Search

Gastric cancer (GC) with microsatellite instability (MSI) has been reported to be sensitive to immunotherapy, however some of GC cases with MSI remain resistant to immunotherapy. Cancer cell lines showing MSI might be useful for the analysis of mechanisms of immunotherapy, while only a few GC cell lines with MSI are available so far. In this study, we established a unique GC cell line with MSI, OCUM-13, from a primary GC with abundant tumor-infiltrating lymphocytes. MSI assay indicated that OCUM-13 cells as well as the primary tumor showed a band shift in more than 3 of 5 microsatellite loci, suggesting that OCUM-13 did have high MSI. The subcutaneous inoculation of OCUM-13 cells into mice performed tumor formation. Insulin-like growth factor 1 receptor inhibitor decreased the growth of OCUM-13 cells. The newly established cell line with MSI, OCUM-13, might be useful for the analysis of cancer therapy for GC with MSI.

Scirrhous-type gastric cancer (SGC), one of the most intractable cancer subtypes, is characterized by rapid cancer cell proliferation and infiltration accompanied by extensive stromal fibrosis. One of the reasons for its poor prognosis may be the lack of molecular target drugs for SGC, because of the unknown driver genes. Exploration of somatic mutations in the human samples of SGC using next-generation sequencing (NGS) has been hampered by abundant fibrous tissues in these samples. Therefore, this study aimed to determine a novel oncogene by RNA-sequencing using SGC cell lines, avoiding contamination with fibrosis.In silico analysis of RNA-sequencing public data of the gastric cancer cell line, and RNA- sequencing using five of our unique SGC cell lines, OCUM1, OCUM2MLN, OCUM8, OCUM12, and OCUM14 were performed.We found three differentially expressed genes, ARHGAP4, NOS3, and OR51B5 that are significantly over-expressed in SGC cells. Immunohistochemical analysis indicated that the protein expression levels of these three genes were significantly higher in SGC than in other types of gastric cancer. The prognosis of patients with positive expression of these three genes was significantly poorer than those with negative expression. In particular, ARHGAP4 expression was an independent predictor of poor prognosis and recurrence.ARHGAP4, NOS3, and OR51B5 may be candidate driver genes for SGC. ARHGAP4 may be a promising molecular target for SGC.

It has been reported that circulating tumor cells (CTCs) are beneficial for predicting tumor stage or treatment response. Although epithelial cell adhesion molecules (EpCAMs) and cytokeratin (CK) have been often used for the identification of CTCs, other tumor markers have not been fully investigated as detecting tools for CTCs. Thus, this study aims to clarify the significance of carcinoembryonic antigen (CEA, CD66e)‐positive CTCs in patients with gastric cancer. A total of 150 patients with gastric cancer were enrolled in this study. The mononuclear fraction of peripheral blood was enriched by Ficoll. The number of cells was enumerated depending on the positivity of EpCAM and CEA or CK by flow cytometry. The association of these cells with clinicopathologic characteristics was investigated. The mean age was 70 (range 28–92). The macroscopic type of gastric cancer was classified as 0/1/2/3/4/5 in 59/11/22/38/16/4 patients, respectively. Seventy‐one patients (47.3%) were diagnosed with intestinal‐type cancer, while 76 patients (50.7%) were diagnosed with the diffuse type. The mean numbers of cells with EpCAM−CK+, EpCAM+CK−, EpCAM+CK+, EpCAM−CEA+, EpCAM+CEA−, and EpCAM+CEA+ were 618, 237, 19.9, 1147, 291, and 7.41, respectively. The number of EpCAM−CEA+cells was significantly higher in patients with stage II–III and IV than in patients with stage I. The 3‐year RFS rate in patients with a high number of EpCAM−CEA+cells (>=622) was 57.5%, while it was 79.3% in patients with a low number of EpCAM−CEA+cells (, This study aims to clarify the significance of epithelial cell adhesion molecules (EpCAMs) and carcinoembryonic antigen (CEA) expressing circulating tumor cells (CTCs) in patients with gastric cancer. The number of EpCAM−CEA+ cells was significantly associated with recurrence free survival. Thus, we conclude that CEA‐positive CTCs will be a clinically beneficial biomarker in patients with gastric cancer.

Background Although the role of Lipocalin-2 (LCN2) in cancer development has been focused on recent studies, the molecular mechanisms and clinical relevance of LCN2 in gastric cancer (GC) still remain unclear. Methods Transcriptome analysis of GC samples from public human data was performed according to Lauren’s classification and molecular classification. In vitro, Western blotting, RT-PCR, wound healing assay and invasion assay were performed to reveal the function and mechanisms of LCN2 in cell proliferation, migration and invasion using LCN2 knockdown cells. Gene set enrichment analysis (GSEA) of GC samples from public human data was analyzed according to LCN2 expression. The clinical significance of LCN2 expression was investigated in GC patients from public data and our hospital. Results LCN2 was downregulated in diffuse-type GC, as well as in Epithelial–Mesenchymal Transition (EMT) type GC. LCN2 downregulation significantly promoted proliferation, invasion and migration of GC cells. The molecular mechanisms of LCN2 downregulation contribute to Matrix Metalloproteinases-2 (MMP2) stimulation which enhances EMT signaling in GC cells. GSEA revealed that LCN2 downregulation in human samples was involved in EMT signaling. Low LCN2 protein and mRNA levels were significantly associated with poor prognosis in patients with GC. LCN2 mRNA level was an independent prognostic factor for overall survival in GC patients. Conclusions LCN2 has a critical role in EMT signaling via MMP2 activity during GC progression. Thus, LCN2 might be a promising therapeutic target to revert EMT signaling in GC patients with poor outcomes.

Fibroblast growth factor receptor (FGFR) is associated with proliferation, migration, and angiogenesis of carcinomas, and FGFR signaling inhibitors are considered a key drug for the treatment of solid tumors with FGFR overexpression. Amplification of FGFR2 is reportedly identified in 3%‐10% of gastric cancers (GCs). The aim of this study is to clarify whether the identification of the circulating tumor cells (CTCs) with FGFR2 overexpression is useful to detect patients with FGFR2‐overexpressing GC. One hundred GC patients who underwent gastrectomy were enrolled. A total volume of 8 mL of peripheral blood was collected from each patient just before gastrectomy, and mononuclear cells were enriched by Ficol density gradient centrifugation. These cells were immunostained with PI/CD45/EpCAM/FGFR2. The number of CTCs with FGFR2 expression in each sample was enumerated by FACScan. The FGFR2 expression level of the resected primary tumor was assessed by immunohistochemistry. The number of FGFR2‐positive CTCs in the GC patients' peripheral blood was significantly correlated with the FGFR2 expression level of the primary GC. The relapse‐free survival of the patients with FGFR2‐positive CTCs (≥5 cells/10 mL blood) was significantly poorer (P = .018, log‐rank) than that of the patients without FGFR2‐positive CTCs (, We aim to clarify the clinical significance of FGFR2‐positive CTCs in gastric cancer. FGFR2‐positive CTCs were correlated with the FGFR2 expression level of primary GC. The detection of FGFR2‐positive CTCs might help identify an existing FGFR2‐positive tumor.

BACKGROUND/AIM We have previously reported that chemokine (C-X-C motif) receptor 2 (CXCR2) signaling was associated with the malignant progression of gastric cancer (GC). We thus examined the clinicopathological significance of CXCR2 ligands, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8, in GC. PATIENTS AND METHODS The expression of CXCR2 ligands in 590 GC cases was investigated by immunohistochemistry. RESULTS The expression was as follows: CXCL1, 46.2% (257/557); CXCL2, 20.7% (122/590); CXCL3, 17.1% (101/589); CXCL5/CXCL6, 2.9% (17/589); CXCL7, 36.4% (215/590); and CXCL8 1.7% (10/585) of the cases. High invasion depth was correlated with CXCL1 expression. Lymph node metastasis and peritoneal cytology positivity were correlated with high expression of CXCL1 and CXCL7. The prognoses of the CXCL1-positive patients were significantly poorer than those of the CXCL1-negative patients (p

Cancer-associated fibroblasts (CAFs) may promote the malignancy of human scirrhous gastric cancer (SGC) cells. We conducted the present study to identify novel growth factors from CAFs.OCUM-12 and 2 CAF cell lines were used. The proliferation of cancer cells was determined by the number of cancer cells or the MTT assay. The growth factor(s) were purified and characterized by the gel filtration chromatography and protein array.The molecular weight of the growth-stimulating factor was estimated to be approximately 66-669 kDa. Protein array of conditioned medium (CM) from CAFs indicated that dipeptidyl peptidase-4 (DPP-4) was one of the growth factors. The addition of CM increased the phosphorylation of C-X-C chemokine receptor 4 (CXCR4). The DPP-4 inhibitor significantly inhibited the growth-stimulating activity of CM.DPP-4 from CAFs might be one of the growth-stimulating factors for SGC through CXCR4.

Background We reported that chemokine C-X-C motif receptor 2 (CXCR2) signaling appears to play an important role in the pathogenic signaling of gastric cancer (GC), and although CXCR2 may have a role in other solid cancers, the significance of CXCR2 in cholangiocarcinoma (CCA) has not been evaluated. Herein, we determined the clinicopathologic significance of CXCL1-CXCR2 signaling in CCA. Materials and methods Two human CCA cell lines, OCUG-1 and HuCCT1, were used. CXCR2 expression was examined by western blotting. We investigated the effects of CXCL1 on the proliferation (by MTT assay) and migration activity (by a wound-healing assay) of each cell line. Our immunohistochemical study of the cases of 178 CCA patients examined the expression levels of CXCR2 and CXCL1, and we analyzed the relationship between these expression levels and the patients’ clinicopathologic features. Results CXCR2 was expressed on both CCA cell lines. CXCL1 significantly inhibited both the proliferative activity and migratory activity of both cell lines. CXCL1 and CXCR2 were immunohistochemically expressed in 73% and 18% of the CCA cases, respectively. The CXCL1-positive group was significantly associated with negative lymph node metastasis (p = 0.043). The CXCR2-positive group showed significantly better survival (p = 0.042, Kaplan-Meier). A multivariate logistic regression analysis revealed that CXCR2 expression (p = 0.031) and lymph node metastasis (p = 0.004) were significantly correlated with the CCA patients’ overall survival. Conclusion CXCR2 signaling might exert a tumor-suppressive effect on CCA cells. CXCR2 might be a useful independent prognostic marker for CCA patients after surgical resection.

Background: It has been reported that cancer stem cells (CSCs) play an important role in the progression of carcinoma and have a high potential for survival in various stress environments such as starvation and hypoxia. However, the mechanisms responsible for the capacity of CSCs to survive under stresses have been unclear. The aim of this study was to clarify the significance of the autophagy systems of CSCs under stress environments.Methods: Four human gastric cancer cell line, OCUM-12, OCUM-2MD3, MKN-45 and MKN-74 were used. Side population (SP) cells were sorted from the parent OCUM-12 and OCUM-2MD3, as CSC rich cells. The expression of stem cell markers was examined by RT-PCR. The viability of cancer cells under starvation and hypoxia (1%O2) was evaluated by MTT assay with or without the autophagy inhibitor, chloroquine. The expression level of the autophagy molecule LC3-II was examined by western blot. The numbers of autophagosomes and autolysosomes were counted by electron microscope.Results: SP cells of OCUM-12 showed a higher expression of stem cell markers and higher viability in starvation and hypoxia. Western blot and electron microscope examinations indicated that the autophagy was more induced in SP cells than in parent cells. The autophagy inhibitor significantly decreased the viability and the proportion of SP cells under the stress environments.Conclusions: Cancer stem cells of gastric cancer might maintain their viability under stress environments of starvation and hypoxia via the autophagy system. Autophagy inhibitors might be a promising therapeutic agent for gastric cancer.

Peritoneal metastasis of gastric cancer (GC) results in extremely poor prognoses. The peritoneal cavity is covered by a monolayer of peritoneal mesothelial cells (PMCs). Interactions between GC cells and PMCs might play a pivotal role in peritoneal metastasis. Extracellular vesicles (EVs) correlate with intercellular communication. Although intercellular communication between cancer cells and PMCs might be associated with the peritoneal metastatic process, the role of EVs from PMCs remains unclear. We investigated the effects of EVs from PMCs on GC cells. Three GC cell lines (OCUM-12, NUGC-3, and MKN74) and four mesothelial cell lines were used. The effects of EVs derived from the PMCs on the invasion and migration of GC cells were evaluated by Matrigel invasion assay. Factors contained in the PMC EVs were analyzed; extra-cellular matrix metalloproteinase inducer (EMMPRIN) was detected in the EVs. The effects of an EMMPRIN inhibitor on the invasion-stimulating activity of EVs were examined. The EMMPRIN expressions of 110 GCs were evaluated by immunohistochemistry. PMC EVs significantly promoted the invasion of diffuse-type GC cells, i.e., OCUM-12 and NUGC-3 cells. EMMPRIN in the EVs stimulated the invasion of OCUM-12 and NUGC-3 cells. The invasion-stimulating activity of PMC EVs was inhibited by the EMMPRIN inhibitor. A high EMMPRIN expression in PMCs was significantly associated with worse cancer-specific survival and peritoneal-recurrence-free survival. EMMPRIN in EVs from PMCs might stimulate the malignant progression of diffuse-type GC. EMMPRIN might be a useful prognostic marker of recurrence in GC patients.

The prognosis of scirrhous gastric carcinoma (SGC), which is characterized by rapid infiltration and proliferation of cancer cells accompanied by extensive stromal fibrosis, is extremely poor. In this study, we report the establishment of a unique SGC cell line from a gastric cancer patient in whom an autopsy was performed. A new SGC cell line, OCUM-14, was established from malignant ascites of a male patient with SGC. A postmortem autopsy was performed on the patient. Characterization of OCUM-14 cells was analyzed by microscopic examination, reverse transcription polymerase chain reaction, fluorescence in situ hybridization analysis, immunohistochemical examination, CCK-8 assay, and in vivo assay. OCUM-14 cells grew singly or in clusters, and were floating and round-shaped. Most OCUM-14 cells had many microvilli on their surfaces. The doubling time was 43.1 h, and the subcutaneous inoculation of 1.0 × 107 OCUM-14 cells into mice resulted in 50% tumor formation. mRNA expressions of fibroblast growth factor receptor 2 (FGFR2) and human epidermal growth factor receptor 2 (HER2) were observed in OCUM-14 cells. FGFR2, but not HER2, overexpression was found in OCUM-14 cells. The heterogeneous overexpression of FGFR2 was also found in both the primary tumor and metastatic lesions of the peritoneum, lymph node, bone marrow, and lung of the patient. The FGFR2 inhibitors AZD4547 and BGJ398 significantly decreased the growth of OCUM-14 cells, while paclitaxel and 5-fluorouracil significantly decreased the proliferation of OCUM-14 cells, but cisplatin did not. A new gastric cancer cell line, OCUM-14, was established from SGC and showed FGFR2 overexpression. OCUM-14 might be useful for elucidating the characteristic mechanisms of SGC and clarifying the effect of FGFR2 inhibitors on SGC.

Background Gastric cancer (GC) patients frequently develop peritoneal metastasis. Recently, it has been reported that peritoneal mesothelial cells (PMCs) activated by GC cells acquire a migratory capacity and promote GC cell invasion. The invasiveness of PMCs reportedly depends on the activity of Tks5, an adaptor protein required for invadopodia formation. However, the relationship between clinicopathologic features and Tks5 expression in PMCs has been poorly documented. In this study, we evaluated the clinicopathologic significance of the Tks5 expression of PMCs in GC patients. Materials and methods A total of 110 GC patients who underwent gastrectomy were enrolled in this study. Tks5 expressions in PMCs from the greater omentum, lesser omentum and retroperitoneum were evaluated by immunohistochemistry. We analyzed the correlation between Tks5 expressions in PMCs and the patients’ clinicopathologic features. Results Tks5 expression was found in 71 (64.5%) of the 110 patients, while 39 (35.5%) were Tks5-negative. Tks5 positivity was significantly (p = 0.038) associated with a greater tumor depth (i.e., T3/4 compared with T1/T2). Peritoneal recurrence was found in 12 of 98 cases within 3 years of surgery. The 3-year peritoneal recurrence-free survival (PRFS) rate in Tks5-positive cases was significantly poorer than that in Tks5-negative cases (80.1% vs 97.4%, p = 0.024). Multivariate analysis revealed that Tks5 positivity and lymph node metastasis were independent factors for PRFS. Conclusion Tks5 is frequently expressed in PMCs in advanced-stage gastric cancer. Tks5 might be a useful predictor for peritoneal recurrence in GC patients.

Background: Clinical sequencing using a multiplex gene panel has recently been applied worldwide for patients with refractory solid tumors, but the significance of clinical sequencing using multiplex gene panel testing remains uncertain. Here we sought to clarify the feasibility and utility of clinical sequencing in the treatment of refractory tumors at our hospitalMethods: A total of 28 patients with advanced solid tumors treated at our hospital between 2018 and 2020 were enrolled in the clinical sequencing. Among them, we identified 26 patients whose tissue samples were of suitable quality for clinical sequencing, and we analyzed the genomic profiles of these tumors.Results: Pathogenic alterations were detected in 20 (77%) of the 26 patients. The most common mutation was TP53 (6/26, 35%), followed by KRAS (5/26, 19%), and the highest frequency of gene amplification was ERBB2 (5/26, 19%). Five of the 26 patients were identified as candidates for novel molecular-targeted therapy based on their actionable gene alterations, but none of the cases had an advantage for novel targeted therapy following the genetic tests; the reasons were clinical deterioration, refusal for off-label use, and complexity of clinical trial access.Conclusions: Our current results suggested that clinical sequencing might be useful for the detection of pathogenic alterations and the management of additional cancer treatment. Both improved optimal timing of clinical sequencing and a consensus about its off-label use might help patients receive greater benefit from clinical sequencing.Trial registration: This study was performed as a part of clinical study approved by Ethical Committee of Osaka City University Graduate School of Medicine (Permission number: 4199, 3925)

A 71-year-old man underwent right hemi-hepatectomyfor a hepatocellular carcinoma(HCC)measuring 18 cm in diameter. The pathological diagnosis was poorlydifferentiated HCC. Ten months after the surgery, computed tomographyrevealed a nodule 12mm in diameter in the right lung as well as 2 nodules measuring 19 and 11mm in diameter in the retroperitoneum at the cranial aspect of the left kidney. Four months later, the nodule in the right lung had enlarged to 44 mm, while the 2 nodules in the retroperitoneum had enlarged to 68mm and 34 mm. These nodules were resected and histopathologicallydiagnosed as metastasis from HCC. Twenty-one months after liver resection, computed tomographyrevealed nodules 16 and 25mm in diameter in the retroperitoneum around the urinarybladder and jejunum, respectively. One month later, intussusception resulted from the jejunal tumor. Laparoscopic surgerywas performed for both tumors, which were diagnosed as metastases from HCC. Twenty-five months after liver resection, metastasis from the HCC appeared in the left adrenal grand, at the site of the jejunal anastomosis, and in the fattytissue around the right scapula. Twenty-nine months after liver resection, the patient died of respiratoryfailure from multiple metastases in the left lung.

Background: Peritoneal dissemination is one of common causes of poor prognosis of patients with gastric cancer (GC). The mechanism of peritoneal metastasis has not been fully elucidated. Recent studies have reported that extracellular vesicles (EVs) play an important role for the intercellular communication in GC microenvironment. The peritoneal dissemination possesses some intercellular communication steps between cancer cells and hostile cells including peritoneal mesothelial cells (PMCs), however, the effect of EVs derived from PMCs on GC cells has not been validated. In the present study, to clarify the intercellular communication we evaluated the effect of EVs from PMCs on the proliferation, migration and invasion of GC cells. Materials and Methods: Four GC cell lines (OCUM12, NUGC3, MKN45, MKN74) and 4 PMCs cell lines were used. PMCs cell lines were primary cultured from ascites of gastric cancer patients. The EVs derived from PMCs were isolated from conditioned medium of PMCs by ultracentrifugation. The EVs derived from PMCs were confirmed by the transmission electron microscope (TEM) using uranyl acetate and the western blotting using EV markers such as CD9, CD63, and CD81. The EVs derived from PMCs were fluorescently labelled using PKH26 and the uptakes of EVs were viewed under a fluorescence microscope. The effect of EVs from PMCs on the proliferation, migration and invasion of GC cells was evaluated by MTT assay, wound healing assay and Matrigel invasion assay respectively. Results: The EVs derived from PMCs showed typical morphology of EVs by TEM, and were positive for EV markers such as CD9, CD63, and CD81 in western blotting. The EVs derived from PMCs significantly suppressed the proliferation of GC cells. On the other hand, the EVs derived from PMCs significantly promoted the migration of OCUM12 and NUGC3 cells, but not MKN45 and MKN74 cells. Furthermore, The EVs derived from PMCs significantly promoted the invasion of OCUM12 and NUGC3 cells. PKH26 labelled EVs derived from PMCs were found in cytoplasm of OCUM12 and NUGC3 cells. Conclusion: The EVs derived from PMCs significantly promoted the migration and invasion of GC cells. The EVs derived from PMCs might be one of factors which associate with the intercellular communication steps in peritoneal dissemination possesses in GC. Citation Format: Atsushi Sugimoto, Masakazu Yashiro, Tomohisa Okuno, Yuichiro Miki, Gen Tsujio, Yurie Yamamoto, Tomohiro Sera, Shuhei Kushiyama, Sadaaki Nishimura, Kenji Kuroda, Shingo Togano, Masaichi Ohira. Extracellular vesicles derived from mesothelial cells promotes the migration and invasion of gastric cancer cells [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 2895.

Background/aim Multiplex gene panel tests using next-generation sequencing (NGS) are clinically available for gastric cancer (GC). The NGS tests can reveal unexpected pathogenic variants to be associated with hereditary diseases, i.e., secondary genetic findings. We investigated whether GC patients at high risk of having hereditary gastric cancer (HGC) can be identified by their clinicopathological variables before they undergo NGS cancer gene panel tests. Patients and methods The cases of 2,286 patients with GC treated at our hospital during the years 1999-2017 were retrospectively analyzed; of them, 143 patients were identified as being at high risk of having HGC (HR-HGC), and the remaining 2,143 patients were classified as having sporadic gastric cancer (SGC). Results Compared to the SGC group, the HR-HGC status was significantly associated with younger age, female gender, macroscopic type IV and a histologically diffuse type. In a multivariate analysis, being young (i.e., ≤50 years old) was an independent risk factor for HR-HGC. Conclusion Female and young patients with diffuse-type GC are closely associated with a high risk of having HGC, and these factors might predict the detection of secondary genetic findings by NGS testing.

A 68-year-old man presented with generalfatigue due to anemia. Gastrointestinalendoscopy showed a tumor approximately 60mm in diameter in the lesser curvature of the angle of the stomach. Large cell neuroendocrine carcinoma was diagnosed by biopsy of a tumor specimen. Computed tomography revealed bulky regional lymph node metastases, solitary liver metastasis, and left adrenal metastasis. After three courses of combined chemotherapy with S-1 and CDDP, the gastric lesion and lymph node metastases shrunk, but the liver metastasis and left adrenal metastasis enlarged. After three courses of combined chemotherapy with ramucirumab and paclitaxel, the liver metastasis and left adrenal metastasis also shrunk, and no other new metastatic lesions appeared. Accordingly, total gastrectomy with lymph nodes dissection, partial resection of the liver and left adrenalectomy were performed for a curative resection. The histological findings revealed neuroendocrine carcinoma and adenocarcinoma of the stomach. The lesions of the liver and lymph nodes were all burned out, and a tiny metastatic lesion of neuroendocrine carcinoma was left in the left adrenal gland. S-1 was administrated for 3 months after surgery. The patient has been alive for 16months without recurrence after surgery.

Stromal cell-derived factor 1α (SDF1α) and its receptor C-X-C chemokine receptor type 4 (CXCR4) have been reported to form an important chemokine signaling pathway. Our previous study reported that SDF1α from tumor stromal cells may stimulate the proliferation of gastric cancer (GC) cells through the CXCR4 axis in a hypoxic microenvironment. However, a limited number of studies have addressed the clinicopathological significance of the expression of SDF1α and CXCR4 in GC, particularly at hypoxic regions. Immunohistochemistry was used to investigate the expression levels of SDF1α, CXCR4 and the hypoxic marker carbonic anhydrase 9 (CA9) in 185 patients with stage II and III GC. The results demonstrated that CA9 was expressed on cancer and stromal cells in hypoxic lesions, CXCR4 was mainly expressed in cancer cells, and SDFα was mainly expressed in stromal cells. CXCR4 expression in cancer cells and SDFα expression in stromal cells were associated with the hypoxic regions with CA9 expression. The CA9 and CXCR4 expression in the cancer cells, and the SDF1α expression in the stromal cells (CA9/CXCR4/SDF1α) was significantly associated with macroscopic type 4 tumor (P=0.012) and the pattern of tumor infiltration into the surrounding tissue (P

Purpose: Hypoxic environments are randomly scattered throughout carcinomas. Although hypoxia is cytotoxic to both cancer cells and normal cells, some types of cancer cells acquire characteristics that allow them to survive and progress in a hypoxic environment. Hypoxia is considered to be associated with aggressive tumor phenotypes of gastric carcinomas, including the ability to metastasize. Hypoxia is clinically associated with metastasis and poor patient outcome, however the underlying processes remain unclear. It has been reported that lysyl oxidase (LOX) is a hypoxia-responsive factor and is associated with the malignant progression of carcinoma. The aim of this study was to clarify the relationship between the epithelial–mesenchymal transition (EMT) and LOX in gastric cancer cells under hypoxia. Methods: Two gastric cancer cell lines, OCUM-2MD3 and OCUM-12, were used in an in vitro study. The effect of siLOX on the EMT and motility of gastric cancer cells under hypoxic condition was analyzed by RT-PCR, western blot, wound-healing assay, and invasion assay. Correlations between LOX expression and the clinicopathological features of 544 patients with gastric carcinoma were examined immunohistochemically. Results: Hypoxic conditions increased the number of polygonal or spindle-shaped cells of EMT in gastric cancer cells. The EMT of cancer cells induced by hypoxia was inhibited by treatment with LOX siRNA. Taken together, the number of migrating and invading gastric cancer cells in hypoxia was significantly decreased by LOX knockdown. LOX siRNA significantly increased the E-cadherin level and decreased the vimentin level of gastric cancer cells. LOX expression was significantly associated with invasion depth, tumor differentiation, lymph node metastasis, lymphatic invasion, venous invasion, and peritoneal metastasis. Multivariable analysis revealed that LOX was an independent parameter for overall survival. Conclusion: HIF-1α might increase the expression of LOX, which affects the EMT of gastric cancer cells in hypoxic conditions via down-regulation of E-cadherin level and up-regulation of vimentin level. LOX expression may be a useful prognostic factor for patients with gastric cancer. Citation Format: Masakazu Yashiro, Hiroaki Kasashima, Yurie Yamamoto, Kenji Kuroda, Tomohiro Sera, Atsushi Sugimoto, Shuhei Kushiyama, Shingo Togano, Tomohisa Okuno, Yuichiro Miki, Masaichi Ohira. Lysyl oxidase is associated with the epithelial–mesenchymal transition of gastric cancer cells in hypoxia [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 3566.

Introduction: The human trophoblast cell-surface antigen TROP-2 is a cell surface glycoprotein receptor involved in tight junctions at the epithelial barrier. Although TROP-2 is reportedly overexpressed in various epithelial tumors and its expression correlates with aggressive tumor behavior, there is no report of the clinicopathological features of gastric cancer (GC) with TROP-2 and phosphorylated TROP-2 (pTROP-2) expression. We evaluated the clinicopathologic significance of TROP-2 and pTROP-2 expression in GC patients. Materials and methods: We retrospectively analyzed the cases of 720 GC patients who underwent resection for GC. The expressions of TROP-2 and pTROP-2 in each tumor were evaluated by immunohistochemistry. We then divided the patients into the TROP-2 high group, TROP-2 low group, pTROP-2 high group, and pTROP-2 low group. The association between TROP-2 or pTROP-2 expression and clinicopathological variables were assessed by the chi-square test. Overall survival was analyzed by Kaplan-Meier method, and compared by log-rank test. The Cox proportional hazards model was used for multivariate analysis. Results: TROP-2 high group was found to be overexpressed in 330 (46.7%) tumor samples. Significantly higher expression of TROP-2 was significantly correlated with intestinal type, tumor invasion depth, lymph node metastasis, lymphatic invasion, and venous invasion. pTROP-2 high group was found to be overexpressed in 306 (43.4%) tumor samples. Significantly higher expression of pTROP-2 was correlated with intestinal type, low tumor invasion depth, no lymph node metastasis, and no lymphatic invasion. TROP2 overexpression was predictive for poor overall survival of patients with GC (p = 0.0002, log rank test), while multivariate analysis to overall survival revealed that TROP-2 overexpression was not an independent prognostic marker (p=0.06). In contrast, pTROP-2 overexpression was predictive for good overall survival of patients with GC (p = 0.004, log rank test). Conclusion: TROP-2 might be associated with metastatic ability of gastric cancer cells, resulting poor prognosis of patients with GC. Citation Format: Syuhei Kushiyama, Masakazu Yashiro, Sadaaki Nishimura, Shingo Togano, Kanji Kuroda, Atsushi Sugimoto, Tomohiro Sera, Yurie Yamamoto, Tomohisa Okuno, Yuichiro Miki, Hiroshi Nakada, Masaichi Ohira. Clinicopathological features of TROP-2 and pTROP-2 in gastric cancer [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 6465.

Introduction: In recent years, certain single-nucleotide polymorphisms (SNPs) have been reported to be associated with increased risk of cancer and prognosis. A Fibroblast growth factor receptor 4 (FGFR4) Gly388Arg polymorphism (rs351855) which caused the substitution of arginine for glycine in the transmembrane domain of the receptor, was reported to increase cancer risk or not. The meta-analysis results showed that FGFR4 Gly388Arg polymorphism (rs351855) might be a cancer risk factor for majority Asians, especially in breast cancer and prostate cancer. There is also a report that FGFR4 Gly388Arg polymorphism (rs351855) may be a prognostic biomarker for lung squamous-cell carcinoma patients with lymph node metastasis and uterine cervical cancer. But few studies have been conducted in gastric cancer. And the Isocitrate Dehydrogenase 1 (IDH1) mutations in patients with acute myelogenous leukemia (AML) who have a normal karyotype are associated with a poor prognosis. IDH1 rs11554137:C>T, which is located in codon 105 in the same exon as the IDH1 codon 132 arginine (IDH1R132) mutation, has been described in patients with AML and has been identified as an adverse prognostic factor. It has also been reported that IDH1 rs11554137:C>T has a negative effect on glioblastoma patients in terms of both progression free survival and overall survival (OS). We investigated the association of the gastric cancer with two SNPs. Methods: This study included 30 cases and the tumor samples obtained from gastric cancer patients by surgery between October 2010 and December 2018. We evaluated all 30 samples by a panel testing (OncoGuide NCC Oncopanel system). And we searched for the expression of FGFR4 Gly388Arg polymorphism (rs351855) and the IDH1 rs11554137. Univariate analysis and Kaplan-Meier analysis was performed based on the OS. Comparison between two groups was evaluated using chi-squared test and Fisher's exact test. The stage evaluation of the cancer adopted Japanese Gastric Cancer Association the 15th Edition. Results: Median age is 70 (34-86) years, gender is Male/Female=18/12 cases, histology is Intestinal/diffuse/Scirrhous=8/13/9 cases. There were 21 cases of FGFR4 Gly388Arg polymorphism (rs351855) expression and 2 cases of the IDH1 rs11554137 expression in 30 cases of gastric cancer. The expression of FGFR4 Gly388Arg polymorphism (rs351855) and the IDH1 rs11554137 was compared with various clinic-pathologic factors, however, no significant correlation was found between clinic-pathologic factors and FGFR4 Gly388Arg polymorphism (rs351855) and the IDH1 rs11554137 expression. Conclusion: Our results suggest that neither FGFR4 Gly388Arg polymorphism (rs351855) nor IDH1 rs11554137 is not associated with the progression of gastric cancer. Citation Format: Tomohiro Sera, Masakazu Yashiro, Yurie Yamamoto, Yukako Kushitani, Atsushi Sugimoto, Syuhei Kushiyama, Kenji Kuroda, Shingo Togano, Tomohisa Okuno, Mami Yoshii, Tatsuro Tamura, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Masaichi Ohira. Association between FGFR4 Gly388Arg polymorphism rs351855 or the IDH1 rs11554137 and Japanese gastric cancer [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 5856.

Background: Cancer progression has been recognized as not only the proliferation of tumor cells but also an interaction between cancer cells and the surrounding stroma in the tumor microenvironment. Among the tumor stromal cells, fibroblasts (especially cancer-associated fibroblasts) have been reported to be closely associated with tumor development in various solid carcinomas. We reported that chemokine (C-X-C motif) receptor 2 (CXCR2) signaling might play an important role in the pathogenic construction of tumor fibroblasts in the gastric tumor microenvironment, suggesting that gastric cancer cells might alter their adjacent stroma to form a permissive environment for tumor progression. Following those findings, we reported that chemokine (C-X-C motif) ligand 1 (CXCL1) from cells stimulated the recruitment of bone marrow cells into the gastric cancer (GC) microenvironment via chemokine (C-X-C motif) receptor 2 (CXCR2) signaling, resulting in the malignant progression of GC. The reported CXCR2 ligands are not only CXCL1 but also CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8. However, there has been only a single in vitro study of significance of these CXCR2 ligands in GC. We thus examined the clinicopathological significance of these members of the CXCL family in GC. Method: We retrospectively analyzed the cases of 590 GC patients. The expressions of CXCR2 ligands were investigated by immunohistochemistry, and we then analyzed the correlation between the clinicopathological features of the patients and the expressions of CXCR2 ligands. Result: The expressions were as follows: CXCL1, 46.2% (257/557); CXCL2, 20.7% (122/590); CXCL3, 17.1% (101/589); CXCL5/CXCL6, 2.9% (17/589); CXCL7, 36.4% (215/590); and CXCL8 1.7% (10/585) of the cases. High invasion depth was correlated with CXCL1 expression. Lymph node metastasis was correlated with high expressions of CXCL1 and CXCL7. Lymphatic invasion and venous invasion were correlated with CXCL1 and CXCL7 expression. Peritoneal cytology positivity was correlated with high expressions of CXCL1 and CXCL7. The prognoses of the CXCL1-positive patients were significantly poorer than those of the CXCL1-negative patients (p Conclusion: Among the CXCR2 ligands, CXCL1 and CXCL7 were frequently expressed in GC cells. CXCL1 was closely associated with malignant potential of GC and was an independent prognostic factor. CXCL1 might play an important role in the malignant progression of GC via CXCL1/CXCR2 signaling. Citation Format: Yurie Yamamoto, Kenji Kuroda, Tomohiro Sera, Atsushi Sugimoto, Syuhei Kushiyama, Sadaaki Nishimura, Shingo Togano, Tomohisa Okuno, Mami Yoshii, Tatsuro Tamura, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Masaichi Ohira, Masakazu Yashiro. The clinicopathologic significance of the CXCR2 ligands, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8 in gastric cancer [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 6193.

Background: Scirrhous-type gastric carcinoma (SGC), which is characterized by the rapid proliferation of cancer cells accompanied by extensive fibrosis, shows extremely poor survival. No driver gene responsible for the malignant potential of SGC has not been identified. Next-generation sequencing (NGS) technology has defined as the genomic landscape of various types of cancers, and has elucidated a number of novel driver mutations of gastric cancer. RHOA and CDH1 are characteristic mutations for SGC, but these mutations are not druggable. Novel molecular target therapy has been necessary for SGC based on its biological behavior. Objective: We sought to identify the characteristic driver gene which is responsible for SGC. Methods: DNA was isolated from six human SGC cell lines: OUCM-1, OCUM-2M, OCUM-8, OCUM-9, OCUM-12, and OCUM-14. The mutational status of 90 cancer-associated genes was examined by next-generation sequencing. The expression level of serine threonine kinase 11/liver kinase B1 (STK11/LKB1) in 708 gastric cancers was examined immunochemically, and the correlations between STK11/LKB1 expression and clinicopathologic features of gastric cancer were analyzed. Results: The nonsynonymous mutations of STK11/LKB1 gene were found in three of the six SGC cell lines. Nonsense mutation of STK11/LKB1, which was associated with STK11/LKB1 loss (Y253*), was found in OCUM-12 cells, and missense mutation of STK11/LKB1 (F354L) was found in OCUM-2M cells and OCUM-14 cells. Rapamycin, an inhibitor of STK11/ AMP-activated protein kinase (AMPK)/mammalian target of rapamycin signaling (mTOR), showed inhibitory effects of SGC cells harboring STK11/LKB1 mutation. The low STK11/LKB1 expression group was significantly associated with SGC, high invasion depth, and nodal involvement, in compared to the high STK11/LKB1 expression group. The 5-year survival rate of patients with low STK11/LKB1 expression was significantly poorer compared to those with high STK11/LKB1 expression (p Conclusion: SGC cells frequently showed the nonsynonymous mutations of STK11/LKB1. STK11/LKB1 mutation might be responsible for the malignant potential of SGC. STK11/AMPK/mTOR signaling might be one of the therapeutic targets for patients with SGC.1 Citation Format: Sadaaki Nishimura, Masakazu Yashiro, Tomohiro Sera, Yurie Yamamoto, Atsushi Sugimoto, Shuhei Kushiyama, Shingo Togano, Kenji Kuroda, Tomohisa Okuno, Masaichi Ohira. Serine threonine kinase 11/liver kinase B1 mutation in sporadic scirrhous-type gastric cancer 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 1217A.

Background Peritoneal recurrence is one of the most frequent recurrent diseases in gastric cancer. Although the exposure of cancer cells to the serosal surface is considered a common risk factor for peritoneal recurrence, there are some cases of peritoneal recurrence without infiltration to the serosal surface even after curative surgery. This study sought to clarify the risk factors of peritoneal recurrence in the absence of invasion to the serosal surface. Materials and methods Ninety-six patients with gastric cancer who underwent curative surgery were enrolled. In all 96 cases, the depth of tumor invasion was subserosal (T3). The microscopic distance from the tumor invasion front to the serosa (DIFS) was measured using tissue slides by H&E staining and pan-cytokeratin staining. E-cadherin expression was evaluated by immunohistochemical staining. Results Among the 96 patients, 16 developed peritoneal recurrence after curative surgery. The DIFS of the tumors with peritoneal recurrence (156±220 μm) was significantly shorter (p = 0.011) than that without peritoneal recurrence (360±478 μm). Peritoneal recurrence was significantly correlated with DIFS ≤234 μm (p = 0.023), but not with E-cadherin expression. The prognosis of DIFS ≤234 μm was significantly poorer than that of DIFS >234 μm (log rank, p = 0.007). A multivariate analysis of the patients' five-year overall survival revealed that DIFS ≤234 μm and lymph node metastasis were significantly correlated with survival (p = 0.005, p = 0.032, respectively). Conclusion The measurement of the DIFS might be useful for the prediction of peritoneal recurrence in T3-gastric cancer patients after curative surgery.

Introduction Thrombospondin-4 [1] is an extracellular glycoprotein involved in wound healing and tissue remodeling. Although THBS4 is reportedly frequently expressed in solid tumors, there are few reports of the clinicopathological features of carcinomas with THBS4 expression. We evaluated the clinicopathologic significance of THBS4 expression in gastric carcinoma (GC). Materials and methods We retrospectively analyzed the cases of 584 GC patients. The expression of THBS4 in each tumor was evaluated by immunohistochemistry. We then divided the patients into the THBS4-high (n = 223, 38.2%) group and THBS4-low (n = 361, 61.8%) group. THBS4 expression in cancer-associated fibroblasts (CAFs), normal-associated fibroblasts (NFs) and gastric cancer cell lines was examined by western blotting. Results THBS4 is expressed on stromal cells with αSMA or Podoplanin expression in the GC microenvironment, but not expressed on cancer cells with cytokeratin expression. The western blot analysis results showed that CAFs (but not NFs and cancer cells) expressed THBS4. Compared to the THBS4-low expression status, the THBS4-high expression status was correlated with higher αSMA expression, higher invasion depth, lymph-node metastasis, lymphatic invasion, peritoneal cytology, peritoneal metastasis, larger tumor size, microscopic diffuse type, and the macroscopic diffuse infiltrating type. The THBS4-high group's 5-year overall survival rate was significantly poorer than that of the THBS4-low group. A multivariate analysis revealed that THBS4 expression was an independent prognostic factor. Conclusion THBS4 is expressed on CAFs in the gastric cancer microenvironment. THBS4 from CAFs is associated with the metastasis of cancer cells, and is a useful prognostic indicator for gastric cancer patients.

Purpose: Scirrhous gastric cancer (SGC), diffusely infiltrating carcinoma, proliferate rapidly with fibrosis, when the cancer cells invade into the submucosa of the stomach. It has been reported that the interactions between the cancer cells and stromal cells, such as cancer-associated fibroblasts (CAFs), surrounding them advance the proliferation and invasion of some types of solid carcinomas. We previously clarified that fibroblast growth factor-7 (FGF7) produced by CAFs promotes the proliferation and tumor progression of SGC. However, the FGF receptor type 2 (FGFR2), which is a specific receptor of FGF7, is just expressed on only 20% of SGC, suggesting that about 80% of growth-stimulating signaling by the interaction between the SGC cells and CAFs might be different from FGF7/FGFR2 signaling. Materials and Methods: In this study, we aim to clarify the interaction between SGC cells and CAFs except FGF7/FGFR2 signaling in the tumor progression of SGC. We used 4 SGC cell lines, OCUM-1, OCUM-8, OCUM-9, and OCUM-12, which do not overexpress FGFR2. Five CAF cell lines, CAF82, CAF88, CAF90, CAF91, and CAF92, were established by a primary culture from individual gastric carcinoma specimens. Serum-free conditioned medium (SF-CM) from CAFs was added to each SGC cell lines and evaluate them. The interaction of SGC and SF-CM was analyzed by CCK assay and the wound healing assay. The humoral factors in SF-CM which stimulate the proliferation of SGC is divided according to the size of the molecular weight and evaluated by the CCK assay. Results: CCK assay revealed that CAF82 SF-CM promoted the proliferation of OCUM-9 and OCUM-12 by each 27 % and 20 %. CAF88 SF-CM promoted the proliferation of OCUM-9 by 17 %. CAF90 SF-CM promoted the proliferation of OCUM-1 and OCUM-9 by each 33 % and 21 %. CAF91 SF-CM promoted the proliferation of OCUM-1 and OCUM-9 by each 29 % and 25 %. CAF92 SF-CM promoted the proliferation of OCUM-1 and OCUM-9 by each 37% and 5 %. According to the wound healing assay, the invasion activity of OCUM-12 was promoted by SF-CM from CAF82, CAF90, CAF91, and CAF92 by each 56%, 37%, 34%, and 52%. CAF82 SF-CM which contain humoral factors that molecular weight is more than 30kDa promote the proliferation of OCUM-12 by 16%. Conclusion: The factors over 30kDa from CAF might stimulate the progression of SGC cells without FGFR2 expression. Citation Format: Syuhei Kushiyama, Masakazu Yashiro, Tomohisa Okuno, Kenji Kuroda, Ryota Tanaka, Shingo Togano, Sadaaki Nishimura, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Kosei Hirakawa, Masaichi Ohira. A factor (> MR 30 kDa) from cancer-associated fibroblasts may stimulate the progression of schirrous gastric cancer without FGFR2 overexpression [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 2045.

Background: Cancer stem cells (CSCs) play an important role for the progression of carcinoma. And, CSCs have a potential of resistance to various stresses such as starvation and chemotherapy. Autophagy is an intracellular degradation system which is induced various kinds of cells under various stress. In cancer cells, autophagy is activated and has involved in cancer survival. However, the significance of autophagy in CSCs has still remained to be unknown, especially in starvation and chemotherapy stress. The aim of this study is to clarify the role of autophagy in CSCs under starvation condition. Materials and Methods: Two scirrhousgastric cancer cell lines, OCUM-2MD3 and OCUM-12 (parent cells) were used. Side population cells (SP cells) which include many CSCs, was known to the fraction of low dyeability of Hoechst 33342 identified by FACScan. Stemness of SP cells, OCUM-2MD3/SP and OCUM-12/SP, were examined by sphere colony assay and RT-PCR using stemness markers, Nanog, Oct3/4, CD44. Then we examined the effect of autophagy in CSCs under starvation condition, as follows.Parents cells and SP cells were evaluated about autophagy. Parents cells and SP cells were incubated in the presence and absence of glucose for various time. The expression level of autophagy molecule, LC3 was examined by western blotting. The effect of an autophagy inhibitor, Chloroquine, to SP frequency was examined by FACScan. Results: The expression of Nanogand Oct3/4, were significantly higher in OCUM-2MD3/SP rather than parent cells OCUM-2MD3. Also, these stemness markers were significantly higher in OCUM-12/SP cells rather than parent cells. The number of sphere colony was greater in both SP cells rather than that in parent cells. The expression level of LC3 was high in both SP cells, in comparison to that in parent cells under glucose deficient condition.The autophagy inhibitor,Chloroquine, significantly decreased the SP fraction in both cells. Conclusion: CSCs might have high autophagy activity under starvation stress and sustain their stemness by autophagy system. The autophagy inhibitor,Chloroquine, might be a promising agent for gastric cancer. Citation Format: Shingo Togano. Gastric cancer stem cells under starvation stress might sustain their stemness via autophagy system [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 4275.

Purpose: It has been reported that the interaction between cancer cells and cancer-associated fibroblasts (CAFs) is closely associated with the progression of various types of cancer. The aim of this study is to clarify the role of bone marrow-derived stromal cells (BM-SCs), which are supposed to be the origin of CAFs in the tumor microenvironment, on the development of gastric cancer. Methods: The effect of human BM-SCs which frequently express CD271 on the proliferation and motility of six gastric cancer cell lines, OCUM-2M, OCUM-2MD3, OCUM-12, KATO-III, NUGC-3, and MKN-74, was examined. CD271 expression levels in BM-SCs were analyzed by flow cytometry. We also generated a gastric tumor model by orthotopic inoculation of OCUM-2MD3 cells in mice that had received transplantation of bone marrow from CAG-EGFP mice. The correlation between the clinicopathological features of 279 primary gastric carcinomas and CD271 expression in tumor stroma was examined by immunohistochemistry. Results: Numerous BM-SCs infiltrated the gastric tumor microenvironment; CD271 expression was found approximately 25% of BM-SCs. Conditioned medium from BM-SCs significantly increased the proliferation of gastric cancer cell lines. Furthermore, conditioned medium from gastric cancer cells significantly increased the number of BM-SCs, while migration of OCUM-12 and NUGC-3 cells was significantly increased by conditioned medium from BM-SCs. CD271 expression in stromal cells was significantly associated with macroscopic type-4 cancers, diffuse-type tumors, and tumor invasion depth. The overall survival of patients (n=279) with CD271-positive stromal cells was significantly worse than that of patients with CD271-negative stromal cells. This is the first report of the significance of BM-SCs in gastric cancer progression. Conclusions: BM-SCs were abundant in the tumor microenvironment of gastric cancer in vivo. In vitro study also indicated that the crosstalk between BM-SCs and gastric cancer cells might play an important role for the development of gastric cancer. CD271-positive tumor stromal cells might be a useful predictive prognostic factor for patients with gastric cancer. BM-SCs, therefore, might be one of therapeutic targets for cancer treatment. Citation Format: Masakazu Yashiro, Hiroaki Kasashima, Syuhei Kushiyama, Sadaaki Nishimura, Kenji Kuroda, Shingo Togano, Tomohisa Okuno, Yuichiro Miki, Tatsunari Fukuoka, Hirohisa Nakamae, Masayuki Hino, Masaichi Ohira. Bone marrow-derived stromal cells in the tumor microenvironment might be associated with the progression of gastric cancer [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 2742.

Background: Human scirrhous gastric carcinoma (SGC) is also known as diffusely infiltrating carcinoma, type 4 on Borrmann’s criteria, and linitis plastica-type carcinoma. SGC is characterized by frequent peritoneal metastasis, high proliferation and infiltration activity with extensive fibrosis in the tumor stroma. We previously reported that a soluble factor(s) from SGC cells changed the peritoneum to pre-metastatic niche such as round shape of mesothelial cells and peritoneal fibrosis, which is a favorable environment for cancer cells to metastasize. Extracellular vesicles (EVs; Exosomes) might be one of soluble factor(s) which might change the peritoneal component to pre-metastatic niche. In this study, we investigated the effect of EVs from SGC cells on the pre-metastatic niche formation of the peritoneum. Methods: Four SGC cell lines, OCUM-2M, OCUM-2MD3, OCUM-12, KATO-III, 2 non-SGC cell lines, MKN74, MKN45, and peritoneal mesothelial cells (PM cells) were used. The effect of EVs from gastric cancer cells on the peritoneum was examined in vitro and in vivo, as follows. PKH26-labeled EVs from OCUM-2MD3 cells were intravenously injected into nude mice for 3 weeks, then we examined the distribution of PKH26-labeled EVs by a fluorescence microscope. Next, we injected EVs intraperitoneally into nude mice, and investigated the morphology of peritoneum. Effect of EVs on the gene expression of PM cells was examined by RT-PCR. Clinical significance of EVs markers, CD9 and CD63, was evaluated by immunohistochemistry using 63 human gastric cancer specimens. Results: PKH26-labeled EVs frequently distributed to peritoneum and the liver. The morphology of PM cells changed from round shape to spindle shape by EVs addiction in vitro and in vivo. mRNA expression level of ZEB and N-cadherin of PM cells was significantly increased following the addiction of EVs. The high expression of EVs markers, CD9 and CD63, was significantly correlated with frequent peritoneal metastasis. Conclusion: EVs from SGC cells might frequently distribute to mesothelial cells of peritoneum. EVs from SGC cells might play an important role for the formation of a pre-metastatic niche on peritoneum by the morphologic change of peritoneal mesothelial cells. Citation Format: Tomohisa Okuno, Masakazu Yashiro, Shuhei Kushiyama, Sadaaki Nishimura, Shingo Togano, Kenji Kuroda, Tatsuro Tamura, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Kosei Hirakawa, Masaichi Ohira. Extracellular vesicles from scirrhous gastric cancer cells induce premetastatic niche formation for peritoneal metastasis [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 5160.

Background It has been reported that CD63, an exosome marker, is expressed in solid cancer tissues. However, its significance in patients with gastric cancer has not been clarified. Exosomes derived from cancer cells and stromal cells might play an important role in the intracellular communications involved in the development of carcinoma. This study aimed to clarify the relationship between CD63 expression in cancer cells and stromal cells and clinical-pathologic factors. Methods A total of 595 gastric cancer patients were enrolled in this study. CD63 expression in cancer cells and stromal cells was analyzed by immunohistochemistry. The correlations between CD63 expression and several clinicopathological factors were investigated. Results CD63 expression was mainly observed on the cell membranes of cancer cells, and in the cytoplasm of stromal cells. Of 595 patients, 247 cases had CD63-positive cancer cells, and 107 cases had CD63-positive stromal cells. Cases with CD63-positive cancer cells were significantly correlated with scirrhous-type gastric cancer, tumor depth, lymph node metastasis, lymphatic invasion, and tumor size. Cases with CD63-positive stromal cells were significantly correlated with age (≥65), tumor depth (T3-4), lymphatic invasion, and tumor size (≥ 5 cm). The 5-year survival rate was significantly lower (p

[Background]Fibroblast Growth Factor Receptor 2(FGFR2) amplification occuers flequently in undifferentiated gastric cancer(GC), and accounts for 3-10% of primay GC. FGFR2 shows two isoforms, IIIb-type expressed in epithelial cells, and IIIc-type expressed in mesenchymal cells. The aim of this study is evaluating clinicopathological significance of FGFR2-IIIb and IIIc expression on GC cells. [Method]562 patients who had undergone GC surgery were retrospectively examined. Immunohistochemistry of resected specimen was used to investigate the expression of FGFR2-IIIb and IIIc. [Result]FGFR2-IIIb and FGFR2-IIIc were positive in 28 cases (4.9%) and 4 cases (0.7%) of 562 gastric cancers. All of FGFR2-IIIc positive tumors were also positive for FGFR2-IIIb in a same tumor, but both were not positive in same cells. 5-year overall survival rate of FGFR2 positive group is significantly poorer (p=0.0011, log-lank) than that of FGFR2 negative. Moreover, the prognosis of FGFR2-IIIc positive group was significantly poorer (p=0.003, log-lank) than that of FGFR2-IIIb positive group. In univariate analysis, the overall survival was significantly correlated to FGFR2-IIIb positive, FGFR2-IIIc positive, invasion depth, macroscopic type, histological type, lymph node metastasis, peritoneal dissemination, lymphatic invasion, and cytology. In multivariate analysis, FGFR2IIIc was significantly correlated to overall survival (p=0.045) [Conclusion] The expression of FGFR2IIIc and/or FGFR2IIIb was observed in 5% of gastric cancer. FGFR2 amplification is associated with poor overall survival. As a result, treatment targeting FGFR2 amplification has the potential to provide clinical benefit for FGFR2 positive GC. Citation Format: Kenji Kuroda, Masakazu Yashiro, Go Masuda, Yuichiro Miki, Tomohisa Okuno, Shingo Togano, Takahiro Toyokawa, Hiroaki Tanaka, Kazuya Muguruma, Kosei Hirakawa, Masaichi Ohira. Clinicopathologic significance of fibroblast growth factor 2 type IIIb and type IIIc in gastric 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 2510.

Background: Gastric cancer is a major cause of cancer death, particularly on the basis of peritoneal metastasis. Human scirrhous gastric carcinoma (SGC), diffusely infiltrating carcinoma, or Borrman type 4 also known as linitis plastica-type carcinoma is characterized by rapid cancer cell infiltration and proliferation accompanied by extensive stromal fibrosis. SGC account for about 10% of all gastric carcinomas, and carry an extremely worse prognosis than other types of gastric carcinoma, reflecting their frequent peritoneal metastasis. We previously reported that SGC cells (“seed”) might change the peritoneum to favorable environment, so-called pre-metastatic niche (“soil”), (Cancer 77:1668-75, 1996). Recently, it has been clarified that extracellular vesicles (EVs) play an important role for cell-cell interactions. In this study, we investigated the effect of EVs from SGC cells on the pre-metastatic niche formation of the peritoneum. Methods: A SGC cell line, OCUM-2MD3, OCUM-12, KATO-III, gastric cancer cell line, MKN74, MKN45, and peritoneal mesothelial cells (PM cells) were used. PKH26-labeled EVs from OCUM-2MD3 cells were intravenously injected into nude mice, and the distribution of EVs was examined by a fluorescence microscope. The morphology of PM cells was investigated in the addiction of EVs from various gastric cancer cell lines using a phase-contrast microscope. And gene expression was examined by RT-PCR in the presence or absence of EVs from OCUM-2MD3. Clinical significance of EVs markers CD9 and CD63 was immunohistochemically evaluated using 63 human gastric cancer specimens. Results: PKH26-labeled EVs were frequently found in the peritoneum, stomach and liver, but rare in lung and brain of mice. Peritoneal mesothelial cells uptake EVs of gastric cancer cells and change morphology from cobblestone shape to spindle shape in vitro. mRNA expression level of MMPs and integrins in PM cells was increased following the addiction of EVs, in compared with the control. The high expression of CD9 and CD63 was significantly correlated with distant metastasis. Conclusion: EVs from gastric cancer cells might induce a pre-metastatic niche at peritoneum by the morphologic and genetic changes of mesothelial cells. Citation Format: Tomohisa Okuno, Masakazu Yashiro, Shingo Togano, Kenji Kuroda, Yuichiro Miki, Hiroaki Kasashima, Kosei Hirakawa, Masaichi Ohira. Effect of extracellular vesicles from gastric cancer cells on the morphologic and genetic changes of peritoneal mesothelial 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 5219.