| Abstract|| |
Background: Cyclin D1, a member of the cyclin protein family, is instrumental in the cell cycle due to its influence on the progression from G1 to the S phase. Its overexpression causes reduced doubling time and is also associated with clonogenic growth. The purpose of the present study was to assess cyclin D1 expression in patients with simple hyperplasia (SH), endometrial intraepithelial neoplasia (EIN) and endometrioid endometrial carcinoma, and to evaluate whether there was an association between cyclin D1 expression and the clinicopathological features of endometrioid endometrial carcinoma. Methods: Retrospective data were available for 193 patients (30 SH, 40 EIN, and 123 endometrioid endometrial carcinoma cases). To detect cyclin D1 expression, immunohistochemistry staining was performed with tissue microarrays. Results: The percentage of cases with positive cyclin D1 staining were 30%, 60% and 78%, for SH, EIN and endometrioid endometrial carcinoma, respectively (P < 0.001). Carcinomas with higher nuclear grade, histological grade, and FIGO grade displayed higher mean cyclin D1 expression compared to lower grade carcinomas. In addition, patients with lymphovascular invasion (P = 0.006), myometrial invasion (P < 0.001) and lymph node invasion (P < 0.001) had higher mean cyclin D1 expression compared to those without invasion. There was a significant correlation between cyclin D1 expression and clinicopathological features of endometrioid endometrial carcinoma including tumor grade, FIGO grade, lymphovascular invasion, lymph node invasion and myometrial invasion (P < 0.05 for each). Conclusion: Cyclin D1 expression is significantly higher in patients with endometrioid endometrial carcinoma compared to that of the SH and EIN. The extent of cyclin D1 expression is strongly correlated with nuclear and histological grade, myometrial invasion, lymphovascular invasion and lymph node invasion in patients with endometrioid endometrial carcinoma. These findings contribute in several ways to our understanding of cyclin D1 expression and provide a basis for future research on this topic.
Keywords: Cyclin D1, endometrial intraepithelial neoplasia, endometrioid endometrial carcinoma, FIGO grade, simple hyperplasia, tumor invasion
|How to cite this article:|
Yildirim HT, Nergiz D, Sadullahoglu C, Akgunduz Z, Yildirim S, Dogan S, Sezer C. The extent of cyclin D1 expression in endometrial pathologies and relevance of cyclin D1 with the clinicopathological features of endometrioid endometrial carcinoma. Indian J Pathol Microbiol 2020;63:412-7
|How to cite this URL:|
Yildirim HT, Nergiz D, Sadullahoglu C, Akgunduz Z, Yildirim S, Dogan S, Sezer C. The extent of cyclin D1 expression in endometrial pathologies and relevance of cyclin D1 with the clinicopathological features of endometrioid endometrial carcinoma. Indian J Pathol Microbiol [serial online] 2020 [cited 2020 Sep 24];63:412-7. Available from: http://www.ijpmonline.org/text.asp?2020/63/3/412/291677
| Background|| |
Endometrial carcinoma is one of the most common invasive tumors of the female genital tract, particularly in developed countries. It represents around 3% of all neoplasms and is reportedly responsible for almost 2% of all cancer-related deaths in adult females. Various potential risk factors have been identified for the development of endometrial cancers, including postmenopausal noncontraceptive use of hormones, use of combination oral contraceptives, higher body-mass index, metabolic syndrome, diabetes, smoking, and sedentary lifestyle.,,,, The most frequently observed histological subtype of endometrial cancer is endometrioid carcinoma (up to 85–90% of cases), which is usually preceded by endometrial hyperplasia.
The cell cycle is the process by which cells duplicate their genome and transfer one copy into two daughter cells. The four main phases of the cell cycle can be briefly described as follows: the G1 phase is the phase in which metabolites required for replication are accumulated; in the S phase, DNA is replicated; the G2 phase can be thought of as a silent interval during which the integrity of the replication process is ensured; and finally, the M phase is the phase in which actual cell division occurs. Several promoting and inhibiting factors that have a role in the regulation of proliferation and differentiation in transformed cells have been identified. Cyclins and cyclin-dependent kinases are one such group that induce transitions through the cell cycle. Cyclin D1, a member of the cyclin protein family, is responsible for the transition from G1 to S. The overexpression of cyclin D1 has been found to reduce doubling time and increase clonogenic growth in transformed cells.
A significant body of evidence has revealed that cyclin D1 overexpression has a role in gastrointestinal tract carcinomas, breast cancer, renal cell carcinoma, and lung adenocarcinoma.,, However, current evidence concerning the relation of cyclin D1 overexpression with endometrioid carcinoma and data comparing cyclin D1 overexpression concerning the grade, local invasion, and invasion pattern of endometrioid carcinoma is limited. Therefore, the present study aimed to evaluate cyclin D1 expression in patients with hyperplasia without atypia, endometrial intraepithelial neoplasia (EIN), and endometrioid endometrial carcinoma. Furthermore, to elucidate whether cyclin D1 expression has a role in the development or progression of endometrioid endometrial carcinoma, we also aimed to investigate the association between cyclin D1 expression and the grade and invasion of endometrioid endometrial carcinoma.
| Materials and Methods|| |
The present study was based on the retrospective analysis of endometrial samples received from patients who underwent hysterectomy or curettage in a tertiary care center from January 2014 and March 2018. The study was approved by a tertiary care center ethical committee (17/12, 2018). Clinicopathological data required for statistical analyses were retrieved from the institutional digital database. Sample preparation was performed as follows: After fixation with 10% formaldehyde, all samples were embedded with paraffin. H&E staining were applied to all samples for histopathological evaluations which were carried out by the same clinical pathologists who had experience with gynecological pathologies (HTY, CSA). Specimens without sufficient tissue and those with inconclusive results were excluded. FIGO grading and histological grading of endometrioid carcinoma samples were carried out according to the criteria of the International Federation of Gynecology and Obstetrics (FIGO).
Tissue microarray production and immunohistochemistry method
Tissue microarrays were prepared from FFPE tissue sections of the tumors. Manual slicing resulted in slices of approximately 2 mm thickness that ideally represents the tumor and the invasion area was used for immunohistochemistry which was performed with an automated immunostainer. Positive control samples were defined as representative sections of the lymph node. Slides that displayed brown nuclear regions of the tumor cells were accepted to show positive results. The extent of staining was performed in ten 40 x fields and was defined as the percentage of cells with positive immunostaining. Cases were defined as positive when cyclin D1 expression was over 5%.
Tissue microarray technique was designed to place small pieces of tissue taken with a hollow needle into a single paraffin block in a certain order and order from the foci thought to best reflect the previously identified tumor from multiple paraffin blocks. Thus, it was provided to evaluate a large number of tissues in a single preparation. Basically, for the application of the tissue microarray technique, (1) donor paraffin tissue blocks, (2) tissue punch tool or metal spacer tool which may be considered as a kind of punch, and (3) paraffin blocks containing receiver holes are required.
In the immunohistochemical study performed with the tissue microarray technique, the fact that the procedures take place under the same conditions, that there is control tissue in each preparation, and that each case is evaluated in a separate preparation gives more reliable results than single applications. Tissue microarray technique is a method with a reduced workload, time usage, and financial advantages.
All analyses were performed on SPSS v21 (IBM, Armonk, NY, USA). For the normality check, the Shapiro-Wilk test was used. Continuous data were described as mean ± standard deviation. Comparison of cyclin D1 expression concerning age, pathological diagnosis, nuclear and histological grade, FIGO grade, and myometrial invasion pattern was performed with one-way ANOVA. Tukey's honest significance difference test was used for post hoc analysis. Categorical variables were compared using the Chi-square test. Pearson and Spearman correlation methods were used to identify the association between cyclin D1 expression and selected variables including tumor grade and local invasion. P value < 0.05 was accepted as statistically significant.
| Results|| |
A final total of 193 patients with a mean age of 54 ± 10 years were included in the study. Thirty patients (16%) were diagnosed as hyperplasia without atypia, 40 (20%) had EIN while the remaining 123 (64%) patients had endometrial endometrioid cancer. Nearly 131 out of 193 (68%) cases were positive for cyclin D1 (Mean percentage 26.3 ± 19.4%). The percentage of positive cases for cyclin D1 was 30% in those with hyperplasia without atypia, 60% in those with EIN, and 78% in those with endometrial endometrioid cancer [Figure 1]a, [Figure 1]b, [Figure 1]c. The difference between groups was significant (P < 0.001). Patients with endometrial endometrioid carcinoma displayed higher mean cyclin D1 expression compared to those with EIN and hyperplasia without atypia (33.6 ± 27.3% vs 20.6 ± 18.5% and 3.96 ± 2.8%, respectively, P < 0.001). Carcinomas with a higher nuclear grade, histological grade, and FIGO grade displayed higher mean cyclin D1 expression compared to lower grade and lower stage carcinomas [Table 1] and [Figure 2]. In addition, patients with lymphovascular invasion (45.4 ± 31.1% vs. 24.9 ± 25.2, P = 0.006), myometrial invasion (39.2 ± 29.3% vs. 19.6 ± 11.2%, P < 0.001) and lymph node invasion (55.2 ± 29.4% vs. 25.1 ± 24.6, P < 0.001) had higher mean cyclin D1 expression compared to those without.
|Figure 1: (a) Cyclin D1 expression in hyperplasia without atypia (×20). (b) Cyclin D1 expression in endometrial intraepithelial neoplasia (×40). (c) Cyclin D1 expression pattern in endometrial carcinoma: A strong positivity immunohistochemical staining (×40)|
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|Table 1: Assessment of Cyclin D1 expression regarding clinicopathological features|
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|Figure 2: The extent of cyclin D1 expression in association with diagnosis, grade, and stage of the endometrial pathology|
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Correlation analysis revealed that cyclin D1 expression was strongly correlated with the presence of endometrial endometrioid carcinoma (r = 0.448; P < 0.001), nuclear grade (r = 0.604; P < 0.001), histological grade (r = 0.627; P < 0.001), and advanced FIGO grade (r = 0.652; P < 0.001). Lymphovascular invasion (r = 0.338; P = 0.002), lymph node invasion (r = 0.410, P < 0.001), and myometrial invasion (r = 0.411, P < 0.001) were also significantly correlated with cyclin D1 expression [Table 2].
|Table 2: Correlation between mean Cyclin D1 expression and selected variables|
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| Discussion|| |
The present study aimed to compare cyclin D1 expression in hyperplasia without atypia, EIN, and endometrial endometrioid carcinoma. Our findings reveal that cyclin D1 expression is significantly higher in patients with endometrial endometrioid carcinoma compared to those with hyperplasia without atypia and EIN. Our results also demonstrate that extent of cyclin D1 expression is strongly correlated with nuclear and histological grade, FIGO grade, myometrial invasion, lymphovascular invasion, and lymph node invasion in patients with endometrial endometrioid carcinoma.
Cyclin D1 has been identified as a proto-oncogene in recent years and it is critical in the regulation of transition from the G1 phase to the S phase of the cell cycle., Three- dimensional structure of cyclin D1 provides an appropriate binding interface for cyclin-dependent kinases (CDK). Activation of cyclin D1 with CDK-4 or CDK-6 leads to the phosphorylation of a series of proteins and consequently promotes the transition from G1 to S. Expression of cyclin D1 also stimulates various growth factors including endothelial growth factor (EGF). Increased activity of EGF is associated with poor prognosis in a variety of human cancers including breast, lung, and colorectal tumors. Given the action of cyclin D1, it is reasonable to assume that overexpression of cyclin D1 might contribute to the dysregulation of the cell cycle, subsequently leading to neoplastic growth.
Several studies have attempted to determine the link between cyclin D1 expression and the clinicopathological features of endometrial carcinoma. Even though some of them have not reported a clear association between cyclin D1 expression and clinicopathologic parameters, many have consistently reported that cyclin D1 expression is associated with advanced stage, histological grade, prognosis, and survival in patients with endometrial carcinoma.,,,, Our findings indicating higher expression of cyclin D1 in cases with endometrial endometrioid carcinoma compared to cases with EIN and hyperplasia without atypia, and also the increase in expression in cases with higher histological grade and stage are compatible with the majority of published data.
Cyclin D1 may induce proliferation, cell attachment, and migration either dependent or independent of the cyclin-dependent kinases and consequently contribute to tumor invasion and spread. Fusté and colleagues explained this phenomenon through the Ral-GTPase activation which is promoted by the attachment of cyclin D1 in the membrane. They reported that activation of Ral-GTPase enhanced cell motility and therefore invasion capacity of the tumor cells. However, evidence demonstrating the role of cyclin D1 in proliferative activity and resultant tumor invasion is still limited. Our findings demonstrated a significant difference in cyclin D1 expression between cases concerning the presence or absence of myometrial, lymphovascular and lymph node invasion. These findings indicate that cyclin D1 not only plays a role in the development of endometrial endometrioid carcinoma but is also critical in the proliferation and invasion of tumor cells.
Our study contributes to our understanding of cyclin D1 expression in endometrial endometrioid cancer and provides a basis for future research. We consider that our findings support the opinion that cyclin D1 expression plays a crucial role in endometrial carcinogenesis. Moreover, our results may suggest that cyclin D1 expression contributes to myometrial, lymphovascular, and lymph node invasion.
The present study has some limitations to be mentioned. Firstly, the lack of a control group consisting of cases without hyperplasia complicates the comparison of normal and neoplastic cells. However, previous studies showing clear differences in cyclin D1 expression between neoplastic and ordinary endometrial cells make a direct comparison unnecessary. Another limitation concerning the present study is the lack of prognostic data. Therefore, we could not provide information regarding the role of cyclin D1 on the prognosis of the patients included in our study.
| Conclusion|| |
The present study demonstrates a significant difference in the level of cyclin D1 expression among patients with endometrial endometrioid carcinoma, EIN, and hyperplasia without atypia; indicating that cyclin D1 expression might play a critical role in endometrial carcinogenesis. Our results also revealed a significant correlation between cyclin D1 expression and the stage and grade of the endometrial endometrioid carcinomas. Moreover, the present study shows that the extent of cyclin D1 expression is significantly associated with myometrial, lymphovascular, and lymph node invasion in patients with endometrial endometrioid carcinoma.
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Conflicts of interest
There are no conflicts of interest.
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Hulya Tosun Yildirim
Department of Pathology, Health Sciences University, Antalya Education and Research Hospital, Antalya 07050
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2]