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The regimen of nanoliposomal irinotecan plus 5-fluorouracil and leucovorin (Nal-IRI/FL) was approved in Japan as second-line chemotherapy after gemcitabine-based treatment for pancreatic ductal adenocarcinoma (PDAC) in 2020. We examined the difference in outcome between patients treated with second-line folinic acid, fluorouracil, irinotecan hydrochloride and oxaliplatin (FOLFIRINOX) and those treated with nal-IRI/FL after first-line gemcitabine and nab-paclitaxel (GnP).The outcomes of 34 patients with PDAC who received second-line FOLFIRINOX (n=21) or nal-IRI/FL (n=13) after GnP at our Department from January 2016 to June 2021 were reviewed retrospectively.Patient backgrounds did not differ between the groups. Dose reduction was more frequently required for treatment with FOLFIRINOX than with nal-IRI/FL (86% vs. 46%, p=0.022). Pegfilgrastim and aprepitant were used more frequently in the FOLFIRINOX group (both p0.01). Progression-free survival (5.9 vs. 8.3 months) and overall survival (9.1 vs. 11.2 months) did not differ significantly between the groups. The frequency of grade 3 (Common Terminology Criteria for Adverse Events) or higher adverse events was similar between the groups. All-grade peripheral neuropathy was more common in the FOLFIRINOX group (100% vs. 77%, p=0.048).FOLFIRINOX and nal-IRI/FL as second-line therapy after GnP provided similar prognoses, although supportive treatment and dose reduction were more frequently required for FOLFIRINOX.

Specific primers for PCR and small interfering RNA

Supplemental Methods

Gene set enrichment analysis of ASGR2 expression in GSE62254

Hematogenous recurrence is a challenging clinical finding that often leads to fatalities of patients with gastric cancer. Therefore, the identification of specific biomarkers and potential therapeutic target molecules for hematogenous recurrence is required to improve the outcomes of these patients. Here, transcriptome and bioinformatics analyses were conducted to uncover candidate molecules differentially expressed in patients with hematogenous recurrence of gastric cancer. One potential candidate identified was asialoglycoprotein receptor 2 (ASGR2), and siRNA experiments were conducted to determine the effect of manipulating ASGR2 expression has on cell phenotypes. ASGR2 mRNA expression analysis using quantitative real-time reverse-transcription PCR was conducted with stage II/III gastric cancer clinical specimens (n = 95). Transcript levels were increased in gastric cancer cells as compared with a control nontumorigenic epithelial cell line. Knockdown of ASGR2 decreased the adhesion and migration potential. Thus, although gastric cancer cell–invasive activity was significantly decreased by knockdown, forced expression of ASGR2 promoted invasive activity. Using a mouse hepatic metastasis model, knockdown of ASGR2 resulted in the absence of hepatic metastasis formation. High ASGR2 expression in primary gastric cancer tissues was an independent predictor of shorter disease-free and overall survival. Finally, patients with high ASGR2 expression were more likely to have a high cumulative rate of hematogenous recurrence but not peritoneal or nodal recurrence.Implications: ASGR2 expression is associated with the malignant phenotypes in gastric cancer and represents a specific biomarker of hematogenous recurrences after curative resection for gastric cancer. Mol Cancer Res; 16(9); 1420–9. ©2018 AACR.

The axon guidance gene family, SLIT/ROBO pathway, controls neural network formation, which correlates with the development of several cancers.We found through analysis of the public database that ROBO4, one of the axon guidance molecules among the SLIT/ROBO family, is significantly downregulated in primary pancreatic cancer tissues compared with adjacent normal tissues. We carried out transfection experiments using three pancreatic cancer cell lines (MiaPaCa-2, BxPC-3, and SW1990) and one pancreatic duct epithelial cell line (HPDE6c7). A total of 51 clinical samples were then examined by immunohistochemical staining to find an association between ROBO4 expression at the protein level, clinical characteristics, and surgical outcomes.ROBO4 overexpression suppressed the invasion and migration abilities in MiaPaCa-2 and BxPC-3, while ROBO4 siRNA transfection to SW1990 and HPDE6c7 enhanced those activities. PCR-based profiling detected MMP-9 as a candidate downstream target of ROBO4, which was validated by decreased MMP-9 activity after the ROBO4 overexpression assay. High ROBO4 expression clinical samples had significantly better overall survival rather than low ROBO4 cases (P = 0.048).These findings suggest that decreased ROBO4 expression activates malignant phenotypes in cancer cells and is correlated with poor survival outcomes in pancreatic cancer.

MicroRNAs (miRNAs) are abnormally expressed and involved in the pathogenesis of various carcinomas. The present study aimed to identify novel miRNA genes associated with the pathogenesis and prognosis of oesophageal squamous cell carcinoma (ESCC).The miRNA profiling of 873 genes was performed using surgically resected oesophageal tissues from 35 patients with ESCC to identify candidate miRNAs. To examine the biological activities of candidate miRNAs, their proliferative, invasive, and migratory abilities were evaluated in ESCC cells subjected to miRNA mimic-mediated over-expression. The miRNA expression levels of the selected candidate miRNAs were analysed in the resected oesophageal tissues of 76 patients with ESCC from the two cohorts and correlated with the clinicopathological parameters.Among the four candidate miRNAs identified by miRNA profiling, miR-877-3p was selected for subsequent analyses. In vitro analyses showed that the over-expression of miR-877-3p significantly suppressed the proliferation, invasion, and migration of ESCC cell lines compared with those of control cells. In the analyses of clinical specimens, the expression of miR-877-3p was down-regulated in ESCC tissues compared with that in adjacent normal oesophageal tissues. The down-regulation of miR-877-3p expression in ESCC tissues was significantly associated with advanced local progression and lymphatic involvement. The miR-877-3p down-regulation was also significantly associated with poor disease-free and disease-specific survival.miR-877-3p acts as a tumour suppressor gene in ESCC cells, and its down-regulation in ESCC tissues is associated with a poor prognosis. Thus, miR-877-3p may serve as a novel prognostic marker and promising therapeutic target.

Anastomotic leakage (AL) after esophagectomy is one of the major complications after esophagectomy. The aim of this study was to evaluate anastomotic procedure to reduce AL after esophagectomy with retrosternal gastric pull up. A total of 157 consecutive patients underwent thoracoscopic esophagectomy followed by retrosternal reconstruction with gastric tube in our hospital between January 2008 and December 2021. The anastomotic procedure was revised at January 2016. After cervical esophagogastric anastomosis using circular stapler, gastric tube was pulled down to linearize and place the anastomotic site below the suprasternal notch (SN). The sternum tracheal distance (STD) and the level of esophagogastric anastomosis (LEA) were measured using CT image. In comparison between the former (-December 2015, 65 cases) and later (January 2016-, 92 cases) group, AL was significantly reduced from 11/65 (16.9%) to 0/92 (0%) cases (p , p = 0.02) and LEA (0>, p Surgical procedure which avoids excessive compression on the gastric tube at the thoracic inlet may take an important role in reduction of AL.

BACKGROUND Distal pancreatectomy with en bloc celiac artery resection (DP-CAR) is a curative surgical method for locally advanced pancreatic body cancer; however, arterial reconstruction remains controversial in this procedure. This report presents the case of a 47-year-old man with advanced distal pancreatic carcinoma and initial partial response to chemotherapy who required celiac axis reconstruction of the common hepatic artery and left gastric artery. CASE REPORT A 47-year-old man had loss of appetite. He had a 40-mm hypovascular tumor extending from the pancreatic body to the tail, invading around the celiac artery, common hepatic artery, left gastric artery, and splenic artery. We initiated chemotherapy concurrent with chemo-radiotherapy with S-1 administration. After chemo-radiotherapy, computed tomography (CT) showed tumor shrinkage, indicating partial response, but soft tissue CT density surrounding the celiac axis arteries persisted. We conducted conversion surgery. When the common hepatic artery was clamped during surgery, the intrahepatic arterial blood flow reduced; thus, we reconstructed the middle hepatic artery to the common hepatic artery. The left gastric artery was also reconstructed using the second jejunal artery to prevent ischemic gastropathy. Histopathologic examination showed no tumor cells in the specimen; thus, R0 resection was achieved. CONCLUSIONS Arterial reconstruction can be an option for R0 resection in DP-CAR when hepatic arterial blood flow is reduced due to an intraoperative common hepatic artery clamping test.

Background The liver is the most common site for haematogenous metastasis of gastric cancer, and liver metastasis is fatal. Methods We conducted a transcriptomic analysis between metastatic foci in the liver, primary tumour and adjacent tissues from gastric cancer patients with metastasis limited to the liver. We determined mRNA expression levels in tumour tissues of 300 patients with gastric cancer via quantitative RT-PCR. The oncogenic phenotypes of GNG4 were determined with knockdown, knockout and forced expression experiments. We established and compared subcutaneous and liver metastatic mouse xenograft models of gastric cancer to reveal the roles of GNG4 in tumorigenesis in the liver. Results GNG4 was upregulated substantially in primary gastric cancer tissues as well as liver metastatic lesions. High levels of GNG4 in primary cancer tissues were associated with short overall survival and the likelihood of liver recurrence. Functional assays revealed that GNG4 promoted cancer cell proliferation, the cell cycle and adhesiveness. Tumour formation by GNG4-knockout cells was moderately reduced in the subcutaneous mouse model and strikingly attenuated in the liver metastasis mouse model. Conclusions GNG4 expression may provide better disease monitoring for liver metastasis, and GNG4 may be a novel candidate therapeutic target for liver metastasis.

Background Gastric cancer (GC) with hepatic metastasis has a poor prognosis. Understanding the molecular mechanisms involved in hepatic metastasis may contribute to the development of sensitive diagnostic biomarkers and novel therapeutic strategies. Methods We performed transcriptome analysis of surgically resected specimens from patients with advanced GC. One of the genes identified as specifically associated with hepatic metastasis was selected for detailed analysis. GC cell lines with knockout of the candidate gene were evaluated in vitro and in vivo. Expression of the candidate gene was analysed in GC tissues from 300 patients. Results Ethanolamine kinase 2 (ETNK2) was differentially upregulated in GC patients with hepatic metastasis. ETNK2 expression was elevated in GC cell lines derived from haematogenous metastases. ETNK2 knockout significantly suppressed proliferation, invasion, and migration; increased apoptosis; reduced Bcl-2 protein expression; and increased phosphorylated p53 expression. In mouse xenograft models, ETNK2 knockout virtually abolished hepatic metastasis. Stratification of GC patients based on ETNK2 mRNA level revealed significant associations between high ETNK2 tumour expression and both hepatic recurrence and worse prognosis. Conclusions Upregulation of ETNK2 in GC enhances hepatic metastasis, possibly via dysregulation of p53–Bcl-2-associated apoptosis. ETNK2 expression may serve as a biomarker for predicting hepatic recurrence and a therapeutic target.

680 Background: Multidisciplinary treatment of borderline resectable (BR)/unresectable locally advanced (UR-LA) pancreatic adenocarcinoma (PDAC) has not yet been established. The purpose of this study is to explore factors that improve prognosis in radical surgery after multidisciplinary treatment for pancreatic cancer. Methods: We evaluated the following prognostic factors in 240 PDAC patients who underwent radical resection after multidisciplinary treatment. Patients were classified into 3 groups according to NCCN guidelines (BR PDAC invading the portal vein (BR-PV), BR pancreatic cancer in contact with the major arteries such as the hepatic artery, celiac axis and superior mesenteric artery (BR-A), and UR-LA), and prognostic factors were investigated. Patients with BR PDAC were treated with chemotherapy followed by surgery, while radiation therapy was added preoperatively in most cases with arterial invasion. All patients with UR-LA underwent surgery after nab-paclitaxel plus gemcitabine (GnP) followed by chemoradiotherapy (CRT) with S-1. Results: BR-PV/BR-A/UR-LA patients were 88/111/41, respectively. Prognosis was significantly better in the NAT group than in the upfront surgery group for both BR-PV/A (P=0.004/42.5 (P=0.022). In univariate analysis in 39 patients with BR-A, the following factors were significantly favorable prognostic factors; TM normalization (P=0.033), preoperative PNI>42.5 (P=0.013), intraoperative blood loss>830 ml (P=0.013). Multivariate analysis revealed that high preoperative PNI was an independent prognostic factor (hazard ratio 0.15 [0.02-0.85]; P=0.014) in BR-A patients. In patients with UR-LA who underwent radical resection after GnP and subsequent CRT, median duration of NAT was 8.8 months, and R0 resection was achieved in 36 patients (88%). 3-year OS was 77.4%, and 5-year OS 58.6%. Multivariate analysis revealed that CA19-9 normalization (hazard ratio 0.23 [0.02-0.88]; P=0.032) and PNI≥41.7 (HR 0.05 [0.01-0.62]; P=0.021) were independent prognostic factors. Conclusions: In both BR/UR-LA pancreatic cancer, normalization of TM and maintenance of good nutritional status during NAT until surgery may contribute to prolonged prognosis.

674 Background: Positive peritoneal cytology has been reported to indicate a poor prognosis in patients with pancreatic cancer even if the primary tumor is surgically resected. This study investigated the clinical implications of peritoneal cytology by staging laparoscopy for patients with potentially resectable pancreatic cancer for whom initial treatment will be started. Methods: We retrospectively reviewed 113 consecutive patients with pancreatic cancer diagnosed as resectable by computed tomography in whom peritoneal cytology was evaluated by staging laparoscopy between December 2018 and August 2022. Patients with positive cytology received induction chemotherapy, and those in whom cytology converted to negative underwent surgical resection as needed when possible. We set best tumor marker cutoff values for predicting positive cytology by maximizing the Youden index. Results: Seventy-three patients were men and the mean age was 72 years. Thirty patients (26.5%) had positive cytology at initial staging laparoscopy. Minimal peritoneal metastases were detected in seven of these patients and liver metastases in two. Larger tumor diameter ( > 30 mm), location in the pancreatic body or tail, an elevated CA19-9 level ( > 138.5 U/ml), an elevated CA125 level ( > 13.5 U/ml), and an elevated CEA level ( > 5.1 ng/ml) were associated with a significantly increased risk of positive cytology (odds ratio 4.71 [confidence interval 1.87–12.2] P = 0.001, 2.49 [1.07–6.05] P = 0.038, 2.95 [1.26–7.12] P = 0.014, 3.89 [1.57-10.7] P = 0.005, and 3.52 [1.23–10.2] P = 0.018, respectively). Eighteen patients (60%) who received induction chemotherapy converted from positive to negative cytology; seven (38%) of these patients underwent surgery and all remain alive without recurrence. Interestingly, median overall survival in patients with negative cytology was not necessarily inferior to that in those with positive cytology (23.4 months vs. 24.2 months, P = 0.33). Conclusions: Over a quarter of patients with pancreatic cancer that is diagnosed as resectable by computed tomography may have positive peritoneal cytology at the initial assessment. These patients tend to have higher CA19-9, CA125, and CEA levels, larger tumors, and tumors located in the body or tail of the pancreas. A more favorable prognosis may be achieved by administering induction chemotherapy until cytology converts to negative than by upfront surgery.

745 Background: Serum CA125 is a potential biomarker for diagnosis of peritoneal metastasis in gastric cancer. However, its significance of pancreatic cancer (PC) has not been investigated. Therefore, we aimed to clarify association between serum CA125 and ascites burden, and its kinetics during first-line chemotherapy for PC. Methods: This multicenter retrospective study comprised PC patients who received FOLFIRINOX or gemcitabine plus nab-paclitaxel in first-line setting between Jan 2014 and Dec 2021. Patient background and treatment outcome was assessed in CA125 elevated and non-elevated group before chemotherapy. The CA125 kinetics after chemotherapy was calculated based on baseline and first measure of CA125. Further, the association between early CA125 change and clinical response during chemotherapy were evaluated based on optimal cut off value calculated receiver operating characteristic (ROC) curve analysis. Results: A total 109 patients from 3 hospitals were assessable. The overall survival (OS) was significantly shorter in elevated group than that in non-elevated group (median, 10.7 vs. 21.3 months, p = 0.0002). The median value of CA125 before chemotherapy was elevated according to ascites burden (Non, 36U/ml; mild, 173U/ml; moderate/severe, 575U/ml; p < 0.0001). CA125 elevation, CA19-9 elevation, poor performance status and poor glasgow prognostic score were independent prognostic factors in multivariate analysis. After chemotherapy, the median first-time measure of CA125 was performed in day 41. Among patients with peritoneal dissemination, the median change of CA125 was correlated with clinical response (CR/PR, -0.55%/day; SD, -0.24%/day; PD, 3.35%/day, p = 0.004). After chemotherapy, first-time measure of CA125 was performed in a median of day 41. According to the ROC curve analysis, the optimal cut-off value of increase in CA125 for progressive disease was 1.56 %/day (specificity 94.1%, sensitivity 80%). The median PFS and OS were 1.4 and 6.6 months in increased group and 3.3 months and 14.0 months in non-increased group, respectively (p = 0.016 and p = 0.028). Conclusions: CA125 is considered a clinically useful marker in unresectable PC treated with first-line chemotherapy because CA125 above the ULN is a poor prognostic factor for OS and an increased CA125 can be an early predictor of progression in patients with peritoneal dissemination.

Liver metastasis is often fatal in patients with gastric cancer, therefore, we aimed to identify genes associated with the mechanisms of liver metastasis of gastric cancer (GC) and to investigate their potential to predict recurrence and to serve as targets of therapy. Recurrence pattern-specific transcriptome analysis was performed to identify liver metastasis-associated genes. A stable knockout cell line was generated to investigate metabolic pathways that contribute to the malignant phenotype in vitro and vivo. Three hundred GC patients were analyzed to demonstrate an association between gene expression levels and clinicopathological parameters. As a results extracellular matrix complex subunit 1 (FRAS1) was identified as a liver metastasis-associated gene. Pathway analysis revealed that FRAS1 expression was significantly correlated with the expression of genes encoding TGFB1, MAP1B, AHNAK, BMP2, MUC1, BIRC5, MET, CDH1, RB1 and MKI67. FRAS1 expression was associated with the activation of the EGFR and PI3K signaling pathways. The proliferation ability of FRAS1 knockout cell line (FRAS1-KO) was inhibited compared to that of the parent cell line through caspase activity increment and cell cycle alteration. FRAS1-KO cells exhibited increased responsiveness to oxygen stress and diminished stemness, invasiveness, and migration. Mouse models of GC revealed decreases in tumor formation and generation of metastasis by FRAS1-KO cells. Moreover, the cumulative liver recurrence rate was significantly increased in patients with GC with high FRAS1 expression levels. We concluded that FRAS1 contributes to the malignant phenotype of GC, especially liver metastasis, and may therefore serve as a predictive marker or a target for treating liver metastasis.

We consider a perturbed system ($$\Sigma_A^+$$,ϕ(ϵ, ·)) with topologically transitive subshift of finite type $$\Sigma_A^+$$ and Holder continuous functions ϕ(ϵ, ·) defined on $$\Sigma_A^+$$ endowed with small parameter ϵ > 0. Through our choice of ϕ(ϵ, ·), we realize the situation that the perturbed system has a unique Gibbs measure μϵ of the potential ϕ(ϵ, ·) for each ϵ > 0 and on the other hand the unperturbed system possesses several Gibbs measures μ1, μ2,…, μm of the limit potential at ϵ = 0. In this paper, we give a necessary and sufficient condition for convergence of the measure μϵ using the notion of Perron complements of Ruelle operators. Our results can be applied also to the problems of convergence of stationary distributions of perturbed Markov chains with holes.

Background and Aims Serum diagnostic markers of early-stage pancreatic ductal adenocarcinoma (PDAC) are needed, especially for stage I disease. As tumors grow and cause pancreatic atrophy, markers derived from pancreatic parenchyma such as serum carboxypeptidase A (CPA) activity lose diagnostic performance. We evaluated, with CA19-9, serum CPA as a marker of early pancreatic cancer. Methods Serum CPA activity levels were measured in 345 controls undergoing pancreatic surveillance, divided into 2 sets, set 1 being used to establish a reference range. Variants within the CPA1 locus were sought for their association with pancreatic CPA1 expression to determine if such variants associated with serum CPA levels. A total of 190 patients with resectable PDAC were evaluated. Results Among controls, those having 1 or more minor alleles of CPA1 variants rs6955723 or rs2284682 had significantly higher serum CPA levels than did those without (P = .001). None of the PDAC cases with pancreatic atrophy had an elevated CPA. Among 122 PDAC cases without atrophy, defining serum CPA diagnostic cutoffs by a subject’s CPA1 variants yielded a diagnostic sensitivity of 18% at 99% specificity (95% confidence interval [CI], 11.7–26) (vs 11.1% sensitivity using a uniform diagnostic cutoff); combining CPA with variant-stratified CA19-9 yielded a sensitivity of 68.0% (95% CI, 59.0–76.2) vs 63.1% (95% CI, 53.9– 71.7) for CA19-9 alone; and among stage I PDAC cases, diagnostic sensitivity was 51.9% (95% CI, 31.9–71.3) vs 37.0% (95% CI, 19.4–57.6) for CA19-9 alone. In the validation control set, the variant-stratified diagnostic cutoff yielded a specificity of 98.2%. Conclusion Serum CPA activity has diagnostic utility before the emergence of pancreatic atrophy as a marker of localized PDAC, including stage I disease.

The detection of molecules and mechanisms affecting the malignant phenotype of gastric cancer cells may contribute to the identification of biomarkers for metastasis and recurrence, and such molecules may serve as targets of therapy. For this purpose, in this study transcriptome analysis was performed using surgically resected specimens from patients with gastric cancer with synchronous metastasis. We identified homeobox C10 (HOXC10) as the most highly expressed gene in gastric cancer tissues compared with the adjacent noncancerous gastric mucosa. Polymerase chain reaction (PCR) array analysis was performed to identify genes coordinately expressed with HOXC10. The effects of inhibiting HOXC10 on malignant phenotype was evaluated using HOXC10 knockout gastric cancer cell lines, and antibody array analysis was performed to assess the effect of HOXC10 knockout on intracellular signaling. We used a mouse subcutaneous xenograft model to evaluate the tumorigenicity. HOXC10 expression was determined in gastric cancer tissues acquired from 300 patients with gastric cancer. PCR array analysis revealed that the levels of HOXC10 messenger RNA positively correlated with those of FGFBP1 and SOX10. The phosphorylation of ERK1/2 was decreased in HOXC10 knockout cells. HOXC10 knockout significantly suppressed proliferation by increasing apoptosis and reducing the migration and invasiveness of gastric cancer cells. Mouse xenograft models revealed that the tumorigenicity of HOXC10 knockout cells was attenuated compared with the parental cells. The relatively high expression levels of HOXC10 in gastric cancer tissues were significantly associated with hepatic and peritoneal recurrence, as well as worse prognosis. Our results indicated that HOXC10 enhances the malignant phenotype of gastric cancer cells. The expression levels of HOXC10 may therefore serve as a prognostic biomarker and the products of HOXC10 may provide targets of therapy.

We study perturbations of topological pressures, Gibbs measures and measure-theoretic entropies of these measures concerning perturbed potentials defined on topologically transitive subshift of finite type. The subshift with respect to non-perturbed system is not assumed to be topologically transitive. Therefore, the subshift of the perturbed systems and the subshift of the unperturbed system are different. We reduce this situation to a perturbation problem of certain irreducible nonnegative matrices generated by Ruelle operators. Moreover, under suitable conditions of potentials, we characterize the limit points of those thermodynamics and give a necessary and sufficient condition for convergence of Gibbs measures and the measure-theoretic entropy of this measure when the subshift of the non-perturbed system has or transitive components with the maximal pressure. Finally we apply our results to a problem of collapse of perturbed graph iterated function systems.

Localized gastric cancer (GC) becomes fatal once recurring. We still have room for improving their prognoses. Firstly, a transcriptomic analysis was done on surgically resected specimens of 16 patients with UICC stage III GC who underwent curative gastrectomy and adjuvant oral fluoropyrimidine monotherapy. Four of them were free from disease for longer than 5 years, and the others experienced 15 metachronous metastasis at either liver, peritoneum, or distant lymph nodes (n = 4 each) within 2 years after surgery. CPLX1 was identified as a novel oncogene candidate for recurrence among 57,749 genes. Secondary, we tested alteration of malignant phenotypes including drug resistance of gastric cancer cell lines by small interfering RNA-mediated CPLX1 knockdown. Inhibiting CPLX1 expression decreased the proliferation, motility, and invasiveness of GC cells, and increased apoptosis and sensitivity to fluorouracil. Thirdly, we validated the clinical significance of CPLX1 expression in GC by quantitative RT-PCR on 180 primary gastric cancer tissues of which patients underwent gastric resection for stage II and III GC without preoperative treatment between 2001 and 2014. Increased expression of CPLX1 mRNA in gastric cancer tissues correlated with worse prognoses and was an independent risk factor for peritoneal recurrence in subgroups receiving adjuvant chemotherapy. CPLX1 may represent a biomarker for recurrence of gastric cancer and a target for therapy.Brief descriptionTranscriptomic analysis identified CPLX1 gene as a novel oncogene candidate for gastric cancer. CPLX1 may promote epithelial-mesenchymal transition and evading apoptosis of gastric cancer cells even under a cytotoxic agent, and also be a predictor for recurrence after surgery for UICC Stage II-III gastric cancer.

Here, we evaluated the therapeutic potential of antibodies (Abs) targeting cholinergic receptor nicotinic beta 2 subunit (CHRNB2) in gastric cancer. To investigate the effects of these Abs on malignant phenotypes in vitro and in mouse xenograft models, we generated gene knockouts through genome editing, performed RNA interference-mediated knockdown of gene expression, and ectopically expressed CHRNB2 in gastric cancer cells. The effects of anti-CHRNB2 Abs on the proliferation of cancer cells were evaluated both in vitro and in vivo. We determined the effects of Chrnb2 deficiency on mice and the clinical significance of CHRNB2 expression in gastric cancer clinical specimens. Knockdown of CHRNB2 attenuated gastric cancer cell proliferation, whereas forced overexpression of CHRNB2 increased cell proliferation. Knockout of CHRNB2 significantly influenced cell survival and functions associated with metastasis. The effects of polyclonal Abs targeting the C- and N-termini of CHRNB2 guided the development of anti-CHRNB2 monoclonal Abs that inhibited the growth of gastric cancer cells in vitro and in vivo. Pathway analysis revealed that CHRNB2 interfered with signaling through the PI3K-AKT and JAK-STAT pathways. Chrnb2-deficient mice exhibited normal reproduction, organ functions, and motor functions. CHRNB2 regulates multiple oncological phenotypes associated with metastasis, and blockade of CHRNB2 expression using specific Abs shows promise for controlling metastasis in gastric cancer.

530 Background: For borderline resectable pancreatic ductal adenocarcinoma (BR-PDAC), upfront surgery was standard in the past, and the usefulness of neoadjuvant treatment (NAT) has been reported in recent years. However, few studies have been conducted to date on whether there is a difference in optimal treatment between BR-PDAC invading the portal vein (BR-PV) or abutting major arteries (BR-A). The objective of this study was to investigate the optimal treatment for BR-PV and BR-A. Methods: We retrospectively analyzed 199 patients with BR-PDAC (88 BR-PV and 111 BR-A). For each BR-PV and BR-A, we analyzed the following points. 1) Comparison of prognosis of upfront surgery vs. NAT, 2) Comparison of regimens in patients who underwent NAT, 3) Prognostic factors in patients who underwent resection after NAT. Results: 1) In BR-PV patients who underwent upfront surgery (n = 46)/NAT (n = 42), survival was significantly better in the NAT group (3-year overall survival (OS): 5.8%/35.5%, p = 0.004). In BR-A patients who underwent upfront surgery (n = 48)/NAT (n = 63), survival was also significantly better in the NAT group (3-year OS:15.5%/41.7%, p < 0.001). 2) The prognosis tended to be better in patients who received newer chemotherapeutic regimens, such as FOLFIRINOX and gemcitabine with nab-paclitaxel than older regimens such as gemcitabine and/or S-1, in each BR-PV and BR-A patients. The R0 rate was significantly higher (100%) when radiotherapy was used in combination with chemotherapy, regardless of the chemotherapeutic regimen. 3) In 36 BR-PV patients who underwent surgery after NAT, univariate analysis revealed that normalization of tumor marker levels ( p = 0.028) and preoperative high prognostic nutritional index (PNI) ( p = 0.022) were significantly associated with a favorable prognosis. In 39 BR-A patients who underwent surgery after NAT, multivariate analysis revealed that preoperative PNI > 42.5 was an independent prognostic factor (hazard ratio: 0.15, p = 0.014). The length of NAT was not a prognostic factor for either BR-PV or BR-A. Conclusions: NAT using newer chemotherapy is essential for improving the prognosis of BR pancreatic cancer. These findings suggest that prognosis may be improved by maintaining good nutritional status during preoperative treatment, not by the length of preoperative treatment. In addition, surgery after normalization of tumor markers levels by preoperative treatment contributes to the prolongation of survival.

Liver metastasis of gastric cancer is the most common for hematogenous metastases and so fatal, that the identification of novel markers and targets for therapy are crucial. We conducted transcriptome analyses between synchronous liver metastasis, primary tumor, and adjacent tissues from four patients with metastasis confined to the liver to discover thatGNG4upregulated substantially in primary gastric cancer tissues. Quantitative RT-qPCR assay for 300 gastric cancer patients revealed that higher levels ofGNG4in primary cancer were associated with shorter overall survival and a higher risk of liver recurrence. The oncogenic phenotypes ofGNG4were determined by knockout and forced expression ofGNG4. Tumor formation byGNG4knockout cells was more strikingly attenuated in a liver metastasis mouse model compared with a subcutaneous model.GNG4is a candidate for a therapeutic target for liver metastasis, and its expression may enable us to provide better disease monitoring for liver metastasis.

Background Controlling metastasis is essential for improving the prognosis of patients with gastric cancer (GC). Here, we aimed to identify a molecule required for GC metastasis and to investigate its potential utility as a target for the development of therapeutic antibodies (Abs). Methods Transcriptome and bioinformatics analyses of human GC cell lines identified the neuronal pentraxin receptor (NPTXR) as a candidate molecule. NPTXR function was probed by modulating its expression in GC cells and assessing the effects on intracellular signaling and malignant behaviors in vitro and in mouse xenograft models. We also generated anti-NPTXR Abs and Nptxr−/− mice, and assessed the clinical significance of NPTXR expression in GC specimens. Results NPTXR mRNA expression in clinical specimens was associated with disease progression and was significantly higher in tissues from GC patients with distant metastasis compared with those without. NPTXR regulated expression of genes involved in metastatic behaviors as well as activation of the PI3K–AKT–mTOR, FAK–JNK, and YAP signaling pathways. NPTXR silencing promoted caspase-mediated apoptosis and attenuated GC cell proliferation, cell cycle progression, migration, invasion, adhesion, stem cell-like properties, and resistance to 5-fluorouracil in vitro, and also inhibited the tumorigenicity of GC cells in vivo. Anti-NPTXR Abs inhibited GC peritoneal metastasis in mice. Nptxr−/− mice showed no abnormalities in reproduction, development, metabolism, or motor function. Conclusions NPTXR plays an essential role in controlling the malignant behavior of GC cells in vitro and in vivo. NPTXR-targeting Abs may thus have utility as novel diagnostic tools and/or treatment modalities for GC.

Additional file 5: Figure S2. Verification of NPTXR knockdown or overexpression in GC cells. a-c qRT-PCR analysis of NPTXR mRNA expression in MKN1, N87, and/or NUGC3 cells with siRNA-mediated knockdown (a), shRNA-mediated knockdown (b), or overexpression (c) of NPTXR. *P

Additional file 7: Figure S4. Effects of NPTXR-mAb-1 on gastric cancer cells. CCK-8 proliferation assay of MKN45 (a) and N87 (b) cells incubated with NPTXR-mAb-1 in dose-dependent manner.

Additional file 2: Table S1. Nucleotide sequences.

Additional file 12: Table S4. Prognostic factors of patients with resectable gastric cancer.

Additional file 3: Figure S1. Extraction diagram of candidate genes identified by global expression analysis. Sixteen patients were categorized into four groups (4 patients for each) according to clinical courses as follows: group 1, no recurrences for > 5 years; group 2, hepatic-confined metastasis within 2 years after surgery; group 3, peritoneal metastasis within 2 years after surgery; and group 4, nodal metastasis within 2 years after surgery. Global expression profiling was conducted to compare the expression levels of 57,749 genes between each cluster. Among them, 14 we considered 14 genes as candidates shown in Supplemental Table 2.

Additional file 8: Figure S5. a qRT-PCR analysis of NUDT13, MAP 1B, MMP9 mRNA levels in parental MKN1, NPTXR-KO-1 and -KO-2 cells. b Flow cytometric analysis of the frequency parental and NPTXR-KO MKN1 cells with phosphorylated ERK1/2. c Pull-down of small GTPases (Ras and Arf6) in parental and NPTXR-KO MKN1 cells.

Additional file 6: Figure S3. Characterization of polyclonal anti-NPTXR Abs effects in vitro and in vivo. a CCK-8 proliferation assay of MKN1 cells incubated with pAbs in vitro. b Therapeutic effects of intraperitoneal administration of anti-NPTXR polyclonal antibodies. Macroscopic appearance of peritoneal nodules 6 weeks after injection of MKN1 cells and treatment with control IgG or NPTXR-pAbs. c Total volumes of peritoneal nodules collected from BALB/c nu/nu mice 6 weeks after injection of MKN1 cells and treatment with control IgG or NPTXR-mAb-1, − 2, or − 3. *P

Additional file 9: Figure S6. a Body weights of Nptxr+/+, Nptxr+/−, and Nptxr−/− mice at 4 and 8 weeks after birth. b and c Metabolic (b) and hematological (c) tests in Nptxr+/+, Nptxr+/−, and Nptxr−/− mice.

Hematogenous recurrence is a challenging clinical finding that often leads to fatalities of patients with gastric cancer. Therefore, the identification of specific biomarkers and potential therapeutic target molecules for hematogenous recurrence is required to improve the outcomes of these patients. Here, transcriptome and bioinformatics analyses were conducted to uncover candidate molecules differentially expressed in patients with hematogenous recurrence of gastric cancer. One potential candidate identified was asialoglycoprotein receptor 2 (ASGR2), and siRNA experiments were conducted to determine the effect of manipulating ASGR2 expression has on cell phenotypes. ASGR2 mRNA expression analysis using quantitative real-time reverse-transcription PCR was conducted with stage II/III gastric cancer clinical specimens (n = 95). Transcript levels were increased in gastric cancer cells as compared with a control nontumorigenic epithelial cell line. Knockdown of ASGR2 decreased the adhesion and migration potential. Thus, although gastric cancer cell–invasive activity was significantly decreased by knockdown, forced expression of ASGR2 promoted invasive activity. Using a mouse hepatic metastasis model, knockdown of ASGR2 resulted in the absence of hepatic metastasis formation. High ASGR2 expression in primary gastric cancer tissues was an independent predictor of shorter disease-free and overall survival. Finally, patients with high ASGR2 expression were more likely to have a high cumulative rate of hematogenous recurrence but not peritoneal or nodal recurrence. Implications: ASGR2 expression is associated with the malignant phenotypes in gastric cancer and represents a specific biomarker of hematogenous recurrences after curative resection for gastric cancer. Mol Cancer Res; 16(9); 1420–9. ©2018 AACR.

Background/aim Establishment of mouse xenograft models is necessary for oncological research and depends on the characteristics of the cell lines and the immune system of the host. In this study, we describe the development of mouse xenograft models using human gastric cancer (GC) cell lines. Materials and methods MKN1 stably-expressing luciferase (MKN1-Luc), N87, KATO III, MKN45 stably-expressing luciferase (MKN45-Luc), NUGC4, and OCUM-1 human GC cell lines were injected intraperitoneally into mice to establish peritoneal metastasis models. MKN45-Luc were injected into subcutaneously implanted spleen, and MKN1-Luc and MKN45-Luc were injected directly into the portal veins of mice for the establishment of hepatic metastasis models. Results Peritoneal metastasis was formed after implantation of MKN1-Luc, N87, KATO III, MKN45-Luc, and NUGC4 in nude mice, but not formed in OCUM-1 even in NOD/SCID mice. After intrasplenic injection of MKN45-Luc, we found no hepatic metastasis formation. We identified hepatic metastasis formation after direct injection of MKN45-Luc and MKN1-Luc into the portal veins of NOD/SCID mice. Conclusion Peritoneal and hepatic metastasis mouse xenograft models were successfully established using several human GC cell lines.

Liver metastasis remains a serious problem in the management of gastric cancer (GC). Our aims were to identify through transcriptome analysis a molecule that mediates hepatic metastasis in GC, and to evaluate its potential as a diagnostic marker and a therapeutic target. The effects of knocking out a relevant molecule using genome editing were evaluated in vitro experiments and in mouse xenograft models. Expression levels of candidate molecule in 300 pairs of gastric tissues were determined to assess whether differentially expressed genes predicted hepatic recurrence, metastasis, or both. Transcriptome data identified the overexpression of synaptotagmin VII (SYT7) in GC tissues with hepatic metastasis. Its expression in the GC cell lines was high, particularly in those that exhibited a differentiated phenotype, and positively correlated with the expression of SNAI1 and TGFB3, and inversely with RGS2. SYT7 knockout inhibited the proliferation of GC cells, indicated by increased apoptosis with activated caspase and loss of mitochondria membrane potential, G2/M cell-cycle arrest and attenuated cell migration, invasion, and adhesion. The tumorigenicity of SYT7-knockout cells was moderately reduced in a mouse model of subcutaneous metastasis in which the levels of BCL2 and HIF1A were decreased and was more strikingly attenuated in a model of hepatic metastasis. The SYT7 levels in the primary GC tissues were significantly associated with hepatic recurrence, metastasis, and adverse prognosis. SYT7 represents a tool for prediction and monitoring of hepatic metastasis from GC as well as being a promising therapeutic target.

Although peritoneal metastasis is a serious concern in patients with gastric cancer, no acceptable and specific biomarker is available. We aimed to identify a candidate biomarker to predict peritoneal metastasis of gastric cancer. Metastatic pathway-specific transcriptome analysis was conducted by comparison of patient groups with no recurrence and with peritoneal, hepatic, and nodal recurrence. Fifteen cell lines and 262 pairs of surgically resected gastric tissues were subjected to messenger RNA (mRNA) expression analysis. Polymerase chain reaction array analysis was performed to explore coordinately expressed cancer-related genes. To evaluate the in situ protein localization and expression patterns, immunohistochemical staining was performed. From transcriptome data, troponin I2 (TNNI2) was identified as a candidate molecule specifically overexpressed in gastric cancer prone to peritoneal metastasis. TNNI2 mRNA was expressed at differential levels, independent of differentiated phenotype of cell lines. Epithelial to mesenchymal transition-related genes, tumor inhibitor of metalloproteinase 1 (TIMP1), and vacuolar protein sorting 13 homolog A (VPS13A) were expressed with TNNI2 at correlation coefficient > 0.7. The optimal cutoff of TNNI2 expression was determined as 0.00017. High TNNI2 expression was significantly and specifically associated with peritoneal metastasis and served as an independent risk marker for peritoneal recurrence after curative gastrectomy. Prevalence of peritoneal recurrence increased in parallel with staining intensity of TNNI2. TNNI2 expression in gastric tissues may serve as a specific biomarker for prediction of peritoneal metastasis of gastric cancer and contribute to improvement of patient management.

// Mitsuro Kanda 1 , Kenta Murotani 2 , Haruyoshi Tanaka 1 , Takashi Miwa 1 , Shinichi Umeda 1 , Chie Tanaka 1 , Daisuke Kobayashi 1 , Masamichi Hayashi 1 , Norifumi Hattori 1 , Masaya Suenaga 1 , Suguru Yamada 1 , Goro Nakayama 1 , Michitaka Fujiwara 1 and Yasuhiro Kodera 1 1 Department of Gastroenterological Surgery (Surgery II), Nagoya University Graduate School of Medicine, Nagoya, Japan 2 Clinical Research Center, Aichi Medical University Hospital, Nagakute, Japan Correspondence to: Mitsuro Kanda, email: m-kanda@med.nagoya-u.ac.jp Keywords: gastric cancer; expression panel; prognosis; biomarker Received: January 05, 2018 Accepted: February 27, 2018 Published: April 10, 2018 ABSTRACT Most of the proposed individual markers had limited clinical utility due to the inherent biological and genetic heterogeneity of gastric cancer. We aimed to build a new molecular-based model to predict prognosis in patients with gastric cancer. A total of 200 patients who underwent gastric resection for gastric cancer were divided into learning and validation cohorts using a table of random numbers in a 1:1 ratio. In the learning cohort, mRNA expression levels of 15 molecular markers in gastric tissues were analyzed and concordance index (C-index) values of all single and combinations of the 15 candidate markers for overall survival were calculated. The multigene expression panel was designed according to C-index values and the subpopulation index. Expression scores were determined with weighting according to the coefficient of each constituent. The reproducibility of the panel was evaluated in the validation cohort. C-index values of the 15 single candidate markers ranged from 0.506–0.653. Among 32,767 combinations, the optimal and balanced expression panel comprised four constituents ( MAGED2, SYT8, BTG1 , and FAM46 ) and the C-index value was 0.793. Using this panel, patients were provisionally categorized with scores of 1–3, and clearly stratified into favorable, intermediate, and poor overall survival groups. In the validation cohort, both overall and disease-free survival rates decreased incrementally with increasing expression scores. Multivariate analysis revealed that the expression score was an independent prognostic factor for overall survival after curative gastrectomy. We developed an integrated multigene expression panel that simply and accurately stratified risk of patients with gastric cancer.

Objective: To develop novel diagnostic and therapeutic targets specific for peritoneal metastasis of gastric cancer (GC). Background: Advanced GC frequently recurs because of undetected micrometastases even after curative resection. Peritoneal metastasis has been the most frequent recurrent pattern after gastrectomy and is incurable. Methods: We conducted a recurrence pattern-specific transcriptome analysis in an independent cohort of 16 patients with stage III GC who underwent curative gastrectomy and adjuvant S-1 for screening candidate molecules specific for peritoneal metastasis of GC. Next, another 340 patients were allocated to discovery and validation sets (1:2) to evaluate the diagnostic and predictive value of the candidate molecule. The results of quantitative reverse-transcription PCR and immunohistochemical analysis were correlated with clinical characteristics and survival. The effects of siRNA-mediated knockdown on phenotype and fluorouracil sensitivity of GC cells were evaluated in vitro, and the therapeutic effects of siRNAs were evaluated using a mouse xenograft model. Results: Synaptotagmin VIII (SYT8) was identified as a candidate biomarker specific to peritoneal metastasis. In the discovery set, the optimal cut-off of SYT8 expression was established as 0.005. Expression levels of SYT8 mRNA in GC tissues were elevated in the validation set comprising patients with peritoneal recurrence or metastasis. SYT8 levels above the cut-off value were significantly and specifically associated with peritoneal metastasis, and served as an independent prognostic marker for peritoneal recurrence-free survival of patients with stage II/III GC. The survival difference between patients with SYT8 levels above and below the cut-off was associated with patients who received adjuvant chemotherapy. Inhibition of SYT8 expression by GC cells correlated with decreased invasion, migration, and fluorouracil resistance. Intraperitoneal administration of SYT8-siRNA inhibited the growth of peritoneal nodules and prolonged survival of mice engrafted with GC cells. Conclusions: SYT8 represents a promising target for the detection, prediction, and treatment of peritoneal metastasis of GC., ファイル公開：2019-03-01

Background: Gastric cancer (GC) relapse can occur even if curative resection is achieved. Biomarkers predicting recurrence are needed to provide appropriate postoperative surveillance and perioperative therapeutic strategy. Methods: A global expression profiling was performed using tissues from GC patients with synchronous liver-confined metastasis. Family with sequence similarity 46, member C (FAM46C), was identified as a candidate biomarker. mRNA expression analysis, direct nucleotide sequencing, bisulfite sequencing and copy number assays for FAM46C were performed with eleven GC cell lines. Expression levels of FAM46C in primary GC tissues from 129 patients who underwent curative GC resection were determined and correlated with clinicopathological factors, including postoperative outcome. Results: Levels of FAM46C mRNA differed among GC cell lines. Point mutations in FAM46C were detected in five GC cell lines accompanied with reduced FAM46C transcription. No hypermethylation was found in the promoter region of FAM46C. Copy number alterations were found in six GC cell lines with differing FAM46C transcription levels. Reduced FAM46C mRNA expression levels were detected in 117 (91 %) GC specimens compared with adjacent noncancerous tissues. Low FAM46C expression levels were significantly associated with larger macroscopic GC tumor sizes. The low FAM46C expression group was likely to have shorter disease-free survival than the high group and low FAM46C level was identified as an independent risk factor for recurrence after curative resection. FAM46C expression levels were low in all cases that were later found to have hepatic recurrence. Conclusions: Reduced GC expression of FAM46C is a potential biomarker to predict hepatic recurrence after curative gastrectomy.

Background Molecular biomarkers capable of predicting recurrence patterns and prognosis are helpful for risk stratification and providing appropriate treatment to patients with hepatocellular carcinoma (HCC). In this study, we focused on G protein-coupled receptor 155 (GPR155), a cell surface signaling protein, as a candidate biomarker. Methods We analyzed GPR155 expression, DNA methylation, and copy number in HCC cell lines. The clinical significance of GPR155 expression was evaluated using 144 pairs of surgically resected liver and normal tissues with subgroup analysis based on hepatitis virus infection. Results GPR155 mRNA expression levels were differential and were decreased in 89% of HCC cell lines. No DNA methylation was detected, whereas copy number alterations were present in five (56%) HCC cell lines. GPR155 mRNA expression level was independent of background liver status and significantly lower in HCC tissues than corresponding normal liver tissues. The expression patterns of GPR155 protein by immunohistochemical staining were significantly associated with those of GPR155 mRNA. Downregulation of GPR155 was significantly associated with more aggressive HCC phenotypes including high preoperative α-fetoprotein, poor differentiation, serosal infiltration, vascular invasion, and advanced disease stage. Patients with downregulation of GPR155 were more likely to have worse prognosis after curative resection irrespective of hepatitis virus infection. Patients who experienced extrahepatic (distant) recurrences had significantly lower GPR155 expression than those with intrahepatic (liver confined) recurrences. Conclusions Downregulation of GPR155 may serve as a prognosticator that also predicts initial recurrence sites independent of hepatitis virus infection. Electronic supplementary material The online version of this article (10.1186/s12885-017-3629-2) contains supplementary material, which is available to authorized users.