High prevalence of vitamin B-12 deficiency before and early after gastrectomy in patients with gastric cancer.

BACKGROUND AND OBJECTIVES: Gastrectomy causes vitamin B-12 deficiency since vitamin B-12 requires gastric acid and intrinsic factor for its absorption. Vitamin B-12 deficiency is considered to develop years after gastrectomy because of large hepatic storage. However, most gastric cancer develops after long-standing atrophic gastritis with vitamin B-12 malabsorption. METHODS AND STUDY DESIGN: We have investigated vita-min B-12 status in 22 patients before gastrectomy and 53 patients after gastrectomy due to gastric cancer, also with consideration on post-gastrectomy anemia. RESULTS: Blood vitamin B-12, folic acid, homocysteine concentrations, parameters of anemia, and dietary intake were evaluated. Percentage of patients with severe vitamin B-12 deficiency (serum vitamin B-12 < 150 pmol/L), vitamin B-12 deficiency (150 pmol/L to < 258 pmol/L) was 19.0 %, and 52.4 % respectively in patients gastrectomized within three years. Before gastrectomy, three and seven patients exhibited severe deficiency and deficiency, respectively. In gastrectomized patients, plasma homocysteine concentration was inversely associated with serum vitamin B-12 concentration, and vitamin B-12 deficiency- and iron deficiency- anemia coexisted with their mean corpuscular volume within the reference range. CONCLUSIONS: Vitamin B-12 deficiency is prevalent in patients early after and before gastrectomy. Coexistence of vitamin B-12 and iron deficiency obscures the diagnosis of post-gastrectomy anemia, and necessitates the blood vitamin B-12 measurement.

Establishment of practical recellularized liver graft for blood perfusion using primary rat hepatocytes and liver sinusoidal endothelial cells.

Tissue decellularization produces a three-dimensional scaffold that can be used to fabricate functional liver grafts following recellularization. Inappropriate cell distribution and clotting during blood perfusion hinder the practical use of recellularized livers. Here we aimed to establish a seeding method for the optimal distribution of parenchymal and endothelial cells, and to evaluate the effect of liver sinusoidal endothelial cells (LSECs) in the decellularized liver. Primary rat hepatocytes and LSECs were seeded into decellularized whole-liver scaffolds via the biliary duct and portal vein, respectively. Biliary duct seeding provided appropriate hepatocyte distribution into the parenchymal space, and portal vein-seeded LSECs simultaneously lined the portal lumen, thereby maintaining function and morphology. Hepatocytes co-seeded with LSECs retained their function compared with those seeded alone. Platelet deposition was significantly decreased and hepatocyte viability was maintained in the co-seeded group after extracorporeal blood perfusion. In conclusion, our seeding method provided optimal cell distribution into the parenchyma and vasculature according to the three-dimensional structure of the decellularized liver. LSECs maintained hepatic function, and supported hepatocyte viability under blood perfusion in the engineered liver graft owing to their antithrombogenicity. This recellularization procedure could help produce practical liver grafts with blood perfusion.

Analysis of Deep Posterior Anal Fistulas by Magnetic Resonance Imaging: Site of Primary Abscess and Extension Patterns According to the Primary Abscess Depth.

OBJECTIVES: The aim of this study was to use magnetic resonance imaging (MRI) to elucidate the site and depth of the primary abscesses associated with deep posterior anal fistulas and their extension patterns. METHODS: We analyzed 176 consecutive patients with deep posterior anal fistulas and classified the fistulas according to whether the MRI-detected site of the primary abscess was at a superficial or a deep external anal sphincter (EAS) level. RESULTS: The distance between the anal center and the primary abscess center was significantly shorter than the length of the EAS and radius at an angle of 45°. In addition, deep posterior anal fistulas with primary abscesses located at the deep EAS level penetrated the EAS significantly more laterally and made external openings at a significantly more lateral site than when the primary abscess was located at a superficial EAS level. CONCLUSIONS: Primary abscesses associated with deep posterior anal fistulas are located in the posterior intersphincteric space or in the EAS muscle itself, not in Courtney's space, as had previously been claimed.

Identification of keratin 19-positive cancer stem cells associating human hepatocellular carcinoma using CYFRA 21-1.

The current lack of an easily measurable surrogate marker of cancer stem cells (CSCs) prevents the clinical application of CSCs for hepatocellular carcinoma (HCC). We previously reported that keratin 19 (K19) is a novel HCC-CSC marker associated with transforming growth factor beta (TGFß)/Smad signaling, and that K19+ HCC-CSCs could be a new therapeutic target of TGFß receptor 1 inhibitor LY2157299. In this study, we examined whether K19+ HCC-CSCs can be tracked using cytokeratin 19 fragment CYFRA 21-1. In 147 HCC patients who underwent curative resection and evaluated K19 expression by immunohistochemistry, preoperative serum CYFRA 21-1 levels were significantly higher in K19+ patients than in K19- patients (P < 0.01). Receiver operating characteristic analyses revealed that serum CYFRA 21-1 was the statistically significant and the most sensitive predictor of tumor K19 expression among preoperative laboratory test values (P < 0.001). In HCC cells encoding with a K19 promoter-driven enhanced green fluorescent protein, fluorescence-activated cell sorting (FACS)-isolated K19+ cells displayed significantly higher levels of supernatant CYFRA 21-1 than K19- cells (P < 0.01). Gain/loss of K19 function experiments confirmed that CYFRA 21-1 levels were regulated by K19 function in HCC cells. Furthermore, CYFRA 21-1 levels reflected the treatment efficacy of LY2157299 in K19+ cells. In conclusion, CYFRA 21-1 can be used to predict K19 expression in HCC, and should thereby aid in the development of novel therapeutic strategies targeting K19+ HCC-CSCs.

A novel three-dimensional culture system maintaining the physiological extracellular matrix of fibrotic model livers accelerates progression of hepatocellular carcinoma cells.

Liver fibrosis is characterized by the progressive accumulation of extracellular matrix (ECM) and is a strong predictor of hepatocellular carcinoma (HCC) development and progression. However, the effect of ECM in fibrotic livers on HCC cells is poorly understood. The aims of this study were to create a new culture system that retained the natural ECM of fibrotic model livers and to establish whether natural ECM regulated the characteristics of HCC cells. Using an organ decellularization technique, we created a new culture system that preserved the tissue-specific ECM of fibrotic model livers from CCl4-treated rats. The content of ECM in fibrotic model liver scaffolds was increased and the ECM microstructure was distorted. Quantitative polymerase chain reaction and immunofluorescence assays of HCC cells cultured in fibrotic model liver scaffolds for 7 days showed an epithelial-mesenchymal transition phenotype. Moreover, the ECM of fibrotic model livers promoted proliferation and chemoresistance of HCC cells. These results showed a novel effect of natural ECM in fibrotic model livers on the malignant behaviour of HCC cells. This new culture system will be useful for both understanding the cell biology of fibrotic livers and developing novel anti-cancer drugs.

Generation of non-viral, transgene-free hepatocyte like cells with piggyBac transposon.

Somatic cells can be reprogrammed to induced hepatocyte-like cells (iHeps) by overexpressing certain defined factors in direct reprogramming techniques. Of the various methods to deliver genes into cells, typically used genome-integrating viral vectors are associated with integration-related adverse events such as mutagenesis, whereas non-integrating viral vectors have low efficiency, making viral vectors unsuitable for clinical application. Therefore, we focused on developing a transposon system to establish a non-viral reprogramming method. Transposons are unique DNA elements that can be integrated into and removed from chromosomes. PiggyBac, a type of transposon, has high transduction efficiency and cargo capacity, and the integrated transgene can be precisely excised in the presence of transposase. This feature enables the piggyBac vector to achieve efficient transgene expression and a transgene-free state, thus making it a promising method for cell reprogramming. Here, we attempted to utilize the piggyBac transposon system to generate iHeps by integrating a transgene consisting of Hnf4a and Foxa3, and successfully obtained functional iHeps. We then demonstrated removal of the transgene to obtain transgene-free iHeps, which still maintained hepatocyte functions. This non-viral, transgene-free reprogramming method using the piggyBac vector may facilitate clinical applications of iHeps in upcoming cell therapy.

Identification of Keratin 19-Positive Cancer Stem Cells Associating Human Hepatocellular Carcinoma Using 18F-Fluorodeoxyglucose Positron Emission Tomography.

Purpose: The current lack of tools for easy assessment of cancer stem cells (CSC) prevents the development of therapeutic strategies for hepatocellular carcinoma (HCC). We previously reported that keratin 19 (K19) is a novel HCC-CSC marker and that PET with 18F-fluorodeoxyglucose (18F-FDG) is an effective method for predicting postoperative outcome in hepatocellular carcinoma. Herein, we examined whether K19+ HCC-CSCs can be tracked using 18F-FDG-PET.Experimental Design: K19 and glucose transporter-1 (GLUT1) expression was evaluated by IHC in 98 hepatocellular carcinoma patients who underwent 18F-FDG-PET scans before primary tumor resection. Standardized uptake values (SUV) for primary tumors and tumor-to-nontumor SUV ratios (TNR) were calculated using FDG accumulation levels, and values were compared among K19+/K19- patients. Using hepatocellular carcinoma cell lines encoding with a K19 promoter-driven enhanced GFP, 18F-FDG uptake and GLUT1 expression were examined in FACS-isolated K19+/K19- cells.Results: In hepatocellular carcinoma patients, K19 expression was significantly correlated with GLUT1 expression and FDG accumulation. ROC analyses revealed that among preoperative clinical factors, TNR was the most sensitive indicator of K19 expression in hepatocellular carcinoma tumors. In hepatocellular carcinoma cells, FACS-isolated K19+ cells displayed significantly higher 18F-FDG uptake than K19- cells. Moreover, gain/loss-of-function experiments confirmed that K19 regulates 18F-FDG uptake through TGFß/Smad signaling, including Sp1 and its downstream target GLUT1.Conclusions:18F-FDG-PET can be used to predict K19 expression in hepatocellular carcinoma and should thereby aid in the development of novel therapeutic strategies targeting K19+ HCC-CSCs. Clin Cancer Res; 23(6); 1450-60. ©2016 AACR.

Efficient recellularisation of decellularised whole-liver grafts using biliary tree and foetal hepatocytes.

A whole-organ regeneration approach, using a decellularised xenogeneic liver as a scaffold for the construction of a transplantable liver was recently reported. Deriving suitable scaffolds was the first step towards clinical application; however, effective recellularisation remains to be achieved. This report presents a strategy for the improvement of the recellularisation process, using novel cell-seeding technique and cell source. We evaluated recellularised liver grafts repopulated through the portal vein or the biliary duct with mice adult hepatocytes or E14.5 foetal hepatocytes. More than 80% of the cells seeded through the biliary tree entered the parenchyma beyond the ductule-lining matrix barrier and distributed throughout the liver lobule. In contrast, about 20% of the cells seeded through the portal tree entered the parenchyma. The gene expression levels of foetal hepatocyte albumin, glucose 6-phosphatase, transferrin, cytokeratin 19, and gamma-glutamyl transpeptidase were increased in three-dimensional cultures in the native liver-derived scaffolds, and the activation of liver detoxification enzymes and formation of biliary duct-like structures were supported. The metabolic functions of liver grafts recellularised with different cell types were similar. These results suggest that biliary tree cell-seeding approach is promising, and that liver progenitor cells represent a good cell source candidate.

SOX9 is a novel cancer stem cell marker surrogated by osteopontin in human hepatocellular carcinoma.

The current lack of cancer stem cell (CSC) markers that are easily evaluated by blood samples prevents the establishment of new therapeutic strategies in hepatocellular carcinoma (HCC). Herein, we examined whether sex determining region Y-box 9 (SOX9) represents a new CSC marker, and whether osteopontin (OPN) can be used as a surrogate marker of SOX9 in HCC. In HCC cell lines transfected with a SOX9 promoter-driven enhanced green fluorescence protein gene, FACS-isolated SOX9(+) cells were capable of self-renewal and differentiation into SOX9(-) cells, and displayed high proliferation capacity in vitro. Xenotransplantation experiments revealed that SOX9(+) cells reproduced, differentiated into SOX9(-) cells, and generated tumors at a high frequency in vivo. Moreover, SOX9(+) cells were found to be involved in epithelial-mesenchymal transition (EMT) and activation of TGFb/Smad signaling. Gain/loss of function experiments showed that SOX9 regulates Wnt/beta-catenin signaling, including cyclin D1 and OPN. Immunohistochemistry of 166 HCC surgical specimens and serum OPN measurements showed that compared to SOX9(-) patients, SOX9(+) patients had significantly poorer recurrence-free survival, stronger venous invasion, and higher serum OPN levels. In conclusion, SOX9 is a novel HCC-CSC marker regulating the Wnt/beta-catenin pathway and its downstream target, OPN. OPN is a useful surrogate marker of SOX9 in HCC.

The Protective Effect of Transplanting Liver Cells Into the Mesentery on the Rescue of Acute Liver Failure After Massive Hepatectomy.

Postoperative liver failure is one of the most critical complications following extensive hepatectomy. Although transplantation of allogeneic hepatocytes is an attractive therapy for posthepatectomy liver failure, transplanting cells via the portal veins typically causes portal vein embolization. The embolization by transplanted cells would be lethal in patients who have undergone massive hepatectomy. Thus, transplant surgeons need to select extrahepatic sites as transplant sites to prevent portal vein embolization. We aimed to investigate the mechanism of how liver cells transplanted into the mesentery protect recipient rats from acute liver failure after massive hepatectomy. We induced posthepatectomy liver failure by 90% hepatectomy in rats. Liver cells harvested from rat livers were transplanted into the mesenteries of hepatectomized rats. Twenty percent of the harvested cells, which consisted of hepatocytes and nonparenchymal cells, were transplanted into each recipient. The survival rate improved significantly in the liver cell transplantation group compared to the control group 7 days after hepatectomy (69 vs. 7%). Histological findings of the transplantation site, in vivo imaging system study findings, quantitative polymerase chain reaction assays of the transplanted cells, and serum albumin measurements of transplanted Nagase analbuminemic rats showed rapid deterioration of viable transplanted cells. Although viable transplanted cells deteriorated in the transplanted site, histological findings and an adenosine-5'-triphosphate (ATP) assay showed that the transplanted cells had a protective effect on the remaining livers. These results indicated that the paracrine effects of transplanted liver cells had therapeutic effects. The same protective effects were observed in the hepatocyte transplantation group, but not in the liver nonparenchymal cell transplantation group. Therefore, this effect on the remnant liver was mainly due to the hepatocytes among the transplanted liver cells. We demonstrated that transplanted liver cells protect the remnant liver from severe damage after massive hepatectomy.

Keratin 19, a Cancer Stem Cell Marker in Human Hepatocellular Carcinoma.

PURPOSE: Keratin 19 (K19) is a known marker of poor prognosis and invasion in human hepatocellular carcinoma (HCC). However, the relationship between K19 and cancer stem cells (CSCs) is unclear. Here, we determined whether K19 can be used as a new CSC marker and therapeutic target in HCC. EXPERIMENTAL DESIGN: HCC cell lines were transfected with a K19 promoter-driven enhanced green fluorescence protein gene. CSC characteristics, epithelial-mesenchymal transition (EMT), and TGFb/Smad signaling were examined in FACS-isolated K19(+)/K19(-) cells. K19 and TGFb receptor 1 (TGFbR1) expression in 166 consecutive human HCC surgical specimens was examined immunohistochemically. RESULTS: FACS-isolated single K19(+) cells showed self-renewal and differentiation into K19(-) cells, whereas single K19(-) cells did not produce K19(+) cells. K19(+) cells displayed high proliferation capacity and 5-fluorouracil resistance in vitro. Xenotransplantation into immunodeficient mice revealed that K19(+) cells reproduced, differentiated into K19(-) cells, and generated large tumors at a high frequency in vivo. K19(+) cells were found to be involved in EMT and the activation of TGFb/Smad signaling, and these properties were suppressed by K19 knockdown or treatment with a TGFbR1 inhibitor. The TGFbR1 inhibitor also showed high therapeutic effect against K19(+) tumor in the mouse xenograft model. Immunohistochemistry of HCC specimens showed that compared with K19(-) patients, K19(+) patients had significantly poorer recurrence-free survival and higher tumor TGFbR1 expression. CONCLUSIONS: K19 is a new CSC marker associated with EMT and TGFb/Smad signaling, and it would thus be a good therapeutic target for TGFbR1 inhibition.

Rapid progression of submucosal invasive micropapillary carcinoma of the colon in progressive systemic sclerosis: report of a case.

A 71-year-old woman presented with hematochezia and narrowing of the stool. She suffered from progressive systemic sclerosis for 12 years and underwent home oxygen therapy due to pulmonary fibrosis and moderate pulmonary hypertension. Colonoscopy revealed a pedunculated, cauliflower-like polyp with a depressed surface in the sigmoid colon. The polyp was regarded as early colon cancer with possible submucosal invasion, and subsequent computed tomographic (CT) scans showed no evidence of lymph node involvement or distant metastases. Because of perioperative risks due to moderate pulmonary hypertension, she underwent an endoscopic resection of the early colon cancer. Pathological examination of the resected specimen of 20 mm diameter revealed the peculiar morphology of an adenocarcinoma with moderate lymphatic invasion. Immunohistochemical analysis for epithelial membrane antigen showed the specific 'inside-out growth pattern' indicative of invasive micropapillary carcinoma (IMPC). Taking the perioperative risks into consideration, she opted to undergo close follow-ups without an additional sigmoidectomy. At 6 months after the resection, the follow-up colonoscopy revealed a local recurrence of the colon cancer, and subsequent CT scans revealed multiple distant metastases including the lung, liver, lymph nodes and spleen. This is a rare case of a pure, submucosal IMPC of the colon. Furthermore, pure IMPC of the colon may represent a reliable predictor of lymphogenous and/or hematogenous metastases. Therefore, one should recommend an additional colectomy after endoscopic mucosal resection treatment when pathological findings confirmed IMPC of the colon and should continue a close follow-up for IMPC patients even when curative resections were performed at an early stage.