Endometrial Carcinoma Samples Research Articles

Lack of Estrogen Receptor-α Is Associated with Epithelial–Mesenchymal Transition and PI3K Alterations in Endometrial Carcinoma

We hypothesized that estrogen receptor-α (ER-α) status in endometrial carcinomas, associated with poor prognosis, is reflected in transcriptional signatures suggesting targets for new therapy. Endometrial carcinoma samples in a primary investigation cohort (n = 76) and three independent validation cohorts (n = 155/286/111) were analyzed through integrated molecular profiling. Biomarkers were assessed by immunohistochemistry (IHC), DNA oligonucleotide microarray, quantitative PCR (qPCR), single-nucleotide polymorphism (SNP) array, and Sanger sequencing in the cohorts, annotated for comprehensive histopathologic and clinical data, including follow-up. ER-α immunohistochemical staining was strongly associated with mRNA expression of the receptor gene (ESR1) and patient survival (both P < 0.001). ER-α negativity associated with activation of genes involved in Wnt-, Sonic Hedgehog-, and TGF-β signaling in the investigation cohort, indicating epithelial-mesenchymal transition (EMT). The association between low ER-α and EMT was validated in three independent datasets. Furthermore, phosphoinositide 3-kinase (PI3K) and mTOR inhibitors were among the top-ranked drug signatures negatively correlated with the ER-α-negative tumors. Low ER-α was significantly associated with PIK3CA amplifications but not mutations. Also, low ER-α was correlated to high expression of Stathmin, a marker associated with PTEN loss, and a high PI3K activation signature. Lack of ER-α in endometrial cancer is associated with EMT and reduced survival. We present a rationale for investigating ER-α's potential to predict response to PI3K/mTOR inhibitors in clinical trials and also suggest EMT inhibitors to ER-α-negative endometrial carcinomas.

Aberrant Expression of Hypoxia-inducible Factor 1 , TWIST and E-cadherin is Associated with Aggressive Tumor Phenotypes in Endometrioid Endometrial Carcinoma

Intratumoral hypoxia promotes angiogenesis, invasion and epithelial-mesenchymal transition, a pivotal event in tumor metastasis. TWIST is a master regulator of multiple developmental processes and has recently been shown to be the key factor responsible for cancer metastasis via the inhibition of E-cadherin expression, a hallmark of epithelial-mesenchymal transition. This study aimed to determine the expression of hypoxia-inducible factor 1α, TWIST and E-cadherin in patients with endometrioid endometrial carcinoma and to examine their clinical significance in endometrioid endometrial carcinoma progression. Using immunohistochemical and tissue microarray approaches, we evaluated the expression of hypoxia-inducible factor 1α, TWIST and E-cadherin in normal endometrial (n = 35), atypical hyperplasia (n = 28) and endometrioid endometrial carcinoma samples (n = 124). Furthermore, we statistically analyzed the association between these markers, as well as their correlation with clinicopathologic variables. The expression of hypoxia-inducible factor 1α and TWIST were markedly increased, whereas E-cadherin was decreased, as lesions progressed from normal endometrium to atypical hyperplasia to carcinoma (P < 0.01). Among various clinical parameters, the expression of hypoxia-inducible factor 1α and TWIST was strikingly elevated with aggressive tumor characteristics, including higher pathologic grade, deep myometrial invasion and lymph node involvement (P < 0.05). More importantly, overexpression of hypoxia-inducible factor 1α positively correlated with enhanced TWIST expression in endometrioid endometrial carcinoma samples (r = 0.249, P < 0.01); however, statistical analysis showed a negative relationship between TWIST upregulation and E-cadherin downregulation (r = -0.183, P = 0.042). These results demonstrated for the first time that the hypoxia-inducible factor 1α/TWIST/E-cadherin pathway may play a critical role in invasion and metastasis of endometrioid endometrial carcinoma. The combined evaluation of these markers may be useful in predicting aggressive phenotypes and thus prognosis in patients with endometrioid endometrial carcinoma.

Expression of E-cadherin and β1-integrin mRNA in endometrial cancer

The metastatic ability of tumors is characteristic for malignant neoplasms and constitutes the main cause of therapeutics failures. Metastasis formation involves the sequence of processes such as proteolytic degradation of the basement membrane, migration, intravasation, extravasation, proliferation and angiogenesis. Cadherins and integrins are groups of proteins directly involved in these processes. In the present study we analyzed the mRNA expression of CDH1 and ITGB1 genes by real-time polymerase chain reaction (RT-PCR). The study included 106 endometrial carcinomas. CDH1 and ITGB1 mRNA expression was found in all of the studied samples. Generally the CDH1 and ITGB1 mRNA expression was significantly higher in well-differentiated rather than poorly differentiated tumors. The mRNA expression levels of CDH1 and ITGB1 in series of 83 samples of endometrial carcinoma were studied by real time RT-PCR method. Statistical analysis of the obtained results was performed. CDH1 and ITGB1 gene expression was observed in all examined tissues and was correlated with cancer malignancy (G). In high grade malignant tumors (G1), CDH1 and ITGB1 gene expression was the highest, in G2 and G3 tumors the expression of both genes was gradually lowering. Moreover the statistically significant correlation between CDH1 and ITGB1 gene expression was observed. (Spearman test, r=0.29, p<O.01). CDH1 and ITGB1 mRNA expression seems to be an important marker of cancer progression and metastases in endometrial malignant tumors.

Matrix metalloproteinase (MMP)-2 and MMP-9 Expression in Tumor Infiltrating CD3 Lymphocytes From Women With Endometrial Cancer

In this study, we hypothesized that not only endothelial malignant cells but also lymphocytes infiltrating tumor epithelium, in patients with endometrial cancer, could be an important source of the gelatinases (matrix metalloproteinase [MMP]-2 and MMP-9) extensive production, which in turn, may facilitate tumor cells infiltration and progression due to the extracellular matrix degradation. First, we isolated lymphocytes from the endometrial carcinoma samples taken from 41 patients who were operated on and from healthy endometrial tissue taken of the same patients after histological verification. Then, we detected the level of CD3-positive cells in endometrial tissues by flow cytometry. Simultaneously, we studied the messenger RNA expression of MMP-2 and MMP-9 in the isolated cells from malignant and unchanged endometrial tissues. Using immunohistochemistry, we compared the protein expression of MMP-2, MMP-9, and CD3 in the studied samples. We showed the enhanced abundance of CD3 lymphocytes both by flow cytometry and immunohistochemistry in the samples from malignant tissues. The expression of MMP-9 in the endometrial carcinoma was increased significantly at the protein level but not at the messenger RNA level. We could not observe any differences concerning MMP-2 expression in both methods of detection. CD-3 lymphocytes significantly infiltrate endometrial cancer tissue, but they do not seem to be the source of enhanced metalloproteinases 2 and 9 expression in the tumor environment. Still, owing to the immunohistochemistry staining, we could show the significant increase of MMP-9 protein in the very close vicinity of tumor-infiltrating CD3 lymphocytes. Could it be the result of CD3 lymphocyte action, or is it just the imperfection of the detecting method we used? This remains unclear. Further studies explaining the role of tumor infiltrating lymphocytes in mediating the endometrial cancer milieu are needed.

FGFR2 alterations in endometrial carcinoma

Fibroblast growth factor receptor 2 (FGFR2) is a tyrosine kinase receptor involved in many biological processes such as embryogenesis, adult tissue homeostasis and cell proliferation. Mutations in FGFR2 have been reported in up to 10–12% of endometrial carcinomas identical to those found in congenital craniofacial disorders. Inhibition of FGFR2 could be a new therapeutic target in endometrial carcinoma. FGFR2 immunostaining was assessed in three tissue microarrays: one constructed from paraffin-embedded blocks of 60 samples of normal endometrium in different phases of menstrual cycle, and two tissue microarrays containing endometrial carcinoma samples (95 and 62 cases). FGFR2 expression was correlated with stage, histological type and grade as well as with immunostaining of PTEN, RASSF1A, estrogen and progesterone receptors, KI67, Cyclin D1, STAT-3 and SPRY2. FGFR2 mutations were assessed by PCR and direct sequencing, with DNA obtained from 31 paraffin-embedded endometrial carcinoma samples. In normal endometrium, FGFR2 expression was higher in the secretory than in the proliferative phase (P=0.001), with an inverse correlation with Ki67 (P=0.00032), suggesting a tumor-suppressor role for FGFR2 in normal endometrium. Cytoplasmic expression of FGFR2 was higher in endometrial carcinoma when compared with the atrophic endometrium from the same patients (P=0.0283), but was lower in comparison with normal endometrium from women in the menstrual cycle. Interestingly, nuclear staining was observed in some cases, and it was less frequent in endometrial carcinoma when compared with the adjacent atrophic endometrium (P=0.0465). There were no statistical differences when comparing superficial and myoinvasive endometrial carcinoma samples. Endometrioid endometrial carcinomas showed higher expression of FGFR2 than nonendometrioid endometrial carcinomas (fold change 2.56; P=0.0015). Grade III endometrioid endometrial carcinomas showed decreased FGFR2 expression when compared with grade II endometrioid endometrial carcinomas (P=0.0055). No differences were found regarding pathological stage. Two missense mutations of FGFR2 gene were detected in exons 6 and 11 (S252W and N549K, respectively; 6.45%). Results support the hypothesis that FGFR2 has a dual role in the endometrium, by inhibiting cell proliferation in normal endometrium during the menstrual cycle, but acting as an oncogene in endometrial carcinoma.

Tumor Expression of Human Growth Hormone and Human Prolactin Predict a Worse Survival Outcome in Patients with Mammary or Endometrial Carcinoma

Evidence suggests that human GH (hGH) and human prolactin (hPRL) possess an autocrine or paracrine oncogenic role in mammary and endometrial carcinoma. However, especially for hGH, the prognostic relevance of tumor expression of these hormones is not well defined. We investigated the potential association of tumor mRNA and protein expression of hGH and hPRL with the clinicopathological features of mammary and endometrial carcinoma. The prognostic relevance of the individual or combined expression of hGH and hPRL in mammary and endometrial carcinoma was also determined. The expression of hGH and hPRL was analyzed in histopathological samples of mammary and endometrial carcinoma, and the respective normal tissues, by in situ hybridization and immunohistochemistry. Kaplan-Meier and Cox regression analysis was performed to examine the association of tumor hGH and hPRL expression with relapse-free survival and overall survival of patients. hGH expression was significantly associated with lymph node metastasis, tumor stage, human epidermal growth factor receptor-2 status, and proliferative index in mammary carcinoma and with International Federation of Gynecology and Obstetrics grade, myometrial invasion, and ovarian metastases in endometrial carcinoma. hPRL expression was associated with lymph node metastasis, tumor grade, and tumor stage in mammary carcinoma and with International Federation of Gynecology and Obstetrics stage and myometrial invasion in endometrial carcinoma. Both hGH and hPRL expression, individually and combined, are associated with worse relapse-free survival and overall survival in patients with mammary or endometrial carcinoma. Tumor expression of both hGH or hPRL in mammary or endometrial carcinoma is associated with a large and significant difference in survival outcome for patients with these tumors.

A Robust Immunohistochemical Assay for Detecting PTEN Expression in Human Tumors

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of the phosphoinositol-3-kinase (PI3K)/AKT signaling pathway that controls cell cycle progression, growth and inhibition of apoptosis. Loss of PTEN protein expression has been associated with tumorigenesis, cancer progression and drug resistance, but conflicting results exist which may be due in part to difficulties inherent in PTEN immunohistochemistry (IHC). We sought a robust PTEN IHC assay. Human tumor cell lines with PTEN status verified by copy number analysis were formalin fixed and paraffin embedded for use as positive and negative controls. PTEN antibodies were optimized on tumor cell lines. Five optimized antibodies were analyzed on 10 molecularly characterized endometrial carcinoma samples. Four antibodies (CST, Millipore, Abcam, Novus) stained 3/10 positive and 7/10 negative, however, all but CST exhibited nonspecific nucleolar staining of negative controls. One antibody (Dako) stained 5/10 positive and 5/10 negative but with areas (≤10%) of positivity. The 4 samples predicted to be negative by sequencing were negative with the CST antibody, however, one was positive with Dako; as a result we chose the CST antibody for our assay. The assay was validated on an automated platform using 50 formalin fixed and paraffin embedded colon, lung, prostate and breast adenocarcinoma cases. Tumor cell lines served as external controls; endothelial cells and peripheral nerves served as internal positive controls. Dichotomous scoring achieved 100% concordance between three independent pathologists. This reproducible PTEN assay (PREZEON) has been implemented in a CLIA certified laboratory.

Promoter hypermethylation and expression of sprouty 2 in endometrial carcinoma

Sprouty 2 is a key antagonist regulator of receptor tyrosine kinases, and downstream signaling pathways, like fibroblastic growth factor (FGF) and Ras-mitogen-activated protein kinase (RAS-MAPK). By controlling these pathways, sprouty 2 is involved in regulation of cell proliferation, differentiation, and angiogenesis. Alterations in fibroblastic growth factor receptor (FGFR) and members of the RAS-MAPK pathway are frequent in endometrial carcinoma. The expression of sprouty 2 has been found to be decreased in several types of human cancer, by mechanisms of promoter methylation. In the present study, we have assessed the expression of sprouty 2 in endometrial carcinoma, in correlation with sprouty 2 promoter methylation. Sprouty 2 immunohistochemical expression was assessed using 3 different tissue microarrays: one constructed from paraffin blocks of 80 samples of normal endometrium and 2 tissue microarrays containing samples of 157 endometrial carcinoma (1 tissue microarray constructed with 95 endometrial carcinomas previously studied for microsatellite instability and alterations in phosphatase and tensin homolog (PTEN), k-ras, and b-catenin, and 1 tissue microarray containing 62 endometrial carcinoma, which were also subjected to sprouty 2 promoter methylation analysis). The immunohistochemical expression of sprouty 2 was correlated with cellular proliferation (Ki67) and clinicopathologic data. Sprouty 2 promoter methylation was assessed by methylation-specific polymerase chain reaction, with DNA obtained from fresh-frozen samples of endometrial carcinoma and corresponding normal tissues, and correlated with promoter methylation of RAS association domain family-1A (RASSF1A). A highly significant decrease in sprouty 2 immunoexpression was seen in the proliferative phase of normal endometrium (P < .001). Differences were detected between types I and II endometrial carcinoma, but they were not statistically significant. Reduced immunoexpression of sprouty 2 was seen in 19.85% of endometrial carcinoma and was strongly and inversely associated with increased cell proliferation (Ki67; r = -0.367; P = .001). Sprouty 2 promoter methylation was detected in 31 (53.4%) of 58 endometrial carcinomas. Results from our study show that alterations in sprouty 2 may be involved in endometrial carcinogenesis by controlling cell proliferation.

Inhibin/activin-betaC subunit does not represent a prognostic parameter in human endometrial cancer

Inhibins, dimeric peptide hormones composed of an α subunit and one of two possible β subunits (betaA or betaB), exhibit substantial roles in human reproduction and in endocrine-responsive tumors. Recently, two novel inhibin-beta subunits, defined as betaC and betaE, have been identified in humans. However, the prognostic significance and clinical implications of the novel inhibin-betaC subunit in endometrial cancers is still quite unclear. A series of 296 uterine endometrial carcinomas were immunohistochemically analyzed with specific antibody against the inhibin-betaC subunit. The staining reactions were correlated with several clinicopathological characteristics and the clinical outcome. Endometrial cancer tissue demonstrated an immunolabelling against the inhibin-betaC subunit. The inhibin-betaC expression in endometrial carcinoma samples revealed a significant association with hemangiosis. However, the expression of this inhibin subunit did not affect patients' progression-free, cause-specific and overall survival. Overall, inhibin-betaC subunit was demonstrated in endometrial cancer tissue. This novel betaC subunit demonstrated a significant association with hemangiosis although without any impact on the patients' survival. Moreover, the inhibin-betaC subunits did not constitute an independent prognostic parameter in endometrial cancer patients. Therefore, the isolated analysis of this subunit might be of minor prognostic value in identifying high-risk patients.

Identification of a 0.4 Kb deletion region in 10q26 associated with endometrial carcinoma

We have identified an allelic deletion common region in the q26 region of chromosome 10 in endometrial carcinomas, which has been reported previously as a potential target of genetic alterations related to this neoplasia. An allelotyping analysis of 19 pairs of tumoral and non-tumoral samples was accomplished using seven microsatellite polymorphic markers mapping in the 10q26 chromosomal region. Loss of heterozygosity for one or more loci was detected in 29% of the endometrial carcinoma samples. The observed pattern of loss enabled the identification of a 3.5 Mb common deleted region located between the D10S587 and D10S186 markers. An additional result from an endometrial sample with evidence of a RER phenotype may suggest a more centromeric region of loss within the above-mentioned interval. This 401.84 Kb interval flanked by the D10S587 and D10S216 markers may be a plausible location for a putative suppressor gene involved in early stage endometrial carcinogenesis.

DcR1 expression in endometrial carcinomas

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family, which mediates apoptosis by the extrinsic pathway. Up-regulation of decoy receptors, DcR1 and DcR2, may result in diminished binding of TRAIL to their functional receptors. DcR1 expression was assessed in normal endometrial tissue (NE) and endometrial carcinoma (EC) samples by immunohistochemistry (IHC) and quantitative real-time polymerase chain reaction (PCR). IHC was performed in two tissue microarrays; one composed of 80 samples of NE and a second one constructed from paraffin-embedded blocks of 62 EC. For quantitative real-time RT-PCR analysis, RNA was obtained from 19 NE and 28 EC samples using Trizol. mRNA expression of DcR1 was assessed with Taqman-based assays in an Abi-Prism 700 SDS. Results were correlated with stage, histological type, and grade. By IHC, cytoplasmic expression of DcR1 was frequently seen in NE (79.6%) and varied according to the menstrual cycle. Positive DcR1 immunostaining was also detected in EC (98.1% of the cases) without any specific statistical association with histological type, grade, and stage. By quantitative real-time PCR, all NE had similar levels of DcR1expression (0.8-1.7 RQ), which were considered the basal levels of DcR1 expression in NE. Increased DcR1 expression (> or =5-fold higher than the basal levels) was detected in 13 of 28 EC (46.4%). High DcR1 expression levels were found in ECs of different stages: IA, four of 12 (33%); IB, two of four (50%); IC, four of six (66%); and IIA and IIB three of six (50%). Results suggest that DcR1 expression occurs in a subset of EC and may contribute to resistance to TRAIL-induced apoptosis.

Evidence for Cancer Stem Cells in Human Endometrial Carcinoma

Emerging evidence indicates that the highly regenerative human endometrium harbors rare populations of epithelial progenitor cells. In tumors of other regenerative epithelial tissues, rare cancer stem cells (CSC) have been identified that may have originated from normal epithelial stem/progenitor cells. We hypothesized that CSC are responsible for epithelial neoplasia associated with endometrial carcinoma, the most common gynecologic malignancy in women. Stem cell characteristics of single cells isolated from endometrial carcinoma tissues from women ages 62 +/- 11.8 years (n = 34) were assessed. Twenty-five of 28 endometrial carcinoma samples contained a small population of clonogenic cells [0.24% (0-1.84%)], with no significant difference in cloning efficiency between the three grades of endometrial carcinoma or between endometrial carcinoma and normal endometrial epithelial samples. Isolated endometrial carcinoma cells transplanted under the kidney capsule of immunocompromised mice in serial dilution (2 x 10(6)-1 x 10(4) cells) generated tumors in 8 of 9 samples with morphologies similar to the parent tumors. These tumors recapitulated cytokeratin, vimentin, estrogen receptor-alpha, and progesterone receptor expression of the parent tumor, indicating that tumor-initiating cells likely differentiated into cells comprising the endometrial carcinoma tissue. Individual clones underwent serial clonal subculture 2.5 to 4 times, with a trend of increasing number of subclonings with increasing tumor grade, indicating increasing self-renewal with greater malignancy. Clonally derived endometrial carcinoma cells also expressed the self-renewal genes BMI-1, NANOG, and SOX-2. Isolated cells from primary tumors were serially transplanted 3 to 5 times in nonobese diabetic/severe combined immunodeficient mice, showing self-renewal in vivo. This evidence of cells with clonogenic, self-renewing, differentiating, and tumorigenic properties suggests that a CSC population may be responsible for production of endometrial carcinoma tumor cells.

EphA2 overexpression is associated with lack of hormone receptor expression and poor outcome in endometrial cancer

EphA2 is a tyrosine kinase receptor in the ephrin family that is implicated in oncogenesis and angiogenesis. The objective of the current investigation was to study the role of EphA2 in endometrial cancer and its relation to steroid hormone receptor expression. EphA2, estrogen receptor (ER), progesterone receptor (PR), and Ki-67 expression levels were evaluated using immunohistochemistry in 139 endometrioid endometrial carcinoma (EEC) samples and in 10 benign endometrial samples. Samples were scored by 2 investigators who were blinded to clinical outcome. The results were correlated with clinicopathologic characteristics using univariate and multivariate analysis. A P value <.05 was considered statistically significant. High expression of EphA2 was detected in 48% of EEC samples versus 10% of benign samples. EphA2 overexpression was associated significantly with high disease stage (P = .04), high tumor grade (P = .003), increased depth of myometrial invasion (P = .05), low ER expression (P = .01), low PR expression (P = .006), and high Ki-67 expression (P = .04). Low ER and PR expression levels were associated with high tumor grade, positive lymph nodes, high Ki-67 expression, and high EphA2 expression. On univariate analysis of all patients, high EphA2 expression was associated significantly with shorter disease-specific survival (DSS) (P < .001). On multivariate analysis, age (P < .001), high disease stage (P = .002), and high EphA2 expression (P = .04) were independent predictors of poor DSS. EphA2 overexpression was associated with aggressive phenotypic features in EEC and was associated inversely with ER and PR expression. Thus, EphA2 may be an important therapeutic target, especially in patients with hormone receptor-negative endometrial carcinoma.

Epigenetic Alteration of the Metallothionein 1E Gene in Human Endometrial Carcinomas

Aberrant expression of metallothioneins (MTs) has been observed in several human tumors. In our microarray analysis, MT-1E was found to have much lower expression in endometrial cancer cells as compared with other types of cancer cells generated from the cervix, ovary or prostate. The result was confirmed by quantitative RT-PCR analysis of the MT-1E levels in individual cancer cells. Treatment of endometrial cancer cells with 5-azacytidine could reactivate MT-1E expression. We further analyzed the DNA methylation status of the promoter region of MT-1E using methylation-sensitive restriction enzymes HhaI and HpaII, followed by PCR. Promoter hypermethylation was detected in 42.4% (53/125) of the endometrial carcinoma samples, whilst none of the 38 normal tissues or hyperplasia samples were methylated. The mRNA levels of MT-1E were significantly lower in the methylation-positive than in the methylation-negative samples. Endometrial carcinoma samples with low MT-1E expression coincidently had low levels of estrogen receptor-α expression and vice versa. This phenomenon was not observed in the expression pattern between estrogen receptor-β and MT-1E. There was no significant correlation between MT-1E methylation and any clinical parameters. In conclusion, a high frequency of cancer-specific hypermethylation of MT-1E was found in endometrial carcinomas. Its functional consequence in the development of endometrial cancer warrants further investigation.

CK2β Is Expressed in Endometrial Carcinoma and Has a Role in Apoptosis Resistance and Cell Proliferation

Protein kinase CK2 (CK2) is a serine/threonine kinase that participates in important cellular processes. We have recently demonstrated that CK2 plays a role in resistance to TRAIL/Fas-induced apoptosis in endometrial carcinoma (EC) by regulating FLIP. Here, we assessed the immunohistochemical expression of CK2beta in EC and checked its role in cell proliferation and anchorage-independent cell growth. CK2beta immunostaining was assessed in two tissue microarrays, one constructed from paraffin-embedded blocks of 95 ECs and another from 70 samples of normal endometrium. CK2beta expression was correlated with histological type; grade and stage; cell proliferation (Ki-67) and apoptotic index; immunostaining for cyclin D1, PTEN, AKT, beta-catenin, and FLIP. Moreover, the Ishikawa EC cell line was subjected to down-regulation of CK2 by shRNA. CK2beta expression was frequent in EC (nuclear, 100%; cytoplasmic, 87.5%). The staining was more intense in EC than in normal endometrium (P = 0.000), and statistically correlated with AKT, PTEN, beta-catenin, and FLIP. In EC, CK2beta expression correlated with cell proliferation. Knock-down of CK2beta blocked colony formation of EC in soft agar, and also resulted in decreased expression of cyclin D1 and ERK phosphorylation. The results confirm that CK2beta is widely expressed in EC, and suggest a role in cell proliferation and anchorage-independent cell growth.

Midkine and its clinical significance in endometrial carcinoma

Midkine (MK) is a secreted heparin-binding growth factor. Several types of human cancer have increased MK expression with elevated serum levels. The purpose of this study was to determine whether MK was expressed in endometrial carcinoma and to evaluate the clinicopathological significance of serum MK in patients with endometrial carcinoma. Immunohistochemical expression of MK was evaluated in 85 endometrial carcinoma samples and 33 controls. MK expression was significantly higher in the carcinomas than in normal endometrium (P < 0.001). Interestingly, MK expression was highest at the margins of invasion and low in the superficial areas of the tumor samples. Using ELISA, we compared serum MK concentration in 120 endometrial carcinoma patients with the concentration in 46 patients with benign gynecologic tumors. Serum MK value in patients with cancer was significantly higher than that in the patients with benign diseases (P = 0.01). Patients with positive lymph node metastasis or recurrence, or cancer death, had a higher serum MK level (P = 0.008, P = 0.009, respectively). In conclusion, MK immunoreactivity in endometrial carcinoma is significantly higher than in normal endometrium. Additionally, preoperative serum MK levels are significantly correlated with prognosis and the presence of lymph node metastasis. Thus, MK may be a useful serum biomarker for identifying high risk patients of endometrial carcinoma.

Total tissue lactate dehydrogenase activity in endometrial carcinoma

Lactate dehydrogenase (LDH) is essential for continuous glycolysis necessary for accelerated tumor growth. The aim of this study was to reconsider if assay of total tissue activity of this enzyme could be useful as marker for endometrial carcinoma (EC). Activity of LDH was measured spectrophotometrically in homogenate supernatants of uterine tissue samples of 40 patients (10 normal endometria, 27 normal myometria, and 33 EC), including 30 matched pairs. Data obtained were analyzed in relation to clinical and histopathologic findings and compared with our previously published results on the tissue levels of the same enzyme in ovarian cancer and on the proteolytic activity of dipeptidyl peptidase III (DPP III) in EC (suggested biochemical indicator of this malignancy). Significantly increased (1.8-3.0 times; P < 1 x 10(-4)) LDH activity was observed in EC samples if compared with normal uterine tissues. This rise was not related to the clinicopathologic findings, however. In contrast to previous results on LDH in ovarian carcinomas, a significant rise in LDH activity was found already in grade 1 EC. Using the cutoff value of 1.06 U/mg, diagnostic sensitivity of 82%, specificity of 100%, and accuracy of 91% for total tissue LDH assay have been calculated. A correlation of tissue's LDH and DPP III activities was found, and their combined assay for EC showed increased diagnostic sensitivity (94%) and accuracy (96%).

417. In vivo and in vitro evidence for cancer stem cells in human endometrial cancer

Cancer stem cells (CSCs) have been identified in solid human cancers, including breast, colon, and ovary. Recent evidence suggests that the highly regenerative human endometrium harbors rare populations of epithelial stem/progenitor cells1. We hypothesised that CSCs are responsible for the epithelial neoplasia associated with endometrial carcinoma (EC), the most common gynaecological malignancy in women. The aim of this study was to demonstrate that a rare population of EC cells posses CSC properties. Stem cell characteristics were assessed in 25 EC and 2 endometrial hyperplasia tissues obtained from women aged 62 ± 9 yrs. Samples were cultured at clonal densities (100–500 cells/cm2) for 3–5 wks to determine cloning efficiency. Individual clones were serially subcloned (&lt;10 cells/cm2) every 2–4 wks to determine self renewal capacity. Isolated cells in serial dilution (103–106 cells) were placed under the kidney capsule of immunocompromised mice for 12–16 wks to examine for the presence of tumour initiating cells (TIC). Resulting tumours and original parent tumours were examined for markers by immunohistochemistry. Most samples (23/26) contained rare colony forming cells. The cloning efficiency was 0.23% ± 0.28% (n = 11) in G1, 0.78% ± 0.67% (n = 8) in G2, 0.22% ± 0.21% (n = 3) in G3, 0.03% (n = 2) in type II tumours, and 0.14% (n = 2) in hyperplasia samples, and did not differ significantly between grades or between type I EC and normal endometrial epithelial samples 1. Single cell derived clones subcloned 2.5 ± 1.4 (n = 11), 3.2 ± 0.4 (n = 5), 3.5 (n = 2), 3.0 ± 1.7 (n = 3), and 2.5 (n = 2) times in G1, G2, G3, type II tumours and hyperplasia samples respectively, indicating increasing self renewal capacity with increasing tumour grade. Transplanted EC single cell suspensions initiated tumour growth with similar morphology, ERα, PR, EpCAM, cytokeratin, and vimentin expression as the parent tumour, indicating the presence of TIC. This evidence suggests that rare cells possessing the CSC properties of clonogenicty, self renewal, and tumorigenicity, may be responsible for the initiation and progression of EC. (1) Chan RWS et al. (2004). Biology of Reproduction. 70:1738–1750

Expression of metalloproteinases 2 and 9 and cyclooxygenase 2 in endometrial carcinoma

0020-7292/$ see front matter D 2006 International Federation of All rights reserved. doi:10.1016/j.ijgo.2005.12.028 * Corresponding author. Tel.: +39 055 4478138; fax: +39 055 4379868. E-mail address: mariarosaria.raspollini@unifi.it (M.R. Raspollini). and metastasis [1] while cyclo-oxygenase (COX)-2 expression has been related to immunomodulation [2] and neoangiogenesis [3]. The aim of this study was to investigate the roles of MMPs and their correlation with COX-2 expression in endometrial carcinoma. mRNA expression of the MMP-2, MMP-9, and COX-2 genes was assessed in paraffin-embedded samples of endometrial carcinoma from 30 consecutive patients and non-neoplastic endometrial tissue from 20 matched controls. Endometrial carcinoma tissue samples were obtained from the uteruses of patients who had undergone total abdominal hysterectomy and bilateral salpingooophorectomy at the Department of Gynecology, Perinatology and Reproductive Medicine of the University of Florence School of Medicine, Florence, Italy. The patients, aged from 50 to 82 years (median, 62.5 years; mean, 62.1 years), had complete records of their clinical history and follow-up. Amplification of mRNA was detected by reversetranscription polymerase chain reaction assay and measured quantitatively. mRNAwas successfully extracted from 15 (75%) of the 20 control samples and 18 (60%) of the 30 tumor International Journal of Gynecology and Obstetrics (2006) 93, 64—66

The role of relaxin in endometrial cancer

Relaxin (RLN) is a naturally occurring hormone that is known to modulate connective tissue remodeling in the uterus and cervix. Our goal was to investigate the role of RLN in endometrial cancer. RLN expression was evaluated using immunohistochemistry in 57 samples of invasive endometrial carcinoma (EC) and 10 benign endometria. 67% of high-stage (III/IV) tumors demonstrated strong RLN expression compared to 37% of low-stage (I/II) cases. Strong RLN expression associated significantly with high-grade and depth of myometrial invasion. Notably, strong RLN expression was associated with a significantly shorter overall survival (p