Tsz-Lun Yeung

Systematic Identification of Druggable Epithelial-Stromal Crosstalk Signaling Networks in Ovarian Cancer

Abstract Background Bulk tumor tissue samples are used for generating gene expression profiles in most research studies, making it difficult to decipher the stroma–cancer crosstalk networks. In the present study, we describe the use of microdissected transcriptome profiles for the identification of cancer–stroma crosstalk networks with prognostic value, which presents a unique opportunity for developing new treatment strategies for ovarian cancer. Methods Transcriptome profiles from microdissected ovarian cancer–associated fibroblasts (CAFs) and ovarian cancer cells from patients with high-grade serous ovarian cancer (n = 70) were used as input data for the computational systems biology program CCCExplorer to uncover crosstalk networks between various cell types within the tumor microenvironment. The crosstalk analysis results were subsequently used for discovery of new indications for old drugs in ovarian cancer by computational ranking of candidate agents. Survival analysis was performed on ovarian tumor–bearing Dicer/Pten double-knockout mice treated with calcitriol, a US Food and Drug Administration–approved agent that suppresses the Smad signaling cascade, or vehicle control (9–11 mice per group). All statistical tests were two-sided. Results Activation of TGF-β-dependent and TGF-β-independent Smad signaling was identified in a particular subtype of CAFs and was associated with poor patient survival (patients with higher levels of Smad-regulated gene expression by CAFs: median overall survival = 15 months, 95% confidence interval [CI] = 12.7 to 17.3 months; vs patients with lower levels of Smad-regulated gene expression: median overall survival = 26 months, 95% CI = 15.9 to 36.1 months, P = .02). In addition, the activated Smad signaling identified in CAFs was found to be targeted by repositioning calcitriol. Calcitriol suppressed Smad signaling in CAFs, inhibited tumor progression in mice, and prolonged the median survival duration of ovarian cancer–bearing mice from 36 to 48 weeks (P = .04). Conclusions Our findings suggest the feasibility of using novel multicellular systems biology modeling to identify and repurpose known drugs targeting cancer–stroma crosstalk networks, potentially leading to faster and more effective cures for cancers.

Abstract 4799: Identification and characterization of stromal factors with clinical significance in the ovarian tumor microenvironment

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ovarian cancer is the most lethal gynecologic malignancy in the United States. While the tumor microenvironment has been shown to play important roles in cancer pathogenesis, large-scale transcriptome profiles of the stromal component of ovarian tumors and the identification of stromal prognostic markers and therapeutic targets are lacking. This study seeks to identify secreted stromal factors and evaluate their clinical significance. Transcriptome profiling and clustering analysis on microdissected ovarian cancer associated fibroblasts (CAFs) samples from 83 patients revealed a heterogeneous gene expression pattern. Since CAFs can be derived from mesenchymal stem cells (MSCs), we compared transcriptome profiles of MSC samples obtained from healthy individuals with those from CAFs. Results showed that CAF expression profiles could be classified into two major subtypes. The MSC subtype, which has gene expression pattern highly resembling to that of undifferentiated MSCs, is significantly associated with poorer patient survival when compared to the non-MSC subtype. Further analyses identified two expression clusters within the MSC subtype: 1) the CAF-C cluster which expressed high level of MSC chondrogenic differentiation associated genes including CSPG2, SFRP2 and COMP, and 2) the CAF-O cluster which expressed high level of osteogenic differentiation associated genes including RUNX2 and BGN. Survival analysis showed that patients with the CAF-O expression subtype had overall survival rates comparable to those with the non-MSC subtypes, while the CAF-C subtype is associated with worst clinical outcomes. Pathway analyses revealed strong interactions among these stromal genes. Among them, SFRP2, which has been shown to inhibit osteogenic differentiation of MSCs by inhibiting Wnt signaling and subsequently suppress RUNX2 expression, was selected for further validation studies. Stromal SFRP2 and RUNX2 protein expression levels were evaluated by immunohistochemistry on paraffin tissue sections from 94 patients. Results showed that high levels of stromal SFRP2 were significantly associated with poor patient survival (p < 0.001), while high levels of stromal RUNX2 were significantly associated with improved patient survival (p < 0.001). A significant inverse correlation between SFRP2 and RUNX2 expression levels was also observed (r = -0.279, p = 0.037), suggested that the interactions among these genes may generate different landscapes in the tumor microenvironment, which modulate MSC differentiation and subsequently cancer cell aggressiveness. This study presents a new paradigm of which osteogenic-like and chondrogenic-like CAF signatures are associated with better and poorer patient survival respectively. Understanding the relationship between the expression patterns of stromal factors and MSC differentiation would provide us with new insights into ovarian cancer pathogenesis. Citation Format: Tsz-Lun Yeung, Cecilia S. Leung, Kwong-Kwok Wong, Samuel C. Mok. Identification and characterization of stromal factors with clinical significance in the ovarian tumor microenvironment. [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 4799. doi:10.1158/1538-7445.AM2014-4799

Abstract LB-214: The immunosuppressive roles of ovarian stromal tumor microenvironment

Ovarian cancer is the most lethal gynecologic malignancy in the US. Our group and others has shown that CD8+ lymphocyte infiltration in the ovarian tumor epithelium is associated with prolonged survival in patients with high-grade serous ovarian cancer. Despite the increasing evidence on stromal involvement in tumor progression, the interactions between stromal fibroblasts and cancer cells, as well as the underlying genetic composition of the stromal cells that could regulate the infiltration and activation of CD8+ cytotoxic T lymphocytes (CTLs) and affect patient survival is not fully understood. The present study seeks to evaluate the roles and to delineate the underlying mechanisms by which stromal cancer associated fibroblasts (CAFs) modulates immune response in ovarian cancer, particularly immune suppression by CAF-derived protein factors. By laser microdissection and transcriptome profiling of tumor tissue samples from ovarian cancer patients, we identified CAF-specific gene signatures for ovarian cancer that are associated with the survival duration of patients and with the differential tumor immune response. Among the differentially expressed genes identified, validation study by qPCR and immunohistochemistry indicated that stromal MFAP5 expression is significantly higher in patients with short survival duration and in patients with lower intratumoral CD8+ T cell density. Survival analysis result suggested that high stromal MFAP5 expression indicates poor disease prognosis (Hazard ratio=2.722, P Delineating the molecular mechanism by which MFAP5 modulates the progression and immune responses of ovarian cancer will shed light on the design of novel treatment modalities based on the blockade of tumor stroma-derived factors, which could promote activation and trafficking of cytotoxic CD8+ T cells and improve patient survival rates. Citation Format: Tsz-Lun Yeung, Cecilia S. Leung, Kwong-Kwok Wong, Michael J. Birrer, Stephen T. Wong, Karen H. Lu, Samuel C. Mok. The immunosuppressive roles of ovarian stromal tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-214. doi:10.1158/1538-7445.AM2017-LB-214

Abstract 4145: Stromal heterogeneity and immune response in ovarian cancer

Ovarian cancer is the most lethal gynecologic malignancy in the US. Our group and others has shown that CD8+ lymphocyte infiltration in the ovarian tumor epithelium is associated with prolonged survival in patients with high-grade serous ovarian cancer (HGSOC). Despite the increasing evidence on stromal involvement in tumor progression, the underlying genetic composition of the stromal cells that could regulate the infiltration and activation of CD8+ cytotoxic T lymphocytes (CTLs) in ovarian cancer is not fully understood. How tumor heterogeneity contributes to a different in immune response remains a challenge question that needs to be addressed. The present study seeks to evaluate the roles and to delineate the underlying mechanisms by which stromal cancer associated fibroblasts (CAFs) modulates immune response in ovarian cancer, particularly immune suppression by CAF-derived protein factors. By laser microdissection of tumor tissue samples from HGSOC patients, we generated cell type specific expression profiles and identified a CAF-specific gene signature for ovarian cancer. Genes expressed exclusively by CAFs that are associated with differential immune response were identified by comparing CAF expression profiles from HGSOC patients with high and low tumor-infiltrated CD8+ T cell densities. Among the differentially expressed genes identified, immunostaining results showed a significant inverse correlation between stromal MFAP5 expression and intratumoral CD8+ T cell density in HGSOC tissue samples (p = 0.006). The results were further confirmed by correlating stromal MFAP5 protein expression and intratumoral CD8+ T cell density. Our recent studies showed that increased stromal MFAP5 expression is associated with poorer survival in HGSOC patients and MFAP5 modulates ovarian caner invasion and motility potential. Together with our preliminary data showing that MFAP5 modulates the expression of immune-related genes, we hypothesize that stromal MFAP5 may generate an immunosuppressive microenvironment through suppressing CD8+ T cell activation and trafficking in the ovarian tumor tissue. Cell culture experiment showed that recombinant MFAP5 protein treatment induced expression of CD47, an immune checkpoint protein, in cancer cells and downregulated CXCL13, a chemokine essential for immune cell adhesion, in endothelial cells. Using animal models, these findings were further validated. Based on our data, animal studies will be performed to further evaluate the efficacy of immune activation by targeting stromal MFAP5 in ovarian cancer treatment. Delineating the molecular mechanism by which MFAP5 modulates the immune responses in ovarian cancer will help to design novel treatment modalities based on stromal MFAP5 blockade, which will promote activation and trafficking of cytotoxic CD8+ T cells and improve patient survival rates. Citation Format: Tsz-Lun Yeung, Cecilia S. Leung, Kwong-Kwok Wong, Samuel C. Mok. Stromal heterogeneity and immune response in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4145.

Abstract 715: Adipose tissues derived exosomal microRNAs and their variants in ovarian cancer progression

Most ovarian cancers are diagnosed at an advanced stage when the tumor is widely metastatic. The 5-year survival drops to 50% for the cancer cases that spread beyond the pelvis to the omentum. However, the mechanisms underlying the effect of omental adipose tissue on ovarian cancer progression are poorly understood. Recent studies showed that exosomes also contain non-coding RNAs such as microRNAs (miRNAs). Thus, we hypothesize that the transfer of microRNAs and their variants from ovarian cancer-associated omental adipose tissues to ovarian cancer cells via exosomes may contribute to the nearby microenvironment for ovarian cancer metastasis and cancer progression. Ion Torrent next generation sequencing was performed on miRNAs isolated and enriched from exosomes and cell lysates of ovarian cancer cell lines (OVCA), the epithelial component of microdissected omental ovarian cancer tissues (CT), normal omental adipose tissues (OMN) and ovarian cancer-associated omental adipose tissues (OMT). By integrating the miRNA expression profiles, 65 miRNAs were expressed at significant higher levels in OMT-derived exosomes compared with those in OMN-derived exosomes and OVCA-derived exosomes. A set of miRNAs (miR-32a, miR-221 and miR320a), which had been implicated in controlling cell growth and chemoresistance, was identified. Also, the Ion Torrent results were validated and exosomal transfer of OMT-derived miRNAs was confirmed in vitro. The exosomal communication between adipose tissues and ovarian cancer cells in the omental tumor microenvironment is verified. The transferable miRNAs and their variants may remain functional in the recipient ovarian cancer cells and confer more aggressive phenotypes in these cells. Citation Format: Chi Lam Au Yeung, Tetsushi Tsuruga, Ngai Na Co, Tsz-Lun Yeung, Cecilia S. Leung, Kwong K. Wong, Samuel C. Mok. Adipose tissues derived exosomal microRNAs and their variants in ovarian cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 715.

Abstract 1535: Omentin: A novel adipokine linking visceral obesity to ovarian cancer progression

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Advanced stage serous ovarian cancer metastasizes preferentially to the omentum, which is a major site of intra-abdominal fat accumulation, suggesting that the omental microenvironment is a favorable niche for ovarian cancer cells. The 5-year survival drops to below 50% for the cancer cases that spread beyond the pelvis to the omentum. It has also become apparent that obesity contributes to a poor clinical outcome. The mechanisms by which omental adipose tissue promotes tumor growth and disease progression are not entirely clear. Using transcriptome profiling analysis on the microdissected adipose tissue from patients with benign gynecologic diseases and from patients with advanced high-grade serous ovarian cancer (HGSOC), we found that a novel adipokine called omentin (Intestinal Lactoferrin Receptor ITLN1) was significantly down-regulated in ovarian cancer associated adipose tissue compared with the normal adipose tissue. Survival correlation studies demonstrated that patients with serum ITLN1 levels of >350 ng/mL at the time of first treatment experienced longer survival times than those with lower levels of ITLN1. We showed that ITLN1 suppressed the ovarian cancer cell migration ability and invasion potential in vitro. To delineate the underlying molecular mechanisms, RNA sequencing and pathway analyses were performed on ITLN1 treated ovarian cancer cells and we identified MMP1 as one of the potential mediators. It has been shown that MMP1 expression was induced by lactoferrin, which is abundant in ascites. We hypothesized ITLN1 could abrogate the effect of lactoferrin on ovarian cancer motility and invasion potential. Our preliminary data suggested that secreted ITLN1 may sequester lactoferrin in the ascites, thereby preventing it from binding to the low-density-lipoprotein-receptor-related-protein-1 (LRP-1) on ovarian cancer cell surface and thus inactivate downstream signaling pathways that control MMP1 expression. The study provides the first evidence that ovarian cancer cells modify the visceral adipose tissue through down-regulation of omentin to facilitate their growth in the omental microenvironment. Citation Format: Chi Lam Au Yeung, Ngai Na Co, Michaela Onstad, Tsz-Lun Yeung, Cecilia S. Leung, Rosemarie Schmandt, Karen H. Lu, Samuel C. Mok. Omentin: A novel adipokine linking visceral obesity to ovarian cancer progression. [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 1535. doi:10.1158/1538-7445.AM2015-1535

Abstract 4184: CAF-derived MFAP5 promotes tumor angiogenesis and confers paclitaxel resistance in high-grade serous ovarian cancer

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy. Although most patients respond to chemotherapy, relapse often occurs as chemoresistance disease. We previously reported the tumor-promoting role of Microfibrillar-Associated Protein 5 (MFAP5) in ovarian cancer. Follow up studies suggested a pro-angiogenic and chemo-protective role of MFAP5. Based on these findings, the present study aims at delineating the mechanism by which stromal MFAP5 modulates tumor angiogenesis and chemoresistance, and evaluating the potential of targeting MFAP5 as therapy for ovarian cancer. Immunostaining of HGSOC tissue sections revealed a significant correlation between stromal MFAP5 expression and intratumoral microvessel density. Further endothelial cell motility and tube formation assays on human endothelial cells demonstrated the pro-angiogenic roles of exogenous MFAP5. Furthermore, anti-αVβ3 integrin antibody and cell permeant calcium chelator, BAPTA, attenuated MFAP59s effects, suggesting that MFAP5 exerts its biological activity on endothelial cells through the αVβ3 integrin receptor and calcium signaling. In vivo studies showed that extensive tubular network was formed in the matrigel mixed with MFAP5 injected into mice (p In addition, analysis on stromal gene expression profiles revealed that stromal MFAP5 expression is significantly increased in the group of patients with shorter progression-free survival when compared to those with longer progression-free survival. Further cell survival assays showed that pretreating to HGSOC cell lines with recombinant MFAP5 confers resistance to ovarian cancer cells towards paclitaxel treatment. RNA-Seq analysis was performed to delineate the underlying mechanism of such induced chemoresistance. To conclude, stromal MFAP5 contributes to ovarian tumor angiogenesis and chemoresistance. Hence, targeting stromal MFAP5 could be a potential therapy for ovarian cancer treatment. Citation Format: Cecilia S. Leung, Tsz-Lun Yeung, Kay-Pong Yip, Kwong-Kwok Wong, Anil K. Sood, Michael J. Birrer, Samuel C. Mok. CAF-derived MFAP5 promotes tumor angiogenesis and confers paclitaxel resistance in high-grade serous ovarian cancer. [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 4184. doi:10.1158/1538-7445.AM2015-4184

Abstract 1029: CAF-derived MFAP5 promotes ovarian tumor angiogenesis through calcium dependent LPP signaling pathway

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ovarian cancer is the most lethal gynecologic malignancy. Although anti-angiogenic therapies look promising, no major strides have been made to improve ovarian cancer patient survival over the past decade. Therefore, finding new molecular targets that can improve therapeutic benefit from anti-angiogenic strategies are of paramount importance. Microfibrillar-Associated Protein 5 (MFAP5) was previously reported to be associated with prognosis and promoted ovarian cancer progression. In ovarian tumor samples, we identified a positive correlation between stromal MFAP5 and CD31 expressions which suggested a pro-angiogenic role of MFAP5. This study aims at delineating the mechanism of stromal MFAP5 in modulating tumor angiogenesis and investigating the potential of targeting MFAP5 as an anti-angiogenic therapy for ovarian cancer treatment. Increased motility and tube formation were observed in the presence of exogenous MFAP5 in both human microvascular endothelial cell 1 (HMEC-1) and telomerase immortalized microvascular endothelial cells (TIME). Besides, both anti-αVβ3 integrin antibody and cell permeant calcium chelator, BAPTA, attenuated MFAP5 stimulated motility. These suggested that MFAP5 exerts its biological activity through the αVβ3 integrin receptor and calcium signal transduction. In vivo, extensive CD34 positive tubular network was formed in the matrigel mixed with recombinant MFAP5 injected subcutaneously into mice. Further analysis using the Leica Metamorph software confirmed that there were significantly increases in the number of nodes, total tube area, number of tube segment and branch points in MFAP5 containing matrigel compared to control (p<0.01). Co-injection of MFAP5 overexpressing fibroblasts and cancer cells subcutaneously into mice led to increased tumor size and number of CD34 positive intratumor microvessels. In an orthotopic mouse model, silencing of stromal MFAP5 by siRNA delivered by nanoparticles demonstrated a significant reduction in tumor weight (p<0.001) and intratumor microvessel densities. Finally, microarray analysis on endothelial cells invaded into Matrigel plug, identified a set of motility promoting genes that were associated with calcium signaling. Among them, upregulation of Lipoma-Peferred Partner (LPP), an actin cytoskeleton protein, was validated by qRT-PCR and western blot analyses. Silencing LPP by siRNA abolished MFAP5 stimulated motility in both HMEC-1 and TIME. Further western blot analyses showed that MFAP5 modulated endothelial cell motility and invasion potential through calcium dependent ERK/CREB/LPP signaling pathways. To conclude, stromal MFAP5 acts on αVβ3 integrin receptor and modulates endothelial cell behavior through calcium signaling. It contributes to ovarian tumor angiogenesis and tumor progression. Hence, targeting stromal MFAP5 could be a potential anti-angiogenic therapy for ovarian cancer treatment. Citation Format: Cecilia S. Leung, Tsz-Lun Yeung, Kay-Pong Yip, Kwong-Kwok Wong, Anil K. Sood, Michael J. Birrer, Samuel C. Mok. CAF-derived MFAP5 promotes ovarian tumor angiogenesis through calcium dependent LPP signaling pathway. [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 1029. doi:10.1158/1538-7445.AM2014-1029

Abstract 4887: Omentin: A novel adipokine in the omental microenvironment associated with ovarian cancer progression

Advanced stage serous ovarian cancer metastasizes preferentially to the omentum, which is a well-vascularized fold of peritoneal tissue and is a major site of intra-abdominal fat accumulation, suggesting that the omental microenvironment is a favorable niche for ovarian cancer cells. The 5-year survival drops to below 50% for the cancer cases that spread beyond the pelvis to the omentum. It has also become apparent that obesity contributes to a poor clinical outcome. The mechanisms by which omental adipose tissue promotes tumor growth and disease progression are not entirely clear. Using transcriptome profiling analysis on the microdissected adipose tissue from patients with benign gynecologic diseases and from patients with advanced high-grade serous ovarian cancer (HGSOC), we identified a gene signature for ovarian cancer associated omental adipose tissue, suggesting that alteration of these genes in the ovarian cancer associated omental adipose tissue may generate a permissive microenvironment to support ovarian cancer growth. Among genes that are significantly up- or down-regulated in ovarian cancer associated adipose tissue compared with the normal adipose tissue, we seek to focus on evaluating the role of omentin (Intestinal Lactoferrin Receptor ITLN1) in ovarian cancer progression since it is a novel adipokine that is predominantly expressed and secreted by visceral adipose tissue and is barely detectable in subcutaneous fats. Our data showed for the first time that omentin was expressed predominantly by the mesothelial cells covering the visceral adipose tissue but not by other cell types in the omental adipose tissue. Interestingly, we showed that circulating omentin level is significantly lower in patients with HGSOC compared with those in the BMI matched healthy individuals. In addition, using monolayer culture models, we demonstrated that omentin suppressed ovarian cancer motility and invasion potential directly and ovarian cancer growth only in the presence of adipocytes. We also showed that omentin can increase insulin-dependent glucose up-take in adipocytes and omentin expression can be down-regulated by co-culturing with ovarian cancer cells and in the presence of TNF-α. The study provides the first evidence that ovarian cancer cells modify the visceral adipose tissue through down-regulation of omentin to facilitate their growth in the omental microenvironment. Citation Format: Chi Lam Au Yeung, Ngai Na Co, Michaela Onstad, Tsz-Lun Yeung, Cecilia S. Leung, Rosemarie Schmandt, Karen H. Lu, Samuel C. Mok. Omentin: A novel adipokine in the omental microenvironment associated with ovarian cancer progression. [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 4887. doi:10.1158/1538-7445.AM2014-4887

Abstract 1939: Identification of microRNAs related to chemosensitivity in ovarian cancer using Next Generation Sequencing.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Ovarian cancer is the most common cause of gynecologic malignancy related mortality in women. The combination of tumor reductive surgery and chemotherapy is the main therapeutic strategy for ovarian cancer. However, especially in the most of advanced cases, complete site reduction is impossible to achieve. In those cases, the sensitivity to chemotherapy strongly affects their prognoses. MicroRNAs (miRNAs), which are approximately 22-nucleotide non-coding RNAs, regulate protein expression by translational inhibition and degradation of their target mRNAs. The expression of miRNAs is dysregulated in various types of cancers, suggesting that miRNAs are involved in carcinogenesis and cancer progression. The purpose of present work was to identify miRNAs which are related to chemo-sensitivity in ovarian cancer. Using Next Generation Sequencing (NGS), miRNA profiling was performed in 38 advanced high-grade serous ovarian cancers (HGSOCs). RNAs including miRNAs were procured from the epithelial component of the laser microdissected tumor tissue removed from patients undergoing primary surgery without neo-adjuvant chemotherapy. A total of 1935 miRNAs and miRNA variants were detected by NGS and normalized expression data of each miRNAs and miRNA variants were used for further analyses. Comparing the copy numbers of each miRNAs between chemo-refractory group (patients with disease progression in the period of first line chemotherapy, n=7) and chemo-sensitive group (patients with disease progression in more than 6 months after completion of first line chemotherapy, n=13) by Mann-Whitney U test, 77 miRNAs and miRNA variants were found to be dysregulated with significant differences (p<0.05). The 10 most significant miRs identified by NGS were selected for further validation studies by real-time reverse transcription PCR (RT-PCR) analysis on the same sample set. To determine whether miRNAs could be used as a prognostic marker HGSOC, expression levels of those 10 miRNAs were quantified by RT-PCR using 95 HGSOC samples including the 38 samples used in the discovery set. The results showed that mir-625-3p expression levels were significantly reduced in the chemo-refractory group compared with the chemo-sensitive group as indicated by the NGS data (n=20, p=0.029), and by the RT-PCR data (n=63, p=0.004). Furthermore, Kaplan-Meier survival analysis with log-rank test indicated that low expression of mir-625-3p (less than mean expression) was significantly associated with shorter progression-free survival (n=95, p=0.007) and shorter over-all survival (n=95, p<0.001) as compared with the high expression group (more than mean expression). Our results strongly suggest that mir-625-3p might be a prognostic marker that can be used to predict chemo-sensitivity in patients with HGSOC. Further characterization of mir-625-3p as a therapeutic target for HGSOC is in progress. Citation Format: Tetsushi Tsuruga, Chi Lam Au Yeung, Cecilia S. Leung, Tsz-Lun Yeung, Kwong K. Wong, Rosemarie Schmandt, Ngai Na Co, Karen H. Lu, Samuel Mok. Identification of microRNAs related to chemosensitivity in ovarian cancer using Next Generation Sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1939. doi:10.1158/1538-7445.AM2013-1939