THE ONCOGENIC POTENTIAL OF ASYMPTOMATIC POSTMENOPAUSAL ENDOMETRIAL POLYPS

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY

Lina Adomaitienė

THE ONCOGENIC POTENTIAL OF

ASYMPTOMATIC POSTMENOPAUSAL

ENDOMETRIAL POLYPS

Doctoral Dissertation

Medical and Health Sciences, Medicine (M 001)

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Dissertation has been prepared at the Department of Obstetrics and Gynae-cology of Medical Academy of Lithuanian University of Health Sciences during the period of the 2014–2020.

Dissertation is defended extramurally. Scientific consultant

Prof. Dr. Rūta Jolanta Nadišauskienė (Lithuanian University of Health Sciences, Medical and Health Sciences, Medicine – M 001).

Dissertation is defended at the Medical Research Council of the Lithuanian University of Health Sciences:

Chairperson

Prof. Habil. Dr. Angelija Valančiūtė (Lithuanian University of Health Sciences, Medical and Health Sciences, Medicine – M 001).

Members:

Prof. Dr. Sonata Jarmalaitė (National Cancer Institute, Natural Sciences, Biology – N 010);

Assoc. Prof. Dr. Rasa Jančiauskienė (Lithuanian University of Health Sciences, Medical and Health Sciences, Medicine – M 001);

Prof. Dr. Brigita Šitkauskienė (Lithuanian University of Health Sciences, Medical and Health Sciences, Medicine – M 001);

Prof. Dr. Dace Rezeberga (Ryga Stradins University (Latvia), Medical and Health Sciences, Medicine – M 001).

Dissertation will be defended at the open session of the Medical Research Council on the 23rd of June 2020 at 12 a.m. in the Great auditorium of the Department of Obstetrics and Gynaecology of Lithuanian University of Health Sciences.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS MEDICINOS AKADEMIJA

Lina Adomaitienė

BESIMPTOMIŲ ENDOMETRIUMO

POLIPŲ SUPIKTYBĖJIMO

POTENCIALAS POMENOPAUZĖJE

Daktaro disertacija Medicinos ir sveikatos mokslai,

medicina (M 001)

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Disertacija rengta 2014–2020 metais Lietuvos sveikatos mokslų universitete Medicinos akademijos Akušerijos ir ginekologijos klinikoje.

Disertacija ginama eksternu. Mokslinė konsultantė

prof. dr. Rūta Jolanta Nadišauskienė (Lietuvos sveikatos mokslų univer-sitetas, medicinos ir sveikatos mokslai, medicina – M 001).

Disertacija ginama Lietuvos sveikatos mokslų universiteto medicinos mokslo krypties taryboje:

Pirmininkė

prof. habil. dr. Angelija Valančiūtė (Lietuvos sveikatos mokslų universi-tetas, medicinos ir sveikatos mokslai, medicina – M 001).

Nariai:

prof. dr. Sonata Jarmalaitė (Nacionalinis vėžio institutas, gamtos mokslai, biologija – N 010);

doc. dr. Rasa Jančiauskienė (Lietuvos sveikatos mokslų universitetas, medicinos ir sveikatos mokslai, medicina – M 001);

prof. dr. Brigita Šitkauskienė (Lietuvos sveikatos mokslų universitetas, medicinos ir sveikatos mokslai, medicina – M 001);

prof. dr. Dace Rezeberga (Rygos Stradinio universitetas (Latvija), medi-cinos ir sveikatos mokslai, medicina – M 001).

Disertacija ginama viešame Medicinos mokslo krypties tarybos posėdyje 2020 m. birželio 23 d. 12 val. Lietuvos sveikatos mokslų universiteto Aku-šerijos ir ginekologijos klinikos Didžiojoje auditorijoje.

Disertacijos gynimo vietos adresas: Eivenių g. 2, LT-50161 Kaunas, Lietuva.

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ABBREVIATIONS

AEH – atypical endometrial hyperplasia AUB – abnormal uterine bleeding BMI – body mass index

CAH – complex atypical hyperplasia CDK – cyclin-dependent kinase CRBP-1 – cellular retinol binding factor DAB – 3,3’ diaminobenzidine

DPE – disordered proliferative endometrium E/P – estrogen/progesterone

EC – endometrial cancer

EEC – endometrioid endometrial carcinoma EGFR – epidermal growth factor receptor EIN – endometrioid intraepithelial neoplasia EP – endometrial polyp

ER –estrogenreceptors

FIGO – the International Federation of Gynecology and Obstetrics H&E – hematoxylin-eosin

HNPCC – Human – non-polyposis colon cancer HRT – hormonal replacement therapy H-score – histologic score

IFN – interferon

IGF – insulin growth factor

IGF-BPI – insulin growth factor binding protein-1 IHC – immunohistochemistry

Il-1 – interleukin-1 IUD – intrauterine device

K-ras – Kirsten ras proto-oncogene LI – labelling index

LVSI – lympho-vascular space invasion MMP – matrix metalloproteinases O – observational

P – prospective

PBS – phosphate buffered saline PCOS – polycystic ovary syndrome PostM – postmenopausal

PR – progesterone receptors PreM – premenopausal

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RT – room temperature

SERM – selective estrogen receptor modulators SH – simplex hyperplasia

TGF – transforming growth factor TMX – tamoxifen

TNF – tumour necrosis factor TVUS – transvaginal ultrasonography VEGF – vascular endothelial growth factor

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1. INTRODUCTION

Endometrial polyps (EP) are outgrowths of endometrial tissue and are composed of varying amounts of glands and fibrotic stroma containing thick-walled blood vessels covered by epithelium [1]. The prevalence of EP in a general population is approximately 8% affecting up to 20% of postmeno-pausal women [2-4].

Improved performances in gynaecological ultrasonography have enabled an increasing number of often asymptomatic endometrial polyps to be detected [5]. Most of these polyps are removed surgically, as a precautionary measure, so as not to miss a case of endometrial cancer [5]. Accidentally diagnosed EPs are often asymptomatic, as they are also a common cause of abnormal uterine bleeding in both premenopausal and postmenopausal women [6]. There is no wide consensus in the literature on the clinical significance and management of asymptomatic polyps [7].

Hysteroscopic polypectomy is the gold standard to treat endometrial polyps and to obtain specimens for histological evaluation. But still there is a continuing debate as to when to offer hysteroscopic polypectomy, and how to best manage asymptomatic postmenopausal women with incidentally identi-fied endometrial polyps. In this situation, gynaecologists must decide whether to stand the risk of malignancy associated with endometrial polyps against the health care costs and complications associated with invasive procedures [6].

Within the endometrial polyps, there is differential expression of estro-genic and progestoestro-genic hormonal receptors, that leads to increased respon-siveness to an estrogenic environment. Estrogen stimulation activates growth factors and leads to belief that there could be an association with some kinds of endometrial cancer, particularly type I or endometrioid, which is estrogen-related [8,9]. Such cancers are often preceded by precursor lesions such as endometrial hyperplasias [9].

However, it appears that the risk of endometrial carcinoma in postmeno-pausal women with asymptomatic endometrial polyps is low [10]. The majo-rity of postmenopausal polyps are benign but a small proportion, i.e. 4.47% among symptomatic postmenopausal women in comparison to 1.51% of asymptomatic women of polyps are malignant [11].

Carcinogenesis is a multistep process that involves the induction of muta-tional activation in tumour suppressor genes, increased cellular proliferation, and angiogenesis for tumour growth [12]. Immunohistochemical methods are useful to detect each step of carcinogenesis using the biomarkers of possible prognostic importance for a number of cancer types, and can improve our

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understanding of the pathogenesis of gynaecologic malignancies. Therefore, among the proteins that might be used as biological markers of endometrial cancer, those responsible for proliferation (Ki-67), tumour suppression (PTEN), and cell-cycle dysregulation (p21) were examined for a better under-standing in a molecular level.

Given the lack of data on the potential and predictors for malignant transformation in asymptomatic postmenopausal polyps, we conducted the present study to determine the malignant potential, immunohistochemical biomarkers and predictors of malignancy in postmenopausal women with endometrial polyps.

1.1. The aim of the study

To investigate the proliferative activity, tumour suppression and cell-cycle regulation in endometrial polyps as an indicator of their oncogenic potential in asymptomatic postmenopausal women.

1.2. The objectives of the study

1. To evaluate the prevalence of malignancy among postmenopausal endo-metrial polyps found by ultrasonography or hysteroscopy.

2. To investigate a set of clinical parameters that are linked with malignancy in postmenopausal endometrial polyps.

3. To evaluate the expression of three genetic markers (Ki-67, PTEN, and p21), linked to endometrial carcinogenesis, in asymptomatic postmeno-pausal endometrial polyps:

a. To analyse the expression of the proliferation marker, Ki-67, in benign, asymptomatic postmenopausal endometrial polyps, malignant polyps and endometrial carcinoma (type I) in comparison with the atrophic endometrium and premenopausal endometrial polyps;

b. To investigate whether the aberrant expression of PTEN is associated with tumorigenesis in postmenopausal endometrial polyps and to com-pare the results between the groups;

c. To explore the role and expression of cell-cycle protein, p21, in benign, asymptomatic postmenopausal endometrial polyps, malignant polyps and endometrial carcinoma (type I) in comparison with the atrophic endometrium and premenopausal endometrial polyps.

4. To determine whether asymptomatic postmenopausal endometrial polyps have a genetic profile, concerning Ki-67, PTEN and p21, similar to that of endometrial cancer.

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1.3. A research hypothesis

Asymptomatic postmenopausalpolyps have a molecular profile dissimilar to that of endometrial cancer, consequently showing low potential of mali-gnancy.

1.4. Novelty of the study

The main objective of this study was to find out the oncogenic potential of asymptomatic postmenopausal endometrial polyps. Even though many researchers worked on endometrial cancer molecular profile, very few of them reported any comprehensive results about endometrial polyps.

If endometrial polyps are the precursors of endometrial cancer, then no wide and evidence based consensus could be found in the literature to answer the clinician “if to resect” or “when to resect” a polyp. Thus, for a better and deeper understanding, our study offers a new approach towards method and material.

New approach towards method. According to the author’s knowledge, this research is the one of the very few analysingthe potential for malignnacy of endometrial polyps not only from the clinical point of view, but primarily, in a molecular level. Uglietti [6], Ferrazzi [13], Lenci [14], Golan [15], Wething-ton [16] published the largest observational studies each encountering more than thousand patients evaluating the risk of malignancy of endometrial polyps in premenopausal and postmenopausal symptomatic and asympto-matic women. Two most recent systeasympto-matic reviews and meta-analysis concerning the oncogenic potential of endometrial polyps were conducted by Lee et al. and Uglietti et al. and involved a total of 10,572 and 35,345 patients, respectively, who underwent polypectomy with histopathologic analysis, showed an overall 3.57 and 2.73% malignancy of endometrial polyps. With respect to abnormal uterine bleeding, Lee et Uglietti revealed next results, respectively, 4.15 and 5.14% of women with symptomatic bleeding had neoplastic polyps (RR 1.97; 95% CI 1.24–3.14) compared with 2.16 and 1.89% of women without abnormal bleeding [11,13]. These findings are similar to those of other studies mentioned above showing a prevalence rate of malignancy in endometrial polyps ranging from 0.8% to 8%. These studies are observational and summarizes the association of menopausal status, uterine bleeding, polyp size and the risk of malignancy among women under-going polyp resection. Whilst immunohistochemistry is not a new method generally, our study offers a new approach towards the same problem, pro-viding a combined evaluation of malignancy potential and biological beha-viour of endometrial polyps using immunohistochemistry.

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Our methodology also includes the definable, reproducible and producing meaningful results of histopathological scoring tool (H-score) by which semi-quantitative data was obtained from tissues. Literature review reveals that many authors apply an ordinal scoring system and present their results in vague terms, such as “mild”, “moderate”, “severe” or as 0, 1, 2, 3 points, etc. This addresses a very real issue faced by researchers and practitioners in histo-pathological area and can reduce interobserver and intraobserver repeatability [17], and even compromise result comparing between researchers. Using H-score tool in our study is also relevant for researchers designing new implementations or optimizations.

With regard to the role of IHC in this study, it was designed not to provide new prognostic biomarkers for endometrial pathology differentiation, but to exploit three biomarkers for the evaluation of specific genetic characteristics of asymptomatic postmenopausal EPs and applying these results to a larger population.

New approach towards material. In addition to introducing the new way of exploring postmenopausal endometrial polyps in a genetic level, the literature search confirms that our research is the first to study such specific selected cases of endometrial lesions, e.g. malignant polyps (having EC focus inside), benign polyps and concurrent endometrial cancer in the same case, postmenopausal endometrial polyps and later developed endometrial cancer, and asymptomatic postmenopausal polyps.

We included all these rare above mentioned cases found in our database through 10 years to get a better understanding if an endometrial cancer focus inside the polyp has the same profile as a typical endometrioid endometrial cancer or differs in origin. Moreover, cases where benign polyp was found with coexisting EC inquire whether this polyp was a precursor of malignant disease or found incidentally. Ultimately, 4 cases of polyps and later deve-loped endometrial carcinoma for the same woman were extremely rare and informative giving a possibility to examine both previously resected endo-metrial polyp and later acquired EC.

Regarding the new approach towards material, the key contribution of this dissertation is the immunohistochemical profiling of directly polyps them-selves (not only EC). Mostly, researchers focus on the hormonal or molecular profile of endometrial cancer or endometrial hyperplasia and very few investigations analyse particularly the polyp tissue [9, 18–21]. Therefore we constructed the study analysing very tissue specific material that is associated directly with the main aim of this study.

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Overall, our study investigating very specific material in a molecular modality not only enables us to reflect and perform in new ways, but also adds novel and valuable information for the deeper understanding of oncogenic potential of asymptomatic postmenopausal EP.

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2. A LITERATURE REVIEW

2.1. Endometrial polyps: the scale of the problem

Endometrial polyps (EP) are outgrowths of endometrial tissue composed of varying amounts of glands and fibrotic stroma containing thick-walled blood vessels covered by epithelium [1]. EPs are the one of the most common etiologies of abnormal genital bleeding in both premenopausal and postmeno-pausal women [11, 22]. The prevalence is reported to be between 7.8% and 34.9%, depending on the population studied [2, 3, 23, 24].

The accurate incidence of the endometrial polyps is difficult to assess, because the data is retrospective, usually not primarily reporting the preva-lence and ultimately, many polyps are asymptomatic. Different diagnostic techniques can be used to diagnose endometrial polyps. Older past studies had nothing else to rely on, but only a histological diagnosis and material of endometrial polyps obtained at dilatation and curettage procedure. Obviously, blind dilation and curettage or endometrial biopsy is inaccurate in diagnosing endometrial polyps [25]. Later the introduction of invasive (hysteroscopy) and non-invasive (transvaginal ultrasonography (TVUS)) visual methods guided to a higher reported prevalence of uterine focal lesions [26, 27]. TVUS has a reported sensitivity of 19% to 96%, specificity of 53% to 100%, positive predictive value of 75% to 100%, and negative predictive value of 87% to 97% to diagnose endometrial polyps compared with hysteroscopy with guided biopsy [28]. The addition of colour-flow or power Doppler respecti-vely may improve the diagnostic capability of TVUS. Colour-flow Doppler may demonstrate the single feeding vessel typical of endometrial polyps [28]. Hysteroscopy with guided biopsy is the most common comparator for other techniques to diagnose polyps as it offers the highest sensitivity and speci-ficity for conservative measures. When hysteroscopy with guided biopsy is compared with diagnostic hysteroscopy alone, the latter only allows subject-tive assessment of the characteristic of the lesion with reported diverse sensitivity of 58% to 99%, specificity of 87% to 100%, positive predictive value of 21% to 100%, and negative predictive value of 66% to 99% [28–31]. Increasing age is the most common risk factor for the presentation of an endometrial polyp [32]. Among clinically recognized polyps, the prevalence appears to rise steadily with increasing age, and to be the highest in peri-menopausal and postperi-menopausal women. Among women undergoing endo-metrial biopsy or hysterectomy, the prevalence of endoendo-metrial polyps is 10 to 24 percent [33].

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Clinical impact and features of endometrial polyps is not enough discussed and is associated with their natural history, risk of malignancy and effects on fertility and pregnancy. Literature concerning the natural history of the polyps is sparse and insufficient, because expectant management is chosen rarely compared with treatment. Wong et al. conducted a retrospective study aiming to know more about the natural history of endometrial polyps in women who were managed expectantly and tried to answer if polyps continue to grow or regress. Their conclusion was that the growth rates of expectantly managed polyps vary considerably and cannot be accurately predicted. However, a small proportion of polyps do regress spontaneously. The study found no correlation between the growth rate of polyps and the subsequent develop-ment of abnormal uterine bleeding. Moreover, Wong et al. found that polyps may regress more frequently in premenopausal women and in those who presented with abnormal uterine bleeding [34].

Another clinical consequence of EP regarding their potential of malig-nancy will be discussed separately. EP effect on fertility and pregmalig-nancy will not be discussed broadly as this study is designated for postmenopausal women health. According to the research, EPs do not appear to be associated with an increased risk of spontaneous abortion or adverse obstetric outcomes, moreover, women evaluated for infertility have the same prevalence of EPs as generally in population [35–37].

To sum it up, as EPs are one of the most common aetiologies of abnormal genital bleeding and are associated with the uncertain potential of malig-nancy, they definitely become a problem in everyday clinician’s work and a material for the research in this field.

2.2. Risk factors for endometrial polyps

Several risk factors are linked with an increased risk of developing endometrial polyps. Assuming the fact that the highest risk to diagnose polyp is in the perimenopausal age, and they are rarely found before menarche, undoubtedly sex steroids are related with their etiology.Below discussed risk factors for endometrial polyps involve increased levels or activity of endo-genous or exoendo-genous estrogen.

2.2.1. Tamoxifen

Tamoxifen (TMX) (ICI 46 474), trans-1-(4-β-dimethylaminoethoxyphe-nyl)-1,2-diphenylbut-1-ene, is the most commonly used drug for the treat-ment of estrogen receptor positive breast cancer and has been saving lives worldwide for the past four decades. Tamoxifen due to its universal use in

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both treatment and chemoprevention of breast cancer is considered a pio-neering drug and is also eligible for novel selective estrogen receptor modu-lators (SERMs) research [38].

TMX acts mainly via estrogen receptors (ER), but also displays anti-tumour activity in breast cancer negative to ERs, suggesting other targets. Actually, tamoxifen has effects on several transduction pathways and diverse ion channels [39].

Despite the successful use of TMX, this drug produces some non-desirable side-effects by acting on different targets [39]. Tamoxifen exhibits ER agonist activity in the uterus and is associated with an increased risk of endometrial hyperplasia and malignancy. Endometrial safety has been an important consideration in the clinical development of SERMs, with improved benefit-risk profiles [40].

In the healthy women population (participants of Breast Cancer Prevention Trial (BCPT)), the relative risk of developing EC in the tamoxifen arm was 2.54, although when stratified by age, in women over 50, the risk increased to 4.01. Consequently, the risk appears to be restricted to women over 50, because, compared with women under 50, there was no statistically signi-ficant increase of endometrial carcinoma [41].

A case control study that enrolled 1,875 patients from three different countries revealed that patients with EC after breast cancer who received TMX treatment for five years for breast cancer have greater endometrial cancer mortality risk than those who did not receive TMX. According to study authors, this can be attributed to non-endometrioid histological subtypes with poorer prognosis among long term tamoxifen users [42].

TMX is associated with an increased incidence of benign endometrial lesions such as polyps and hyperplasia [42–44]. Polyps develop in 2.1 to 36 percent of postmenopausal women treated with tamoxifen and the incidence of EPs is higher in symptomatic than in asymptomatic women [45–47].

Systematic review and meta-analysis estimating factors associated with malignancy in hysteroscopically resected EPs showed that TMX use (PR, 1.53; 95% CI, 1.06–2.21; I2, 0.0%) was associated with endometrial polyp malignancy [48].

The clinician should be alerted when evaluating various endometrial pathologies described in association with postmenopausal TMX treatment.

2.2.2. Metabolic syndrome

Metabolic anomalies such as obesity, type 2 diabetes, hypertension and dyslipidaemia play an important role in abnormal endometrial proliferation [49].

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Bueloni-Dias et al. conducted a cross-sectional study to answer if meta-bolic syndrome is a predictor of endometrial polyps in postmenopausal wo-men, and finds out that a higher percentage of women with polyps were obese (72%) when compared with control (39%) (p< 0.0001). Waist circumference was greater among women with polyps (p = 0.0001). The incidence of dia-betes, hypertension, and dyslipidaemia was higher among women with endometrial polyps (p < 0.0001) [50].

Kaya et al. in their study attempted to assess the relation between benign endometrial pathologies (polyp and/or hyperplasia without atypia) and the metabolic status (insulin resistance and metabolic syndrome) of the patients. A total of 168 cases were enrolled in the study. The study observed a significant relationship between an insulin resistance and benign endo-metrial pathologies, and the conclusion that an insulin resistance may play an important role in the development of benign endometrial pathologies was made [49].

Also, above mentioned metabolic anomalies have been known as risk factors for type I endometrial cancer [49,51].

Systematic review and meta-analysis estimating factors associated with malignancy in hysteroscopically resected EPs showed that diabetes mellitus (PR, 1.76; 95% CI, 1.43–2.16; I2, 0.0%), systemic arterial hypertension (PR, 1.50; 95% CI, 1.20–1.88; I2, 75.9%) and obesity (PR, 1.41; 95% CI:1.13– 1.76; I2, 41.2%) were associated with endometrial polyp malignancy [48].

Data suggest that metabolic syndrome is associated not only with a higher risk for endometrial polyps development but with their malignancy as well.

2.2.3. Hormone replacement therapy

There is an evidence for an association between development of endo-metrial polyps and postmenopausal hormone replacement therapy.

Oguz et al. evaluated the iatrogenic effect of different protocols of hor-mone replacement therapy (HRT) on EP formation in 2685 menopausal women and made a conclusion that EP genesis may be dependent on the composition of medication used, e.g. type and dosage of the estrogen and progestogen. Especially a progestogen with high anti-estrogenic activity may play an important preventive role in the development of endometrial polyps [52]. However, a case of malignant endometrial polyp in woman with a levonorgestrel intrauterine system was reported by Kuzel et al. [53].

Iatrakis et al. evaluated the effect of different doses of hormone repla-cement therapy (HRT) on endometrial polyp formation in 398 menopausal women and comes to the conclusion that endometrial polyp formation may be related to HRT dosage [54].

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Risk factors for endometrial polyps are evidently the same as for EC, thus leading to the speculations that EPs are the precursors of EC [55-57].

2.3. Pathogenesis of endometrial polyps

A number of molecular mechanisms of endometrial polyp pathogenesis have been proposed to play a role. These incorporate monoclonal endometrial hyperplasia, overexpression of endometrial aromatase and gene mutations [58].

Indraccolo et al. conducted a semi-quantitative review of the pathogenesis of endometrial polyps. Their used log-linear model resulted significantly in the correspondence found with the following factors: causative link (ageing, bcl-2 protein, excess weight/obesity, tamoxifen regardless of timing, relation-ship between estrogen receptors and progestinics, unbalanced estrogen thera-py, estrogen-like effect, and unbalance between estrogens and progestinics), protective link (progestinics, anti-estrogenic action), and unclear link (meno-pause, Ki-67 protein, angiogenesis, tamoxifen for a short time, tamoxifen for a long time, hormone replacement therapy, endometritis/inflammation) [59] (Fig. 2.3.1). The perceptual map of this review is presented in Fig. 2.3.1.

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Fig. 2.3.1. Factors implied in endometrial polyps pathogenesis:

perceptual map from correspondence analysis.

Adapted from [59], written permission for republishing was obtained

Causative link, unclear link, and protective link are highlighted as the squared points of a bi-dimensional map. 58 factors are reported as smaller circular points. The closer the factors are to the squared points, the stronger the association is. E/P – estrogen/progesterone. CRBP-1: Cellular Retinol Binding Factor. HNPCC: Human Non-Polyposis Colon Cancer. HRT: Hormonal Replacement Therapy. IFN: Interferon. IGF: Insulin Growth Factor. IGF-BPI: Insulin Growth Factor Binding Protein-1. Il-1: Interleuchin-1. IUD: Intrauterine Device. PCOS: Polycystic Ovary Syndrome. TGF: Transforming Growth Factor. TNF: Tumour Necrosis Factor. VEGF: Vascular Endothelial Growth Factor.

2.3.1. Monoclonal endometrial hyperplasia

Monoclonal endometrial hyperplasia is one of the molecular mechanisms explaining the growth of endometrial polyps. Cytogenetic studies revealed that approximately 50% of uterine leiomyomas were characterized by clonal chromosomal alterations [60–62]. Identical monoclonal mutations have been found also in endometrial polyps. These karyotypic deviations are dominated

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by rearrangements involving a particular part of chromosome 12, i.e. region 12q13-15 [63]. The investigation explored that the multiple aberration region on chromosome 12q15 harbours recurrent breakpoints frequently found in a variety of benign solid tumours [63].

Walter et al. cytogenetically investigated postmenopausal endometrial polyp and found a single clonal karyotypic anomaly, inv(12)(p11.2q13), in about 30% of cells analysed after short-term culture. This finding contributes further to the hypothesis that the chromosomal segment 12q13-q14, which is also involved in chromosomal rearrangements in uterine leiomyomas, pleo-morphic adenomas of the salivary glands, lipomas, and myxoid liposarcomas, contains a gene or genes that are related to cellular proliferation rather than to malignant transformation [64].

Fletcher et al. karyotyped a large endometrial polyp in which 19 of 25 metaphase cells contained a t(1;6;4)(q21;p21;q13). Subsequent combined immunohistochemical/cytogenetic analysis showed all aberrant metaphase cells to be of mesenchymal derivation, whereas epithelial cells from the polyp were diploid. These studies indicated that rearrangement of chromo-some band 6p21 is a characteristic cytogenetic aberration in the stromal component of endometrial polyps [65].

To sum it up, cytogenetic studies revealed that the presence of clonal chromosomal changes were associated with pathogenesis of endometrial polyps.

2.3.2. Overexpression of endometrial aromatase

Aromatase is the key enzyme for estrogen biosynthesis. It is normally expressed in the human ovary, skin, adipose tissue and brain. Aroma-tase activity is not detectable in normal endometrium [66]. Exposure to excessive estrogen can cause hyperproliferation and neoplastic transfor-mation of breast and endometrial cells [67]. The current studies support a role of dysregulated aromatase expression in proliferative endometrial pathologies including EPs.

The CYP19A1 gene encodes the enzyme aromatase, which is responsible for the final step in the biosynthesis of estrogens and is a major contributor to circulating estrogen in the post-menopausal women [66,68].

A pathogenic role for an exaggerated local aromatase activity within a spectrum of estrogen-dependent proliferative disorders including breast cancer, mammary ductal hyperplasia, endometrial cancer, endometriosis, adenomyosis and uterine leiomyomas is suggested [69]. Pal et al. report that an overexpression of an endometrial aromatase may underlie pathogenesis of

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EP after evaluating CYP191A1 mRNA within the seven premenopausal EPs [69].

Maia et al. also concludes that the presence of aromatase expression is significantly higher in endometrial polyps than in disease-free endometria [70].

In conclusion, aromatase protein is overexpressed within EPs compared with normal endometrium.

2.3.3. Gene mutations

Kirsten ras proto-oncogene (K-ras) mutation is thought to occur at an early stage of neoplastic progression in the endometrium [71].

Mutations in codon 12 of K-ras were observed in 7 of 11 tamoxifen-related endometrial polyps in Hashisuga et al. conducted investigation [71]. K-ras mutation-positive cases exhibited in a significantly high number of endometrial polyps Takeda et al. study revealed [72]. Banno et al. concludes that if mutations occur, K-ras excessive signalling causes cell proliferation and induces carcinogenesis. Thus, K-ras mutations have been detected in 6– 16% of cases of endometrial hyperplasia and 10–31% cases of endometrial cancer. These results suggest an idea that K-ras is involved in two stages of carcinogenesis: a shift from endometrial hyperplasia to endometrial cancer and invasive proliferation of well-differentiated tumour cells [73].

Defects of tumour suppression may have an important role in tumori-genesis and in the formation of multiple endometrial polyps. A disintegrin-like and metalloproteinase domain with thrombospondin-type 1 motifs (ADAMTS) proteins in a very recent study are described as novel molecular mediators contributing to EPs physiopathology [74].

2.4. Oncogenic potential of endometrial polyps 2.4.1. Are EPs true cancer precursors?

The majority of postmenopausal polyps are benign but a small proportion is malignant.

Hysteroscopic resection has become the gold standard in the management of EPs as it enables the entire uterine cavity to be examined, full resection of the lesion is allowed with subsequent histological analysis [75]. Therefore, not to miss a case of endometrial cancer when the exact risk of malignancy in endometrial polyp certainly is not known, such strategy is usually suggested [76].

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Observational studies evaluating the risk of malignancy of endometrial polyps in premenopausal and postmenopausal symptomatic and asympto-matic women were analysed. Studies were identified using computerized databases (PubMed [National Library of Medicine, Bethesda, MD], MEDLINE and Cochrane Databases). Searches were done for any published and unpublished literature in English. Key words used “endometrial polyp,” “oncogenic potential,” “malignancy,” “postmenopausal,” “asymptomatic polyps”.

Inclusion criteria included: observational studies of women with endo-metrial polyps who underwent polypectomy with histopathologic diagnosis. Polyps were diagnosed either by transvaginal ultrasonography, saline in-fusion sonohysterography, or hysteroscopy. Intraepithelial changes occurring in endometrial polyps include atrophy, various metaplasias, hyperplasias, and adenocarcinoma. Cytologic atypia is the most common feature in diagnostic disagreements [77] and diagnostic subgroups often cannot be combined with certainty. Mutter et al. have considered a new classification system which refers to carcinoma precursors as endometrial intraepithelial neoplasia (EIN), [78] although there is no evidence concerning the less reproducibility of WHO 94’s classification than others [79].

The lack of agreement in the diagnoses of complex hyperplasia and atypical hyperplasia and the lack of reproducibility in the recognition of the histologic feature of stromal alterations to differentiate atypical hyperplasia from well-differentiated adenocarcinoma suggest that the histologic classify-cation should be simplified by including a combined category for simple and complex hyperplasia, called hyperplasia, and a combined category for aty-pical hyperplasia and well-differentiated adenocarcinoma, called endomet-rioid neoplasia [80].

Thus, in this review endometrial polyps were divided into two groups: benign and malignant. Polyps with simple or complex hyperplasia without atypia were considered as benign polyps; and polyps with simple or complex hyperplasia and atypia were considered as premalignant or malignant polyps.

Postmenopausal status was defined as the absence of menstruation for a period ≥1 year.

In this review, one systematic review and meta-analysis [11] (which encountered 17 observational studies from the year 1980 to 2010), and later observational studies were analysed (Table 2.4.1.1).

A total of 19,381 women were used to review the overall percentage of malignant polyps. Table 2.4.1.2 excluded 251 patients out of Wethington et al. and all of Lenci et al. studies because they did not present the results of premenopausal and postmenopausal women separately. Out of 19,381 endo-metrial polyps resected, 651 appeared to be malignant, accounting for 3.39%.

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The prevalence of malignant lesions among postmenopausal patients were more frequent than in premenopausal (4.79% vs. 1.53%) (Table 2.4.1.2).

Table 2.4.1.1. Review study characteristics and overall malignancy

Reference

(Lead Author) Year Country Patients, N

Patients with premalignant and malignant polyps, N Lee [11] 2010 USA 10,572 377 Ferrazzi [14] 2009 Italy 1922 64 Golan [16] 2010 Switzerland 1124 23

Wethington SL [6] 2011 United States 1011 18

Costa-Paiva [23] 2011 United States 844 36

Uglietti A [81] 2014 Italy 1284 36

Lenci [15] 2014 Brazil 1020 26

Ricciardi [82] 2014 Italy 973 41

Bel [75] 2017 France 631 30

Total: 19,381 651

Table 2.4.1.2. Risk of malignancy and menopausal status

Reference (Lead Author) Postmenopausal malignant polyps, N Postmenopausal malignant polyps, % Premenopausal malignant polyps, N Premenopausal malignant polyps, % Lee [11] 214/3946 5.42 68/3997 1.70 Ferrazzi [14] 64/1152 5.56 – – Golan [16] 16/641 2.50 8/483 1.66 Wethington SL [6] 12/361* 3.32 4/399* 1.0 Costa-Paiva [23] 32/665 4.67 4/205 1.95 Uglietti A [81] 30/481 6.2 6/803 0.7

Lenci [15] Not counted separately

Ricciardi

[82] 26/973 1.54 – –

Bel [75] 30/631 4.75 – –

Total: 424/8850 4.79 90/5887 1.53

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Lee et al. conducted a systematic review and meta-analysis that involved a total of 10,572 patients who underwent polypectomy with histopathologic analysis, and showed an overall 3.57% malignancy of endometrial polyps. With respect to menopausal status, endometrial neoplasia was identified in 5.42% of women with endometrial polyps who were postmenopausal (RR 3.86; 95% CI 2.92–5.11) compared with 1.70% of premenopausal women. With respect to abnormal uterine bleeding, 4.15% of women with symptom-matic bleeding had neoplastic polyps (RR 1.97; 95% CI 1.24–3.14) compared with 2.16% of women without abnormal bleeding (Tables 2.4.1.1; 2.4.1.2) [11].

Still no consensus approved if the size of an EP matters. While Lee et al. suggests to remove polyps gretater than 1 cm in diameter [11], Namazov et al. finds that the incidence of premalignant or malignant lesions among various cut-offs of polyp size (10, 15, 20 mm) was not significantly different [83].

Other recent studies involved in this review showed the same distribution of benign and malignant endometrial polyps within premenopausal and postmenopausal women compared with Lee et al. systematic review (Tables 2.4.1.1; 2.4.1.2).

All studies noted that the prevalence of women with abnormal uterine bleeding was significantly higher in the malignancy group. Moreover, obviously postmenopausal status in women with endometrial polyps is associated with an increased risk of endometrial malignancy. However, no evidence proves that EP itself develop into cancer.

2.4.2. Endometrial cancer pathogenesis

Endometrial cancer as the most common gynaecologic malignancy is increasing worldwide, and the number of patients with this disease is likely to continue to grow, including younger patients [73,84]. Current concepts of endometrial cancer successfully integrate traditional histopathology with pathogenetic mechanisms [85]. Many endometrial cancers show estrogen-dependent proliferation, but the carcinogenic mechanisms are unknown or not completely explained beyond mutations of single oncogenes and tumour suppressor genes [73].

Below enhanced understanding of dualistic EC model and novel early genetic events of endometrial tumorigenesis are described.

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25 2.4.2.1. Dualistic model

A dualistic model of endometrial tumorigenesis is historically recognized and used, broadly termed type I and type II, based on a classification system hypothesized by Bokhman in 1983, who was the first to describe two clinico-pathological types of endometrial carcinoma based on epidemiological studies [84,86,87].

Type I endometrial cancer typically develops in premenopausal or peri-menopausal women and occurs with unopposed estrogen exposure via aty-pical endometrial hyperplasia [73, 84]. Risk factors include obesity, anovu-lation, nulliparity, and exogenous estrogen exposure [84]. The tumour is positive for the estrogen receptor and progesterone receptor, exhibits well-differentiated endometrioid adenocarcinoma, has a lower frequency of lymph node metastasis, shows little muscular invasion, and often has a relatively favourable prognosis [73].

In contrast, type II endometrial cancer is less common, accounting for 10% to 20% of EC, tends to develop in postmenopausal women in an estrogen-independent manner, and is thought to be due to de novo carcinogenesis that develops directly from the normal (atrophic) endometrium, rather than via endometrial hyperplasia or undiagnosed precancerous lesions [73, 84]. The tissue type is specific, including extremely poorly differentiated endometrioid adenocarcinoma and papillary serous or clear cell carcinoma. Clinically, type 2 cancers are marked by an aggressive clinical course, and they have a pro-pensity for early spread and poor prognosis [73, 84, 88].

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Table 2.4.2.1.1. Characteristics of type I and II endometrial cancer

Type I endometrial cancer Type II endometrial cancer

Type Endometrioid cancer Non-endometrioid cancer

Development Perimenopausal age

Chronic estrogen stimulation Anovulatory cycles

Obesity Hypertension Diabetes

Age over 65 years No estrogen stimulation Atrophic endometrium

Incidence 80% of endometrial cancers 20% of endometrial cancers The initial

change Endometrial intraepithelial neoplasia (EIN) Atypicalhyperplasia

Endometrial intraepithelial carcinoma (EIC)

Method of

spreading Infiltration of the myometrium Spreading through the lymphatic vessels

Deep infiltration of the myometrium

Spreading through the lymphatic vessels

Course of the

disease Slow and stable High five-year survival rate (80–85%) Aggressive Peritoneal and lymph nodes metastases

Five-year survival rate is low (30–70%)

Grade Low grade and good prognosis High grade and poor prognosis Receptors ER+

PR+ ER– PR–

Molecular

disorders DNA repair genes PTEN (40–80%) K–ras (20–35%) microsatellite instability (20–40%) PIK3CA β–catenin (30–40%) TP53 (5–10%) E–catherin (10–15%) p16 (10%)

Microsatellite instability (up to 5%) PTEN (10%) K–ras (up to 5%) TP53 (90%) β–catenin (up to 5%) E–catherin (80–90%) p16 (40%)

ER: estrogen receptor. PR: progesterone receptor. DNA: deoxyribonucleic acid. PTEN: phosphatase and tensin homolog. TP53: tumour protein 53.

PIK3CA: phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha. Adapted from [89].

2.4.2.2. Molecular aspects of endometrial carcinogenesis

Recently, improved access to molecular and genetic level research, has focused on the identification of molecular changes leading to different histo-logical subtypes of endometrial cancer than only dualistic model.

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Environmental factors, including estrogen, an abnormal mismatch repair (MMR) system, genetic abnormalities, and aberrant methylation of DNA and microRNA, are currently brought forward as major mechanisms of carcino-genesis in endometrial cancer [73].

Besides morphologic and clinical aspects, type I and type II endometrial carcinomas are different by genetic mutations (Fig. 2.4.2.2.1).

Endometrioid and nonendometrioid cancers are associated with mutations from independent sets of genes [84,90]. Endometrioid endometrial carcino-mas involve mutations in PTEN, Kras, and β-catenin, as well as defects in DNA mismatch repair (Fig. 2.4.2.2.1; Table 2.4.2.2.1). Nonendometrioid endometrial cancers frequently show aneuploidy and p53 mutations [84].

Fig. 2.4.2.2.1. Gene mutations in the carcinogenesis of endometrial cancer.

Adapted from [73], written permission for republishing was obtained.

Table 2.4.2.2.1. Genetic alterations in endometrial cancer: percentage

fre-quency of genetic mutations identified in type I and II endometrial cancers Genetic Alteration Type I Carcinoma (%) Type II Carcinoma (%)

PTEN inactivation 50–80 10 K-ras mutation 15–30 0–5 β-catenin mutation 20–40 0–3 Microsatellite instability 20–40 0–5 p53 mutation 10–20 80–90 HER-2/neu 10–30 40–80 p16 inactivation 10 40 E-cadherin 10–20 60–90

HER-2/neu: human epidermal growth factor receptor. Adapted from [84].

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The Cancer Genome Atlas (TCGA) Research Network performed an integrated genomic, transcriptomic, and proteomic characterization of 373 endometrial carcinomas using array- and sequencing-based technologies in 2013 [91]. This research provided novel key molecular insights into tumour classification and classified EC into four categories: polymerase ε (POLE) ultramutated, microsatellite unstable, copy number low/microsatellite stable, and copy number high/’serous-like’ [91, 92]. TCGA based classification of EC has shown promise in refining endometrial carcinoma classification and more accurately reflecting patient outcome [87].

Advanced understanding of the molecular and genetic mechanisms of endometrial cancer carcinogenesis not only increases better understanding of the cancer itself development but also can propose individualized treatment for patients.

2.5. Biomarkers of endometrial cancer

2.5.1. Why are biomarkers of endometrial cancer not implemented so far?

In the last years, plentiful studies have investigated a broad spectrum of tumour biomarkers including those of EC.

Biomarkers Definitions Working Group describe a biological marker (biomarker) as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Biomarkers may have the greatest value in early efficacy and safety evaluations such as in vitro studies in tissue samples, in vivo studies in animal models, and early-phase clinical trials to establish “proof of concept.” Biomarkers have many other valuable applications including (1) use as a diagnostic tool for the identi-fication of those patients with a disease or abnormal condition; (2) use as a tool for staging of disease use as an indicator of disease prognosis; (3) use for prediction and monitoring of clinical response to an intervention [93].

Biomarkers include not only protein routinely used as tumour markers but also genes and chromosomes. The limiting factor in the use of markers in the diagnosis of endometrial cancer is their lack of specificity. However, specific markers for endometrial cancer are the subject of much research attention [89].

Significant biomarkers in the diagnosis of EC are given in Table 2.5.1.1 [89].

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Table 2.5.1.1. Biomarkers in the diagnosis of endometrial cancer

Biomarker group Biomarker

Serum Ca 125, Ca 15.3, HE4, VEGF

Genetic DNA ploidy

Suppressor genes PTEN, TP53 gene, p53 protein, p21, p16

Oncogenes HER2/neu, PI3K-AKT-mTOR, FGFR2, K-ras, MSI, ER and PR, Ki-67, Cox-2

Adhesive molecules E-cadherin, β-catenin

Clinical BMI, comorbidity, germline mutations

Imaging Ultrasound, CT-scan, MRI, PET

VEGF: vascular endothelial growth factor. BMI: body mass index. CT: computerized tomography. MRI: magnetic resonance imaging. PET: positron emission tomography.

The question is “why biomarkers of EC are not implemented so far?”. There are several issues to be mentioned:

•_{Many different biomarkers but only few validated. As it can be noticed} in the Table 2.5.1.1, biomarkers that are being investigated are of different groups and numerous. Only few of them are already validated and implicated in clinical practice (e.g. Ca 125), the vast majority have been proven to be unsatisfactory because cannot be applied for all types of EC, for example, serum Ca 15.3 levels are increased in 24-32% of patients with endometrial cancer [89].

• Which biomarker(s)? If different biomarkers are of potential use for different types of EC included in a molecular level, then how a clinician should be aware which biomarker fits the best for the exact patient from the beginning of diagnosis and treatment. Nature and financial resources are limited and, thus, there is no possibility to check every biomarker for every case, moreover, to use them for monitoring the patient. Further studies are needed to validate specific biomarkers or their groups for individual patient.

•_{Changes in management: from dualistic (clinic pathologic)} classify-cation to molecular. Understanding that EC is nowadays more than only type I and type II, complicates the implication of biomarkers in diagnosis. There is no clarity whether to proceed using dualistic model or to rely on molecular profiling of EC, or to combine both. This confusion is about to resolve knowing that so many investigations are in process.

• Clinical applicability. As some of biomarkers are serum and can be comfortably implicated in clinical practice for diagnosis and monitoring the patient as well, while others are genetic or immunohistochemical

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and can be applied only on pathistological material. This means that latter biomarkers are not always convenient and sustainable in prog-nostic and surveillance practical use.

Below three biomarkers, Ki-67, PTEN and p21, associated with different EC carcinogenesis mechanisms, are presented, likewise their literature review. This dissertation also includes immunohistochemical results of the same above mentioned biomarkers.

2.5.2. Ki-67

Uncontrolled proliferation is a hallmark of cancer [94]. Ki-67 pertains to non-histone proteins and is an affirmative proliferation marker at present. In cell cycle its expression begins to appear in phase G1, increases in phases S and G2, reaches to the peak in phase M, and disappears rapidly in the advanced stage of cell division, but it is not expressed in phase G0 [95]. During the proliferative phase of the menstrual cycle, the expression of Ki-67 is normally enhanced [43]. Proliferation status of tumours is most widely measured using Ki-67 which becomes an important marker in clinical treat-ment decisions. An accurate estimation of the tumour proliferation is of high importance to exclude patients with slowly proliferating tumour cells and to avoid overtreatment [96].

Ki-67 becomes the most reliable index in the detection of tumour cell proliferation activity due to short half-life period [95], thus the expression of Ki-67 reflects the tumour proliferation rate and correlates with initiation, progression, metastasis and prognosis of a number of types of tumours, e.g. correlation between Ki-67 expression and patients survival is proven in cervical, uterine, breast cancer, non Hodgkin’s lymphoma and large bowel cancer [97]. Unsurprisingly, the number of studies suggesting that Ki-67 may be an important factor in cancer grading and prognostic evaluation, is in-creasing.

Observational studies immunohistochemically evaluating the proliferative activity with Ki-67 of endometrial polyps, endometrial cancer, other endo-metrium lesions or normal endoendo-metrium in premenopausal and postmeno-pausal symptomatic and asymptomatic women were analysed. Studies were identified using computerized databases (PubMed [National Library of Medicine, Bethesda, MD], MEDLINE and Cochrane Databases). Searches were done for any published and unpublished literature in English. Key words used “Ki-67”, “proliferative activity”, “MIB-1”, “endometrial polyp”, “endometrium”, “profile”, “endometrial cancer”. Last time the data were collected 2nd_{Jan, 2019.}

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A total of 19 studies were included for systematic review (Table 2.5.2.1). Two of them were prospective, two – observational, others – retrospective studies.

Quantitative analysis was impossible due to heterogeneity of Ki-67 expression analysis methods used. Many authors applied an ordinal scoring system and presented their results in not quantitative terms, such as “low” or “high”, or as 0, 1, 2, 3 points, or negative , (+), (++), (+++), (++++). Nine studies presented their results in quantitative manner, the percentage of Ki-67. Nevertheless, the results of these later studies were incompatible with the method used to acquire the field of Ki-67 evaluation. Some of them while microscoping selected these fields randomly, the rest – used “hot spot” areas. Thus, only qualitative analysis was possible and authors’ key findings are presented in the Table 2.5.2.1.

Only two of reviewed studies investigated EPs [18,20] and none of them malignant ones. Most of the studies were exploring EC or endometrial hyperplasia. Most of these studies come to conclusion that IHC findings of Ki-67 may help to differentiate benign hyperplastic endometrium from low grade EC, and may serve as informative biomarker to recognize subsets of lesions that may be precancerous.

Among the proteins that are responsible for cellular proliferation Ki-67 is used as a cellular proliferation marker and might be used as a biological marker of endometrial cancer.

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Table 2.5.2.1. Studies examining Ki-67 expression in polyps and endometrium.

Author Year

[Ref] Country

Study

design N Specimens Markers Ki-67 expression analysis method Author’s conclusions

1 2 3 4 5 6 7 8

Apostolou 2013 [98]

Greece P 104 35 low grade EEC; 10 high grade EC; 10 DPE; 13 benign hyperplasia; 36 normal EM Ki-67, p53, Bcl-2, Cox-2 Percentage in randomly selected fields (low expression <40%, high – ≥40%)

IHC findings from a combination of Ki-67, p53, Bcl-2, Cox-2 may differentiate DPE/benign hyperplastic EM from low grade EEC

Cao 2002 [99]

USA R 40 10 benign EM; 6 CH;

24 EEC Ki-67, cyclin D1, cdk inhibitor, p21

Semiquantitative

(score 0-3) Ki-67 was positive in all proliferative and neoplastic EM Dahmoun

2004 [100]

Sweden P 43 92 PostM EM

biopsies before and during HRT

Ki-67, Apoptotic index, ER, PR

Percentage

(Ki-67 index) The unaffected homeostasis in EM epithelium contributes to EM safety and is in accordance with the histopathological findings of no hyperplasia Geels

2018 [101]

Netherlands R 53 EEC (53 cases) b-catenin, E-cadherin, ER, PR, PTEN, p16, MLH1, PMS2, L1CAM, p53, p21, MIB1

Percentage IHC of metastatic lesions in addition to primary EECs might be of value in managing EC

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Table 2.5.2.1 continued

1 2 3 4 5 6 7 8

Gurda 2014 [102]

USA R 74 44 EM lesions with

secretory features; 30 secretory EM

Ki-67 Percentage in “hot

spot” areas Ki-67 labelling index is a useful technique to distinguish secretory EM from cancer precursors Horree 2007 [103] Netherlands O 78 Inactive EM (16 cases); SH (23 cases); EEC (39 cases) Cyclin A, cyclin B1, cyclin D1, cyclin E, cdk2, p16, p21, p27, p53 and Ki-67

Percentage During endometrial carcinogenesis, there is increasing proliferation paralleled by progressive derailment of cyclin B1, cyclin D1, cyclin E, p16, p21, p27, p53, and cdk2.

Kahyaoglu 2012 [104]

Turkey R 59 19 eutopic and ectopic EM of endometriosis stage I-II; 19 eutopic and ectopic EM of endometriosis stage III-IV; 21 normal EM

Ki-67 Nuclear staining evaluated as negative , (+), (++), (+++), (++++)

The increase in proliferation activity as the severity of endometriosis increases is shown by the increase of Ki-67 index

Kato 2003 [105]

Japan R 92 23 normal EM; 9

endometrial hyperplasias; 60 EC

Ki-67, MCM2,

MCM3, ER, PR Percentage (LI) in randomly selected fields

The expression of MCM2 and MCM3 directly reflects cell proliferation in normal and hyperplastic EM. The replication-licensing system may be aberrant in EC

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Table 2.5.2.1 continued 1 2 3 4 5 6 7 8 Kitson 2016 [106] UK R 179 179 EC Ki-67 Percentage (proliferation index)

Hot spot scoring of whole sections is an optimal method to quantify Ki-67 in EC window study specimens Koi

2015 [107]

Japan R 82 82 PostM EM Ki-67, p53, ER Percentage (LI) Overexpression of p53 may be responsible for the high proliferative activity of PostM EM associated with conditions of low apoptotic cell death Maia Jr

2004 [18]

Brazil R 196 78 PreM polyps; 118

normal EM biopsies Ki-67, Bcl-2, p53 Percentage EPs undergo cyclic changes in the expression of their proliferation and apoptosis during the menstrual cycle Ozuysal

2005 [108]

Turkey R 89 30 EC; 14 AEH,

15 SH; 30 proliferative EM

Cyclin D1,

Ki-67 Percentage (LI) in “hot spot” areas Cyclin D1 expression in EC is higher than in proliferative EM and SH

Palazzo 1997 [109]

USA R 97 10 mormal EM;

9 cervical squamous dysplasias; 10 EH; 17 endocervical adenocarcinomas; 31 EC; 10 leiomyomas; 10 leiomyosarcomas Ki-67, p21 Semiquantitative: nuclear staining evaluated as negative , (1+), (2+), (3+), (4+) p21 expression is inversely correlated with proliferation in endometrial hyperplasias and EC as determined by Ki-67 staining Peres 2018 [20] Brazil Cross-sectional comparative study 90 30 EP without atypia; 30 EEC; 30 normal EM ER, PR, Ki-67, endoglin CD105, clau-dins 3 and 4, MMP-2 and -9 Percentage (proliferation index) in “hot spot” areas

Malignant potential of EP could not be determined by assessing the immuneexpres-sion of Ki-67

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Table 2.5.2.1 continued 1 2 3 4 5 6 7 8 Shevra 2015 [110] India R 142 38 EC; 26 CH;

78 SH Ki-67, Cyclin D1 Percentage in “hot spot” areas Cyclin D1 and Ki-67 may serve as informative biomarkers to recognize precancerous lesions and help to categorize these lesions

Shih 2003 [111] Japan R 102 82 cases of EC; 20 normal EM Ki-67, cyclins (D1, E, A, and B1), cdk2, cdk4, cdc2, and tumour-suppressor gene products (p53, p21, and p27) Positivity index

(PI) Various cell-cycle regulators are involved in activated cell growth of EC. Positive staining for cyclin A could be a useful marker for unfavourable patient prognosis. Sivridis

2004 [112]

Greece R 134 84 PostM EM;

50 EEC Ki-67, ER, PR, EGFR Percentage (semiquantitatively) At least half of the disease free PostM atrophic EM show a weak proliferative pattern.

Yamawaki 2017 [113]

Japan R 258 258 cases of

EEC SOX2, p21, p53, Ki-67 Semiquantitative (score 0-3), IHC-positive, IHC-negative

SOX2 expression stimulates cell cycle progression via p21 inhibition Yu 2015 [95] China R 173 67 EC; 53 normal EM; 53 AEH

ER, PR, C-erbB-2,

Ki-67 Percentage (semiquantitatively) Abnormal expression of Ki-67 might play an important role in EM malig-nant transformation and cell differen-tiation

P: prospective. R: retrospective. O: observational. EM: endometrium. PostM: postmenopausal. PreM: premenopausal. EEC: endometrioid endometrial carcinoma. EH: endometrial hyperplasia. SH: simple hyperplasia. CH: complex hyperplasia. AEH: atypical endometrial hyperplasia. H-score: histologic score. DPE: disordered proliferative endometrium. EIN: endometrioid intraepithelial neoplasia. LI: labelling index. EGFR: epidermal growth factor receptor. MMP: matrix metalloproteinases. ER: estrogen receptor. PR: progesterone receptor. Bcl: B-cell lymphoma protein. MLH: human mutL homolog. L1CAM: L1 B-cell adhesion molecule. CD105: cluster of differentiation 105. Cdk – cyclin dependent kinases. Cox-2: cyclooxygenase-2. MCM: minichromosome maintenance complex component 2. SOX2: SRY-box transcription factor 2. ErbB-2: Erb-B2 receptor tyrosine kinase 2.

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36 2.5.3. PTEN

The tumour suppressor, PTEN, encodes a phosphatase and acts through an Akt-dependent pathway to suppress cell division and promote apoptosis and is located on chromosome 10q23. PTEN mutations and deletions are the most frequent genetic alterations seen in endometrial cancer; genetic inactivation is found in approximately 80% of cases [114, 115]. Moreover, tissues that lose PTEN expression are susceptible to a higher cellular proliferation rate, leading to errors in DNA replication and a greater number of mutations, and potentially to carcinogenesis [9].

Observational studies immunohistochemically evaluating the activity with PTEN of endometrial polyps, endometrial cancer, other endometrium lesions or normal endometrium in premenopausal and postmenopausal symptomatic and asymptomatic women were analysed. Studies were identified using computerized databases (PubMed [National Library of Medicine, Bethesda, MD], MEDLINE and Cochrane Databases). Searches were done for any published and unpublished literature in English. Key words used “PTEN”, “tumour suppression”, “endometrial polyp”, “endometrium”, “profile”, “and endometrial cancer”.Last time the data were collected was the 2nd_{Jan, 2019.} A total of 21 studies were included for systematic review (Table 2.5.3.1). One of them was prospective, one – epidemiological, others – retrospective. Quantitative analysis was impossible due to heterogeneity of PTEN expression analysis methods used. Many authors applied an ordinal scoring system and presented their results in not quantitative terms, such as “absence”, “moderate”, or “strong”, or as colour intensity points 0, 1, 2, 3, or PTEN-null glands/heterogeneous/negative. And only 4 studies presented their results in quantitative manner, using H-scoring system. Thus, only qualitative analysis was possible and authors’ key findings are presented in the Table 2.5.3.1.

Only one of reviewed studies investigated EPs, and they were post-menopausal [9] and none of the studies explored malignant ones. Most of the studies were exploring EC or endometrial hyperplasia. The vast majority of the studies come to conclusion that PTEN is involved in the early stages of endometrial carcinogenesis and might improve discrimination between complex atypical hyperplasia (CAH) and EC, between simplex hyperplasia (SH) and CAH. Also might be used to predict the potential for progression from endometrial hyperplasia to invasive EC and presence and location of EC metastases. Troncon et al. study reveals no evidence that symptomatic EPs have a similar phenotype to type 1 EC [9].

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Table 2.5.3.1. Studies examining PTEN expression in polyps and endometrium.

Author

Year [Ref] Country design Study N Specimens Markers PTEN expression analysis method Author’s conclusions

1 2 3 4 5 6 7 8 Ayhan 2015 [116] USA Japan Taiwan R 114 114 AEH/EIN PTEN, ARID1A, Ki-67

PTEN loss ARID1A prevents PTEN inactivation from promoting cellular proliferation in the transition of precancerous lesions to EC

Brucka 2013 [117]

Poland R 49 20 EC; 29

normal EM PTEN, MMP-2 Quantitative, % Simultaneous evaluation of PTEN and MMP-2 immunoexpression in ectopic EM foci cannot be used to identify women with an increased risk of neoplastic transformation Cirpan

2006 [118]

Turkey R 37 37 EH PTEN Complete loss/partial

loss/present PTEN expression showed no differences among the cases of EIN, EC and proliferative phase EM.

El-Maqsoud 2009 [119]

Egypt R 67 12 normal EM; 12 SH; 8 AEH; 35 EC

PTEN, ER, PR H-score PTEN is involved in the early stages of EM carcinogenesis.

Erkanli 2006 [120]

Turkey R 77 29 EC; 38 EH; 10

proliferative EM

Survivin, PTEN, p27

+2 , +1 and negative Survivin overexpression might be an important mechanisms in the

development of EEC along with lost or decreased activity of PTEN and p27 Feng

2012 [121]

China R 187 35 normal EM; 28 AEH; 124 EC

PTEN,

PTTG1 PTEN positive/ PTEN negative High expression of PTTG1 and low expression of PTEN may be involved in pathogenesis and development of EC

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Table 2.5.3.1 continued

1 2 3 4 5 6 7 8

Geels 2018 [101]

Netherlands R 53 53 EEC b-catenin, E-cadherin, ER, PR, PTEN, p16, MLH1, PMS2, L1CAM, p53, p21, MIB1 Semiquantitative

score (0-9) IHC predicts both presence and location of EEC metastases

Geels 2015 [122]

Netherlands R 64 43 EEC; 21

Serous EC b-catenin,E-cadherin, ER, PR, PTEN, p16, MLH1, PMS2, L1CAM, p53, p21, MIB1

Semiquantitative

score (0–9) EEC arising from atrophic background EM were shown to be comparable to EEC arising from hyperplastic background EM

Huang 2015 [123]

Taiwan R 42 42 EC PTEN,

ARID1A PTEN positive/ PTEN negative The different frequencies of molecular genetic alterations between EC and ovarian endometrioid adenocarcinomas imply that distinct processes may be involved in their tumorigenesis Kapucuoglu

2007 [124]

Turkey R 95 23 normal EM;

37 EH; 35 EC PTEN, ER, PR, bcl-2, bax

H-score PTEN is involved in the early phases of EM tumorigenesis. Decreased PTEN expression with loss of differentiation in EC can contribute to the emergence of tumours with a more aggressive phenotype Kikalishvili 2018 [125] Georgia R 83 EH, EM dysplasia, EEC grade 1, EEC grade 2, EEC grade 3 Ki67, CD146,

PTEN Semiquantitative (<10% negative, 10–50% heterogenic, >50% positive)

Negativity of PTEN protein is increasing in parallel with the malignancy

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Table 2.5.3.1 continued 1 2 3 4 5 6 7 8 Kimura 2004 [126] Japan R 168 117 EC; 19 normal EM; 9 SH; 4 CH; 7 AEH

PTEN LI score and

staining intensity (1, 2, 3, 4)

Disturbed PTEN expression occurs in an early phase of the tumorigenesis of well-differentiated EC

Lee 2012 [127]

Korea R 75 10 normal EM; 21 SH; 22 AEH; 22 EC PTEN, six miRNAs (miR-21, 182, 183, 200a, 200c and 205) PTEN-null glands/ heterogeneous PTEN-null glands/PTEN negative

PTEN improve discrimination between CAH and EC, between SH and CAH and might be used to predict the progression from EH to invasive EC

Pieczynska 2011 [128] Poland R 98 59 SH; 20 CH; 19 AEH; 43 DPE

PTEN, ER, PR H-score PTEN presents as a strong prognosticator which may help in determining the risk of progression in advanced stages of EH Sarmadi 2009 [129] Iran R 87 29 normal proliferative EM; 21 SH; 8 AEH; 29 EC PTEN H-score, Colour intensity 0, +1, +2,

PTEN expression was significantly higher in cyclical EM than in atypical hyperplasia and EEC

Shawana 2016 [130]

Pakistan R 53 23 EC; 6 AEH (6 cases); 14 CH; 6 SH; 4 proliferative EM

PTEN,

Cyclin D1 PTEN loss Loss of PTEN, expression, and cyclin D1 overexpression was seen in a well differentiated EC and CAH, suggesting both as an early event in EM

carcinogenesis Skrzypczak

2013 [131]

Germany

Poland R 90 12 normal cyclical EM; 18 PostM EM; 60 EC

PTEN,

SCUBE2 Genes calculated according to the 2-delta delta Ct method

Decline of SCUBE2 expression in high-grade EC and its association with expression of ERa, PR and PTEN suggest that expression of this gene might be beneficial in EC

THE ONCOGENIC POTENTIAL OF ASYMPTOMATIC POSTMENOPAUSAL ENDOMETRIAL POLYPS

Lina Adomaitienė