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Detection of microRNA in urine to identify patients with endometrial cancer: a feasibility study
  1. Hannah Donkers1,
  2. Marc Hirschfeld2,3,
  3. Daniela Weiß2,
  4. Thalia Erbes2,
  5. Markus Jäger2,
  6. Johanna Pijnenborg4,
  7. Ruud Bekkers5,6 and
  8. Khadra Galaal1
  1. 1 Royal Cornwall Hospital NHS Trust, Truro, UK
  2. 2 Department of Obstetrics and Gynecology, University of Freiburg, Freiburg im Breisgau, Germany
  3. 3 Institute of Veterinary Medicine, Georg-August-University Goettingen, Goettingen, Germany
  4. 4 Department of Obstetrics & Gynecology, Radboud Institute for Health Science, Radboud University Medical Centre, Nijmegen, The Netherlands
  5. 5 Department of Obstetrics and Gynaecology, Catharina Hospital, Eindhoven, The Netherlands
  6. 6 Department of Obstetrics and Gynaecology and GROW - School for Oncology and Developmental Biology, Maastricht University Medical Centre+, Maastricht, The Netherlands
  1. Correspondence to Dr Hannah Donkers, Royal Cornwall Hospital, Truro, TR1 3LJ, UK; hannahdonkerss{at}gmail.com

Abstract

Objective To find dysregulated urinary microRNAs associated with endometrial cancer as a first step in finding a non-invasive new diagnostic biomarker. The second objective is to determine the correlation of urinary microRNAs with clinicopathological characteristics.

Methods A prospective cohort study of patients presenting with abnormal bleeding between March and November 2019 was performed at the Royal Cornwall Hospital Trust Truro. Urine samples were obtained from women diagnosed with endometrial cancer and benign endometrial sampling. MicroRNA was isolated and quantitative real time PCR was used to detect expression levels of microRNAs.

Results A total of 61 women were included in this study: 24 endometrial cancer patients, and 37 controls. Median age was 64 years (range 45–94) and median body mass index was 29 kg/m2 (range 17–54). MiR-223 was significantly up-regulated in urine of endometrial cancers patients (p=0.003). Furthermore, let7-i, miR-34a, and miR-200c were significantly down-regulated and miR-424 was up-regulated in obese women. In addition, miR-148a and miR-222 were significantly down-regulated in elderly women, and miR-16, miR-26b, and miR-200c were significantly deregulated in women with multiple comorbidities.

Conclusion MicroRNA expression levels in urine can potentially be used as a non-invasive diagnostic test for endometrial cancer. Furthermore, aberrant microRNA expression in urine is associated with patient characteristics. Further research in larger trials is needed to validate the potential utility of urinary microRNAs.

  • uterine neoplasms
  • uterine cancer
  • endometrial neoplasms

Data availability statement

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

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HIGHLIGHTS

  • MicroRNA expression levels in urine can potentially be used as a non-invasive diagnostic test for endometrial cancer.

  • MicroRNAs are associated with body mass index, age, and comorbidities.

  • Further research is needed to validate the potential of urinary microRNA in diagnosing endometrial cancer

Introduction

Endometrial cancer is the sixth most common gynecologic cancer in developed countries, accounting for more than 2% of deaths due to cancer worldwide.1 The prevalence of endometrial cancer in developed countries has increased in recent years due to the rising prevalence of obesity.1 Obesity is associated with an increased risk of endometrial cancer by a variety of mechanisms including increased levels of estrogen and chronic inflammation leading to hyperinsulinemia and hyperglycemia, both stimulating endometrial proliferation.2

The primary symptoms of endometrial cancer are postmenopausal bleeding and abnormal premenopausal bleeding. However, the risk of endometrial cancer in premenopausal women with abnormal uterine bleeding is low and can be mainly attributed to hormonal imbalances, fibroids, and benign endometrial or cervical polyps.3 Postmenopausal bleeding occurs in approximately 90% of women with endometrial cancer; however, only 9% of women with postmenopausal bleeding are diagnosed with endometrial cancer, with the vast majority being diagnosed with benign conditions such as atrophy, endometrial polyps, or unscheduled bleeding in women using hormone replacement therapy.4 As patient age increases after menopause, the probability that postmenopausal bleeding is caused by endometrial cancer progressively rises.5–7

Currently, abnormal uterine bleeding is evaluated with transvaginal ultrasound scan and endometrial biopsy mainly performed by pipelle endometrial sampling. Pipelle endometrial biopsy is an invasive and uncomfortable procedure, with a Visual Analog Scale pain score of 6.5 in postmenopausal women.8 Furthermore, referring all women with abnormal uterine bleeding for transvaginal ultrasound scan and/or endometrial biopsy carries a considerable cost for healthcare systems. Dilation and curettage has been found to more accurately reflect final FIGO (International Federation of Gynecology and Obstetrics) grade than pipelle endometrial biopsy; however, even with dilation and curettage, a higher grade will be found in 8.7% of the cases at the time of hysterectomy.9 The low positive predictive value of postmenopausal bleeding and inaccuracy in grading of endometrial biopsy emphasizes the need for additional triage tests with high specificity to improve management of abnormal uterine bleeding and avoid unnecessary biopsies in low-risk women. Therefore, novel non-invasive diagnostic biomarkers are needed to improve management, patient care, and acceptability of women presenting with abnormal uterine bleeding.

In the past years, microRNAs have gained interest in cancer research. MicroRNAs are a class of small non-coding RNA molecules that regulate several key cellular processes including developmental timing, stem cell division, and apoptosis.10 They play an important role in regulating gene expression; it has been proposed that overexpressed microRNAs may function as oncogenes while those that are underexpressed behave as tumor suppressor genes.11 Abnormal expression of microRNA has been found in various disorders including numerous cancer sites.12 MicroRNA expression profiles are becoming promising and useful tools in cancer screening, diagnosis, and prognosis and may play a future role in cancer therapeutics.

A distinct microRNA panel has been shown to be promising in the detection of endometrial cancer in tissue, plasma, or serum samples.13 In addition, microRNAs have been shown to be stable in other body fluids such as Pap smear and urine.14

MicroRNAs have emerged as key regulators of lipid and glucose metabolism and play pivotal roles in the onset of obesity and obesity-related diseases.15 The role of microRNAs as a potential link between obesity and endometrial cancer, however, is unknown. Therefore, the aim of this study is to analyze urinary microRNA expression profiles in endometrial cancer to find dysregulated microRNAs associated with endometrial cancer as a first step in finding a non-invasive new diagnostic biomarker. In addition, we aim to determine the correlation of urinary microRNAs with clinicopathological characteristics.

Methods

Study Design and Participants

A prospective explorative cohort study was performed including women diagnosed with primary endometrial cancer between March 2019 and November 2019 at the Royal Cornwall Hospital Trust, Truro, UK. This study was approved by the Health Research Authority and Health and Care Research Wales with reference number 19/ES/0007. The study was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients.

Recruitment

Women attending their general practitioner with abnormal uterine bleeding were referred to postmenopausal bleeding clinics for suspected endometrial cancer. All consecutive women attending postmenopausal bleeding clinics were considered eligible and were approached to participate in the study. Detailed information was given and written informed consent was obtained. Those women attending postmenopausal bleeding clinics within the described timeframe with histological confirmed benign endometrial tissue served as controls. Urine samples were collected from participants after informed consent following standard protocols and stored at −80°C within 30 min after collection until further processing. All women had a transvaginal ultrasound scan followed by pipelle endometrial sampling.

Exclusion criteria were prior or coexisting other malignancies, presence of autoimmune disorders other than diabetes mellitus, inability to provide a urine sample, or no endometrial sampling performed. Standard treatment for women diagnosed with endometrial cancer was hysterectomy with bilateral salpingo-oophorectomy, and adjuvant therapy (radiotherapy and/or chemotherapy) when indicated. The histopathology results were confirmed by surgical resection of tumors in those patients treated with surgery and cancer stage was defined according to the FIGO staging criteria.16 For the five (20%) patients who did not undergo surgery, the FIGO stage was purely based on clinical examination and imaging results (computed tomography (CT) scan or magnetic resonance imaging scan and/or positron emission tomography-CT scan). The clinical and pathologic variables of the women were extracted from patient records and included age, parity, body mass index, comorbidities, FIGO stage, histologic subtype, and grade.

In accordance with the journal’s guidelines, we will provide our data for the reproducibility of this study in other centers if such is requested.

Sampling and Storage

Urine samples were collected in 100 mL sterile urine sampling cups and frozen in 10 mL aliquots at −80°C until further processing.

Sample Preparation, RNA Isolation, Reverse Transcription and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) of MicroRNAs

Urine microRNA preparation, RNA isolation, and reverse transcription method was described in detail by this group in a previous publication.17 A total of 41 microRNAs were investigated, based on previous literature.13 Supplementary Table 1 provides detailed information on the microRNA types studied, including their target sequences.

Statistical Analysis

For normal distributed data, continuous outcomes were presented as medians and IQR, and categorical data were presented as frequencies and proportions. For analysis purposes, body mass index was categorized into normal (<25 kg/m2), overweight (25–29.9 kg/m2), obese (≥30 kg/m2), and morbidly obese (≥40 kg/m2). For the data obtained by qRT-PCR, the non-parametric Mann-Whitney U test was used for the comparison between endometrial cancer and benign controls, and the Benjamini-Hochberg correction was applied to adjust for multiple comparisons with a critical value for false discovery rate of 0.25.18 P values <0.05 were considered significant for all tests. Data were analyzed with International Business Machines Corp (IBM) Statistical Package for the Social Sciences (SPSS) statistics version 26.0.

Results

Demographics

All consecutive women who consulted postmenopausal bleeding clinics for abnormal bleeding were approached, of whom 77 women met the inclusion criteria (Figure 1). Of these, 74 women consented to participate in the trial. Only three women declined participation due to the following reasons: not interested, no time, and fear of identity theft. Subsequently, 13 women were excluded due to histological diagnosis of simple or complex atypical hyperplasia (n=6), cervical cancer (n=2), or sarcoma (n=1), due to insufficient RNA extraction (n=2) or inconclusive histology (n=2), resulting in a final total of 61 patients suitable for analysis (Figure 1).

Figure 1

Flowchart of recruitment for study.

The median age of patients included was 64 years (Q1 55, Q3 74) and median body mass index was 29 kg/m2 (Q1 26, Q3 34) (Table 1). There were 24 women diagnosed with endometrial cancer and 37 women with confirmed benign histology. The majority of women with endometrial cancer had endometrioid endometrial carcinoma (n=19, 79%), and were diagnosed with FIGO stage I (n=16, 67%) and grade 1 (n=13, 54%). Most women diagnosed with endometrial cancer were treated surgically (n=20, 83%) and four women with advanced-stage disease received hormonal treatment (17%).

Table 1

Baseline characteristics

Histology of women in the control group included atrophic (n=22) and proliferative endometrium (n=15). Women in the control group were younger with a median age of 59 years versus 70 years in the endometrial cancer group (Table 2).

Table 2

Differences between cancer patients and benign control patients

MicroRNA Expression Profiles in Endometrial Cancer

To establish whether urinary microRNA can distinguish women with endometrial cancer from benign endometrium, the differences in expression profiles were assessed. MiR-10b, miR-15a, miR-135b, and miR-223 showed significantly altered expression profiles in endometrial cancer patients (Table 3); however, only miR-223 remained significantly up-regulated after correction for multiple testing.

Table 3

Up-regulated and down-regulated microRNAs

MicroRNA and Patient Characteristics

To explore the relationship between microRNA and patient characteristics, urinary microRNA expression levels were analyzed in relation to body mass index, age, and comorbidities in the complete study population. All 41 microRNAs were examined, of which eight showed significant different expression patterns (Table 4). Let7-i, miR-34a, and miR-200c were significantly down-regulated in obese women (body mass index ≥30 kg/m2), whereas miR-424 was up-regulated. In addition, only lower let7-i expression was associated with morbid obesity (body mass index ≥40 kg/m2).

Table 4

MicroRNA expression levels and patient characteristics

The impact of age on microRNA expression was analyzed; miR-148a and miR-222 were significantly down-regulated in elderly (age ≥70 years) women. Moreover, miR-16, miR-26b, and miR-200c were significantly deregulated in women with multiple (two or more) comorbidities.

The difference in microRNA expression levels between women with atrophic and proliferative endometrium was assessed, which showed a significant different expression level of let-7a (p=0.011), with a median expression level of 1.04 (range 1.50) in proliferative endometrium and a median of 1.50 (range 3.26) for atrophic endometrium (data not shown).

Discussion

Summary of Main Results

In this study, higher expression levels of miR-223 were observed in the urine of women with endometrial cancer. Furthermore, urinary microRNAs were significantly different in obese women (let7-i, miR-34a, miR-200c, miR-424), in elderly women (miR-148a, miR-222), and in women with multiple comorbidities (miR-16, miR-26b, miR-200c).

Results in the Context of Published Literature

MiR-223 has previously been found to be deregulated in endometrial cancer tissue, plasma, and serum samples13 and has a key role in the development and homeostasis of the immune system, and its involvement has been demonstrated for many types of cancers, inflammatory diseases, and autoimmune diseases.19 Furthermore, miR-223 is an inflammatory microRNA and is involved in the adipocyte inflammation associated with morbid obesity.20 In addition, plasma levels of miR-223 were found to be low in patients with type 2 diabetes.21 Interestingly, we did not observe an association of miR-223 with obesity in our study cohort. This enhances the potential of urinary miR-223 as a diagnostic biomarker for endometrial cancer, even in obese women. Further research should examine the pathophysiology of miR-223 in endometrial cancer and determine if increased miR-223 levels are directly related to endometrial tumors or rather reflect an inflammatory response against the tumor.

In this study, an association between let-7i, miR-34a, miR-200c, miR-424, and obesity was seen. Previous studies have shown that these microRNAs are associated with insulin production and resistance pathways, pancreatic development, and glucose metabolism.22 To our knowledge, the association between urinary microRNAs and obesity in endometrial cancer has not been investigated so far. Therefore, the results of our study may help in understanding the role of microRNAs in obesity, metabolism, inflammation, and insulin resistance pathways. Urinary microRNAs are promising biomarkers, not only in the early detection and screening of endometrial cancer, but are also potentially useful to identify women at risk of excess body fat accumulation and related metabolic abnormalities.

Currently, literature regarding urinary microRNA expression in endometrial cancer is scarce. A study by Ritter et al showed a tendency of miR-10b-5p up-regulation and miR-205-5 p down-regulation in urine of a small set of endometrial, ovarian, and breast cancer patients.23 They commented that in these limited number of endometrial cancer cases, microRNAs could not be confirmed as a diagnostic marker in urine samples. In addition, Záveský et al found urinary miR-106b to be down-regulated in 10 endometrial cancer patients and proposed more research should focus on confirming the diagnostic potential of urinary microRNAs.24

Lee et al examined tissue specimens of women with normal endometrial tissue, simple hyperplasia, complex atypical hyperplasia, and endometrial cancer; they suggested that miR-182, miR-183, miR-200a, miR-200c, and miR-205 are involved in progression from complex atypical hyperplasia to endometrial cancer, whereas PTEN has a role in the progression from simple hyperplasia to complex atypical hyperplasia.25 To our knowledge, our study is the first to explore the differences in urinary microRNA expression levels between women with atrophic and proliferative endometrium. In this study, we found a different expression pattern for let-7a between women with atrophic and proliferative endometrium. A further understanding is needed of the role of microRNAs in the development of endometrial hyperplasia and endometrial cancer carcinogenesis.

There is limited knowledge on the exact origin of urinary microRNAs. Due to different identification methods and normalization strategies, the microRNA expression patterns found in studies in other specimens are not easily comparable to urine.26 Furthermore, the presence of microRNAs in urine differ from those in other bodily fluids such as blood or serum, with lower numbers of detectable microRNA species in the urine.26 This suggests that the majority of circulating microRNAs are either ‘picked up’ by kidneys through an unknown mechanism or are destroyed in urine.27 Transrenal passage of the microRNAs from the blood could be suspected along with other sources involved in cell–cell communication or passive leakage from the injured or dead cells. Urinary microRNAs might originate from the tumor itself, or could derive from the cells of the immune system as a form of response to the presence of a malignancy.28 Further research is needed to understand this complex process and comprehend microRNA expression patterns in urine samples of endometrial cancer patients in more depth.

Strengths and Weaknesses

The strengths of this study include the prospective inclusion of postmenopausal bleeding patients suspected to have endometrial cancer and the standardized validation of the urinary microRNA expression patterns. However, the study is limited by the small sample size, the inclusion of different subtypes of endometrial cancer, and small groups regarding proliferative or atrophic endometrium. Furthermore, due to the small number of non-endometrioid tumors, we were unable to compare endometrioid versus non-endometrioid tumors. Of note, there were differences in baseline characteristics (age and comorbidities) between the cancer and control group. However, we showed that these characteristics were not associated with significant altered expression profiles of miR-223, and therefore we assume this has not affected our results. In addition, microRNA expression patterns can be correlated with stage at diagnosis; however, due to small numbers we were unable to account for stage.

Implications for Practice and Future Research

This study was performed to establish the feasibility of urinary microRNA testing in endometrial cancer as a first step in finding a non-invasive new diagnostic biomarker to improve the management of women presenting with abnormal uterine bleeding. Ideally, a urinary screening test for those women can easily be done by the general practitioner and only women with an abnormal screening test can then be sent for further investigations, thereby improving patient care and early diagnosis. Further research in larger prospective trials should focus on validating the potential of urinary microRNA as a diagnostic biomarker. In addition, expression values of miR-223 should be tested in a cohort of inflammatory conditions of the endometrium such as endometritis and endometriosis to assess the pathogenesis of this inflammatory microRNA in endometrial tissue.

Conclusion

In the current study, we demonstrated the feasibility of using microRNA expression levels in urine as a possible non-invasive diagnostic test for endometrial cancer. The microRNA levels can contribute to improved understanding of the carcinogenesis and its relation to body mass index and comorbidity. Further research in larger trials is needed to validate the potential of these microRNAs.

Supplemental material

Data availability statement

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics statements

Ethics approval

This study was approved by the Health Research Authority (HRA) and Health and Care Research Wales (HCRW) with REC reference number 19/ES/0007. The study was performed in accordance with the Declaration of Helsinki.

References

Footnotes

  • Contributors HD aided the design of the study and was responsible for data collection, analysis of the data and wrote the manuscript with input from all authors. MH and DW contributed to the design of the study, performed experiments and analyzed and interpreted the miRNA data. MJ performed the experiments. TE contributed to the design of the study, analysis and interpretation of the miRNA data. RB and JP revised and contributed to segments of the manuscript. KG was responsible for the conception and design of the study, data analysis, and contributed to the writing and revision of the manuscript.

  • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.