of the study, analysis of obtained results.
YW drafted the manuscript. All authors have read and approved the final
We are grateful to Jin Liu, Ming-ming Chu and Ke-fei Wu for their excellent
technical assistance in making sections. This study was supported by the
Department of Pathology of the 2nd Affiliated Hospital of the Harbin
Received: 20 December 2012 Accepted: 15 January 2013
Published: 18 January 2013
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Cite this article as: Wang et al.: An analysis of Cyclin D1, Cytokeratin 5/6
and Cytokeratin 8/18 expression in breast papillomas and papillary
carcinomas. Diagnostic Pathology 2013 8:8.
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