Benign Endometrial Tissue Research Articles

The Impact of High Adiposity on Endometrial Progesterone Response and Metallothionein Regulation.

Obesity is a disease with deleterious effects on the female reproductive tract, including the endometrium. We sought to understand the effects of excess adipose on the benign endometrium. A physiologic in vitro coculture system was developed, consisting of multicellular human endometrial organoids, adipose spheroids, and menstrual cycle hormones. Native human endometrial tissue samples women with and without obesity were also analyzed. Academic institution. Benign endometrial tissues from premenopausal women were obtained following written consent. Gene expression, protein expression, chromatin binding, and expression of DNA damage and oxidative damage markers were measured. Under high-adiposity conditions, endometrial organoids downregulated endometrial secretory phase genes, suggestive of an altered progesterone response. Progesterone specifically upregulated the metallothionein (MT) gene family in the epithelial cells of endometrial organoids, while high adiposity significantly downregulated the MT genes. Silencing MT genes in endometrial epithelial cells resulted in increased DNA damage, illustrating the protective role of MTs. Native endometrium from women with obesity displayed increased MT expression and oxidative damage in the stroma and not in the epithelium, indicating the cell-specific impact of obesity on MT genes. Taken together, the in vitro and in vivo systems used here revealed that high adiposity or obesity can alter MT expression by decreasing progesterone response in the epithelial cells and increasing oxidative stress in the stroma.

Expression of netrin-1 in uterine serous carcinoma and its association with prognosis.

At present, there are few biomarkers used to predict the prognosis of uterine serous carcinoma (USC). Netrin-1 may be a promising biomarker candidate. We investigated netrin-1 expression in USC tissues and healthy endometrial tissues to determine its relevance to disease prognosis. Netrin-1 expression was examined in the tissues of 48 patients with USC and 30 patients with healthy benign endometrial tissues via immunohistochemistry. None of the healthy tissues were stained with netrin-1. In tumor tissues, the overall positivity rate of netrin-1 was 75%, detected as high expression in 17 patients (35%) and low in 19 (40%). Patients who had tumors with no netrin-1 expression (n = 12) had a median overall survival (OS) of 60.0 months (95% confidence interval [CI], 47-98), whereas patients who had tumors with low to strong netrin-1 expression (n = 33) had a lower median OS of 50 months, but the difference was not statistically significant (95% CI, 58-108; P = 0.531). Disease-free survival (DFS) was not statistically significant between the groups (95% CI, 67.7-115.9; P = 0.566). Patients with a tumor diameter ≥2 cm had higher netrin-1 expression than those with a tumor diameter of 2 cm (P = 0.027). We did not find any difference in overall and DFS when age, tumor stage, histology, tumor diameter, p53 status, lymphovascular space invasion, myometrial invasion, and lymph node metastasis were compared according to netrin-1 expression (P > 0.05). Netrin-1 was expressed in USC but not in healthy tissues. Its expression was not associated with OS or DFS.

Abstract A004: TP53 mutational landscape in non-cancerous endometrium during the lifespan of Black and White individuals

Abstract Background TP53 somatic mutations accumulate and clonally expand in histologically normal tissues with aging, however, little is known about this process and how it influences cancer development. Within endometrial carcinoma, TP53 mutations tend to be found in aggressive, non-endometrioid histologies associated with poor survival outcomes, more commonly affecting Black women. We seek to understand the TP53 mutational burden in benign endometrium by characterizing mutations acquired throughout a lifespan with the integration of social determinants of health in Black and White women. Methods Endometrial tissue was collected at autopsy or hysterectomy, confirmed with H&E staining, and macrodissected. Ultra-deep duplex sequencing (~10,000x) of isolated DNA was used to sequence TP53somatic mutations with high resolution. Each individual’s TP53 mutation frequency was calculated as the number of unique TP53 variants divided by the total duplex nucleotides sequenced. Mutations were classified as pathogenic if insertion-deletion, splice, or nonsense in addition to all pathogenic substitutions identified by the Seshat TP53 database. Socioeconomic metrics were acquired using zip codes and the 2021 US Census Bureau American Community Survey. Individuals with a history of endometriosis, prior malignancy, or prior chemotherapy were excluded. Results Benign endometrial tissue was collected from 69 individuals (24 Black, 45 White), including 22 aged 0-19 years, 25 aged 20-39 years, 17 aged 40-59 years, and 5 aged over 60 years. 174 TP53 variants were identified. Variant types included 66.7% missense (n=116), 13.2% insertion-deletion (n=23), 12.1% synonymous (n=21), 4.6% nonsense (n=8), and 3.4% splice (n=6). 102 TP53 mutations (58.6%) were pathogenic. TP53 mutation frequency increased linearly with age (Spearman correlation p<0.01). 12.6% of mutations were identified in more than one duplex mutant read (large clone) beginning at age 29 through age 81. The frequency of large TP53 mutant clones increased exponentially with age. In this limited dataset, there was no statistical association between socioeconomic metrics and the mutational burden of Black and White individuals. Conclusions The number and size of TP53 mutant clones in benign endometrium are associated with older age, indicating clonal evolution through life. Most somatic mutations identified were pathogenic substitutions or loss-of-function variants, confirming positive selection of cancer-like mutations. Additional data are needed to determine the role of socioeconomic factors in TP53 genomic burden. Citation Format: Eric Rios-Doria, Elizabeth U. Parker, Brendan F. Kohrn, Elena Latorre-Estevez, Jeanne Fredrickson, Elizabeth M. Swisher, Kemi M. Doll, Rosana Risques. TP53 mutational landscape in non-cancerous endometrium during the lifespan of Black and White individuals [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A004.

Endometrial Pipelle Biopsy Computer-Aided Diagnosis: A Feasibility Study

Endometrial biopsies are important in the diagnostic workup of women who present with abnormal uterine bleeding or hereditary risk of endometrial cancer. In general, approximately 10% of all endometrial biopsies demonstrate endometrial (pre)malignancy that requires specific treatment. As the diagnostic evaluation of mostly benign cases results in a substantial workload for pathologists, artificial intelligence (AI)-assisted preselection of biopsies could optimize the workflow. This study aimed to assess the feasibility of AI-assisted diagnosis for endometrial biopsies (endometrial Pipelle biopsy computer-aided diagnosis), trained on daily-practice whole-slide images instead of highly selected images. Endometrial biopsies were classified into 6 clinically relevant categories defined as follows: nonrepresentative, normal, nonneoplastic, hyperplasia without atypia, hyperplasia with atypia, and malignant. The agreement among 15 pathologists, within these classifications, was evaluated in 91 endometrial biopsies. Next, an algorithm (trained on a total of 2819 endometrial biopsies) rated the same 91 cases, and we compared its performance using the pathologist’s classification as the reference standard. The interrater reliability among pathologists was moderate with a mean Cohen’s kappa of 0.51, whereas for a binary classification into benign vs (pre)malignant, the agreement was substantial with a mean Cohen’s kappa of 0.66. The AI algorithm performed slightly worse for the 6 categories with a moderate Cohen’s kappa of 0.43 but was comparable for the binary classification with a substantial Cohen’s kappa of 0.65. AI-assisted diagnosis of endometrial biopsies was demonstrated to be feasible in discriminating between benign and (pre)malignant endometrial tissues, even when trained on unselected cases. Endometrial premalignancies remain challenging for both pathologists and AI algorithms. Future steps to improve reliability of the diagnosis are needed to achieve a more refined AI-assisted diagnostic solution for endometrial biopsies that covers both premalignant and malignant diagnoses.

Endometrial telomerase activity in women with either endometrial cancer or hyperplasia: A systematic review and meta-analysis.

We performed a systematic review and meta-analysis to assess whether endometrial telomerase activity is associated with endometrial cancer or hyperplasia. PubMed, Web of Science, Embase, Scielo, LILAC, and CNKI databases were searched to obtain relevant literature for articles published through June 2022, following PRISMA guidelines and a registered PROSPERO protocol. We included observational studies reporting endometrial telomerase activity in patients with either endometrial cancer or hyperplasia compared with benign endometrial tissue (control women). The Newcastle-Ottawa Scale was used to evaluate the quality of studies. Data were expressed as the odds ratios (OR) and 95 % confidence intervals (CI). Random effects and inverse variance methods were used to meta-analyze associations. The I2 test was used to assess heterogeneity. There were significant associations between endometrial telomerase activity and either endometrial cancer (20 studies, OR = 10.65, 95 % CI 6.39, 17.75, p = 0.00001, I2 = 21 %) or endometrial hyperplasia (nine studies, OR = 3.62, 95 % CI 1.61, 8.13, p = 0.002, I2 = 36 %) compared to women without endometrial cancer and hyperplasia. There was not a significant difference in telomerase activity in women with endometrial cancer compared to those with endometrial hyperplasia (seven studies, OR = 1.03; 95 % CI 0.31, 3.37, p = 0.96, I2 = 49 %). In subgroup analyses, there were no significant differences in telomerase activity in patients with endometrial cancer by type of observational studies and by countries of the studies. Endometrial telomerase activity is higher in women with either endometrial cancer or endometrial hyperplasia compared to control women without those lesions.

Comparative Study of Immunohistochemical Expression of ER and PR as Prognostic Markers and Correlation with Clinicopathological Parameters in Human Endometrial Adenocarcinoma

In human steroid sensitive tissue, progesterone receptors (PRs) and estrogen receptors (ERs) have been demonstrated at the mRNA and/or protein levels by using molecular biology and immunohistochemistry techniques. Estrogen and progesterone receptors are present both in endometrial hyperplasia and in neoplastic endometrium. The aim of the present work was to compare the expression of ER and PR as prognostic biomarkers in human endometrial adenocarcinoma versus benign tumors and normal endometrial tissues as well as their correlation with different pathological and histological parameters. Immunohistochemical technique was used to examine the expression of ER and PR in normal, benign as well as in endometrial adenocarcinoma. Present results showed higher expression of ER and PR in endometrial adenocarcinoma comparing to normal and benign endometrial tissues.

MiRNA-576-5p promotes endometrial cancer cell growth and metastasis by targeting ZBTB4

PurposeMicroRNAs (miRNAs) have already been shown to have a strong correlation with the invasion and metastasis capacity of tumor cells. The present research examined the function of miRNA-576-5p (miR-576-5p) in the development of endometrial cancer (EC).MethodsmiR-576-5p and ZBTB4 expression in EC and benign endometrial tissues was measured using quantitative real-time PCR (qRT-PCR) and western blot. To evaluate the proliferation ability of tumor cells in vitro, 2,5-diphenyl-2H-tetrazolium bromide (MTT) and colony formation assays were carried out. The effect of miR-576-5p on the proliferation ability of EC cells in vivo was measured by the tumor formation in nude mice. The migration and invasion ability of tumor cells was determined using the transwell assay. To confirm the association between expressions of miR-576-5p and zinc finger and BTB domain containing four (ZBTB4), western blot, qRT-PCR, and luciferase assay were carried out.ResultsmiR-576-5p expression increased significantly in EC samples than in benign endometrial tissues. The level of miR-576-5p was significantly higher in the polymerase ε (POLE) ultramutated subgroup compared to the other three subgroups. High levels of miR-576-5p expression were linked to a shorter progression-free interval time in the copy number high subgroup. Furthermore, upregulated miR-576-5p facilitated EC cell invasion and migration in vitro and promoted the proliferation of EC tumor cell lines both in vitro and in vivo. Moreover, this study showed that the expression of ZBTB4 decreased in patients with EC, and the dual-luciferase reporter assay confirmed that miR-576-5p binds directly to the 3′-UTR of ZBTB4 and inhibits the expression of ZBTB4. An increase in miR-576-5p expression leads to a decrease in the mRNA and protein expression level of ZBTB4. The effects of miR-576-5p can be reversed by overexpression of ZBTB4.ConclusionmiR-576-5p promoted proliferation and metastasis capacity of EC cells by inhibiting ZBTB4 expression. We hypothesized that miR-576-5p could be a prospective therapeutic target for EC.

Aberrant upregulation of CDK1 contributes to medroxyprogesterone acetate (MPA) resistance in cancer-associated fibroblasts of the endometrium

The response to medroxyprogesterone acetate (MPA) decreases as endometrial disease progresses from the benign to malignancy. In a mouse model, progesterone receptor (PR) expression in normal fibroblasts is accountable for the MPA's inhibitory effects in cancer cells. However, it is still unclear, if and how, fibroblasts from human tumors respond to MPA. In this study, three benign-associated fibroblasts (BAFs) and four cancer-associated fibroblasts (CAFs) were isolated from human benign and cancerous endometrial tissues, respectively, to examine MPA activation on PR signaling. PR-B protein expression were heterogeneously expressed in both CAFs and BAFs, despite a lower mRNA expression in the former. In a luciferase reporter assay, MPA treatment stimulated some PR DNA-binding activity in BAFs but not in CAFs. Yet, activation of PR target gene was generally more pronounced in MPA-treated CAFs compared to BAFs. Cyclin-dependent kinase 1 (CDK1) was exclusively upregulated by 10 nM MPA in CAFs (5.1-fold vs. 1.1-fold in BAFs, P < 0.05), leading to a higher CDK1 protein expression. Subsequently in a dose-response study, CAFs showed an average of ∼20% higher cell viability when compared to BAFs, indicative of drug resistance to MPA. MPA resistance was also observed in EC-CAFs co-culture, when MPA-treated cells showed greater tumor spheroid formation than in EC-BAFs co-culture (2-fold, P < 0.01). The increased cell viability observed in CAFs was reversed with mifepristone (RU486), a PR antagonist which suppressed MPA-induced CDK1 expression. This indicates that MPA-induced abnormal upregulation of CDK1 may contribute to the enhanced CAFs cell proliferation, suggesting a new mechanism of MPA resistance within endometrial cancer microenvironment.

Metabolomic analysis of endometrial cancer by high-resolution magic angle spinning NMR spectroscopy.

To analyze endometrial metabolite profiles between patients with endometrial cancer and controls. Seventeen (17) women with endometrium cancer and 18 controls were enrolled in this study. 1H HR-MAS (High Resolution-Magic Angle Spinning) NMR (Nuclear Magnetic Resonance) spectroscopy data obtained from endometrial tissue samples of patients with endometrial cancer and control group were analyzed with bioinformatics methods. Principal component analysis (PCA) and the partial least squares discriminant analysis (PLS-DA) score plots obtained with the multivariate statistical analysis of pre-processed spectral data shows a separation between the samples from patients with endometrial cancer and controls. Analysis results suggest that the levels of lactate, glucose, o-phosphoethanolamine, choline, glycerophosphocholine, phosphocholine, leucine, isoleucine, valine, glutamate, glutamine, n-acetyltyrosine, methionine, taurine, alanine, aspartate and phenylalanine are increased in patients with endometrial cancer compared to the controls. The metabolomics signature of patients with endometrial cancer is different from that of benign endometrial tissue.

Cancer-associated fibroblasts as cellular vehicles in endometrial cancer cell migration.

Cell motility is a critical step in the metastasis cascade. However, the role of cancer-associated fibroblasts (CAFs) in facilitating endometrial cancer (EC) cell motility remains unclear. The present study aimed to investigate the role of CAFs in EC motility in a 3D environment. A co-culture model was established using an EC cell line (ECC-1) and CAFs on a Matrigel® matrix and compared to the respective individual monocultures. It was demonstrated that endometrial CAFs increased the motility of the EC cell line, compared with the monoculture. Using live cell imaging, CAFs were observed to form cell projections that served as contact guidance for ECC-1 cell locomotion in the spheroid formation process. These effects were specific to CAFs, as fibroblasts isolated from benign endometrial tissue samples did not form cell projections. Molecular analysis revealed that RhoA/Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) signaling activation partly contributed to CAF-mediated ECC-1 cell migration. The presence of Matrigel® increased the mRNA expression of RhoA, and the mRNA and protein expression levels of its downstream effectors, ROCK1 and p-MLC, respectively, in the ECC-1 and CAF co-culture, as well as the ECC-1 and CAF monocultures. Interestingly, high phosphorylation levels of myosin light chain mediated the activation of RhoA/ROCK1 signaling in the ECC-1 and CAF co-culture. The ROCK1 inhibitor Y-27632 attenuated the motility of tumor cells in ECC-1 and CAF co-cultures. However, similar treatment led to a significant inhibition in the motility of the CAF monoculture, but not the ECC-1 monoculture. Moreover, tumor spheroid formation was inhibited due to a reduction in stress fiber formation in ECC-1 and CAF co-cultures. Altogether, these findings suggest that the regulation of the RhoA/ROCK1 signaling pathway is required for CAFs to serve as cellular vehicles in order for EC cells to migrate and form spheroids in a 3D environment.

A Case Study on Ayurvedic Management of Ovarian Endometrioma

Menstruation is purely a natural process occurring in a woman’s body each month. It is the visible manifestation of cyclic uterine bleeding due to shedding of endometrium. Endometriosis is a common health problem for women. It is the occurrence of benign ectopic endometrial tissues outside the uterus. Ovarian endometrioma is the most common form of endometriosis. It increases the risk of ovarian cancer in reproductive age group among woman. Although most of the endometriomas are benign, some of them may undergo malignant changes. This is the case report of a 38 year old lady who presented with increased menstrual bleeding with excessive clots, dysmenorrhoea, burning micturition, and dyspareunia. On USG she was detected to have bilateral hydrosalpinx and ovarian endometrioma. Based on the clinical features presented by the patient amapachana, kaphapittasamana and vataanulomana mode of management was given. After six months of internal medications symptoms reduced considerably and there was no endometrial cysts and features of hydrosalpinx in USG.

Abstract PO030: Understanding the influence of adiposity on the endometrium using human primary endometrial organoids

Abstract Endometrial cancer is the most commonly diagnosed type of gynecological cancer, and obesity is a major risk factor for this disease. Despite rising rates of obesity and endometrial cancer, it is still unclear how obesity alters the benign endometrium and the endometrial hormonal response to promote carcinogenesis. We used an endometrial organoid model to investigate how adiposity predisposes women to develop endometrial cancer. We hypothesized that increased adiposity would directly alter endometrial gene expression and diminish the protective progesterone response of the benign endometrium. Scaffold-free, three-dimensional endometrial organoids consisting of both epithelial and stromal cells from benign endometrial tissue were generated. Organoids were cocultured with different numbers of adipocyte spheroids to represent different levels of adiposity and were treated with a menstrual cycle level of hormones (E2 + P4 +T) over 14 days. Endometrial organoid gene expression was investigated using RNA-seq. Coculturing endometrial organoids with 30 adipocyte spheroids in the presence of E2, P4, and T caused significant differential expression of pathways involved in metabolism, angiogenesis, chromatin modification, and metal ion response, compared to endometrial organoids cultured without adipocyte spheroids. We investigated the metal ion response pathways that were altered and found that many of the most significantly differentially regulated genes in the metal ion response pathways belonged to the metallothionein (MT) family of genes. The expression of MT1A, MT1M, MT1X, and MT2A decreased approximately 50% in endometrial organoids cocultured with 30 adipocyte spheroids. Furthermore, although the results were not statistically significant, we observed a downregulation of the progesterone-responsive genes HSD17B2, IGFBP1, PAEP, PRL, FOXO1, and SPP1 in the endometrial organoids cultured with 30 adipocyte spheroids. Our results show that culturing endometrial organoids in conditions of increased adiposity alters endometrial gene expression and may change the way the endometrium responds to progesterone. Metallothioneins, which are downregulated in the presence of adipocyte spheroids, are important modulators of zinc and copper ions and reactive oxygen species in the cell. Progesterone plays an important protective role against endometrial cancer. In summary, our data show that adipocytes may influence the progesterone response of the benign endometrium. Further investigation into the role of progesterone-driven metallothioneins in the endometrium is currently underway. Citation Format: Alina R. Murphy, J. Julie Kim. Understanding the influence of adiposity on the endometrium using human primary endometrial organoids [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO030.

Adenomyosis and Coexisting Gynaecological Pathologies

Introduction: Adenomyosis is defined as presence of benign endometrial tissue comprising of both endometrial glands and stroma into the myometrium leading on to diffuse enlargement of the uterus. Adenomyosis co-exists with various pathologies such as leiomyomas, endometriosis, endometrial polyp, endometrial hyperplasia and carcinoma suggesting hyperestrogenism to be a factor in pathogenesis of adenomyosis. Aim: To study the associated pathological conditions and histopathological patterns of endometrium in patients presenting with Abnormal Uterine Bleeding (AUB) due to adenomyosis. Materials and Methods: This retrospective study was conducted in a tertiary care institute of Punjab for a period of two years (August 2018 to August 2020). All the patients who underwent hysterectomy were re-evaluated and cases diagnosed with adenomyosis were included in the study. Clinical details were recorded. A total of 101 patients were diagnosed with adenomyosis. These cases were reviewed for presenting symptoms and correlated with histological features of endometrium and other associated gynaecological pathologies. The quantitative variables were expressed as mean and qualitative variables as percentages. Results: The age of the patients who had adenomyosis ranged from 29 to 79 years, majority were in the age group of 41-50 years. AUB was the most common symptom. The histopathological examination revealed associated pattern of endometrium from proliferative endometrium to endometrial hyperplasia and endometrial carcinoma. Other gynaecological pathologies include leiomyoma, adenocarcinoma ovary, serous adenomas of ovary and Cervical Intraepithelial Neoplasia (CIN) grade 3. Co-existence with leiomyoma is most common. Hyperestrogenemia can be considered as a risk factor as it is associated with leiomyomas, endometrial hyperplasia, endometrial carcinoma or polyps. Conclusion: Adenomyosis is one of the causes of AUB and this decreases the quality of life in women. It is also considered as a cause of infertility. The associated histopathological findings vary from leiomyoma, endometrial hyperplasia, endometrial polyps and rarely adenocarcinoma of endometrium and ovary. Meticulous and careful examination of gross and microscopic foci of adenomyosis and associated pathologies can help in better management of patients.

Could Obesity be a Triggering Factor for Endometrial Tubal Metaplasia to be a Precancerous Lesion?

Background & Aims Endometrial tubal metaplasia (ETM) is mostly described in conjunction with unopposed estrogen levels, and its association with endometrial hyperplasia and endometrial carcinoma (EC) is striking. Obesity is a risk factor for endometrial hyperplasia and EC development. The aim of this study is to investigate the impact of BMI and serum estradiol level on expression of PAX-2, H-TERT, P16, Ki-67, and P53 in studied ETM in reference to benign endometrium and EC. Methods The study was conducted on the following groups: group (1) consists of 57 cases that had endometrial biopsies with histologically demonstrable ETM (typical or atypical) and all were subjected to serum estradiol levelling and body mass index (BMI) evaluation; group (2) had adjacent benign endometrial tissue as control; group (3) consists of 52 cases of conventional endometrial carcinoma and 16 serous carcinoma paraffin blocks which were collected and reevaluated. All included groups were immunostained for PAX-2, H-TERT, p16, ki67, and p53. Results The relation between BMI and serum estradiol level in group 1 and PAX-2, H-TERT, P16, and p53 was statistically significant, while their relation with atypia and ki67 expression was insignificant. Twenty-three ETM cases (40.4%) out of group 1 were all (100%) obese, 87% had high serum estradiol level, and 73.9% were postmenopausal and had a similar immunohistochemical profile as EC cases (group 3). Conclusions The presence of ETM regardless of the histologic atypia in obese postmenopausal patients with high serum estradiol level is an alarming sign. This implies that ETM might not be as benign as generally accepted, as under certain clinical conditions, it may turn into a potential premalignant lesion.

Frequent loss of mutation-specific mismatch repair protein expression in nonneoplastic endometrium of Lynch syndrome patients.

Lynch syndrome is most often caused by a germline mutation in one of four DNA mismatch repair (MMR) genes (MLH1, PMS2, MSH2, or MSH6) or EPCAM and is associated with a significantly increased risk of endometrial cancer in affected women. Although universal screening of endometrial cancer for Lynch syndrome is becoming increasingly common by various algorithms using MMR immunohistochemistry and/or microsatellite instability testing by PCR, establishing the diagnosis of Lynch syndrome can be still challenging. MMR-deficient nonneoplastic colonic crypts have been recently described in Lynch syndrome patients with colorectal carcinoma, and have been proposed to be a novel indicator of Lynch syndrome. Presence of MMR-deficient nonneoplastic endometrial glands have not yet been systematically evaluated in Lynch syndrome patients. We performed MMR protein immunohistochemistry in prophylactic hysterectomies and endometrial curettings/biopsies from 27 patients with known Lynch syndrome confirmed by germline mutation analysis. A total of 56 control benign endometrial tissues were also analyzed, and included benign endometrium adjacent to MMR-deficient sporadic (MLH1 promoter hypermethylated) endometrial carcinoma (n = 9), adjacent to MMR-intact sporadic endometrial carcinoma (n = 27), and normal endometrium from hysterectomies performed for benign disease (n = 20). MMR protein deficient nonneoplastic endometrial glands were identified in 70% (19 of 27) of Lynch syndrome patients. In all 19 cases the MMR protein loss was specific for the patients' known germline mutation. None of the control cases showed loss of MMR protein expression in nonneoplastic endometrium. Our findings suggest that MMR-deficient nonneoplastic endometrial glands may be a unique, specific marker of Lynch syndrome, and may provide an important insight into the pathogenesis of Lynch syndrome-associated endometrial cancer. Evaluation of MMR protein expression of benign background endometrium in endometrial cancer patients may be further explored as a possible useful addition to the Lynch syndrome screening algorithm.

Proteomic analysis of malignant and benign endometrium according to obesity and insulin-resistance status using Reverse Phase Protein Array

Obesity and hyperinsulinemia are known risk factors for endometrial cancer, yet the biological pathways underlying this relationship are incompletely understood. This study investigated protein expression in endometrial cancer and benign tissue and its correlation with obesity and insulin resistance. One hundred and seven women undergoing hysterectomy for endometrial cancer or benign conditions provided a fasting blood sample and endometrial tissue. We performed proteomic expression according to body mass index, insulin resistance, and serum marker levels. We used linear regression and independent t test for statistical analysis. Proteomic data from 560 endometrial cancer cases from The Cancer Genome Atlas (TCGA) databank were used to assess reproducibility of results. One hundred and twenty seven proteins were significantly differentially expressed between 66 cancer and 26 benign patients. Protein expression involved in cell cycle progression, impacting cytoskeletal dynamics (PAK1) and cell survival (Rab 25), were most significantly altered. Obese women with cancer had increased PRAS40_pT246; a downstream marker of increased PI3K-AKT signaling. Obese women without cancer had increased mitogenic and antiapoptotic signaling by way of upregulation of Mcl-1, DUSP4, and Insulin Receptor-b. This exploratory study identified a number of candidate proteins specific to endometrioid endometrial cancer and benign endometrial tissues. Obesity and insulin resistance in women with benign endometrium leads to specific upregulation of proteins involved in insulin and driver oncogenic signaling pathways such as the PI3K-AKT-mTOR and growth factor signaling pathways which are mitogenic and also disruptive to metabolism.

Nicotinamide N-methyltransferase expression and its association with phospho-Akt, p53 expression, and survival in high-grade endometrial cancer

Background/aimNicotinamide N-methyltransferase (NNMT) is an enzyme that is overexpressed in malignancies. NNMT expression has not been previously studied in endometrial cancer (EC). Increased phospho-Akt (pAkt) levels in response to NNMT overexpression have been reported in in vitro studies of different cancer types. We assayed NNMT expression in primary and metastatic high-grade EC and investigated the relationship of NNMT with p53, pAkt, and survival.Materials and methodsNNMT, pAkt, and p53 expressions were assayed in 100 tissue samples of benign endometria, primary EC, and metastatic EC by immunohistochemistry.ResultsThe NNMT immunoreactivity score was significantly higher in primary high-grade EC than benign endometrial tissue (P = 0.001). NNMT expression in metastatic tissue was significantly higher than in primary cancer (P < 0.001). Metastatic stromal NNMT expression was significantly higher than that of the adjacent tumor and stroma adjacent to the primary tumor. p53 expression in the primary tumor showed a significant positive correlation with omental NNMT and pAkt expression. NNMT expression was also correlated with pAkt expression in metastatic tissue. NNMT overexpression in metastatic tissue was associated with decreased survival (P = 0.039).ConclusionThis study suggests that NNMT may promote cancer progression and that NNMT overexpression is associated with aberrant p53 expression, pAkt, and poor survival. NNMT’s role in cancer progression could make it a target of EC therapy.

Expression of CD44 variant 6 and its prognostic value in benign and malignant endometrial tissue.

CD44 expression in both the early and metastatic phases of many epithelial and non-epithelial cancers is strongly prognostic. The objective of the study is to evaluate whether there is any relationship between the expression of CDD44v6 and endometrial cancer (EC) staging and prognosis. This retrospective study included 60 EC patients for whom surgical staging was performed between 2000 and 2006. Twenty-eight randomly selected patients with normal endometria served as the control group. We immunohistochemically evaluated membranous and cytoplasmic CD44v6 staining in tissue paraffin blocks. The results were graded as positive or negative. Membranous staining in both advanced and early stage EC patients was significantly higher than that in the control group (p=0.002). The extent of either membranous or cytoplasmic staining in both advanced- and early stage patients did not differ significantly by age, tumor grade, stage, extent of myometrial invasion, lymph node involvement, cytology, adnexal involvement, or omental spreading. In advanced-stage patients, neither papillary serous not clear cell cancers exhibited cytoplasmic staining. CD44v6 membranous staining can be useful for differentiating malignant from benign endometrial tissue. However, staining is not associated with EC staging or prognosis.

GLUT-1 Expression in Proliferative Endometrium, Endometrial Hyperplasia, Endometrial Adenocarcinoma and the Relationship Between GLUT-1 Expression and Prognostic Parameters in Endometrial Adenocarcinoma.

Malignant cells show increased glucose uptake in in vitro and in vivo studies. This uptake is mediated by glucose transporter proteins. GLUT-1 is the most common transporter protein, and its expression is reported to be increase in many human cancers. The aim of this study is to determine the GLUT-1 overexpression in benign, hyperplastic, and malignant endometrial tissues, to evaluate the usefulness of GLUT-1 expression in endometrial hyperplasia, and to determine its role in the neoplastic progression to endometrioid type adenocarcinoma. We also aimed to analyze prognostic clinical parameters, predict prognosis, and survival. We examined immunohistochemical expression of GLUT-1 in 91 cases of endometrial hyperplasia, 100 cases of endometrioid type adenocarcinoma, and 10 proliferative endometrial tissues. The percentage of positive cells and staining intensity were assessed in a semi quantitative fashion and scored (1+ to 3+). GLUT-1 immunoreactivity was not present in proliferative endometrium. Twenty-nine (31.9%) of 91 endometrial hyperplasia cases showed positive immunoreactivity, of which only six were cases of hyperplasia without atypia while 23 of them were cases with atypia. We found GLUT-1 positivity of 95% in endometrioid type adenocarcinoma. GLUT-1 overexpression was not significantly correlated with any of the clinicopathological parameters except histological grade in endometrioid adenocarcinoma; the survival was not found to be correlated with GLUT-1 expression. GLUT-1 immunostaining may be useful in distinguishing hyperplasia without atypia from hyperplasia with atypia; GLUT-1 overexpression is a consistent feature of endometrioid adenocarcinoma. A correlation between GLUT -1 expression and tumor grade has been found, although other prognostic parameters and survival has no meaningful correlation.

PTEN expression in benign human endometrial tissue and cancer in relation to endometrial cancer risk factors.

Clonal loss of PTEN expression occurs frequently in endometrial carcinoma and endometrial hyperplasia. Limited data from immunohistochemical studies suggest that PTEN-null appearing endometrial glands are detectable in women without pathologic abnormalities, but the relationship of PTEN expression to endometrial cancer risk factors has not been extensively explored. We evaluated relationships between endometrial cancer risk factors and loss of PTEN expression in a set of benign endometrial samples prospectively collected from women undergoing hysterectomy and in endometrial cancer tissues from a population-based case-control study. We used a validated PTEN immunohistochemical assay to assess expression in epidemiological studies designed to assess benign endometrium [Benign Reproductive Tissue Evaluation Study (n = 73); Einstein Endometrium Study (n = 19)], and endometrial cancer [Polish Endometrial Cancer Study (n = 148)] tissues. Associations between endometrial cancer risk factors (collected via study-specific risk factor questionnaires) and PTEN expression in endometrial tissues were determined using Fisher's exact tests. PTEN loss was detected in 19% of benign endometrial tissues versus 55% in endometrial cancers. NSAID use was statistically significantly associated with PTEN loss in the benign endometrium (p = 0.02). Our data demonstrate that PTEN loss is detectable in endometrial tissues that are benign and malignant, with substantially more frequent loss in endometrial cancer compared with benign endometrium. However, alterations in expression were unrelated to most risk factors in this analysis, except for the association with NSAID use, which may represent a chance finding or reverse causality among patients with endometriosis who may have PTEN pathway abnormalities in eutopic endometrium. Further evaluation of factors associated with PTEN loss and long-term follow-up of women with PTEN-null endometrial glands may be useful in understanding early events in endometrial carcinogenesis.