Identification of a novel diagnostic gene expression signature to discriminate uterine leiomyoma from leiomyosarcoma.

Leiomyosarcomas are rare, aggressive tumors, which exhibit a poor prognosis regardless of stage. Pre-operative diagnosis can be difficult as leiomyosarcoma can mimic features of the more common, benign uterine leiomyoma. The goal of this study was to identify specific molecular markers to discriminate between uterine leiomyosarcomas and leiomyomas to facilitate timely, accurate diagnosis and treatment. Gene expression profiles of three leiomyosarcomas, leiomyomas, and normal myometrial tissue samples were analyzed using the Affymetrix Human Gene 1.0 ST Array. GC-robust multiarray average calculation and ANOVA statistical testing were used to identify differentially expressed genes. Sixty genes, with functional roles in tumor progression or suppression, exhibited divergent expression profiles in leiomyosarcomas and leiomyomas, compared to normal myometrium. Differential RNA and protein levels of seven genes, with the most discriminatory expression patterns, were confirmed by RTPCR and immunohistochemistry in an additional 10 leiomyosarcoma and 20 leiomyoma independent samples. CHI3L1, MELK, PRC1, TOP2A, and TPX2 were overexpressed in leiomyosarcomas, while HPGD and TES were overexpressed in leiomyomas. Distinguishing leiomyosarcomas from leiomyomas represents a diagnostic challenge, particularly in the context of minimally invasive surgery. The unique gene expression signatures identified in this study may accurately differentiate between these tumor types at the earliest stage and provides potential prognostic factors and novel therapeutic targets for the treatment of leiomyosarcoma.

A method for extracting and characterizing RNA from urine: For downstream PCR and RNAseq analysis.

Readily accessible samples such as urine or blood are seemingly ideal for differentiating and stratifying patients; however, it has proven a daunting task to identify reliable biomarkers in such samples. Noncoding RNA holds great promise as a source of biomarkers distinguishing physiologic wellbeing or illness. Current methods to isolate and characterize RNA molecules in urine are limited. In this proof of concept study, we present a method to extract and identify small noncoding RNAs in urine. Initially, quantitative reverse transcription PCR was applied to confirm the presence of microRNAs in total RNA extracted from urine. Once the presence of micro RNA in urine was confirmed, we developed a method to scale up RNA extraction to provide adequate amounts of RNA for next generation sequence analysis. The method described in this study is applicable to detecting a broad range of small noncoding RNAs in urine; thus, they have wide applicability for health and disease analyses.

Fascin Is Associated With Aggressive Behavior and Poor Outcome in Uterine Carcinosarcoma.

OBJECTIVE: The mechanisms underlying the histogenesis and aggressiveness of uterine carcinosarcoma (UCS) are poorly understood; however, previous studies implicate epithelial-mesenchymal transition (EMT). Fascin is a proinvasive, actin-bundling protein and an important component of EMT. It is associated with poor outcomes in human carcinoma, especially in estrogen receptor (ER)-negative tumors arising in organs normally expressing ER. We sought to evaluate fascin expression in UCS and its relationship to ER status, clinicopathologic indicators of tumor aggressiveness, and survival outcomes. METHOD: Forty-four surgically staged cases of UCS were immunohistochemically evaluated for fascin and estrogen receptor-α expression and correlated with clinicopathologic parameters derived from electronic medical records and pathology reports. RESULTS: Fascin was only expressed in malignant epithelium and mesenchyma and was uniformly absent in background benign counterparts. Increased expression was associated with extrapelvic disease (P = 0.028), higher stage (P = 0.021), larger tumor size (P = 0.032), shorter progression-free interval (P = 0.035), and reduced estrogen receptor-α expression (P = 0.04). CONCLUSION: Fascin is aberrantly expressed in both elements of UCS and is associated with aggressive behavior and worse outcome. As a component of EMT and mediator of invasion, fascin may serve as a target in future therapies.

Claudin-4 activity in ovarian tumor cell apoptosis resistance and migration.

BACKGROUND: Claudin-4 is a transmembrane protein expressed at high levels in the majority of epithelial ovarian tumors, irrespective of subtype, and has been associated with tumor cells that are both chemoresistant and highly mobile. The objective of this study was to determine the functional role that claudin-4 plays in apoptosis resistance and migration as well as the therapeutic utility of targeting claudin-4 activity with a small mimic peptide. METHODS: We examined claudin-4 activity in human ovarian tumor cell lines (SKOV3, OVCAR3, PEO4) using in vitro caspase and scratch assays as well as an in vivo mouse model of ovarian cancer. Claudin-4 activity was disrupted by treating cells with a small peptide that mimics the DFYNP sequence in the second extracellular loop of claudin-4. Claudin-4 expression was also altered using shRNA-mediated gene silencing. RESULTS: Both the disruption of claudin-4 activity and the loss of claudin-4 expression significantly increased tumor cell caspase-3 activation (4 to 10-fold, respectively) in response to the apoptotic inducer staurosporine and reduced tumor cell migration by 50 %. The mimic peptide had no effect on cells that lacked claudin-4 expression. Female athymic nude mice bearing ZsGreen-PEO4 ovarian tumors showed a significant decrease in ovarian tumor burden, due to increased apoptosis, after treatment with intraperitoneal injections of 4 mg/kg mimic peptide every 48 h for three weeks, compared to control peptide treated mice. CONCLUSION: Claudin-4 functionally contributes to both ovarian tumor cell apoptosis resistance and migration and targeting extracellular loop interactions of claudin-4 may have therapeutic implications for reducing ovarian tumor burden.

Progestin treatment decreases CD133+ cancer stem cell populations in endometrial cancer.

OBJECTIVES: Endometrial cancer is a hormonally responsive malignancy. Response to progestins is associated with estrogen receptor (ER) and progesterone receptor (PR) status. CD133 is a marker of endometrial cancer stem cells. We postulated that CD133+ cells express ER and PR and that progestin therapy differentially regulates CD133+ cells. METHODS: The Ishikawa (ER/PR positive) and KLE (ER/PR negative) cell lines were examined for the presence of CD133 populations. Cell lines were treated with 30.4µM medroxyprogesterone 17-acetate (MPA) for 6days. After treatment, cell counts, apoptosis assays and CD133+ populations were examined. In a clinical project, we identified 12 endometrial cancer patients who were treated with progestin drugs at our institution. Using immunohistochemistry, CD133, ER, PR, and androgen receptor (AR) expression was scored and evaluated for change over time on serial biopsies. RESULTS: CD133+ populations were identified in Ishikawa and KLE cell lines. MPA treatment resulted in a significant reduction in the percentage of live cells (Ishikawa, P=0.036; KLE, P=0.0002), significant increase in apoptosis (Ishikawa, P=0.01; KLE, P=0.0006) and significant decrease in CD133+ populations (Ishikawa, P<0.0001; KLE, P=0.0001). ER, PR, AR and CD133 were present in 96.4%, 96.4%, 89.3% and 100% of patient samples respectively. Paralleling the in vitro results, CD133 expression decreased in patients who had histologic response to progestin treatment. CONCLUSION: CD133+ populations decreased after treatment with MPA in an in vitro model and in patients responding to treatment with progestins. Progestin treatment differentially decreases CD133+ cells.

Interactions with industry under the Sunshine Act: an example from gynecologic oncology.

THE PROBLEM: Clinicians may be unaware that industry payments to physicians are now publicly searchable under the Physician Payments Sunshine Act. Furthermore, the extent of industry's financial involvement in subspecialty practice has not been previously accessible. As an example, 6948 direct, research-unrelated payments totaling $1,957,004 were made to 765 gynecologic oncologists in 2014, the first full year of data available. A total of 153 companies reported at least 1 payment; however, the 10 manufacturers reporting the highest total payment amount accounted for 82% of all payments to physicians. In all, 48 gynecologic oncologists received >$10,000 from manufacturers, accounting for $1,202,228, or 61%, of total payments. A SOLUTION: Obstetrician-gynecologists, including gynecologic oncologists, should be aware of their publicly reported payments from industry and ensure reports' accuracy. Professional organizations, including the Society of Gynecologic Oncology (SGO), should strongly consider proactively developing guidelines regarding interactions with industry for their general memberships.

Cytokeratin 5-Positive Cells Represent a Therapy Resistant subpopulation in Epithelial Ovarian Cancer.

OBJECTIVE: Cytokeratin 5 (CK5) is an epithelial cell marker implicated in stem and progenitor cell activity in glandular reproductive tissues and endocrine and chemotherapy resistance in estrogen receptor (ER)(+) breast cancer. The goal of this study was to determine the prevalence of CK5 expression in ovarian cancer and the response of CK5(+) cell populations to cisplatin therapy. MATERIALS AND METHODS: Cytokeratin 5 expression was evaluated in 2 ovarian tissue microarrays, representing 137 neoplasms, and 6 ovarian cancer cell lines. Cell lines were treated with IC(50) (half-maximal inhibitory concentration) cisplatin, and the prevalence of CK5(+) cells pretreatment and posttreatment was determined. Proliferation of CK5(+) versus CK5(-) cell populations was determined using 5-bromo-2'-deoxyuridine incorporation. Chemotherapy-induced apoptosis in CK5(+) versus CK5(-) cells was measured using immunohistochemical staining for cleaved caspase-3. RESULTS: Cytokeratin 5 was expressed in 39.3% (42 of 107) of epithelial ovarian cancers with a range of 1% to 80% positive cells. Serous and endometrioid histologic subtypes had the highest percentage of CK5(+) specimens. Cytokeratin 5 expression correlated with ER positivity (38 of 42 CK5(+) tumors were also ER(+)). Cytokeratin 5 was expressed in 5 of 6 overall and 4 of 4 ER(+) epithelial ovarian cancer cell lines ranging from 2.4% to 52.7% positive cells. Cytokeratin 5(+) compared with CK5(-) cells were slower proliferating. The prevalence of CK5(+) cells increased after 48-hour cisplatin treatment in 4 of 5 cell lines tested. Cytokeratin 5(+) ovarian cancer cells compared with CK5(-) ovarian cancer cells were more resistant to cisplatin-induced apoptosis. CONCLUSIONS: Cytokeratin 5 is expressed in a significant proportion of epithelial ovarian cancers and represents a slower proliferating chemoresistant subpopulation that may warrant cotargeting in combination therapy.

A phase II trial of brivanib in recurrent or persistent endometrial cancer: an NRG Oncology/Gynecologic Oncology Group Study.

PURPOSE: Brivanib, an oral, multi-targeted tyrosine kinase inhibitor with activity against vascular endothelial growth factor (VEGF) and fibroblast growth factor receptor (FGFR) was investigated as a single agent in a phase II trial to assess the activity and tolerability in recurrent or persistent endometrial cancer (EMC). PATIENTS AND METHODS: Eligible patients had persistent or recurrent EMC after receiving one to two prior cytotoxic regimens, measurable disease, and performance status of ≤2. Treatment consisted of brivanib 800 mg orally every day until disease progression or prohibitive toxicity. Primary endpoints were progression-free survival (PFS) at six months and objective tumor response. Expression of multiple angiogenic proteins and FGFR2 mutation status was assessed. RESULTS: Forty-five patients were enrolled. Forty-three patients were eligible and evaluable. Median age was 64 years. Twenty-four patients (55.8%) received prior radiation. Median number of cycles was two (range 1-24). No GI perforations but one rectal fistula were seen. Nine patients had grade 3 hypertension, with one experiencing grade 4 confusion. Eight patients (18.6%; 90% CI 9.6%-31.7%) had responses (one CR and seven PRs), and 13 patients (30.2%; 90% CI 18.9%-43.9%) were PFS at six months. Median PFS and overall survival (OS) were 3.3 and 10.7 months, respectively. When modeled jointly, VEGF and angiopoietin-2 expression may diametrically predict PFS. Estrogen receptor-α (ER) expression was positively correlated with OS. CONCLUSION: Brivanib is reasonably well tolerated and worthy of further investigation based on PFS at six months in recurrent or persistent EMC.

Outcomes of women with atypical glandular cells on preoperative cytology and endometrial cancer.

OBJECTIVE: This study aims to examine the prognostic importance of preoperative cervical cytologic diagnosis with atypical glandular cells (AGC) or malignant cells (MC) as a predictor of poor outcomes in endometrial cancer. MATERIALS AND METHODS: A total of 563 patients were surgically staged for endometrial adenocarcinoma from 2002 to 2012 at our institution. Of these patients, 106 were included to perform a case-control study (39 patients with AGC or MC and 67 controls). Included patients were not significantly different from excluded patients and were matched for age, race, and body mass index. Outcome variables included presence of extrauterine disease (International Federation of Gynecology and Obstetrics stage ≥II) and high intermediate risk (HIR) disease. Further analysis sought to improve the prediction combining AGC or MC with other factors, such as grade and CA-125 levels. Standard statistical analyses were used. RESULTS: Among the patients with AGC or MC, 53.8% had HIR disease compared with 30.3% with normal cervical cytologic diagnosis (odds ratio [OR], 2.68; 95% confidence interval [CI], 1.18-6.09; P = 0.02). Extrauterine disease was found in 43.6% of patients with AGC or MC compared with that of 15.2% in patients with normal cervical cytologic diagnosis (OR, 4.33; 95% CI, 1.72-10.90; P < 0.01). Multivariate analysis confirmed that AGC or MC was an independent predictor of HIR disease (OR, 8.41; 95% CI, 1.34-52.78; P = 0.02) and extrauterine disease (OR, 4.78; 95% CI, 1.26-18.1; P = 0.02). The combination of elevated CA-125 levels with AGC or MC cervical cytologic diagnosis increased the statistical prediction of extrauterine disease (OR, 13.3; 95% CI, 3.1-56.8; P < 0.01) and HIR disease (OR, 5.83; 95% CI, 1.44-23.71; P = 0.02). CONCLUSIONS: Patients with AGC or MC on preoperative cervical cytology are at risk for extrauterine and HIR disease. These preoperative findings should warn surgeons of the potential of extrauterine or occult metastatic disease.

Copy neutral loss of heterozygosity is more frequent in older ovarian cancer patients.

Loss of heterozygosity (LOH) is a common type of genomic alterations in ovarian cancer. Analyzing 74,415 copy neutral LOH events in 513 serous ovarian adenocarcinomas samples from the Cancer Genome Atlas, we report that the frequency of LOH events increases with age. Similar trend is observed for LOH involving chromosome 17, which is frequently implicated in ovarian cancer. The results are consistent when we analyze data from the Boston high-grade serous cancer cohort. We further show that germ line and somatic mutations in BRCA1 (in chromosome 17) and BRCA2 (in chromosome 13) loci are not necessary to establish the pattern. We also report significant age-related changes in expression patterns for several genes in the homologous recombination (HR) pathway, such as BRCA1, RAD50, RAD52, XRCC2, XRCC3, and MRE11A in these patient samples. Furthermore, we develop a metric for pathway-level imbalance, and show that increased imbalance in the HR pathway, i.e., increase in expression of some HR genes and decrease in expression of others, is common and correlates significantly with the frequency of LOH events in the patient samples. Taken together, it is highly likely that aging and deregulation of HR pathway contribute to the increased incidence of copy-neutral LOH in ovarian cancer patients.

Claudins play a role in normal and tumor cell motility.

BACKGROUND: Claudins are key integral proteins of the tight junction. Although they play an essential role in controlling paracellular diffusion in epithelia, increasing evidence supports a role for these proteins in non-barrier forming activities. To elucidate a potential function for claudins outside of their traditional role in tight junctions, subcellular localization of claudin-4 was determined in normal mammary epithelial cells as well as breast and ovarian cancer cell lines and the effects of a claudin mimic peptide on cell motility were determined. RESULTS: Immunofluorescence revealed that claudin-4 was localized along cellular projections. Using a fluorescent peptide that mimics a conserved sequence in the second extracellular loop of a set of claudin subtypes, that includes claudin-4, exposure of this loop to the extracellular environment was confirmed in non-polarized cells. This peptide inhibited cell motility when normal mammary epithelial cells as well as breast and ovarian tumor cells were subjected to a wound healing assay. Knockdown of claudin-4 also inhibited cell motility and the mimic peptide had no effect on motility in the claudin-4 knockdown cells. This effect on motility was seen when cells were grown on collagen, but not when cells were grown on non-physiological cell adhesive or fibronectin. CONCLUSION: The second extracellular loop of claudins is able to interact with the extracellular environment to promote normal and tumor cell motility when it is not associated with tight junction structures.

Estrogen switches pure mucinous breast cancer to invasive lobular carcinoma with mucinous features.

Mucinous breast cancer (MBC) is mainly a disease of postmenopausal women. Pure MBC is rare and augurs a good prognosis. In contrast, MBC mixed with other histological subtypes of invasive disease loses the more favorable prognosis. Because of the relative rarity of pure MBC, little is known about its cell and tumor biology and relationship to invasive disease of other subtypes. We have now developed a human breast cancer cell line called BCK4, in which we can control the behavior of MBC. BCK4 cells were derived from a patient whose poorly differentiated primary tumor was treated with chemotherapy, radiation and tamoxifen. Malignant cells from a recurrent pleural effusion were xenografted in mammary glands of a nude mouse. Cells from the solid tumor xenograft were propagated in culture to generate the BCK4 cell line. Multiple marker and chromosome analyses demonstrate that BCK4 cells are human, near diploid and luminal, expressing functional estrogen, androgen, and progesterone receptors. When xenografted back into immunocompromised cycling mice, BCK4 cells grow into small pure MBC. However, if mice are supplemented with continuous estradiol, tumors switch to invasive lobular carcinoma (ILC) with mucinous features (mixed MBC), and growth is markedly accelerated. Tamoxifen prevents the expansion of this more invasive component. The unexpected ability of estrogens to convert pure MBC into mixed MBC with ILC may explain the rarity of the pure disease in premenopausal women. These studies show that MBC can be derived from lobular precursors and that BCK4 cells are new, unique models to study the phenotypic plasticity, hormonal regulation, optimal therapeutic interventions, and metastatic patterns of MBC.

Risk-reducing salpingectomy as preventative strategy for pelvic serous cancer.

The systemic failure to detect early-stage ovarian cancer may be attributed to a significant amount of pelvic serous cancers arising from the fallopian tube rather than the ovarian surface epithelium. This article reviews the possibility of applying risk-reducing salpingectomy as a new paradigm for the prevention of pelvic serous cancer in both high- and low-risk women.

Unliganded progesterone receptors attenuate taxane-induced breast cancer cell death by modulating the spindle assembly checkpoint.

Whether the presence of steroid receptors in luminal breast cancers renders them resistant to taxanes remains uncertain. Here we assess the role of progesterone receptors (PR) on taxane-induced cell death. We previously showed that estrogen receptor (ER)-positive human breast cancer cells that inducibly express PR-A or PR-B isoforms were protected from taxane-stimulated apoptosis when compared to the identical cells lacking PR. Surprisingly, PR-dependent protection occurred in the absence of progesterone, demonstrating that the unliganded receptors were biologically active. The present studies demonstrate that unliganded PR, focused on PR-A, protect breast cancer cells from taxane-stimulated apoptosis. The studies identify genes regulated by taxanes in isogenic ER-positive cells that either lack or express PR-A. We show that unliganded PR-A alters the gene expression pattern controlled by taxanes, especially multiple genes involved in the spindle assembly checkpoint, a group of proteins that insure proper attachment of microtubules to kinetochores during mitosis. Importantly, taxanes and unliganded PR regulate many of these genes in opposite directions. As a result, mitotic slippage is exacerbated by the presence of PR, leading to an increase in the number of multinucleated cells both in vitro and in xenograft tumors. We describe a simple new assay for assessing multinucleation in paraffin sections. We speculate that rather than inducing cell death, unliganded PR exploits multinucleation to promote cell survival from taxane therapy. This can be prevented with antiprogestin.

Specificity and prognostic validation of a polyclonal antibody to detect Six1 homeoprotein in ovarian cancer.

OBJECTIVE: The presence of Six1 mRNA gene portends a poor prognosis in ovarian cancer. We describe validation of a Six1 specific antibody and evaluate its association with tumorigenicity and prognosis in ovarian cancer. METHODS: A Six1 antibody (Six1cTerm) was raised to residues downstream of the Six1 homeodomain, representing its unique C-terminus as compared to other Six family members. Cells were transfected with Six1-Six6 and Western blot was performed to demonstrate Six1 specificity. Ovarian cancer cell lines were analyzed for Six1 mRNA and Six1cTerm and tumorigenicity was evaluated. Ovarian cancer tissue microarrays (OTMA) were analyzed for Six1cTerm by immunohistochemistry and scored by two blinded observers. The metastatic tumors of 15 stage IIIC high grade serous ovarian cancers were analyzed with Six1 mRNA and Six1cTerm and expression was compared to clinical factors and survival. RESULTS: The Six1cTerm antibody is specific for Six1. Cell line tumorigenicity in SCID mice correlates with Six1 levels both by mRNA(p=0.001, Mann-Whitney U test) and by protein (presence vs. absence, p=0.05 Fischer's Exact test). Six1 protein was present in up to 54% of OTMA specimens. Six1 protein expression in omental/peritoneal metastases correlated with worsened survival in a sample (n=15) of high grade serous stage IIIC ovarian cancers (p=0.001). CONCLUSIONS: The Six1cTerm antibody is specific and able to detect Six1 in cell lines and tumor tissue. Six1 protein detection is common in ovarian cancer and is associated with tumorigenicity and poor prognosis in this group of patient samples. Six1cTerm antibody should be further validated as prognostic tool.

DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination.

OBJECTIVES: Cell lines derived from human ovarian and endometrial cancers, and their immortalized non-malignant counterparts, are critical tools to investigate and characterize molecular mechanisms underlying gynecologic tumorigenesis, and facilitate development of novel therapeutics. To determine the extent of misidentification, contamination and redundancy, with evident consequences for the validity of research based upon these models, we undertook a systematic analysis and cataloging of endometrial and ovarian cell lines. METHODS: Profiling of cell lines by analysis of DNA microsatellite short tandem repeats (STR), p53 nucleotide polymorphisms and microsatellite instability was performed. RESULTS: Fifty-one ovarian cancer lines were profiled with ten found to be redundant and five (A2008, OV2008, C13, SK-OV-4 and SK-OV-6) identified as cervical cancer cells. Ten endometrial cell lines were analyzed, with RL-92, HEC-1A, HEC-1B, HEC-50, KLE, and AN3CA all exhibiting unique, uncontaminated STR profiles. Multiple variants of Ishikawa and ECC-1 endometrial cancer cell lines were genotyped and analyzed by sequencing of mutations in the p53 gene. The profile of ECC-1 cells did not match the EnCa-101 tumor, from which it was reportedly derived, and all ECC-1 isolates were genotyped as Ishikawa cells, MCF-7 breast cancer cells, or a combination thereof. Two normal, immortalized endometrial epithelial cell lines, HES cells and the hTERT-EEC line, were identified as HeLa cervical carcinoma and MCF-7 breast cancer cells, respectively. CONCLUSIONS: Results demonstrate significant misidentification, duplication, and loss of integrity of endometrial and ovarian cancer cell lines. Authentication by STR DNA profiling is a simple and economical method to verify and validate studies undertaken with these models.

Large cell neuroendocrine carcinoma of the cervix: prognostic factors and survival advantage with platinum chemotherapy.

OBJECTIVE: Large cell neuroendocrine carcinoma of the cervix (LCNEC) is a rare cervical neoplasm associated with poor survival. Our objective was to identify treatments associated with improved survival. METHODS: Relevant data were abstracted from an English literature MEDLINE search, SEER database, and a patient treated at our institution. Multivariate analysis was performed by generating Cox proportional hazard ratios. RESULTS: We identified 62 patients with LCNEC: 49 cases from the English literature, 12 patients in the SEER database and our patient. Out of the 62 women, median age was 37 (range, 21-75). FIGO stage was as follows: 58% had stage I disease, 16% had stage II, 2% had stage III, 8% had stage IV disease and 16% had no stage documented. Of all patients, 73% underwent primary surgery, 4.7% underwent primary radiation, 4.7% underwent chemotherapy, 8% had chemoradiation, and 9.6% had no primary treatment. Of all patients, 58% died of disease, 26% had no evidence of disease, 3% were alive with disease, and 13% had no survival data. The overall median survival was 16.5 months (0.5-151 months). Median overall survival for stage I, II, III, and IV cancers was 19, 17, 3, and 1.5 months, respectively. In a multivariate analysis, earlier stage (p<0.00001) and the addition of chemotherapy (p=0.04) were associated with improved survival. Both platinum agents (p=0.034) and platinum and etoposide together (p=0.027) were associated with improved survival. CONCLUSIONS: Perioperative chemotherapy, in particular platinum with or without etoposide, improves survival in the rare LCNEC.