Serum HE4 predicts progestin treatment response in endometrial cancer and atypical hyperplasia: A prognostic study

To investigate serum human epididymis‐4 (HE4) as a predictive biomarker of intrauterine progestin response in endometrial cancer and atypical endometrial hyperplasia (AEH).


| I N TRODUC TION
Endometrial cancer is the commonest gynaecological malignancy in the UK, affecting approximately 9700 women annually. 1 Around 70% are endometrioid histology subtypes, 2 of which atypical endometrial hyperplasia (AEH) is a precursor lesion, progressing to endometrial cancer in around one-third. 3 Obesity is a major risk factor for both AEH and endometrioid endometrial cancer, with every 5 kg/m 2 increase in body mass index (BMI) increasing the risk of endometrial cancer by 60%. 4 Other risk factors include advancing age, polycystic ovary syndrome, nulliparity and family history, and there has been a rise in the incidence of premenopausal women with AEH and endometrial cancer. 5 The mainstay of treatment for AEH and endometrial cancer is surgical, including a total hysterectomy with or without bilateral salpingo-oophorectomy. 6 However, hysterectomy is not always appropriate for women with class III obesity and associated co-morbidities, because of increased surgical and anaesthetic risk. Furthermore, surgical management is unacceptable to young women wishing to retain their fertility. Conservative management of AEH and early stage (FIGO [the International Federation of Gynecology & Obstetrics] Stage IA), low grade (WHO [World Health Organization] Grade 1 + 2) endometrial cancer using intrauterine progestins is recommended for those unable to undergo hysterectomy, 6,7 but although response rates are good, there are still a significant number of women who do not respond. 8,9 Currently, there is no biomarker that predicts response to conservative therapy beyond standard surveillance imaging and endometrial biopsies. 10 The rising number of premenopausal and obesity-related endometrial cancer cases means that demand for conservative management is likely to increase. 11 However, around a third do not respond 12 and inevitably undergo hysterectomy following a period of repeated invasive and painful surveillance biopsies, with a risk of disease progression. A biomarker that can predict response to intrauterine progestins is needed to improve clinical care and patient experience.
Human epididymis-4 (HE4) has shown promise as a prognostic biomarker in endometrial cancer, correlating with histopathological markers of disease severity and survival. [13][14][15] We previously showed that serum HE4 may be a good predictive biomarker for the success of conservative management in a small population of women with AEH and early-stage endometrial cancer using a manual HE4 enzyme immunoassay (EIA). 16 In the present study, we tested serum HE4 using a fully automated chemiluminescent EIA (CLEIA) technique to enable straightforward clinical translation in women undergoing conservative management of AEH and endometrial cancer with intrauterine progestin. Our aim was to establish whether serum HE4 is an independent predictor of progestin treatment response in a larger cohort of women.

| Patient and public involvement
The research question was informed by collaborative work through the James Lind Alliance Womb Cancer Priority Setting Partnership. 17 'Can we predict, at the time of diagnosis, which endometrial cancers and precancerous lesions will respond to hormone treatments' was voted one of the top ten most important research questions in endometrial cancer by clinicians, patients and the public.

| Study population
Serum samples were obtained from women undergoing conservative management of AEH or endometrial cancer as part of the 'MIrena for the Reduction of Endometrial Neoplastic Abnormalities' (MIRENA) study from June 2014 to March 2021, at St Mary's Hospital, Manchester, UK. In this prospective observational study, women with histologically and radiologically confirmed AEH and endometrial cancer who were unsuitable for surgical management, either due to co-morbidities (including obesity) or for fertility-sparing reasons, were treated with a levonorgestrel-releasing intrauterine system (LNG-IUS). Women were eligible for inclusion in the study if they were aged 18 years or over with biopsyproven AEH or low-grade (WHO Grade 1 or 2) endometrioid endometrial cancer, with less than 50% myometrial invasion on magnetic resonance imaging (MRI) (FIGO 2009 Stage IA). Recruitment was temporal. Women were excluded if central pathology review concluded high-grade or nonendometrioid histology; central radiology review showed myometrial invasion of at least 50% or extrauterine disease; or women chose to undergo standard surgical management after discussion with specialists. All participants gave written informed consent for inclusion in the study. The study followed reporting recommendations for tumour marker prognostic studies (REMARK) guidance 18 (Table S1).

| Study procedures
Women were screened for eligibility at baseline. The index biopsy from the referring hospital was reviewed centrally by two specialist gynaecological pathologists, and the diagnosis was confirmed according the WHO classification system. 19 All participants underwent a baseline MRI and images were reviewed by specialist gynaecological radiologists. A computed tomography scan was performed to assess for extrauterine disease where an MRI was contraindicated.
At baseline review, clinical data were collected, including age, BMI, ethnicity, menopausal status, parity, smoking, comorbidities and medication. A baseline serum sample was obtained by routine venepuncture. All women underwent a pretreatment endometrial biopsy before having the LNG-IUS (52 mg intrauterine progestin, Mirena®) fitted in the outpatient department. Where the biopsy or LNG-IUS placement failed or was poorly tolerated, women underwent the procedure either in the outpatient hysteroscopy clinic or under general anaesthetic.
Where there were concerns about LNG-IUS displacement, women underwent a transvaginal ultrasound scan and review in the outpatient clinic for LNG-IUS replacement. If the LNG-IUS was displaced more than once, women were treated using oral medroxyprogesterone acetate 200 mg twice daily.
Women were seen in the outpatient clinic every 3 months for the first year. At follow up, symptoms were reviewed and an endometrial biopsy was taken. An MRI was performed at 3 months and/or 6 months to assess for interval change in women with endometrial cancer at baseline, and again at 12 months. At 12 months, the LNG-IUS was removed for a 6-week washout period. A final endometrial biopsy was obtained 6 weeks following removal, and the LNG-IUS was replaced. Women were withdrawn from conservative management if there was evidence of progressive disease on any surveillance endometrial biopsy or imaging, and treated either surgically or with radiotherapy depending on performance status and preference.

| Outcomes
Complete response to progestin therapy was defined as both a 12-month endometrial biopsy showing no hyperplasia or cancer, and a 12-month MRI that was normal. Partial response was defined as substantial improvement on the endometrial biopsy and a normal MRI. Stable disease was defined as similar appearance of the 12-month endometrial biopsy and MRI to baseline. Progressive disease was defined as worsening morphology on any endometrial biopsy or MRI taken during the surveillance period.
Those who had a complete or partial response to LNG-IUS on the end of treatment investigations were considered responders, whereas those who had stable or progressive disease were considered non-responders.

| Laboratory assay
Baseline serum samples were processed on the day of sampling at the Manchester University NHS Foundation Trust (MFT) Biobank. Samples were centrifuged at 1500 g for 15 minutes, and then stored in aliquots at −80°C until testing. Samples were thawed to room temperature before analysis. Serum HE4 analysis was carried out using the Fujirebio Lumipulse® G600II automated analyser (Fujirebio Europe N.V.), using a CLEIA technique. In brief, this is a two-step sandwich immunoassay, and the luminescence produced in the final enzyme reaction reflects the amount of analyte in the sample, which is calculated using the calibration curve. The Lumipulse® G HE4 immunoreaction cartridges (234068; Fujirebio Europe N.V.) has reported limits of detection of 20-1500 pmol/L and a total coefficient of variation between 3.4 and 5.5%. Quality controls were run before and after each batch of assays and the protocol for testing was in line with the manufacturer's instructions.

| Statistical analysis
Data analyses were performed using STATA (StataCorp, 2015, Stata Statistical Software: Release 14; StataCorp LLC, College Station, TX, USA). A prospective sample size calculation was not performed because the sample size was fixed by the MIRENA study. There were no missing data.
Numeric data are reported as medians and interquartile ranges (IQR) and categorical data as number and percentages. As the data were considered to be non-normal, comparison between groups was done using a Mann-Whitney U test. To compare categorical variables, a chi-square test was used. A p value less than 0.05 indicated significance. Univariable and multivariable logistic regression were used to evaluate the association between variables, including age, BMI, parity, menopausal status, type 2 diabetes mellitus, histological diagnosis, smoking, pretreatment serum HE4 and LNG-IUS treatment response. Logistic regression analyses are reported as odds ratios (OR) and 95% CI. A multivariable analysis adjusted for age and histology type, which are known predictors of progestin response, was performed to determine whether HE4 was an independent prognostic factor. All data were analysed in the form in which they were recorded (categorical or continuous). Nonlinear associations with the logit of the probability of progestin response were also explored using fractional polynomials of degree 1 for the univariable models. 20

| Study population
In total, 115 patients underwent baseline study investigations and 76 were included in the final analysis. Thirty-nine were excluded for the following reasons: no pretreatment serum available (n = 15), upstaged on baseline tests (n = 15), declined LNG-IUS (n = 3), death from unrelated causes (n = 5) and loss to follow up (n = 1; Figure S1A).

| Baseline serum HE4
The median pretreatment serum HE4 of the whole cohort was 79.  Table S2).

| Serum HE4 as a predictive biomarker of progestin treatment response
In the univariable logistic regression analysis, increasing pretreatment HE4 was associated with a lower likelihood of T A B L E 1 Baseline characteristics and human epididymis-4 (HE4) of study participants.

| Main findings
In this study, pretreatment serum HE4 was independently associated with progestin treatment failure in women with AEH and early-stage, low-grade endometrial cancer. We tested samples using a fully automated CLEIA to enable rapid clinical translation. If confirmed in large external cohorts, serum HE4 may offer a useful predictive biomarker of progestin treatment response, and could be considered for use in a predictive model that may facilitate individualised counselling and shared decision-making. Serum HE4 could identify women who are less likely to respond to conservative management, enabling early recourse to hysterectomy to ensure the best oncological outcomes.

| Strengths and limitations
Strengths of the study include its prospective design with recruitment of consecutive patients, minimising selection bias. We measured HE4 on all serum samples using an automated immunoassay for standardised results. There were 76 participants with evaluable clinical responses, comparing favourably with the three other prospective studies of intrauterine progestins for endometrial neoplasia, which reported outcomes on 46, 21 36 22 and 108 participants, 23 respectively. Our robust protocol with clearly defined clinical end points is another strength of our study. Our clinical outcomes (84% and 55% response rates in AEH and endometrial cancer, respectively) are equivalent to those in the literature. 24 Further, our cohort is representative of the diverse and complex population who undergo conservative management, including women with class III obesity, severe co-morbidities and poor performance status, as well as young women wishing to preserve their fertility. Regarding limitations, this was a single-centre study with few events (non-responders), emphasising the challenge of predictive biomarker studies of progestin response within a reasonable time frame without multicentre collaboration. Our results should be interpreted with caution given the wide confidence intervals surrounding our estimates of diagnostic accuracy, related to lack of statistical power. Our cohort lacked racial and ethnic diversity; therefore, we are unable to comment on the usefulness of serum HE4 as a predictive biomarker of progestin treatment response in non-White populations.

| Interpretation
There are only two studies evaluating the use of HE4 for the prediction of progestin treatment response in women undergoing conservative management of AEH and endometrial cancer. 16,25 Previous work by our group showed that pretreatment serum HE4 was significantly higher in nonresponders than responders to the LNG-IUS and was more predictive of treatment success than tissue HE4, but this was based on data from a small number of women and used a manual EIA-based assay that does not lend itself to routine clinical translation. 16 In the current study, we confirmed that pretreatment serum HE4 is an independent predictor of progestin treatment response in a larger cohort, with every 1 pmol/L increase in serum HE4 reducing the likelihood of response by 3%. Our findings suggest that baseline serum HE4 could offer clinical utility in a predictive model of progestin treatment response. However, the predictive power of single factors is limited, 26 and several factors in combination within a multivariable model may provide a more accurate and useful individualised risk estimate that could be used to aid clinical management decisions and facilitate informed shared decision-making. 26,27 Further work is required to develop and validate a multivariable model in a suitable sample size. This would require a collaborative multicentre effort, due to the small numbers undergoing conservative treatment at individual centres. The literature is conflicted regarding clinical predictors of response to progestin therapy; however, most studies suggest no association with age, menopausal status or BMI. 28 Our study also demonstrated no association between BMI and progestin therapy response, but conversely found that increasing age was associated with progestin treatment failure. This may be in part explained by the fact that women over the age of 50 years had a higher rate of endometrial cancer (25/41, 61%) compared with those younger than 50 years (19/35, 54%). Endometrial cancer is less likely to respond than AEH, 29 and histopathological features that indicate more aggressive disease are predictive of treatment failure. 28,30 Serum HE4 is associated with clinical markers of disease severity including myometrial invasion of 50% or more, FIGO stage, lymphovascular space invasion and tumour size, 13,15,[31][32][33][34][35][36] offering a potential explanation for its association with treatment failure and highlighting the importance of identifying non-responders before treatment. Several tissue biomarkers have shown promise as predictors of progestin treatment response, 37,38 but serum biomarkers like HE4 offer numerous advantages over these, including low-cost, high-throughput, fully automated testing with rapid turnaround times and the potential for straightforward incorporation into routine diagnostic workstreams.
There is a growing need for conservative management options for AEH and endometrial cancer due to the rising incidence of premenopausal women with the disease. 5 Primary surgical management is the recommended treatment for most women with AEH and early-stage endometrial cancer, and it is often curative. However, women with class III obesity and associated medical co-morbidities are challenging to operate on safely due to considerable surgical and anaesthetic risks. 39 Polycystic ovary syndrome, anovulatory cycles and subfertility are major risk factors for endometrial cancer, making hysterectomy unacceptable to a significant proportion of young women with the disease. Although progestin treatment is an option for those in whom surgical management is declined or contraindicated, there are no randomised controlled trials evaluating its effectiveness and safety compared with hysterectomy. It is essential that high-risk women are identified and counselled accordingly, because conservative management is likely to fail and delayed hysterectomy risks clinically significant progression and poorer clinical outcomes. Hence, women undergoing conservative management must be selected carefully and monitored according to standardised protocols by expert teams. A predictive biomarker that could accurately identify women at risk of treatment failure would help to tailor intrauterine progestin treatment appropriately and ensure that women are protected from undue risks of progression during conservative management.

| CONCLUSION
Serum HE4 shows promise as a potential predictive biomarker of progestin treatment response in women undergoing conservative management of AEH and low-grade, early-stage endometrial cancer. Pretreatment serum HE4 could aid informed decision-making, offer a more personalised approach to management and ensure that those women at greatest risk of treatment failure are counselled appropriately. Further large prospective studies are required to confirm our findings, and develop and validate a predictive model to establish the most appropriate serum HE4 thresholds for clinical use.

AU T HOR C ON T R I BU T ION S
EJC was principal investigator for the study and is its guarantor. EJC designed the study and supervised its execution. EJC, CEB and HJA recruited participants, collected clinical data and performed all study procedures. CEB performed the serum HE4 assays. RJMV and JB reviewed and reported the endometrial biopsies. CEB performed statistical analyses. JCS advised on the design, interpretation and reporting of statistical analyses. CEB and EJC wrote the manuscript.
All authors provided critical comment, edited the manuscript and approved its final version.

AC K NO W L E D GE M E N T S
We would like to thank the women who participated in this study and the clinical team who cared for them. We would like to thank Fujirebio for the loan of the Lumipulse® G600II and the gift of its reagents.

F U N DI NG I N FOR M AT ION
The funder had no role in the design and conduct of the study; collection, management, analysis and interpretation of the data; preparation, review or approval of the manuscript; and decision to submit the manuscript for publication. CEB and HJA were supported by Manchester University NHS Foundation Trust Clinical Research Fellowships. EJC was supported through a National Institute for Health and Care Research (NIHR) Clinician Scientist (NIHR-CS-012-009) and this article presents independent research funded by the NIHR, supported by the NIHR Manchester Biomedical Research Centre (IS-BRC-1215-20 007) and facilitated by the Greater Manchester Local Clinical Research Network. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.

C ON F L IC T OF I N T E R E S T S TAT E M E N T
None declared. Completed disclosure of interests form available to view online as supporting information. Fujirebio provided access to the Lumipulse® G600II and reagents for the CA125 and HE4 testing but had no role in study design, conduct, interpretation of results or the decision to publish them.

DATA AVA I L A BI L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

E T H IC A L A PPROVA L
The 'MIrena for the Reduction of Endometrial Neoplastic Abnormalities' (MIRENA) study was sponsored by MFT and approved by the North West Research Ethics Committee (14/NW/0056). The study was prospectively registered on the UK (ISRCTN 31662931) clinical trial database. All women gave written, informed consent to participate, and the study was conducted in accordance with Good Clinical Practice guidelines.