The relationship between KRAS LCS6 polymorphism and endometrium cancer

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The relationship between KRAS LCS6

polymorphism and endometrium cancer

Feyza Nur İncesu Çintesun, Özlem Seçilmiş Kerimoğlu, Ersin Çintesun,

Süleyman Nergiz, Hasan Acar & Çetin Çelik

To cite this article: Feyza Nur İncesu Çintesun, Özlem Seçilmiş Kerimoğlu, Ersin Çintesun, Süleyman Nergiz, Hasan Acar & Çetin Çelik (2020) The relationship between KRAS LCS6 polymorphism and endometrium cancer, Journal of Obstetrics and Gynaecology, 40:7, 988-993, DOI: 10.1080/01443615.2019.1678576

To link to this article: https://doi.org/10.1080/01443615.2019.1678576

Published online: 02 Dec 2019.

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ORIGINAL ARTICLE

The relationship between KRAS LCS6 polymorphism and endometrium cancer

a

Department of Obstetrics and Gynecology, University of Health Sciences Konya Training and Research Hospital, Konya, Turkey;

b

Department of Obstetrics and Gynecology, Selc¸uk University Medicine Faculty, Konya, Turkey;cDepartment of Genetics, Selc¸uk University Medicine Faculty, Konya, Turkey

ABSTRACT

The aim of this study was to investigate the relationship betweenKRAS LCS6 mutation and endometrial cancer (EC). The study included 105 patients who had hysterectomy for benign reasons and 99 EC patients. The patients with Type 1 EC were classified according to histological properties, cancer stage, grade, tumour dimension, myometrial invasion (MMI), lymphovascular invasion (LVI), cytology, and number of positive lymph nodes.KRAS LCS6 mutation was examined in blood samples taken from all patients in both groups. No statistically significant difference was determined between the EC patients and the control group in demographic features. Weight and the Body Mass Index (BMI) values were higher in EC group (p < .001). While the incidence of this polymorphism is 5.8% throughout the world, the polymorphism rate was found to be 16.2% in the EC group and 12.4% in the control group, with no statistically significant difference determined (p > .05). Despite the higher rate of LCS6 polymorph-ism incidence in EC patients in this study conducted on a relatively large sample, there was not found to be a statistically significant difference in comparison with the control group. In addition, the pres-ence of LCS6 polymorphism was not determined to have an effect on EC histopathological characteristics.

IMPACT STATEMENT

What is already known on this subject? Endometrial cancer (EC) is a genital system cancer which is one of the most widespread gynecological cancers seen in the USA and other developed coun-tries, In EC, the most frequently seen gene mutations are PTEN tumour suppressor gene, KRAS,b1 catenin, BCL-2, CTNNB and P53 mutations. KRAS LCS6(let-7 miRNA binding region polymorphism) polymorphism has a worldwide incidence of 5.8% (Chin et al. 2008).There are studies shown that KRAS LCS6 polymorphism has an effect on developing EC (Lee et al. 2014), ovarian cancer(Ratner et al.2010)and endometriosis in women (Grechukhina et al.2012).

What do the results of this study add? In our study, LCS6 located on KRAS 3’-UTR was found at the rate of 16.2% in Type 1 EC patients. This increase is noticeable when it is considered that the incidence of this polymorphism is 5.8% in the general population. The results of the current study supports the preliminary findings of Lee et al.

What are the implications of these findings for clinical practice and/or further research? These new genetic markers could help to develop gene-targeted therapies, identify genetic basis of the disease and the factors that could affect the EC prognosis.

KEYWORDS

Genetic; mutation; KRAS LCS6; endometrium cancer

Introduction

Endometrial cancer (EC) is a genital tract cancer that is often seen in postmenopausal women. It is one of the most wide-spread gynaecological cancers seen in the USA and other developed countries, with >60,000 new cases per year and >10,000 EC-related deaths reported in the USA (Siegel et al. 2016). The annual mortality rate is 1.7–2.4% (Siegel et al. 2016). After breast, colon and lung cancers, it is the most commonly encountered cancer in females, and the 7th most common cause of cancer-related deaths (Mutter2000).

EC risk factors include advanced age, nulliparity, tamoxifen treatment, early menarche, late menopause, polycystic ovary syndrome, obesity, diabetes, oestrogen-secreting tumours,

Lynch syndrome, and familial female genital tract cancers, breast and colon cancers (Pellerin and Finan2005; Iqbal et al. 2012; Renaud et al. 2013). The role of oestrogen in the aeti-ology of EC has been definitively shown and all the factors that increase exposure to an imbalance of oestrogen and progesterone increase the risk.

Vaginal bleeding is an early symptom of premalignant lesions of EC, and with the widespread use of ultrasound and endometrial sampling, the diagnosis of EC can be easily made under polyclinic conditions.

Generally, there are two sub-types of EC; Type 1 is an oes-trogen -dependent adenocarcinoma which is seen more fre-quently, while Type 2 is known as non-endometrioid

CONTACTFeyza Nur _Incesu C¸intesun feyzanurincesu@gmail.com Department of Obstetrics and Gynecology, University of Health Sciences Konya Training and Research Hospital, Konya, Turkey

ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2020, VOL. 40, NO. 7, 988_–993

adenocarcinoma and shows oestrogen independent autono-mous development. It is seen in thin, elderly, postmeno-pausal women who do not have the capacity to produce oestrogen and develops on the basis of atrophic endomet-rium. Many genetic studies have been conducted in investi-gations about aetiological causes of both Type 1 and Type 2

endometrium cancers (Berchuck and Boyd 1995; Inoue 2001;

Tsugawa et al.2002). In Type 1 EC, the most frequently seen

gene mutations are PTEN tumour suppressor gene, KRAS, b1

catenin, BCL-2 and CTNNB, whereas in Type 2 EC, P53 muta-tion is the most commonly seen genetic abnormality (Berchuck and Boyd1995; Dobrzycka et al.2012).

KRAS is encoded in the short arm of proto-oncogene 12th chromosome, and is a protein which functions in normal cell proliferation signal transmission. KRAS which has undergone mutation becomes a potent oncogene. Single gene poly-morphism has been determined in the LCS6 section which controls the down-regulation ofKRAS gene (Chin et al. 2008). In a study which examined the relationship between

endo-metriosis and KRAS LCS6 polymorphism, there was

deter-mined to be increased KRAS mRNA and protein expression,

and increased proliferation and invasion of endometrial

stro-mal cells in women with KRAS LCS6 polymorphism positivity

(Grechukhina et al.2012).

The aim of the this study was to investigate the

relation-ship between LCS6 polymorphism in the KRAS gene in

patients with Type 1 EC and the additional histopathological characteristics of the effect increasing invasion.

Material and method Patient selection

This prospective study included a total of 204 patients, 99 Type 1 EC patients who underwent hysterectomy for EC after confirmed histopathological diagnosis and 105 patients who underwent hysterectomy for benign gynecological com-plaints and reported a benign diagnosis from endometrial sampling (proliferative endometrium, secretory endometrium, endometritis etc). The study was conducted at our clinic between January 2012 and January 2014. Approval for the study was granted by the Local Ethics Committee and informed consent was obtained from all the patients included in the study.

The age, parity, height and weight values of all the patients were recorded. For the EC patients, a record was also made of tumour stage, histological grade, lymphovascu-lar invasion (LVI), the number of positive pelvic and

para-aor-tic lymph nodes collected during surgery, myometrial

invasion (MMI) and cytology results. The EC patient group included those with endometrioid histological type (Type 1). The patients were classified as stage 1, 2, or 3 according to the 2009 FIGO surgical staging criteria.

Patients were excluded if they had endometrial hyperpla-sia, a diagnosis of any organ cancer other than EC, or if they did not wish to participate in the study. The genetic studies of the patients in this study were conducted in the Molecular

Genetic Laboratory of the Selcuk University Medical

Genetic Department.

DNA isolation

From the blood samples obtained, DNA isolation was made under sterile conditions using a DNA isolation kit (Vivantis, Malaysia) with the spin column method as follows. Lysis buf-fer (200mL) was placed in a 1.5 mL sterile, empty Eppendorf tube, then 200mL pellet was added. A further 20 mL protein-ase-K was added to the pellet and lysis buffer mixture and was mixed. This mixture was incubated on a hot plate at 56C for 10 mins, after which the mixture was vortexed. At

the end of the incubation, 200mL 100% ethyl alcohol was

added to the mixture, was pipetted and transferred to the spin column. By transferring to a new Eppendorf tube each

time and adding 500mL washing solution, the spin column

washing procedure was made at 500 g at 1-min intervals. 200mL distilled water previously heated to 56C in the incu-bator was added to the spin column and the mixture was centrifuged at 5000 g for 1 min and the DNA held in the spin column filter was extracted. The DNA obtained was stored at 20_{C until PCR analysis.}

Polymerase chain reaction (PCR)

The T> G base change is located in the 4th position of the 30UTR (untranslated region) of theKRAS gene. Analysis of this polymorphism was made by examining the change in length of the fragment products obtained using the PCR-restriction enzyme fragment method (PCR-RFLP, PCR-restriction frag-ment length polymorphism). While enzyme fragfrag-mentation occurs if thymine nucleotide is found in the gene region, fragmentation does not occur in the presence of guanine

nucleotide. Using Reverse: 50-AGTACCTAGGATTAT-30 and

Forward: 50-CGTGTGCACTCACTA -3’primers for this, the

region which could be polymorphic was proliferated. The pri-mers were designed by the Medical Genetic Department of our university. The method which was used for the reaction

was the same method as Grechukhina et al used

(Grechukhina et al.2012).

Agarose gel electrophoresis

Agarose of 2 g was weighed and placed in a 250 mL Erlenmayer flask and 100 mL 1X TAE (Tris, Acetic acid and EDTA) buffer was added. This was placed in a microwave oven for 2 min until the agar melted and boiled. The melted agar solution was kept at room temperature and cooled to

approximately 75C, then 100mL was added from 0.5 mg/mL

ethidium bromide. The prepared agarose gel was poured into the electrophoresis cup, which had been previously placed with an approximately 1 mm gap on the grooved

base, and was kept at room temperature for 20–30 min to

freeze. Holding the grooves from both ends, the gel cup was carefully removed and placed in the electrophoresis tank. Appropriate DNA markers and PCR products as a total of

12mL (10 lL PCR productþ 2 lL 6X loading dye buffer) were

transferred to the wells which emerged in the gel.

Electrophoresis was applied at 125 V (12.5 V/cm2) for

20–30 min. By comparing the resulting bands with markers in

the UV illuminator (image analysis system), the DNA walking distance was determined (Figure 1).

Restriction enzyme fragment studies

The Restriction Fragment Length Polymorphism (RFLP)

method is a method which allows the examination of DNA profiles formed by separation in the agarose gel electrophor-esis system of DNA segments and fragmentation with a restriction endonuclease enzyme specific to genomic DNA. By identifying specific sequences in the DNA of these enzymes, which are found in bacteria, the fragmentation process occurs in the identified region or in another specific sequence outside this region. Genotyping of single nucleo-tide polymorphisms occurs in two steps with this method. The first step is based on PCR product sequencing or enzyme fragmentation after gel electrophoresis for the genotyping of the polymorphic region by hundreds of thousands of prolifer-ations with PCR of approximately 200 base pairs around the single nucleotide polymorphism (SNP) in the DNA region (Linn and Arber1968).

In the RFLP method, after fragmentation of the sample DNA with one or more restriction endonuclease enzymes, the molecular weight of the DNA fragments obtained can be determined by molecular weight standard (marker) using gel electrophoresis. After treatment with ethidium bromide, the gel renders the fragments into a form which can be seen under UV light, and genotyping can be made by examining the number and size of bands in the gel profile, and the fre-quency of genotype/allele of the locus can be determined.

In the current study, genotype analysis was made by

Hinf1 enzyme in 1.5mL for 50 mL reaction with RFLP method

of the LCS6 (let-7 complementary sites) polymorphic regions in the 3’UTR of the KRAS gene.

The mixture was left in a water bath at 65C for 5–10 min. At the end of this period, after treatment of the obtained enzyme fragmentaation products in gel, evaluation was made using a UV illuminator and photographs were taken (Figure 2).

Statistical analysis of the data

All statistical analyses were performed using SPSS 20 statis-tical software package (SPSS Inc., Chicago, IL). The categorical data was compared using Chi-square analysis, the Mann

Whitney U-test was used for the comparisons of two groups

of numerical data and in the comparisons of three or more groups, the Kruskal WallisH-test was used. A value of p < .05 was accepted as statistically significant.

Results

The demographic data of the patients with EC subtypes and

the control group are shown inTable 1. The mean age was

56.9 ± 8.1 years in the Type 1 EC patients, 58.3 ± 7.7 years in the control group. No statistically significant difference was determined between the two groups in respect of mean age (p ¼ .156). No statistically significant difference was found between the groups in respect of height and parity. The weight and BMI values were determined to be statistically significantly higher in the Type 1 EC group than in the con-trol group (p < .005).

The correlations between KRAS LCS6 polymorphism and

type 1 EC patients are presented in Table 2. Comparisons

were made between 105 patients in the control group and 99 patients in the EC group. The polymorphism rate was found to be 16.2% in the Type 1 EC group and 12.4% in the control group, with no statistically significant difference determined (p > .05).

The pathological characteristics of the Type 1 EC patients are shown in Table 3. A total of 99 EC patients were

eval-uated in respect of KRAS LCS6 polymorphism. No difference

was found between KRAS LSC6 variant and heterozygote/

homozygote patients in respect of stage, grade, MMI, LVI and cytology (p > .05). Although there was no istatistically signifi-cant difference, the MMI rate was observed to be higher in the wild type patient group than in theKRAS variant patient group (86.7% vs. 68.8%). The presence of LVI was determined

to be lower in wild type patients than in the KRAS variant

patients (9.6% vs. 18.8%). The number of positive para-aortic

and pelvic lymph nodes were found to be similar in the wild type and variant patient groups.

Discussion

EC is the most common gynaecologic malignancy in devel-oped countries and the second most common in developing countries after cervical cancer (Siegel et al.,2016). All women with apparently resectable EC should undergo surgical ther-apy. In especially type 1 EC patients with less than 50%

myo-metrial invasion, histologic grade 1 or 2 and no

lymphovascular space invasion, a hysterectomy with bilateral adnexectomy, but without lymphadenectomy is sufficient (Burke et al. 2014, Mariani et al. 2000b). For patients with a biopsy-proven diagnosis of grade 1 endometrial cancer who wish to preserve future fertility may be candidates for conser-vative treatment using progestin therapy (Jozwik et al.2015). The recommended therapy of type 2 EC cancers or Type 1 EC patients with a myometrial invasion of more than 50% or grade 3 EC patients or tumour dimension is greater than 2 cm is a hysterectomy with bilateral adnexectomy with a pelvic and paraaortic lymphadenectomy (Mariani et al. 2008, 2000a). Radiotherapy is often recommended for patients greater than stage 1 (Nout et al.2010).

Let-7 miRNA is an RNA molecule which functions in the

down-regulation of KRAS gene expression. Normal LCS6 Let

-7 binding occurs together with an increase in loss of KRAS transcription and translation of the region. The regulating effects of RAS proteins are important in the tyrosine kinase mitogenic and oncogenic effect. RAS activation increases cell life and proliferation (Khosravi-Far and Der 1994; Schubbert et al.2007).

Figure 2. Image of the PCR gel electrophoresis for the LCS6 polymorphism T> G change in the 3’ UTR of the KRAS gene.

Table 1. The demographic data of the patients with Type 1 EC and the con-trol group.

Type 1 EC Group Control Group p Age (years) 57 (42–77) 59 (41–79) .156 Height (cm) 158 (141–169) 159 (144–170) .369 Weight (kg) 72 (50–127) 65 (45–81) <.001 Parity 2 (0–9) 2 (0–8) .263 BMI (kg/m2_{) 30 (20–50) 26.5 (18–32.4) <.001}

Values are stated as median (minimum–maximum). EC: Endometrial cancer; BMI: Body mass index. Obtained from Mann-Whitney U Test.

Table 2. The relationship of _{KRAS LCS6 polymorphism with EC} histo-logical type. Wildtype n (%) Homozygote þ Heterozygote mutationn1þn2 ¼ n (%) p Endometrioid Adenocarcinoma (Type 1) 83 (83.8) 5þ 11 ¼ 16 (16.2) .440 Control group 92 (87.6) 2þ 11 ¼ 13 (12.4)

Values are stated as number and percentage (%). Obtained from Pearson’s Chi-square analysis.

In a 2008 study by Chin et al, the frequency of LCS6 poly-morphism allele was investigated in 2433 healthy individuals in 46 different populations, and variant G allele was deter-mined in the LCS6 polymorphism region at the rate of 5.8%. This polymorphism in East Asian and Native American Indian populations is so low as to be negligible, it is extremely rare in African populations, and has been determined at the rate of 7% in European populations (Chin et al.2008).

In a study published by Grechukhina et al. in 2012, the relationship was investigated between endometriosis and the let-7 miRNA binding region (LCS6) polymorphism located on KRAS 3’-UTR, and the presence of this variant allele in cell cul-ture was shown to increase the proliferation and invasion rates in endometrial cells. This polymorphism was found at a higher rate (31%) in patients diagnosed with endometriosis compared to the control group. These findings were one of the sources of inspiration for the current study. In another

study that examined the relationship ofKRAS LCS6 SNP with

endometriosis, no significant difference was found between the endometriosis patient group and the control group (Farahani et al.2015).

The relationship between the presence of KRAS variant

and non-gynaecological cancers was investigated in an American population and the variant was determined at a higher rate in patients with non-small-cell lung cancer com-pared to the control group (18–20% vs. 12–14%). It has been accepted as a genetic marker of poor prognosis in head and neck cancers. (Christensen et al.2009). It is noticeable that in these studies, nearly all the KRAS variants were heterozygote in both patient and healthy control tissues (Chin et al.2008).

In the current study, 77% of the KRAS variants were

deter-mined to be heterozygote. Just as this may be related to the studies having been conducted on different cancer groups, it could also be attributed to the study data being formed from different ethnic groups.

In a 2010 study by Ratner et al, the frequency ofKRAS variant allele was determined as 25% in ovarian cancer cases, and it was concluded that theKRAS variant allele was a factor increasing the risk of the development of ovarian cancer (Ratner et al.2010).

In a study which investigated the frequency of LCS6 morphism and the clinical effect in EC patients, LCS6 poly-morphism was determined in 16.7% of Type 1 EC patients, in 24.3% of Type 2 EC patients and in 13.9% of the control cases (p ¼ .19) (Lee et al. 2014). According to these results, it was stated that LCS6 polymorphism did not generally increase the risk of EC, but could be a genetic marker for Type 2 EC risk. In this study, tumour specimen expression profiles were examined of 46 Type 1 EC patients and miRNA and DNA were isolated from genotypes, and the relationships were examined between the miRNA expression levels and

the patient characteristics of KRAS variant genotype, the

histopathological characteristics and survival rates. The rela-tionship between LCS6 polymorphism and age, disease grade, histology, MI depth and LVI was examined, and it was stated that these parameters did not change with the pres-ence of variant allele or non-variant allele. The 3-year survival rate was reported as 100% in patients with KRAS variant and 77% in patients with non-variant allele (HR0.3, p ¼ .24), but no statistically significant difference was determined. The authors attributed this to the low number of patients and the short follow-up period. As it was considered that just as KRAS variant could change gene expression, the miRNA expression might also be changed, the miRNA expression lev-els in tumour tissues were examined, and it was shown that miRNA expression changed with age, LVSI and the presence ofKRAS variant (Lee et al.2014).

In the current study, let-7 miRNA binding region

poly-morphism (LCS6) located on KRAS 3’-UTR was found at the

rate of 16.2% in Type 1 EC patients. Despite the frequency of the variant allele determined in the EC patients, that this was

Table 3. Pathological characteristics of the EC patients. Endometrioid adenocarcinoma Wild type (Normal) TT HomozygoteKRAS LCS6 PolymorphismGG and TG HeterozygoteKRAS LCS6 PolymorphismGG and TG _p1 p2 STAGE 1A,1B,2 74 (86.0%) 4 (4.7%) 8 (9.3%) .284 .216 3A,3B,3C 9 (69.2%) 1 (7.7%) 3 (23.1%) GRADE 1,2 76 (83.5%) 5 (5.5%) 10 (11.0%) .791 _>.99 3 7 (87.5%) 0 1 (12.5%) MMI 50% 72 (86.7%) 4 (4.8%) 7 (8.4%) .143 .129 50% 11 (68.8%) 1 (6.3%) 4 (25.0%) LVI Absent 75 (85.2%) 5 (5.7%) 8 (9.1%) .156 .378 Present 8 (72.7%) 0 3 (27.3%) Cytology Negative 81 (8.35%) 5 (5.2%) 11 (11.3%) .821 >.99 Positive 2 (100%) 0 0

Positive Pelvic Lymph Nodes 0 (0–11) 0 (0–3) 0 (0–3) .822 .903 Positive Para-Aortic Lymph Nodes 0 (0_–15) 0 0 .488 .231 Values are stated as number and percentage (%) and median (minimum–maximum).

MMI: myometrial invasion. LVI: lymphovascular invasion. p1_{: value between three groups.}

p2

: value between normal and homoþ hetero groups.

not statistically significant when compared to the rate of 13% determined in the control group can probably be attrib-uted to the small sample. When it is considered that the inci-dence of this polymorphism is 5.8% throughout the world in general, the elevation of the polymorphism rates determined in the EC patients is noticeable (Chin et al.2008). When the distribution of the histopathological variables is examined, the results of the current study are similar to the findings of Lee et al. with no statistically significant difference deter-mined between the patients carrying the variant and non-variant allele.

Limitations of the current study can be said to be the lim-ited number of patients, that no examination could be made of Type 2 EC patients as a separate group because of the low incidence and that miRNA expression could not be eval-uated because of technical insufficiencies.

In conclusion, although no significant difference was determined in the frequency of LCS6 polymorphism between the EC patients and the control group, it was noticeably that the result is high compared to previous studies with exten-sive case series. Moreover, the presence of LCS6

polymorph-ism was not determined to have an effect on the

histopathological characteristics of EC. Further studies exam-ining the effect of LCS6 polymorphism on EC with a greater number of patients will be of great importance in identifying the genetic basis of the disease, factors that could affect the EC prognosis, individuals at risk of the disease and in deter-mining personalised treatment targets.

Disclosure statement

The authors declare that they have no conflict of interest

Funding

This study was supported by Selcuk University Scientific Projects fund.

References

Berchuck A, Boyd J. 1995. Molecular basis of endometrial cancer. Cancer 76:2034–2040.

Burke WM, Orr J, Leitao M, Salom E, Gehrig P, Olawaiye AB, et al. 2014. Endometrial cancer: a review and current management strategies: part I. Gynecologic Oncology 134:385–392.

Chin LJ, Ratner E, Leng S, Zhai R, Nallur S, Babar I, et al. 2008. A SNP in a let-7 microRNA complementary site in the KRAS 3’ untranslated region increases non-small cell lung cancer risk. Cancer Research 68: 8535–8540.

Christensen BC, Moyer BJ, Avissar M, Ouellet LG, Plaza SL, Mcclean MD, et al. 2009. A let-7 microRNA-binding site polymorphism in the KRAS 3’ UTR is associated with reduced survival in oral cancers. Carcinogenesis 30:1003–1007.

Dobrzycka B, Terlikowski SJ, Garbowicz M, Niklinski J, Chyczewski L, Kulikowski M. 2012. The prognostic significance of the immunohisto-chemical expression of P53 and BCL-2 in endometrial cancer. Folia Histochemica et Cytobiologica 49:631–635.

Farahani MS, Shahbazi S, Moghaddam SA, Mahdian R. 2015. Evaluation of KRAS Gene Expression and LCS6 Variant in Genomic and Cell-Free DNA of Iranian Women With Endometriosis. Reproductive Sciences (Thousand Oaks, Calif.) 22:679_–684.

Grechukhina O, Petracco R, Popkhadze S, Massasa E, Paranjape T, Chan E, et al. 2012. A polymorphism in a let-7 microRNA binding site of KRAS in women with endometriosis. EMBO Molecular Medicine 4:206–217. Inoue M. 2001. Current molecular aspects of the carcinogenesis of the

uterine endometrium. International Journal of Gynecological Cancer 11:339_–348.

Iqbal J, Ginsburg OM, Wijeratne TD, Howell A, Evans G, Sestak I, Narod SA. 2012. Endometrial cancer and venous thromboembolism in women under age 50 who take tamoxifen for prevention of breast cancer: a systematic review. Cancer Treatment Reviews 38:318–328. Jozwik M, Jozwik M, Modzelewska B, Niewinska M, Jozwik M. 2015.

Levonorgestrel-releasing intrauterine system Mirena(R) (Bayer) for the prevention and treatment of endometrial adenocarcinoma and the incidence of other malignancies in women]. Polish Gynaecology 86: 305–310.

Khosravi-Far R, Der CJ. 1994. The Ras signal transduction pathway. Cancer and Metastasis Reviews 13:67_–89.

Lee LJ, Ratner E, Uduman M, Winter K, Boeke M, Greven KM, et al. 2014. The KRAS-variant and miRNA expression in RTOG endometrial cancer clinical trials 9708 and 9905. PLoS One 9:e94167.

Linn S, Arber W. 1968. Host specificity of DNA produced by Escherichia coli, X. In vitro restriction of phage fd replicative form. Proceedings of the National Academy of Sciences of the United States of America U S A 59:1300–1306.

Mariani A, Dowdy SC, Cliby WA, Gostout BS, Jones MB, Wilson TO, Podratz KC. 2008. Prospective assessment of lymphatic dissemination in endometrial cancer: a paradigm shift in surgical staging. Gynecologic Oncology 109:11_–18.

Mariani A, Webb MJ, Galli L, Podratz KC. 2000a. Potential therapeutic role of para-aortic lymphadenectomy in node-positive endometrial cancer. Gynecologic Oncology 76:348–356.

Mariani A, Webb MJ, Keeney GL, Haddock MG, Calori G, Podratz KC. 2000b. Low-risk corpus cancer: is lymphadenectomy or radiotherapy necessary? American Journal of Obstetrics and Gynecology 182: 1506–1519.

Mutter GL. 2000. Endometrial intraepithelial neoplasia (EIN): will it bring order to chaos? The Endometrial Collaborative Group. Gynecologic Oncology 76:287–290.

Nout RA, Smit VT, Putter H, Jurgenliemk-Schulz IM, Jobsen JJ, Lutgens LC, et al. 2010. Vaginal brachytherapy versus pelvic external beam radiotherapy for patients with endometrial cancer of high-intermediate risk (PORTEC-2): an open-label, non-inferiority, randomised trial. The Lancet 375:816–823.

Pellerin GP, Finan MA. 2005. Endometrial cancer in women 45 years of age or younger: a clinicopathological analysis. American Journal of Obstetrics and Gynecology 193:1640–1644.

Ratner E, Lu L, Boeke M, Barnett R, Nallur S, Chin LJ, et al. 2010. A KRAS-variant in ovarian cancer acts as a genetic marker of cancer risk. Cancer Research 70:6509–6515.

Renaud M-C, Le T, Le T, Bentley J, Farrell S, Fortier MP, et al. 2013. Epidemiology and investigations for suspected endometrial cancer. Journal of Obstetrics and Gynaecology Canada 35:380–381.

Schubbert S, Shannon K, Bollag G. 2007. Hyperactive Ras in developmen-tal disorders and cancer. Nature Reviews. Cancer 7:295–308.

Siegel RL, Miller KD, Jemal A. 2016. Cancer statistics, 2016. CA: A Cancer Journal for Clinicians 66:7–30.

Tsugawa K, Jones MK, Sugimachi K, Sarfeh IJ, Tarnawski AS. 2002. Biological role of phosphatase PTEN in cancer and tissue injury heal-ing. Frontiers in Bioscience: a Journal and Virtual Library 7:e245–51.