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Cervical cancer screening rate is extremely low and the governmental recommendation of HPV vaccine has been suspended for 5 years in Japan. Here, we utilized data from the Osaka Cancer Registry, collected between 1976 and 2012, to evaluate cervical cancer trends in Japan. Age-adjusted incidence, relative survival, and conditional survival rates were calculated using multiple imputation methods and period analyses in 25,826 cervical cancer cases. Association of survival rates and clinical factors, including patients' age, clinical stage, and treatment procedures, were also analyzed. A trend for significantly decreasing age-adjusted incidence of cervical cancer (per 100,000) began in 1976 but reversed after 2000, increasing significantly to date (annual percent change = 3.8, 95% confidence interval, 2.7–4.8; age-adjusted rate: 28.0 in 1976, 9.1 in 2000, 14.1 in 2012). The 10-year relative survival rate improved significantly after 2002, especially in cases of “localized” and “adjacent organs” disease; this was likely due to the introduction of concurrent chemotherapy and radiation. The conditional 5-year relative survival rate improved significantly yearly until the fourth survival year. In the surgery-based group, we observed no age-dependent differences in outcomes. Unexpectedly, however, prognosis for younger age groups was poorer in the radiation-based treatment group. These results indicate that although relative survival rates have recently increased, treatment for more advanced cases with distant metastasis requires further improvement. In addition, this study is the first to suggest that age might be an important predictor of radiotherapy resistance in cervical cancer. Significance: A large-cohort analysis of cervical cancer cases reveals that age-adjusted incidence in Japan has increased since 2000 and that age may negatively correlate with resistance to radiotherapy.

Histopathologic analysis through biopsy has been one of the most useful methods for the assessment of malignant neoplasms. However, some aspects of the analysis such as invasiveness, evaluation range, and turnaround time from biopsy to report could be improved. Here, we report a novel method for visualizing human cervical tissue three-dimensionally, without biopsy, fixation, or staining, and with sufficient quality for histologic diagnosis. Near-infrared excitation and nonlinear optics were employed to visualize unstained human epithelial tissues of the cervix uteri by constructing images with third-harmonic generation (THG) and second-harmonic generation (SHG). THG images enabled evaluation of nuclear morphology in a quantitative manner with six parameters after image analysis using deep learning. It was also possible to quantitatively assess intraepithelial fibrotic changes based on SHG images and another deep learning analysis. Using each analytical procedure alone, normal and cancerous tissue were classified quantitatively with an AUC ≥0.92. Moreover, a combinatory analysis of THG and SHG images with a machine learning algorithm allowed accurate classification of three-dimensional image files of normal tissue, intraepithelial neoplasia, and invasive carcinoma with a weighted kappa coefficient of 0.86. Our method enables real-time noninvasive diagnosis of cervical lesions, thus constituting a potential tool to dramatically change early detection. Significance: This study proposes a novel method for diagnosing cancer using nonlinear optics, which enables visualization of histologic features of living tissues without the need for any biopsy or staining dye.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy, but it still lacks effective treatment options. In this study, we utilized proteomic technology to identify lipolysis-stimulated lipoprotein receptor (LSR) as a new tumor antigen of EOC. Immunohistochemical analysis of EOC tissues in conjunction with survival analysis of EOC patients showed that high expression of LSR is associated with poor prognosis. High LSR expression also occurred in tumor metastases including to the lymph node and omentum. To evaluate the possible benefits of blocking this antigen in EOC, we raised a new monoclonal antibody (mAb) to human LSR (hLSR). In mouse xenograft models of hLSR+ EOC (cell lines or patient-derived tumors), we found that administration of anti-hLSR mAb inhibited tumor growth in a manner independent of both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Mechanistic investigations showed that hLSR expression increased incorporation of very-low-density lipoprotein (VLDL) into EOC cells and that anti-hLSR mAb inhibited lipid uptake in vitro and in vivo. Moreover, VLDL promoted cell proliferation in hLSR-positive EOC cells in vitro, and this effect was inhibited by anti-hLSR mAb. While the anti-hLSR mAb studied cross reacted with the mouse antigen, we observed no adverse effects on normal organs and lipid metabolism in murine hosts. Our findings suggest that hLSR plays a key functional role in EOC development and that this antigen can be therapeutically targeted by specific mAb to improve EOC treatment. Significance: These findings offer preclinical evidence of the therapeutic efficacy of a novel targeted antibody therapy against deadly epithelial ovarian cancers. Cancer Res; 78(2); 516–27. ©2017 AACR.

Introduction: Advanced endometrial cancer (EC) still has poor prognosis and requires the development of new therapeutic agents. In ovarian and gastric cancer, we revealed that high expression of lipolysis-stimulated lipoprotein receptor (LSR), a cell-surface membrane protein, was associated with poor prognosis and LSR could be a new therapeutic target. However, in EC, the clinical and cell biological role of LSR is still unclear. We aimed to investigate the functions of LSR in EC. Methods: We evaluated LSR expression by immunohistochemistry and analyzed overall survival (OS) and clinicopathological factors in 228 EC patients. To investigate the mechanism by which LSR affects the prognosis of EC patients, the pathway enrichment analysis and gene ontology analysis were conducted using the publicly available proteogenomic dataset of EC. Cell proliferation was analyzed by WST-8 assay using two LSR-knockdown cell lines (HEC1 and HEC116) developed by transfection of LSR-siRNA, and the activity of several signaling pathways were examined by Western blotting. Results: Patients were divided into two groups based on LSR expression; High (darkly stained cells in ≥25% of the area, n=153) and Low (darkly stained cells in Conclusion: LSR regulated cell proliferation via MEK/ERK pathway, and contributed poor prognosis in EC. LSR may be a new therapeutic target of advanced EC. Citation Format: Yoshikazu Nagase, Kosuke Hiramatsu, Satoshi Nakagawa, Shinya Matsuzaki, Toshihiro Kimura, Satoshi Serada, Yutaka Ueda, Tetsuji Naka, Tadashi Kimura. LSR is a novel prognostic factor by regulating cell proliferation via MEK/ERK pathway in endometrial cancer:Analysis of signal transduction, bioinformatics, and in vitro/in vivo study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1989.

Objective: The development of therapeutic agents for platinum-resistant ovarian carcinoma remains a significant issue. Currently, the use of antibody-drug conjugates (ADCs) represents an innovative therapeutic approach for various cancers. In this study, we examined the antitumor effect of a CD70-ADC on platinum-resistant serous ovarian carcinoma (SOC). Methods: The CD70 expression was examined in A2780 cells (an SOC cell line) by western blotting and fluorescence activated cell sorting analysis. Cisplatin resistant A2780 cell lines (A2780 CisR cells) were generated, and the IC50 values for the A2780 parent and A2780 CisR cells were determined using modified MTT assays. The A2780 CisR cells were transfected with siRNA to suppress the CD70 expression. The suppression was confirmed by western blotting. Next, the CD70 monoclonal antibodies were generated to investigate their antitumor effect on the A2780 parent and A2780 CisR cells. ADCs, which consisted of CD70 monoclonal antibody linked to monomethyl auristatin F (MMAF, the tubulin polymerization inhibitor), were generated, and their antitumor effect against the A2780 CisR cells was investigated in vitro and in vivo. After the A2780 CisR-xenograft tumors were treated with CD70-ADC, immunohistochemistry (IHC) analysis was performed using antibodies against the mitotic marker, anti-phospho-histone H3. Results: The expression of CD70 was observed in the A2780 CisR cells but not in the A2780 parent cells. CD70 silencing in A2780 CisR cells did not significantly reduce the cisplatin IC50 value nor did it inhibit cell proliferation. The CD70 monoclonal antibodies did not inhibit the proliferation of the A2780 parent and CisR cells. These results suggest that inhibiting the expression of CD70 does not affect the sensitivity of cisplatin and the growth of the tumor. Next, we generated the CD70-ADC. The CD70-ADC did not reach IC50 in the A2780 cells; however, the IC50 value was 0.104 nM for the A2780 CisR cells. CD70-ADC successfully exhibited antitumor effects in the CD70-ADC-treated mice in comparison with the control, i.e., the ADC-treated mice (162.4 vs. 534.2 mm3; p Conclusion: Our results revealed that CD70-ADC could be a novel therapeutic agent for platinum-resistant SOC. Citation Format: Mayu Shiomi, Shinya Matsuzaki, Ruriko Nakae, Satoshi Nakagawa, Akiko Okazawa, Eiji Kobayashi, Yutaka Ueda, Satoshi Serada, Tetsuji Naka, Tadashi Kimura. CD70 antibody-drug conjugate: A novel therapeutic agent for platinum-resistant ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3853.

Small cell neuroendocrine carcinoma (SCNEC) of uterine cervix is a rare tumor accounting for approximately 1-2% of uterine cervical tumors. SCNEC of uterine cervix is highly invasive and has an extremely poorer prognosis than other histologic types for which no standard treatment is established. To establish a new management strategy against SCNEC of uterine cervix, we developed a drug sensitivity assay based on a 3D primary culture system. We previously developed the cancer tissue-originated spheroid (CTOS) method, a primary culture method from patients’ tumors. To date, we established a panel of 12 SCNEC CTOS lines with the success rate of 100%. For each line, multiple CTOSs were placed in one well of a 96 well plate and dose response analysis was performed for seven anticancer drugs; paclitaxel, carboplatin, irinotecan, SN-38, cisplatin, etoposide and gemcitabine. After 7 days of culture, the CTOSs viability was evaluated by measuring intracellular ATP levels, corrected by the size of CTOS at day0. All drugs had substantial variations in sensitivity among the lines. Cerv54 was far more sensitive to irinotecan than other lines while was similarly sensitive to SN-38. We revealed that cancer cells of cerv54 had high levels of carboxylesterase(CES)1 expression, which converts irinotecan to SN38. Since cerv54 with CES1 knockdown by pGFP-C-shCES1 lentivirus had lower sensitivity than sh-scramble, CES1 expression is related to the irinotecan sensitivity in cerv54. CES1 expression in cancer cells may be useful for predicting the effect of irinotecan based chemotherapy. In addition, this study indicates that sensitivity test using CTOS might be applicable to precision medicine by selecting sensitive drugs for each patient. Citation Format: Mie Tanaka, Satoshi Kubota, Yu Ito, Yumiko Kiyohara, Hiroko Endo, Jumpei Kondo, Satoshi Nakagawa, Akiko Okazawa, Shinya Matsuzaki, Toshihiro Kimura, Eiji Kobayashi, Yutaka Ueda, Kiyoshi Yoshino, Shoji Kamiura, Tadashi Kimura, Masahiro Inoue. Ex vivo drug sensitivity assay with a panel of cancer patient-derived spheroids of small cell neuroendocrine carcinoma of uterine cervix [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 33.

Background:Previously, we identified lipolysis-stimulated lipoprotein receptor (LSR) as a new target of epithelial ovarian cancer (EOC), and we reported inhibitory effect of our newly developed monoclonal antibody (mAb) against LSR-positive ovarian cancer cells in vitro and in vivo. We also demonstrated that LSR took in triglyceride-rich protein and promoted cell proliferation, however, LSR function in EOC cells is not investigated enough. Therefore, we aimed to reveal the function of LSR in EOC cells focusing on lipid metabolism including beta oxidation and subsequent TCA cycle. Material and Method: Using LSR-positive ovarian cancer cell lines (OVCAR3 and RMG-I), we investigated activation of beta-oxidation by LSR after administration of VLDL which is one of the triglyceride-rich protein. Moreover, we constructed LSR-knockdown EOC cells and investigated the effect of VLDL administration in these cells. In addition, we showed inhibitory effect of our anti-LSR mAb against lipid metabolism. Expression of beta-oxidation related proteins were analyzed by western blot analysis. Production of acetyl CoA and ATP were measured using each assay kit. Cell viability was analyzed by WST-8 assay and thymidine incorporation assay. Results: In LSR-positive EOC cells, LSR promoted uptake of VLDL in glucose restricted environment compared with LSR-knockdown cells (p < 0.05) and VLDL administration promoted acetyl CoA and ATP production (p < 0.05), suggesting that beta-oxidation, TCA cycle and electron transport system were activated by fatty acid from VLDL in LSR-positive cells. Moreover, in LSR-positive EOC cells, VLDL administration increased viable cells compared with LSR-knockdown cells (p < 0.05), however, DNA synthesis was not promoted (p < 0.05). These results showed that VLDL uptake via LSR promoted lipid metabolism and contributed cell viability. Finally, our anti-LSR mAb inhibited these processes. Conclusion: In glucose restricted environment, LSR takes in triglyceride-rich protein, activates lipid metabolism via beta-oxidation and contributes cell viability in LSR-positive EOC cells. Citation Format: Kosuke Hiramatsu, Satoshi Serada, Minoru Fujimoto, Shinya Matsuzaki, Yutaka Ueda, Kiyoshi Yoshino, Tadashi Kimura, Tetsuji Naka. LSR promotes lipid metabolism via beta oxidation and contributes cell viability in epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1434.

Objective: Resistance to platinum drugs remains a significant problem. Our objective is to elucidate the role of ATP7B, a copper transporter which excretes intracellular platinum giving drug resistance, in gynecologic malignancies and to develop a method which can overcome the platinum resistance. Methods: The expression of ATP7B was evaluated in 3 uterine leiomyosarcoma (LMS) cell lines (SK-LMS, SK-UT1 and SKN) and 26 clinical samples of LMS by immunohistochemical (IHC) staining, respectively. The IC50 values of cisplatin were measured by modified MTT assay. Intracellular platinum accumulation was determined by ICP-MS analysis in SK-LMS cells and ATP7B-stably suppressed cells (SK-LMS-7B cells), which transfected the pRS ATP7B shRNA vector. To analyze in vivo platinum sensitivity, SK-LMS and SK-LMS-7B cells were subcutaneously injected into nude mice. Cisplatin (3 mg/kg) or PBS was administered i.p. to xenografted mice twice weekly for 28 days. Further, we attempted to show the improvement of platinum sensitivity by using premedication for CuSO4 before cisplatin treatment in vitro and in vivo model. Results: Expression of ATP7B was observed in all 3 LMS cell lines. In clinical samples, ATP7B was over expressed in 14 of 26 (53.8%) cases. In drug resistance analysis, significantly lower IC50 values of cisplatin were observed in SK-LMS-7B cells (4.3μM, p < 0.01) compared with SK-LMS cells (17μM) indicating knockdown of ATP7B improved platinum sensitivity. Also, significantly higher platinum accumulation was detected in SK-LMS-7B cells (7.2ng/dish, p < 0.01) compared with SK-LMS cells (52ng/dish). In in vivo analyses, a significantly higher anti-tumor effect of cisplatin was observed in SK-LMS-7B-xenografted mice compared with SK-LMS-xenografted mice (56±27 μg vs 214±68 μg, p Conclusion: ATP7B contributes platinum resistance in LMS. Presumably, ATB7B preferentially excrete copper ion thereby platinum can be accumulate in cells. Thus, pretreatment with CuO4 effectively improved platinum sensitivity in vitro and in vivo by blocking ATP7B. Citation Format: Mamoru Kakuda, Shinya Matsuzaki, Ruriko Nakae, Yusuke Tanaka, Eiji Kobayashi, Yutaka Ueda, Kiyoshi Yoshino, Tadashi Kimura. Novel strategy to overcome platinum resistance in uterine leiomyosarcoma; blocking ATP7B by copper ion [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 1200. doi:10.1158/1538-7445.AM2017-1200

Objective:Ovarian cancer is the most lethal gynecological malignancy. Five-year survival of advanced ovarian cancer patients remains less than 50% and the mortality rate has not changed in recent years. This study is aimed to screen for novel ovarian cancer therapeutic targets using quantitative proteomic approach and to develop antibody-based medicine targeting novel ovarian cancer antigens. Method:Exhaustive quantitative proteome analysis focused on cell surface membrane proteins were performed by isobaric tags for relative and absolute quantitation(iTRAQ) using a normal ovarian surface epithelial cell line and 7 ovarian cancer cell lines. By this approach, ovarian cancer-specific membrane proteins were identified. We confirmed the expression of lipolysis-stimulated lipoprotein receptor (LSR) using immunohistochemical staining, western blotting method, and flow cytometry. By modified MTT assay, cell growth inhibition was performed in LSR-knock down cells compared with control cells. Cell adhesion assay was also performed in vitro. Anti-LSR monoclonal antibody (anti-LSR mAb) was generated to evaluate the efficacy of the LSR targeted antibody therapy in vivo. For subcutaneous xenograft experiments, ovarian cancer cell line (RMG-1) was injected subcutaneously into the CB17/SCID mice. Mice were then randomly divided into two groups and administrated intraperitoneally anti-LSR mAb or control IgG antibody twice a week for 6 times. In addition, luciferase transfected ovarian cancer cell line (A2780-luc) was implanted intraperitoneally into the CB17/SCID mice. Mice were administrated anti-LSR mAb or control IgG antibody intraperitoneally twice a week for 5 times. Result:By iTRAQ analysis, we identified 1685 proteins and one of the ovarian cancer candidates protein, LSR was identified. We confirmed the expression of LSR in 8 out of 11 ovarian cancer cell lines and all of ovarian cancer clinical specimens. We performed proliferation assay using siRNA against ovarian cancer cell lines. When expression of LSR was suppressed, a tumor growth was significantly inhibited at day 4(p Conclusion:We showed that the LSR is related to cell proliferation and adhesion in vitro in ovarian cancer cells and proved the antitumor effect of anti-LSR mAb. These results suggested that LSR can be a new therapeutic target of ovarian cancer. Citation Format: Satoko Matsuzaki, Kosuke Hiramatsu, Satoshi Serada, Satoshi nakagawa, Shinya Matsuzaki, Yutaka Ueda, Minoru Fujimoto, Kiyoshi Yoshino, Tadashi Kimura, Tetsuji Naka. Lipolysis-stimulated lipoprotein receptor (LSR) can be a novel therapeutic target of 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 4998.

Background: Platinum drugs are often used for the postoperative adjuvant therapy or treatment in advanced and recurrent ovarian cancer. We have previously reported that ovarian clear cell carcinoma (CCC) has increased expression of Annexin A4 (Anx A4) in comparison with other subtype of ovarian cancer. Enhanced expression of Anx A4 has been shown to induce chemoresistance via extracellular efflux of carboplatin. Purpose: To identify the domain of Anx A4 relevant to chemoresistance. Materials and methods: Anx A4 possesses the NH2-terminal domain and four annexin repeat domains with one Ca2+-binding site in each domain. Deleting annexin repeat domains one by one from C-terminal site, we generated four deletion mutants of Anx A4 named R1, 2, 3, and 4 (arabic figure shows the number of annexin repeat domains). Stable cell lines overexpressing each deletion mutants were established in NUGC3 cells. Chemosensitivity to platinum drugs was evaluated using the MTT assay. Results. Compared with mock transfected cells, all the four deletion mutants induced chemoresistance to both cisplatin and carboplatin. Conclusions. The deletion derivative covering only one annexin repeat domain can induce chemoresistance to platinum drugs. Further study is underway to determine the specific site for chemoresistance of Anx A4. Citation Format: Akiko Morimoto, Takayuki Enomoto, Shinya Matsuzaki, Kousuke Hiramatsu, Yutaka Ueda, Kiyoshi Yoshino, Masami Fujita, Takuhei Yokoyama, Satoshi Serada, Tetsuji Naka, Tadashi Kimura. Functional domain of Annexin A4: which domain is indispensable for chemoresistance to platinum drugs. [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 4057. doi:10.1158/1538-7445.AM2013-4057

Because platinum drugs are often used for the chemotherapy of human cancers, when platinum resistance occurs it is a major issue. We recently reported that enhanced expression of Annexin A4 (Anx A4) increases chemoresistance to Carboplatin via increased extracellular efflux of the drug. However, the precise mechanisms of that chemoresistance, and the relationship of Anx A4 to platinum resistance in vivo, remain unclear. In this report we investigate in vitro the mechanism of platinum resistance induced by Anx A4 in endometrial carcinoma cells (HEC1 cells) which normally have a low level of expression of Anx A4. Forced expression of Anx A4 in HEC1 cells resulted in chemoresistance to platinum drugs. In addition, we compared HEC1 control cells with Anx A4-overexpressing HEC1 cells when both were xenografted to mice, Anx A4-overexpressing xenografted mice presented with significantly greater chemoresistance to Cisplatin in vivo. By immunofluorescence analysis we found that exposure to platinum drugs induced relocation of Anx A4 from the cytoplasm to the cellular membrane, where it became co-localized with ATP7A, a copper transporter also well known to be a platinum effluxer. When the expression of ATP7A was suppressed by small interfering RNA in HEC1 control cells, they showed no change of chemosensitivity to platinum drugs. However, suppressed ATP7A in Anx A4 overexpressing platinum-resistant cells, they showed improved chemosensitivity to platinum drugs, to a level comparable of that of control cells. Our results indicate that enhanced expression of Anx A4 confers platinum resistance by promoting efflux of platinum drugs via ATP7A. Citation Format: Shinya Matsuzaki, Akiko Morimoto, Satoshi Serada, Takuhei Yokoyama, Toshihiro Kimura, Eiji Kobayashi, Yutaka Ueda, Kiyoshi Yoshino, Masami Fujita, Takayuki Enomoto, Tetsuji Naka, Tadashi Kimura. Annexin A4-conferred platinum resistance is mediated by the copper transporter ATP7A. [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 4059. doi:10.1158/1538-7445.AM2013-4059

BACKGROUND: Platinum drugs play the key role for postoperative adjuvant therapy and treatment in progress and recurrent ovarian cancer. We have previously reported that ovarian clear cell carcinoma (CCC) has increased expression of Annexin A4 (Anx A4) in comparison with other subtypes of ovarian cancer. Enhanced expression of Anx A4 has been shown to induce chemoresistance via extracellular efflux of carboplatin. However, it remains unclear whether targeting Anx A4 is effective for improving the sensitization against chemotherapy, like platinum drugs. The aim of this study was to evaluate the role of Anx A4 in chemoresistance in ovarian CCC. MATERIAL AND METHODS: RMG-I cell lines were used to establish the stable knockdown of Anx A4. Plasmids encoding short hairpin RNAs (shRNAs) targeting Anx A4 were transfected. Chemoresistance was assessed by IC50 value in various drugs. RESULTS: Compared with control cells, knockdown of Anx A4 gene expression in RMG-I cells induced chemosentisitzation around 2-fold to cisplatin (IC50: 11.2 μM to 5.8 μM, P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4225. doi:1538-7445.AM2012-4225

Background. Endometrial cancer is one of the most common malignancies of the female genital tract. The identification of proteins for prognostic assessment and therapeutic targets in this disease is of significant clinical importance. Quantitative proteomic analysis provides a powerful approach in screening for alterations in protein levels and post-translational modifications that are associated with tumors. To date, proteomic studies of cancer have focused particularly on sub-cellular compartments or post-translational modifications. An analysis of the differential expression of cell-surface membrane proteins would be of particular interest, since these proteins play key roles in cell function including cell migration and drug resistance. Purpose of study. To identify membrane proteins overexpressed in endometrial cancer which may represent novel biomarkers of disease and therapeutic targets. Procedures. We developed a biotinylation-based approach for cell membrane enrichment combined with iTRAQ (isobaric tags for relative and absolute quantitation) technology using nano-LC-MS/MS analysis, to identify cell surface proteins overexpressed in seven endometrial cancer cell lines compared with a control normal endometrial cell line. Results. 365 proteins were identified, of which, 65 proteins (18%) possessed a trans-membrane domain. We identified overexpression of 24 membrane proteins in endometrial cancer cell lines compared with a normal endometrial cell line. By this proteomic approach, we identified several tumor antigens which included antigens previously associated with endometrial cancer and antigens not previously reported in this disease. Tumorigenic proteins such as neural cell adhesion molecule L1 (NCAML1), previously reported to be overexpressed in endometrial cancer, were identified. Interestingly we also identified Claudin-4, a protein associated with poor prognosis in various cancers, but not previously reported in the pathogenesis of cancer. In addition, we also successfully identified novel antigens associated with endometrial cancer. Conclusions. We developed a high throughput cell-surface membrane proteomic analysis combining biotinylation of cell-surface membrane protein and iTRAQ technology. In addition to the identification of previously reported tumor antigens such as NCAML1 and Claudin-4, we also identified novel tumor antigens in endometrial cancer by this methodology. This iTRAQ based quantitative proteomic analysis represented a useful approach in screening for novel disease biomarkers and therapeutic targets. Further studies on the physiological role of novel tumor antigens overexpressed in endometrial cancer and their application as biomarkers/therapeutic targets are underway. 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 5108. doi:10.1158/1538-7445.AM2011-5108