Follicular cholangitis mimicking a common bile duct cancer: a case report.

BACKGROUND: Follicular cholangitis (FC) is a benign bile duct disease that was first reported 2003. Pathologically, it is characterized by lymphoplasmacytic infiltration with multiple lymphoid follicle formations under the mucosal layer of the biliary tract. However, as this disease is extremely rare, little is known about its etiology and pathogenesis. CASE PRESENTATION: A 77-year-old woman was diagnosed with middle bile duct stenosis and potential increases in alkaline phosphatase (ALP) and γ-glutamyl transpeptidase levels (γ-GTP). Carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9) and IgG4 levels were all within the normal limits. Contrast-enhanced computed tomography (CE-CT) and magnetic resonance imaging (MRI) revealed bile duct dilation from intrahepatic to upper common bile duct and an irregular mass lesion in distal bile duct. Additionally, multiple overlapping leaf-like folds were detected. 18F-fluorodeoxyglucose positron emission tomography-computed tomography (18F-FDG-PET/CT) did not demonstrate fluorodeoxyglucose uptake. Subtotal stomach-preserving pancreaticoduodenectomy with regional lymph node dissection was performed because common bile duct cancer could not be ruled out. The resected specimen showed diffuse homogeneous middle bile duct wall thickening. Microscopically, the lesion exhibited thick fibrosis with several invaded lymphoplasmacytic cells, and lymphoid follicle formations were detected under the mucosal layer. Immunohistochemical staining (IHC) revealed positive for CD3, CD4, CD20 and CD79a, and these findings led to a final diagnosis of FC. The patient has not experienced recurrence to date (42 months postoperatively). CONCLUSIONS: Currently, accurate preoperative diagnosis of FC is difficult. More cases must be accumulated to generate additional knowledge on its precise diagnosis and proper treatment.

[A Long Term Survival Case of Gastric Cancer with Peritoneal Dissemination Responding to Intraperitoneal Chemotherapy].

A 32-year-old woman was admitted our hospital due to epigastric discomfort. The patient diagnosed as having scirrhous carcinoma of the stomach by upper gastrointestinal scope. Peritoneal dissemination and ovarian metastasis were confirmed by the diagnostic laparoscopy. Therefore, combination chemotherapy with S-1 and intraperitoneal chemotherapy(ip)with docetaxel (DTX) was started. After 2 courses chemotherapy, laparoscopy was performed again. Peritoneal dissemination was scarred, but biopsy showed altered AE1/AE3 positive cells, and increased left ovarian metastasis, so systemic chemotherapy was changed to DCS chemotherapy and added DTX ip. After 4 courses chemotherapy and 7 months after the first diagnosis, subtotal gastrectomy, hysterectomy and bilateral adnexectomy were performed because the cytology and tumor marker remained within normal range. In histopathological diagnosis, the effect of chemotherapy was Grade 2 at the primary site and Grade 3 at the metastatic site. Nine years have passed since the initial diagnosis and she has no relapse with postoperative adjuvant chemotherapy.

A typical case of resected pancreatic hamartoma: a case report and literature review on imaging and pathology.

BACKGROUND: Pancreatic hamartomas are rare entities and difficult to diagnose before resection. We report a case of resected pancreatic hamartoma and literature review of typical characteristics of the lesion. CASE PRESENTATION: A 78-year-old man presented with a mass in his pancreas, which was incidentally identified when he experienced pneumonia. No remarkable symptoms were observed, and laboratory tests showed no abnormalities, except a slight carcinoembryonic antigen elevation. Enhanced computed tomography and magnetic resonance imaging showed a well-demarcated solid mass with heterogeneous contrast that was 2 cm in size. A gradual enhancement pattern was also observed. The biopsy revealed no specific findings; therefore, surgical resection was necessitated to confirm the diagnosis. Histopathologically, ducts, acinar cells, and adipose cells without atypia were observed among abundant fibrous stroma, but islets of Langerhans and peripheral nerves were absent. An immunohistochemical examination demonstrated CD34 and c-kit positive staining in the stromal cells, S-100 positivity in the adipose cells, and a lack of elastic fibers in the duct walls. The lesion was diagnosed as a pancreatic hamartoma. CONCLUSION: Asymptomatic pancreatic hamartomas can avoid resection. A careful consideration of imaging and appropriate immunohistochemistry of biopsy specimen may facilitate accurate diagnosis before resection. Therefore, sufficient recognition of the characteristics of pancreatic hamartomas is desirable.

CD90 expression in human intrahepatic cholangiocarcinoma is associated with lymph node metastasis and poor prognosis.

BACKGROUND AND OBJECTIVES: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer. However, its prognosis remains poor. Expression of cluster of differentiation 90 (CD90) has been identified as an indicator of poor prognosis in many cancers. Here, we examined the importance of CD90 expression in ICC. METHODS: We performed immunohistological assays for CD90 in human ICC surgical specimens and assessed its relationship with clinicopathological findings and prognosis. Moreover, we analyzed the characteristics of CD90+/- cells, mainly with respect to metastatic potential, using human ICC cell lines. RESULTS: CD90 expression was significantly associated with lymph node metastasis and was revealed to be an independent prognostic factor. The CD90+ cells present in ICC specimens did not appear to be cancer-associated fibroblasts, as they did not express α-smooth muscle actin. In vitro, CD90 + cells exhibited greater migratory ability and higher expression of epithelial-mesenchymal transition (EMT)-related genes, including CXCR4 and MMP7, than CD90- cells. Wnt/ß-catenin signaling pathway activation was also heightened in CD90+ cells. In such cells, EMT appeared to be induced by CXCR4 and MMP7 expression through activation of Wnt/ß-catenin signaling. CONCLUSION: CD90+ cells demonstrate high metastatic potential owing to Wnt/ß-catenin signaling activation and are associated with poor prognosis in ICC.

[Two-Stage Radical Resection of a Case of Colon Cancer with Abdominal Wall Invasion and Cholangiocarcinoma].

A woman in her 50s was admitted to our hospital with fever and lower abdominal swelling. Abdominal CT/MRI examinations revealed irregular thickening of the transverse colon wall, which was attached to a subcutaneous abscess. An abdominal wall mass, a patent urachus, and a tumor in the 5th segment of the liver were also noted. Colonoscopy revealed type 2 advanced transverse colon cancer. The solitary, sessile tumor was observed at the apex of the bladder under cystoscopy, suggesting the formation of the urachal carcinoma. Transcutaneous liver biopsy obtained from the liver tumor indicated adenocarcinoma, which was morphologically different from the existing transverse colon cancer. Right hemicolectomy with resection of the umbilicus, abdominal wall, urachus, and part of the bladder wall was performed. Diagnosis of the transverse colon cancer invading the abdominal wall and bladder was confirmed by histopathological examination. Hepatectomy was performed in the next surgery, and the tumor was histopathologically diagnosed as an intrahepatic cholangiocarcinoma. Both the transverse colon cancer and the intrahepatic cholangiocarcinoma were radically resected. Radical surgical diagnostic resection may be valuable in cases of multicentric cancers of unknown primary origin, if radical resection of each individual tumor is required.

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.

[A Case of Esophageal Carcinoma with Large Intramural Metastasis to the Stomach].

We report a rare case of esophageal carcinoma with gastric wall metastasis. A 73-year-old man with dysphagia underwent endoscopy and upper GI series and chest-abdominal computed tomography, revealing esophageal carcinoma and gastric cancer, which was diagnosed as squamous cell carcinoma by biopsy. The esophageal carcinoma was located in the lower thoracic esophagus(Lt). Total gastrectomy was performed. The resected specimen showed a type 3 tumor measuring 7×7 cm in the anterior wall of the stomach. Pathologically, the depth of invasion of the stomach was SE. He died 3 months after the operation. Esophageal carcinoma with gastric intramural metastasis is very rare and has a dismal prognosis. We report a rare case of esophageal carcinoma with large intramural metastasis to the stomach.

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.

[A Case of Unresectable Locally Advanced Pancreatic Cancer Resulted in R0 Surgery after Chemotherapy].

A 67-year-old man visited our hospital for jaundice. Abdominal dynamic CT showed the hypovascular tumor at the head of the pancreas that surrounded superior mesenteric artery(SMA)at an angle of 220 degree. No metastasis in lymph nodes and other organs was observed. We diagnosed the tumor unresectable locally advanced(UR-LA)pancreatic cancer. Chemotherapy was administered with gemcitabine and nab-paclitaxel(GEM+nab-PTX)and achieved partial response. Regression in size and in range around SMA to an angle of 150 was observed. We assessed it possible to resect the tumor curatively, and performed subtotal stomach preserving pancreaticoduodenectomy and dissection of the plexus around the SMA, resulted in R0 surgery. Adjuvant chemotherapy was administered, and no recurrence was observed up to present, more than a year. It is suggested that GEM+nab-PTX can be effective as the primary therapy against UR-LA pancreatic cancer.

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.