Katsutoshi Shoda

Plasma level of metastasis‐associated lung adenocarcinoma transcript 1 is associated with liver damage and predicts development of hepatocellular carcinoma

Recent studies have shown that metastasis‐associated lung adenocarcinoma transcript 1 (MALAT1) was overexpressed in many human solid cancers, however, its roles in plasma of hepatocellular carcinoma (HCC) patients were unclear. The aim of this study was to investigate the significance of plasma MALAT1 levels in HCC patients. Plasma samples were collected from pre‐operative HCC, hepatic disease patients, and healthy controls, and tissue samples from HCC patients and colorectal cancer patients with liver metastasis. Plasma and tissue MALAT1 levels were measured. Plasma MALAT1 levels were progressively and significantly higher in HCC patients than hepatic disease patients, and higher in hepatic disease patients than healthy controls. The expression of MALAT1 in HCC tissue was slightly higher than that in paired non‐cancerous liver tissue, but not significant. The expression of MALAT1 in the non‐cancerous liver tissue of 20 HCC patients was significantly higher than that in normal liver tissue of 13 colorectal cancer patients. In contrast, plasma MALAT1 levels were significantly low in HCC patients with hepatitis B infection, and significantly high in patients with liver damage B or liver cirrhosis. In a receiver–operator curve analysis of HCC and hepatic disease patients, the cut‐off value of plasma MALAT1 was 1.60 and the area under the curve was 0.66. Plasma MALAT1 levels were not correlated with α‐fetoprotein or protein induced by vitamin K absence II, whereas sensitivity and specificity for the detection of HCC with the combination of MALAT1, α‐fetoprotein, and protein induced by vitamin K absence II were 88.6% and 75%, respectively. In conclusion, the plasma MALAT1 level is associated with liver damage, and has clinical utility for predicting development of HCC.

HuR Regulates Alternative Splicing of the TRA2β Gene in Human Colon Cancer Cells under Oxidative Stress

ABSTRACT Hu antigen R (HuR) regulates stress responses through stabilizing and/or facilitating the translation of target mRNAs. The human TRA2β gene encodes splicing factor transformer 2β (Tra2β) and generates 5 mRNA isoforms (TRA2β1 to -5) through alternative splicing. Exposure of HCT116 colon cancer cells to sodium arsenite stimulated checkpoint kinase 2 (Chk2)- and mitogen-activated protein kinase p38 (p38MAPK)-mediated phosphorylation of HuR at positions S88 and T118. This induced an association between HuR and the 39-nucleotide (nt) proximal region of TRA2β exon 2, generating a TRA2β4 mRNA that includes exon 2, which has multiple premature stop codons. HuR knockdown or Chk2/p38MAPK double knockdown inhibited the arsenite-stimulated production of TRA2β4 and increased Tra2β protein, facilitating Tra2β-dependent inclusion of exons in target pre-mRNAs. The effects of HuR knockdown or Chk2/p38MAPK double knockdown were also confirmed using a TRA2β minigene spanning exons 1 to 4, and the effects disappeared when the 39-nt region was deleted from the minigene. In endogenous HuR knockdown cells, the overexpression of a HuR mutant that could not be phosphorylated (with changes of serine to alanine at position 88 [S88A], S100A, and T118A) blocked the associated TRA2β4 interaction and TRA2β4 generation, while the overexpression of a phosphomimetic HuR (with mutations S88D, S100D, and T118D) restored the TRA2β4-related activities. Our findings revealed the potential role of nuclear HuR in the regulation of alternative splicing programs under oxidative stress.

HER2 amplification detected in the circulating DNA of patients with gastric cancer: a retrospective pilot study

BackgroundWe used real-time quantitative polymerase chain reaction (rqPCR) to detect human epidermal growth factor receptor 2 (HER2) amplification in the circulating cell-free DNA (cfDNA) of patients with gastric cancer (GC), which shows the spatial and temporal intrinsic heterogeneity of HER2 expression/copy number during progression, for liquid biopsy and treatment monitoring.MethodsWe first enrolled 52 patients with advanced GC who underwent surgery and 40 healthy volunteers. For patients with GC, plasma cfDNA was obtained before surgery (43 patients) and during postoperative treatment (nine of 43 patients). After ribonuclease P RNA component H1 (RPPH1) had been selected as a reference gene for HER2 CN assessment by rqPCR in GC tumours and plasma, plasma HER2-to-RPPH1 ratios were determined retrospectively in a development cohort and an additional independent validation cohort.ResultsThe HER2-to-RPPH1 ratio of GC tissues determined by rqPCR was concordant with routinely determined HER2 status. The plasma HER2-to-RPPH1 ratio was significantly higher for patients with HER2-positive tumours than for those with HER2-negative tumours. The sensitivity and specificity of the plasma HER2-to-RPPH1 ratio test were 0.539 and 0.967, respectively, in the development cohort, and 0.667 and 1.000, respectively, in the validation cohort. HER2 amplifications acquired and lost during tumour progression and treatment, respectively, were apparently detected by repeated assessments of plasma HER2-to-RPPH1 ratios during postoperative treatment.ConclusionOur preliminary data demonstrated the potential clinical use of circulating cfDNA to detect HER2 amplification as a therapeutic marker to detect and monitor HER2 CN status for effective molecular targeted therapy in patients with GC.

Tumor exosome-mediated promotion of adhesion to mesothelial cells in gastric cancer cells

Background Peritoneal metastasis consists of a highly complex series of steps, and the details of the underlying molecular mechanism remain largely unclear. In this study, the effects of tumor-derived exosomes (TEX) on the progression of gastric cancers were investigated in peritoneal metastasis. Results TEX were internalized in both mesothelial and gastric cancer cells in a cellular origin non-specific manner. Internalization of TEX into mesothelial cells promoted significant adhesion between mesothelial and gastric cancer cells, and TEX internalization into gastric cancer cells significantly promoted migratory ability, while internalization of mesothelial cell-derived exosomes did not. Expression of adhesion-related molecules, such as fibronectin 1 (FN1) and laminin gamma 1 (LAMC1), were increased in mesothelial cells after internalization of TEX from gastric cancer cell line and malignant pleural effusion. Methods TEX were extracted from cell-conditioned medium by ultracentrifugation. The effects of TEX on the malignant potential of gastric cancer were investigated in adhesion, invasion, and proliferation assays. PCR array as well as western blotting were performed to determine the underlying molecular mechanisms. The molecular changes in mesothelial cell after internalization of TEX derived from malignant pleural effusion were also confirmed. Conclusions TEX may play a critical role in the development of peritoneal metastasis of gastric cancer, which may be partially due to inducing increased expression of adhesion molecules in mesothelial cells.

Post-hepatectomy survival in advanced hepatocellular carcinoma with portal vein tumor thrombosis

AIM To analyze hepatocellular carcinoma (HCC) patients with portal vein tumor thrombosis (PVTT) using the tumor-node-metastasis (TNM) staging system. METHODS We retrospectively analyzed 372 patients with HCC who underwent hepatectomy between 1980 and 2009. We studied the outcomes of HCC patients with PVTT to evaluate the American Joint Committee on Cancer TNM staging system (7(th) edition) for stratifying and predicting the prognosis of a large cohort of HCC patients after hepatectomy in a single-center. Portal vein invasion (vp) 1 was defined as an invasion or tumor thrombus distal to the second branch of the portal vein, vp2 as an invasion or tumor thrombus in the second branch of the portal vein, vp3 as an invasion or tumor thrombus in the first branch of the portal vein, and vp4 as an invasion or tumor thrombus in the portal trunk or extending to a branch on the contralateral side. RESULTS The cumulative 5-year overall survival (5yrOS) and 5-year disease-free survival (5yrDFS) rates of the 372 patients were 58.3% and 31.3%, respectively. The 5yrDFS and 5yrOS of vp3-4 patients (n = 10) were 20.0%, and 30.0%, respectively, which was comparable with the corresponding survival rates of vp1-2 patients (P = 0.466 and 0.586, respectively). In the subgroup analysis of patients with macroscopic PVTT (vp2-4), the OS of the patients who underwent preoperative transarterial chemoembolization was comparable to that of patients who did not (P = 0.747). There was a significant difference in the DFS between patients with stage I HCC and those with stage II HCC (5yrDFS 39.2% vs 23.1%, P < 0.001); however, the DFS for stage II was similar to that for stage III (5yrDFS 23.1% vs 13.8%, P = 0.330). In the subgroup analysis of stage II-III HCC (n = 148), only alpha-fetoprotein (AFP) > 100 mg/dL was independently associated with DFS. CONCLUSION Hepatectomy for vp3-4 HCC results in a survival rate similar to hepatectomy for vp1-2. AFP stratified the stage II-III HCC patients according to prognosis.

Significance of GSTP1 for predicting the prognosis and chemotherapeutic efficacy in esophageal squamous cell carcinoma

Glutathione S-transferases (GSTs) have been reported to be activated in several types of cancers, including esophageal squamous cell carcinoma (ESCC). The aim of the present study was to investigate whether GSTP1 protein expression is a useful predictor of the clinical outcome or drug resistance in ESCC. Immunohistochemistry was conducted with 75 ESCC resected specimens using a monoclonal antibody against GSTP1. The patients were divided into two groups according to the degree of GSTP1 staining, and the relationship between the GSTP1 level and the clinicopathological features was examined. Seventy-five patients were divided into low (grade 1, n=36) and high (grade 2, n=39) GSTP1 expression groups. The overall survival was significantly worse in the grade 2 patients than in the grade 1 patients (5‑year survival rate, 78.5 vs. 51.2%; p=0.027). Cox proportional hazard analysis revealed that macroscopic type 3 or 4 disease (p=0.001), lymph node metastasis (p=0.010), and high GSTP1 expression (p=0.029) were independent predictors of a poor prognosis. With regard to the subgroup analysis among the 31 patients undergoing adjuvant chemotherapy, the grade 2 patients had a worse prognosis than did the grade 1 patients (5‑year survival rate, 45.0 vs. 81.8%; p=0.081). This tendency was not observed in the subgroup without adjuvant chemotherapy (5‑year survival rate, 51.7 vs. 59.9%; p=0.979). In conclusion, the GSTP1 expression is a good predictor of prognosis, and it may be closely related to the chemotherapeutic efficacy of 5-FU plus cisplatin in ESCC patients.

Clinical utility of circulating cell-free Epstein–Barr virus DNA in patients with gastric cancer

Recent comprehensive molecular subtyping of gastric cancer (GC) identified Epstein–Barr virus (EBV)-positive tumors as a subtype with distinct salient molecular and clinical features. In this study, we aimed to determine the potential utility of circulating cell-free EBV DNA as a biomarker for the detection and/or monitoring of therapeutic response in patients with EBV-associated gastric carcinoma (EBVaGC). The EBV genes-to-ribonuclease P RNA component H1 ratios (EBV ratios) in the GC tumors and plasma samples were determined by quantitative real-time polymerase chain reaction in 153 patients with GC, including 14 patients with EBVaGC diagnosed by the conventional method. Circulating cell-free EBV DNA was detected in 14 patients with GC: the sensitivity and specificity of detection were 71.4% (10/14) and 97.1% (135/139), respectively. Plasma EBV ratios were significantly correlated with the size of EBVaGC tumors, and the plasma EBV DNA detected before surgery in EBVaGC cases disappeared after surgery. Patients with EBVaGC may have a better prognosis, but circulating cell-free EBV DNA had no or little impact on prognosis. In addition, repeated assessment of the plasma EBV ratio in EBVaGC showed a decrease and increase in plasma EBV DNA after treatment and during tumor progression/recurrence, respectively. These results suggest the potential utility of circulating cell-free DNA to reveal EBV DNA for the identification of the EBVaGC subtype and/or for real-time monitoring of tumor progression as well as treatment response in patients with EBVaGC.

Value of Preoperative PET-CT in the Prediction of Pathological Stage of Gastric Cancer

BackgroundPreoperative precise staging is essential for the treatment of gastric cancer (GC); however, the diagnostic accuracy of conventional modalities needs to be increased. The present study investigated the clinical value of positron emission tomography-computed tomography (PET-CT) for the staging of GC.MethodsThis was a retrospective study of 117 patients with a clinical diagnosis of advanced GC who underwent PET-CT followed by gastrectomy. The incidence of FDG uptake in the primary tumor or lymph nodes and its relationship with clinicopathological factors, particularly pathological stage (pStage) III/IV, were examined.ResultsFDG uptake in the primary tumor was noted in 83 patients (70.9%). FDG uptake in the lymph nodes was detected in 21 patients (17.9%), and its sensitivity and specificity for lymph node metastasis were 22.7 and 90.5%, respectively. Multiple logistic regression analyses showed that FDG uptake in the primary tumor (odds ratio (OR) 2.764; 95% confidence interval (CI) 1.104–7.459, p = 0.029) and that in the lymph nodes (OR 4.660; 95% CI 1.675–13.84, p = 0.003) were factors independently associated with pStage III/IV. FDG uptake in the primary tumor detected pStage III/IV with higher sensitivity (80.4%) and that in lymph nodes found pStage III/IV with higher specificity (88.7%) than those of upper endoscopy plus CT (60.9 and 67.6%, respectively).ConclusionsPET-CT appears to be a useful complementary modality in the assessment of pStage III/IV because of the high sensitivity of FDG uptake in the primary tumor and the high specificity of FDG uptake in the lymph nodes.Preoperative precise staging is essential for the treatment of gastric cancer (GC); however, the diagnostic accuracy of conventional modalities needs to be increased. The present study investigated the clinical value of positron emission tomography-computed tomography (PET-CT) for the staging of GC. This was a retrospective study of 117 patients with a clinical diagnosis of advanced GC who underwent PET-CT followed by gastrectomy. The incidence of FDG uptake in the primary tumor or lymph nodes and its relationship with clinicopathological factors, particularly pathological stage (pStage) III/IV, were examined. FDG uptake in the primary tumor was noted in 83 patients (70.9%). FDG uptake in the lymph nodes was detected in 21 patients (17.9%), and its sensitivity and specificity for lymph node metastasis were 22.7 and 90.5%, respectively. Multiple logistic regression analyses showed that FDG uptake in the primary tumor (odds ratio (OR) 2.764; 95% confidence interval (CI) 1.104–7.459, p = 0.029) and that in the lymph nodes (OR 4.660; 95% CI 1.675–13.84, p = 0.003) were factors independently associated with pStage III/IV. FDG uptake in the primary tumor detected pStage III/IV with higher sensitivity (80.4%) and that in lymph nodes found pStage III/IV with higher specificity (88.7%) than those of upper endoscopy plus CT (60.9 and 67.6%, respectively). PET-CT appears to be a useful complementary modality in the assessment of pStage III/IV because of the high sensitivity of FDG uptake in the primary tumor and the high specificity of FDG uptake in the lymph nodes.

Detection of fusion gene in cell-free DNA of a gastric synovial sarcoma

Synovial sarcoma (SS) is genetically characterized by chromosomal translocation, which generates SYT-SSX fusion transcripts. Although SS can occur in any body part, primary gastric SS is substantially rare. Here we describe a detection of the fusion gene sequence of gastric SS in plasma cell-free DNA (cfDNA). A gastric submucosal tumor was detected in the stomach of a 27-year-old woman and diagnosed as SS. Candidate intronic primers were designed to detect the intronic fusion breakpoint and this fusion sequence was confirmed in intron 10 of SYT and intron 5 of SSX2 by genomic polymerase chain reaction (PCR) and direct sequencing. A locked nucleic acid (LNA) probe specific to the fusion sequence was designed for detecting the fusion sequence in plasma and the fusion sequence was detected in preoperative plasma cfDNA, while not detected in postoperative plasma cfDNA. This technique will be useful for monitoring translocation-derived diseases such as SS.

Tumor-promoting function and prognostic significance of the RNA-binding protein T-cell intracellular antigen-1 in esophageal squamous cell carcinoma.

T-cell intracellular antigen-1 (TIA1) is an RNA-binding protein involved in many regulatory aspects of mRNA metabolism. Here, we report previously unknown tumor-promoting activity of TIA1, which seems to be associated with its isoform-specific molecular distribution and regulation of a set of cancer-related transcripts, in esophageal squamous cell carcinoma (ESCC). Immunohistochemical overexpression of TIA1 ectopically localized in the cytoplasm of tumor cells was an independent prognosticator for worse overall survival in a cohort of 143 ESCC patients. Knockdown of TIA1 inhibited proliferation of ESCC cells. By exogenously introducing each of two major isoforms, TIA1a and TIA1b, only TIA1a, which was localized to both the nucleus and cytoplasm, promoted anchorage-dependent and anchorage-independent ESCC cell proliferation. Ribonucleoprotein immunoprecipitation, followed by microarray analysis or massive-parallel sequencing, identified a set of TIA1-binding mRNAs, including SKP2 and CCNA2. TIA1 increased SKP2 and CCNA2 protein levels through the suppression of mRNA decay and translational induction, respectively. Our findings uncover a novel oncogenic function of TIA1 in esophageal tumorigenesis, and implicate its use as a marker for prognostic evaluation and as a therapeutic target in ESCC.