Vanitha N. Sivalingam

Measuring the biological effect of presurgical metformin treatment in endometrial cancer

Background:Preclinical studies in endometrial cancer (EC) show that metformin reduces cellular proliferation by PI3K-AKT-mTOR inhibition. We tested the hypothesis that short-term presurgical metformin reduces cellular proliferation in atypical endometrial hyperplasia (AEH) and endometrioid EC, and assessed the feasibility of using phosphorylated PI3K-AKT-mTOR proteins as tissue end points.Methods:Women with AEH or EC received metformin 850 mg twice a day or no drug in the presurgical window between diagnosis and hysterectomy. Before and after the window, tissue samples were obtained; serum markers of insulin resistance (e.g. homeostasis model of assessment of insulin resistance index) were determined; and anthropometrics measured (e.g. BMI). Cell proliferation (Ki-67) and PI3K-AKT-mTOR phosphostatus were assessed by immunohistochemistry and scored blinded to treatment.Results:Twenty-eight metformin-treated and 12 untreated patients, well matched for age and BMI, completed the study. Metformin treatment (median 20 days, range 7–34) was associated with a 17.2% reduction in tumour Ki-67 (95% CI −27.4, −7.0, P=0.002), in a dose-dependent manner. Tumour PI3K-AKT-mTOR protein phosphostatus varied but the effects were not significant after adjusting for changes in controls.Conclusions:Short-term metformin was associated with reduced Ki-67 expression in EC. Changes in tumour PI3K-AKT-mTOR protein phosphostatus were seen in both groups. Future studies should address the variability attributed to different sampling techniques including devascularisation of the uterus at hysterectomy.

A presurgical window-of-opportunity study of metformin in obesity-driven endometrial cancer.

BACKGROUND Metformin use is associated with reduced cancer risk in several observational studies of patients with type 2 diabetes. Results from preclinical studies in endometrial cancer show that metformin reduces cellular proliferation by inhibition of the PI3K-AKT-mTOR pathway. We tested the hypothesis that metformin would reduce cellular proliferation in vivo in atypical endometrial hyperplasia and endometrial endometrioid adenocarcinoma. METHODS We recruited women attending gynaecological oncology clinics in Manchester, UK, with atypical endometrial hyperplasia or endometrial endometrioid adenocarcinoma. Women received metformin (850 mg twice daily) or no drug (control) during the 1-4 week presurgical window between cancer diagnosis and hysterectomy according to patient preference. Paired blood and tumour samples were obtained at recruitment and hysterectomy. Cellular proliferation was assessed by Ki-67 proliferation index. Automated scoring on two separate occasions provided consistent replicate scores (SD <10%). This study is registered with the ISRCTN register, number ISRCTN81570194. FINDINGS Samples from 40 women have been analysed (28 metformin-treated [median age 64 years, IQR 58-69]; 12 control [70, 64-70]). 24 of the patients (60%) were obese. 22 patients (55%) had either undiagnosed diabetes (fasting glucose >7·0 mmol/L, n=4) or insulin resistance (homoeostatic model assessment of insulin resistance >2·8, n=18). Metformin was taken for a median of 20 days (IQR 17-24), and mild gastrointestinal side-effects were reported by 22 metformin-treated patients. In the metformin-treated group, Ki-67 was 12·9% lower at hysterectomy than at recruitment (95% CI 3·7-22·1, p=0·008) after adjustment for baseline Ki-67, Ki-67 change in controls, age, and body-mass index. No significant changes in phosphorylation of AKT or markers of insulin resistance after adjustment for treatment arm were seen. INTERPRETATION Undiagnosed insulin resistance or diabetes were common in our study population. Short-term presurgical metformin was associated with a reduction in Ki-67 proliferation index. We are now exploring the hypothesis that metformin reduces Ki-67 expression by inducing phosphorylation of AMP-activated kinase and subsequent mTOR proproliferative inhibition, independent of insulin and insulin-like growth factor receptor activation. FUNDING Wellbeing of Women, Wellcome Trust.

Ki-67 in endometrial cancer: Scoring optimization and prognostic relevance for window studies

Ki-67, a marker of cellular proliferation, is increasingly being used in pre-surgical window studies in endometrial cancer as a primary outcome measure. Unlike in breast cancer, however, there are no guidelines standardizing its measurement and its clinical relevance as a response biomarker is undetermined. It is, therefore, imperative that Ki-67 scoring protocols are optimized and its association with patient survival rigorously evaluated, in order to be able to clinically interpret the results of these studies. Using the International Ki-67 in Breast Cancer Working Group guidelines as a basis, whole slide, hot spot and invasive edge scoring protocols were evaluated using endometrial biopsies and hysterectomy specimens from 179 women. Whole sections and tissue microarrays, manual and semi-automated scoring using Definiens Developer software were additionally compared. Ki-67 scores were related to clinicopathological variables and cancer-specific survival in uni- and multivariate analysis. Against criteria of time efficiency, intra- and inter-observer variability and consistency, semi-automated hot spot scoring was the preferred method. Ki-67 scores positively correlated with grade, stage and depth of myometrial invasion (P-values all <0.03). By univariate analysis, higher Ki-67 scores were associated with a significant reduction in cancer-specific survival (P≤0.05); however, this effect was substantially attenuated in the multivariate model. In conclusion, hot spot scoring of whole sections using Definiens is an optimal method to quantify Ki-67 in endometrial cancer window study specimens. Measured this way, it is a clinically relevant marker, though further work is required to determine whether reductions in Ki-67 in neoadjuvant intervention studies translate into improved patient outcome.

BGCS uterine cancer guidelines:: Recommendations for practice

The British Gynaecological Cancer Society has issued the first Endometrial (Uterine) Cancer guidelines as recommendation for practice for the UK.

The unrecognized burden of cardiovascular risk factors in women newly diagnosed with endometrial cancer: a prospective case control study

Highlights • CVD risk was measured in a prospective study of 150 EC patients and 746 controls.• EC patients had significantly more CVD risk factors than other women.• Most CVD risk factors were either unrecognized or inadequately treated.• EC patients had a significantly higher 10-year risk of CVD (QRISK2) than controls.• Identifying and treating CVD risk factors could improve outcomes for EC survivors.

Expression of NAD(P)H quinone dehydrogenase 1 (NQO1) is increased in the endometrium of women with endometrial cancer and women with Polycystic Ovary Syndrome

Women with a prior history of polycystic ovary syndrome (PCOS) have an increased risk of endometrial cancer (EC).

High prevalence of metabolic syndrome in women newly diagnosed with endometrial cancer

Thank you for the opportunity to respond to the letter from Galazis et al. (Galazis et al., 2018). The authors correctly state that women newly diagnosed with endometrial cancer are not routinely screened for cardiovascular risk factors in the UK. This is a missed opportunity for primary prevention; our data point to a very high prevalence of unrecognized and undertreated hypercholesterolemia, hypertension and hyperglyaemia in these women (Kitson et al., 2018). This is somewhat unsurprising given that nearly two-thirds of our patients are obese (BMI > 30 kg/m), and obesity is a known aetiological driver for both endometrial cancer and cardiovascular disease. Adipose-derived estrogen unopposed by progesterone in postmenopausal women is the most accepted theory linking obesity to endometrial carcinogenesis; however, the concept of metabolically unhealthy obesity, rather than excess adiposity per se, is gaining popularity, and goes some way towards explaining why some but not all obese women develop endometrial cancer. Defining metabolically unhealthy obesity is a contentious issue that has yet to be resolved, but most published studies have utilised the closely related concept of the metabolic syndrome. This is ‘a constellation of interrelated risk factors of metabolic origin’ that collectively predispose an individual to cardiovascular disease (Grundy et al., 2005). Individuals meeting the diagnostic criteria for the metabolic syndrome are considered metabolically unhealthy. Several indices have been used to characterise the metabolic syndrome, but the National Cholesterol Education Programs Adult Treatment Panel III (NECP ATP III) is most widely adopted. Using these criteria, we metabolically phenotyped a subset of our endometrial cancer patients. We measured waist circumference and blood pressure, and checked fasting blood glucose, lipid levels and liver enzymes. Those with elevated alanine transaminase (ALT) or alkaline phosphatase levels were referred for an ultrasound scan of their liver. In total, 52% (95% confidence intervals 42.2–61.8%) met the diagnostic criteria for metabolic syndrome (Table 1), with ≥3 of 5 indicative features (Fig. 1). In addition, 14 women were found to have previously unrecognized hepatic steatosis and non-alcoholic fatty liver disease. By contrast, 23.1% of the general US population meet the diagnostic criteria for the metabolic syndrome (Ford et al., 2004). There are no comparable data for a UK population; in our age and ethnicity-matched Health Survey for England (HSE) control women, HbA1c was measured instead of fasting blood glucose, and there were insufficient data to diagnose metabolic syndrome in 15.5%. Nonetheless, just 9.2% (95% confidence intervals 7.3–11.2) of control women met the criteria for metabolic syndrome, more than five-fold fewer than our endometrial cancer patients, a comparison that was highly significant (p < .0001), and mirrors previous work (Rosato et al., 2011). Endometrial cancer patients who met the diagnostic criteria for metabolic syndrome were more likely to be obese than those who did not (82.7% vs. 37.5%, p= .0001). However, the presence of metabolic syndrome was not associated with age, grade or stage of endometrial cancer (data not shown). Whilst these findings lend strong support to cardiovascular risk factor screening and optimization in endometrial cancer survivors, they also hint at common biological mechanisms underpinning the pathogenesis of the two conditions. Further, they offer the very exciting possibility that simple, safe, non toxic drugs with proven activity against elements of the metabolic syndrome may offer women protection against both cardiovascular disease and endometrial cancer. Interventions that reduce the risk of endometrial cancer are urgently needed to limit escalating incidence rates and consequent deaths from the disease (Crosbie and Morrison, 2014). Accumulating evidence highlights the potential anti-cancer effect of aspirin (Takiuchi et al., 2018), statins (Arima et al., 2017) and metformin, an insulin sensitising drug long used in the treatment of type 2 diabetes (Sivalingam et al., 2014). Data supporting an anti-cancer activity of the beta-blocker antihypertensive drugs have been less persuasive (Sanni et al., 2017). It is tantalising to think that adequately