PROgesterone Therapy for Endometrial Cancer prevention in ......2020/12/11 · 1 PROgesterone Therapy for Endometrial Cancer prevention in obese women (PROTEC) trial: a feasibility

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PROgesterone Therapy for Endometrial Cancer prevention in obese women

(PROTEC) trial: a feasibility study

Abigail E. Derbyshire1, Jennifer L. Allen2, Matthew Gittins3, Bhavna Lakhiani2, James Bolton4, Joseph

Shaw4, Philip W. Pemberton5, Michelle Needham6, Michelle L. MacKintosh1, Richard J. Edmondson1,2,

Henry C. Kitchener2, Emma J. Crosbie1,2

1-Division of Gynaecology, Manchester University NHS Foundation Trust, Manchester Academic Health

Science Centre, Manchester, UK

2-Division of Cancer Sciences, Faculty of Biology, Medicine and Health, University of Manchester, St

Mary's Hospital, Manchester, M13 9WL, UK

3-Centre for Biostatistics, School of Health Sciences, University of Manchester, Manchester Academic

Health Science Centre, Manchester, UK

4-Department of Histopathology, Manchester University NHS Foundation Trust, Manchester Academic

Health Science Centre, Manchester, UK

5-Department of Clinical Biochemistry, Manchester University NHS Foundation Trust, Manchester

Academic Health Science Centre, Manchester, UK

6-Sleep Apnoea Service, Salford Royal Hospitals NHS Foundation Trust, Salford, UK

Running title: PROgesterone Therapy for Endometrial Cancer prevention

Keywords: Endometrial cancer; obesity; levonorgestrel intrauterine system (LNG-IUS);

chemoprevention; feasibility trial

Correspondence to: Professor Emma Crosbie, Division of Cancer Sciences, University of Manchester,

School of Medical Sciences, Faculty of Biology, Medicine and Health, 5th Floor Research, St Mary’s

Hospital, Oxford Road, Manchester M13 9PL. Email: [email protected]. Phone. +44

(161) 701 6942.

Conflicts of interest: The authors declare no potential conflicts of interest.

Cancer Research. on July 7, 2021. © 2020 American Association forcancerpreventionresearch.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on September 30, 2020; DOI: 10.1158/1940-6207.CAPR-20-0248

mailto:[email protected]://cancerpreventionresearch.aacrjournals.org/

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Abstract

Obesity is the major aetiological driver for endometrial cancer. The levonorgestrel intrauterine system

(LNG-IUS) reduces the risk of endometrial cancer and its precursor, atypical hyperplasia. We assessed

feasibility and uptake of the LNG-IUS for primary prevention of endometrial cancer in high-risk women

and its impact on endometrial tissue biomarkers. Women with class-III obesity (BMI>40kg/m2) and

histologically normal endometrium were invited to participate in a clinical trial of the LNG-IUS for

endometrial protection. Recruitment, successful LNG-IUS insertion and adherence to trial procedures

were recorded. We measured impact of the LNG-IUS on circulating biomarkers of endometrial cancer

risk, endometrial proliferation (Ki-67, pAKT, PTEN), endometrial hormone receptor status (ER, PR),

mental wellbeing and menstrual function. At six months, women chose to keep their LNG-IUS or have

it removed. In total, 103 women were approached, 54 were offered a participant information sheet, 35

agreed to participate and 25 received a LNG-IUS. Their median age and BMI were 54 years (IQR 52,57)

and 47kg/m2 (IQR 44,51) respectively. Three women (3/35, 9%) were ineligible due to atypical

hyperplasia/endometrial cancer on their baseline biopsy. The LNG-IUS was well tolerated and had a

positive overall effect on bleeding patterns and mental wellbeing. The LNG-IUS was associated with

endometrial morphological change, reduced Ki-67 and PR expression but circulating biomarkers of

endometrial cancer risk were unchanged. All but one woman (96%) kept her LNG-IUS. The LNG-IUS

appears to be acceptable to some women with class-III obesity for primary prevention of endometrial

cancer, which could provide a strategy for a prevention trial.



http://cancerpreventionresearch.aacrjournals.org/

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Introduction

Endometrial cancer is the sixth most common cancer in women, with more than 382,000 new

diagnoses and 89,900 deaths recorded globally in 2018 [1]. The incidence of endometrial cancer is

rising sharply in parallel with escalating obesity rates [2]. Obesity is the strongest risk factor for the

most common histological subtype, endometrioid (type I) endometrial cancer and its precursor lesion,

atypical hyperplasia [3]. Such is the strength of the association that approximately 40% of endometrial

cancers are thought to be directly attributable to obesity [4], and a marked dose-response relationship

bestows higher risk as body mass index (BMI) rises [5]. It has been estimated that women with obesity

class III (BMI>40kg/m2) have a seven-fold increased risk of endometrial cancer compared with normal

weight women (BMI 18.5-25kg/m2)[3]. The biological mechanism responsible for this association

relates to the endometrial stimulatory effect of adipose-derived estrogen, which is unopposed by

progesterone in anovulatory and postmenopausal women, and augmented by the negative

consequences of insulin resistance and chronic inflammation [6]. Weight loss achieved and sustained

through bariatric surgery reduces endometrial cancer risk [7,8] with measurable impact on circulating

biomarkers of adiposity, reproductive hormones and insulin status, accompanied by down-regulation

of pro-oncogenic signalling pathways in the endometrium [9]. Bariatric surgery is neither available,

appropriate nor acceptable to everyone with an elevated BMI however, and cannot be recommended

solely for the purpose of primary prevention of endometrial cancer [10]. Dietary caloric restriction can

facilitate weight loss, particularly if accompanied by increased levels of physical activity, but the

amount of weight lost and duration of benefit is considerably lower than following bariatric surgery

[11]. Alternative strategies are therefore urgently needed to provide protection to women at greatest

risk of endometrial cancer in order to thwart the explosion in incidence rates predicted by modelling

studies [12,13].

The levonorgestrel intrauterine system (LNG-IUS) delivers progestin directly to the endometrium,

counteracting the stimulatory effect of estrogen through stromal decidualisation, down-regulation of

proliferative signalling pathways and glandular atrophy [14]. Epidemiological studies have shown ever-

users of the LNG-IUS have a reduced risk of endometrial cancer [15,16] and several meta-analyses

have demonstrated its effectiveness as a therapeutic agent for women with atypical hyperplasia and

low grade cancers confined to the endometrium [17-19]. Despite strong evidence for its anti-cancer

activity, no previous studies have investigated use of the LNG-IUS as a chemopreventive agent for the

primary prevention of obesity-driven endometrial cancer. It is not known whether women with a

raised BMI are aware of their increased risk of endometrial cancer or whether they would be prepared

to engage in risk reduction with a LNG-IUS. In preparation for a clinical efficacy study, we measured

feasibility, participation rate and compliance with the LNG-IUS for endometrial protection in women




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with class III obesity. We studied its short-term effects on endometrial morphology, proliferation and

hormone receptor status and on circulating biomarkers of endometrial cancer risk. Further, we

explored the impact of the LNG-IUS on menstrual bleeding patterns, as well as mental wellbeing,

through validated questionnaires.

Materials and methods

Study governance

The study was sponsored by Manchester University NHS Foundation Trust (MFT) and approved by the

Cambridge East Research Ethics Committee – (15/EE/0063), Medicine and Healthcare Products

Regulatory Authority (MHRA, reference 21387/0234/001-0001) and local Research and Development

departments. The trial was prospectively registered on the European (EudraCT number 2014-005610-

37) and UK (ISRCTN40940943) clinical trial databases and conducted in accordance with Good Clinical

Practice guidelines and the Declaration of Helsinki.

Study design

This was a single arm feasibility study of the LNG-IUS for endometrial protection in women with class

III obesity. Women attended clinic at baseline (screening visit, T0), 2 ±1 months (LNG-IUS inserted in

clinic, T1) and 8 ±3 months (final assessment, T2)(Figure 1). Serial assessment of anthropometric

measures (weight, BMI, waist:hip ratio), serum biomarkers (hormone status, insulin resistance,

adiposity), endometrial biomarkers (endometrial morphology, hormone receptor status, Ki-67

proliferation index, pro-proliferation signalling molecules), menstrual bleeding patterns and mental

wellbeing was performed at all time points.

Feasibility, willingness to receive and compliance with the LNG-IUS

Willingness to receive the LNG-IUS for endometrial protection was determined as the proportion of

eligible women who agreed to its insertion. The feasibility of using the LNG-IUS in women with class III

obesity was calculated as the proportion of successful LNG-IUS insertions. Complications of insertion,

side effects and adverse events were recorded. At T2 final visit, women chose whether to keep their

LNG-IUS for ongoing endometrial protection or have it removed. Adherence with repeated

endometrial sampling and other trial procedures was recorded. Compliance with the LNG-IUS was

calculated as the proportion of women who chose to keep their LNG-IUS.

Participants




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Eligible women were ≥18 years of age with a BMI ≥40kg/m2 and histologically normal endometrium at

baseline. All participants gave written, informed consent. We advertised the study on the University of

Manchester and MFT websites, Cancer Research UK and UK ISRCTN clinical trials databases, on social

media platforms and by word of mouth. We recruited women who approached the research team for

participation directly and those attending gynaecology and sleep apnoea outpatient clinics at MFT and

Salford Royal Hospitals NHS Foundation Trust, respectively. Exclusion criteria included previous

hysterectomy; LNG-IUS or other intrauterine device within the past 6 months; planning pregnancy,

pregnant or breast feeding; previous endometrial ablation; congenital or acquired uterine anomaly;

history of pelvic inflammatory disease or genital actinomyces; breast cancer; overdue cervical

screening or last screen abnormal; immunodeficiency; actively trying to lose weight; contraindications

to LNG-IUS, including coagulopathy, liver disease, migraine, raised blood pressure, arterial disease,

postpartum endometritis, infected abortion during the past three months or recent trophoblastic

disease with persistently elevated hCG levels; and inability to tolerate endometrial sampling/ LNG-IUS

insertion as an outpatient.

Medical history and baseline safety check

At baseline, we recorded last menstrual period (LMP), menstrual bleeding pattern and contraceptive

use. Postmenopausal status was defined as LMP occurring >1 year before if FSH, LH and oestradiol

levels were confirmatory; the remaining participants were considered premenopausal. A urinary

pregnancy test was performed if indicated. Cervical screening was offered in accordance with the

National Health Service Cervical Screening programme. High vaginal and endocervical swabs were

taken to exclude active lower genital tract infection. Medical history was documented. Screening

bloods, including full blood count, urea and electrolytes and liver function tests were taken to confirm

medical fitness for participation in the trial.

LNG-IUS insertion

The Mirena® LNG-IUS (Bayer plc, Berkshire UK) was inserted in clinic at T1; women were advised to

take paracetamol and non-steroidal anti-inflammatory drugs one hour before insertion, if not

contraindicated. The procedure was carried out on a colposcopy couch using a Winterton speculum

under aseptic conditions, according to the manufacturer’s instructions. Safety monitoring was by

telephone call at 6, 12 and 18 weeks following LNG-IUS insertion. Side effects, adverse events and

complications were recorded. Participants were advised to attend their General Practitioner for a coil

thread check 4 weeks after LNG-IUS insertion.

Anthropometric measurements




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Height was measured using a stadiometer with shoes removed. Weight was measured using electronic

scales following removal of bulky clothing and BMI derived using the formula kg/m2. Waist to hip ratio

was calculated from waist (midpoint between lower margin of last palpable rib and top of the iliac

crest measured with a tape measure) and hip circumference (widest portion of the buttocks).

Blood biomarkers

Serum obtained by venepuncture following a 6 hour fast was used to measure a) reproductive function

(luteinizing hormone, LH; follicle stimulating hormone, FSH; sex hormone binding globulin, SHBG;

testosterone; free androgen index, FAI; oestradiol; progesterone), b) insulin resistance (glucose and

insulin to derive Homeostasis Model Assessment: Insulin Resistance, HOMA-IR [20]; glycosylated

haemoglobin A1c, HbA1c), c) adiposity (adiponectin, leptin) and d) inflammation (C-reactive protein,

CRP). With the exception of adiponectin and leptin, all analytes were measured using automated

routine clinical service protocols in the MFT Clinical Biochemistry Laboratory. Adiponectin and leptin

were measured with a DuoSet ELISA development kit (R&D Systems, Abingdon, UK).

Endometrial histopathology and tissue biomarkers

Endometrial sampling was performed using a Pipelle© (Carefusion, UK) or MedGyn Endosampler©

(MedGyn, IL, USA). Premenopausal participants were sampled on day 12 ±2 of the menstrual cycle,

where possible. Endometrial tissue was formalin-fixed, paraffin embedded, sectioned and stained with

haematoxylin and eosin. Endometrial morphology was assessed by a consultant gynaecological

pathologist. In premenopausal participants, endometrial morphology and reproductive hormone profile

was used alongside LMP to determine menstrual cycle phase. Abnormalities were confirmed by a

second consultant gynaecological pathologist and classified according to WHO guidelines [21,22].

Tissue sections (4 µm) were baked for 30 minutes at 70°C. The automated Ventana BenchMark Ultra

IHC Staining Module (Ventana Co., Tucson, AZ, USA) was used with the Ultraview 3, 3’

diaminobenzidine (DAB) v3 detection system (Ventana Co.). Tissue sections were deparaffinised and

incubated in EZPrep Volume Adjust (Ventana Co.). A heat-induced antigen retrieval protocol was

carried out using a TRIS–ethylenediamine tetracetic acid (EDTA)–boric acid pH 8 buffer, Cell Conditioner

1(CC1). The sections were incubated with ultraviolet inhibitor blocking solution for 4 min, followed by

an optimized concentration of antibody (Table S1). Sections were then incubated with horseradish

peroxidase-linked secondary antibody, DAB chromogen and copper. Counterstain (Haematoxylin II) was

applied for 12 minutes before a 4-minute incubation with bluing reagent. Slides were dehydrated

through three steps of 99% IMS and two changes of Xylene. Sections were coverslipped using ClearVue




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Mount XYL (Thermo Scientific). Negative (isotype control) and positive tissue controls were used for

quality assurance.

The Ki-67 score was the proportion of glandular cells with positive nuclear staining. The Ki-67 score was

determined from >1000 nuclei scored in 3 representative high-powered fields (x20), chosen by the

study pathologists; scanty samples were scored in their entirety [23]. Estrogen (ER) and progesterone

receptor (PR) staining was assessed by modified H-score (0-18), the product of area score (proportion

of positively stained tissue, scored 0-6) and intensity of staining score (0=none, 1=mild, 2=moderate,

3=strong). Phosphorylated (p)AKT staining was scored using the percentage of positively stained tissue

[H = (3 x % strong staining) + (2 x % moderate staining) + (% weak staining)] to account for within tissue

heterogeneity (0-300). PTEN status was scored ‘PTEN null’ if there were endometrial glands negative

for PTEN adjacent to positive stroma. Slides were scored as ‘PTEN positive’ if all endometrial glands

expressed PTEN [24]. Scoring was performed manually by two independent scorers who were blinded

to time point. Discrepant scores (>10% or disagreement as to PTEN status) were reviewed and resolved

by consensus agreement.

Menstrual blood loss and mental wellbeing assessment

Two validated questionnaires, the Hospital Anxiety and Depression Scale (HADS) [25, 26] and Warwick-

Edinburgh Mental Wellbeing Scale (WEMWBS) [27,28], were completed at baseline and follow up to

determine whether the LNG-IUS had an impact on mental wellbeing. For the HADS, different cut-offs

are indicative of a mental health disorder, depending on clinical context [26], but lower scores indicate

absent or lower severity of symptoms. For the WEMWBS, the mean score in the general population is

51, with higher scores reflecting improved mental wellbeing [27]. Premenopausal participants

completed the Menstrual Bleeding Questionnaire [29] at baseline and follow up.

Sample size considerations

This was a preliminary study designed to inform recruitment rates, feasibility of and likely adherence to

a clinical efficacy trial of the LNG-IUS for endometrial protection in women with class III obesity. We

considered that a clinical efficacy trial could be successfully conducted if >50% of eligible women

agreed to participation, >50% of those eligible had a LNG-IUS successfully fitted, and >75% of women

kept their LNG-IUS for >6 months. We also measured LNG-IUS-induced change in circulating and tissue

biomarkers to inform intermediary biomarker endpoints for our definitive study. We did not perform a

formal sample size calculation and planned the pragmatic recruitment of 30-40 women over a six to

twelve month recruitment period.

Statistical analysis




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Statistical analyses were performed using Graphpad Prism 5.0b for Mac (GraphPad Software, San

Diego, USA) and SPSS 23.0 for Mac (IBM Corp, Armonk, NY, USA). Descriptive statistics included mean

and standard deviation (SD) for normally distributed, and median and interquartile range (IQR), for

non-normally distributed data. Within-individual changes over time were compared using paired t-test

and Wilcoxon signed-rank test for normally distributed and non-normally distributed data, respectively.

To assess the short-term impact of the LNG-IUS on endometrial proliferation, a mixed effects

regression model was fitted, with Ki-67 score set as the dependent variable, time point (baseline set as

reference category) as the predictor of interest and the covariates baseline Ki-67 score, age,

menopausal status (pre/post), smoking (never, ever, current), type II diabetes mellitus (yes/no)

baseline BMI, and baseline waist:hip ratio. A further analysis was performed that included weight at

follow up, to determine if change in weight was responsible for change in Ki-67 at outcome. To account

for repeated measures within participants, a random effect intercept was included to account for the

within subject vs between subject variation. To account for possible departures in normality, a cluster

bootstrapping procedure was employed with 1000 replications. In an effort to emphasise clinically over

statistically important effects, data are reported in terms of mean difference effect estimates and 95%

confidence intervals.

Results

Study population

Between October 2015 and September 2016, 103 women were approached, 54 were offered a

participant information sheet, 35 agreed to participate and 25 received a LNG-IUS (Figure 1). Forty nine

women (48%) were ineligible to receive the participant information sheet for the following reasons:

LNG-IUS in situ (n=13); previous hysterectomy (n=12); pending bariatric surgery (n=10); social/ capacity

reasons (n=7); LNG-IUS contraindicated (n=4) or BMI

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46kg/m2) or ethnicity/ race (all except three were White British). Twelve (48%) were premenopausal

but just 4 had regular menstrual cycles; most were either amenorrhoeic (5/12) or experienced irregular

menstrual bleeding (3/12). Four (16%) were using hormone replacement therapy (3/25, 12%) or oral

contraceptives (1/25, 4%) at baseline, which they continued throughout the trial. All had at least one

comorbidity, most commonly type II diabetes (10/25, 40%), hypertension (15/25, 60%) or asthma

(8/25, 32%) and 48% had more than three comorbidities.

Compliance with intervention and study procedures

All 25 women received the LNG-IUS in clinic without complication. There were no insertion failures,

expulsions, uterine perforations or lost devices. One woman (4%) developed mild symptoms of

endometritis following LNG-IUS insertion, which was treated with oral antibiotics. One patient

complained of pelvic discomfort/ mild pain following LNG-IUS insertion that settled with oral analgesia.

Other adverse and serious adverse events, specifically urinary tract infection (1/25, 4%), vasculitis

(1/25, 4%), sciatica (1/25, 4%) and attempted suicide (1/25, 4%) were not thought to be related to the

LNG-IUS. All women kept their LNG-IUS until their final assessment when one woman (4%) chose to

have it removed (“easier now than later”); the remaining 24 women (96%) kept their LNG-IUS for

ongoing endometrial protection. All 25 women were compliant with study procedures, including

sequential endometrial biopsies (all 3 biopsies taken, 25/25).

Endometrial morphology and biomarkers

Three of 35 participants (9%) had an incidental finding of atypical hyperplasia or endometrial cancer on

a pre-LNG-IUS biopsy and were excluded from the study. All other women had histologically normal

endometrium at T0 (baseline) and T1 (time of LNG-IUS insertion). Many of the samples were scanty.

Morphology was consistent with menopausal status and/or reported phase of menstrual cycle, as

appropriate. At follow up (T2), all endometrial biopsies showed stromal decidualisation and glandular

atrophy, consistent with the progesterone effect associated with LNG-IUS treatment.

The LNG-IUS was associated with a significant decrease in endometrial proliferation as assessed by Ki-

67 score. The mean Ki-67 score was 27.1% (SD 23.4) at baseline, 21.8% (SD 14.8) at the time of LNG-IUS

insertion and 12.7% (SD 10.9) at follow up. A mixed effects regression model adjusting for within

participant clustering, potential confounders, and weight change between time points is shown in

Table 2. Between baseline (T0) and time of LNG-IUS insertion (T1) and between baseline (T0) and

follow up (T2) the change in Ki-67 score was -5.4% (95% CI -17.1%, 6.3%) and -14.6% (-25.3%, -3.9%),

respectively. These results were consistent across all three models, indicating that potential sources of

confounding, including change in weight during follow up, had little effect on Ki-67 score. Progesterone




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receptor expression decreased with LNG-IUS treatment (Table 3). There was no significant change in

expression of the other endometrial biomarkers, estrogen receptor, PTEN or pAKT. Interestingly, all

three women excluded because of occult endometrial abnormalities had PTEN null glands and a further

2/25 participants in the study had PTEN null glands before but not after LNG-IUS insertion.

Anthropometric and circulating biomarkers of endometrial cancer risk

Overall, women lost weight during the trial, although this was not clinically significant (median weight

124.4kg (IQR 111, 143), 123.9kg (IQR 111, 142) and 123kg (IQR 111, 144) at T0, T1 and T2, respectively).

There were no clinically significant changes in circulating biomarkers of reproductive function, insulin

resistance, adiposity or inflammation across the three time points (Table 3), with the notable exception

of altered serum FSH, LH and progesterone levels over time, which likely reflect natural reproductive

ageing in our peri-menopausal cohort.

Menstrual bleeding and mental wellbeing

Of the 12 premenopausal participants, 5 (42%) were amenorrhoeic, 4 (33%) had regular and 3 (25%)

irregular menstrual bleeding at baseline. As expected, the 7 women who experienced menstrual

bleeding reported a significant reduction in blood loss with the LNG-IUS; all but two became

amenorrhoeic according to the Menstrual Bleeding Questionnaire. Mental wellbeing improved with

the LNG-IUS according to both the WEMWBS and HADS scales (Table 4). A change in score of 2-3

points is clinically significant, but did not reach statistical significance, most likely because of small

numbers.

Discussion

The PROTEC trial was undertaken to assess the feasibility of a future clinical efficacy trial of the LNG-

IUS for endometrial protection in women with class III obesity. In a twelve-month recruitment period,

we approached 103 women, 54 (52%) of whom met the inclusion criteria, 35 (65%) agreed to

participate and 25 (71%) proceeded to LNG-IUS insertion. There were no insertion failures and all

women were fully compliant with all study procedures, including an endometrial biopsy at the final

visit. There were no related serious adverse events but one case each of endometritis and post-

insertion pain, both recognised complications of LNG-IUS treatment. We observed no detrimental

impact of the LNG-IUS on mental wellbeing and self-reported menstrual bleeding profiles improved for

our premenopausal participants. These data suggest that women at greatest risk of obesity-driven

endometrial cancer are willing to engage in risk reduction with a LNG-IUS and that a clinical efficacy

trial could be feasible. Roughly a quarter of women approached for the trial had a LNG-IUS inserted,




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indicating a relatively high proportion of screen failures and non-continuation rate. These findings

must be factored in to the design of a clinical efficacy trial.

To determine the short-term impact of the LNG-IUS on biomarkers of endometrial cancer risk, we

measured change in anthropometric variables, reproductive hormones, insulin resistance, endometrial

morphology and glandular proliferation status between baseline, two months and eight months. As

predicted, we observed stability in these biomarkers prior to LNG-IUS insertion. Short-term treatment

with the LNG-IUS was associated with changes in endometrial morphology, reduced proliferation and

progesterone receptor expression; there were no associated changes to circulating hormone levels,

measures of insulin resistance or adiposity. An unexpected finding was that women lost an average

2.5kg in weight during the study, however, this was neither clinically nor statistically significant. Given

our conviction that women with class III obesity are at sufficiently high risk of endometrial cancer that

they would benefit from risk reducing measures, it is striking that 3/35 (9%) of our participants had an

incidental finding of atypical endometrial hyperplasia or endometrioid endometrial cancer at baseline,

requiring hysterectomy. This is consistent with our previous study that found 10/72 (14%) women with

class III obesity referred for weight loss management had occult underlying endometrial neoplasia [9].

Endometrial glands are clonal cell populations that frequently harbour driver mutations in cancer

genes [30]. PTEN null glands confer a proliferative advantage, predisposing to endometrial

carcinogenesis and have been shown to persist between menstrual cycles but only a small proportion

progress to endometrial cancer [31]. We found PTEN null glands in all three women with occult

endometrial abnormalities and in two participants with histologically normal endometrium before, but

not after, LNG-IUS insertion. This is consistent with the hypothesis that PTEN null glands in

morphologically normal endometrium represent latent endometrial cancer precursors that regress

with LNG-IUS treatment [24].

Whilst the endometrial impact of the LNG-IUS is well studied [32, 33], this is the first trial to offer the

LNG-IUS to women with class III obesity for the primary prevention of endometrial cancer.

Confirmation that the expected endometrial effects of the LNG-IUS are observed in this population is

important given their defining characteristics, specifically their class III obesity and amenorrhoea or

irregular menstrual bleeding, which distinguish them from regular users of the device. It is known that

Ki-67 score is higher in endometrium harvested from women with obesity compared to that collected

from normal weight women [34]. We considered that the expected change in endometrial

morphology, a reduction in glandular proliferation as assessed by Ki-67 score, and down-regulation of

endometrial progesterone receptors would confirm the utility of the LNG-IUS at standard doses in this

population. We also quantified the Ki-67 drop at six months post-LNG-IUS insertion for the purposes of




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developing an intermediary molecular endpoint for a definitive trial of the LNG-IUS for endometrial

protection upon which a sample size calculation could be based. It is interesting that the 15% Ki-67

drop observed after six months treatment with the LNG-IUS was similar to that observed after an

average bariatric surgery-induced weight loss of 22kg at two months in women with class III obesity

[9]. Bariatric surgery is known to reduce endometrial cancer risk [7, 8, 35], and although the

mechanisms underlying risk reduction are not fully understood, it is thought that down-regulation of

endometrial pro-proliferative signalling pathways could be important [9, 36]. Ki-67 is only expressed by

proliferating cells, a hallmark of cancer; indeed, Ki-67 is known to differentiate benign from malignant

endometrium, with higher Ki-67 scores observed in high grade, advanced stage cancer and correlating

with poor survival outcomes in this group [37]. We considered that a reduction in glandular

proliferation in benign peri- and postmenopausal endometrium could reduce the risk of mutational

events that trigger malignant transformation [10].

We have demonstrated proof of principle that some women at high risk of obesity-driven endometrial

cancer are prepared to engage in risk reduction with a LNG-IUS, paving the way for a clinical efficacy

trial in this population. Despite concerns that LNG-IUS insertion would be challenging in the outpatient

setting in postmenopausal women with class III obesity, we had no insertion failures, consistent with

previous studies [38]. Concerns that uterine instrumentation would be unacceptable to women who

did not have a gynaecological complaint were also unsubstantiated, with 32/35 (91%) of participants

consenting to and undergoing three sequential biopsies, without complication. Further, we found the

LNG-IUS was not associated with a detrimental impact on mental wellbeing using two validated

questionnaires, with even some suggestion that mental wellbeing improved during the trial, possibly

due to improved menstrual bleeding profiles and peace of mind regarding endometrial health; indeed

24/25 (96%) of participants chose to keep their LNG-IUS at the end of the trial for ongoing endometrial

protection. A short-term study of this kind cannot confirm that women will be compliant with the LNG-

IUS in the medium to long-term, however. Nor can it help define the optimal duration of a clinical

efficacy trial. We did not deliberately target peri-menopausal women for trial participation, although

long-term use of the LNG-IUS in a clinical efficacy trial would ideally avoid women whose compliance

could be compromised by future pregnancy plans. The single centre nature of this research is a

limitation of the study, since we cannot necessarily extrapolate feasibility of our approach to other

centres, countries or healthcare settings. The lack of racial and ethnic diversity in our study population

precludes any insight into the acceptability and uptake of the LNG-IUS for uterine protection in non-

White British women. We do not know whether women would consent to randomisation to a no

intervention arm, which would be the ideal clinical efficacy trial design, and would certainly impact

feasibility of the definitive study. Furthermore, our biomarker findings should be interpreted with




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caution given the small sample size and marked heterogeneity of participating women with respect to

age, menopausal status and use of exogenous hormones at baseline.

Whilst invasive, the advantage of the LNG-IUS is that it releases a continuous supply of levonorgestrel

directly to the endometrium, avoiding the peaks and troughs observed with oral administration and

eliminating compliance issues [39]. Apart from insertion problems, there are few contraindications to

its use, at least partly because systemic concentrations of the drug are much lower than those

achieved with oral administration [40]. Serum levonorgestrel levels are 20-fold lower in LNG-IUS users

than levonorgestrel-containing combined oral contraceptive pill users, for example [41]. There is an

inverse correlation between serum levonorgestrel concentrations and body mass index [42],

suggesting even lower systemic levels in our population. A further advantage of the LNG-IUS for this

indication is that it would be expected to eradicate or treat latent endometrial cancer precursors,

atypical hyperplasia and occult obesity-driven endometrial cancer, as previously demonstrated [24, 17-

19]. Regression of established endometrial abnormalities takes 6-12 months or longer and is more

likely in the case of atypical hyperplasia (approx. 90% complete response rate) than early stage

endometrial cancer (67% complete response rate)[43]. There are currently no validated biomarkers

that predict LNG-IUS response to established disease [44], although some show promise [45, 46],

mandating careful assessment of any new bleeding that develops following device-induced

amenorrhoea [47] in an endometrial cancer prevention trial.

Overall, we found the LNG-IUS to be safe and well-tolerated, with no unacceptable side effects in our

study population. This is particularly important if the LNG-IUS is being used for endometrial protection

rather than an established clinical indication, and should be a focus of future work. There is no

evidence that the LNG-IUS increases the risk of cardiometabolic disorders in obese women [48], but a

recent systematic review found LNG-IUS users have a modestly increased breast cancer risk (odds ratio

=1.16 (95% CI 1.06-1.28, I2 =78%, p

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[51]. Here, we demonstrate that a LNG-IUS is acceptable to some women with class III obesity and

that a clinical efficacy trial would be feasible. The specifics of trial design require careful consideration

because a large cohort with sufficient follow up will be challenging and expensive to achieve.

Minimising trial size, duration of follow up and cost is an important goal for women, researchers and

funders of such a trial. Whilst endometrial cancer risk is high in women with class III obesity, absolute

risk is modulated by reproductive, metabolic and genetic factors [13, 52], as well as competing risks for

death. More sophisticated risk prediction models, calibrated for clinical use, must now be developed

to establish the optimal prevention trial target population, to maximise the benefits of participation

and reduce unnecessary harms [53].




15

Acknowledgements

We would like to thank the women who participated in this study. We are grateful to all the clinical

staff involved in their care who helped facilitate recruitment, especially Samantha Johnson and Bryan

Wilson. We would particularly like to thank Linsey Nelson, who contributed to study set up, and Tina

Pritchard, who supported patient recruitment, provided nursing care and helped with administrative

tasks. We are grateful to the independent members of the Trial Steering Committee, Professor Sudha

Sundar, Professor Martin Rutter, Professor Steve Roberts and Mrs Anne Lowry for providing study

oversight.

AED was a Manchester University NHS Foundation Trust Clinical Research Fellow and EJC an NIHR

Clinician Scientist (NIHR-CS-012-009), and their work was supported through the NIHR Manchester

Biomedical Research Centre (IS-BRC-1215-20007) and the William Walter Will Trust. This article

presents independent research funded by the NIHR. The views expressed are those of the authors and

not necessarily those of the NHS, NIHR, or the Department of Health.

Author contributions

EJC was Principal Investigator for the study and is its guarantor. EJC and HCK obtained funding and

designed the study. EJC, MLM and RJE supervised study execution. MN supported study recruitment.

AED recruited women to the study and carried out trial procedures. AED, JLA, MG, JB, JS, PWP and EJC

analyzed the results and interpreted the data. AED and EJC wrote the manuscript. All authors provided

critical comment, edited the manuscript, and approved its final version.




16

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21

Table 1: Baseline characteristics of the study population

Median age (IQR), years 54 (52, 57)

White British, n (%) 24 (96)

Median weight (IQR), kg 124 (111, 143)

Median BMI (IQR), kg/m2 47 (44, 51)

Median waist:hip ratio (IQR) 0.87 (0.83, 0.93)

Menopausal status, n (%)

Pre-menopausal 9 (36)

Post-menopausal 16 (64)

Menstrual cycle, n (%)

Amenorrhoeic 5 (20)

Regular 4 (16)

Irregular 3 (12)

Parity, n (%)

0 1 (4)

1 6 (24)

2 10 (40)

3+ 8 (32)

Exogenous hormones, n (%) 4 (16)

Polycystic ovary syndrome (PCOS), n (%) 6 (24)

Comorbidities, n (%)

Hypertension 15 (60)

Type II diabetes mellitus 10 (40)

Hypercholesterolaemia 5 (20)

Gallbladder/ liver disease 5 (20)

Thromboembolic disease 3 (12)

Asthma/ COPD 8 (32)

Sleep apnoea 9 (36)

Osteoarthritis 15 (60)

Depression/ anxiety 8 (32)




22

Table 2: Mixed effects regression model reporting change in Ki-67 score over time

Ki-67 score (No. observations = 71)

Factor Category Coef (95% C.I.)a Coef (95% C.I.)b Coef (95% C.I.)c

Time point T0 - Baseline (Ref) - - -

T1 - LNG-IUS -5.27(-16.9,6.39) -5.37(-17.1,6.33) -5.76(-17.5,5.99)

T2 - Follow up -14.4(-25.1,-3.62) -14.6(-25.3,-3.91) -15.3(-25.9,-4.58)

Baseline age - 0.69(-0.50,1.85) 0.77(-0.49,2.04)

Baseline weight - -0.26(-0.90,0.38) -0.32(-0.98,0.33)

Baseline BMI - 0.94(-1.00,2.82) 1.09(-0.84,3.03)

Smoker Never (Ref) - -

Ever - 3.54(-7.70,14.8) 3.21(-8.02,14.4)

Yes - 7.34(-4.80,19.5) 7.92(-4.28,20.1)

Type II diabetes mellitus No (Ref) - -

Yes - -2.73(-10.9,5.49) -2.23(-10.8,6.30)

Menopause status Post (Ref) - -

Pre - 5.56(-3.80,14.9) 5.42(-4.16,15.0)

Change in weight - - -0.41(-1.50,0.68)

Constant 27.1(17.8,36.4) -23.82(-107,58.9) -28.5(-117,560.0)

Random Effects (Bootstrapped)

Variance constant 7.17E-12 2.38E-11 2.69E-11

Variance residuals 285.4 290.3 290.8

Intra-class correlation 1.47E-13 8.20E-14 9.25E-14

a. Mixed model with time of assessment only adjusted for within participant clustering b. Mixed model with time of assessment adjusted for within participant clustering and baseline

screening characteristics c. Mixed model with time of assessment adjusted for within participant clustering, baseline

screening characteristics and change in weight from baseline




23

Table 3: Changes in anthropometric, blood and endometrial tissue biomarkers over time

T0 - baseline n=25

T1 - LNG-IUS n=25

T2 - follow up n=25

Anthropometric measures, mean (SD)

Weight, kg 129.1 (19.2) 128.3 (19.4) 126.7 (19.2)

BMI, kg/m2 48.3 (6.3) 48.0 (6.4) 47.5 (6.7)

Waist:hip ratio 0.9 (0.07) 0.88 (0.07) 0.87 (0.07)

Blood biomarkers of reproductive function, mean (SD)

Estradiol, pmol/L 11.8 (6.6) 9.7 (5.5) 10.1 (6.0)

Progesterone, ng/ml 3.1 (2.8) 3.8 (3.8) 1.4 (1.0)

Testosterone, nmol/L 7.1 (3.6) 6.3 (3.4) 6.8 (3.7)

SHBG, nmol/L 43.6 (28.8) 44.6 (24.7) 41.6 (21.9)

FAI 3.1 (2.7) 2.5 (2.2) 2.6 (1.8)

LH, IU/L 18.5 (13.1) 17.1 (12.8) 23.3 (16.1)

FSH, IU/L 28.8 (23.0) 27.9 (21.7) 35.1 (23.8)

Blood biomarkers of insulin resistance, mean (SD)

Glucose, mmol/L 5.5 (0.8) 5.5 (1.0) 5.5 (0.9)

Insulin, mU/L 139.0 (122.6) 123.1 (78.8) 124.9 (110.8)

HOMA 12.5 (7.1) 28.0 (24.3) 33.8 (34.9)

HbA1c, mmol/mol 41.9 (8.2) 41.7 (9.3) 42.2 (9.5)

Blood biomarkers of adiposity, mean (SD)

Adiponectin, mg/L 5.6 (13.4) 3.6 (3.7) 3.2 (4.1)

Leptin, ng/mL 77.3 (35.2) 82.9 (46.0) 83.4 (41.4)

Blood biomarkers of inflammation, mean (SD)

CRP, mg/L 9.0 (4.1) 6.2 (3.3) 7.3 (4.0)

Tissue biomarkers, mean (SD)

Ki-67 score (%) 27.1 (23.4) 21.8 (14.8) 12.7 (10.9)

pAKT H-score 105.5 (49.9) 89.4 (38.3) 93.1 (40.6)

Estrogen receptor (ER) H-score 0.8 (0.1) 0.9 (0.1) 0.8 (0.1)

Progesterone receptor (PR) H-score 0.8 (0.2) 0.9 (0.1) 0.4 (0.2)

Any PTEN-null glands (n, % participants) 2 (8%) 2 (8%) 0 (0%)




24

Table 4: Changes in mental wellbeing and quality of life over time

WEMWBS HADS

All participants

n=25

Subset of participants*

n=17

All participants n=25

Subset of participants*

n=17

Baseline (T0) Mean score (SD)

45.4 (10.6) 47.3 (10.9) 17.1 (10.4) 12.3 (9.3)

Follow up (T2) Mean score (SD)

47.9 (9.1) 52 (10.9) 14.9 (9.5) 11.4 (8.8)

*After excluding women with known depression/ anxiety disorder




25

Figure legend

Figure 1: Study flow chart showing accrual and retention of participants




Published OnlineFirst September 30, 2020.Cancer Prev Res Abigail E Derbyshire, Jennifer L Allen, Matthew Gittins, et al. obese women (PROTEC) trial: a feasibility studyPROgesterone Therapy for Endometrial Cancer prevention in

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