Goli Samimi

A Gene Signature Predictive for Outcome in Advanced Ovarian Cancer Identifies a Survival Factor: Microfibril-Associated Glycoprotein 2

Advanced stage papillary serous tumors of the ovary are responsible for the majority of ovarian cancer deaths, yet the molecular determinants modulating patient survival are poorly characterized. Here, we identify and validate a prognostic gene expression signature correlating with survival in a series of microdissected serous ovarian tumors. Independent evaluation confirmed the association of a prognostic gene microfibril-associated glycoprotein 2 (MAGP2) with poor prognosis, whereas in vitro mechanistic analyses demonstrated its ability to prolong tumor cell survival and stimulate endothelial cell motility and survival via the alpha(V)beta(3) integrin receptor. Increased MAGP2 expression correlated with microvessel density suggesting a proangiogenic role in vivo. Thus, MAGP2 may serve as a survival-associated target.

Methylation of cell-free circulating DNA in the diagnosis of cancer.

A range of molecular alterations found in tumor cells, such as DNA mutations and DNA methylation, is reflected in cell-free circulating DNA (circDNA) released from the tumor into the blood, thereby making circDNA an ideal candidate for the basis of a blood-based cancer diagnosis test. In many cancer types, mutations driving tumor development and progression are present in a wide range of oncogenes and tumor suppressor genes. However, even when a gene is consistently mutated in a particular cancer, the mutations can be spread over very large regions of its sequence, making evaluation difficult. This diversity of sequence changes in tumor DNA presents a challenge for the development of blood tests based on DNA mutations for cancer diagnosis. Unlike mutations, DNA methylation that can be consistently measured, as it tends to occur in specific regions of the DNA called CpG islands. Since DNA methylation is reflected within circDNA, detection of tumor-specific DNA methylation in patient plasma is a feasible approach for the development of a blood-based test. Aberrant circDNA methylation has been described in most cancer types and is actively being investigated for clinical applications. A commercial blood test for colorectal cancer based on the methylation of the SEPT9 promoter region in circDNA is under review for approval by the Federal Drug Administration (FDA) for clinical use. In this paper, we review the state of research in circDNA methylation as an application for blood-based diagnostic tests in colorectal, breast, lung, pancreatic and ovarian cancers, and we consider some of the future directions and challenges in this field. There are a number of potential circDNA biomarkers currently under investigation, and experience with SEPT9 shows that the time to clinical translation can be relatively rapid, supporting the promise of circDNA as a biomarker.

Prognostic and diagnostic significance of DNA methylation patterns in high grade serous ovarian cancer

OBJECTIVE Altered DNA methylation patterns hold promise as cancer biomarkers. In this study we selected a panel of genes which are commonly methylated in a variety of cancers to evaluate their potential application as biomarkers for prognosis and diagnosis in high grade serous ovarian carcinoma (HGSOC); the most common and lethal subtype of ovarian cancer. METHODS The methylation patterns of 10 genes (BRCA1, EN1, DLEC1, HOXA9, RASSF1A, GATA4, GATA5, HSULF1, CDH1, SFN) were examined and compared in a cohort of 80 primary HGSOC and 12 benign ovarian surface epithelium (OSE) samples using methylation-specific headloop suppression PCR. RESULTS The genes were variably methylated in primary HGSOC, with HOXA9 methylation observed in 95% of cases. Most genes were rarely methylated in benign OSE, with the exception of SFN which was methylated in all HGSOC and benign OSE samples examined. Methylation of DLEC1 was associated with disease recurrence, independent of tumor stage and suboptimal surgical debulking (HR 3.5 (95% CI:1.10-11.07), p=0.033). A combination of the methylation status of HOXA9 and EN1 could discriminate HGSOC from benign OSE with a sensitivity of 98.8% and a specificity of 91.7%, which increased to 100% sensitivity with no loss of specificity when pre-operative CA125 levels were also incorporated. CONCLUSIONS This study provides further evidence to support the feasibility of detecting altered DNA methylation patterns as a potential diagnostic and prognostic approach for HGSOC.

Patient-Derived Xenograft Models to Improve Targeted Therapy in Epithelial Ovarian Cancer Treatment

Despite increasing evidence that precision therapy targeted to the molecular drivers of a cancer has the potential to improve clinical outcomes, high-grade epithelial ovarian cancer (OC) patients are currently treated without consideration of molecular phenotype, and predictive biomarkers that could better inform treatment remain unknown. Delivery of precision therapy requires improved integration of laboratory-based models and cutting-edge clinical research, with pre-clinical models predicting patient subsets that will benefit from a particular targeted therapeutic. Patient-derived xenografts (PDXs) are renewable tumor models engrafted in mice, generated from fresh human tumors without prior in vitro exposure. PDX models allow an invaluable assessment of tumor evolution and adaptive response to therapy. PDX models have been applied to pre-clinical drug testing and biomarker identification in a number of cancers including ovarian, pancreatic, breast, and prostate cancers. These models have been shown to be biologically stable and accurately reflect the patient tumor with regards to histopathology, gene expression, genetic mutations, and therapeutic response. However, pre-clinical analyses of molecularly annotated PDX models derived from high-grade serous ovarian cancer (HG-SOC) remain limited. In vivo response to conventional and/or targeted therapeutics has only been described for very small numbers of individual HG-SOC PDX in conjunction with sparse molecular annotation and patient outcome data. Recently, two consecutive panels of epithelial OC PDX correlate in vivo platinum response with molecular aberrations and source patient clinical outcomes. These studies underpin the value of PDX models to better direct chemotherapy and predict response to targeted therapy. Tumor heterogeneity, before and following treatment, as well as the importance of multiple molecular aberrations per individual tumor underscore some of the important issues addressed in PDX models.

Methylated circulating tumor DNA in blood: power in cancer prognosis and response

Circulating tumor DNA (ctDNA) in the plasma or serum of cancer patients provides an opportunity for non-invasive sampling of tumor DNA. This ‘liquid biopsy’ allows for interrogations of DNA such as quantity, chromosomal alterations, sequence mutations and epigenetic changes, and can be used to guide and improve treatment throughout the course of the disease. This tremendous potential for real-time ‘tracking’ in a cancer patient has led to substantial research efforts in the ctDNA field. ctDNA can be distinguished from non-tumor DNA by the presence of tumor-specific mutations and copy number variations, and also by aberrant DNA methylation, with both DNA sequence and methylation changes corresponding to those found in the tumor. Aberrant methylation of specific promoter regions can be a very consistent feature of cancer, in contrast to mutations, which typically occur at a wide range of sites. This consistency makes ctDNA methylation amenable to the design of widely applicable clinical assays. In this review, we examine ctDNA methylation in the context of monitoring disease status, treatment response and determining the prognosis of cancer patients.

Up-regulation of stromal versican expression in advanced stage serous ovarian cancer

OBJECTIVE The purpose of this study is to examine the role of versican (VCAN) in advanced stage serous ovarian cancer by investigating its expression, its function, and its correlation with clinical outcomes. METHODS Microarray analysis was performed on RNA isolated from tumor and stromal components of advanced stage serous ovarian cancer and normal ovarian epithelial tissue to identify genes up-regulated in ovarian tumor stroma. Validation studies using immunohistochemistry and quantitative real-time PCR (Q-RT-PCR) was performed on one of the up-regulated genes, VCAN. Immunolocalization of VCAN (n=111) and CD31 (n=56) was done on serous ovarian tumors. CD31 staining was performed to examine microvessel density (MVD). Q-RT-PCR was performed on 65 samples to evaluate the differential expression of VCAN isoforms. Cell proliferation and invasion assays were performed to examine how V1-treated ovarian cancer cell lines and an endothelial cell line would differ from controls. Univariate survival analyses were done with VCAN expression. Correlation analysis was done with CD31, platinum resistance, and clinical data. RESULTS Validation studies using Q-RT-PCR and immunohistochemistry showed significantly higher VCAN V1 isoform expression in ovarian cancer stroma compared with normal ovarian stroma and ovarian cancer cells. Correlation studies showed stromal VCAN expression was associated with poorer overall and progression-free survival, platinum resistance, and increased MVD. VCAN-treated ovarian cancer and endothelial cells showed increased invasion potential. CONCLUSIONS VCAN overexpression is associated with increased MVD and invasion potential, which may lead to poorer overall and progression-free survival and platinum resistance.

Methylation-capture and Next-Generation Sequencing of free circulating DNA from human plasma

BackgroundFree circulating DNA (fcDNA) has many potential clinical applications, due to the non-invasive way in which it is collected. However, because of the low concentration of fcDNA in blood, genome-wide analysis carries many technical challenges that must be overcome before fcDNA studies can reach their full potential. There are currently no definitive standards for fcDNA collection, processing and whole-genome sequencing. We report novel detailed methodology for the capture of high-quality methylated fcDNA, library preparation and downstream genome-wide Next-Generation Sequencing. We also describe the effects of sample storage, processing and scaling on fcDNA recovery and quality.ResultsUse of serum versus plasma, and storage of blood prior to separation resulted in genomic DNA contamination, likely due to leukocyte lysis. Methylated fcDNA fragments were isolated from 5 donors using a methyl-binding protein-based protocol and appear as a discrete band of ~180 bases. This discrete band allows minimal sample loss at the size restriction step in library preparation for Next-Generation Sequencing, allowing for high-quality sequencing from minimal amounts of fcDNA. Following sequencing, we obtained 37×106-86×106 unique mappable reads, representing more than 50% of total mappable reads. The methylation status of 9 genomic regions as determined by DNA capture and sequencing was independently validated by clonal bisulphite sequencing.ConclusionsOur optimized methods provide high-quality methylated fcDNA suitable for whole-genome sequencing, and allow good library complexity and accurate sequencing, despite using less than half of the recommended minimum input DNA.

Etiology and Pathogenesis of Epithelial Ovarian Cancer

Ovarian cancer is complex disease composed of different histological grades and types. However, the underlying molecular mechanisms involved in the development of different phenotypes remain largely unknown. Epidemiological studies identified multiple exogenous and endogenous risk factors for ovarian cancer development. Among them, an inflammatory stromal microenvironment seems to play a critical role in the initiation of the disease. The interaction between such a microenvironment, genetic polymorphisms, and different epithelial components such as endosalpingiosis, endometriosis, and ovarian inclusion cyst in the ovarian cortex may induce different genetic changes identified in the epithelial component of different histological types of ovarian tumors. Genetic studies on different histological grades and types provide insight into the pathogenetic pathways for the development of different disease phenotypes. However, the link between all these genetic changes and the etiological factors remains to be established.

Epigenetic biomarkers in epithelial ovarian cancer

Ovarian cancer is the most lethal gynecological malignancy and the 5th leading cause of cancer death in women. Women with ovarian cancer are typically diagnosed at late stage, when the cancer has spread into the peritoneal cavity and complete surgical removal is difficult. The 5-year survival time for patients diagnosed at this stage is 30%, in contrast to a 5-year survival of 90% for patients diagnosed at early stage. Cancer screening and early detection have the potential to greatly decrease the mortality and morbidity from cancer. The emerging field of epigenetics offers a valuable opportunity to identify cancer-specific DNA methylation changes that can be used in the clinic to improve early-stage diagnosis and better predict response in treated patients. To date, numerous DNA methylation aberrations have been identified in epithelial ovarian cancer; here we review some candidate genes and pathways with potential clinical utility as biomarkers for diagnosis and/or prognosis. It has become clear that even with the great promise of DNA methylation biomarkers in epithelial ovarian cancer, the identification of highly specific, sensitive and robust panels of markers and the standardization of analysis techniques are still required in order to improve detection, treatment and thus patient outcome.

ZNF300P1 Encodes a lincRNA that regulates cell polarity and is epigenetically silenced in type II epithelial ovarian cancer

BackgroundWe previously identified that the CpG island-associated promoter of the novel lincRNA ZNF300P1 (also known as LOC134466) is frequently hypermethylated and silenced in ovarian cancer tissues. However, the function of ZNF300P1 was unknown. In this report we demonstrate that ZNF300P1 is involved in the regulation of key cell cycle and cell motility networks in human ovarian surface epithelial cells, and may play a role in promoting metastasis in ovarian cancer cells.MethodsWe applied methylated DNA immunoprecipitation on whole genome promoter tiling arrays and Sequenom assays to examine methylation status of ZNF300P1 in multiple ovarian cancer cell lines, as well as in normal ovarian and ovarian tumor tissues. Transcript profiling was used to investigate the effects of ZNF300P1 suppression in ovarian cancer cells. We utilized siRNA knockdown in normal ovarian surface epithelial cells and performed cellular proliferation, migration and adhesion assays to validate and explore the profiling results.ResultsWe demonstrate that ZNF300P1 is methylated in multiple ovarian cancer cell lines. Loss of ZNF300P1 results in decreased cell proliferation and colony formation. In addition, knockdown of the ZNF300P1 transcript results in aberrant and less persistent migration in wound healing assays due to a loss of cellular polarity. Using an ex vivo peritoneal adhesion assay, we also reveal a role for ZNF300P1 in the attachment of ovarian cancer cells to peritoneal membranes, indicating a potential function of ZNF300P1 expression in metastasis of ovarian cancer cells to sites within the peritoneal cavity.ConclusionOur findings further support ZNF300P1 as frequently methylated in ovarian cancer and reveal a novel function for ZNF300P1 lincRNA expression in regulating cell polarity, motility, and adhesion and loss of expression may contribute to the metastatic potential of ovarian cancer cells.