Discovery and validation of methylation markers for endometrial cancer

The prognosis of endometrial cancer is strongly associated with stage at diagnosis, suggesting that early detection may reduce mortality. Women who are diagnosed with endometrial carcinoma often have a lengthy history of vaginal bleeding, which offers an opportunity for early diagnosis and curative treatment. We performed DNA methylation profiling on population‐based endometrial cancers to identify early detection biomarkers and replicated top candidates in two independent studies. We compared DNA methylation values of 1,500 probes representing 807 genes in 148 population‐based endometrial carcinoma samples and 23 benign endometrial tissues. Markers were replicated in another set of 69 carcinomas and 40 benign tissues profiled on the same platform. Further replication was conducted in The Cancer Genome Atlas and in prospectively collected endometrial brushings from women with and without endometrial carcinomas. We identified 114 CpG sites showing methylation differences with p values of ≤10^?7 between endometrial carcinoma and normal endometrium. Eight genes (ADCYAP1, ASCL2, HS3ST2, HTR1B, MME, NPY and SOX1) were selected for further replication. Age‐adjusted odds ratios for endometrial cancer ranged from 3.44 (95%‐CI: 1.33–8.91) for ASCL2 to 18.61 (95%‐CI: 5.50–62.97) for HTR1B. An area under the curve (AUC) of 0.93 was achieved for discriminating carcinoma from benign endometrium. Replication in The Cancer Genome Atlas and in endometrial brushings from an independent study confirmed the candidate markers. This study demonstrates that methylation markers may be used to evaluate women with abnormal vaginal bleeding to distinguish women with endometrial carcinoma from the majority of women without malignancy.     

血脂异常与子宫内膜癌的相关性分析

目的:回顾性分析子宫内膜癌患者中血脂代谢异常发病情况并探讨血脂代谢异常与子宫内膜癌发病风险的相关性。方法:收集2010年至2014年就诊于新疆医科大学第一附属医院的未经过放疗及化疗的且 经病理确诊为雌激素依赖型子宫内膜癌的病例81例。另以子宫内膜正常组,子宫内膜增生组作为对照组、各组分别56例,收集患者和对照者一般资料,既往史,月经史,婚育史,用药史,家族史(包括家族肿瘤史),生化血脂结果,病理结果。结果:子宫内膜正常组、子宫内膜增生组、子宫内膜癌组三组在发病年龄、月经状态、孕产情况、糖尿病发病情况、肿瘤家族史、PCOS/甲减发病率、HRT/TAM使用情况之间差异无统计学意义（P＞0.05）;子宫内膜癌组的体重指数（BMI）、甘油三酯（TG）、总胆固醇（TC）、高血压发病率均高于子宫内膜正常组、增生组,差异有统计学意义（P＜0.05）;子宫内膜癌组的高密度脂蛋白（HDL）明显低于子宫内膜正常组、增生组,差异有统计学意义（P<0.05）。 结论:子宫内膜癌患者的BMI、TG、TC、HDL及高血压发病率明显高于非子宫内膜癌患者,血脂异常很可能是子宫内膜癌发生的关键因素,有望高血脂将可能作为尽早识别子宫内膜癌高危人群的预警指标。     

Importin 13在子宫内膜异位症和子宫内膜癌中的表达及意义

背景与目的:正常情况下的子宫内膜干/祖细胞有助于子宫内膜的生理性修复,而子宫内膜干/祖细胞的异常增殖和异常分化则会导致子宫内膜疾病(如子宫内膜异位症和子宫内膜癌)。Importin 13(IPO13)是importinβ家族新成员的一个核质双向的转运受体蛋白,是角膜上皮干细胞的一个标记,在维持干细胞的性状、高增殖潜力、低分化状态,调节细胞分化以及小鼠生殖细胞减数分裂上具有重要的作用。本文探讨成体干细胞标记IPO13在子宫内膜异位症和子宫内膜癌中的表达及意义。方法:手术取正常子宫内膜组织40例(对照组),其中增生期和分泌期各20例,异位子宫内膜组织20例(异位症组)和子宫内膜癌病灶组织20例(内膜癌组)。采用免疫组化SP法检测IPO13蛋白在细胞内的定位;采用实时荧光定量PCR技术(real-time quantitativepolymerase chain reaction,RT-PCR)检测IPO13 mRNA的表达;Western blot检测IPO13蛋白的表达。结果:IPO13蛋白在内膜癌、异位症及正常对照组中腺上皮细胞和间质细胞的细胞质和细胞核中均有表达。IPO13蛋白在对照组增生期表达量(0.52±0.30)明显高于分泌期(0.25±0.04,P<0.05);IPO13蛋白在异位症组表达量(0.81±0.12)明显高于对照组增生期及分泌期(P<0.05);IPO13蛋白在内膜癌组表达量(1.21±0.11)明显高于异位症组(P<0.05)。IPO13 mRNA在对照组增生期表达量是分泌期的3倍(P<0.05);IPO13 mRNA在异位症组中表达量是对照组分泌期的6倍(P<0.05),增生期的2.5倍(P<0.05);IPO13 mRNA在内膜癌组表达量是异位症组的2倍(P<0.05)。结论:IPO13在子宫内膜癌中及异位症组中表达明显高于对照组,推测其高表达与子宫内膜癌及子宫内膜异位症发病密切相关。     

HERG potassium channels are more frequently expressed in human endometrial cancer as compared to non-cancerous endometrium

HERG K(+)channels, besides contributing to regulate cardiac and neuronal excitability, are preferentially expressed in tumour cell lines of different histogenesis, where their role in the development and maintenance of the neoplastic phenotype is under study. We show here that both herg gene and HERG protein are expressed with high frequency in primary human endometrial cancers, as compared to normal and hyperplastic endometrium. RT-PCR and immunohistochemistry, using specific anti-HERG antibodies developed in our laboratory, were applied to tissue specimens obtained from 18 endometrial cancers and 11 non-cancerous endometrial tissues. herg RNA and HERG protein are expressed in 67% and 82%, respectively, of cancerous, while in only 18% of non-cancerous tissues. In particular, no expression was found in endometrial hyperplasia. Moreover, electrophysiological experiments confirmed the presence of functioning HERG channels on the plasma membrane of tumour cells. On the whole, these data are the first demonstration of the presence of HERG channels in primary human neoplasias, and could candidate HERG as a potential tool capable of marking cancerous versus hyperplastic endometrial growth.     

Early aberrant insulin-like growth factor signaling in the progression to endometrial carcinoma is augmented by tamoxifen

Tamoxifen is an important selective estrogen receptor (ER) modulator for treatment of steroid hormone positive breast cancer. In addition to the beneficial effect, tamoxifen is one risk factor for endometrial carcinoma (EnCa) development. We hypothesized that, (1) dysregulation of gene expression and protein phosphorylation of the insulin-like growth factor (IGF) and steroid hormone receptor-signaling occur early in benign endometrial tissues and (2) signaling differences would be detected between patients with or without tamoxifen treatment. Seventy-eight tissues, including 2 benign cohorts from patients treated with (n = 24) or without tamoxifen (n = 28) (hyperproliferative endometrium, hyperplasia, polyps), EnCa (n = 12) with endometrium controls (n = 14) were analyzed for expression of 15 genes from the IGF and steroid hormone receptor-signaling, including the target genes Syncytin-1, PAX2 and c-myc. Total and phosphorylated protein expression were examined for ERalpha, PTEN, AKT, mTOR and Syncytin-1. Compared to controls similar significant deregulation of IGF and steroid hormone receptor-signaling, Syncytin-1 and PAX2 occurred in both benign cohorts, irrelevant of tamoxifen treatment. Comparing both benign cohorts with and without tamoxifen significant expression differences were noted. Increased total protein and phosphorylation of pERalpha-Ser118, pPTEN-Thr380, pAKT-Thr308, pAKT-Ser473, pmTOR-Ser2448 and Syncytin-1 were noted in early benign tissue stages associating with tamoxifen, especially polyps. Functional kinetic studies following tamoxifen treatment of the PTEN mutated RL95-2 EnCa cell line, demonstrated a doubling of phosphorylation of pERalpha-Ser118 and a 4.2-fold induction of pAKT-Thr308 along with Syncytin-1 induction. This study supports that dysregulated IGF and steroid hormone receptor signaling is prominent in endometrial benign stages and these alterations could represent clinical indicators for the risk of EnCa and also help in development of new therapies.     

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. ( Cancer Sci 2008; 99: 1125–1130)     

Coexpression of growth arrest-specific gene 6 and receptor tyrosine kinases Axl and Sky in human uterine endometrial cancers.

BACKGROUND: Gas6, the protein product of the growth arrest-specific gene 6 (gas6), a member of the vitamin K-dependent protein family, was identified as a ligand for the Axl/Sky family of receptor tyrosine kinases. Gas6 acts as a growth-potentiating factor for thrombin-induced proliferation of vascular smooth muscle cells. The aim of the present study was to test for the presence of Gas6 and its receptors Axl and Sky, related to specific growth in uterine endometrial cancers, and to evaluate their plausible growth potential and mechanism. MATERIALS AND METHODS: Sixty patients underwent curative resection for uterine endometrial cancers. In uterine endometrial cancers, the mRNA levels and histoscores of Gas6, Axl and Sky were determined by competitive RT-PCR using recombinant RNA and immunohistochemical studies, respectively. The rate of proliferation by immunochemistry for Ki67 and the rate of apoptosis by TUNEL were determined. RESULTS: The mRNA levels and the histoscores of Gas6 and Axl in well-differentiated endometrial cancers (G1 EC) were significantly higher than in normal uterine endometrium (NE) and in moderately and poorly differentiated endometrial cancers (G2 + G3 EC). The rate of apoptosis in G1 EC was significantly lower than that in NE and in G2 + G3 EC. CONCLUSIONS: Gas6 and Axl signal transduction is aberrantly stimulated in well-differentiated endometrial cancers, plausibly related to tumor progression due to protection from apoptosis in cancers cells.     

VEGF在子宫内膜癌组织中的表达和意义

目的　探讨血管内皮生长因子 (VEGF)在子宫内膜癌组织中的表达及意义。方法　应用免疫组织化学S -P法对 5 0例子宫内膜腺癌、10例子宫内膜复杂型增生过长、10例正常子宫内膜组织中VEGF表达进行检测。结果　子宫内膜腺癌、子宫内膜复杂型增生过长、正常子宫内膜组织中VEGF的强阳性表达率分别为 74 .0 % (37/5 0 )、2 0 % (2 /10 )、10 % (1/10 ) ,统计学显示癌组织的强阳性表达率明显高于复杂性增生过长和正常组织 (P <0 .0 5 ) ;VEGF的表达与临床分期、组织学分级、肌层浸润程度、患者年龄均无明显相关性(P >0 .0 5 )。结论　VEGF高表达可能与子宫内膜癌的发生有关 ,但不能作为判断其转移及预后的指标。     

Regulation of endometrial cancer cell growth by luteinizing hormone (LH) and follicle stimulating hormone (FSH)

Gonadotrophin releasing hormone analogues (GnRHa) have been used to treat recurrent endometrial cancer. However, the mode of action is uncertain. Our previous studies showed no direct effect of GnRHa on endometrial cancer cell growth in vitro. We have now examined the effect of luteinizing hormone (LH) and follicle stimulating hormone (FSH) on endometrial cancer cell growth. The aim was to determine whether suppression of pituitary LH and FSH by GnRHa could explain the tumour regression seen in up to 44% of patients treated with this drug. We show that recombinant human LH and FSH (rhLH and rhFSH) produce a concentration dependent stimulation of the endometrial cancer cell line HEC-1A, in serum-free medium (maximum increase of 62 and 50% respectively relative to untreated controls). This increase is equivalent to that obtained by addition of 10% newborn calf serum. Growth of the Ishikawa cell line in culture increases in the presence of rhLH (maximum increase of 67%) but not with rhFSH. Using RT-PCR, we show that the Ishikawa cell line intermittently expresses receptor mRNA of LH but not of FSH; there is no expression of either mRNA by HEC-1A. Classically, both LH and FSH act via cAMP linked membrane receptors. However, neither rhLH nor rhFSH elicit cAMP production in either of our endometrial cancer cell lines. Thus, although a growth response to LH and FSH can be shown, and some cells express the LH receptor, stimulation appears to be via a pathway separate from that of the classical gonadotrophin receptor.     

孕激素相关子宫内膜蛋白基因与肿瘤

 孕激素相关子宫内膜蛋白（PAEP）基因是一个主要表达于分泌期子宫内膜和妊娠早期蜕膜的基因。近年来研究表明,PAEP基因在乳腺癌、子宫内膜癌、卵巢癌、胃癌、恶性黑素瘤等多种肿瘤组织中表达上调,对肿瘤发生发展起重要作用。要将PAEP基因作为临床诊断、判断预后及指导治疗的指标应用,还需进一步研究PAEP基因对肿瘤生物学行为影响的作用机制。     

Risk of subsequent endometrial carcinoma associated with endometrial intraepithelial neoplasia classification of endometrial biopsies

BACKGROUND.

Histopathologic diagnosis of endometrial biopsies is used to estimate the risk of progression to carcinoma and guide clinical management. Problems with the widely used World Health Organization (WHO) system for classifying endometrial hyperplasia (EH) have prompted the development of an alternative system based on endometrial intraepithelial neoplasia (EIN). The authors estimated progression risk associated with EIN among endometrial biopsies in a nested case‐control study of EH progression.

METHODS.

Index biopsies with original community pathology diagnoses of disordered proliferative endometrium (DPEM) or EH that were independently confirmed by a panel of pathologists were independently reviewed and assigned EIN classifications (inadequate, benign, EIN, or cancer) by a second panel of pathologists. Cases (N = 138) progressed to carcinoma at least 1 year (median, 6 years) after their index biopsy. Controls (N = 241) also had EH, did not progress to carcinoma, and were individually matched to cases based on age at EH, date of EH, and length of follow‐up. By using conditional logistic regression, the authors estimated relative risks (RRs) with 95% confidence intervals (95% CIs) for progression to carcinoma for EIN versus benign.

RESULTS.

In the EIN system, 71 (52.6%) cases and 159 (66.8%) controls were classified as benign and 42 (31.1%) cases and 65 (27.3%) controls were classified as EIN. The RR for EIN versus benign was 7.76 (95% CI, 3.36‐17.91). In the WHO system, the RR for atypical hyperplasia (AH) versus DPEM, simple hyperplasia, or complex hyperplasia was 9.19 (95% CI, 3.87‐21.83).

CONCLUSIONS.

Among women observed for at least 1 year after receiving a biopsy‐based EH diagnosis, EIN and AH were both found to have similarly increased risks of progression to carcinoma. Cancer 2008. © Published 2008 by American Cancer Society     

beta- Catenin mutations and aberrant nuclear expression during endometrial tumorigenesis

To clarify the possible role of aberrant beta-catenin expression during endometrial tumorigenesis, a total of 199 cases of endometrial carcinomas (endometrioid type), as well as 37 cases of simple/complex and 32 of atypical hyperplasias, was consecutively investigated for immunohistochemistry, along with 141 normal endometrial samples distant from carcinomas. Of 199 carcinoma cases, 73 tumours as well as 44 normal samples were also analysed using a combination of RT-PCR and Southern blot hybridization, Western blot, and mutation gene assays. Cell membrane beta-catenin immunoreactivity showed a stepwise decrease from normal, through atypical hyperplasia, to grade 3 carcinomas. In contrast, the nuclear accumulation in atypical hyperplasias and grade 1 or 2 tumours was higher than in simple/complex hyperplasias. Mutations in exon 3 of the beta-catenin gene involving codons 33, 34, 37, 41, and 45 were observed in 16 (22.9%) of 70 endometrial carcinomas, as well as 3 (12.5%) of 24 atypical hyperplasias, the results being significantly related to low membrane and high nuclear immunoreactivity but not relative mRNA expression levels, suggesting that the gene mutations may be closely associated with changes in subcellular distribution. In addition to significant association between beta-catenin mutation and low grade histological malignancy (P = 0.048), the mutations were detected in none of 15 and 13 (26%) of 50 tumours with or without lymph node metastasis, the difference being significant (P = 0.027). These findings suggest that beta-catenin abnormalities may play an important role in a relatively early event during the endometrial hyperplasia-carcinoma sequence.     

HER2和BRCA1在子宫内膜癌中的表达及其与预后的关系

目的探讨人表皮生长因子受体-2(human epidermal growth factor receptor 2,HER2)和乳腺癌易感基因1(breast cancer susceptibility gene 1,BRCA1)在子宫内膜癌中的表达及其与预后的关系。方法采用免疫组织化学方法检测79例子宫内膜癌、40例非典型增生子宫内膜、30例正常子宫内膜组织中HER2与BRCA1的表达,并结合随访资料分析这2个指标与患者生存时间的关系。结果在正常子宫内膜、非典型增生子宫内膜、子宫内膜癌中HER2阳性表达率分别为6.7%、17.5%、67.1%(P=0.000),BRCA1阳性表达率分别为93.3%、62.5%、31.7%(P=0.000)。子宫内膜癌中,HER2阳性表达与组织分化、手术病理分期、肌层浸润及淋巴结转移有关(均P<0.05),BRCA1阳性表达与组织分化、手术病理分期及淋巴结转移有关(均P<0.05);HER2阳性表达患者的5年生存率低于HER2阴性表达患者(69.8%vs 92.3%,P<0.05),BRCA1阳性表达患者与阴性表达患者的5年生存率差异无统计学意义(84.6%vs 72.2%,P>0.05);BRCA1与HER2蛋白在子宫内膜癌中表达无相关性(r=-0.103,P>0.05)。结论 BRCA1的表达缺失可能与子宫内膜癌的发生、发展有关,HER2蛋白的高表达与子宫内膜癌侵袭性及不良预后有关。     

The role of hepatocyte growth factor activator inhibitor (HAI)‐1 and HAI‐2 in endometrial cancer

Hepatocyte growth factor activator inhibitors (HAI‐1 and HAI‐2) are Kunitz‐type serine protease inhibitors that have a broad inhibitory spectrum against serine proteases. This is the first study to investigate the role of HAI‐1 and HAI‐2 in endometrial cancer. We investigated the biological functions of HAI‐1 and HAI‐2 using KLE and HEC‐251 endometrial cancer cell lines, thus HAI‐1 and HAI‐2 were examined in uterine normal endometrium, endometrial hyperplasia and cancer specimens by immunohistochemistry. HAI‐1 and HAI‐2 showed potential inhibitory effects on cell proliferation, migration and cellular invasion by reduction of matriptase and hepsin expression. This in turn led to an increase in the levels of E‐cadherin and Slug, and a reduction in the levels of Vimentin, SIP1, Snail and Twist, and hence ER and PR signal transduction in endometrial cancer cells. The levels of HAI‐1 and HAI‐2 expression were significantly decreased in endometrial cancer specimens relative to the corresponding normal endometrium specimens. Low HAI‐1 and HAI‐2 expression was a significant predictor for a poor prognosis compared with high HAI‐1 and HAI‐2 expression. These findings indicate that HAI‐1 and HAI‐2 could be considered as therapeutic targets and used as favorable prognosis markers for endometrial cancer.     

Brush border myosin Ia inactivation in gastric but not endometrial tumors

Brush border Myosin Ia (MYO1A) has been shown to be frequently mutated in colorectal tumors with microsatellite instability (MSI) and to have tumor suppressor activity in intestinal tumors. Here, we investigated the frequency of frameshift mutations in the A8 microsatellite in exon 28 of MYO1A in MSI gastric and endometrial tumors and found a high mutation rate in gastric (22/47; 46.8%) but not endometrial (3/48; 6.2%) tumors. Using a regression model, we show that MYO1A mutations are likely to confer a growth advantage to gastric, but not endometrial tumors. The mutant MYO1A^7A protein was shown to lose its membrane localization in gastric cancer cells and a cycloheximide‐chase assay demonstrated that the mutant MYO1A^7A protein has reduced stability compared to the wild type MYO1A. Frequent MYO1A promoter hypermethylation was also found in gastric tumors. Promoter methylation negatively correlates with MYO1A mRNA expression in a series of 58 non‐MSI gastric primary tumors (Pearson's r = ?0.46; p = 0.0003) but not in a cohort of 54 non‐MSI endometrial tumors and treatment of gastric cancer cells showing high MYO1A promoter methylation with the demethylating agent 5‐aza‐2′‐deoxycytidine, resulted in a significant increase of MYO1A mRNA levels. We found that normal gastric epithelial cells, but not normal endometrial cells, express high levels of MYO1A. Therefore, when considered together, our findings suggest that MYO1A has tumor suppressor activity in the normal gastric epithelium but not in the normal endometrium and inactivation of MYO1A either genetically or epigenetically may confer gastric epithelial cells a growth advantage.     

PTEN及p27在子宫内膜腺癌中的表达及临床意义

目的　研究 PTEN及 p2 7蛋白在子宫内膜腺癌中的表达及临床意义。方法　应用免疫组化法检测 11例正常增生期内膜、4 4例子宫内膜增生症及 5 2例子宫内膜腺癌 PTEN及 p2 7蛋白表达。结果　 PTEN及 p2 7蛋白在正常增生期内膜、子宫内膜简单型 /复杂型增生过长 (无不典型增生 )、子宫内膜不典型增生过长及子宫内膜腺癌中表达率分别为 10 0 .0 %、91.4 %、6 6 .7%、6 7.3%和 90 .9%、88.6 %、6 6 .7%、5 9.6 %。子宫内膜腺癌 PTEN及 p2 7蛋白表达率明显低于正常增生期内膜和简单型 /复杂型增生过长 ( P<0 .0 1) ,与不典型增生过长相比差异无显著性( P>0 .0 5 )。 PTEN及 p2 7蛋白表达随组织分化越差呈递减趋势 ,PTEN表达与子宫内膜腺癌差分化相关 ( P<0 .0 5 ) ,与其它临床病理因素无关。在子宫内膜腺癌中 PTEN及 p2 7蛋白表达有相关性 ( P=0 .0 19)。结论　 PTEN及 p2 7在子宫内膜腺癌发生、发展中起重要作用 ,并可能是子宫内膜腺癌发生中的早期事件 ,检测 PTEN及 p2 7蛋白表达有助于子宫内膜腺癌的早期诊断及预后判断