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PDF file - 488K, Supplementary Figure 1: Experimental design. Supplementary Figure 2. Reliability of RNA extraction and reproducibility of RTqPCR techniques. Supplementary Figure 3. Reliability of RT-qPCR assays. Supplementary Figure 4. miR-132 demonstrates consistent CT values (ranging from 27 to 29.5.) across all categories of plasma samples (healthy 0=Normal Healthy Endo1=mEnedtoriosis 2 = SSOerCous 3E=EAAOOCC controls, n=20; endometriosis, n=33; EAOC, n=14; and SOC, n=21), and was used for normalization of RT-qPCR results in this study. Supplementary Figure 5. Unsupervised hierarchical clustering analysis of samples based on the 23-miRNA expression profiles. Supplementary Figure 6. Box plots of top three differentially expressed miRNAs in pair-wise comparisons. Supplementary Figure 7. Lack of correlation for miRNA expression between matching tumor tissue and plasma samples from five tumor-bearing LSLKrasG12D/+/ Ptenloxp/loxp mice.

PDF file - 36K, Supplementary Table 1. Demographic and clinical characteristics of the study cohort. Supplementary Table 2. List of the 24 miRNAs identified by genome-wide plasma miRNA expression profiling from healthy controls (n=6), endometriosis patients (n=7), and EAOC patients (n=7). Supplementary Table 3. The 10 most differentially expressed miRNAs in pair-wise comparisons between SOC patients and healthy controls, endometriosis, or EAOC samples.

Purpose: Endometriosis, a largely benign, chronic inflammatory disease, is an independent risk factor for endometrioid and clear cell epithelial ovarian tumors. We aimed to identify plasma miRNAs that can be used to differentiate patients with endometriosis and ovarian cancer from healthy individuals.Experimental Design: We conducted a two-stage exploratory study to investigate the use of plasma miRNA profiling to differentiate between patients with endometriosis, patients with endometriosis-associated ovarian cancer (EAOC), and healthy individuals. In the first stage, using global profiling of more than 1,000 miRNAs via reverse transcriptase quantitative PCR (RT-qPCR) in a 20-patient initial screening cohort, we identified 23 candidate miRNAs, which are differentially expressed between healthy controls (n = 6), patients with endometriosis (n = 7), and patients with EAOC (n = 7) based on the fold changes. In the second stage, the 23 miRNAs were further tested in an expanded cohort (n = 88) of healthy individuals (n = 20), endometriosis (n = 33), EAOC (n = 14), and serous ovarian cancer cases (SOC; n = 21, included as controls).Results: We identified three distinct miRNA signatures with reliable differential expression between healthy individuals, patients with endometriosis, and patients with EAOC. When profiled against the control SOC category, our results revealed different miRNAs, suggesting that the identified signatures are reflective of disease-specific pathogenic mechanisms. This was further supported by the fact that the majority of miRNAs differentially expressed in human EAOCs were mirrored in a double transgenic mouse EAOC model.Conclusion: Our study reports for the first time that distinct plasma miRNA expression patterns may serve as highly specific and sensitive diagnostic biomarkers to discriminate between healthy, endometriosis, and EAOC cases. Clin Cancer Res; 19(5); 1213–24. ©2013 AACR.

Mucin1 (MUC1) is an epithelial glycoprotein overexpressed in ovarian cancer and actively involved in tumor cell migration and metastasis. Using novel in vitro and in vivo MUC1-expressing conditional (Cre-loxP) ovarian tumor models, we focus here on MUC1 biology and the roles of Kras activation and Pten deletion during cell transformation and epithelial-to-mesenchymal transition (EMT). We generated several novel murine ovarian cancer cell lines derived from the ovarian surface epithelia (OSE) of mice with conditional mutations in Kras, Pten or both. In addition, we also generated several tumor-derived new cell lines that reproduce the original tumor phenotype in vivo and mirror late stage metastatic disease. Our results demonstrate that de novo activation of oncogenic Kras does not trigger increased proliferation, cellular transformation or EMT, and prevents MUC1 upregulation. In contrast, Pten deletion accelerates cell proliferation, triggers cellular transformation in vitro and in vivo, and stimulates MUC1 expression. Ovarian tumor-derived cell lines MKP-Liver and MKP-Lung cells reproduce in vivo EMT and represent the first immune competent mouse model for distant hematogenous spread. Whole genome microarray expression analysis using tumor and OSE-derived cell lines reveal a 121 gene signature associated with EMT and metastasis. When applied to n=542 cases from The Cancer Genome Atlas (TCGA) ovarian cancer dataset, the gene signature identifies a patient subset with decreased survival (P=0.04). Using an extensive collection of novel murine cell lines we have identified distinct roles for Kras and Pten on MUC1 and EMT in vivo and in vitro. The data has implications for future design of combination therapies targeting Kras mutations, Pten deletions and MUC1 vaccines.

The ability of stress to induce immune suppression is widely recognized, but the mechanisms underlying the effects of stress on the adaptive immune system during tumor progression are not completely understood. To study the effect of stress on the immune system in vivo, we used a preclinical immunocompetent mouse model bearing 4T1 mammary adenocarcinoma cells. Mice were randomized into 4 groups, including social isolation (SI), acute restraint stress (aRRS), chronic restraint stress (cRRS), or no stress (NS). We found that SI significantly decreased the number of tumor-bearing mice still alive at the end of protocol (28 days), compared to NS mice. Although we did not detect significant changes in primary tumor volume, we observed a significant increase in the endothelial marker CD31 in primary tumors of SI mice and in lung metastases in SI and RRS mice. Survival decline in SI mice was associated with significant decreases in splenic CD8 cells and in activated T cells. From a mechanistic standpoint, RRS increased expression of FOXP3, CXCL-10, and granzyme B in mouse tumors, and the effects were reversed by propranolol. Our data demonstrate that various forms of stress differentially impact adaptive immunity and tumor angiogenesis, and negatively impact survival.

Purpose: Mechanisms of immune dysregulation associated with advanced tumors are relatively well understood. Much less is known about the role of immune effectors against cancer precursor lesions. Endometrioid and clear-cell ovarian tumors partly derive from endometriosis, a commonly diagnosed chronic inflammatory disease. We performed here a comprehensive immune gene expression analysis of pelvic inflammation in endometriosis and endometriosis-associated ovarian cancer (EAOC). Experimental Design: RNA was extracted from 120 paraffin tissue blocks comprising of normal endometrium (n = 32), benign endometriosis (n = 30), atypical endometriosis (n = 15), and EAOC (n = 43). Serous tumors (n = 15) were included as nonendometriosis-associated controls. The immune microenvironment was profiled using Nanostring and the nCounter GX Human Immunology Kit, comprising probes for a total of 511 immune genes. Results: One third of the patients with endometriosis revealed a tumor-like inflammation profile, suggesting that cancer-like immune signatures may develop earlier, in patients classified as clinically benign. Gene expression analyses revealed the complement pathway as most prominently involved in both endometriosis and EAOC. Complement proteins are abundantly present in epithelial cells in both benign and malignant lesions. Mechanistic studies in ovarian surface epithelial cells from mice with conditional (Cre-loxP) mutations show intrinsic production of complement in epithelia and demonstrate an early link between Kras- and Pten-driven pathways and complement upregulation. Downregulation of complement in these cells interferes with cell proliferation. Conclusions: These findings reveal new characteristics of inflammation in precursor lesions and point to previously unknown roles of complement in endometriosis and EAOC. Clin Cancer Res; 20(23); 6163–74. ©2014 AACR.

Purpose: Endometriosis, a largely benign, chronic inflammatory disease, is an independent risk factor for endometrioid and clear cell epithelial ovarian tumors. We aimed to identify plasma miRNAs that can be used to differentiate patients with endometriosis and ovarian cancer from healthy individuals. Experimental Design: We conducted a two-stage exploratory study to investigate the use of plasma miRNA profiling to differentiate between patients with endometriosis, patients with endometriosis-associated ovarian cancer (EAOC), and healthy individuals. In the first stage, using global profiling of more than 1,000 miRNAs via reverse transcriptase quantitative PCR (RT-qPCR) in a 20-patient initial screening cohort, we identified 23 candidate miRNAs, which are differentially expressed between healthy controls (n = 6), patients with endometriosis (n = 7), and patients with EAOC (n = 7) based on the fold changes. In the second stage, the 23 miRNAs were further tested in an expanded cohort (n = 88) of healthy individuals (n = 20), endometriosis (n = 33), EAOC (n = 14), and serous ovarian cancer cases (SOC; n = 21, included as controls). Results: We identified three distinct miRNA signatures with reliable differential expression between healthy individuals, patients with endometriosis, and patients with EAOC. When profiled against the control SOC category, our results revealed different miRNAs, suggesting that the identified signatures are reflective of disease-specific pathogenic mechanisms. This was further supported by the fact that the majority of miRNAs differentially expressed in human EAOCs were mirrored in a double transgenic mouse EAOC model. Conclusion: Our study reports for the first time that distinct plasma miRNA expression patterns may serve as highly specific and sensitive diagnostic biomarkers to discriminate between healthy, endometriosis, and EAOC cases. Clin Cancer Res; 19(5); 1213–24. ©2013 AACR.

Epithelial ovarian cancer is an aggressive malignancy, with a low 5-year median survival. Continued improvement on the development of more effective therapies depends in part on the availability of adequate preclinical models for in vivo testing of treatment efficacy. Mucin 1 (MUC1) glycoprotein is a tumor-associated antigen overexpressed in ovarian cancer cells, making it a potential target for immune therapy. To create a preclinical mouse model for MUC1-positive ovarian tumors, we generated triple transgenic (Tg) mice that heterozygously express human MUC1(+/-) as a transgene, and carry the conditional K-rasG12D oncoallele (loxP-Stop-loxP-K-ras(G12D/+)) and the floxed Pten gene (Pten/(loxP/loxP)). Injection of Cre recombinase-encoding adenovirus (AdCre) in the ovarian bursa of triple (MUC1KrasPten) Tg mice triggers ovarian tumors that, in analogy to human ovarian cancer, express strongly elevated MUC1 levels. The tumors metastasize loco-regionally and are accompanied by high serum MUC1, closely mimicking the human disease. Compared with the KrasPten mice with tumors, the MUC1KrasPten mice show increased loco-regional metastasis and augmented accumulation of CD4+Foxp3+ immune-suppressive regulatory T cells. Vaccination of MUC1KrasPten mice with type 1 polarized dendritic cells (DC1) loaded with a MUC1 peptide (DC1-MUC1) can circumvent tumor-mediated immune suppression in the host, activate multiple immune effector genes and effectively prolong survival. Our studies report the first human MUC1-expressing, orthotopic ovarian tumor model, reveal novel MUC1 functions in ovarian cancer biology and demonstrate its suitability as a target for immune-based therapies.

Ovarian cancer patients with persistent (platinum-resistant) or progressive (platinum-refractory) disease respond poorly to second line chemotherapy and have low survival expectancy. New and improved therapeutic approaches are needed and immune biologics are one possibility. Interleukin-2 (IL-2) is a T-cell growth factor believed to be important in anti-tumor immunity. We performed a phase II clinical trial with intraperitoneal (IP) recombinant IL-2 administered in weekly infusions of 6 x 10(5) IU/m2. Thirty-one subjects were sequentially entered into the study and clinical responses were surgically confirmed in 24 patients. The primary end point of this study was clinical response with immunologic measurements as secondary end points. The IP regimen was generally well tolerated. Of the 24 patients assessed for response, there were 6 (4 complete, 2 partial) responses for an overall response rate of 25.0% [95% confidence interval (CI) of 11-45]. The median survival of the 31 patient cohort was 2.1 years (95% CI of 1.3-4.4), but for the 6 patients with responses the median survival has not been reached (range 24-120+ months). Eosinophil and lymphocyte numbers were continuously monitored during treatment. Peripheral blood eosinophils were markedly increased at the completion of treatment (p0.0001) and associated with increased circulating eotaxin (p = 0.03). We also found significant associations between changes in CD3 counts and survival (p = 0.05) and between IFNγ- secreting CD8 T cells at early time points and survival (p = 0.04). This study provides important evidence for IP IL-2 in platinum-resistant ovarian cancer and identifies several immune correlates of survival.

Increased expression and activation of receptor tyrosine kinases frequently occur in human brain tumors, mediating a variety of growth-promoting pathways and leading to radioresistance; however, little is known about their motogenic potency relative to one another. In this study, we found co-expression of Insulin like growth factor-1 receptor (IGF-1R) and platelet derived growth factor receptor (PDGFR) in two high-grade gliomas (HGG) cell lines 18 and 38. Dual targeting of IGF-1R and PDGFR increased cell death in both 18 and 38 cell lines in comparison to inhibition of either receptor alone. In addition, co-inhibition of IGF-1R and PDGFR increased radiosensitivity in 18 cells but failed to intensify the effect of radiation in 38 cells. In HGG cells, radiation-induced cell death has been connected to the activation of c-Jun-NH2-terminal kinase-1 (JNK1). We found that JNK1 was weakly expressed in 38 cells while it had an elevated expression in 18 cells. Exposure to ionizing radiation induced JNK1 activation only in 18 cells without affecting the protein activity in 38 cells. These results suggest that in 18 cell line radiation-activated JNK1 may provide an anti-proliferative signaling, parallel to receptors co-targeting. To test this hypothesis, HGG cells were treated with dominant negative JNK1 (dnJNK1) and the response to radiation was assayed in presence or absence of receptors co-inhibition. Indeed dnJNK protected 18 cells against gamma-irradiation-induced cell death. dnJNK treatment did not influence radiation response of the 38 cell line, which expressed low levels of JNK1. In conclusion we found that IGF-1R and PDGFR co-inhibition caused an increased cell death in two HGG cell line and induced the radiosensitization of the JNK1 expressing cell line.

The insulin-like growth factor-1 receptor (IGF-1R) mitogenic signaling mediates malignant cell survival by many complex and redundant pathways. This study compared the effects of IGF-1R inhibition on viability and apoptosis of two NSCLC cell lines, using three different methods for the impairment of IGF-1R function: (IR3, an anti-IGF-1R antibody; tyrphostin AG1024, a tyrosine kinase inhibitor (TKI) and IGF-1R-small interfering RNA (siRNA). IGF-1R inhibition led to a decrease of cell survival and induced apoptosis in a manner depending on the approach used for the receptor inhibition. To find an explanation, we analyzed the effects of these treatments on three major antiapoptotic pathways evoked by IGF-1R signaling: IRS-1, Shc and 14.3.3-dependent mitochondrial translocation of Raf-1 kinase (mitRaf). (IR3 downregulated IRS-1 phosphorylation in A549 cells and Shc phosphorylation in U1810 cells. While in A549 cells AG1024 treatment decreased both IRS-1 and Shc phosphorylation, in U1810 cells the IRS-1 phosphorylation was only slightly affected and the Shc phosphorylation drastically downregulated. Neither (IR3 nor AG1024 had any effect on Raf-1 kinase translocation. Irrespective of the cell line, IGF-1R-siRNA treatment induced downregulation of both IRS-1 and Shc phosphorylation coupled with the abrogation of mitRaf. In addition, the IGF-1R-siRNA proved to be the most potent inducer of apoptosis suggesting that more than one antiapoptotic pathway in IGF-1R signaling should be inhibited to effectively induce apoptosis in lung cancer cells.

Activating mutations of Kras oncogene and deletions of Pten tumor suppressor gene play important roles in cancers of the female genital tract. We developed here new preclinical models for gynecologic cancers, using conditional (Cre-loxP) mice with floxed genetic alterations in Kras and Pten. The triple transgenic mice, briefly called MUC1KrasPten, express human MUC1 antigen as self and carry a silent oncogenic KrasG12D and Pten deletion mutation. Injection of Cre-encoding adenovirus (AdCre) in the ovarian bursa, oviduct or uterus activates the floxed mutations and initiates ovarian, oviductal, and endometrial cancer, respectively. Anatomical site-specific Cre-loxP recombination throughout the genital tract of MUC1KrasPten mice leads to MUC1 positive genital tract tumors, and the development of these tumors is influenced by the anatomical environment. Endometrioid histology was consistently displayed in all tumors of the murine genital tract (ovaries, oviducts, and uterus). Tumors showed increased expression of MUC1 glycoprotein and triggered de novo antibodies in tumor bearing hosts, mimicking the immunobiology seen in patients. In contrast to the ovarian and endometrial tumors, oviductal tumors showed higher nuclear grade. Survival for oviduct tumors was significantly lower than for endometrial tumors (p = 0.0015), yet similar to survival for ovarian cancer. Oviducts seem to favor the development of high grade tumors, providing preclinical evidence in support of the postulated role of fallopian tubes as the originating site for high grade human ovarian tumors.

We describe an ACT-R model for sentence memory that extracts both a parsed surface representation and a propositional representation. In addition, if possible for each sentence, pointers are added to a long-term memory referent which reflects past experience with the situation described in the sentence. This system accounts for basic results in sentence memory without assuming different retention functions for surface, propositional, or situational information. There is better retention for gist than for surface information because of the greater complexity of the surface representation and because of the greater practice of the referent for the sentence. This model's only inference during sentence comprehension is to insert a pointer to an existing referent. Nonetheless, by this means it is capable of modeling many effects attributed to inferential processing. The ACT-R architecture also provides a mechanism for mixing the various memory strategies that participants bring to bear in these experiments.

In the process of cellular carcinogenesis, genetic and epigenetic mechanisms contribute to abnormal expression of genes. Conrad Waddington introduced the term ‘‘epigenetics’’ in 1942 (Goldberg, Allis et al. 2007) to describe heritable changes in gene expression that have no connection with changes in DNA sequence (Yoo and Jones 2006; Goldberg, Allis et al. 2007). The molecular basis of epigenetics is complex and involves DNA methylation, histone modifications, chromatin remodeling and microRNAs (Esteller 2006).

MUC1 (CA15-3) and MUC16 (CA125) tumor-associated antigens are upregulated in ovarian cancer and can be detected in patients’ sera by standardized tests. We postulated that increased MUC1 and MUC16 antigens augment antibody responses in platinum-resistant ovarian cancer patients and that the frequency and intensity of these responses can be used as immune biomarkers of treatment response and disease outcome. We measured MUC1 and MUC16 tumor expression by immunohistochemistry (IHC), assessed serum antigenic levels and quantitated circulating antibodies by ELISA in a cohort of 28 ovarian cancer patients with platinum-resistant or platinum-refractory ovarian cancer, and treated with intraperitoneal (IP) interleukin-2 (IL-2). MUC1 and MUC16 were overexpressed in tumor samples and showed differential distribution profiles. Serum MUC1 (CA15-3) measurements were elevated in all patients and significantly correlated with increased risk of death (P = 0.003). MUC1-specific IgM and IgG anitbodies were found in 92 and 50% of cases, respectively. Patients with progressive disease had higher mean anti-MUC1 IgG than responders at both early (P = 0.025) and late (P = 0.022) time points during IP IL-2 treatment. Anti-MUC1 IgM antibodies inversely correlated with overall survival at both early (P = 0.052) and late (P = 0.009) time points. In contrast to MUC1, neither soluble MUC16 nor MUC16-specific antibodies were significantly associated with clinical response or overall survival in this study. Increased serum MUC1 and high anti-MUC1 antibody levels are prognostic for poor clinical response and reduced overall survival in platinum-resistant or platinum-refractory ovarian cancer.

Stress triggers complex response mechanisms designed to recognize and adapt to perturbations in homeostasis. The immune system is highly responsive to stress, although the complete mechanisms linking stress and immune mediators including T lymphocytes, are not fully understood. Stress exerts its effects on immune effectors through two primary pathways: the sympathetic-adrenal-medullary pathway, and the hypothalamic-pituitary-adrenal pathway which modulate adaptive immunity and lymphocyte migration. In this report we show that stress via release of stress hormones induces early T cell activation and greatly impacts the cytoskeleton by modulating numerous actin-regulating proteins. In particular, proteomic profiling revealed significant decreases in numerous key actin-binding proteins including moesin. Although confocal microscopy showed that moesin and actin were uniformly distributed on the surface of resting T cells, a remarkable polarization and redistribution of moesin and actin was observed following treatment with stress hormones with moesin localizing at the distal pole complex. In addition, the alteration in moesin localization and eventual decrease in expression were accompanied by a loss of CD43; a receptor involved in negatively regulating T cell activation. In conclusion, we have defined a novel molecular mechanism whereby stress hormones negatively impact T cell activation and migration through regulation of key cytoskeletal and plasma membrane factors.

SUMMARYEndometriosis is defined by the presence of tissue implants resembling endometrial glands outside of the uterus, at ectopic sites, frequently on the ovarian surface. The ectopic lesions are often invasive, resistant to therapy, and may predispose to endometrioid and clear cell ovarian tumors. The complex mechanisms leading to chronic endometriosis are mediated partly by impaired immune surveillance in the host. Although innate immunity has been addressed previously, the response of adaptive immune effectors to specific antigens has not been characterized, mostly because very few endometriosis antigens have been defined to date. We postulated that the mucin 1 (MUC1) glycoprotein, which is normally present on eutopic human endometrial glands and overexpressed in endometrioid and clear cell ovarian tumors, is also present in ectopic lesions of ovarian endometriosis. Furthermore, changes in MUC1 expression in endometriosis could promote adaptive anti-MUC1 immunity that might play a role in the malignant progression. To test our hypothesis, we crossed MUC1 transgenic mice, which express human MUC1 under the endogenous promoter, with the loxP-Stop-loxP-KrasG12D/+ (Kras) mice, in which endometriosis can be induced through Cre-loxP recombination. The double transgenic MUC1Kras mice develop benign, MUC1-positive ovarian lesions, closely resembling human endometriosis. Subsequent to disease induction, the mice generate high titers of IgM and IgG antibodies that are specific for MUC1. Antibodies appear early in disease and the predominance of the IgG1 subclass suggests Th2-driven immunity. Immune phenotyping revealed an accumulation of Foxp3+ CD4 regulatory T cells (Tregs) in the draining lymph nodes at late-stage disease. Furthermore, our observations in human endometriosis showed a similar recruitment of FOXP3+ CD4 T cells. Overall, our results reveal a Th2/Treg-dominant natural immunity in endometriosis with potential implications for cancer progression.

Introduction: Patients with ovarian cancer are usually diagnosed late, when tumors have already spread to the surrounding tissues. Hematogenous dissemination of ovarian cancer cells can also occur and lung metastasis can be found in 12% of ovarian cancer patients. Human mucin1 (MUC1) is a large transmembrane glycoprotein with roles in cell adhesion and migration. More than 80% of ovarian cancer overexpress MUC1, regardless of histology. MUC1 is a widely used vaccine target for numerous adenocarcinomas, including ovarian tumors. Approach and results: We have recently described the triple transgenic (Tg) mice with conditional (cre-loxP) mutations in Kras and Pten pathways that progress to human MUC1-expressing endometrioid ovarian tumors. Using orthotopic tumors from these mice we generated here several new murine cell lines. The MKP-L cell line was derived from an ovarian tumor implant attached to the liver surface. Intraperitoneal (IP) injection of MKP-L cells into syngeneic mice triggers IP tumors and multiple lung metastatic masses. Cells isolated from lung metastases (MKP-Lung cell line) triggered peritoneal tumors as well as lung metastases, suggesting that they retain the original homing capacity. Compared to the parental, endometrioid-looking and low-MUC1 expressing MKP-L cells, the metastatic MKP-Lung cells showed morphological changes consistent to epithelial-to-mesenchymal transition (EMT) and are homogeneously high in MUC1. Mouse WG6 microarray analysis of MKP-L and MKP-Lung cells revealed expression changes of several EMT-associated transcription factors as well as their downstream proteins. Western blot confirmed upregulation of ZEB1, vimentin and downregulation of cytokeratins. To dissect the mechanistic roles of Kras and Pten pathways in EMT and MUC1 regulation, we also generated a series of ovarian surface epithelial (OSE)-derived cell lines with conditional (Cre-loxP) oncogenic mutations in Kras (MKOSE), deletion in Pten (MPOSE) or both (MKPOSE). Using these cell lines we recapitulated in vitro the EMT phenotype and MUC1 upregulation acquired by MKP-Lung cells in vivo. Furthermore, small molecule inhibitors of oncogenic Kras (AZD6244) and Pi3K/Pten pathway (BEZ235) demonstrated that MUC1 expression is significantly upregulated in response to Pten deletion and that Pi3K inhibitor (BEZ235) downregulated MUC1. In contrast, Kras activation slightly lowered MUC1 expression while cell exposure to AZD6244 upregulated MUC1. Conclusions: We report here the first transgenic mouse metastatic ovarian cancer model that expresses human MUC1.Usig this model we identified a gene signature consistent with lung metastasis and EMT and identified mechanisms of MUC1 upregulation during these processes. These findings have important consequences for MUC1 immunobiology in metastatic ovarian tumors with mutations in Kras, Pten/Pi3K or both. Citation Format: Lixin Zhang, Tianzhou Ma, Joan Brozick, Kathlene Babalola, Raluca Budiu, George Tseng, Anda Vlad. Lung metastasis of ovarian cancer in a transplantable MUC1.Tg mouse model is accompanied by upregulation of MUC1 expression and epithelial to mesenchymal transition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4081. doi:10.1158/1538-7445.AM2015-4081

In this study we investigate the comprehension of various kinds of anaphoric metaphors in context. We describe an experiment that manipulated the metaphoricity of simple noun + verb + ending sentences by using either a metaphoric noun or a metaphoric verb or both. Our results show that metaphoric nouns affect sentence comprehension to a greater extent than do metaphoric verbs. Thus, even though there were no sentence-reading time differences between metaphoric and literal targets, metaphoric nouns were read more slowly than were literal nouns, and they also affected the reading time of the following verb. Moreover, in trials involving metaphoric-noun targets, participants read the endings of the targets faster and made more mistakes in answering posttrial questions than they did in literal-noun trials. We argue that these results suggest a comprehension deficit for anaphoric noun metaphors even when they are preceded by a context.

Introduction: Ovarian epithelial tumors are highly heterogeneous. Patients are diagnosed late, when tumors have spread to the surrounding tissues, most often to the omentum. Despite optimal removal and response to therapy, the tumors often recur. Personalized medicine approaches often require molecular profiling of tumors, for patient selection and to predict response to therapy. Given intratumor heterogeneity, identification of tumor sites that are best to select for profiling is still up for debate. Approach: We recently developed triple transgenic (MUC1KrasPten.Tg) mice that develop orthotopic MUC1-expressing endometrioid ovarian tumors. The tumors develop in the ovaries and spread throughout the peritoneal cavity, closely mirroring the human disease. Using tumor cells isolated from either the MUC1KrasPten primary ovarian tumor (MKP-T) or the liver implant (MKP-L), we generated several new murine ovarian cancer cell lines which express human MUC1 as self, while simultaneously carrying constitutively active oncogenic Kras and defective Pten tumor suppressor. Results: MKP-T and MKP-L showed variable expression pattern of MUC1, EpCAM, folate receptor, ALDH, cytokeratin and vimentin, demonstrating the relationship between the anatomical location of each originating tumor and the cell line's in vitro phenotype. Unlike the MKP-L cells, the MKP-T cells display loss of K-ras heterozygosity, suggesting that migration to a metastatic site was an early event. Although both cell lines can grow IP tumors in syngeneic triple transgenic MUC1KrasPten.Tg, they carry distinct in vivo phenotypes. MKP-T cells multiply at higher rate and generate larger loco- regional tumors. In contrast, the MKP-L cells, derived from a metastatic site, have a slower growth rate but spread throughout the peritoneal cavity, undergo epithelial-mesenchymal transition (EMT) and trigger distant metastases to the lungs(MKP-Lng). In line with their Kras status, MEK 1/2 inhibitor AZD6244 (currently tested in several Phase II clinical trials) is more effective on MKP-T cells than on MKP-L and MKP-Lng cells in vitro, suggesting that metastatic tumor cells may be more resistant to MEK1/2 inhibitiors. Conclusions: Using novel murine tumor cell populations, we reproduce intratumoral heterogeneity and demonstrate that in vitro and in vivo biological properties vary with the location of originating tumor. Most importantly, we show that cells derived from metastatic sites, which are more likely to reproduce the characteristic of the recurrent tumors, have an increased metastatic potential and reduced response to therapeutic drugs. These results suggest that molecular profiling of tumors needs to focus on multiple tumor sites and that in ovarian cancer, distant tumor implants may be particularly important. Supported by NIH R01 CA163462 and DOD OC093429. Citation Format: Lixin Zhang, Raluca Budiu, Joan Brozick, Anda M. Vlad. Novel ovarian cancer transplantable models generated from MUC1KrasPten tumors show in vitro and in vivo heterogeneity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4976. doi:10.1158/1538-7445.AM2014-4976

Introduction: Endometriosis is a largely benign, chronic inflammatory disease defined by the presence of endometrial-like glands surrounded by stroma. Epidemiologic studies suggest that endometriosis is an independent risk factor for endometrioid and clear cell epithelial ovarian tumors, collectively called endometriosis-associated ovarian cancers (EAOC). Histopathology findings demonstrate that EAOC occur in the presence of atypical endometriosis (AE), often found in direct continuity with the tumor, suggesting AE as the transitioning entity from benign lesions to malignant variants. Although it is widely accepted that chronic inflammation drives cancer, immune deregulation in chronic precursor lesions to ovarian cancer have not been studied in a systematic way. Experimental procedure: We extracted RNA from 135 paraffin tissue blocks comprising of normal endometrium (n=32), benign endometriosis consisting of ovarian and extra ovarian endometriosis cases (n=30,) atypical endometriosis (n=15) and EAOC, (n=43). Serous tumors (n=15) were included as non-endometriosis associated controls. Using Nanostring and the nCounter® GX Human Immunology Kit, we profiled a total of 511 immune genes. Cluster analyses and differential expressions (DE) were calculated using EdgeR. Protein expression of candidate proteins was validated with immunohistochemistry (IHC). To identify mechanisms of complement activation during early stages of genomic instability, we used murine cell lines derived from mice with conditional mutations in Kras and Pten pathways. Results: Nanostring immune gene expression profile eveal the predominant role of adaptive immunity and of the adaptive-innate immune cross-talk. The complement pathway was most prominently expressed, suggesting its roles as one of the major immune pathways involved in the transition from chronic endometriosis to atypical (premalignant) endometriosis and to EAOC. Complement pathway genes were upregulated in human endometriosis and atypical endometriosis. Tissue deposition of several complement components and changes in peripheral blood were confirmed by IHC and ELISA, respectively. Genomic instability in murine ovarian cancer cell lines with conditional mutations resulted in upregulation of complement genes expression in epithelial cells, but without an advantage on cell death. These findings further support a paradigm shift on complement roles in cancer, suggesting its pro-tumorigenic roles. Conclusions: We performed the first comprehensive gene profile of the tissue immune microenvironment in benign endometriosis, EAOC and precursor lesions and identified the previously unrecognized roles for the complement pathway. Complement activation may be an early trigger of inflammation and an early link between epithelium and immune environment. These findings have high translational potential for immune therapy and prevention in EAOC. Citation Format: Swati Maruti Suryawanshi, Xin Huang, Raluca Budiu, SungHwan Kim, George Tseng, Esther Elishaev, Marcia Klein-Patel, Ted Lee, Suketu Mansuria, Robert Edwards, Anda Vlad. Complement roles in endometriosis and endometriosis-associated ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1653. doi:10.1158/1538-7445.AM2014-1653

Endometriosis is a largely benign, chronic inflammatory disease that may predispose to some of the ovarian tumors histotypes, collectively called endometriosis-associated ovarian cancers (EAOC). Although it is widely accepted that chronic inflammation drives cancer, the immune deregulation in chronic precursors of ovarian cancer has not been systematically studied. We performed here the first comprehensive gene profile of the tissue immune microenvironment in normal endometrium, benign endometriosis and EAOC (n=133), using Nanostring and the nCounter® GX Human Immunology Kit comprising probes for 511 immune genes. We found that the complement pathway was most prominently upregulated, suggesting its involvement in the transition from chronic endometriosis to atypical (premalignant) endometriosis and to EAOC. Complement protein expression was confirmed via immunohistochemistry, which identified upregulation of complement in epithelial cells. To explore the roles of complement in early EAOC carcinogenesis, we derived several novel ovarian cell lines with conditional mutations that can be turned on and off, using the Cre-loxP system. Using this in vitro model we demonstrate that genetic instability triggers upregulation of complement genes in epithelial cells, but without an advantage on cell death. These findings reveal an early link between epithelium and innate immune environment and further support a paradigm shift on complement roles in supporting cancer growth.

Introduction: Sustained improvement in the development of new therapeutic approaches in ovarian cancer depends in part on the availability of adequate preclinical models for in vivo testing of treatment efficacy. We recently developed triple transgenic (MUC1KrasPten.Tg) mice expressing human MUC1 oncoprotein as self and carrying the conditional K-rasG12D oncoallele (loxP-Stop-loxP-K-rasG12D/+) and the floxed Pten gene (PtenloxP/loxP). The MUC1KrasPten.Tg mice develop MUC1-expressing endometrioid ovarian tumors that closely mirror the human disease and respond to human MUC1-based immune therapy. Compared to the KrasPten mice, tumors from MUC1KrasPten.Tg mice had a higher potential for loco-regional spread, suggesting active MUC1 involvement in local and distant tumor spread. Approach: In order to study in vitro and in vivo MUC1 roles in tumor growth and metastasis and interactions between MUC1 signaling and Kras and Pten pathways, we generated several new murine ovarian cancer cell lines. These cell lines express human MUC1 as self, while simultaneously carrying constitutively active oncogenic Kras and defective Pten tumor suppressor. The originating cells were derived from either the MUC1KrasPten primary ovarian tumor or the liver implant via trypsinization into single cell suspension. Cells were cultured in vitro for at least 20 passages to generate the stable MKP-T (from primary tumor) and MKP-L (from liver metastasis) ovarian cancer cell lines. Clonal populations were obtained via limiting dilution assays. Results: Although derived from the same host, the MKP-T and MKP-L cells showed different K-ras status reflective of tumor heterogeneity in vivo. Unlike the MKP-L cells, the MKP-T cells display loss of K-ras heterozygosity. MKP-T and MKP-L showed variable expression pattern of MUC1, EpCAM, folate receptor, ALDH, cytokeratin and vimentin, demonstrating how the anatomical location of each originating tumor influences the cell line's in vitro phenotype. Furthermore, although both cell lines can grow IP tumors in syngeneic triple transgenic MUC1KrasPten.Tg, single transgenic MUC1.Tg as well as wild-type C57BL/6J-bgJ mice, they carry distinct in vivo phenotypes. MKP-L cells grow slower in vitro (doubling time 27.5 hours) and in vivo and trigger distant metastases to the lungs. MKP-T cells multiply at higher rate in vitro (doubling time 13.2 hours) and in vivo and generate larger loco- regional tumors. Two sub-lines (MKP-T-2F8-Asc and MKP-L-Lng) derived from MKP-T ascites and MKP-L lung metastasis, respectively, by in vivo clonal selection, showed stable and high MUC1 expression, further demonstrating its in vivo roles for both loco-regional as well as distant spread. In line with their Kras status, MEK 1/2 inhibitor AZD6244 (currently tested in several Phase II clinical trials) showed more inhibition on MKP-T cells than on MKP-L cells in vitro, providing support for its use in this preclinical models. Conclusions: We have generated several novel murine cancer cell lines and characterized the first immune competent transplantable ovarian cancer model expressing human MUC1 as a transgene. The cell lines show various levels of MUC1 and display different metastatic behaviors in vitro and in vivo. MKP-L cells metastasize to the lungs and represent the first in vivo model for distant metastasis in ovarian cancer. Our novel preclinical models are uniquely suited for in vivo combination therapies targeting MUC1, Kras and Pten/PI3K pathways and provide new tools for elucidating the biological behaviors of ovarian cancer cells both in vitro and in vivo. Citation Format: Lixin Zhang, Raluca Budiu, Joan Brozick, Anda Vlad. Novel ovarian cancer transplantable models generated from MUC1KrasPten tumors show different in vitro and in vivo biological behaviors. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B66.

Introduction: Epithelial ovarian cancer (EOC) is detected in late stages, when mortality is high due to the lack of an effective screening modality. One of the possible accepted precursors for endometrioid, clear cell and low grade serous ovarian tumors is endometriosis, a chronic inflammatory disease that can affect up to 10-15% of women in the reproductive age group. MicroRNAs (miRNAs) are important regulators of numerous biological pathways. In the current study, we investigated role of miRNAs in the transition from benign endometriosis to cellular atypia and to malignancies. Experimental procedure: We extracted RNA from 150 paraffin tissue blocks comprising of normal endometrium (n=31), benign endometriosis consisting of ovarian and extra ovarian endometriosis cases (n=32,) atypical endometriosis (n=15) and endometriosis associated ovarian cancer, consisting of clear cell and endometrioid ovarian cancer cases (n=41). Serous tumors (n=15) were included as non-endometriosis associated controls. Using NanoString miRNA Expression Assay, we globally profiled the expression of 800 miRNAs in these samples. Cluster analyses and differential expressions (DE) were calculated using EdgeR. We also validated our NanoString results across all disease categories by miRNA real-time PCR assays. Results: Many miRNAs are dysregulated in atypical endometriosis and cancer samples. Interestingly, several novel miRNAs have been identified that are predicted to regulate genes that may promote carcinogenesis in endometriosis, such as genes involved in estrogen signaling pathways and immune response. Among these miRNAs, differential expression of the miR-200 family between healthy controls and patients with endometriosis and EAOC is particularly intriguing. The miR-200 family of microRNAs consisting of miR-200a, 200b, 200c, 141 and 205, functions in various biological and pathological processes, especially on cellular oxidative stress response and during epithelial to mesenchymal transition (EMT). Our analysis demonstrated that expression of miR-200 family members is downregulated in endometriosis and highly upregulated in EAOC tissue as compared to normal tissues. Conclusion: Our global miRNA profiling results suggest that miR-200 family potentially plays an important role during the transition from benign endometriosis to endometriosis-associated ovarian cancer. Citation Format: Swati Suryawanshi, Anda Vlad, Robert Edwards, Raluca Budiu, Xin Huang. miR-200 family in endometriosis-associated ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A75.

Introduction: Endometriosis is a largely benign, chronic inflammatory disease defined by the presence of endometrial-like glands surrounded by stroma. Epidemiologic studies suggest that endometriosis is an independent risk factor for endometrioid and clear cell epithelial ovarian tumors, collectively called endometriosis-associated ovarian cancers (EAOC). Although it is widely accepted that chronic inflammation drives cancer, immune deregulation in chronic precursor lesions to ovarian cancer have not been studied in a systematic way. Hypothesis: Gene expression traits intrinsic to the premalignant endometriotic epithelium (leading to fields of atypia and eventually invasive cancer) result in highly specific quantitative and qualitative changes of the immune stroma that may differentiate benign from pre-malignant and malignant lesions. Experimental procedure: We extracted RNA from 150 paraffin tissue blocks comprising of normal endometrium (n=32), benign endometriosis consisting of ovarian and extra ovarian endometriosis cases (n=32,) atypical endometriosis (n=15) and EAOC, (n=41). Serous tumors (n=15) were included as non-endometriosis associated controls. Using Nanostring and the nCounter® GX Human Immunology Kit, we profiled a total of 511 immune genes. Cluster analyses and differential expressions (DE) were calculated using EdgeR. ELISAs were done to measure levels of complement factors in plasma samples of healthy controls (n=18), patients with EAOC (n = 12), endometriosis (n=34) and serous ovarian cancer (n = 15). Protein expression of candidate proteins was validated with immunohistochemistry (IHC). Results: Nanostring immune gene expression profile demonstrates that non ovarian and ovarian endometriosis develop in distinct immune microenvironments with distinct cancer risk. Based on all (unsupervised) gene expression, non-ovarian endometriosis resembles that of normal endometrium while the ovarian endometriotic lesions cluster closer to ovarian cancer. Fewer immune genes differentiate atypical from ovarian than extra-ovarian endometriosis. Several of the DE genes demonstrate decreased Th1 and NK cell cytotoxicity, increased B cell activation and antibody production, further suggesting why women with ovarian endometriosis may be at higher risk of ovarian cancer. Overall, the top 3 pathways reveal the predominant role of adaptive immunity and of the adaptive-innate immune cross-talk. The complement pathway was most prominently expressed, suggesting its roles as one of the major immune pathways involved in the transition from chronic endometriosis to atypical (premalignant) endometriosis and to EAOC. Tissue deposition of several complement components and changes in peripheral blood were confirmed by IHC and ELISA, respectively. We are currently investigating the mechanisms by which complement cascades influence the tumor microenvironment, epithelium-immune cell interactions and early development of EAOC. Conclusions: We performed the first comprehensive gene profile of the tissue immune microenvironment in benign endometriosis, EAOC and precursor lesions and identified the previously unrecognized roles for the complement pathway. These findings have high translational potential for immune therapy and prevention in EAOC. Citation Format: Swati Suryawanshi, Xin Huang, Raluca Budiu, Gina Mantia, Robert Edwards, Anda Vlad. Immune mechanisms that drive early carcinogenesis in endometriosis-associated ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B71.

Immune suppression plays a critical role in tumor progression, particularly in triple negative breast cancer (TNBC) and ovarian cancer. However the role of stress in adaptive immunity is still unclear. We hypothesize that psychological stress can regulate T cell mediated immunity, thus contributing to disease outcome. To test this hypothesis, we used female BALB/c mice injected with TNBC 4T1 mammary adenocarcinoma cells into the mammary fat pad and a LSLS-KrasG12D/+PtenloxP/loxp conditional transgenic mouse model for endometrioid ovarian cancer. For the TNBC model, mice were randomized into 4 groups (n = 10); (1) social isolation; (2) restraint stress for 1 h,3x/week; (3) restraint stress for 2 h, daily and (4) no stress. For the ovarian model, mice were restrained for 1 h,3x week. In the TNBC model, the stress groups demonstrated significantly reduced survival compared to non-stressed controls (p = 0.013) with none of the social isolation mice surviving. There was a significant difference across the groups for CD3 (p = 0.042), CD3CD69 (p = 0.047) and CD4FOXP3 (Tregs; p = 0.0014) in mouse spleens. In the ovarian mouse model, we found that stressed mice demonstrated early onset of tumors and a significant decrease in CD3CD69 (p = 0.006). These findings indicate that stress regulates T cell immunity in mice with TNBC and ovarian cancer and demonstrate the complexity of the stress response in tumor-bearing, immune competent mice.

Background: Despite improvements in surgical techniques and chemotherapy approaches, ovarian cancer continues to be associated with a low 5 year survival. Sustained improvement in the development of new therapeutic approaches depends in part on the availability of adequate preclinical models for in vivo testing of treatment efficacy. MUC1 oncoprotein is a tumor -associated antigen expressed by all epithelial ovarian tumors and a valuable target for immunotherapy. Objective: To test the therapeutic efficacy of a MUC1-based vaccine using dendritic cells matured in type 1 polarizing conditions (DC1). This vaccine was administered to novel triple transgenic conditional tumor-bearing mice, which we recently generated and characterized. Methods: Our new triple transgenic mice (MUC1+/−LSLKrasG12D/+PtenloxP/loxP) co-express a Kras oncogenic mutation and a Pten deficiency (previously described) and carry the MUC1 transgene encoding for the human MUC1 tumor-associated antigen, driven by the endogenous Muc1 promoter. We induced tumors in n=21 double and n=18 triple transgenic (Tg) mice by AdCre injection under the ovarian bursa, during survival surgery. Five of the 18 mice were vaccinated with 3x 103 DC1, grown in GM-CSF, matured with IL-4, IFNγ, LPS and Poly I:C (DC1 cocktail) and pulsed with 10ug/ml 100mer MUC1 peptides (DC1-MUC1). The vaccine was administered s.c 3 times, 2 weeks apart starting at week 5 after AdCre injection. We measured natural and vaccine- induced immune responses via ELISA, multicolor flow cytometry, and real-time PCR arrays. Results: Intrabursal administration of AdCre in the double (KrasPten) and triple (MUC1KrasPten) Tg mice leads to de novo ovarian epithelial tumors. The tumors occur in the injected ovary only, spread throughout the abdominal cavity and on the diaphragm and may be accompanied by hemorrhagic ascites. Characterization of the tumor environment revealed increase PGE2 accumulation in ascites and overexpression of COX2 at the tumor site and throughout the injected side of the female genital tract. Despite the histomorphologic similarities of MUC1KrasPten and KrasPten mice neoplasms, only the triple Tg tumors express MUC1, mimicking even closer the human disease. The overall survival was shorter in triple Tg compared to double Tg mice (average of 65 and 82 days, respectively, p = 0.105) pointing out to important MUC1 roles in ovarian tumor biology. DC1-TnMUC1 vaccine significantly increases overall survival (p = 0.003) and decreases CD4+FOXP3+ Treg cells in spleen, compared with non-vaccinated tumor-bearing mice (p < 0.001). Conclusions: MUC1KrasPten mice represent the first preclinical model for orthotopic ovarian carcinogenesis expressing the human MUC1 antigen. This preclinical data provides a platform for using MUC 1 vaccine as a form of immunotherapy in ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 751. doi:10.1158/1538-7445.AM2011-751

Background: Ovarian cancer continues to be associated with a very high mortality rate. Improvements on the development of new therapeutic approaches, including antigen-specific immune therapies, depend in part on the availability of adequate preclinical models for in vivo testing of treatment efficacy. MUC1 mucin is a tumor associated antigen and a potential target for cancer immunotherapy. Human and murine mucin 1 share little homology in the extracellular region and transgenic mouse models with de novo lesions expressing the human antigen are needed. Objective: To identify MUC1 antigen-specific immune regulation in mice with de novo malignant ovarian lesions. Methods: In order to generate a human MUC1 antigen expressing ovarian cancer mouse model, we crossed the MUC1 transgenic mice which express human MUC1 under the endogenous promoter with the previously described KrasG12D/+PtenloxP/loxP mice that develop orthotopic ovarian tumors. Results: The newly generated MUC1+/−KrasG12D/+ PtenloxP/loxP triple transgenic mice constitute a valuable Cre-loxP conditional in vivo model for MUC1 expressing ovarian tumors. Upon injection of Cre-encoding adenovirus under the ovarian bursa, the triple transgenic mice develop aggressive ovarian tumors with clear cell histology. The lesions express high levels of MUC1 and are estrogen receptor and cytokeratin 7-positive, markers classically used to diagnose malignant epithelial proliferation. The tumors are accompanied by ascites and numerous peritoneal implants, mimicking late stage disease clinically seen in women. We identified increased accumulation of Treg cells in the para-aortic (draining) lymph nodes and presence of Th17 cells in ascites, mimicking closely the immune phenotype of the human disease. In addition, using quantitative PCR T cell gene profiler arrays, we identified 5 significantly up- and 16 down-regulated genes in the spleen T cells of tumor-bearing mice. One of the upregulated genes was osteopontin (Spp1), recently validated as a marker in detecting human recurrent ovarian cancer. Among the down-regulated genes we identified, Cd4, Cd40ligand (Cd40lg), Interferon-g (Ifnγ), IL-23 receptor (IL-23ra) all suggestive of a shift in immune effectors, some of which may promote the chronic inflammatory background. Conclusions: Our results reveal important immune regulatory mechanisms in mice with de novo MUC1-expressing ovarian tumors and provide unique opportunities for future in vivo testing of MUC1 vaccines as ovarian cancer therapy and potentially immune prevention. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4793.

Objective: Examine the expression and immunogenicity of a known ovarian tumor-associated antigen, MUC1, in ectopic endometriosis lesions and epithelial ovarian cancer (EOC). Determine whether an imbalance in CD4 T cell subsets contributes towards endometriosis pathogenesis and ovarian carcinogenesis. Methods: Biologic specimens were obtained from women with endometriosis, endometriosis-related (endometrioid and clear cell) or serous EOC, and healthy controls, according to University of Pittsburgh IRB protocols. MUC1 and immune cell expression were determined via immunohistochemistry (IHC). Anti-MUC1 IgM and IgG antibody levels were determined via ELISA. Peripheral blood mononuclear cells and cryopreserved cell suspensions from tissue lesions were immunophenotyped using multicolor flow cytometry. Stroma and epithelial cells from tissue biopsies were collected by laser capture microdissection (LCM) and examined with immune-based quantitative RT-PCR arrays. Results: MUC1 was overexpressed in 67% of endometriosis and 89% of EOC lesions. Women with early or late stages of endometriosis had significantly higher anti-MUC1 IgM responses when compared to those women with endometriosis-related EOC (25.3 % of positive control vs.14.2%, p=0.03; 26.3 vs. 14.2%, p=0.04) and to those with serous EOC (25.3 vs. 14.6%, p=0.04; 26.3 vs.14.6%, p=0.04). Women without disease also had significantly higher anti-MUC1 IgM responses compared to endometriosis-related (29.2 vs.14.2%, p=0.01) and serous EOC (29.2 vs.14.6%, p=0.02). Immune cells, including CD3, CD4, and CD8 T cells, are present in endometriotic lesions and appear more abundant than in eutopic endometrium. Flow cytometric analysis of endometriotic lesion-infiltrating T cells revealed a decrease in the frequency of IFN-γ CD4 and CD8 T cells, similar to that seen in patients with EOC. Conclusions: MUC1 is present and appears to be overexpressed in ectopic endometriotic and EOC lesions. Women with endometriosis-related EOC or with serous EOC had significant lower anti-MUC1 IgM levels, suggesting that a compression of antigen-specific humoral (i.e., Th2 driven) immunity may play a role in carcinogenesis. Endometriotic and EOC lesions also appear to have diminished Th1 immunity, suggesting a possible role for local immune suppression. Quantitative analyses of several immune genes in the lesion microenvironment are currently ongoing and will potentially contribute to immune biomarker discovery and improvements in the early detection and prevention of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1335.