Language：Japanese   Publisher：The Japanese Society of Gastroenterological Surgery  

<p>Herein we present a case of laparoscopic cholecystectomy in a patient who was positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The patient was a 56-year-old man with acute cholecystitis. During preoperative workup, he was revealed to be SARS-CoV-2-positive, but without any signs of pneumonia. As treatment of acute cholecystitis was complicated by asymptomatic coronavirus disease 2019 (COVID-19), nonoperative management with antibiotics was initiated. However, his condition deteriorated to gangrenous cholecystitis and obstructive cholangitis on the 8^th day of symptom onset. A multidisciplinary team discussed the treatment and perioperative risk mitigation strategy against viral transmission. After sharing information and simulation of preventive measures, laparoscopic cholecystectomy and C-tube drainage were performed on the 11^th day of symptom onset, when we thought that infectiousness should have declined significantly. During surgery, protective measures against surgical smoke were applied using pneumoperitoneum devices equipped with an ultralow particulate air filter. We report our experience of perioperative risk mitigation strategies in laparoscopic surgery for a patient who was positive for SARS-CoV-2.</p>

DOI： 10.5833/jjgs.2021.0009

Scopus

CiNii Research

Language：Japanese   Publishing type：Research paper (scientific journal)  

Background: We aim to investigate the utility of serial gene mutation tracking for locally advanced CRC in those who underwent curative resection following neoadjuvant chemotherapy.

Methods: We prospectively collected 10 locally advanced CRC cases for which curative resection was performed following preoperative neoadjuvant chemotherapy. Tissues from the primary tumour, distant metastatic tumours, and blood plasma were obtained during serial treatment. Comprehensive mutation analysis of 47 cancer-associated genes was performed using a pre-designed gene panel and next-generation sequencing.

Results: All cases showed a partial response to neoadjuvant chemotherapy, and pathological R0 resection was accomplished. In primary tumours, non-synonymous mutations were detected at between 1 and 14 sites before chemotherapy and at between 1 and 2 sites after. Founder mutations were precisely detected in blood plasma and metastatic tumours during longitudinal treatment.

Conclusions: Serial mutational analysis indicated that subclonal selection occurs during chemotherapy and that plasma can substitute for tumourous tissue in mutational analysis for drug selection and treatment decisions.

DOI： 10.1038/s41416-018-0208-5.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Advanced colorectal cancer harbors extensive intratumor heterogeneity shaped by neutral evolution; however, intratumor heterogeneity in colorectal precancerous lesions has been poorly studied. We perform multiregion whole-exome sequencing on ten early colorectal tumors, which contained adenoma and carcinoma in situ. By comparing with sequencing data from advanced colorectal tumors, we show that the early tumors accumulate a higher proportion of subclonal driver mutations than the advanced tumors, which is highlighted by subclonal mutations in KRAS and APC. We also demonstrate that variant allele frequencies of subclonal mutations tend to be higher in early tumors, suggesting that the subclonal mutations are subject to selective sweep in early tumorigenesis while neutral evolution is dominant in advanced ones. This study establishes that the evolutionary principle underlying intratumor heterogeneity shifts from Darwinian to neutral evolution during colorectal tumor progression.

DOI： 10.1038/s41467-018-05226-0.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Objective: The involvement of epithelial-mesenchymal transition (EMT) in esophageal squamous cell carcinoma (ESCC) has not been fully elucidated. Here, we aimed to identify EMT-related genes associated with TGF-β in ESCC and to clarify the role of these genes in the progression of ESCC.

Methods: EMT-related genes associated with TGF-β expression were identified in patients with ESCC using microarray analysis and public datasets. The effects of ubiquitin-like with PHD and ring finger domains 2 (UHRF2) expression were analyzed in ESCC cell lines. Cell proliferation and invasion were measured using MTT and invasion assays, respectively. UHRF2 mRNA expression was also analyzed in 75 ESCC specimens to determine the clinical significance of UHRF2 in ESCC.

Results: Treatment of ESCC cell lines with TGF-β increased UHRF2 expression. UHRF2 overexpression increased CDH1 (E-cadherin) expression and decreased invasive capacity. The 75 ESCC specimens were divided into the UHRF2 high-expression group (n = 61) and the UHRF2 low-expression group (n = 14). Low UHRF2 expression was significantly correlated with vascular invasion (p = 0.034) and was an independent prognostic factor for poor prognosis (p = 0.005).

Conclusion: UHRF2 may be a negative regulator of EMT and a novel prognostic biomarker for ESCC.

DOI： 10.1159/000488860.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Background/aim: SLC9A9 plays an oncogenic role in esophageal squamous carcinoma and glioblastoma. Herein, we showed an oncogenic function of SLC9A9 in colorectal cancer (CRC).

Materials and methods: We examined SLC9A9 expression in CRC specimens by immunohistochemistry. In CRC tissues, the relationship between SLC9A9 expression and clinicopathological factors was further elucidated by quantitative real-time polymerase chain reaction (qRT-PCR) and gene set enrichment analysis (GSEA). In vitro, we performed knockdown and overexpression experiments.

Results: SLC9A9 was overexpressed in CRC specimens. In clinicopathological analysis of our cohort, high SLC9A9 expression increased liver metastasis and was correlated with worse prognoses in two cohorts. A significantly positive relationship between SLC9A9 and EGFR was revealed. While knockdown of SLC9A9 suppressed proliferation and anchorage-independent growth, up-regulation of SLC9A9 promoted proliferation and anchorage-independent growth in vitro.

Conclusion: SLC9A9 has an oncogenic function by being related to EGFR signaling, suggesting SLC9A9 may be a novel prognostic indicator and a therapeutic target in CRC.

DOI： 10.21873/anticanres.11562.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Background/aim: Amplification of chromosome 7p (Ch.7p) is common in colorectal cancer (CRC). The aim of this study was to identify potential driver genes on Ch.7p that are overexpressed due to DNA copy number amplification and determine their clinical significance in CRC.

Materials and methods: We identified phosphoserine phosphatase (PSPH) as a potential driver gene using a CRC dataset from The Cancer Genome Atlas (TCGA) using a bioinformatics approach. The expression of PSPH in 124 primary CRCs was examined by quantitative reverse transcription polymerase chain reaction (PCR) and immunohistochemistry. The biological effect of PSPH expression was explored by Gene Set Enrichment Analysis (GSEA) using the TCGA dataset.

Results: PSPH was overexpressed in tumor tissues and PSPH positively correlated with depth of invasion and distant metastasis. On multivariate analysis, high PSPH expression was an independent poor prognostic factor. These results were supported by GSEA.

Conclusion: PSPH could be a novel prognostic biomarker with malignant potential on Ch.7p in CRC.

DOI： 10.21873/anticanres.11574.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Objective: MOB1, a core component of the Hippo signaling pathway, suppresses cell proliferation, and MOB1 liver conditional knockout mice develop intrahepatic cholangiocarcinoma (ICC). However, its clinical significance in human ICC has not been established. The aim of this study was to characterize protein levels and the role of Hippo and TGF pathways in ICCs.

Methods: The protein levels of yes-associated protein 1 (YAP1), MOB1, Smad2, and TGFβ2 in 88 ICC cases were analyzed. Protein level was graded by a scoring system; then, the clinicopathological factors, including prognosis, were analyzed based on protein level.

Results: Nuclear overexpression of YAP1 was seen in 28 cases (31.8%), and it was significantly associated with a poor overall survival rate (p = 0.01). MOB1 expression decreased in 42 cases (47.7%) and was associated with a poor overall survival rate (p = 0.02). SMAD2 nuclear localization was significantly correlated with a high YAP1 level independent of TGFβ2. Multivariate analysis revealed that a high YAP1 level, a low MOB1 level, and lymphatic permeation were independent risk factors for overall survival.

Conclusions: These results showed that key components of the Hippo signaling pathway are aberrantly expressed and associated with the malignant potential of human ICC.

DOI： 10.1159/000463390.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Background: The RND1 gene encodes a protein that belongs to the Rho GTPase family, which regulates various cellular functions. Depletion of RND1 expression activates the oncogenic Ras signaling pathway. In this study, we aimed to clarify the clinical significance of RND1 expression in predicting prognosis and to investigate its biological role in human hepatocellular carcinoma (HCC).

Methods: The association between RND1 expression and clinical outcomes in patients with HCC was analyzed in three independent cohorts: 120 cases resected in our hospital; 370 cases in The Cancer Genome Atlas (TCGA); and 242 cases in GSE14520. Gene set enrichment analysis (GSEA) was also conducted. Finally, knockdown experiments were performed using small interfering RNA (siRNA) in vitro.

Results: In all cohorts, RND1 expression was decreased as cancer progressed, and was affected by promoter methylation. In our HCC cases, the 5-year overall survival (OS) and recurrence-free survival of patients with low RND1 expression was significantly poorer than those of patients with high RND1 expression. TCGA and GSE14520 analyses provided similar results for OS. Multivariate analysis indicated that RND1 expression was an independent prognostic factor for OS in all three cohorts. Additionally, GSEA showed an inverse correlation between RND1 expression and the Ras signaling activity. In vitro, knockdown of RND1 expression resulted in significant increases in proliferation, invasion, and chemoresistance to cisplatin in HCC cells.

Conclusions: Reduced RND1 expression in HCC was associated with cancer progression, likely through regulation of the Ras signaling pathway, and may serve as a novel clinical biomarker for predicting prognosis in patients with HCC.

DOI： 10.1245/s10434-016-5573-9.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Background: Hypermethylation of DNA silences gene expression and is an important event in colorectal cancer (CRC). This study aimed to identify aberrantly methylated genes that contribute to a poor prognosis for patients with CRC.

Methods: The study comprehensively explored DNA methylation microarray profiles from 396 CRC samples and 45 normal control samples in a database and selected aberrantly methylated transcription factors associated with prognosis and metastasis. Using quantitative reverse transcription polymerase chain reaction, the identified genes in 140 patients with CRC were validated to assess the relationship between expression of methylated genes and prognosis.

Results: In the study, FOXE1 was newly identified as a gene associated with prognosis and metastasis in CRC. Expression of FOXE1 in CRC tissues was significantly lower than in normal colorectal tissues (p = 0.01). The survival rate for the patients with low expression of FOXE1 was significantly lower than that for patients with high expression of FOXE1 in uni- and multivariate analyses. Inhibition of DNA methylation recovered FOXE1 expression in CRC cells.

Conclusions: Methylation-mediated silencing of FOXE1 expression was shown to be a potential prognostic factor in CRC.

DOI： 10.1245/s10434-016-5289-x.

Language：Japanese   Publishing type：Research paper (scientific journal)  

miR-146a plays important roles in cancer as it directly targets NUMB, an inhibitor of Notch signaling. miR-146a is reportedly regulated by a G>C polymorphism (SNP; rs2910164). This polymorphism affects various cancers, including colorectal cancer (CRC). However, the clinical significance of miR-146a polymorphism in CRC remains unclear. A total of 59 patients with CRC were divided into 2 groups: a CC/CG genotype (n = 32) and a GG genotype (n = 27), based on the miR-146a polymorphism. cDNA microarray analysis was performed using 59 clinical samples. Significantly enriched gene sets in each genotype were extracted using GSEA. We also investigated the association between miR-146a polymorphism and miR-146a, NUMB expression or migratory response in CRC cell lines. The CC/CG genotype was associated with significantly more synchronous liver metastasis (p = 0.007). A heat map of the two genotypes showed that the expression profiles were clearly stratified. GSEA indicated that Notch signaling and JAK/STAT3 signaling were significantly associated with the CC/CG genotype (p = 0.004 and p = 0.023, respectively). CRC cell lines with the pre-miR-146a/C revealed significantly higher miR-146a expression (p = 0.034) and higher NUMB expression and chemotactic activity. In CRC, miR-146a polymorphism is involved in liver metastasis. Identification of this polymorphism could be useful to identify patients with a high risk of liver metastasis in CRC.

DOI： 10.1371/journal.pone.0165912.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Objectives: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignancies owing to the high frequency of tumor recurrence. The identification of markers for early ESCC diagnosis and prediction of recurrence is expected to improve the long-term prognosis. Therefore, we searched for associations between tumor recurrence and cell-free DNA (cfDNA) mutations in blood plasma, which contains genetic markers for various cancer types.

Experimental design: Genomic DNA from tumors and cfDNA from plasma were obtained from 13 patients undergoing treatment for newly diagnosed ESCC. Next-generation sequencing of cfDNA in plasma was performed to identify mutations in 53 cancer-related genes, in which recurrent mutations were previously detected in ESCC. cfDNA mutational profiles were compared before and after tumor resection in four patients. Furthermore, somatic mutations in serial plasma samples were monitored after treatment in four patients.

Results: We identified multiple concordant somatic mutations in cfDNA and primary tumor samples from 10 patients (83.3%) and in cfDNA and metastatic tumor samples from one patient (100%). Furthermore, the allele frequency of the concordant mutations in cfDNA changed concomitantly with tumor burden and increased approximately 6 months earlier than the detection of tumor recurrences by imaging tests in two patients. Conventional biomarkers, such as SCC and p53-Ab, did not reflect tumor recurrences.

Conclusions: The present multigene panel, which enabled the diagnosis of tumor recurrence with greater accuracy than did using standard tumor markers or imaging methods, is expected to greatly facilitate standard, postoperative follow-up monitoring in ESCC.

DOI： 10.18632/oncotarget.11409

Language：Japanese   Publishing type：Research paper (scientific journal)  

Background: Here, we explored the genetic interactions between diabetes and oncogenic single-nucleotide polymorphisms (SNPs) that determine colorectal cancer (CRC) morbidity.

Methods: 8q24 rs6983267 polymorphism analysis and cDNA microarray were performed in 107 CRCs to identify the genes associated with diabetes and the oncogenic SNP. Then clinical significance of the gene was validated in 132 CRCs. Meta-analysis of microarray data and diabetic comorbidity was performed.

Results: Of genes associated with a minor SNP allele at 8q24, diabetes, and MYC overexpression, apolipoprotein A-IV (ApoA-IV) was associated with oncogenesis and poor prognosis in CRC patients. Patients with high ApoA-IV expression showed significantly poorer prognosis by univariate and multivariate analysis. Meta-analysis revealed lipid metabolism was associated with ApoA-IV-related oncogenesis in diabetic patients.

Conclusions: Changes in lipid metabolism associated with aberrant expression of ApoA-IV were risks for CRC oncogenesis.

DOI： 10.1245/s10434-016-5374-1.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Background/aim: Homeobox B7 (HOXB7) gene is involved in various cellular functions. We investigated the clinical significance of HOXB7 expression in hepatocellular carcinoma (HCC).

Materials and methods: HOXB7 mRNA expression in 103 HCC samples and 58 matched non-cancerous liver tissues were examined by quantitative real-time polymerase chain reaction (qRT-PCR). HOXB7 protein expression was also examined by immunohistochemistry. Gene set enrichment analysis (GSEA) was performed using a public dataset.

Results: HOXB7 expression was significantly higher in HCC tissues than in liver parenchyma. Ten-year overall survival (OS) and 5-year recurrence-free survival (RFS) of cases with higher HOXB7 expression were significantly poorer than those with lower HOXB7 expression. HOXB7 expression was significantly associated with larger tumor size and higher rate of biliary invasion and constituted an independent prognostic factor for OS by multivariate analysis. These results were supported by GSEA.

Conclusion: HOXB7 expression in HCC could be a novel biomarker for long-term prognosis after tumor resection.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Sentinel lymph node biopsy is performed as a standard procedure in breast cancer surgery, and the development of quick and simple methods to detect metastatic lesions is in high demand. Here, we validated a new fluorescent method using γ-glutamyl hydroxymethyl rhodamine green to diagnose metastatic lymph nodes in breast cancer. One hundred and forty-nine lymph nodes from 38 breast cancer patients were evaluated in this study. Comparison of fluorescent and pathological images showed that this fluorescent method was successful for visualizing breast cancer cells in lymph nodes. This method had a sufficiently high sensitivity (97%), specificity (79%) and negative predictive value (99%) to render it useful for an intraoperative diagnosis of cancer. These preliminary findings suggest that this novel method is useful for distinguishing non-cancerous specimens from those in need of careful examination and could help save time and cost for surgeons and pathologists.

DOI： doi: 10.1038/srep27525.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Fructose-1,6-bisphosphatase (FBP1), the rate-limiting enzyme in gluconeogenesis, is reduced in expression in certain cancers where it has been hypothesized to act as a tumor suppressor, including in hepatocellular carcinoma (HCC). Here, we report functional evidence supporting this hypothesis, providing a preclinical rationale to develop FBP1 as a therapeutic target for HCC treatment. Three independent cohorts totaling 594 cases of HCC were analyzed to address clinical significance. Lower FBP1 expression associated with advanced tumor stage, poor overall survival, and higher tumor recurrence rates. In HCC cell lines, where endogenous FBP1 expression is low, engineering its ectopic overexpression inhibited tumor growth and intracellular glucose uptake by reducing aerobic glycolysis. In patient specimens, promoter methylation and copy-number loss of FBP1 were independently associated with decreased FBP1 expression. Similarly, FBP1 downregulation in HCC cell lines was also associated with copy-number loss. HCC specimens exhibiting low expression of FBP1 had a highly malignant phenotype, including large tumor size, poor differentiation, impaired gluconeogenesis, and enhanced aerobic glycolysis. The effects of FBP1 expression on prognosis and glucose metabolism were confirmed by gene set enrichment analysis. Overall, our findings established that FBP1 downregulation in HCC contributed to tumor progression and poor prognosis by altering glucose metabolism, and they rationalize further study of FBP1 as a prognostic biomarker and therapeutic target in HCC patients. Cancer Res; 76(11); 3265-76. ©2016 AACR.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Background/aim: Defects in alternative splicing contribute to carcinogenesis, cancer progression and chemoresistance. The spliceosome pathway, including SF3B4, a component of spliceosomal complex is suggested to play a role in progression of hepatocellular carcinoma (HCC); however, the clinical relevance of SF3B4 in HCC remains unknown.

Patients and methods: SF3B4 expression was evaluated by real-time reverse transcription polymerase chain reaction in 72 HCC samples and non-cancerous liver samples. The relationship between the DNA copy number and SF3B4 expression levels was investigated using TCGA datasets.

Results: SF3B4 expression was significantly higher in cancerous than in non-cancerous tissues and positively correlated with SF3B4 DNA copy number. High SF3B4 expression is significantly associated with intrahepatic metastasis and poor prognosis. These results were consistent with data from the public datasets.

Conclusion: Overexpression of SF3B4, that is due to DNA copy number increase, is suggested to play a role in progression of HCC.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Recent studies have indicated that increased ribosomal activity contributes to cancer progression. Transcription termination factor, RNA polymerase I (TTF1) acts as a transcription factor for RNA polymerase I. However, the role which TTF1 plays in cancer progression still remains unknown. The present study aimed to determine whether TTF1 plays a critical role in the progression of human liver hepatocellular carcinoma (HCC). In the present study, quantitative real-time reverse transcription polymerase chain reaction was conducted to evaluate TTF1 mRNA expression in 60 HCC tissue samples in order to determine the clinicopathological significance of TTF1. To investigate whether the expression levels of TTF1 were associated known gene signatures which represented ribosomal activity, we applied gene set enrichment analysis (GSEA) to HCC cases in The Cancer Genome Atlas (TCGA) a. We also performed in vitro proliferation assays using TTF1‑overexpressing HCC cells. TTF1 expression was significantly higher in HCC tumor tissues than in adjacent liver tissues (P<0.001). The overall survival (OS) of patients with high TTF1 expression levels was significantly shorter than that of patients with low TTF1 expression (P=0.027). Multivariate analysis indicated that TTF1 expression was an independent prognostic factor for OS (P=0.020). GSEA revealed significant associations between TTF1 expression and gene sets involved in ribosomal function. In vitro, cell proliferation and rRNA transcription were significantly promoted by overexpression of TTF1 in the HCC cell lines HuH-7 and HepG2. From these results, it was suggested that TTF1 participate in poor prognoses and play a role in tumor cell growth in HCC, possibly by upregulating ribosomal activity. In conclusion, we first propose that TTF1 may be a novel biomarker and therapeutic target in HCC. Increased expression of TTF1 was significantly associated with poor prognosis in two independent sets of HCC cases. Furthermore, in vitro experiments provided an explanation for clinical data showing that overexpression of TTF1 contributed to the proliferation of cancer cells.

DOI： 10.3892/or.2016.4593.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Background: The nephroblastoma overexpressed (NOV) gene, which belongs to the cysteine-rich, angiogenic inducer 61/connective tissue growth factor/nephroblastoma overexpressed (CCN) family, is located in the 8q24 region and promotes migration and invasiveness in several types of malignancies. We explored the clinical significance of NOV expression in colorectal cancer (CRC).

Materials and methods: NOV expression in CRC specimens and CRC cell lines were evaluated. The association between the clinicopathlogical factors and NOV mRNA expression of tumor tissues was assessed in 126 patients with CRC. We assessed the relationships between NOV expression and overall survival in public databases. We performed overexpression experiments in vitro.

Results: CRC specimens and CRC cell lines showed high NOV expression. High NOV mRNA expression was correlated with poorer overall survival and higher Union for International Cancer Control (UICC) T factor. In public databases, high NOV expression was associated with poorer prognoses. Overexpression of NOV promoted invasiveness of CRC cells.

Conclusion: NOV may be an indicator of poor prognosis and a therapeutic target in CRC.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Background: A recent study reported that long non-coding RNA activated by TGF-β (lncRNA-ATB) induced epithelial-mesenchymal transition (EMT) through the transforming growth factor-β (TGF-β)/miR-200s/ZEB axis in hepatocellular carcinoma. Herein, we focused on the clinical significance of lncRNA-ATB in gastric cancer (GC) patients.

Materials and methods: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to examine expression of lncRNA-ATB, miR-200b, and miR-200c in GC tissues (n = 183). Patients were divided into high and low lncRNA-ATB expression groups using a cutoff of lncRNA-ATB/GAPDH ≥0.60 or <0.60 to determine the clinicopathological significance of lncRNA-ATB in GC. Moreover, we evaluated the expression of TGF-β, lncRNA-ATB, miR-200s, and ZEB1 in GC cell lines by qRT-PCR. GC cell lines were treated by recombinant TGF-β1 or TGF-β receptor inhibitor to examine morphologic changes and genetic alterations, such as lncRNA-ATB, miR-200s, and ZEB1 levels, with respect to the EMT phenotype.

Results: The high lncRNA-ATB group experienced a lower overall survival rate compared with the low lncRNA-ATB group, and multivariate analysis indicated that lncRNA-ATB was an independent prognostic factor (hazard ratio 3.50; 95 % CI 1.73-7.44; p = 0.0004). miR-200c levels were lower and ZEB1 levels were higher in the high lncRNA-ATB group than in the low lncRNA-ATB group. Treatment with TGF-β in GC cell lines resulted in morphological EMT changes, upregulation of lncRNA-ATB and ZEB1, and downregulation of miR-200c and CDH1. SB431542 reduced lncRNA-ATB expression.

Conclusion: LncRNA-ATB plays an important role in EMT to promote invasion and metastasis through the TGF-β/miR-200s/ZEB axis, resulting in a poor prognosis in GC. LncRNA-ATB is a novel biomarker of lncRNA, indicative of a poor prognosis in GC patients.

DOI： 10.1245/s10434-015-4554-8.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Background: Downregulation of paired related homeobox 1 (PRRX1) is associated with the acquisition of cancer stem cell (CSC)-like properties and poor prognosis in cancers. The purpose of this study is to clarify the role of PRRX1 expression in predicting prognosis and mediating CSC-like properties in hepatocellular carcinoma (HCC).

Methods: The association between PRRX1 expression and overall survival (OS) of patients with HCC was analyzed in three independent datasets: 62 resected primary cases, 242 cases from GSE14520, and 162 cases from The Cancer Genome Atlas (TCGA). A cell line expressing PRRX1 (HuH7) was established for the functional analyses. The ability to form spheres, the expression levels of the hepatic CSC surface markers (CD13, CD133, and EpCAM), in vitro chemosensitivity to 5-fluorouracil (FU), and radiosensitivity were evaluated.

Results: Univariate and multivariate analyses showed that the 5-year OS of the low PRRX1 expression group was significantly poorer than that of the high PRRX1 expression group (P = 0.024 and P = 0.045, respectively). Consistent with this, the low PRRX1 expression group in GSE14520 and TCGA datasets showed significantly shorter OS (P = 0.027 and P = 0.010, respectively). Gene set enrichment analysis on GSE14520 and TCGA datasets indicated that downregulation of PRRX1 was correlated with the stemness signature. The number of spheres and the expression levels of CSC markers were significantly decreased when PRRX1 was expressed. Moreover, PRRX1 impaired resistance to 5-FU and radiation.

Conclusions: Downregulation of PRRX1 expression contributes to the poor prognosis of patients with HCC through acquisition of CSC-like properties.

DOI： 10.1245/s10434-014-4242-0.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Background: RNA polymerase 1 transcription termination factor (TTF1) mediates the transcription of ribosomal RNA (rRNA). In the current study, we investigated the clinical and biological significance of the TTF1 gene in colorectal cancer (CRC).

Methods: The expression of TTF1 messenger RNA (mRNA) in tumor and normal tissues from 136 patients with CRC was examined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). We also performed in vitro cell proliferation and migration assays in TTF1-expressing CRC cells. The biological role of TTF1 in CRC was further elucidated using gene set enrichment analysis (GSEA) with CRC samples.

Results: TTF1 expression was significantly higher in tumor tissues than in corresponding normal tissues (p = 0.016). In clinicopathological analysis, the high-TTF1 expression group showed a higher incidence of liver metastasis and lymphatic invasion than the low-TTF1 expression group (p < 0.05), and tended to have more frequent venous invasion than the low-TTF1 expression group. Furthermore, the high-TTF1 expression group had a significantly poorer prognosis than the low-TTF1 expression group (p = 0.011). Moreover, overexpression of TTF1 enhanced the proliferation and migration capacity of CRC cells in vitro. GSEA revealed that TTF1 was significantly associated with the RAS and MYC pathways, and this observation was confirmed in samples from 136 patients with CRC.

Conclusion: TTF1 was involved in cancer progression via the RAS and MYC pathways in CRC, suggesting that TTF1 may be a prognostic indicator and therapeutic target in CRC.

DOI： doi: 10.1245/s10434-015-4652-7.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Background: Few driver genes have been well established in esophageal squamous cell carcinoma (ESCC). Identification of the genomic aberrations that contribute to changes in gene expression profiles can be used to predict driver genes.

Methods: We searched for driver genes in ESCC by integrative analysis of gene expression microarray profiles and copy number data. To narrow down candidate genes, we performed survival analysis on expression data and tested the genetic vulnerability of each genes using public RNAi screening data. We confirmed the results by performing RNAi experiments and evaluating the clinical relevance of candidate genes in an independent ESCC cohort.

Results: We found 10 significantly recurrent copy number alterations accompanying gene expression changes, including loci 11q13.2, 7p11.2, 3q26.33, and 17q12, which harbored CCND1, EGFR, SOX2, and ERBB2, respectively. Analysis of survival data and RNAi screening data suggested that GRB7, located on 17q12, was a driver gene in ESCC. In ESCC cell lines harboring 17q12 amplification, knockdown of GRB7 reduced the proliferation, migration, and invasion capacities of cells. Moreover, siRNA targeting GRB7 had a synergistic inhibitory effect when combined with trastuzumab, an anti-ERBB2 antibody. Survival analysis of the independent cohort also showed that high GRB7 expression was associated with poor prognosis in ESCC.

Conclusion: Our integrative analysis provided important insights into ESCC pathogenesis. We identified GRB7 as a novel ESCC driver gene and potential new therapeutic target.

DOI： 10.1371/journal.pone.0139808.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Hepatocellular carcinoma (HCC) is a poor-prognosis cancer due to its high rate of recurrence. microRNAs (miRNAs) are a class of small non-coding RNA molecules that affect crucial processes in cancer development. The objective of this study is to identify the role of miRNAs in patient bone marrow (BM) and explore the function of these molecules during HCC progression. We purified miRNAs from bone marrow cells of seven HCC patients, and divided them into three fractions by cell surface markers as follows: CD14(+) (macrophage), CD14(-)/CD45(+) (lymphocyte), and CD14(-)/CD45(-)/EpCAM(+) (epithelial cell). We employed microarray-based profiling to analyze miRNA expression in the bone marrow of patients with HCC. Differentially expressed miRNAs were significantly different between fractions from whole bone marrow, macrophages, and lymphocytes, and depended on stages in tumor progression. Differences in expression of miRNAs associated with cell proliferation also varied significantly between HCC patients with recurrence, multiple tumors, and advanced clinical stages. These results suggest that miRNA profiles in separated fractions of BM cells are associated with HCC progression.

DOI： 10.3390/jcm4081600.

Language：Japanese   Publishing type：Research paper (scientific journal)  

MicroRNA-29b (miR-29b) targets numerous important genes that mediate carcinogenesis and tumor development in breast cancer in vitro and in vivo. The aim of the present study was to determine the clinical significance of miR-29b expression in primary breast cancer patients. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) of miR-29b and certain target genes of miR-29b, such as DNA methyltransferase 3A (DNMT3A), ten-eleven translocation 1 (TET1) and thymine DNA glycosylase (TDG), was performed in 94 primary breast cancer samples. Low expression of miR-29b in primary tumors was significantly associated with poorer disease-free survival (DFS) (P=0.0075) and overall survival (OS) (p=0.0012). Multivariate analysis indicated that miR-29b expression was an independent prognostic factor for OS [relative risk=15.6 (2.33-348), P=0.0026]. In addition, a significant inverse correlation was identified between the expression levels of DNMT3A and miR-29b in estrogen receptor-positive breast cancer patients (P=0.027). To the best of our knowledge, this is the first study to investigate the clinicopathological significance of miR-29b in breast cancer cases and miR-29b is shown to act as a tumor suppressive microRNA in breast cancer and as a potential marker for recurrence and metastasis in breast cancer patients.

DOI： 10.3892/mco.2015.565.

Language：Japanese   Publishing type：Research paper (scientific journal)  

We previously developed γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) as a tool to detect viable cancer cells, based on the fact that the enzyme γ-glutamyltranspeptidase (GGT) is overexpressed on membranes of various cancer cells, but is not expressed in normal tissue. Cleavage of the probe by GGT generates green fluorescence. Here, we examined the feasibility of clinical application of gGlu-HMRG during breast-conserving surgery. We found that fluorescence derived from cleavage of gGlu-HMRG allowed easy discrimination of breast tumors, even those smaller than 1 mm in size, from normal mammary gland tissues, with 92% sensitivity and 94% specificity, within only 5 min after application. We believe this rapid, low-cost method represents a breakthrough in intraoperative margin assessment during breast-conserving surgery.

DOI： 10.1038/srep12080.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Background/aim: A long noncoding RNA (lncRNA) activated by transforming growth factor (TGF)-β (lncRNA-ATB) was recently described to promote the invasion-metastasis cascade in hepatocellular carcinoma. The aim of the present study was to clarify the clinicopathological role and prognostic relevance of lncRNA-ATB in colorectal cancer (CRC).

Materials and methods: lncRNA-ATB expression was evaluated by real-time reverse transcription polymerase chain reaction in 124 patients with CRC. Patients were divided into two groups based on the median lncRNA-ATB expression.

Results: High lncRNA-ATB expression was significantly associated with greater tumor size, depth of tumor invasion, lymphatic invasion, vascular invasion, and lymph node metastasis. Patients of the high-lncRNA-ATB expression group had significantly poorer outcomes than those of the low-expression group. Additionally, levels of lncRNA-ATB expression were significantly higher in patients with hematogenous metastases.

Conclusion: lncRNA-ATB may be involved in the progression of CRC and be a novel indicator of poor prognosis in patients with CRC.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Background/aim: MiR-15a targets Cyclin E1 (CCNE1), which regulates the cell cycle and promotes cell proliferation and progression. Herein, we investigated the clinicopathological significance of miR-15a as a prognostic marker in breast cancer (BC) cases.

Materials and methods: We collected primary tumor samples of 230 BC cases, including 68 triple-negative cases. The expression levels of miR-15a in primary tumors were measured by qRT-PCR assay.

Results: Low expression of miR-15a in primary tumors was significantly correlated with shorter disease-free survival (p=0.0012) and overall survival (p=0.005) compared to the high miR-15a expression in triple-negative BC cases. Multivariate analysis indicated that low miR-15a expression was an independent prognostic factor for overall survival [RR=2.56(1.03-7.18), p=0.04].

Conclusion: MiR-15a expression levels could be a promising biological and prognostic marker for overall survival especially in triple-negative BC cases.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Background: Human CDCP1 gene, located on chromosome 3p21.3, is a transmembrane glycoprotein widely expressed in epithelial tissues, and its role in cancer remains to be understood.

Methods: Using microarray profiles of gene expression and copy number data from 69 esophageal squamous cell carcinoma (ESCC) samples, we performed informatics analyses to reveal the significance of CDCP1 expression. We also performed migration and invasion assays of siRNA-targeted CDCP1-transfected cells and CDCP1-overexpressing cell in vitro. Moreover, we evaluated the clinical magnitude of CDCP1 expression in esophageal squamous cell cancer cases.

Results: Allelic loss of chromosome 3p was confirmed by copy number analysis. The expression level of CDCP1 in tumor tissue was significantly lower than that in corresponding normal tissue. siRNA targeting of CDCP1 promoted the migratory and invasive abilities of esophageal cancer cell lines, whereas both abilities were reduced in CDCP1-overexpressing cells. Gene set enrichment analysis showed that expression levels of CDCP1 were associated with tumor differentiation and metastasis, consistent with the result of clinicopathologic analyses. Finally, multivariate analysis revealed that the expression level of CDCP1 was an independent prognostic factor for survival.

Conclusions: Loss of CDCP1 expression may be a novel indicator for biological aggressiveness in ESCC.

DOI： 10.1245/s10434-014-3740-4.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Purpose: Recent studies indicated that the scaffolding adaptor protein GAB2 (GRB2-associated binding protein 2) plays a critical role in the proliferation and migration of various cancers. This study aimed to determine the role of aberrant GAB2 expression in human colorectal cancer (CRC).

Methods: Quantitative real-time reverse transcription polymerase chain reaction was used to evaluate GAB2 mRNA expression in 152 CRC tissues samples to determine the clinicopathological significance of GAB2 expression. We also performed in vitro proliferation assays using siGAB2-transfected CRC cells.

Results: GAB2 expression in tumor colorectal tissues was significantly higher than in normal colorectal tissues (p = 0.0212). High GAB2 expression levels were associated with malignant clinicopathologic potential factors, including lymphatic invasion (p = 0.0003), venous invasion (p = 0.0170), and liver metastasis (p = 0.0144). The survival rate of patients with high GAB2 expression levels was significantly lower than that of patients with low GAB2 expression (p = 0.0074). Multivariate analysis indicated that GAB2 expression was a factor affecting lymph node metastasis. Cell proliferation was significantly suppressed by siGAB2 expression in CRC cells in vitro.

Conclusions: GAB2 expression was associated with lymph node metastasis and may play a role in the growth and metastasis of CRC. These results suggest that GAB2 is a potential therapeutic target in CRC.

DOI： 10.1245/s10434-014-3889-x.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Background: High mobility group 1 (HMGB1) is a highly conserved non-histone nucleosomal protein in mammals. We investigated the clinical significance of HMGB1 expression in colorectal cancer (CRC).

Patients and methods: The expression of HMGB1 mRNA in 140 tumor and normal tissues from CRC patients was examined by quantitative real-time polymerase chain reaction (PCR). We immunohistochemically investigated HMGB1 expression in tumor and metastatic lymph nodes in CRC.

Results: HMGB1 expression was significantly higher in tumor than in normal tissues. High HMGB1 expression was associated with larger tumor volumes, higher rates of lymphatic invasion, more frequent lymph node metastases and poorer prognoses for overall survival. Multivariate analyses showed that HMGB1 expression was an independent prognostic indicator of overall survival. Immunocytochemical analysis revealed that HMGB1 was overexpressed in both CRC tissues and regional lymph node metastases.

Conclusion: Investigating HMGB1 expression may be a predictor of postoperative lymph node metastasis and prognosis in CRC.

Language：Japanese   Publishing type：Research paper (scientific journal)  

Event date： 2024.2

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2023.9

Language：Japanese  

Venue：奈良   Country：Japan  

Event date： 2023.9

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2023.9

Language：Japanese  

Venue：福岡   Country：Japan  

Event date： 2023.3

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2022.5

Language：Japanese  

Venue：長崎   Country：Japan  

Event date： 2021.2

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2021.2

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2017.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：大分   Country：Japan  

【背景】近赤外線酸素モニター（NIRS）は連続的かつ非侵襲的に装着部位直下の組織内酸素需給バランスが予測可能なため、頭部に装着することで脳低灌流の早期に発見に有用であると考えられている。しかし今回、NIRSを前額部に装着しモニタリングしたにもかかわらず広範な脳梗塞を予測できなかった症例を経験したので報告する。
【症例】６５歳、男性。１７２cm、８６kg。胸背部痛、左下肢脱力を主訴とする上行大動脈から右総腸骨動脈まで大動脈解離に対して、緊急での弓部置換術の方針となった。術前のCT検査で右総頚動脈まで解離が進行し、意識レベルの低下が進行してきたため、麻酔導入後に両側前額部にNIRSを装着し脳の脳局所酸素飽和度（rSO2）をモニタリングしたが値に左右差なく、術中も大きな変化なく40~50を推移した。また右橈骨動脈と左浅側頭動脈でモニタリングした観血的動脈圧からも脳低灌流を疑う所見はなかった。しかしICUで鎮静薬の投与を中止したところ患者の覚醒が悪く、術後２日目のCT検査で右中大脳動脈灌流領域の広範な脳梗塞と脳浮腫所見を認めたため緊急で減圧開頭術が行われた。
【考察】NIRSはセンサー装着部直下の状況を反映するため、前額部に装着した場合は脳組織の一部しか灌流状態を評価することができない。本症例ではrSO2をモニタリングできていたと思われる部位はCT上で脳梗塞を発症しておらず、おそらく左内頚動脈から前交通動脈を介して灌流されていた可能性が考えられる。そのため本症例のように内頚動脈由来の血流を評価したい場合はセンサー装着部位を側頭部に移動させるなどの工夫が必要になる。
【まとめ】NIRSにより広範な脳梗塞を予測できなかった症例を経験した。NIRSは低侵襲で有用なモニターであるものの評価可能な範囲は限定的であり、そのことを十分理解した上で使用しなければならない。

Event date： 2017.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：大分   Country：Japan  

【はじめに】心臓血管外科手術において経食道心エコー（TEE）は必要不可欠であり、術中管理や修復後の評価、術式変更の考慮に非常に有用である。今回、人工心肺離脱後の低血圧の原因検索、その後の術式追加にTEEが非常に有用であった症例を経験したので報告する。
【症例】６５歳、男性。１７２cm、８６kg。胸背部痛、左下肢脱力を主訴に当院救急搬送され、CT検査で上行大動脈から右総腸骨動脈まで大動脈解離を認め、右内頚動脈の解離進展に伴う意識障害が進行してきたため、緊急での弓部置換術の方針となった。
【麻酔経過】一般的なモニタリングに加え、観血的動脈圧、中心静脈酸素飽和度、前額部の局所組織酸素飽和度、TEEをモニタリングした。両側腋窩動脈、左大腿動脈送血、右房脱血で人工心肺を開始し、冷却後に循環停止、選択的脳分離循環下に弓部置換を行った。人工心肺離脱後、循環動態に問題がなかったためプロタミンを投与したところ、投与終了５分後より収縮期血圧50mmHg台の低血圧、完全房室ブロックを認めた。再ヘパリン化を行い、人工心肺を再開しつつTEEで観察したところ、前壁中隔から側壁の一部にかけての無収縮、大動脈解離のフラップによる左冠動脈主幹部閉塞と左前下行枝の血流途絶を認めた。心臓外科医と協議し大伏在静脈を用いた左冠動脈前下行枝の冠動脈バイパスを追加した。左冠動脈前下行枝と左冠動脈回旋枝の血流再開とともに、徐々に心筋の壁運動異常は改善した。循環動態が落ち着いた後に再度人工心肺から離脱し、その後は大きな問題なく手術を終了した。
【まとめ】人工心肺離脱後に大動脈解離のフラップによる左冠動脈急性閉塞を起こした症例を経験した。大動脈解離に対する弓部置換術においては人工心肺離脱後にもこのような冠動脈閉塞をきたすことがあり、またプロタミン投与後の低血圧に対する原因検索にTEEが非常に有用であった。

Event date： 2017.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：大分   Country：Japan  

【背景】血中乳酸値は、酸素需給バランスの評価や全身性の組織代謝失調の検出に広く用いられている。しかし、周術期に高乳酸血症を来す原因の鑑別は容易ではないことがある。今回頸部手術中に進行性の高乳酸血症を来した症例を経験したので報告する。
【症例】68歳、男性。169cm、85kg。左頸部リンパ節転移を伴う甲状腺癌と右耳下腺のWarthin腫瘍に対して、甲状腺全摘、左頸部リンパ郭清及び右耳下腺腫瘍摘出が予定された。既往として高血圧、糖尿病があり内服加療されていた。術前検査では運動誘発性冠動脈攣縮も疑われていたが、概ね心機能は良好であった。
【麻酔経過】プロポフォールとレミフェンタニルを用いた全静脈麻酔で管理し、観血的動脈圧をモニターすることとした。術中に大きな循環変動は無かったが、動脈血ガス分析で進行する高乳酸血症を認め、導入 10 時間後にはpH 7.300、BE -6.7 mmol/L、乳酸値 61.0 mg/dl となった。薬剤性高乳酸血症を疑い、疑われる原因薬剤の一つであるプロポフォールを中止しデスフルランに変更した。その後動脈血ガス所見は改善を認め、退室時の乳酸値は37.0 mg/dl であった。
【考察】周術期の高乳酸血症は、酸素需給バランスの破綻によるものが多いと思われるが、それ以外の様々な原因により起こりうる。中でも薬剤性にミトコンドリア機能が障害されて起こるものは、細胞レベルでの酸素利用障害を伴っているため注意が必要である。本症例では循環不全を疑う所見が無いため薬剤性を疑い、プロポフォールの投与を中止という対応をした。
【まとめ】薬剤性が疑われる高乳酸血症を来した症例を経験した。術中使用という比較的短時間の投与でも、プロポフォールが乳酸アシドーシスを惹起しうる可能性があり注意が必要である。

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Language：Japanese   Publishing type：Internal/External technical report, pre-print, etc.  

65歳男性。Stanford A型急性大動脈解離に対し緊急に弓部人工血管置換術を行った。人工心肺は問題なく離脱したが、プロタミン緩徐静注開始約8分後(人工心肺離脱約10分後)に血圧が突然低下した。経食道心エコー(TEE)を行い、解離フラップによる急性冠動脈閉塞と診断した。左大伏在静脈を用いた左冠動脈前下行枝の肝動脈バイパス術を行い、再TEEで左冠動脈の血流を確認した。TEEが人工心肺離脱後の急性冠動脈閉塞の診断に有用であった。

Language：Japanese   Publishing type：Internal/External technical report, pre-print, etc.  

65歳男。胸背部痛と左下肢の脱力感を主訴に救急搬送され、Stanford A型急性大動脈解離の診断で弓部大動脈置換術を施行した。術後1日目に鎮痛薬を中止したが覚醒不良で、2日目に頭部CTを施行したところ右中大脳動脈領域に広範なlow density areaと左への著明なmidline shiftを認めた。緊急開頭減圧術を施行し、その後の経過は良好であった。本例は術中に近赤外線脳酸素モニター(NIRS)を装着していたが、rSO2の大きな変動はみられなかった。術後2日目の頭部CTで右の前頭葉に一部脳梗塞を逃れた領域を認め、この領域は、右内頸動脈解離に伴う右中大脳動脈の低灌流により左前大脳動脈から前交通動脈を介して灌流された可能性がある。この脳特有の血流支配のために、NIRS装着部位直下の血流はある程度保たれていたことで、実際に脳梗塞を起こしていた部分の低灌流を正確にモニタリングできていなかったものと考えられた。

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publishing type：Internal/External technical report, pre-print, etc.  

81歳女。大動脈弁狭窄兼閉鎖不全症、僧帽弁閉鎖不全症の既往があり、保存的治療をしていた。突然誘因なく腹痛をきたし、救急搬送された。搬送時ショック状態で、CTにて脾出血と診断されたが、高齢、既往歴などの理由により侵襲的治療不能と判断した。加療後も出血コントロール困難であった。心エコードップラー所見は大動脈弁閉鎖不全症(AR)中等度、僧帽弁閉鎖不全症(MR)高度で、腹部造影CT検査は脾臓の腫大を認め、破裂に伴った出血を疑った。拡張した静脈を有する領域の栄養動脈に選択的にマイクロコイル、脾動脈本幹にスポンゼルにてTAEを施行した。TAE施行2日後に確定診断、出血コントロールのため脾臓摘出術を施行した。術後心不全、肺水腫を発症したが、保存的加療にて軽快した。術後26日目に肺炎球菌ワクチンを接種した。全身状態は良好に回復し、術後41日目に転院した。

Language：Japanese   Publishing type：Internal/External technical report, pre-print, etc.  

症例は29歳女性で、出生直後に臍帯の止血困難を認め、先天性無フィブリノゲン血症と診断された。24歳時より卵巣出血予防、妊娠目的にフィブリノゲン(Fbg)製剤投与下に妊娠が成立し、右卵管楔状切除術既往があるため、妊娠38週2日に選択的帝王切開が予定された。プロトロンビン時間(PT)、活性化部分トロンボプラスチン時間(APTT)は正常で、臨床的出血傾向もなかった。麻酔は全身麻酔で行い、チアミラール、スキサメトニウムで導入し、気管挿管後、亜酸化窒素、酸素で維持し、児娩出後、ペンタゾシン、ベクロニウムを投与した。手術直後の改良型トロンボエラストメトリ(ROTEM)は正常であった。術後はFbg製剤を投与し血漿Fbg値80mg/dl前後とやや低めに推移したが術後出血所見はなかった。術後4日目のROTEMのMLが35%と全身性の線溶系亢進を示唆する所見であったが、臨床的には血栓症や出血傾向などの所見はなく、術後6日目の検査ではこの所見も改善し退院した。