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Label-free image identification of circulating rare cells, such as circulating tumor cells within peripheral blood nucleated cells (PBNCs), the vast majority of which are white blood cells (WBCs), remains challenging. We previously described developing label-free image cytometry for classifying live cells using computer vision technology for pattern recognition, based on the subcellular structure of the quantitative phase microscopy images. We applied our image recognition methods to cells flowing in a flow cytometer microfluidic channel, and differentiated WBCs from cancer cell lines (area under receiver operating characteristic curve = 0.957). We then applied this method to healthy volunteers' and advanced cancer patients' blood samples and found that the non-WBC fraction rates (NWBC-FRs), defined as the percentage of cells classified as non-WBCs of the total PBNCs, were significantly higher in cancer patients than in healthy volunteers. Furthermore, we monitored NWBC-FRs over the therapeutic courses in cancer patients, which revealed the potential ability in monitoring the clinical status during therapy. Our image recognition system has the potential to provide a morphological diagnostic tool for circulating rare cells as non-WBC fractions.

Background Pancreatic juice is constantly activated by contaminated bile in patients with pancreaticobiliary maljunction (PBM). Here, we report a case of laparoscopic distal pancreatectomy for a patient with PBM and sphincterotomized papilla, resulting in fatal pancreatic fistula. Case presentation A 79-year-old man was diagnosed with pancreatic intraductal papillary mucinous neoplasm and common bile duct stones. Endoscopic sphincterotomy was performed prior to surgery. The pancreatic duct was simultaneously visualized when the contrast agent was injected into the common bile duct. Sudden bleeding was observed from the abdominal drain on postoperative day (POD) 6. Emergent stent graft placement and coil embolization were performed for bleeding from the splenic artery. On POD 9, the drainage fluid changed to yellowish in color with bile contamination. For internal drainage of the digestive fluid, endoscopic retrograde biliary tube and pancreatic drainage tube were placed. On POD 24, second emergent coil embolization was performed for bleeding from the left gastric artery. On POD 25, open abdominal drainage was performed. On POD 32, third emergent coil embolization was performed for bleeding from the gastroduodenal artery. Subsequently, remnant pancreatic resection was performed. On POD 39, massive bleeding was again observed from the abdominal drain. Emergency arterial portography revealed bleeding in the right wall of the superior mesenteric vein. The patient died of hemorrhagic shock on the same day. Conclusions The extreme risk of severe pancreatic fistula after distal pancreatectomy should be considered in patients with PBM and sphincterotomized papilla. In this extraordinary situation, surgeons should promptly decide whether to resect the remnant pancreas to prevent losing the patient.

Early diagnosis of anastomotic leakage (AL) after esophagectomy is essential to minimize postoperative complications. In this study, we hypothesized that drain amylase levels may be useful for early AL detection, and measuring drain amylase levels could reduce severe postoperative AL incidence. We, therefore, analyzed the usefulness of measuring drain fluid amylase levels after esophagectomy, in esophageal cancer patients. From January 2016 to March 2020, 134 patients with esophageal cancer who underwent surgical resection with esophagogastric anastomosis in the cervical region were included. The patients were divided into a group whose cervical drain fluid amylase levels were not measured (No-AMY Group) and a group whose cervical drain fluid amylase levels were measured daily until postoperative day (POD) 7 (AMY Group). The incidence of severe AL was compared between groups. In the AMY Group, we also investigated the association between AL and drain amylase levels. Drain amylase levels were significantly higher in AL-positive cases than in AL-negative cases (P

Thoracoscopic esophagectomy (TE) is becoming a common surgical method for esophageal cancer. TE is performed with the patient the left lateral decubitus position, prone position, or hybrid position combining the left lateral decubitus and prone positions. However, only few studies have compared the clinical utility of these TE positions. Methods In our institute, we introduced TE in the prone position (prone TE) in 2014, and have performed TE in the hybrid position (hybrid TE) since March 2017. The present study compared the short-term outcomes of prone TE versus hybrid TE. One-hundred-and-three patients with esophageal or esophagogastric junction cancer who underwent TE between March 2014 and December 2019 were included. Patients were divided into those who underwent prone TE (prone TE group; n = 43) and those who underwent hybrid TE (hybrid TE group; n = 60). Clinicopathological data were retrospectively reviewed and compared between groups. Results There were no differences between groups in age, tumor histology, and tumor location. Compared with the hybrid TE group, the prone TE group had a smaller tumor depth (P Conclusion The most significant differences between prone TE and hybrid TE involved the upper mediastinal procedures. In hybrid TE, the motion of the assistant’s forceps causes less interference with the operative field, and the angle at which the operator's forceps approach the upper mediastinal lymph nodes enables the maintenance of appropriate traction. These advantages of hybrid TE appeared to result in a shorter operation time and less recurrent laryngeal nerve paralysis compared with prone TE.

Background: Tumour stroma has important roles in the development of colorectal cancer (CRC) metastasis. We aimed to clarify the roles of microRNAs (miRNAs) and their target genes in CRC stroma in the development of liver metastasis. Methods: Tumour stroma was isolated from formalin-fixed, paraffin-embedded tissues of primary CRCs with or without liver metastasis by laser capture microdissection, and miRNA expression was analysed using TaqMan miRNA arrays. Results: Hierarchical clustering classified 16 CRCs into two groups according to the existence of synchronous liver metastasis. Combinatory target prediction identified tenascin C as a predicted target of miR-198, one of the top 10 miRNAs downregulated in tumour stroma of CRCs with synchronous liver metastasis. Immunohistochemical analysis of tenascin C in 139 primary CRCs revealed that a high staining intensity was correlated with synchronous liver metastasis (P

Background: Circulating tumor cells (CTCs) are used as a liquid biopsy target in the treatment of cancer patients and are important for personalized medicine. Most CTC detection technologies are based on their surface markers such as EpCAM and cytokeratin. However, those positive selection methodologies appear to count only some CTCs because invasive tumor cells tend to change their surface markers during progression. Because the vast majority of nucleated cells circulating in the bloodstream are white blood cells (WBCs), eliminating WBCs from circulation is an efficient method to purify CTCs by negative selection. In this study, we demonstrate the development of cell recognition by computer vision technologies for pattern recognition based on the morphological features of live cells. Materials and methods: Blood samples obtained from healthy volunteers, and five cancer cell lines, SW480, DLD-1, HCT116, Panc-1, and HepG2 were imaged by quantitative phase microscopy (QPM), which provides morphological information without staining quantitatively in terms of optical thickness of cells. Subcellular features were then extracted from the obtained images as training data sets for the machine learning. WBC recognition algorithm was developed using computer vision technologies for pattern recognition based on the histogram-oriented gradient (HOG) features extracted from QPM images (QPIs). Results: We observed WBCs and five cell line cells by QPM, extracted certain features from the obtained images as training data for pattern recognition, and created an algorithm to differentiate WBCs from cell lines. The obtained algorithm successfully differentiated WBCs from cell lines (AUC=0.996). This image recognition methods was then applied to QPIs of the cells flowing in the chamber, and properly differentiated WBCs from cell line cells flowing in the chamber (AUC=0.99). Finally, we applied this method to blood samples obtained from a healthy volunteer and patients with advanced cancer, and identified CTC fractions which were much higher in blood of a cancer patient than that of a healthy volunteer. In addition, in esophageal cancer patients who received neoadjuvant chemotherapy followed by surgery, CTC fractions changed over time consistently with therapeutic effects. Conclusions: We developed a novel method for label-free image identification of live cells by computer vision technologies for pattern recognition based on the features of QPIs. Our novel method is expected to apply to detecting CTCs in a noncytotoxic manner, thus providing good opportunities for studying intact CTCs Citation Format: Hirotoshi Kikuchi, Amane Hirotsu, Yusuke Ozaki, Hidenao Yamada, Daisuke Yamashita, Shigetoshi Okazaki, Yukio Ueda, Tomohiro Matsumoto, Yoshihiro Hiramatsu, Kinji Kamiya, Yoshifumi Morita, Takanori Sakaguchi, Hiroyuki Konno, Hiroya Takeuchi. Label-free classification of cells based on supervised machine learning of subcellular structures for negative selection of circulating tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5376.

It is demonstrated that cells can be classified by pattern recognition of the subcellular structure of non-stained live cells, and the pattern recognition was performed by machine learning. Human white blood cells and five types of cancer cell lines were imaged by quantitative phase microscopy, which provides morphological information without staining quantitatively in terms of optical thickness of cells. Subcellular features were then extracted from the obtained images as training data sets for the machine learning. The built classifier successfully classified WBCs from cell lines (area under ROC curve = 0.996). This label-free, non-cytotoxic cell classification based on the subcellular structure of QPM images has the potential to serve as an automated diagnosis of single cells.

We propose a line-field quantitative phase-imaging flow cytometer for analyzing large populations of label-free cells. Hydrodynamical focusing brings cells into the focus plane of an optical system while diluting the cell suspension, resulting in decreased throughput rate. To overcome the trade-off between throughput rate and in-focus imaging, our cytometer involves digitally extending the depth-of-focus on loosely hydrodynamically focusing cell suspensions. The cells outside the depth-of-focus range in the 70-µm diameter of the core flow were automatically digitally refocused after image acquisition. We verified that refocusing was successful with our cytometer through statistical analysis of image quality before and after digital refocusing.

287 Background: As recent advances in chemotherapy improved prognosis of gastric cancer (GC) patients with peritoneal metastasis (PM), accurate diagnosis of PM has become more important. However, the sensitivity of conventional imaging modalities such as CT or PET is not satisfactory. Staging laparoscopy (SL) is often used to diagnose PM in advanced GC patients, but accurate detection of PM can be difficult. In this study, we evaluate the usefulness of laparoscopic narrow-band imaging (NBI) versus conventional laparoscopic white-light imaging (WLI) for the diagnosis of PM and for the evaluation of therapeutic effect of chemotherapy. Methods: We excised 54 white nodules from the parietal peritoneum of 40 GC patients. Among them, 9 patients received chemotherapy for advanced or recurrent GC before SL, and 31 did not receive chemotherapy except for adjuvant chemotherapy with S-1. The WLI and NBI findings were compared with the pathological findings. Results: Intranodular vessels were evaluated by WLI and NBI for dilatation, tortuousness, heterogeneity, and brown spots. Detection of any one abnormal finding on NBI plus clear demarcation of the peritoneal nodules on WLI more properly diagnosed 42 peritoneal nodules of 31 patients who did not receive chemotherapy (sensitivity, 100%; specificity, 88.9%; accuracy, 95.2%). In contrast, diagnosis ability was poor for 12 peritoneal nodules of 9 patients who received chemotherapy before SL (sensitivity, 66.7%; specificity, 33.3%; accuracy, 58.3%). Conclusions: Laparoscopic NBI is a useful tool for the diagnosis of PM in advanced GC before chemotherapy, and disappearance of dilated vessels on laparoscopic NBI could be useful to evaluate the therapeutic effect.

Highlights • A 78-year-old man was diagnosed with stage cIB primary gastric choriocarcinoma. • Total gastrectomy with spleen-preserving D2 lymphadenectomy was performed. • Early recurrence was diagnosed 3 months postoperatively. • The patient underwent a standard nongestational choriocarcinoma chemotherapy., Introduction Primary gastric choriocarcinoma (PGC) is a rare and rapidly invasive tumor. We report a case of PGC diagnosed by endoscopic biopsy and treated with gastrectomy and chemotherapy. Presentation of case A 78-year-old man was referred to our hospital because esophagogastroduodenoscopy showed a tumor at the fornix of the stomach. Pathologic examination of biopsy specimens revealed choriocarcinoma. Abdominal computed tomography (CT) revealed no enlarged abdominal lymph nodes or distant metastases. Robot-assisted total gastrectomy with spleen-preserving D2 lymphadenectomy was performed on the basis of a diagnosis of cT2N0M0, stage cIB PGC. The pathologic diagnosis was pT2, ly0, v1, pN0, PM0, DM0, stage pIB PGC. The postoperative course was uneventful, and the patient was followed carefully without adjuvant chemotherapy. Three months after gastrectomy, blood tests indicated that serum β-human chorionic gonadotropin (β-hCG) levels had increased, and CT revealed multiple liver metastases. The patient underwent a standard nongestational choriocarcinoma chemotherapy regimen with etoposide, methotrexate, actinomycin D, cyclophosphamide, and vincristine. Although β-hCG levels temporarily decreased with chemotherapy, the patient experienced tumor recurrence with ascites and his serological test demonstrated an elevated level of β-hCG (120 ng/mL). The patient died 10 months postoperatively. Conclusion We report a case of stage pIB PGC with poor prognosis, recurring at only 3 months postoperatively despite curative surgery and chemotherapy.

Background: Tenascin C, a glycoprotein in the extracellular matrix is reportedly associated with poor clinical outcome in several malignancies. We have previously reported that tenascin C in colorectal cancer stroma is a predictive marker for liver metastasis (Br J Cancer 117:1360-70, 2017). In this study, we evaluated the clinicopathological and prognostic significance of tenascin C expression in gastric cancer stroma. Materials and methods: Expression levels of tenascin C protein were immunohistochemically analyzed in 176 primary gastric cancer samples. Tenascin C staining intensity and relativity were classified into four scores and two groups, respectively. The correlation of the tenascin C staining intensity and relativity with clinicopathological factors, postoperative overall survival (OS) and disease-free survival (DFS) were analyzed. Results: There were positive correlations between the tenascin C staining intensity or relativity and tumor depth, lymphatic invasion, venous invasion, lymph node metastasis, pathological stage, high preoperative carcinoembryonic antigen (CEA)>5ng/ml, and high preoperative carbohydrate antigen (CA19-9) >37U/ml. Multivariate analysis revealed that high tenascin C staining intensity significantly correlated with tumor invasion into the muscularis propria (p=0.015) and venous invasion (p=0.042), and that high tenascin C staining relativity correlated with venous invasion (p=0.013). Gastric cancer patients with high tenascin C staining intensity had significantly shorter postoperative OS (p Conclusions: Tenascin C protein expression in primary tumor stroma was identified as a predictive marker for postoperative prognosis in patients with gastric cancer. Citation Format: Hirotoshi Kikuchi, Tomohiro Murakami, Sanshiro Kawata, Amane Hirotsu, Tomohiro Matsumoto, Yusuke Ozaki, Yoshihiro Hiramatsu, Kinji Kamiya, Yoshifumi Morita, Takanori Sakaguchi, Hiroyuki Konno, Hiroya Takeuchi. Tenascin C in gastric cancer stroma is a predictive marker for postoperative prognosis [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 4028.

Background Single-port mediastinoscope-assisted transhiatal esophagectomy for thoracic esophageal cancer is a minimally invasive surgery and can be applied to patients with low pulmonary function. However, special skills are needed to safely perform this surgical procedure as a curative operation without complications such as recurrent laryngeal nerve paralysis. To date, no animal training models for this surgical procedure has not been established. In this study, we developed a new animal training model for mediastinal lymphadenectomy with a single-port mediastinoscopic cervical approach. Methods A domestic pig was intubated, with a normal tracheal tube, and placed in a spine position. Because pigs do not have clavicles, skin incision was made on the cephalic side of the left forelimb. Anterior cervical muscles were cut and cephalic parts of thymus and left thyroid were resected to deploy the operation field. Esophagus and left recurrent laryngeal nerve were identified behind the trachea. A wound edge protector (Lap-Protector) and EZ Access was attached to the wound. An artificial pneumomediastinum was created using 5–6 mmHg of CO2 gas and three 5mm ports were inserted to EZ Access. Operator dissected connective tissues around esophagus and vessels mainly using a bipolar sealing device. At the right side of the esophagus, fat tissue with 106recL lymph nodes was dissected from tracheal wall. After dissection was reached to the carina level, esophagotracheal ligament was cut and dissection on the left side of esophagus was carried out to the level of aortic arch. By displacing the trachea to the front side, 109R, 107 and 109L lymph node were dissected. Results Because pig's tracheal cartilage is sub-circumferential, mobility of trachea is relatively poor, and risks of tracheal membranous portion damage are low. Although there are some anatomical differences between pigs and human, this animal training model reproduces surgical procedures of mediastinal lymphadenectomy in human such as forceps operation techniques in narrow fields and effects of artificial pneumomediastinum. Conclusion We established a new animal training model for mediastinal lymphadenectomy with a single-port mediastinoscopic cervical approach. This model reproduces surgical procedures in human and is useful for training surgeons who wish to start performing mediastinal lymphadenectomy by mediastinoscopic cervical approach. Disclosure All authors have declared no conflicts of interest.

Background: Liver metastasis in colorectal cancer (CRC) is still one of the major determinants of survival. Tumor stroma has important roles in the development of colorectal cancer (CRC) metastasis. MicroRNAs (miRNAs) are known to regulate the expression of genes involved in many physiological conditions, including cancer. Previous studies reported important roles of miRNAs in cancer cells in CRC development and metastasis. However, only few studies have focused on miRNA expression in CRC stroma. In this study, we revealed the miRNA expression profiles in tumor stroma by miRNA array analysis using Laser captured microdissection (LCM) to identify miRNAs and their target genes as novel molecular markers involved in CRC liver metastasis. Methods: Tumor stroma was isolated from formalin-fixed, paraffin-embedded tissues of primary CRCs with or without liver metastasis by LCM, and miRNA expression was analyzed using TaqMan miRNA arrays. Results: Hierarchical clustering classified 16 CRCs into two groups according to the existence of synchronous liver metastasis. Combinatory target prediction identified tenascin C (TNC) as a predicted target of miR-198, one of the top 10 miRNAs downregulated in tumor stroma of CRCs with synchronous liver metastasis. Immunohistochemical analysis of TNC in 139 primary CRCs revealed that a high staining intensity was correlated with synchronous liver metastasis (P Conclusions: Alterations of miRNAs in CRC stroma appear to form a metastasis-permissive environment that can elevate TNC to promote liver metastasis. TNC in primary CRC stroma has the potential to be a novel biomarker to predict postoperative prognosis. Citation Format: Tomohiro Murakami, Hirotoshi Kikuchi, Hisahito Ishimatsu, Sanshiro Kawata, Amane Hirotsu, Tomohiro Matsumoto, Yusuke Ozaki, Yoshihiro Hiramatsu, Kinji Kamiya, Megumi Baba, Satoshi Baba, Masatoshi Kitagawa, Takanori Sakaguchi, Hiroyuki Konno, Hiroya Takeuchi. Tenascin C in colorectal cancer stroma is a predictive marker for liver metastasis and is a potent target of miR-198 as identified by microRNA analysis [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 1067.

Background: Circulating tumor cells (CTCs) are used as a liquid biopsy target in the treatment of cancer patients and are important for personalized medicine. Most CTC detection technologies are based on their surface markers such as EpCAM and cytokeratin. However, those positive selection methodologies appear to count only some CTCs because invasive tumor cells tend to change their surface markers during progression. Because the vast majority of nucleated cells circulating in the bloodstream are white blood cells (WBCs), eliminating WBCs from circulation is an efficient method to purify CTCs by negative selection. In this study, we demonstrate the development of cell recognition by computer vision technologies for pattern recognition based on the morphological features of live cells. Materials and methods: Blood samples were obtained from healthy volunteers. Five cancer cell lines, SW480, DLD-1, HCT116, Panc-1, and HepG2 were used as CTC models. We applied quantitative phase microscopy (QPM) that detects the small delay of the phase and image optical path lengths and provides quantitative morphological information of live cells with high contrast without staining. WBC recognition algorithm was developed using computer vision technologies for pattern recognition based on the histogram-oriented gradient (HOG) features extracted from QPM images (QPIs) for negative selection of CTCs. Results: We observed WBCs and five cell line cells by QPM, extracted certain features from the obtained images as training data for pattern recognition, and created an algorithm to differentiate WBCs from cell lines. The obtained algorithm successfully differentiated WBCs from cell lines (AUC=0.98). To simulate WBCs and cell lines with a small and strong change, respectively, in optical thickness (OT) at the center of the cell, we created a homogeneous hemi-ellipsoid model and a heterogeneous hemi-ellipsoid model, respectively. In these models, OT gradually increased from the edge to the center, but fluctuated around the center to simulate intracellular heterogeneity of a cancer cell. Based on these simulations, the classifier was interpreted to recognize intracellular heterogeneity, especially in the center of the cell. Finally, we applied our image recognition methods to QPIs of the cells flowing in the chamber, and properly differentiate WBCs from cell line cells flowing in the chamber (AUC=0.99). Conclusions: We developed a novel method for label-free image identification of live cells by computer vision technologies for pattern recognition based on the features of QPIs. Our study makes it possible to sort CTCs in a non-cytotoxic manner, thus providing good opportunities for molecular biological approaches to isolate CTCs. Citation Format: Hirotoshi Kikuchi, Yusuke Ozaki, Amane Hirotsu, Hidenao Yamada, Shigetoshi Okazaki, Tomohiro Murakami, Tomohiro Matsumoto, Yoshihiro Hiramatsu, Kinji Kamiya, Takanori Sakaguchi, Hiroyuki Konno, Hiroya Takeuchi. Label-free classification of live cells using quantitative phase microscopy images for negative selection of circulating tumor cells [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 2588.

Background: Field-emission scanning electron microscopy (FE-SEM) enables us to observe nano-sized objects with great depth of focus and high resolution. However, the observation of biological samples including colorectal tissues using an FE-SEM has been difficult because it requires to evacuate its inside to prevent electron scattering, therefore all organisms containing ca. 70% water are rapidly evaporated and consequently caused structural disruption and collapse. To overcome the limitations of the conventional SEM, equipment such as low-vacuum scanning electron microscopy and environmental scanning electron microscope was developed. However, they are not reliable enough to investigate living organisms or wet tissues with high resolution level. We have recently reported that a simple surface modification consisting of a thin extra layer, coined the term ‘NanoSuit®’, can keep organisms alive in the high vacuum (10−3 to 10−7 Pa) of the SEM. We now modified the technique and developed a new solution, which enables FE-SEM observations of wet tissues. In this study, we utilized this technique to observe real images of colorectal cancers and their adjacent normal mucosae at high resolution. Materials and methods: Colorectal cancer tissues and their adjacent normal mucosa were cut with a scalpel from specimens surgically resected. All patients enrolled in this study provided written informed consent. Observations were carried out with an FE-SEM (S-4800, Hitachi or JEM-7100F, JEOL, Japan) at acceleration voltage of 1.0 kV. The vacuum level of the observation chamber was 10-3 - 10-7 Pa. The newly developed surface shield enhancer (SSE) solution was used to make NanoSuit® for wet tissue observation. To form the NanoSuit®, the specimens were dipped for 1 min into the SSE solution and blotted briefly thereafter put on dry filter paper to remove excess solution. Specimens were then introduced directly into an FE-SEM to form a NanoSuit® following irradiation of the electron beam. Results: Fine structures of intestinal crypt and villi were observed in normal colon mucosa using FE-SEM with a NanoSuit®, whereas fixed specimens prepared with conventional method showed obvious structural damage. Comparing with the region of normal colon mucosa, colorectal cancer lesion had relatively amorphous surface, therefore the border between non-cancerous mucosa and cancer lesions in colon tissues are able to be distinguished under high magnification. In addition, fiber-like structure was observed at the border between noncancerous mucosa and cancer lesions, suggesting an invasive front of colorectal cancer. Conclusions: We successfully observed the real mucosal surfaces and cancer lesions of colorectum with high resolution by an FE-SEM using a newly developed vacuum-proof suit, the “NanoSuit®”. This novel technique will enable us to investigate further physiopathology of GI tract including cancers. Citation Format: Hirotoshi Kikuchi, Tomohiro Matsumoto, Takanori Hiraide, Yusuke Ozaki, Amane Hirotsu, Tomohiro Murakami, Toshiki Kawabata, Yoshihiro Hiramatsu, Kinji Kamiya, Takanori Sakaguchi, Yasuharu Takaku, Isao Ohta, Takahiro Hariyama, Hiroyuki Konno. Direct observation of colorectal cancers using field-emission scanning electron microscopy with a thin polymer membrane, the NanoSuit [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 1862. doi:10.1158/1538-7445.AM2017-1862

Background: Liver is the most common site for colorectal cancer (CRC) metastasis, and mechanisms underlying colorectal liver metastasis need to be explored. The tumor microenvironment consists of many stromal cells which are known to interact with cancer cells and to promote metastasis. Recently many microRNAs (miRNAs) specifically expressed in the stroma is receiving attention. In this study, we analyzed the differences in miRNA expression profiles between peritumoral stroma of CRCs with and without liver metastasis, and sought to identify liver metastasis-promoting molecules that can be regulated by the altered expression of miRNAs in the peritumoral stroma. Materials and methods: The formalin-fixed paraffin-embedded (FFPE) specimens of primary CRCs from 8 patients without metastasis, 4 patients with synchronous liver metastasis and 4 patients with metachronous liver metastasis were used for miRNA array analysis. None of the patients had received any treatment prior to surgery. Tumor stroma was isolated from FFPE samples using laser capture microdissection without contamination of cancer cells, and total RNAs were extracted. The quality of total RNA was assessed, and miRNA expression was analyzed using TaqMan miRNA arrays. Protein expression of Tenascin C (TNC) was immunohistochemically analyzed in 139 primary CRCs. Results: Unsupervised hierarchical clustering classified 16 samples analyzed into two groups according to their miRNA expressions. CRCs with synchronous liver metastasis showed differential miRNA expression profiles from those without liver metastasis or with metachronous liver metastasis. Top10 miRNAs up-regulated in CRCs with synchronous liver metastasis vs. CRCs without liver metastasis or with metachronous liver metastasis were hsa-miR-518b, 618, 186, 520f, 29c, 520e, 155, 30c, 518e and 106b, and those down-regulated in CRCs with synchronous liver metastasis were hsa-miR-302a, 551b, 627, 628-5p, 19a, 372, 302b, 384, 198 and 636. The predictive targets of the top 10 down-regulated miRNAs were analyzed in silico using on-line data base such as .miRDB. TNC was identified as a candidate for metastasis-promoting molecule which can be upregulated in the peritumoral stroma followed by the downregulation of miRNAs. Immunohistochemical analysis revealed that protein expression of TNC was higher in the stroma of primary CRCs with synchronous liver metastasis compared with those without liver metastasis or with metachronous liver metastasis. Furthermore, high expression of TNC protein in the primary CRC stroma was correlated with shorter overall survival and liver metastasis-free survival. Conclusions: Alterations of miRNAs in the CRC stroma appear to create a metastasis permissive environment in which TNC plays an important role. Expression of TNC in primary CRC stroma has the potential to be a novel biomarker to predict the risk of postoperative liver metastasis in CRC patients. Citation Format: Tomohiro Murakami, Hisahito Ishimatsu, Hirotoshi Kikuchi, Amane Hirotsu, Tomohiro Matsumoto, Yusuke Ozaki, Toshiki Kawabata, Yoshihiro Hiramatsu, Kinji Kamiya, Megumi Baba, Takanori Sakaguchi, Hiroyuki Konno. MiRNA expression analysis in the tumor stroma predicts Tenascin C to promote colorectal cancer liver metastasis [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 4438. doi:10.1158/1538-7445.AM2017-4438