Esther Elishaev

Angiopoietin-2 Stimulates Breast Cancer Metastasis through the α5β1 Integrin-Mediated Pathway

Acquisition of a metastatic phenotype by breast cancer cells includes alternations of multigenic programs that permit tumor cells to metastasize to distant organs. Here, we report that angiopoietin-2 (Ang2), a known growth factor, is capable of promoting breast cancer cell invasion leading to metastasis. Analysis of 185 primary human breast cancer specimens that include 97 tumors showing lymph node and/or distant metastasis reveals a significant correlation between the expression of Ang2 and E-cadherin, Snail, metastatic potential, tumor grade, and lymph-vascular invasion during breast cancer progression. Using a xenograft model, we show that overexpression of Ang2 in poorly metastatic MCF-7 breast cancer cells suppresses expression of E-cadherin and induces Snail expression and phosphorylation of Akt and glycogen synthase kinase-3beta (GSK-3beta) promoting metastasis to the lymph nodes and lung. In cell culture, Ang2 promotes cell migration and invasion in Tie2-deficient breast cancer cells through the alpha(5)beta(1) integrin/integrin-linked kinase (ILK)/Akt, GSK-3beta/Snail/E-cadherin signaling pathway. Inhibition of ILK and the alpha(5)beta(1) integrin abrogates Ang2 modulation of Akt, GSK-3beta, Snail, and E-cadherin and Ang2-stimulated breast cancer cell migration and invasion. Together, these results underscore the significant contribution of Ang2 in cancer progression, not only by stimulating angiogenesis but also by promoting metastasis, and provide a mechanism by which breast cancer cells acquire an enhanced invasive phenotype contributing to metastasis.

TLR4 signaling induced by lipopolysaccharide or paclitaxel regulates tumor survival and chemoresistance in ovarian cancer.

Toll-like receptors (TLRs) expressed on immune cells trigger inflammatory responses. TLRs are also expressed on ovarian cancer (OvCa) cells, but the consequences of signaling by the TLR4/MyD88 pathway in these cells are unclear. Here, TLR4 and MyD88 expression in OvCa tissues (n=20) and cell lines (OVCAR3, SKOV3, AD10, A2780 and CP70) was evaluated by reverse transcriptase–PCR, western blots and immunohistochemistry. Cell growth, apoptosis, nuclear factor-κB (NF-κB) translocation, IRAK4 and TRIF expression and cJun phosphorylation were measured following tumor cell exposure to the TLR4 ligands, lipopolysaccharide (LPS) or paclitaxel (PTX). Culture supernatants were tested for cytokine levels. TLR4 was expressed in all tumors, tumor cell lines and normal epithelium. MyD88 was detectable in tumor tissues and in 3/5 OvCa lines but not in normal cells. In MyD88+ SCOV3 cells, LPS or PTX binding to TLR4 induced IRAK4 activation and cJun phosphorylation, activated the NF-κB pathway and promoted interleukin (IL)-8, IL-6, vascular endothelial growth factor and monocyte chemotactic protein-1 production and resistance to drug-induced apoptosis. Silencing of TLR4 in SCOV3 cells with small interference RNA resulted in phosphorylated-cJun (p-cJun) downregulation and a loss of PTX resistance. In PTX-sensitive, MyD88neg A2780 cells, TLR4 stimulation upregulated TRIF, and TLR4 silencing eliminated this effect. Thus, TLR4/MyD88 signaling supports OvCa progression and chemoresistance, promoting immune escape.

Plasma MicroRNAs as Novel Biomarkers for Endometriosis and Endometriosis-Associated Ovarian Cancer

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.

Sentinel lymph node biopsy in early-stage cervical cancer: Utility of intraoperative versus postoperative assessment

OBJECTIVE To determine the diagnostic accuracy of sentinel lymph node (SLN) detection using lymphoscintigraphy, intraoperative blue dye, and radiocolloid in patients with early-stage cervical cancer. METHODS Intra-cervical injection of technetium-99 sulfur colloid and lymphoscintigraphy were performed preoperatively. Isosulfan blue was injected intra-cervically immediately prior to surgery. SLNs were excised and examined intraoperatively (imprint cytology and frozen section) and postoperatively (H and E histology and immunohistochemistry (IHC) for cytokeratin). RESULTS Thirty eight patients were evaluable. Laparoscopy and laparotomy were performed in 28.9% and 71.1%, respectively. Subjects had squamous cell carcinoma (n=26), adenocarcinoma (n=10) or adenosquamous (n=2) histologies. 55.3% had cervical tumors <2 cm. The overall SLN detection rate was 92.1%. The external iliac region just distal to the common iliac bifurcation was the most common SLN location. A mean of 2.1 SLNs were detected per patient with bilateral SLNs observed in 47.4%. On final pathology, metastatic nodal disease was identified in 15.7% of patients. Of these, 83.3% were detected in the SLNs. Sensitivity of SLN detection of metastasis was 100% for patients with cervical tumors <2 cm. However intraoperative evaluation by imprint cytology and frozen section correctly identified lymph node metastasis in only 33.3%. CONCLUSIONS SLN detection is feasible and accurately reflects pelvic nodal basin status when performed in early-stage cervical cancer patients. However, while current intraoperative pathology techniques for assessing nodal metastases reliably detect metastases larger than 2 mm, they lack sufficient sensitivity to detect micrometastasis and isolated tumor cells.

Introduction of the Thin Prep Imaging System (TIS): experience in a high volume academic practice.

Objective Since the introduction of the liquid-based ThinPrep testing in 1996, most cytology laboratories across the country have adopted the liquid-based cytology (LBC) for Pap test screening. Subsequent to wide-spread adoption of the ThinPrep Pap test, the ThinPrep Imaging System (TIS) Cytyc Corp, Marlborough, MA was introduced to improve the accuracy and efficiency of screening interpretation. We report our initial experience with the TIS at Magee Womens Hospital. We introduced the TIS in December 2004. Methods The imager assisted Pap test results over the first 12 months (December 2004 to December 2005) of implementation were reviewed and analyzed. Our implementation protocol included each cytotechnologist manually prescreening 200 negative slides to gain experience with the imager slides and serve as a quality check for the TIS. We re-screened 3400 slides (200 slides each for 17 cytotechnologists) manually which were initially determined to be negative using the TIS. 104,457 Pap tests were imaged on the TIS. 95,899 manually screened Pap tests, 12 months prior to the introduction of the TIS (December 2003–November 2004) are taken as the historic control group for our study. Results The mean ASC-US rate employing the automated imager was 8.70% [9088/104,457]. The mean LSIL detection rate was 4.22% [4409/104,457]. The imager did not miss any detectible high-grade lesions during these months, with a HSIL (+) detection rate of 0.68% in comparison to 0.60% by manual screening confirmed by follow-up biopsies. The difference is statistically significant with a p value of 0.022. The definition of false negative rate for purposes of this study is calculated as the number of false negative cases identified out of number of negatives re-screened. The TIS false negative rate was estimated at 0.012% [4/3400]. Conclusion The overall performance of the TIS in our lab appears to be highly satisfactory in terms of improving sensitivity in screening cervical precursor lesions. The increased accuracy of detection of HSIL indicates a positive impact of the TIS in our laboratory.

Very low human papillomavirus DNA prevalence in mature women with negative computer-imaged liquid-based Pap tests

The prevalence of high‐risk Human Papillomavirus DNA (hrHPV DNA) in women with negative Papanicolaou (Pap) test results provides a measure of residual risk for cervical neoplasia after cytology screening. The purpose of this study was to document the prevalence of hrHPV DNA in several thousand women ages ≥30 years with negative ThinPrep Imaging System (TIS)‐imaged Pap test results in a large academic hospital cytology laboratory.

NY-BR-1 and PAX8 Immunoreactivity in Breast, Gynecologic Tract, and Other CK7+ Carcinomas

The distinction between breast and müllerian carcinomas from each other and from tumors with a similar cytokeratin profile can be difficult. We tested the usefulness of 2 new markers, NY-BR-1 and PAX8, by staining a variety of breast and gynecologic carcinomas, along with tumors of pancreas, bile ducts, stomach, and gastroesophageal junction. NY-BR-1 expression (ie, H score >10) was seen in 58.4% of breast carcinomas (111/190), 5.6% of müllerian carcinomas (8/142), 7% of pancreatic tumors (1/15), 0% of cholangiocarcinomas (0/22), 0% of gastric tumors (0/36), and 0% of gastroesophageal carcinomas (0/25). All 188 breast carcinomas were negative for PAX8. PAX8 expression was seen in 72.4% of müllerian tumors (105/145). All pancreatic tumors (n = 15), cholangiocarcinomas (n = 23), and gastric (n = 35) and gastroesophageal junction (n = 25) carcinomas were negative for PAX8. Addition of NY-BR-1 and PAX8 in a panel would be useful in distinguishing breast cancer, gynecologic tumors, and tumors of the upper gastrointestinal tract.

Complement Pathway Is Frequently Altered in Endometriosis and Endometriosis-Associated Ovarian Cancer

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.

Significance of histologic pattern of carcinoma and sarcoma components on survival outcomes of uterine carcinosarcoma

BACKGROUND To examine the effect of the histology of carcinoma and sarcoma components on survival outcome of uterine carcinosarcoma. PATIENTS AND METHODS A multicenter retrospective study was conducted to examine uterine carcinosarcoma cases that underwent primary surgical staging. Archived slides were examined and histologic patterns were grouped based on carcinoma (low-grade versus high-grade) and sarcoma (homologous versus heterologous) components, correlating to clinico-pathological demographics and outcomes. RESULTS Among 1192 cases identified, 906 cases were evaluated for histologic patterns (carcinoma/sarcoma) with high-grade/homologous (40.8%) being the most common type followed by high-grade/heterologous (30.9%), low-grade/homologous (18.0%), and low-grade/heterologous (10.3%). On multivariate analysis, high-grade/heterologous (5-year rate, 34.0%, P = 0.024) and high-grade/homologous (45.8%, P = 0.017) but not low-grade/heterologous (50.6%, P = 0.089) were independently associated with decreased progression-free survival (PFS) compared with low-grade/homologous (60.3%). In addition, older age, residual disease at surgery, large tumor, sarcoma dominance, deep myometrial invasion, lymphovascular space invasion, and advanced-stage disease were independently associated with decreased PFS (all, P < 0.01). Both postoperative chemotherapy (5-year rates, 48.6% versus 39.0%, P < 0.001) and radiotherapy (50.1% versus 44.1%, P = 0.007) were significantly associated with improved PFS in univariate analysis. However, on multivariate analysis, only postoperative chemotherapy remained an independent predictor for improved PFS [hazard ratio (HR) 0.34, 95% confidence interval (CI) 0.27-0.43, P < 0.001]. On univariate analysis, significant treatment benefits for PFS were seen with ifosfamide for low-grade carcinoma (82.0% versus 49.8%, P = 0.001), platinum for high-grade carcinoma (46.9% versus 32.4%, P = 0.034) and homologous sarcoma (53.1% versus 38.2%, P = 0.017), and anthracycline for heterologous sarcoma (66.2% versus 39.3%, P = 0.005). Conversely, platinum, taxane, and anthracycline for low-grade carcinoma, and anthracycline for homologous sarcoma had no effect on PFS compared with non-chemotherapy group (all, P > 0.05). On multivariate analysis, ifosfamide for low-grade/homologous (HR 0.21, 95% CI 0.07-0.63, P = 0.005), platinum for high-grade/homologous (HR 0.36, 95% CI 0.22-0.60, P < 0.001), and anthracycline for high-grade/heterologous (HR 0.30, 95% CI 0.14-0.62, P = 0.001) remained independent predictors for improved PFS. Analyses of 1096 metastatic sites showed that carcinoma components tended to spread lymphatically, while sarcoma components tended to spread loco-regionally (P < 0.001). CONCLUSION Characterization of histologic pattern provides valuable information in the management of uterine carcinosarcoma.

Hormone receptor expression patterns in the endometrium of asymptomatic morbidly obese women before and after bariatric surgery

OBJECTIVE Obesity increases risk for endometrial neoplasia, but neither the pathophysiology nor the effects of weight loss on the risk are well established. We attempted to characterize the molecular profile of the endometrium of asymptomatic women with morbid obesity before and following bariatric surgery-induced weight loss. METHODS 59 asymptomatic, morbidly obese women underwent endometrial sampling before bariatric surgery; 46 (78%) of these returned one year later for re-biopsy (median weight loss of 41kg). Duplicate samples from these specimens were scored for expression of estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), and Ki-67 by two independent, blinded pathologists using an H-score [staining intensity (0-3)×(percent of tissue involved)]. RESULTS The prevalence of hyperplasia pre-operatively was 7% overall and 10% among patients not on an anti-estrogen. ER H-scores were similar before and after surgery overall (median 190 and 196 respectively, p=0.82), but patients with hyperplasia had higher pre-operative H-scores (median 256, p<0.001) and experienced greater H-score drops, than those without hyperplasia (-112 vs +50, p=0.028). In two patients with persistent hyperplasia at one year, ER H-scores fell to levels that were similar to those without pathology. One patient who developed hyperplasia during the study period had a rising ER H-score. Patients with hyperplasia had higher median PR H-scores pre-operatively (284 vs 188, p=0.01), which normalized through greater drops (75 vs 0, p=0.053). AR H-scores dropped significantly after surgery (13 vs 2, p=0.015), but were similar between patients with and without hyperplasia (p=0.33). Weight loss did not affect Ki-67 proliferation index. CONCLUSION Asymptomatic morbidly obese patients have a high prevalence of occult hyperplasia, characterized by relatively high hormone receptor expression. These profiles appear to normalize with weight loss and in advance of pathologically identifiable changes. These data suggest a potential role for screening this population as well as the possibility that weight loss may be a valid treatment strategy for risk reduction.