Abstract
Background/Aim: To investigate tumor suppression as an indicator of malignization potential within endometrial polyps in asymptomatic postmenopausal women. Materials and Methods: Immunohistochemical studies of the phosphatase and tensin homolog (PTEN) were performed. Cases included 52 benign postmenopausal polyps, 19 endometrioid carcinomas with coexisting benign polyps, and 12 polyps with foci of carcinoma. Controls included 31 atrophic endometria and 32 benign premenopausal polyps. PTEN was scored by quantitative methods according to staining intensity. Results: The mean epithelial and stromal PTEN H-score in postmenopausal benign endometrial polyps (193.8 and 123.2, respectively) was significantly higher than that in the atrophic endometrium (135.5 and 90.2, p=0.008), and premenopausal benign endometrial polyps (100.7 and 198.7, p<0.001). Significant difference between postmenopausal endometrial polyps and endometrial carcinoma was noticed in the epithelial compartment (193.8 vs. 65.7, respectively, p<0.001). Conclusion: Asymptomatic benign postmenopausal polyps have a distinctively high tumor suppression compared with endometrial cancer, suggesting low malignization potential.
Endometrial polyps (EPs) are localized projections of endometrial tissue found in approximately 30% of women in the western world and are commonly considered benign endometrial lesions (1-4).
Hysteroscopic polypectomy is the gold standard to treat symptomatic endometrial polyps, to obtain specimens for histological evaluation, and to provide a relatively safe and cost-effective secondary prevention of endometrial cancer (EC) (5, 6). Widespread use of gynaecological ultrasonography has led to improved detection of asymptomatic endometrial polyps (6, 7). The management of asymptomatic postmenopausal women with incidentally identified EPs is still a matter of debate, as the association of these EPs with malignancy is low and accounts for 0.1-1.51%, compared with 4.47% in symptomatic EPs (4, 6, 8-11).
Phosphatase and tensin homolog (PTEN) mutations and deletions are the most frequent genetic alterations seen in endometrioid endometrial cancer with genetic inactivation found in approximately 80% of cases (12, 13). Immunohistochemical (IHC) analysis is a widely used technique capable to detect specific antigens in tissue samples, predict the course of the disease, and help in treatment decisions. Therefore, among the proteins that might be used as biological markers of endometrial cancer, one responsible for tumor suppression (PTEN), is expected to give a better understanding at the molecular level.
Accordingly, considering the high incidence of PTEN alterations in EC and given the lack of data on the potential for malignant transformation in asymptomatic postmenopausal polyps, the present study was conducted to investigate tumor suppression in postmenopausal endometrial polyps as one of the indicators of their malignization potential in asymptomatic women.
Materials and Methods
Design. A retrospective study done in the Lithuanian University of Health Sciences, Kaunas, Lithuania.
Patients and tissue collection. The selected cases were obtained from the Pathology Department at the Hospital of Lithuanian University of Health Sciences and included 52 benign postmenopausal endometrial polyps with no history of bleeding, 19 endometrial carcinomas which had coexisting benign endometrial polyps, and 12 endometrial polyps with foci of endometrial carcinoma. The controls included 31 cases of atrophic endometrium and 32 premenopausal benign endometrial polyps. Women were considered postmenopausal if they reported a period of amenorrhea of at least 12 months duration after the age of 45 years. Endometrial samples were submitted either as endometrial curetting, polypectomy or hysterectomy specimens of the patients diagnosed with endometrial polyps, endometrial carcinoma or endometrial atrophy. Two independent expert pathologists reviewed haematoxylin-eosin sections in order to confirm the histologic diagnosis. In the endometrial carcinoma cases, stage was determined by The International Federation of Gynaecology and Obstetrics (FIGO) surgical staging, 2009 (14).
Ethical approval. Kaunas Regional Biomedical Research Ethics Committee, Lithuanian University of Health Sciences, Lithuania, provided a consent waiver and approved the study (BE-2-14, 17 February 2016). All samples were coded using unique identifiers.
Immunohistochemistry. Paraffin-embedded blocks were obtained and 4 μm sections cut and placed on slides. Immunohistochemistry was performed on sections using the avidin-biotin-peroxidase complex method for the identification of the antigen binding sites. Monoclonal mouse anti-human PTEN antibody (Anti-PTEN clone 6H2.1; Dako Corp., Glostrup, Denmark) was used for the identification of the PTEN protein. Sections were heated at 62°C for 1 h, de-waxed in xylene and rehydrated in graded alcohol, and boiled in buffer (pH 9.0) for 3 min at 120°C. After blocking of endogenous peroxidase, they were incubated with the pre-diluted primary antibody Anti-PTEN clone 6H2.1 (Dako Corp., Glostrup, Denmark) using 1:100 dilution. After 30 min incubation, the slides were treated with anti-mouse IgG as the secondary antibody. Reactions were visualized using an avidin-biotin-peroxidase complex with 3,3’ diaminobenzidine (DAB, Sigma Chemical Co., St. Louis, MO, USA) as a chromogen. Counterstaining was performed with Mayer's haematoxylin. Finally, the sections were dehydrated and mounted. For each batch of slides, when staining with the primary antibody, negative and positive controls were used according to the manufacturer's recommendation.
Scoring of immunohistochemical staining. Image capture and analysis was performed using an Olympus BX 53F (Tokyo, Japan) light microscope and the digital camera QImaging EXi AQUA (Surrey, CO, Canada). Digital image analysis was performed using the image analysis software Image-Pro Plus (version 7.0). The overall stain intensity and the proportion of positive and negative glandular and stromal cells in the captured fields were counted under 40× magnification, and the semi-quantitative histo-score (H-score) was obtained per field. H-score was obtained by applying the following formula: H-score=1×(% light staining)+2×(% moderate staining)+3×(% strong staining).
Statistical analysis. Calculations were carried out using Statistical Package of Social Science, Windows version 23 (Statistical Package for the Social Sciences (SPSS), IBM, Brøndby, Denmark). As data were distributed normally, pairwise comparisons were performed by the Student's t-test. The numerical data are presented with mean and standard deviation and the results were considered significant when p-value was less than 0.05.
Results
PTEN immunoreactivity was heterogeneous (Figure 1). Some cells within a gland or some glands were negative for PTEN staining (PTEN null glands). The least proportion of PTEN null glands cases was noted among benign postmenopausal EP patients (4%), and this proportion was significantly lower than that in endometrial atrophy (19%, p<0.05), premenopausal polyps (28%, p<0.05) and EC (39%, p<0.05) (Figure 2).
The lowest epithelial PTEN immunoreactivity (mean H-score 65.7) was detected in EC and the differences were significant compared with postmenopausal (p<0.001) and premenopausal (p=0.006) EPs and atrophic endometria (p<0.001) (Figure 2, Table I). The mean epithelial and stromal PTEN H-score in postmenopausal benign EPs (193.8 and 123.2, respectively) was significantly higher than in atrophic endometrium (135.5 and 90.2, p=0.008), and premenopausal benign EPs (100.7 and 198.7, p<0.001), however, a significant difference between postmenopausal EPs and EC was noticed only in the epithelial compartment (193.8 versus 65.7, respectively, p<0.001) with no difference between stromal components (Table I).
When benign EPs were found in association with EC, epithelial PTEN expression was significantly lower in the cancer tissue, than in the polyp, at 76.8 versus 138.9; p=0.005 (Figure 3).
The group of EPs with foci of EC revealed a statistically significantly higher PTEN level in the polyp tissue itself compared with the EC focus, with overall H-scores of PTEN in epithelial and stromal components at 107.4 and 118.4 versus 48.1 and 68.8, respectively, p<0.05 (Table I, Figure 3).
Interestingly, the tissue of malignant polyps (including polyp tissue of cases where polyp was found with coexisting EC or had EC focus inside) in the epithelial compartment showed high tumor suppression (126.7), which had insignificant difference from the atrophic endometrium (135.5, p=0.543) or premenopausal polyps (100.7, p=0.089), and was significantly lower than that in benign postmenopausal polyps (193.8, p<0.0001) and higher compared with EC (65.7, p<0.0001).
Discussion
Asymptomatic postmenopausal endometrial polyps are a frequently reported incidental finding. Data concerning the malignization potential of asymptomatic postmenopausal EPs, however, remain unclear and sparse. This study was conducted to pursue evidence whether asymptomatic postmenopausal EPs show any early and the most common carcinogenetic alteration characteristic to endometrial cancer precursors. To the best of the authors' knowledge, in this article, we report the largest series of IHC of asymptomatic benign postmenopausal EPs and the first IHC investigation of malignant EPs.
PTEN over the spectrum of endometrium lesions. A: A case of endometrial cancer showing PTEN-null glands and PTEN positive stroma. B: A case of postmenopausal benign polyp showing PTEN positive glands and stroma. C: A case of atrophic endometrium showing PTEN positive glands and heterogeneous stroma. D: A case of premenopausal benign polyp showing PTEN positive glands and stroma. Original magnification ×10 (A, B, C, D).
Differential expression of PTEN over the spectrum of endometrium lesions. *p<0.05.
Mean values of H score for PTEN in different endometrial lesions.
PTEN is the second most frequently mutated tumor suppressor gene in human cancers (15, 16). Loss of PTEN expression is considered an early event in endometrial pathogenesis and the most common genetic defect in endometrioid carcinoma, seen in up to 80-83% of tumors that are preceded by a histologically discrete premalignant phase (17, 18). In 2016, a multidisciplinary panel of 40 leading experts of European Society for Medical Oncology (ESMO), European SocieTy for Radiotherapy & Oncology (ESTRO) and European Society of Gynaecological Oncology (ESGO) developed a consensus in the management of endometrial cancer and recommended immunohistochemical assessment of PTEN to distinguish premalignant endometrial changes from benign mimics (19). As the new era of personalized medicine is on the horizon, postmenopausal patients with asymptomatic EPs have a great perspective to benefit from a less aggressive treatment and, thus, reduction in morbidity and complications related with invasive procedures could be achieved.
Patterns of tumor suppression in postmenopausal endometrial polyps. In the present study, we found that PTEN expression was significantly reduced in endometrioid endometrial cancer compared to benign endometrial lesions. This is consistent with the findings from previous studies (20-27).
A surprising finding was that malignant polyps (including polyps from cases where a polyp was found with coexisting endometrioid cancer or had a focus of cancer inside) showed high tumor suppression in the epithelial compartment of the polyp tissue, which was not significantly different from that in atrophic endometrium or premenopausal polyps but was significantly lower than that in benign postmenopausal polyps and higher compared with endometrioid cancer. These results demonstrate a different expression of tumor suppressors in polyp tissue compared with endometrioid EC regardless of the proximity of the EP to the surrounding malignant endometrium.
Box and whisker plot showing epithelial expression of PTEN over the spectrum of endometrial lesions in selected cases. *p<0.05. PostM: Postmenopausal polyps; EP: endometrial polyp; EC: endometrial carcinoma. a: Cases of endometrial carcinoma with coexisting benign endometrial polyps; b: Cases of endometrial polyps with foci of endometrial carcinoma.
Additionally, our previous study revealed a very low proliferative activity in postmenopausal EPs, nevertheless they were benign, asymptomatic or a polyp tissue surrounding an EC focus (28).
Accordingly, we have two types of speculations - either our findings do not provide evidence that EPs have any genetic changes associated with early carcinogenesis, or EP is not a precursor lesion to EC, but has the same causes and risk factors as EC (epidemiological association).
Conclusion
Asymptomatic benign postmenopausal polyps have a distinctive profile, indicating high levels of tumor suppression compared with endometrial cancer. Our study findings support the idea that resection of incidentally found endometrial polyps in asymptomatic postmenopausal women should not be recommended routinely. Further studies are needed to determine whether endometrial polyps are true premalignant precursors or whether they are benign endometrial disease with the potential for endometrial cancer as any other locus of benign endometrium. The establishment of a wider genetic panel and new biomarkers could shed more light upon this disease and clarify the detailed carcinogenetic mechanism and the potential for it.
Acknowledgements
The Authors thank Angelija Valančiūtė and dedicated laboratory staff Rūta Curkūnavičiūtė and Laura Ribokaitė (Department of Histology and Embryology, Lithuanian University of Health Sciences, Kaunas, Lithuania) who made a measurable impact on this project and provided all technical assistance needed.
Footnotes
Authors' Contributions
Conceptualization: L.A.; Data curation: A.C., J.P.; Formal analysis: L.A.; Investigation: L.A., J.P., M.N.A.; Methodology: L.A., R.N., A.C., C.H, M.W.S., M.N.A, J.P.; Supervision: M.W.S., R.N.; Writing original draft: L.A.; Writing, review, editing: J.P., M.W.S., C.H.
Conflicts of Interest
The Authors declare no conflicts of interest regarding this study.
- Received December 3, 2019.
- Revision received December 12, 2019.
- Accepted December 17, 2019.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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