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Aim To identify predictors of poor prognosis of patients with colon cancer (CC) who underwent surgery with curative intent, we investigated the association between the albumin to globulin ratio (AGR) with clinicopathological findings such as overall (OS) and disease-free (DFS) survival. Patients and methods We conducted a retrospective study of clinicopathological findings, including preoperative laboratory data, for 248 patients with stage I-III CC. Results Patients with low AGR had shorter DFS and OS compared to those with high AGR. Multivariate analyses identified low AGR as an independent variable independently associated with recurrence and poor prognosis of patients with CC who underwent surgery with curative intent regardless of lymphnode metastasis. Conclusion The preoperative AGR was an independent predictor of recurrence and poor prognosis of patients with CC who underwent surgery with curative intent. The AGR indicates that these patients may benefit from intensive adjuvant therapy.

Although patients with metastatic colorectal cancer (CRC) are often unable to undergo treatment after resection of primary tumors, identifying such patients before surgery is not easy. In this study, we evaluated the association among clinicopathological findings, survival outcomes, and ability to undergo multimodal therapy after primary tumor resection in patients with Stage IV CRC. We collected clinicopathological findings and preoperative laboratory data, including carcinoembryonic antigen (CEA) and systemic inflammatory response markers for 92 patients who were treated for Stage IV CRC between 2005 and 2014. We used multivariate analysis on factors that affect prognosis and ability to undergo postoperative treatment. Postoperative multimodal therapy improved overall survival (OS) significantly. Among serum markers, elevated CEA, neutrophil-to-lymphocyte ratio, and modified Glasgow prognosis score (mGPS) were significant indicators of shorter OS. In multivariate analysis, low performance status (P = 0.003), undifferentiated histology type (P = 0.019), and elevated mGPS (P = 0.042) were independent predictors of worse prognosis; and older age (P = 0.016), right-sided colon cancer (P = 0.043), and elevated mGPS (P = 0.031) were independent risk factors for difficulty of introducing postoperative multimodal therapy. Preoperative mGPS is a useful objective indicator for CRC patients with multiple metastases who are able to undergo primary site resection followed by postoperative multimodal therapy.

A 63-year-old man visited the clinic with numbness in the right hand. Magnetic resonance imaging demonstrated multiple low-intensity lesions in the cervical vertebrae and sacrum, which was suspicious of cervical bone metastasis. Fluorodeoxyglucose positron emission tomography/computed tomography revealed areas of increased fluorodeoxyglucose uptake in the thoracic esophagus, sternum and sacrum. A flat, elevated esophageal cancer was identified by upper gastrointestinal endoscopy, and the macroscopic appearance indicated early-stage disease. From the cervical, thoracic and abdominal computed tomography images, there were no metastatic lesions except for the bone lesions. To confirm whether the bone lesions were metastatic, we performed bone biopsy. The histopathological diagnosis was bone marrow hyperplasia. It was crucial for treatment planning to establish whether the lesions were distant metastases. Here, we report a case of esophageal cancer with bone marrow hyperplasia mimicking bone metastasis.

Metastasis is a major cause of death in patients with gastric cancer (GC). MicroRNAs (miRNAs) relating to the epithelial–mesenchymal transition (EMT) control GC progression and metastasis. The aim of this study was to evaluate serum EMT-associated miRNAs for metastatic and prognostic noninvasive biomarkers in GC. In the first step of this study (preliminary experiments), we selected candidate miRNAs associated with metastasis by analyzing the expression of the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) and miR-203 in serum samples from stage I (n = 12) and stage IV (n = 12) GC patients. The second phase involved the independent validation of candidate miRNAs in serum specimens from 130 patients with GC and 22 controls. Based on the preliminary experiments, miR-203 was selected as the candidate serum miRNA that was most closely associated with metastasis. Validation analysis revealed that serum miR-203 levels were significantly lower in stage IV than stage I–III GC patients. Serum miR-203 expression was significantly lower in GC patients with a higher T stage, vessel invasion, and lymph node, peritoneal, and distant metastases. Low expression of serum miR-203 was significantly associated with poor disease-free and overall survival. Multivariate analysis revealed that low serum miR-203 expression was an independent predictive marker for lymph node, peritoneal, and distant metastases and a poor prognosis in patients with GC. Serum miR-203 has the potential to serve as a noninvasive biomarker for prognosis and to predict metastasis in patients with GC.

// Koichiro Mori 1 , Yuji Toiyama 1 , Kohei Otake 1 , Shozo Ide 1 , Hiroki Imaoka 1 , Masato Okigami 1 , Yoshinaga Okugawa 1 , Hiroyuki Fujikawa 1 , Susumu Saigusa 1 , Junichiro Hiro 1 , Minako Kobayashi 1 , Masaki Ohi 1 , Koji Tanaka 1 , Yasuhiro Inoue 1 , Yuhko Kobayashi 2 , Yasuhiko Mohri 1 , Issei Kobayashi 2 , Ajay Goel 3 and Masato Kusunoki 1 1 Department of Gastrointestinal and Pediatric Surgery, Mie University Graduate School of Medicine, Tsu, Mie, Japan 2 Center for Molecular Biology and Genetics, Mie University, Mie, Japan 3 Center for Gastrointestinal Research & Center for Epigenetics, Cancer Prevention and Cancer Genomics, Baylor Scott & White Research Institute and Charles A Sammons Cancer Center, Baylor University Medical Center, Dallas, TX USA Correspondence to: Yuji Toiyama, email: ytoi0725@clin.medic.mie-u.ac.jp Ajay Goel, email: Ajay.Goel@BSWHealth.org Keywords: iTRAQ-based quantitative proteomic analysis; ezrin; lymph node metastasis; colorectal cancer; biomarker Received: February 02, 2017 Accepted: June 29, 2017 Published: October 30, 2017 ABSTRACT Colorectal cancer (CRC)-associated mortality is primarily caused by lymph node (LN) and distant metastasis, highlighting the need for biomarkers that predict LN metastasis and facilitate better therapeutic strategies. We used an I sobaric T ags for R elative and A bsolute Q uantification (iTRAQ)-based comparative proteomics approach to identify novel biomarkers for predicting LN metastasis in CRC patients. We analyzed five paired samples of CRC with or without LN metastasis, adjacent normal mucosa, and normal colon mucosa, and differentially expressed proteins were identified and subsequently validated at the protein and/or mRNA levels by immunohistochemistry and qRT-PCR, respectively. We identified 55 proteins specifically associated with LN metastasis, from which we selected ezrin for further analysis and functional assessment. Expression of ezrin at both the protein and mRNA levels was significantly higher in CRC tissues than in adjacent normal colonic mucosa. In univariate analysis, high ezrin expression was significantly associated with tumor progression and poor prognosis, which was consistent with our in vitro findings that ezrin promotes the metastatic capacity of CRC cells by enabling cell invasion and migration. In multivariate analysis, high levels of ezrin protein and mRNA in CRC samples were independent predictors of LN metastasis. Our data thus identify ezrin as a novel protein and mRNA biomarker for predicting LN metastasis in CRC patients.

We previously visualized in vivo responses to chemotherapy in a colorectal liver metastatic xenograft model using in vivo real-time and time-series intravital two-photon laser scanning microscopy (TPLSM). In this study, we established the method for evaluating the response of peritoneal xenografts to chemotherapy of metastatic gastric cancer using intravital TPLSM.Red fluorescent protein-expressing gastric cancer cells (NUGC4) were inoculated into the peritoneal cavity of green fluorescent protein nude mice.Laparotomy revealed that 2 weeks after inoculation, macroscopic peritoneal metastatic nodules were formed. The first intravital TPLSM session revealed that they were composed of red tumor cell clusters and green surrounding stroma. Paclitaxel was administered intraperitoneally after the first TPLSM three times a week for 7 days in the treatment group. At the second laparotomy, there were significantly fewer and smaller nodules in the treated mice than in the controls. The second intravital TPLSM session showed tumor cell fragmentation, swelling, and nuclear condensation in the metastatic nodules--a response to chemotherapy. There were multinuclear tumor cells in the paclitaxel-treated living mice.Our method may become a powerful tool for evaluating the efficacy of novel anti-gastric cancer drugs in a preclinical murine model with minimum interindividual variation.

Cancer research is currently focused on blocking the metastatic process at its early steps. Some particularly attractive targets are metastasis suppressor genes, which control cancer cell dissemination. The aim of this study was to clarify the relationship between the expression of KiSS1, a metastasis suppressor gene, and disease progression in colorectal cancer patients. One-hundred and seventy-five patients who underwent surgery for colorectal cancer were enrolled in this study. We analyzed KiSS1 mRNA expression by real-time reverse transcription PCR in colorectal cancer tissue and paired adjacent normal mucosa. KiSS1 protein expression in early- and advanced-stage colorectal cancer samples was determined by immunohistochemical analysis. Decreased KiSS1 expression was significantly associated with lymph node metastasis and was an independent prognostic factor. Logistic regression analysis revealed that decreased KiSS1 expression was an independent risk factor for lymph node metastasis. Immunohistochemical analysis indicated that KiSS1 was highly expressed in the cell cytoplasm of early-stage colorectal cancer cells. The loss of KiSS1 appears to correlate with the progression of lymph node metastasis. An assessment of KiSS1 expression may assist in the accurate colorectal cancer diagnosis and may contribute to predict clinical outcomes.

In vivo real-time visualization of chemotherapy response at the cellular level provides us with direct evidence of what happens on the tumor microenvironment of metastatic organs. We imaged the response of metastatic tumor cells and host stromal cells to chemotherapeutics on liver metastatic xenografts in living mice using intravital two-photon laser scanning microscopy (TPLSM). Red fluorescent protein-expressing human colorectal cancer cells (HT29) was inoculated to the spleen of green fluorescent protein-expressing nude mice. 5-Fluorouracil or irinotecan was intraperitoneally administered after the formation of macroscopic liver metastases. Intravital TPLSM was performed at multiple time-points for time-series imaging of liver metastatic xenografts in the same mice. Under the 1st TPLSM, HT29 cells were visualized in hepatic sinusoids at the single cell level. Liver metastatic nodules consisting of viable cancer cells and surrounding stroma with tumor vessels were visualized under the 2nd TPLSM. After chemotherapy, tumor cell fragmentation, condensation, swelling and intracellular vacuoles were observed under the 3rd TPLSM. There was no obvious morphological difference in tumor response between these chemotherapeutics. Time-series intravital TPLSM imaging on the metastatic tumor xenografts may be useful for screening and evaluating new chemotherapeutics with less interindividual variability.

A 39-year-old man received a diagnosis of unresectable multiple liver metastases from multiple colorectal cancers with familial adenomatous polyposis. After construction of an ileostomy, modified FOLFOX6 (mFOLFOX6) with panitumumab was administrated because rectal cancer and sigmoid colon cancer are KRAS wild type. The 13 courses of chemotherapy resulted in a marked reduction in the size of liver metastases and sigmoid colon cancer. Consequently, curative resection with total colectomy, ileal pouch anal anastomosis, and liver metastasis resection with radiofrequency ablation was performed. Progression of KRAS wild-type rectal cancer after chemotherapy suggested that each clone from rectal and sigmoid colon cancer might have a different sensitivity to epidermal growth factor receptor antibody. Immunohistochemical analysis revealed loss of PTEN expression in rectal cancer compared with liver metastases from sigmoid colon cancer, showing that the difference of mFOLFOX6 with panitumumab might be related to activation of the PI3K-AKT pathway.

546 Background: Accumulating evidences reveal that overexpression of Heat shock protein 47 (HSP47) increase cancer progression, and that HSP47 expression in the tumor-associated stroma serve as diagnostic marker in various cancers. In addition, we recently performed immunohistochemistry to evaluate HSP47 expression in surgical colorectal cancer (CRC) specimens, and showed that the count of HSP47 positive spindle cell in cancer stroma was significantly associated with lymph node (LN) metastasis, early recurrence and poor prognosis in CRC patients. Thus, evaluating HSP47 expression in CRCs preoperatively may be valuable for planning the treatment of patients with CRC. In this study, we quantified the messenger RNA(mRNA) expression of HSP47 in CRCs by using preoperative biopsy samples, and analyzed the association between HSP47 mRNA expression and clinico-pathological factors including prognosis in patients with CRC. Methods: A total of 139 CRC samples, which were taken by biopsy before surgery at Mie University Hospital from 2000 to 2005, were enrolled. HSP47 gene expression was determined by quantitative real-time reverse transcription–PCR using Power SYBR Green PCR methods. Results: All CRC patients were classified according to the tumor-node-metastasis (TNM) classification system as follows: stage I (n = 33); stage II (n = 44); stage III (n = 33), and stage IV (n = 29). High HSP47 expression in CRC was significantly associated with higher T stage (p = 0.0163), LN metastasis (p = 0.0186), vessel invasion (p = 0.0328) and higher TNM staging (p = 0.0115). Kaplan-Meier analysis showed that patients with high HSP47 expression had significantly poorer overall survival (OS) than those with low (p = 0.0003). Furthermore, multivariate analyses revealed that HSP47 expression was an independent predictive marker for LN metastasis and poor OS in CRC patients, respectively (LN metastasis; OR; 2.3946, p = 0.0249, OS; HR; 2.7407, p = 0.00224). Conclusions: In conclusion, quantification of HSP47 expression using biopsy samples can identify the CRC patients who may suffer from LN metastasis and poor prognosis, preoperatively.

655 Background: We often encounter the colorectal cancer (CRC) patients with multiple distant metastases who rapidly progress after resection of primary tumor site due to preventing bowel obstruction, tumor bleeding and perforation. However, there has been few knowledge for identification of these patients at the present time. In this study, we evaluated the association between clinicopathological findings, including with preoperative laboratory data and survival outcome, and possibility of multimodality therapy in CRC patients with multiple metastases after resection of primary tumor lesion. Methods: Clinicopathological findings and preoperative laboratory data, including carcinoembryonic antigen (CEA) and systemic inflammatory response markers, neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and modified Glasgow Prognosis Score (mGPS) for 92 CRC patients with multiple metastases from 2005 to 2014 were collected. We performed multivariate analysis for overall survival (OS) in cox proportional hazard model. In addition, we performed multivariate logistic regression analysis to evaluate the factors influencing possibility of postoperative multimodality therapy. Results: Postoperative multimodality therapy improved overall survival significantly. Among serum markers, elevated CEA, NLR, and mGPS were significant indicators of poorer OS. Multivariate analysis for OS revealed that elevated CEA (P = 0.026), mGPS (P = 0.038), and undifferentiated histology type (P = 0.02) were independent poorer prognostic factors. In addition, multivariate logistic regression analysis showed that elevated mGPS (P = 0.029) and higher age (P = 0.013) were independent risk factors for difficulty of introducing postoperative multidisciplinary therapy. Conclusions: Preoperative mGPS is useful objective marker for selection of colorectal cancer patients with multiple metastases who can undergo primary resection followed by multidisciplinary therapy.

647 Background: Albumin to globulin ratio (AGR) has been reported to predict long term mortality in patients with several cancers. However, prognostic impact of preoperative AGR in colon cancer patients with curative intent has not yet been fully addressed. Therefore, we, for the first time, investigated the association between AGR and clinico-pathological findings including overall survival (OS) and disease free survival (DFS) in stage I-III colon cancer patients. Methods: Clinicopathological findings including preoperative laboratory data (carcinoembryonic antigen [CEA] and AGR) from 251 curative colon cancer patients were assessed as indicators of early recurrence and poor prognosis in this retrospective study. AGR was calculated as [AGR = albumin/ (total protein - albumin)]. The cut-off value of AGR was 1.32 in current study. Results: Several clinicopathological categories related with tumor progression such as lymph node metastasis, T4 tumor, large tumor size, undifferentiated tumor, venous and lymphatic invasion, and high CEA were significantly associated with low AGR level. The patients with low AGR were significantly poorer OS (P = 0.001) and DFS (P = 0.003) than those with high AGR, respectively. In addition, multivariate analyses demonstrated that low AGR was independently associated with early recurrence (HR = 2.87, P = 0.007) and poor prognosis (HR = 2.56, P = 0.008), respectively. On the other hand, sub analysis of survival curves revealed that stage III colon cancer patients with low AGR were significantly poorer OS (P = 0.007) and DFS (P = 0.02) than those with high AGR, respectively. Furthermore, significantly poorer OS and DFS were also shown in stage I-II colon cancer patients with low AGR, respectively (OS: P = 0.02, DFS: P = 0.01). Conclusions: Preoperative AGR was an independent predictor of early recurrence and poor prognosis in curative colon cancer patients. AGR may represent a simple, potentially useful predictive biomarker for selecting stage I-II colon cancer patients who might need adjuvant chemotherapy. Furthermore, AGR may select candidates who are better to introduce more intensive adjuvant chemotherapy after curative operation in stage III colon cancer patients.

5-fluorouracil (5FU) is often used in the treatment of colorectal cancer. 5FU improves the median overall and disease-free survival rates and reduces recurrence rates in patients who have undergone curative surgical resection. However, in the adjuvant setting, whether 5FU eradicates clinically undetectable micrometastases in target organs such as the liver, or whether 5-FU inhibits the adhesion of circulating tumor cells has not yet been established. In the present study, 5FU was administered following the inoculation of red fluorescent protein-expressing HT29 cells into green fluorescent protein (GFP)-transgenic nude mice to examine its inhibitory effect. 2-photon laser scanning microscopy was performed at selected time points for time-series imaging of liver metastasis of GFP-transgenic mice. The cell number in vessels was quantified to evaluate the response of the tumor microenvironment to chemotherapy. HT29 cells were visualized in hepatic sinusoids at the single-cell level. A total of 2 hours after the injection (early stage), time-series imaging revealed that the number of caught tumor cells gradually reduced over time. In the 5FU treatment group, no significant difference was observed in the cell number in the early stage. One week after the injection (late stage), a difference in morphology was observed. The results of the present study indicated that 5FU eradicated clinically undetectable micrometastases in liver tissues by acting as a cytotoxic agent opposed to preventing adhesion. The present study indicated that time-series intravital 2-photon laser scanning microscopic imaging of metastatic tumor xenografts may facilitate the screening and evaluation of novel chemotherapeutic agents with less interindividual variability.

A phase I clinical study was conducted to determine the maximum tolerated dose (MTD) and recommended dose (RD) of the standard treatment of 5-fluorouracil/l-leucovorin (5-FU/LV) with bevacizumab, in combination with radiation therapy in patients with locally advanced rectal cancer. Eligible patients had previously untreated stage T3 or T4 locally advanced rectal cancer. Patients received radiotherapy to the pelvis, at a total dose of 45 Gy in 25 fractions. During radiotherapy, patients received three courses of a simplified LV and 5-FU regimen (sLV5FU2), in combination with bevacizumab. Bevacizumab was infused at a fixed dose of 5 mg/kg on Days 1, 15 and 29. The sLV5FU2 regimen consisted of 200 mg/m2 of LV administered by continuous intravenous (i.v.) infusion over 2 h, followed by 400 mg/m2 of 5-FU administered by i.v. bolus injection, delivered at an initial loading dose of 2,000 mg/m2 over 46 h. The dose was gradually increased to determine the MTD and RD of this regimen. Of the patients enrolled in the study, two developed Grade 3 diarrhea and one developed Grade 3 neutropenia. Since dose-limiting toxicity (DLT) occurred in two out of the six patients, 5-FU at a dose of 2,000 mg/m2 over 46 h was determined as the MTD and designated as the RD, taking into consideration the toxicities in matched patients who received standard preoperative chemoradiotherapy with sLV5FU2 during the same period. The combination of 5-FU, LV, bevacizumab and radiotherapy in patients with locally advanced rectal cancer was found to be tolerable, with encouraging response rates. Further investigation is required in a phase II setting.

13 Background: Albumin to globulin ratio (AGR) has mainly been used as a clinical indicator for multiple myeloma or other immunoproliferative diseases. Recently, AGR has been reported to predict long term mortality in patients with nasopharyngeal carcinoma, colorectal cancer, lung cancer and breast cancer. However, clinical impact of preoperative AGR in gastric cancer (GC) has not yet been addressed. Therefore, we, for the first time, investigated the association between AGR and clinico-pathological findings including overall survival (OS) and disease free survival (DFS) in GC patients with curative intent. Methods: Clinicopathological findings including preoperative laboratory data (carcinoembryonic antigen [CEA], carbohydrate antigen [CA] 19-9 and AGR) from 384 curative GC patients were assessed as indicators of early recurrence and poor prognosis in this retrospective study. AGR was calculated as [AGR = albumin/ (total protein - albumin)]. The cut-off value of AGR was 1.4 in current study. Results: Several pathological categories related with tumor progression such as lymph node metastasis, tumor serosal invasion, large tumor size, venous invasion were significantly associated with lower AGR levels. In addition, significant negative correlation between AGR and age was found. Among preoperative serum markers, AGR was an independent predictor of early recurrence in curative GC patients after adjusted by age and sex. On the other hand, 2 pathological factors such as lymph node metastasis and serosal invasion were independent factors to identify early recurrence in curative GC patients among postoperative categories. Furthermore, sub analysis revealed that GC patients with AGR < 1.4 showed significant poorer DFS in both lymph node metastasis and serosal invasion group, respectively. Conclusions: Preoperative AGR may represent a simple, potentially useful predictive biomarker for selecting GC patients who might need neoadjuvant chemotherapy. Furthermore, AGR may select candidates who are better to introduce more intensive adjuvant chemotherapy after curative operation in stage II-III GC.

14 Background: Although Gastric cancer (GC) mortality has been reduced by advances in new treatments and in chemotherapy, it still has a poor prognosis and high mortality. One of the reasons for poor prognosis is that at the time of diagnosis advanced GC with metastatic disease is often detected and it is frequently accompanied by peritoneal metastasis. Imaging studies are frequently used to predict peritoneal metastasis, however, these modalities did not obtain consistently high sensitivity and specificity in assessing peritoneal metastasis. Therefore, we investigated whether the combination of several serum markers and clinical factors could be used for preoperative prediction of peritoneal metastasis in GC as an indicator for neoadjuvant treatment. Methods: A total of 493 patients with GC were enrolled in our Hospital. Preoperative serum tumor markers [carcinoembryonic antigen (CEA) and carbohydrate antigen (CA)19-9], systemic inflammatory marker C-reactive protein (CRP), host immune markers [neutrophil and lymphocyte counts and their ratio (NLR)], albumin as a nutritional marker, and objective preoperative clinical factors were available as indicators of postoperative peritoneal metastasis. Results: This study included a total of 344 men and 149 women who were classified according to UICC TNM Classification: 264 patients had stage I disease, 79 stage II, 78 stage III and 72 stage IV. Specific clinical factors, including tumor size, histopathology of biopsy sample, and tumor morphology, were significantly correlated with peritoneal metastasis. CA19-9, lymphocyte count and NLR were also predictive factors for peritoneal metastasis. Multivariate analysis identified the clinical factors tumor morphology and histopathology, and laboratory markers CA19-9 and lymphocyte count as independent factors predictive for peritoneal metastasis. Next, a combination of independent predictive factors achieved high predictive accuracy (0.882) for peritoneal metastasis preoperatively. Conclusions: A combination of preoperative tumor features and serum parameters is an alternative method to preoperatively discriminate patients with GC with peritoneal metastasis from those without.

73 Background: Angiopoietin-like protein 2 (ANGPTL2) is a secreted glycoprotein with homology to the angiopoietins and overexpression of ANGPTL2 is known to act as a causative mediator of chronic inflammatory carcinogenesis. Recently, expression in tumor cells which are associated with tumor progression has been recognized in lung, breast, colon and gastric cancer. However, to our knowledge, functional and clinical significance of ANGPTL2 expression has not been investigated in esophageal cancer (EC). Aim: We investigated the functional roles of ANGPTL2 in vitro assay and evaluated the clinical significance of ANGPTL2 expression in both primary tumor and matched serum in patients with EC. Materials and Methods: First, in vitro assays were performed for functional analysis of ANGPTL2 using small interfering RNA (siRNA). Next, ANGPTL2 expression in EC tissues was evaluated by immunohistochemically (IHC) in 71 EC patients. Finally, we investigated serum ANGPLT2 levels from EC patients (n=71) and healthy controls (n=35) by ELISA. Results: Knockdown of ANGPTL2 expression decreased proliferative, invasive and migrated capacity in EC cell lines. ANGPTL2 expression in EC tissues was significantly elevated in EC patients with high T stage, squamous cell carcinoma, and high TNM stage. Kaplan–Meier analysis showed that patients with high expression of ANGPTL2 had significantly poorer overall (p

Background: c-MET (hepatocyte growth factor receptor) gene is overexpressed in approximately 20% of gastric cancer. Onartuzumab, a MET-selective humanized one-armed monoclonal antibody, has been studied in metastatic Met-positive non-small cell lung cancer and expected to be introduced to MET-positive gastric cancer. Two-photon laser scanning microscopy (TPLSM) has revolutionized in vivo real-time imaging because of the benefits of higher resolution, increased tissue penetration, and reduced photodamage. We established a method of intravital imaging for intra-abdominal organs using TPLSM with an organ stabilizing system (Japanese Patent No.5268282). Aim: To visualize the binding of fluorescence labeled-antibody to its receptor of cancer cells in peritoneal metastatic xenografts of living mice using a TPLSM. Method: Human gastric cancer cell lines (NUGC4) were inoculated into the peritoneal cavity of green fluorescent protein (GFP) expressing nude mice to make macroscopic peritoneal metastases. In vitro study, the binding of Zenon Alexa Fluor 594 labeled anti-c-MET antibody to viable NUGC4 cells was imaged using fluorescence microscopy. In vivo study, the binding of fluorescence labeled-antibody to viable NUGC4 cells of peritoneal metastatic xenografts was imaged using a TPLSM. Result: Zenon Alexa Fluor 594 labeled anti-c-MET antibody (red) was bound to the surface of viable NUGC4 cells, and clearly visualized under a fluorescence microscopy. Peritoneal metastases with label-free NUGC4 cells (black) and murine stromal cells (green) were imaged under TPLSM in vivo real-time. Fluorescence labeled-antibody could be visualized within tumor vessels of peritoneal metastases after its intravenous administration. Thereafter, label-free NUGC4 cells were identified by the binding of fluorescence labeled- anti-c-MET antibody to c-MET of NUGC4 cells. Conclusion: We could visualize fluorescence labeled-antibody in peritoneal metastases of gastric cancer in living mice using a TPLSM. The binding of fluorescence labeled- anti-c-MET antibody to c-MET of viable NUGC4 cells was clearly imaged at high magnification and high resolution in vivo real-time. It is valuable to develop a method of in vivo optical pathology for therapeutic monoclonal antibodies using MPM on metastatic tumor xenografts. Citation Format: Shozo Ide, Koji Tanaka, Tadanobu Shimura, Masato Okigami, Satoru Kondo, Takahito Kitajima, Hiroki Imaoka, Yoshinaga Okugawa, Susumu Saigusa, Yuji Toiyama, Hiromi Yasuda, Masaki Ohi, Yasuhiro Inoue, Toshimitsu Araki, Keiichi Uchida, Yasuhiko Mohri, Masato Kusunoki. In vivo visualization using two-photon laser scanning microscopy of anti-c-MET antibody in peritoneal metastatic gastric cancer. [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 4938. doi:10.1158/1538-7445.AM2014-4938

Background: Gastric carcinogenesis is regarded as a multistep process with an intestinal metaplasia-dysplasia-carcinoma sequence, which is initiated by Helicobacter pylori infection that causes a chronic active inflammation in the gastric mucosa. Angiopoietin-like protein 2 (ANGPTL2) is known to act as a causative mediator of chronic inflammation and inflammatory carcinogenesis. However, the biological role and clinical significance of ANGPTL2 expression remains poorly understood in human cancer. We investigated the functional role of ANGPTL2 and evaluated the clinical significance of its expression in both primary tumor and matched serum specimens in patients with gastric cancer (GC). Methods: The function of ANGPTL2 in GC was investigated by siRNA using GC cell lines (MKN1 and KATO III). Next, we examined ANGPTL2 expression in GC tissues (n=192) by immunohistochemistry (IHC) to evaluate associations between its expression and various clinicopathological features. Finally, we determined serum ANGPTL2 levels from 32 GC and 23 normal controls (NC), and validated its expression levels using 194 serum samples from GC and 45 from NC to evaluate its utility as a biomarker by ELISA. Results: Knockdown of ANGPTL2 resulted in significant induction of anoikis (p Conclusion: Our study first reports that overexpression of ANGPTL2 in GC cells results in increased malignant potential and metastasis. Serum ANGPTL2 expression emerged as a novel, non-invasive biomarker for the earlier diagnosis, recurrence and prognosis in patients with gastric cancer. Citation Format: Yuji Toiyama, Takahito Kitajima, Tadanobu Shimura, Hiroki Imaoka, Satoru Kondo, Shozo Ide, Masato Okigami, Hiromi Yasuda, Susumu Saigusa, Masaki Ohi, Koji Tanaka, Yasuhiro Inoue, Yasuhiko Mhori, Ajay Goel, Masato Kusunoki. Angiopoietin-like protein 2, a driver of cancer cell metastasis, is a novel serum biomarker for the diagnosis and prognosis in patients with gastric cancer. [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 883. doi:10.1158/1538-7445.AM2014-883

e22062 Background: The optical imaging using a bioluminescent or fluorescent probe can visualize, characterize, and measure the biological process of anticancer drugs at the molecular and cellular ...

9 Background: Angiopoietin-like protein 2 (ANGPTL2) is a secreted glycoprotein with homology to the angiopoietins and is known to act as a causative mediator of chronic inflammation and inflammatory carcinogenesis. Recently, we demonstrated that ANGPTL2 overexpresses in gastric cancer (GC) compared to normal gastric mucosa and high ANGPTL2 is associated with poor prognosis. Therefore, if tumor-derived ANGPTL2 over-secretes systemically, focusing ANGPTL2 in blood is logical to evaluate its ability as a biomarker. Accordingly, we quantified serum ANGPTL2 level and assessed its clinical significance in GC patients. Methods: We screened serum ANGPTL2 levels from 32 GC and 24 normal controls (NC) by ELISA (cohort 1). Next, we validated 194 serum samples from GC and 48 NC (cohort 2). Finally, we examined ANGPL2 expression in matched GC tissues of cohort 2 (n=194) by immunohistochemistry (IHC). The IHC score of ANGPTL2 was determined on the basis of both staining intensity and the percentage of positive cells. Results: In cohort 1,serum ANGPTL2 levels in GC were significantly higher than that in NC (p

e22205 Background: Peritoneal metastasis shows an extremely poor prognosis in patients with gastric cancer. Clinically, the tumor response to chemotherapeutics depends on anatomical location of metastasis. Metastatic tumor xenografts have been shown to be more resistant to chemotherapy than subcutaneous non-metastatic tumor xenografts in preclinical murine model. We have reported a method of in vivo optical pathology using multiphoton microscopy (MPM) in colorectal liver metastatic tumor xenograft model. Aim: We established a method of time-series in vivo optical pathology of peritoneal metastatic xenografts of gastric cancer using MPM. Then, we imaged and evaluated paclitaxel efficacy in the tumor microenvironment with regard to both tumor cell itself and intravascular change in tumor vessels. Methods: Red fluorescent protein (RFP) expressing human gastric cancer cell line (NUGC4) was inoculated into the peritoneal cavity of green fluorescent protein (GFP) expressing nude mice. Paclitaxel (10 mg/kg) was administered three times a week for more than three weeks. Intravital MPM was performed before and after paclitaxel treatment for the exteriorized peritoneal metastatic lesion in the same living mouse. Results: Four to six weeks later, RFP-NUGC4 cells formed macroscopic peritoneal metastases. Red-colored cancer cells and green-colored surrounding stroma with tumor vessels were clearly imaged at the cellular level (in vivooptical pathology). Their cross-sectional images were obtained from the tissue surface to the area of depth of 200 μm (z-stacks imaging). After paclitaxel treatment, tumor cell fragmentation, condensation, swelling and intracellular vacuoles were observed. Within the tumor vessels, platelet aggregation and platelet adhesion to endothelial cells were observed. Conclusions: In vivo optical pathology using MPM provides histopathological information about three-dimensional tissue microarchitecture without tissue shrinkage by fixation and tissue destruction by microtome-sectioning. Our method may become a powerful tool to evaluate the tumor response to new chemotherapeutics on ‘metastatic site’ in preclinical tumor xenograft model.

e21053 Background: Tumor endothelium is a distinct target for anticancer treatments of metastatic colorectal cancer. Various chemotherapeutics themselves have anti-angiogenic effects on tumor microenvironment by targeting proliferative endothelial cells during tumor angiogenesis. In this study, we imaged the dynamics of tumor microcirculation of colorectal liver metastasis in living mice in vivo real-time using two-photon laser scanning microscopy (TPLSM), and evaluated the microcirculatory alterations in tumor microenvironment by chemotherapy. Methods: Red fluorescent protein expressing human colorectal cancer cell line (HT29) was inoculated to the spleen of green fluorescent protein expressing nude mice. 5-fluorouracil or irinotecan was administered three times a week for more than three weeks for metronomic scheduling. Intravital TPLSM was performed at multiple time points for time-series imaging of liver metastatic xenografts in the same mice. The alterations in tumor microcirculation during chemotherapy was evaluated by measuring blood flows at tumor vessels of colorectal liver metastasis and hepatic sinusoids of adjacent normal liver. Results: At the first TPLSM imaging, the blood flow, as determined from the movement of platelets, was heterogeneous and non-directional in the tumor vessels of liver metastatic xenografts. The blood flow was relatively slower in tumor vessels than normal hepatic sinusoids. At the second TPLSM imaging after chemotherapy, platelet aggregation was observed in tumor vessels of the same mice. Aggregated platelets were frequently adhered to the tumor endothelium, suggesting tumor vessel damage or intratumoral coagulation abnormality by chemotherapy. There was no difference in chemotherapeutics with regard to these findings. Conclusions: Intravital TPLSM imaging of tumor microcirculation at metastatic tumor xenografts is a useful tool to evaluate anti-angiogenic drugs in preclinical models.