Tissue Thickness Effects on Immunohistochemical Staining Intensity of Markers of Cancer.

High-quality patient samples are required for reliable immunohistochemistry test outcomes that provide a significant benefit for patient care. Among the preanalytic variables in tissue handling, tissue thickness is thought to be easily controlled; however, whether the thickness of the tissue effects the staining intensity for antibody immunohistochemistry has not been quantitatively demonstrated. To investigate, we cut multiblock tissue sections of tonsil, liver, and kidney at 2, 4, 6, and 8 µm thicknesses. Interferometry measurements of the sectioned paraffin showed a <1 µm variation within a preset microtome thickness. Sections were then immunostained with antibodies targeting different cellular localizations; Ki-67 and BCL6 (nuclear), CD7 (membranous), and cytokeratin (cytoplasmic). A pathologist annotated regions of interest for each marker and performed brightfield and whole-slide visual scoring. Then a pixel-wise processing algorithm determined intensity of each pixel in these regions of interest and binned them into predetermined 0, 1+, 2+, or 3+ intensities. Visual scores from brightfield and whole-slide images were highly correlated to the percentage of pixels in each intensity bin. A stepwise increase was observed in pathologist scores and algorithmically defined percentage of pixels in each bin with increasing thickness demonstrating that changes in preset section thickness impacts staining intensity. The use of tissue thickness outside vendors' recommendations might change the intensity including the proportion of positive and negative cells and eventually the overall diagnosis outcome. Therefore, we recommend that tissue be consistently cut within the middle of thickness range specified by the assay manufacturer.

CGRRF1 as a novel biomarker of tissue response to metformin in the context of obesity.

OBJECTIVE: Obesity-associated hyperestrogenism and hyperinsulinemia contribute significantly to the pathogenesis of endometrial cancer. We recently demonstrated that metformin, a drug long used for treatment of type 2 diabetes, attenuates both insulin- and estrogen-mediated proliferative signaling in the obese rat endometrium. In this study, we sought to identify tissue biomarkers that may prove clinically useful to predict tissue response for both prevention and therapeutic studies. We identified CGRRF1 (cell growth regulator with ring finger domain 1) as a novel metformin-responsive gene and characterized its possible role in endometrial cancer prevention. METHODS: CGRRF1 mRNA expression was evaluated by RT-qPCR in the endometrium of obese and lean rats, and also in normal and malignant human endometrium. CGRRF1 levels were genetically manipulated in endometrial cancer cells, and its effects on proliferation and apoptosis were evaluated by MTT and Western blot. RESULTS: CGRRF1 is significantly induced by metformin treatment in the obese rat endometrium. In vitro studies demonstrate that overexpression of CGRRF1 inhibits endometrial cancer cell proliferation. Analysis of human endometrial tumors reveals that CGRRF1 expression is significantly lower in hyperplasia, Grade 1, Grade 2, Grade 3, MMMT, and UPSC endometrial tumors compared to normal human endometrium (p<0.05), suggesting that loss of CGRRF1 is associated with the presence of disease. CONCLUSION: CGRRF1 represents a novel, reproducible tissue marker of metformin response in the obese endometrium. Furthermore, our preliminary data suggests that up-regulation of CGRRF1 expression may prove clinically useful in the prevention or treatment of endometrial cancer.

Critical tumor suppressor function mediated by epithelial Mig-6 in endometrial cancer.

Endometrial cancer is preceded by endometrial hyperplasia, unopposed estrogen exposure, and genetic alterations, but the precise causes of endometrial cancer remain uncertain. Mig-6, mainly known as a negative regulator of the EGF receptor, is an important mediator of progesterone signaling in the uterus, where it mediates tumor suppression by modulating endometrial stromal-epithelial communications. In this study, we investigated the function of Mig-6 in the uterine epithelium using a tissue-specific gene knockout strategy, in which floxed Mig-6 (Mig-6(f/f)) mice were crossed to Wnt7a-Cre mice (Wnt7a(cre+)Mig-6(f/f)). Wnt7a(cre+)Mig-6(f/f) mice developed endometrial hyperplasia and estrogen-dependent endometrial cancer, exhibiting increased proliferation in epithelial cells as well as apoptosis in subepithelial stromal cells. We documented increased expression of NOTCH1 and BIRC3 in epithelial cells of Wnt7a(cre+)Mig-6(f/f) mice and decreased expression of the progesterone receptor (PR) in stromal cells. Progesterone therapy controls endometrial growth and prevents endometrial cancer, but the effectiveness of progesterone as a treatment for women with endometrial cancer is less clear. We noted that the hyperplasic phenotype of Wnt7a(cre+)Mig-6(f/f) mice was prevented by progesterone treatment, whereas this treatment had no effect in PR(cre/+)Mig-6(f/f) mice where Mig-6 was deleted in both the epithelial and stromal compartments of the uterus. In contrast, activation of progesterone signaling in the stroma regulated proliferation and apoptosis in the epithelium via suppression of ERα signaling. In summary, our results establish that epithelial Mig-6 functions as a critical tumor suppressor that mediates the ability of progesterone to prevent the development of endometrial cancer.

Chemopreventive effects of metformin on obesity-associated endometrial proliferation.

OBJECTIVE: Obesity is a significant contributing factor to endometrial cancer risk. We previously demonstrated that estrogen-induced endometrial proliferation is enhanced in the context of hyperinsulinemia and insulin resistance. In this study, we investigate whether pharmacologic agents that modulate insulin sensitivity or normalize insulin levels will diminish the proliferative response to estrogen. STUDY DESIGN: Zucker fa/fa obese rats and lean controls were used as models of hyperinsulinemia and insulin resistance. Insulin levels were depleted in ovariectomized rats following treatment with streptozotocin, or modulated by metformin treatment. The number of BrdU-incorporated cells, estrogen-dependent proliferative and antiproliferative gene expression, and activation of mTOR and ERK1/2 MAPK signaling were studied. A rat normal endometrial cell line RENE1 was used to evaluate the direct effects of metformin on endometrial cell proliferation and gene expression in vitro. RESULTS: Streptozotocin lowered circulating insulin levels in obese rats and decreased the number of BrdU-labeled endometrial cells even in the presence of exogenous estrogen. Treatment with the insulin-sensitizing drug metformin attenuated estrogen-dependent proliferative expression of c-myc and c-fos in the obese rat endometrium compared to untreated controls and was accompanied by inhibition of phosphorylation of the insulin and IGF1 receptors (IRß/IGF1R) and ERK1/2. In vitro studies indicated metformin inhibited RENE1 proliferation in a dose-dependent manner. CONCLUSION: These findings suggest that drugs that modulate insulin sensitivity, such as metformin, hinder estrogen-mediated endometrial proliferation. Therefore, these drugs may be clinically useful for the prevention of endometrial cancer in obese women.

Sex hormone regulation of survivin gene expression.

Survivin (BIRC5) is a cell survival gene that is overexpressed in endometrial cancer and has been implicated to have a physiological role in normal endometrial function. To determine whether survivin gene expression is regulated by reproductive steroid hormones in the human endometrium, RNA was prepared from normal cycling women in the proliferative and secretory phases of the menstrual cycle. RNA was also isolated from 21 endometrial biopsies from premenopausal women at baseline and following 3 months of treatment with depot medroxyprogesterone acetate. Finally, RNA was isolated from endometrial biopsies from ten healthy postmenopausal women participating in a clinical trial of estrogen replacement therapy at baseline and following 6 months of treatment with conjugated equine estrogen. Quantitative RT-PCR analysis was used to determine survivin, insulin-like growth factor binding protein 1 (IGFBP1), Ki67, and IGF1 gene expression levels. Survivin gene expression was highest in the proliferative phase of the menstrual cycle and showed a statistically significant 4-fold increase in expression following chronic treatment with estrogens; this was strongly correlated with increased Ki67, a marker of proliferation. Survivin gene expression decreased 4.6-fold following chronic progestin treatment in the human endometrium. These data suggest that survivin transcript is regulated by estrogens and progestins in the disease-free human endometrium. The data also suggest that survivin transcript may be used as a biomarker of estrogen and progestin treatment efficacy, but validation studies must be conducted to support this conclusion.

Loss of inhibitory insulin receptor substrate-1 phosphorylation is an early event in mammalian target of rapamycin-dependent endometrial hyperplasia and carcinoma.

Insulin-like growth factor-I receptor signaling contributes to the development of endometrial hyperplasia, the precursor to endometrioid-type endometrial carcinoma, in humans and in rodent models. This pathway is under both positive and negative regulation, including S6 kinase (S6K) phosphorylation of insulin receptor substrate-1 (IRS-1) at S636/639, which occurs downstream of mammalian target of rapamycin (mTOR) activation to inhibit this adapter protein. We observed activation of mTOR with a high frequency in human endometrial hyperplasia and carcinoma, but an absence of IRS-1 phosphorylation, despite high levels of activated S6K. To explore when during disease progression mammalian target of rapamycin (mTOR) activation and loss of negative feedback to IRS-1 occurred, we used the Eker rat (Tsc2(Ek/+)) model, where endometrial hyperplasia develops as a result of loss of Tsc2, a "gatekeeper" for mTOR. We observed mTOR activation early in progression in hyperplasias and in some histologically normal epithelial cells, suggesting that event(s) in addition to loss of Tsc2 were required for progression to hyperplasia. In contrast, whereas IRS-1 S636/639 phosphorylation was observed in normal epithelium, it was absent from all hyperplasias, indicating loss of IRS-1 inhibition by S6K occurred during progression to hyperplasia. Treatment with a mTOR inhibitor (WAY-129327) significantly decreased hyperplasia incidence and proliferative indices. Because progression from normal epithelium to carcinoma proceeds through endometrial hyperplasia, these data suggest a progression sequence where activation of mTOR is followed by loss of negative feedback to IRS-1 during the initial stages of development of this disease.

Molecular clustering of endometrial carcinoma based on estrogen-induced gene expression.

Identification of biomarkers potentially provides prognostic information that can help guide clinical decision-making. Given the relationship between estrogen exposure and endometrial cancer, especially low grade endometrioid carcinoma, we hypothesized that high expression of genes induced by estrogen would identify low risk endometrioid endometrial cancers. cDNA microarray and qRT-PCR verification were used to identify six genes that are highly induced by estrogen in the endometrium. These estrogen-induced biomarkers were quantified in 72 endometrial carcinomas by qRT-PCR. Unsupervised cluster analysis was performed, with expression data correlated to tumor characteristics. Time to recurrence by cluster was analyzed using the Kaplan-Meier method. A receiver operating characteristic (ROC) curve was generated to determine the potential clinical utility of the biomarker panel to predict prognosis. Expression of all genes was higher in endometrioid carcinomas compared to non-endometrioid carcinomas. Unsupervised cluster analysis revealed two distinct groups based on gene expression. The high expression cluster was characterized by lower age, higher BMI, and low grade endometrioid histology. The low expression cluster had a recurrence rate 4.35 times higher than the high expression cluster. ROC analysis allowed for the prediction of stage and grade with a false negative rate of 4.8% based on level of gene expression in endometrioid tumors. We have therefore identified a panel of estrogen-induced genes that have potential utility in predicting endometrial cancer stage and recurrence risk. This proof-of-concept study demonstrates that biomarker analysis may play a role in clinical decision making for the therapy of women with endometrial cancer.

Comparison of human and rat uterine leiomyomata: identification of a dysregulated mammalian target of rapamycin pathway.

Uterine leiomyomata, or fibroids, are benign tumors of the uterine myometrium that significantly affect up to 30% of reproductive-age women. Despite being the primary cause of hysterectomy in the United States, accounting for up to 200,000 procedures annually, the etiology of leiomyoma remains largely unknown. As a basis for understanding leiomyoma pathogenesis and identifying targets for pharmacotherapy, we conducted transcriptional profiling of leiomyoma and unaffected myometrium from humans and Eker rats, the best characterized preclinical model of leiomyomata. A global comparison of mRNA from leiomyoma versus myometrium in human and rat identified a highly significant overlap of dysregulated gene expression in leiomyomata. An unbiased pathway analysis using a method of gene-set enrichment based on the sigPathway algorithm detected the mammalian target of rapamycin (mTOR) pathway as one of the most highly up-regulated pathways in both human and rat tumors. To validate this pathway as a therapeutic target for uterine leiomyomata, preclinical studies were conducted in Eker rats. These rats develop uterine leiomyomata as a consequence of loss of Tsc2 function and up-regulation of mTOR signaling. Inhibition of mTOR in female Eker rats with the rapamycin analogue WAY-129327 for 2 weeks decreased mTOR signaling and cell proliferation in tumors, and treatment for 4 months significantly decreased tumor incidence, multiplicity, and size. These results identify dysregulated mTOR signaling as a component of leiomyoma etiology across species and directly show the dependence of uterine leiomyomata with activated mTOR on this signaling pathway for growth.

Enhanced estrogen-induced proliferation in obese rat endometrium.

OBJECTIVE: We tested the hypothesis that the proliferative estrogen effect on the endometrium is enhanced in obese vs lean animals. STUDY DESIGN: Using Zucker fa/fa obese rats and lean control, we examined endometrial cell proliferation and the expression patterns of certain estrogen-regulated proproliferative and antiproliferative genes after short-term treatment with estradiol. RESULTS: No significant morphologic/histologic difference was seen between the obese rats and the lean rats. Estrogen-induced proproliferative genes cyclin A and c-Myc messenger RNA expression were significantly higher in the endometrium of obese rats compared with those of the lean control. Expression of the antiproliferative gene p27Kip1 was suppressed by estrogen treatment in both obese and lean rats; however, the decrease was more pronounced in obese rats. Estrogen more strongly induced the antiproliferative genes retinaldehyde dehydrogenases 2 and secreted frizzled-related protein 4 in lean rats but had little or no effect in obese rats. CONCLUSION: Enhancement of estrogen-induced endometrial proproliferative gene expression and suppression of antiproliferative gene expression was seen in the endometrium of obese vs lean animals.

Developmental reprogramming of IGF signaling and susceptibility to endometrial hyperplasia in the rat.

In rodents, a brief neonatal exposure of the developing reproductive tract to the xenoestrogen, diethylstilbestrol (DES) reprograms developing tissues to increase susceptibility to tumorigenesis in adult animals, including uterine adenocarcinoma. Progression from a normal endometrium to carcinoma occurs via the intermediate stage of endometrial hyperplasia. We previously reported that endometrial hyperplasia in postmenopausal women is linked to abnormal insulin-like growth factor-I (IGF-I) signaling. To identify early events involved in the development of hyperplasia in the endometrium, we examined expression and activation of IGF-I pathway components in endometrium of rats exposed to DES. By 5 months of age, 36/60 (60%) of rats exposed to DES on days 3-5 after birth developed endometrial hyperplasia compared to 0% of vehicle-treated controls. Consistent with activation of a mitogenic signaling pathway, Ki67-positive cells increased in DES-exposed endometrium despite compromised ovarian function and hypoestrogenic milieu characteristic of DES-exposed animals. The endometrium of DES-exposed rats overexpressed IGF-II and insulin receptor substrate-1 (IRS-1) and exhibited elevated Akt expression and activation (as judged by phosphorylation) and mTOR signaling (phosphorylation of S6) compared to vehicle-treated endometrium. In contrast to vehicle-treated endometrium, in which negative feedback to IRS-1 was observed (phosphorylation of S636/639), negative feedback to IRS-1 was absent in DES-exposed endometrium. These data support a central role for IGF-I signaling in the development of both human and rodent endometrial hyperplasia. Furthermore, both global activation of IGF-IR signaling and abrogation of negative feedback to IRS-1 appear to be reprogrammed by DES in endometrial hyperplasia, implicating for the first time loss of negative feedback to IRS-1 in development of a preneoplastic lesion.

Overexpression of the insulin-like growth factor I receptor and activation of the AKT pathway in hyperplastic endometrium.

PURPOSE: Although there is considerable information on the molecular aberrations associated with endometrial cancer, very little is known of the changes in gene expression associated with endometrial hyperplasia. EXPERIMENTAL DESIGN: To address this, we have compared the level of expression of estrogen-regulated genes and components of the insulin-like growth factor I (IGF-I) signaling pathway in endometrial biopsies from subjects with normal endometrium, complex atypical endometrial hyperplasia, and endometrial adenocarcinoma (type I). RESULTS: There was a significant increase in the expression of the IGF-I receptor (IGF-IR) in biopsies from hyperplastic endometrium and endometrial carcinoma compared with the proliferative endometrium. The receptor was also activated, as judged by increased tyrosine phosphorylation. In addition, in endometrial hyperplasia and carcinoma, the downstream components of the IGF-IR pathway are activated, as reflected in increased Akt phosphorylation. Loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) expression in endometrial hyperplasia did not correlate with increased activation of IGF-IR. However, the simultaneous loss of PTEN expression and increased IGF-IR activation in hyperplasia was associated with an increased incidence of endometrial carcinoma. CONCLUSIONS: These results suggest that up-regulation of IGF-IR and loss of PTEN may be independent events that give rise to complementary activation of the IGF-I pathway and increase the probability of the development of cancer. These studies suggest that increased expression of IGF-IR may be an important contributor to the risk of endometrial hyperplasia and cancer.

Identification of a novel estrogen-regulated gene, EIG121, induced by hormone replacement therapy and differentially expressed in type I and type II endometrial cancer.

PURPOSE: The identification of genes and pathways that are affected by estrogenization may shed light on the mechanisms of estrogen action. Here, we describe the expression pattern of a novel estrogen-induced gene, EIG121, in distinct types of endometrial cancer. EXPERIMENTAL DESIGN: EIG121 was identified by cDNA microarray analysis of endometrial RNA from women receiving either placebo or estrogen replacement therapy. The expression level of EIG121 was then measured by real-time quantitative reverse transcription-PCR in benign, hyperplastic, and malignant endometrial samples. A polyclonal antibody was used to detect EIG121 protein by immunohistochemistry. RESULTS: In postmenopausal endometrium, estrogen replacement therapy with Premarin and synthetic estrogen sulfate conjugates induced the expression of EIG121 2- and 3-fold, respectively. In premenopausal endometrium, the expression of EIG121 was higher in the estrogen-dominated proliferative phase than the secretory phase. In endometrial complex, hyperplasia, and endometrioid adenocarcinoma, neoplastic proliferations associated with estrogen excess, the expression of EIG121 was significantly elevated (on average 3.8-fold in hyperplasias and 21-fold in grade 1 tumors). Although the level of EIG121 mRNA in grade 3 endometrioid carcinoma was still 3.5-fold of that in benign endometrium, EIG121 expression tended to decline with increasing tumor grade and disease stage. Immunohistochemistry showed faint staining of normal endometrial epithelium, but intense staining of endometrioid tumors. In sharp contrast, EIG121 expression was significantly suppressed in both uterine papillary serous carcinoma and uterine malignant mixed mullerian tumor, two tumors not associated with estrogen exposure, to <5% of the level in benign endometrium. CONCLUSIONS: Our results suggest that EIG121 is a good endometrial biomarker associated with a hyperestrogenic state and estrogen-related type I endometrial adenocarcinoma.