Identification of highly connected and differentially expressed gene subnetworks in metastasizing endometrial cancer.

We have identified nine highly connected and differentially expressed gene subnetworks between aggressive primary tumors and metastatic lesions in endometrial carcinomas. We implemented a novel pipeline combining gene set and network approaches, which here allows integration of protein-protein interactions and gene expression data. The resulting subnetworks are significantly associated with disease progression across tumor stages from complex atypical hyperplasia, primary tumors to metastatic lesions. The nine subnetworks include genes related to metastasizing features such as epithelial-mesenchymal transition (EMT), hypoxia and cell proliferation. TCF4 and TWIST2 were found as central genes in the subnetwork related to EMT. Two of the identified subnetworks display statistically significant association to patient survival, which were further supported by an independent validation in the data from The Cancer Genome Atlas data collection. The first subnetwork contains genes related to cell proliferation and cell cycle, while the second contains genes involved in hypoxia such as HIF1A and EGLN3. Our findings provide a promising context to elucidate the biological mechanisms of metastasis, suggest potential prognostic markers and further identify therapeutic targets. The pipeline R source code is freely available, including permutation tests to assess statistical significance of the identified subnetworks.

HER2 expression patterns in paired primary and metastatic endometrial cancer lesions.

BACKGROUND: Despite successful implementation of drugs targeting the human epidermal growth factor receptor 2 (HER2) receptor in breast and gastric cancers, the potential of HER2 as a therapeutic target in other cancers has been less studied, including endometrial cancer. We investigated expression levels of HER2 (ERBB2) in a large cohort of endometrial cancer lesions, also including complex atypical hyperplasia and metastatic lesions. METHODS: 67 precursor lesions, 790 primary endometrial cancers and 383 metastatic lesions were investigated for HER2 expression in relation to clinicopathologic features and outcome. Protein levels were assessed by immunohistochemistry (using the HercepTest and staining index (SI) criteria), mRNA levels by microarrays and amplification status by chromogenic in situ hybridisation. RESULTS: High HER2 protein levels were significantly associated with features of aggressive disease and increased mRNA ERBB2 levels. HER2 expression defined by the SI proved to be a better predictor of survival compared with the HercepTest. A discordant HER2 expression pattern between paired primary and metastatic lesions was detected, revealing substantial reduction in HER2 expression from primary to metastatic disease. CONCLUSIONS: Loss of HER2 expression is common in metastatic endometrial cancer lesions and assessment of HER2 levels in the metastatic lesions may be important to define the potential benefit of anti-HER2 treatments in endometrial cancer patients.

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.

Preoperative imaging markers and PDZ-binding kinase tissue expression predict low-risk disease in endometrial hyperplasias and low grade cancers.

PURPOSE: Distinguishing complex atypical hyperplasia (CAH) from grade 1 endometrioid endometrial cancer (EECG1) preoperatively may be valuable in order to prevent surgical overtreatment, particularly in patients wishing preserved fertility or in patients carrying increased risk of perioperative complications. MATERIAL AND METHODS: Preoperative histological diagnosis and radiological findings were compared to final histological diagnosis in patients diagnosed with CAH and EECG1. Imaging characteristics at preoperative magnetic resonance imaging (MRI) and fluorodeoxyglucose positron emission tomography/computer tomography (FDG-PET/CT) were compared with tumor DNA oligonucleotide microarray data, immunohistochemistry findings and clinicopathological annotations. RESULTS: MRI assessed tumor volume was higher in EECG1 than in CAH (p=0.004) whereas tumor apparent diffusion coefficient value was lower in EECG1 (p=0.005). EECG1 exhibited increased metabolism with higher maximum and mean standard uptake values (SUV) than CAH (p≤0.002). Unsupervised clustering of EECG1 and CAH revealed differentially expressed genes within the clusters, and identified PDZ-binding kinase (PBK) as a potential marker for selecting endometrial lesions with less aggressive biological behavior. CONCLUSION: Both PBK expression and preoperative imaging yield promising biomarkers that may aid in the differentiation between EECG1 and CAH preoperatively, and these markers should be further explored in larger patient series.

Type of vascular invasion in association with progress of endometrial cancer.

Vascular invasion (VI) is a well-established marker for lymph node metastasis and outcome in endometrial cancer. Our study explored whether specific types of VI, defined as lymphatic (LVI) or blood vessel invasion (BVI), predict pattern of metastasis. From a prospectively collected cohort, we conducted a case-control study by selecting three groups of endometrial cancer patients (n = 183): 52 with positive lymph nodes at primary surgery, 33 with negative nodes at primary surgery and later recurrence and death from disease, and 98 with negative nodes and no recurrence. All patients underwent hysterectomy with lymphadenectomy. Immunohistochemical staining with D2-40 and CD31 antibodies was used to differentiate between BVI and LVI. By immunohistochemical staining, detection of VI increased from 24.6 to 36.1% of the cases. LVSI was significantly more often seen in patients with positive lymph nodes compared with patients with negative nodes (p = 0.001). BVI was significantly more often seen in node-negative patients with recurrence compared with node-negative patients without recurrence (p = 0.011). In multivariable analysis, BVI, age, and tumor grade were predictors separating patients with and without recurrence. Lymph node-positive patients showed more often LVI compared with lymph node-negative patients, while BVI seems to be a predictor for recurrent disease.

PIK3CA exon9 mutations associate with reduced survival, and are highly concordant between matching primary tumors and metastases in endometrial cancer.

Mutations of the phosphoinositide-3-kinase (PI3K) catalytic subunit alpha gene (PIK3CA) are frequent in endometrial cancer. We sequenced exon9 and exon20 of PIK3CA in 280 primary endometrial cancers to assess the relationship with clinicopathologic variables, patient survival and associations with PIK3CA mRNA and phospho-AKT1 by gene expression and protein data, respectively. While PIK3CA mutations generally had no impact on survival, and were not associated with clinicopathological variables, patients with exon9 charge-changing mutations, providing a positive charge at the substituted amino acid residue, were associated with poor survival (p = 0.018). Furthermore, we characterized PIK3CA mutations in the metastatic setting, including 32 patients with matched primary tumors and metastases, and found a high level of concordance (85.7%; 6 out of 7 patients), suggesting limited heterogeneity. PIK3CA mRNA levels were increased in metastases compared to the primary tumors (p = 0.031), independent of PIK3CA mutation status, which rather associated with reduced PIK3CA mRNA expression. PIK3CA mutated tumors expressed higher p-AKT/AKT protein levels, both within primary (p < 0.001) and metastatic lesion (p = 0.010). Our results support the notion that the PI3K signaling pathway might be activated, both dependent- and independently of PIK3CA mutations, an aspect that should be considered when designing PIK3 pathway targeting strategies in endometrial cancer.

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.

Proteomic profiling of endometrioid endometrial cancer reveals differential expression of hormone receptors and MAPK signaling proteins in obese versus non-obese patients.

Endometrial cancer development is strongly linked to obesity, but knowledge regarding the influence of excess weight on endometrial tumor signaling pathways remains scarce. We therefore analyzed reverse phase protein array (RPPA) data for obesity-related protein expression patterns, using one training (n=272) and two test cohorts (n=68; n=178) of well-annotated samples from women treated for endometrioid endometrial cancer. Gene expression profiling and immunohistochemistry were used for cross-platform validation. Body mass index (BMI) was significantly correlated with progesterone receptor (PR) expression and a hormone receptor protein signature, across all cohorts. In two of the cohorts, BMI was negatively correlated with RTK- and MAPK-pathway activation, particularly phosphorylated MAPK T202 Y204 (p-MAPK) level. Using stepwise selection modelling, a BMI-associated protein signature, including phosphorylated estrogen receptor α S118 (p-ERα) and p-MAPK, was identified. In the subset of FIGO stage 1, grade 1-2 tumors, obese patients (BMI≥30) had better survival compared to non-obese patients in the two cohorts with longest follow-up time (p=0.042, p=0.058). Non-obese patients had higher p-MAPK levels, whereas obese patients had higher p-ERα levels and enrichment of gene signatures related to estrogen signaling, inflammation, immune signaling and hypoxia. In subgroup analysis of non-obese patients with FIGO stage 1 tumors, low PI3K-activation was associated with reduced survival (p=0.002, training cohort). In conclusion, increasing BMI is associated with increased PR and p-ERα levels and reduced MAPK signaling, both in all patients and in subsets with predicted excellent prognosis. The MAPK-pathway represents a potential therapeutic target in non-obese patients with low stage and low grade tumors.

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.

The genomic landscape and evolution of endometrial carcinoma progression and abdominopelvic metastasis.

Recent studies have detailed the genomic landscape of primary endometrial cancers, but the evolution of these cancers into metastases has not been characterized. We performed whole-exome sequencing of 98 tumor biopsies including complex atypical hyperplasias, primary tumors and paired abdominopelvic metastases to survey the evolutionary landscape of endometrial cancer. We expanded and reanalyzed The Cancer Genome Atlas (TCGA) data, identifying new recurrent alterations in primary tumors, including mutations in the estrogen receptor cofactor gene NRIP1 in 12% of patients. We found that likely driver events were present in both primary and metastatic tissue samples, with notable exceptions such as ARID1A mutations. Phylogenetic analyses indicated that the sampled metastases typically arose from a common ancestral subclone that was not detected in the primary tumor biopsy. These data demonstrate extensive genetic heterogeneity in endometrial cancers and relative homogeneity across metastatic sites.

Clinical value of dna content assessment in endometrial cancer.

Endometrial carcinoma (EC) is the most common gynecologic cancer in industrialized countries. Traditional prognostic markers include FIGO stage, histologic subtype and histologic grade. DNA ploidy was introduced as a prognostic marker 30 years ago, and the majority of published literature demonstrates significant associations between tumor aneuploidy and poorer prognosis in EC. However, ploidy analysis is not routinely implemented in the clinic. We reviewed the literature on clinical value of ploidy measured by DNA content as a prognostic marker, and its potential role as a predictive marker in EC. PubMed was searched for papers evaluating the prognostic or predictive role of ploidy in EC. Search criteria were "DNA ploidy prognosis/predictive value endometrial cancer/carcinoma". Only articles written in English, published year 2000 or later were included. The majority of the studies demonstrated highly significant correlation between DNA index (DI) and survival, in univariate analysis including stages I-IV, and in subgroup analysis of stage I and stage I-II EC. Several studies also showed significant association between DI and survival in multivariate analysis. Few studies have evaluated DI as a prognostic marker in a prospective setting. No studies evaluating DI as a predictive marker in EC were identified. In other cancer types, ploidy has been linked to prediction of response to hormonal therapy and chemotherapy. Ploidy assessment in EC by DI is a strong prognostic marker. Still, its clinical applicability needs validation in a routine diagnostic, prospective setting with sufficient number of patients, characterized by state of the art histopathological evaluation and surgical staging. © 2014 Clinical Cytometry Society.

Clinical value of DNA content assessment in endometrial cancer.

Endometrial carcinoma (EC) is the most common gynecologic cancer in industrialized countries. Traditional prognostic markers include FIGO stage, histologic subtype, and histologic grade. DNA ploidy was introduced as a prognostic marker 30 years ago, and the majority of published literature demonstrates significant associations between tumor aneuploidy and poorer prognosis in EC. However, ploidy analysis is not routinely implemented in the clinic. We reviewed the literature on clinical value of ploidy measured by DNA content as a prognostic marker, and its potential role as a predictive marker in EC. PubMed was searched for papers evaluating the prognostic or predictive role of ploidy in EC. Search criteria were "DNA ploidy prognosis/predictive value endometrial cancer/carcinoma". Only articles written in English, published year 2000 or later were included. The majority of the studies demonstrated highly significant correlation between DNA index (DI) and survival, in univariate analysis including stages I-IV, and in subgroup analysis of stage I and stage I-II EC. Several studies also showed significant association between DI and survival in multivariate analysis. Few studies have evaluated DI as a prognostic marker in a prospective setting. No studies evaluating DI as a predictive marker in EC were identified. In other cancer types, ploidy has been linked to prediction of response to hormonal therapy and chemotherapy. Ploidy assessment in EC by DI is a strong prognostic marker. Still, its clinical applicability needs validation in a routine diagnostic, prospective setting with sufficient number of patients, characterized by state of the art histopathological evaluation and surgical staging.

High phospho-Stathmin(Serine38) expression identifies aggressive endometrial cancer and suggests an association with PI3K inhibition.

PURPOSE: High Stathmin expression has recently been associated with clinical progress in endometrial cancers. Stathmin protein activity is modulated by phosphorylation, and the Serine38 site is one of four Stathmin phospho-sites. The presence and significance of pStathmin(S38) is largely unknown in human cancers, and we here examined the associations between this marker and tumor cell proliferation, clinicopathologic phenotype, and survival impact in endometrial cancer. A relationship with possible treatment targets was explored by integrated analysis of transcriptional alterations. EXPERIMENTAL DESIGN: Primary endometrial cancers from two independent patient series (n = 518/n = 286) were analyzed. Biomarkers were assessed by immunohistochemistry, FISH, flow cytometry, DNA oligonucleotide microarray, single-nucleotide polymorphism array, and Sanger sequencing, and related to clinicopathologic annotations and follow-up information. RESULTS: High pStathmin(S38) level was associated with poor prognosis, independent of other features, and correlated to increased tumor cell proliferation as well as high Stathmin levels. On the basis of transcriptional differences between high/low pStathmin(S38) tumors, phosphoinositide 3-kinase (PI3K)/mTOR/HSP90 were suggested as possible targets in pStathmin(S38)-high cases. High pStathmin(S38) was associated with several PI3K pathway alterations: amplification of the 3q26 region, increased PIK3CA copy number (FISH) and a PI3K activation score (all P < 0.05). CONCLUSIONS: High pStathmin(S38) is a novel biomarker of increased tumor cell proliferation and impaired prognosis as reported here for independent cohorts of endometrial cancer and not previously shown in human cancer. Our data support a rationale for further studies exploring effects of drugs inhibiting the PI3K signaling pathway in pStathmin(S38)-high endometrial cancer, including a potential value of pStathmin(S38) in predicting response to PI3K/mTOR/HSP90 inhibitors.

Improved survival related to changes in endometrial cancer treatment, a 30-year population based perspective.

OBJECTIVE: Age adjusted incidence rate for uterine cancers in Norway has increased over last three decades from 12.2/100.000 (1981-90) to 16.0 (2001-2010). Corresponding 5-year survival increased nationally from 76.3% to 83.3%. METHODS: We wanted to investigate how changes in therapeutic strategies during a 30-year period are reflected in survival changes through careful characterization of a population-based series of 1077 endometrial carcinoma patients from Hordaland County, Norway. RESULTS: In concordance with increase in endometrial cancer nationally, the number of patients treated from Hordaland County rose from 286 (1981-1990) through 307 (1991-2000) to 484 (2001-2010). Main treatment changes included increase in routine pelvic lymphadenectomy from 0% through 9% to 77%, adjuvant chemotherapy from 0% through 3% to 9% and a dramatic reduction in adjuvant radiotherapy from 75% through 48% to 12% (all P<0.001). Body mass index increased significantly during this 30-year period, as did the 5-year disease-specific survival: from 75.8 through 80.2 to 86.9% (P=0.002) and overall survival from 67.8 through 71.7 to 77.8% (P=0.03). CONCLUSION: Improved overall and disease specific survival for endometrial carcinoma patients over the last 30 years is demonstrated in a population-based setting. Increasing BMI among patients and a change in treatment strategy with reduction in adjuvant radiotherapy and more extensive surgery is demonstrated for the same period.