Context-related biomarkers in endometrial cancer. A study with focus on obesity and genomic alterations

Background: Endometrial cancer is the most common female pelvic gynaecologic malignancy in industrialised countries, and incidence has been increasing over the past decades. This has partly been ascribed to the increasing obesity epidemic seen worldwide, and particularly in affluent countries. Whereas increasing body mass index (BMI, kg/m2) is a known risk factor for endometrial cancer, less is known about its influence on tumour development and prognosis. Aims: The aim of this study was to increase the understanding about how contextrelated factors, including obesity (assessed by BMI and imaging methods) and genomic alterations (assessed by DNA ploidy status), are related to molecular tumour markers and outcome in endometrial cancer. By exploring gene and protein expression data from tumours arising in different settings, we aimed to shed light on potential contextrelated alterations, that may improve prognostication and represent relevant targets for therapy in future clinical trials. Materials and methods: For the studies included in this thesis (Paper I-IV), cohorts of patients treated for primary endometrial cancer at Haukeland University Hospital with thorough follow-up data and clinicopathological characterisation were used. For subsets of the patients, FFPE tissue was available for IHC analysis (Paper I-IV), fresh ethanol fixed tissue was used for DNA ploidy analysis (Paper II and III), and fresh frozen tissue was used for gene expression microarray (Paper II-IV) and RPPA analyses (Paper IV). Preoperative CT scans were used to study body fat distribution (Paper III). Results: High BMI was significantly associated with low FIGO stage, endometrioid histology and a high level of PR expression, but not ERα expression. Women with BMI≥25 had significantly better endometrial cancer survival compared to women with BMI<25 in univariable analysis, however not significant in multivariable analysis. Applying overall survival as outcome measure, increasing BMI independently predicted worse survival (Paper I). Aneuploidy was significantly associated with high age, high FIGO stage and high grade, non-endometrioid histology and ER/PR negativity, and independently predicted reduced survival. In ER/PR negative tumours, aneuploidy independently predicted recurrence and lymph node metastasis. A nine-gene prognostic ‘aneuploidy signature’, linked to low expression of chromosome 15q genes, was identified and validated in TCGA data (Paper II). Abdominal fat volumes were strongly positively correlated with BMI and waist circumference, and inversely correlated with liver density. High fat volumes and BMI were associated with low grade endometrioid tumours and PR and AR positivity, but not ERα positivity. The visceral fat percentage, VAV%, was not correlated with BMI or total abdominal fat volume, however, high VAV% was associated with high age and aneuploidy, and independently predicted reduced survival. Tumours from patients with low VAV% showed enrichment of gene signatures related to inflammatory and immunogenic signalling (Paper III). In endometrioid endometrial cancers, BMI was significantly correlated with a signature of hormone receptor expression, as well as PR and phospho-ERα (S118) levels. BMI was negatively correlated with RTK- and MAPK-pathway activation, and particularly phospho-MAPK (T202 Y204) level. In the subset of FIGO stage 1, grade 1-2 tumours, non-obese patients had significantly reduced survival compared to obese patients, associated with higher level of MAPK- and RTK-pathway activation. The obese patients had higher phospho-ERα (S118) levels, and showed enrichment of gene signatures related to oestrogen signalling, inflammation, immune signalling and hypoxia (Paper IV). Conclusions: BMI and imaging based estimates of obesity are associated with clinicopathological markers of less aggressive endometrial cancer (Paper I, III and IV). High BMI is associated with PR and AR but not ERα expression (Paper I, III and IV). Obese patients with endometrioid endometrial cancer have higher levels of phosphorylated ERα. Non-obese patients have higher levels of phosphorylated MAPK (Paper IV). High BMI is associated with improved DSS in univariable, but not multivariable analysis, and worse OS in multivariable analysis (Paper I). Increasing VAV% independently predicts reduced DSS (Paper III). Obesity is associated with improved DSS in patients with assumed excellent prognosis (Paper IV). Gene sets linked to inflammation and immune activation are enriched in tumours arising in patients with low VAV%, and equally in tumours arising in obese patients with FIGO stage 1, grade 1-2 tumours (Paper III and IV). DNA ploidy is a robust prognostic marker in endometrial cancer, and aneuploidy independently predicts reduced DSS. In patients with ER/PR negative tumours, aneuploidy independently predicts increased risk of lymph node metastases and recurrence (Paper II). A nine-gene aneuploidy signature is associated with reduced survival and low expression of chromosome 15q genes (Paper II)

Context-related biomarkers in endometrial cancer. A study with focus on obesity and genomic alterations

Background: Endometrial cancer is the most common female pelvic gynaecologic malignancy in industrialised countries, and incidence has been increasing over the past decades. This has partly been ascribed to the increasing obesity epidemic seen worldwide, and particularly in affluent countries. Whereas increasing body mass index (BMI, kg/m2) is a known risk factor for endometrial cancer, less is known about its influence on tumour development and prognosis. Aims: The aim of this study was to increase the understanding about how contextrelated factors, including obesity (assessed by BMI and imaging methods) and genomic alterations (assessed by DNA ploidy status), are related to molecular tumour markers and outcome in endometrial cancer. By exploring gene and protein expression data from tumours arising in different settings, we aimed to shed light on potential contextrelated alterations, that may improve prognostication and represent relevant targets for therapy in future clinical trials. Materials and methods: For the studies included in this thesis (Paper I-IV), cohorts of patients treated for primary endometrial cancer at Haukeland University Hospital with thorough follow-up data and clinicopathological characterisation were used. For subsets of the patients, FFPE tissue was available for IHC analysis (Paper I-IV), fresh ethanol fixed tissue was used for DNA ploidy analysis (Paper II and III), and fresh frozen tissue was used for gene expression microarray (Paper II-IV) and RPPA analyses (Paper IV). Preoperative CT scans were used to study body fat distribution (Paper III). Results: High BMI was significantly associated with low FIGO stage, endometrioid histology and a high level of PR expression, but not ERα expression. Women with BMI≥25 had significantly better endometrial cancer survival compared to women with BMI<25 in univariable analysis, however not significant in multivariable analysis. Applying overall survival as outcome measure, increasing BMI independently predicted worse survival (Paper I). Aneuploidy was significantly associated with high age, high FIGO stage and high grade, non-endometrioid histology and ER/PR negativity, and independently predicted reduced survival. In ER/PR negative tumours, aneuploidy independently predicted recurrence and lymph node metastasis. A nine-gene prognostic ‘aneuploidy signature’, linked to low expression of chromosome 15q genes, was identified and validated in TCGA data (Paper II). Abdominal fat volumes were strongly positively correlated with BMI and waist circumference, and inversely correlated with liver density. High fat volumes and BMI were associated with low grade endometrioid tumours and PR and AR positivity, but not ERα positivity. The visceral fat percentage, VAV%, was not correlated with BMI or total abdominal fat volume, however, high VAV% was associated with high age and aneuploidy, and independently predicted reduced survival. Tumours from patients with low VAV% showed enrichment of gene signatures related to inflammatory and immunogenic signalling (Paper III). In endometrioid endometrial cancers, BMI was significantly correlated with a signature of hormone receptor expression, as well as PR and phospho-ERα (S118) levels. BMI was negatively correlated with RTK- and MAPK-pathway activation, and particularly phospho-MAPK (T202 Y204) level. In the subset of FIGO stage 1, grade 1-2 tumours, non-obese patients had significantly reduced survival compared to obese patients, associated with higher level of MAPK- and RTK-pathway activation. The obese patients had higher phospho-ERα (S118) levels, and showed enrichment of gene signatures related to oestrogen signalling, inflammation, immune signalling and hypoxia (Paper IV). Conclusions: BMI and imaging based estimates of obesity are associated with clinicopathological markers of less aggressive endometrial cancer (Paper I, III and IV). High BMI is associated with PR and AR but not ERα expression (Paper I, III and IV). Obese patients with endometrioid endometrial cancer have higher levels of phosphorylated ERα. Non-obese patients have higher levels of phosphorylated MAPK (Paper IV). High BMI is associated with improved DSS in univariable, but not multivariable analysis, and worse OS in multivariable analysis (Paper I). Increasing VAV% independently predicts reduced DSS (Paper III). Obesity is associated with improved DSS in patients with assumed excellent prognosis (Paper IV). Gene sets linked to inflammation and immune activation are enriched in tumours arising in patients with low VAV%, and equally in tumours arising in obese patients with FIGO stage 1, grade 1-2 tumours (Paper III and IV). DNA ploidy is a robust prognostic marker in endometrial cancer, and aneuploidy independently predicts reduced DSS. In patients with ER/PR negative tumours, aneuploidy independently predicts increased risk of lymph node metastases and recurrence (Paper II). A nine-gene aneuploidy signature is associated with reduced survival and low expression of chromosome 15q genes (Paper II)

High visceral fat percentage is associated with poor outcome in endometrial cancer

Despite evidence of increased endometrial cancer (EC) risk in obese women, the impact of obesity on clinical and histological phenotype is poorly understood. This study explored abdominal fat volumes and fat distribution quantified by computed tomography (CT), in relation to tumor characteristics and outcome. 227 EC patients with preoperative abdominal CT scans were included. Total abdominal fat volume (TAV), subcutaneous abdominal fat volume (SAV) and visceral abdominal fat volume (VAV) were quantified, and visceral fat percentage calculated (VAV%=[VAV/TAV]x100). Waist circumference (WC) and liver density (LD) were measured, and body mass index (BMI) calculated. Data for estrogen, progesterone and androgen receptor (ERα/PR/AR) expression by immunohistochemistry were available for 149 tumors, and global gene expression data for 105 tumors. High BMI, TAV, SAV, VAV and WC, and low LD, were associated with low grade endometrioid tumors and PR and AR positivity (all p≤0.03). High VAV% was associated with high age (p<0.001), aneuploidy (p=0.01) and independently predicted reduced disease-specific survival (HR 1.05, 95% CI 1.00-1.11, p=0.041). Tumors from patients with low VAV% showed enrichment of gene sets related to immune activation and inflammation. In conclusion, high VAV% independently predicts reduced EC survival. Tumors arising in patients with low VAV% show enrichment of immune and inflammation related gene sets, suggesting that the global metabolic setting may be important for tumor immune response

Aneuploidy related transcriptional changes in endometrial cancer link low expression of chromosome 15q genes to poor survival

Aneuploidy is a widely studied prognostic marker in endometrial cancer (EC), however, not implemented in clinical decision-making. It lacks validation in large prospective patient cohorts adjusted for currently standard applied prognostic markers, including estrogen/progesterone receptor status (ER/PR). Also, little is known about aneuploidy-related transcriptional alterations, relevant for understanding its role in EC biology, and as therapeutic target. We included 825 EC patients with available ploidy status and comprehensive clinicopathologic characterization to analyze ploidy as a prognostic marker. For 144 patients, gene expression data were available to explore aneuploidy-related transcriptional alterations. Aneuploidy was associated with high age, FIGO stage and grade, non-endometrioid histology, ER/PR negativity, and poor survival (p-values<0.001). In patients with ER/PR negative tumors, aneuploidy independently predicted poor survival (p=0.03), lymph node metastasis (p=0.007) and recurrence (p=0.002). A prognostic ‘aneuploidy signature’, linked to low expression of chromosome 15q genes, was identified and validated in TCGA data. In conclusion, aneuploidy adds prognostic information in ER/PR negative EC, identifying high-risk patients that could benefit from more aggressive therapies. The ‘aneuploidy signature’ equally identifies these aggressive tumors and suggests a link between aneuploidy and low expression of 15q genes. Integrated analyses point at various dysregulated pathways in aneuploid EC, underlining a complex biology

Tissue and imaging biomarkers for hypoxia predict poor outcome in endometrial cancer

Hypoxia is frequent in solid tumors and linked to aggressive phenotypes and therapy resistance. We explored expression patterns of the proposed hypoxia marker HIF-1α in endometrial cancer (EC) and investigate whether preoperative functional imaging parameters are associated with tumor hypoxia. Expression of HIF-1α was explored both in the epithelial and the stromal tumor component. We found that low epithelial HIF-1α and high stromal HIF-1α expression were significantly associated with reduced disease specific survival in EC. Only stromal HIF-1α had independent prognostic value in Cox regression analysis. High stromal HIF-1α protein expression was rare in the premalignant lesions of complex atypical hyperplasia but increased significantly to invasive cancer. High stromal HIF-1α expression was correlated with overexpression of important genes downstream from HIF-1α, i.e. VEGFA and SLC2A1 (GLUT1). Detecting hypoxic tumors with preoperative functional imaging might have therapeutic benefits. We found that high stromal HIF-1α expression associated with high total lesion glycolysis (TLG) at PET/CT. High expression of a gene signature linked to hypoxia also correlated with low tumor blood flow at DCE-MRI and increased metabolism measured by FDG-PET. PI3K pathway inhibitors were identified as potential therapeutic compounds in patients with lesions overexpressing this gene signature. In conclusion, we show that high stromal HIF-1α expression predicts reduced survival in EC and is associated with increased tumor metabolism at FDG-PET/CT. Importantly; we demonstrate a correlation between tissue and imaging biomarkers reflecting hypoxia, and also possible treatment targets for selected patients