Keywords carcinoma - cyclin D1 - endometrium - hyperplasia - immunohistochemistry
Introduction
Endometrial carcinoma is often preceded by endometrial hyperplasia. Hyperplasia is
usually associated with exogenous estrogen stimulation and thus, estrogen is considered
as an endometrial carcinogen. There is a continuum of changes evolving to endometrial
carcinoma.[1 ]
Many of the genes involved in cell cycle progression are frequently mutated in human
cancers leading to uncontrolled cell division and tumor growth. Endometrial carcinogenesis
includes a mutation in p53, PTEN tumor suppressor gene, and overexpression of cyclin
D1. Mutation in p53 has been detected in serous endometrial carcinoma. Mutation/overexpression
of PTEN has been reported in most of the endometrial carcinoma. Many of the genes
involved in cell cycle progression are frequently mutated in human cancers leading
to uncontrolled cell division and tumor growth. Cyclin D1 is a member of the cyclin
G1 family and controls the transition from G1 to S phase in the cell cycle. The cyclin
D1 protein is encoded by the CCND1 proto-oncogene localized on chromosome 11q13. Binding
of cyclin D1 to the cyclin-dependent kinases 4 and 6 (CDK4/6) results in the formation
of active complexes that phosphorylate the retinoblastoma tumor suppressor gene during
the G1 phase.[2 ] Mutation, amplification, and overexpression of the CCND1 gene, which can alter cell
cycle progression, are observed frequently in a variety of tumors and may contribute
to tumorigenesis. Multiple studies have shown cyclin D1 overexpression as a potential
biomarker for precancerous and cancerous endometrial lesions, however, whether it
participates in a causative or incidental manner in tumor progression is still to
be determined.[3 ]
[4 ]
This study was aimed to check the relationship between cyclin D1 expression in various
endometrial lesions and clinicopathological parameters to explore the possibility
for cyclin D1 as a potential diagnostic marker to distinguish these endometrial lesions.
Material and Methods
This study was a prospective case–control study; 50 cases of endometrial biopsy/hysterectomy
specimens were selected randomly for the study from the period of October 2013 to
September 2015. Uncooperative and nonconsenting patients were excluded. The study
was done after approval from the Institutional Ethical Committee. The histological
diagnosis and staging were assigned based on the criteria established by the International
Federation of Gynecology and Obstetrics Staging of Endometrial Cancer, 2009.[5 ] Out of 50, 16 hysterectomy specimens and 34 endometrial biopsies were included in
the study. The samples included simple hyperplasia without atypia (n = 10), complex hyperplasia without atypia (n = 6), complex hyperplasia with atypia (n = 4), and endometrial carcinoma (n = 20). Ten cases of normal proliferative and secretory endometrium were selected
as control. Special histochemical stains were applied as per standard techniques.
Immunohistochemistry was performed using rabbit monoclonal antibody technology (clone:
EP12 manufactured by DAKO Epitomics Inc.).
The streptavidin-biotin method was performed on the paraffin sections to study the
expression of cyclin D1. Sections of 4 microns size were deparaffinized with xylene
and rehydrated through graded alcohol. Antigen retrieval was done using Tris EDTA
in the microwave. Sections were rinsed in tris-buffered saline (TBS) and then treated
with FLEX peroxidase blocking reagent (DM821). Then sections were incubated with FLEX
monoclonal rabbit anti-human cyclin D1 antibody (Code ISO83). The slides were washed
and incubated with FLEX/HRP (Code DM822) and then washed with TBS. An avidin-biotin-peroxidase
complex (DAKO, North America, United States) with DAB (3,3’-diaminobenzidine) as chromogen
was used for detecting antibody binding. Slides were counterstained with Mayer’s hematoxylin.
Representative samples of breast carcinoma served as a positive control for cyclin
D1 antibody. Cyclin D1 staining was assessed in the glandular epithelium according
to the number, distribution, and intensity of the positively stained cells.
Cyclin D1 was evaluated for intensity and extent of nuclear positivity. Due to heterogeneity
of the reaction, the area with the highest level of nuclear staining was selected
and the number of positively stained nuclei in 1,000 glandular cells was counted under
40×magnification and results were expressed as percentage staining. Staining of cyclin
D1 was evaluated according to the number, distribution, and intensity of the positively
stained cells. The intensity was graded as 0 = no staining; 1+ = weak nuclear staining
(faint chromatin pattern); 2+ = moderate nuclear staining (chromatin clearly defined);
and 3+ = marked nuclear staining (chromatin obscured by staining) and the extent of
staining was graded on a scale of 1 to 100 and scoring was done as < 10% = 0; 11 to
30% = 1+; 31 to 60% = 2+, and 60 to 100% = 3+.
Results
The age of the patients was in the range of 26 to 75 years. Out of 20 cases, 15 (75%)
with endometrial carcinoma were in the post-menopausal age group. In complex hyperplasia,
four cases were of atypia while six cases were complex hyperplasia without atypia
([Fig. 1 ]). [Table 1 ] shows the distribution of different endometrial lesions according to age groups.
Table 1
Distribution of various endometrial lesions according to age (n = 50)
Age group
(y)
No. of
cases
Simple hyperplasia
(n = 10)
Complex hyperplasia
(n = 10)
Malignant endometrial tumor
(n = 20)
N
%
N
%
N
%
N
%
21–30
01
2%
–
–
–
–
–
–
31–40
16
32%
3
30%
5
50%
2
10%
41–50
15
30%
6
60%
2
20%
4
20%
51–60
13
26%
1
10%
3
30%
9
45%
61–70
04
8%
–
–
–
–
4
20%
> 70
01
2%
–
–
–
–
1
5%
Mean age
46.1
42.0
43.0
54.25
Fig. 1 (A ) Low power (10×) view showing simple hyperplasia without atypia (H&E) inset showing
negative cyclin D1 expression, intensity 1+, extent 0 (< 5%). (B ) Low power (10×) view showing complex hyperplasia without atypia (H&E) inset showing
negative cyclin D1 expression, intensity 1+, extent 0 (5–10%). (C ) High power (40×) view showing simple hyperplasia with atypia (H&E) inset showing
positive cyclin D1 expression, intensity 2+, extent 3+ (70–80%). H&E, hematoxylin
and eosin.
Amongst endometrial carcinoma, 15 cases (60%) were of endometrioid type (two cases
revealed villoglandular pattern while squamous morules were seen in three cases),
three cases were reported to be clear cell type and two cases were papillary serous
carcinoma ([Figs. 2 ]
[3 ]). Ten cases (50%) of endometrial carcinoma were found to be in grade I, three cases
(15%) were in grade II, and seven cases (35%) including clear cell and papillary serous
carcinoma were in grade III. Nine cases of endometrial carcinoma were considered for
staging as only hysterectomy specimens could be assessed. Six cases (66.7%) were found
to be in Stage IA, two cases (22.2%) were in Stage II, and one case (11.1%) in Stage
III. Out of nine, six cases (66.67%) showed less than half myometrial invasion and
three cases (33.33%) showed more than half myometrial invasion.
Fig. 2 (A ) Low power (10×) view showing endometrioid carcinoma in a glandular pattern (H&E)
inset showing positive cyclin D1 expression, intensity 2+, extent 1+ (10–15%). (B ) Low power (10×) view showing endometrioid carcinoma villoglandular type (H&E). (C ) Low power (10×) view showing endometrioid carcinoma villoglandular type (H&E) positive
cyclin D1 expression, intensity 3+, extent 2+ (50%). H&E, hematoxylin and eosin.
Fig. 3 (A ) High power (40×) view showing endometrioid carcinoma arranged in glands with squamous
morules (adenoacanthoma) (H&E) inset showing positive cyclin D1 expression, intensity
2+, extent 1+ (20%). (B ) High power (40×) view showing clear cell carcinoma of endometrium, a cell having
abundant clear cytoplasm with high-grade nuclei and hob-nailing, inset showing prominent
hob-nail appearance (H&E). (C ) High power (40×) view showing clear cell carcinoma of endometrium, positive for
cycleinD1 expression, intensity 2+, extent 2+ (30–40%). H&E, hematoxylin and eosin.
Expression of intensity and extent of cyclin D1 in endometrial diseases : [Table 2 ] shows the nuclear intensity and extent of immuno-expression of cyclin D1 in normal
proliferative, hyperplastic, and neoplastic endometrial samples. It was observed that
all the cases of clear cell carcinoma and serous carcinoma gave positivity for cyclin
D1 while 11 out of 15 (73.33%) endometrioid carcinoma were positive for cyclin D1.
Table 2
Immunoreactivity of cyclin D1 (extent and intensity) in normal and various endometrial
lesions
Lesion
No.
of cases
Extent
Intensity
0
1
2
3
0
1
2
3
N
%
N
%
N
%
N
%
N
%
N
%
N
%
N
%
N
%
Proliferative endometrium
05
10%
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Secretory endometrium
05
10%
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Simple hyperplasia
10
20%
7
70%
3
30%
–
–
–
–
3
30%
7
70%
–
–
–
–
Complex hyperplasia
10
20%
2
20%
3
30%
4
40%
1
10%
–
–
5
50%
5
50%
–
–
Endometrial carcinoma
20
40%
4
20%
4
20%
11
55%
1
5%
–
–
6
30%
10
50%
4
20%
Statistical comparison of cyclin D1 expression in endometrial diseases : Statistical analysis showed a significant difference in extent as well as the intensity
of cyclin D1 immunoreactivity between simple hyperplasia and carcinoma (p = 0.015 and p = 0.030, respectively). No significant difference was found between simple hyperplasia
and complex hyperplasia ([Table 3 ]).
Table 3
Comparison of extent and intensity of cyclin D1 expression
Parameter a vs. parameter b
Positives
(Parameter a )
Positives
(Parameter b )
χ
2
p- Value
N
%
N
%
Abbreviation: NA, not applicable; NS, not specified.
Simple hyperplasia
vs. complex hyperplasia
Extent
3/10
30%
8/10
80%
5.051
0.070 NS
Intensity
7/10
70%
10/10
100%
3.529
0.211 NS
Simple hyperplasia
vs. carcinoma
Extent
3/10
30%
16/20
80%
7.177
0.015 S
Intensity
7/10
70%
20/20
100%
6.667
0.030 S
Complex hyperplasia vs. carcinoma
Extent
8/10
80%
16/20
80%
0
1.000 NS
Intensity
10/10
100%
20/20
100%
NA
NA
Correlation of cyclin D1 with various clinicopathological features : Various clinicopathological parameters were correlated with cyclin D1 expression.
Statistical analysis showed a significant difference with menopausal status (p = 0.033). No significant correlation was found with other parameters including myometrial
invasion, grade, and stage of the tumor.
Discussion
Endometrial cancer is the fourth most common cancer occurring in women after breast,
bowel, and lung cancers and is derived from the endometrial epithelial lining of the
uterine corpus.[6 ] Endometrial carcinoma is often preceded by endometrial hyperplasia. Hyperplasia
is usually associated with exogenous estrogen stimulation and thus, estrogen is considered
as an endometrial carcinogen. There is a continuum of changes evolving to endometrial
carcinoma.[1 ] Clinicopathological, immunohistochemical, and molecular genetic studies during the
last two decades have generated substantial data to support the development of endometrial
carcinoma.[7 ]
[8 ]
Immunohistochemical methods have been useful for detecting several biomarkers of possible
prognostic importance for several cancer types. Cyclin D1 is a key protein in the
regulation of the cell cycle at the G1 to S phase transition and is essential for
the regulation of proliferation, differentiation, and transcriptional control. Overexpression
of cyclin D1 induces excessive cellular proliferation and is often overexpressed in
human neoplasias, e.g., in situ and infiltrating ductal breast carcinoma, colorectal
carcinoma, bladder carcinoma, head and neck, lung and prostate cancers by rearrangement,
amplification, and mutation of the genes.[9 ]
Overexpression of cyclin D1 in endometrial cancer has been reported in previous studies
but very few studies have compared the expression of cyclin D1 in various endometrial
conditions. In the present study, none of the cases of proliferative and secretory
endometrium showed positivity for cyclin D1. Three out of ten cases (30%) of simple
hyperplasia were positive for cyclin D1. In complex hyperplasia, eight out of ten
cases (80%) gave positivity for cyclin D1. All these findings correspond to studies
done by Ruhul Quddus et al,[10 ] Özuysal et al,[11 ] Choudhary and Bansal,[12 ] Liang et al,[13 ] Stewart et al,[14 ] and Shevra et al.[15 ] Increased expression by cyclin D1 was seen in few cases of simple hyperplasia and
complex hyperplasia without atypia. The reason could be explained by the fact that
as the normal proliferative cell enters the cell cycle from G0 phase, cyclin D1 is localized in the nucleus early in the G1 phase, and exits as the cell progresses into the S phase. One case of complex hyperplasia
with atypia showed foci of extensively crowded glands showing atypia with cyclin D1
expression in almost 70% of nuclei, strongly suggesting that the lesion is potentially
premalignant.
In endometrial carcinoma, out of 20 cases, 16 cases gave cyclin D1 positivity. Four
cases (20%) showed an extent of 1+, 11 cases (55%) showed an extent of 2+, and one
case (05%) showed an extent of 3+. Shawana et al revealed strong staining in > 30%
of nuclei in eight (44.4%) cases of endometrial adenocarcinoma.[3 ]
Consistent with previous findings, we observed a gradual and progressive increase
in expression of cyclin D1 levels in normal proliferative, hyperplastic endometrium,
atypical hyperplastic proliferation, and endometrial carcinoma. [Table 4 ] shows a comparison of cyclin D1 reactivity in simple, complex hyperplasia, and endometrial
carcinoma. The mechanism of cyclin D1 dysregulation is likely to contribute to an
increase in the proportion of cells in the transition from G1 to S phase.
Table 4
Comparison of cyclin D1 immunoreactivity
Study
Year
Cyclin D1 expression in
Simple hyperplasia
Complex hyperplasia
Carcinoma endometrium
Ruhul Quddus et al[10 ]
2002
57%
71%
68%
Özuysal et al[11 ]
2005
0%
1%
26.6%
Choudhary et al[12 ]
2006
0%
33%
50%
Liang et al[13 ]
2013
30%
49%
67%
Our study
2015
30%
80%
80%
Statistical analysis of the extent of cyclin D1 immunoreactivity showed that in simple
hyperplasia and carcinoma endometrium, the difference was statistically significant
(p = 0.015) while in simple hyperplasia versus complex hyperplasia and complex hyperplasia
versus carcinoma, the difference was not statistically significant. In our study,
no statistical correlation was found in simple hyperplasia versus complex hyperplasia
and complex hyperplasia versus carcinoma while in other studies there was a significant
statistical difference.[10 ] This discordance may be due to the small sample size in our study.
Ruhul Quddus et al analyzed immunoreactivity in five groups (proliferative, secretary,
simple hyperplasia, complex hyperplasia with or without atypia, and endometrial carcinoma)
and have shown that they are statistically different groups. Their observations were
similar to ours and have documented no difference between complex hyperplasia and
carcinoma and between proliferative, secretory, and simple hyperplasia. They did not
segregate atypical hyperplasia as a separate category. Instead, they merged it with
the complex hyperplasia category in their study. Significant overexpression was also
noted in papillary, syncytial, and squamous metaplasia compared with normal surface
epithelium or epithelium with tubal metaplasia. They suggested that maximal dysregulation
occurs at the complex hyperplasia state and concluded that some alterations might
be responsible for the different morphologic features and behavior of complex hyperplasia
and carcinoma. Authors have also suggested that cyclin D1 overexpression might be
an early event in endometrial carcinogenesis.[10 ]
Liang et al checked the relationship between its expression and clinicopathological
variables of endometrial lesions to explore the possibility for cyclin D1 as a potential
diagnostic and prognostic marker. The results of this research showed the expression
of cyclin D1 increased in the order of simple hyperplasia, complex hyperplasia, endometrioid
carcinoma, clear cell carcinoma except for endometrial serous carcinoma; the statistical
comparisons of these types with each other demonstrated no statistical significance
(p > 0.005).[13 ]
Shevra et al evaluated and compared the expression profile of cyclin D1 and Ki -67 expressions in 61 endometrial samples. There was increased expression of cyclin
D1 and Ki -67 in patients with endometrial carcinoma relative to proliferative endometrium and
simple hyperplasia, but there was no such difference between cases of atypical hyperplasia
and endometrial carcinoma.[15 ]
Kala et al evaluated the expression of cyclin D1 in hyperplasia and endometrial carcinoma
and compared the variation of cyclin D1 expression in various tumor types, especially
serous and endometrioid carcinomas. A statistical difference was found in the extent
of cyclin D1 positivity of complex hyperplasia and carcinoma of the endometrium. None
of the cases of serous carcinoma showed cyclin D1 immunoreactivity. No correlation
was seen between cyclin D1 expression and clinicopathologic parameters in endometrioid
carcinomas. It was inferred that cyclin D1 expression is a quantitative molecular
dysregulation that is increasing progressively from complex hyperplasia to carcinoma
of the endometrium.[16 ]
Various clinicopathological parameters were correlated with cyclin D1 expression.
Contrary to various studies our findings revealed significant statistical difference
with menopausal status (p = 0.033).[13 ]
[17 ] No significant correlation was found with other parameters including grade, stage,
and myometrial invasion of the tumor similar to study done by Liang et al and Kala
et al.[13 ]
[16 ]
[18 ]
Conclusion
Our findings suggest that cyclin D1 is dysregulated and therefore may have a role
in endometrial carcinogenesis. Since there was a statistically significant difference
in cyclin D1 expression between simple hyperplasia and carcinoma, it appears that
the dysregulation is maximal at the level of complex hyperplasia and that cyclin D1
overexpression may be an early event in endometrial carcinogenesis. Our findings support
the significance of complex hyperplasia as a precursor lesion and to some extent,
simple hyperplasia is also precancerous. Thus, cyclin D1 overexpression may be an
informative biomarker to recognize subsets of endometrial lesions that may be precancerous
and therefore amenable to surgical therapy.
