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Discovery of Non-Invasive Biomarkers for the Diagnosis of Endometriosis

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Discovery of Non-Invasive Biomarkers for the Diagnosis of Endometriosis Irungu et al. Clin Proteom (2019) 16:14 https://doi.org/10.1186/s12014-019-9235-3 Clinical Proteomics RESEARCH Open Access Discovery of non-invasive biomarkers for the diagnosis of endometriosis Stella Irungu1, Dimitrios Mavrelos2, Jenny Worthington1, Oleg Blyuss1, Ertan Saridogan2 and John F. Timms1* Abstract Background: Endometriosis is a common gynaecological disorder afecting 5–10% of women of reproductive age who often experience chronic pelvic pain and infertility. Defnitive diagnosis is through laparoscopy, exposing patients to potentially serious complications, and is often delayed. Non-invasive biomarkers are urgently required to accelerate diagnosis and for triaging potential patients for surgery. Methods: This retrospective case control biomarker discovery and validation study used quantitative 2D-diference gel electrophoresis and tandem mass tagging–liquid chromatography–tandem mass spectrometry for protein expression profling of eutopic and ectopic endometrial tissue samples collected from 28 cases of endometriosis and 18 control patients undergoing surgery for investigation of chronic pelvic pain without endometriosis or prophylactic surgery. Samples were further sub-grouped by menstrual cycle phase. Selected diferentially expressed candidate markers (LUM, CPM, TNC, TPM2 and PAEP) were verifed by ELISA in a set of 87 serum samples collected from the same and additional women. Previously reported biomarkers (CA125, sICAM1, FST, VEGF, MCP1, MIF and IL1R2) were also validated and diagnostic performance of markers and combinations established. Results: Cycle phase and endometriosis-associated proteomic changes were identifed in eutopic tissue from over 1400 identifed gene products, yielding potential biomarker candidates. Bioinformatics analysis revealed enrichment of adhesion/extracellular matrix proteins and progesterone signalling. The best single marker for discriminating endo- metriosis from controls remained CA125 (AUC 0.63), with the best cross-validated multimarker models improving the AUC to 0.71–0.81, depending upon menstrual= cycle phase and control group. Conclusions: We have identifed menstrual cycle- and endometriosis-associated protein changes linked to various cellular processes that are potential biomarkers and that provide insight into the biology of endometriosis. Our data indicate that the markers tested, whilst not useful alone, have improved diagnostic accuracy when used in combina- tion and demonstrate menstrual cycle specifcity. Tissue heterogeneity and blood contamination is likely to have hindered biomarker discovery, whilst a small sample size precludes accurate determination of performance by cycle phase. Independent validation of these biomarker panels in a larger cohort is however warranted, and if successful, they may have clinical utility in triaging patients for surgery. Keywords: Endometriosis, Biomarkers, Eutopic tissue, Ectopic tissue, Serum *Correspondence: [email protected] 1 Department of Women’s Cancer, Institute for Women’s Health, University College London, Cruciform Building 1.1, Gower Street, London WC1E 6BT, UK Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Irungu et al. Clin Proteom (2019) 16:14 Page 2 of 16 Introduction additionally validate biomarkers reported in the litera- Endometriosis is estimated to afect 5–10% of women ture [4]. of reproductive age. Although some women with endo- metriosis may be asymptomatic, it can cause signif- Materials and methods cant pain symptoms and infertility. Te diagnosis of Patient recruitment endometriosis can be difcult to achieve. Various stud- Patients and samples were sourced from the Univer- ies worldwide have documented a delay of 7–11 years sity College London Hospital Gynaecology Depart- from appearance of symptoms until diagnosis [1–3]. ment following ethical approval and informed consent. Tis delay is partly due to the fact that an invasive lap- Patients were those referred by their general practition- aroscopy is needed to establish the diagnosis in some ers or other clinicians for investigation of pelvic pain or women. Imaging techniques are sensitive for diagnosis for diagnosis and/or treatment of endometriosis. Tose of ovarian endometriomas and deep endometriosis in who agreed to undergo laparoscopic surgery for inves- experienced hands, but are less accurate for diagnosing tigation and treatment of endometriosis, pelvic pain or other forms of endometriosis [4]. bilateral salpingoophorectomy for strong family his- While eforts have been made to develop blood- tory of breast and ovarian cancer were approached and based diagnostic tests for endometriosis, few reported recruited. Inclusion criteria were as follows: ‘endome- markers have been independently validated and none triosis cases’ were defned as women diagnosed with are in clinical use. As a result, defnitive diagnosis of endometriosis at laparoscopy and confrmed histologi- endometriosis remains surgical, requiring laparoscopy cally; ‘controls with pain’ were defned as symptomatic under general anaesthetic, exposing patients to poten- women with pelvic pain of unknown cause or chronic tially serious complications. Difculty in establishing a pelvic infammatory disease without surgical evidence diagnosis leads to patients spending years on visits to of endometriosis; ‘controls without pain’ were regularly their doctors and unnecessary investigations, adding cycling women with no known disease undergoing bilat- to the signifcant economic burden of the disease [5]. eral tubal ligation and/or prophylactic bilateral salpin- While early stage endometriosis is not without conse- goophorectomy due to familial risk of breast and ovarian quence, delay in diagnosis may allow disease progres- cancer and with no visual evidence of endometriosis at sion with reduced treatment efciency and poorer laparoscopy. Te following women were excluded from outcomes. Tere is clearly an urgent and unmet need the study; post-menopausal women, women with a posi- for a minimally invasive diagnostic test for endometrio- tive pregnancy test or unknown pregnancy status on day sis, though clinically useful blood-borne markers have of surgery, those with other benign conditions or malig- yet to be found [6, 7]. Te aim of a non-invasive test nancies (particularly patients with fbroids and/or cancer of endometriosis should be to expedite clinical diagno- were excluded as these conditions may compromise the sis by achieving a high positive predictive value (> 90%) integrity of the endometrium), women on any hormonal or to triage patients for laparoscopic investigation for medication < 3 months prior to surgery and those whose conclusive pathological diagnosis. A lower sensitiv- surgical fndings and pathological reports were inconsist- ity may be tolerated for the latter if specifcity is high ent. Cycle phase was determined by a triple approach to (> 90%). Ideally, a test would also need to function ensure accuracy; chronologically, by histological dating independently of menstrual cycle phase. Early diagno- and by sex steroid hormone determination. Women with sis of endometriosis may allow preventative measures unconfrmed menstrual cycle stage were excluded. Addi- that delay progression of the disease such as use of the tional patient data was collected including age, fertility progesterone only pill or Mirena coil. Women hop- history, treatment history (oral contraceptive and GnRH ing to conceive or those who are refractory to medical analogue use), menstrual cycle phase, pain history, histo- management can be put forward for surgical treatment pathology fndings and anatomic characteristics of dis- earlier. ease lesions. All patient records were handled per NHS Te frst aim of our study was to identify potential confdentiality practices. Samples were anonymised and biomarkers through proteomic profling of eutopic sequentially numbered. A lab coding system and data- and ectopic endometrial tissue specimens taken from base were developed for recording anonymised patient women with a confrmed diagnosis of endometriosis and sample information. and relevant controls undergoing exploratory surgery for chronic pelvic pain without endometriosis or pro- Sample collection phylactic surgery due to strong familial risk of cancer. Endometrial tissue biopsies (eutopic endometrium) were Te second aim was to test these potential biomarkers obtained by Pipelle cannula or curettage from women in sera from a larger case control set of women, and undergoing laparoscopy. Ectopic endometrial tissue Irungu et al. Clin Proteom (2019) 16:14 Page 3 of 16 (endometriosis) samples were obtained from the same Ground tissue was lysed in 2D lysis bufer (8 M urea, 4% women at laparoscopy. Superfcial lesions and deep infl- w/v CHAPS and 10 mM Tris–HCl pH 8.3) using a ratio trating endometriosis samples only were used in this of 5 μL of lysis bufer per 1 mg of tissue at room tem- study, the rationale being
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