Strategies for earlier diagnosis of endometrial cancer: targeting endometrial hyperplasia

Abstract

Endometrial cancer (EC) is the most common gynaecological malignancy in the developed world, with approximately 9000 new cases reported annually within the UK (2013-2015). Incidence rates of EC have been steadily climbing over the last decade, with a notably steep increase described in the 40 to 49-year-old age group, most likely as a consequence of rising rates of obesity. Endometrial hyperplasia (EH) is a uterine pathology which is characterised by an increase in the endometrial gland-to-stroma ratio when compared to endometrium from the proliferative phase of the menstrual cycle. The clinical significance of EH lies in its association with progression to endometrioid endometrial cancer and ‘atypical’ forms of EH are widely considered to be premalignant lesions.

The overarching objective of the studies described in this thesis was to use cellular and molecular approaches to improve our capacity for earlier diagnosis of EC, through targeting and enhancing our understanding of EH. The following aims were addressed:

1. To develop a human EH tissue resource and utilise this to evaluate the current methods used to classify EH and predict its progression to EC.

2. To characterise key molecular changes within EH lesions so that they can be used to extend and enhance pathological classification of EH.

3. To explore in vitro models of the endometrium and investigate the role of PTEN and ARID1A in endometrial epithelial cell proliferation.

The results obtained herein provide novel insight into the diagnostic reproducibility of the two prominent EH pathological classification systems; i) the well-known and widely used World Health Organisation 1994 (WHO94) classification and ii) the more recent Endometrioid Intraepithelial Neoplasia (EIN)/WHO2014 iteration. Following an extensive retrospective review of patient medical records, a human tissue resource was established from samples held within The Lothian NRS Human Annotated Bioresource. Archival tissue sections from n=125 individual patient samples, that were pathologically diagnosed and coded as EH lesions based on the WHO94 criteria, were identified. A dual, blinded, expert gynaecological pathologist review was subsequently carried out. Interobserver percentage agreement for each of the expert pathologists and the original WHO94 based diagnosis was 56.0 % (n=70) and 48.8 % (n=61) respectively. Upon reclassification using the EIN/WHO2014 classification system, EIN lesions were identified in 52/125 patients, with increased interobserver agreement noted between the expert pathologists (67.2 %, n=84). The EIN/WHO2014 classification system also appeared to improve upon the WHO94 system when predicting progression to EC. Investigation of EH lesions for molecular changes pertinent to endometrial carcinogenesis revealed significant differences in the immunohistochemical expression pattern of the tumour suppressor PTEN, and the transcription factors PAX2 and HAND2 between EIN and benign EH lesions. These data may, pending further validation studies, lend favourably to their use as a diagnostic pathological aid. Somewhat unexpectedly, the frequency of defects in mismatch repair protein (dMMR) expression was considerably less than hypothesised amongst EIN lesions. This was surprising given that dMMR are reported in approximately 25-30 % of somatic ECs.

An accepted risk factor for the development of both EH and EC is exposure to ‘unopposed oestrogens’ i.e. oestrogen without progesterone opposition. A novel in vitro model was created utilising EC cell lines to investigate the proliferative effects of silencing two commonly mutated genes within both EHs and ECs, namely PTEN and ARID1A, and also the overexpression of oestrogen receptor alpha (ERα). Cellular manipulation of gene expression for ERα, PTEN and ARID1A was performed. Findings demonstrated a significant increase in EC cell proliferation with knockdown of PTEN and to a lesser extent ARID1A, when compared to EC cells transfected with a scrambled sequence. The addition of a functional ERɑ to the knockdown models did not appear to increase cell proliferation in this context.

In conclusion, novel data described herein highlights the current difficulties in achieving a reproducible diagnosis for EH. The use of immunohistochemistry identified changes in protein expression, which together with automated tissue analysis and in vitro studies, were used to complement and extend our understanding of premalignant changes in the endometrium. These findings have implications for the clinical management of women diagnosed with EH, as well as the development of a personalised approach to monitoring of women at increased risk of progression to EC.