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Purpose:Few studies have investigated prognostic biomarkers in patients with intrahepatic cholangiocarcinoma (ICC). Nardilysin (NRDC), a metalloendopeptidase of the M16 family, has been suggested to play important roles in inflammation and several cancer types. We herein examined the clinical significance and biological function of NRDC in ICC.Experimental Design: We measured serum NRDC levels in 98 patients with ICC who underwent surgical resection in two independent cohorts to assess its prognostic impact. We also analyzed NRDC mRNA levels in cancerous tissue specimens from 43 patients with ICC. We investigated the roles of NRDC in cell proliferation, migration, gemcitabine sensitivity, and gene expression in ICC cell lines using gene silencing.Results:High serum NRDC levels were associated with shorter overall survival and disease-free survival in the primary (n = 79) and validation (n = 19) cohorts. A correlation was observed between serum protein levels and cancerous tissue mRNA levels of NRDC (Spearman ρ = 0.413; P = 0.006). The gene knockdown of NRDC in ICC cell lines attenuated cell proliferation, migration, and tumor growth in xenografts, and increased sensitivity to gemcitabine. The gene knockdown of NRDC was also accompanied by significant changes in the expression of several epithelial–mesenchymal transition (EMT)-related genes. Strong correlations were observed between the mRNA levels of NRDC and EMT-inducing transcription factors, ZEB1 and SNAI1, in surgical specimens from patients with ICC.Conclusions:Serum NRDC, a possible surrogate marker reflecting the EMT state in primary tumors, predicts the outcome of ICC after surgical resection.

Supplementary Tables S1-S7; Supplementary Figures S1-S7

This study assessed the significance of measuring liver stiffness using virtual touch quantification before hepatectomy to predict posthepatectomy refractory ascites. A total of 267 patients with hepatocellular carcinoma who underwent hepatectomy were prospectively analyzed. Liver stiffness was defined as the median value of the virtual touch quantification (Vs; m/s) by acoustic radio-force-impulse-based virtual touch. A multivariate analysis showed that Vs and the aspartate aminotransferase-to-platelet ratio index were independent risk factors for postoperative refractory ascites (odds ratio = 3.27 and 3.08, respectively). The cutoff value for Vs was 1.52 m/s (sensitivity: 59.5%, specificity: 88.6%) as determined by the analysis of the receiver-operating characteristic curve, and the area under the receiver-operating characteristic curve was 0.79. The cutoff value for the aspartate aminotransferase-to-platelet ratio was 0.952 (sensitivity: 65.5%, specificity: 82.9%), and the area under the receiver-operating characteristic curve was 0.75. Vs is an independent risk factor for refractory ascites after hepatectomy. The measurement of liver stiffness by virtual touch quantification before hepatectomy can help estimate the risk of postoperative refractory ascites. Nonsurgical treatments should be considered for the management of patients who are at high risk for refractory ascites.

This study aimed to clarify what hepatocellular carcinoma (HCC) phenotype, as categorized by intraoperative contrast-enhanced ultrasonography (CEUS), showed a high risk of recurrence after hepatic resection. Patients who underwent initial curative hepatectomy with intraoperative CEUS for a single HCC nodule were retrospectively assigned to three patterns of fine (FI), vascular (VA), and irregular (IR) according to the maximum intensity projection pattern based on intraoperative CEUS. Staining was performed for Ki-67, pyruvate kinase type M2 (PKM2), and vascular endothelial growth factor (VEGF) to assess the tumor proliferative activity, tumor glucose metabolism, and angiogenesis, respectively. Of 116 patients, 18, 50, and 48 were assigned to the FI, VA and IR patterns, respectively. IR patients demonstrated a significantly worse prognosis for both the recurrence-free survival (RFS) and overall survival (OS) (P = 0.0002, 0.0262, respectively) than did patients with other patterns. A multivariate analysis revealed an IR pattern in intraoperative CEUS to be an independent predictive factor for a poor RFS, and major hepatectomy and an IR pattern were independent predictive factors for a poor OS. An IR pattern was closely related to the tumor size (≥ 3.3 cm) and poor histological differentiation and showed a high Ki-67 index, low VEGF expression, and high PKM2 expression. IR-pattern HCCs as classified by intraoperative CEUS may be associated with a higher risk of recurrence and worse outcomes in HCC patients after hepatic resection than other patterns.

Purpose: Few studies have investigated prognostic biomarkers in patients with intrahepatic cholangiocarcinoma (ICC). Nardilysin (NRDC), a metalloendopeptidase of the M16 family, has been suggested to play important roles in inflammation and several cancer types. We herein examined the clinical significance and biological function of NRDC in ICC. Experimental Design: We measured serum NRDC levels in 98 patients with ICC who underwent surgical resection in two independent cohorts to assess its prognostic impact. We also analyzed NRDC mRNA levels in cancerous tissue specimens from 43 patients with ICC. We investigated the roles of NRDC in cell proliferation, migration, gemcitabine sensitivity, and gene expression in ICC cell lines using gene silencing. Results: High serum NRDC levels were associated with shorter overall survival and disease-free survival in the primary (n = 79) and validation (n = 19) cohorts. A correlation was observed between serum protein levels and cancerous tissue mRNA levels of NRDC (Spearman ρ = 0.413; P = 0.006). The gene knockdown of NRDC in ICC cell lines attenuated cell proliferation, migration, and tumor growth in xenografts, and increased sensitivity to gemcitabine. The gene knockdown of NRDC was also accompanied by significant changes in the expression of several epithelial–mesenchymal transition (EMT)-related genes. Strong correlations were observed between the mRNA levels of NRDC and EMT-inducing transcription factors, ZEB1 and SNAI1, in surgical specimens from patients with ICC. Conclusions: Serum NRDC, a possible surrogate marker reflecting the EMT state in primary tumors, predicts the outcome of ICC after surgical resection.

Cancer-associated fibroblasts (CAFs) are an important constituent of the cancer stroma. In intrahepatic cholangiocarcinoma (ICC), the features of CAFs at the primary site and in the metastatic lymph nodes (Met-LNs) and their origin have been unclear. In the present study, we characterized CAFs at the primary site (n = 42) and in the Met-LNs (n = 10) of human ICC by immunohistochemistry using potential molecular markers of CAFs, portal fibroblasts (PFs), hepatic stellate cells (HSCs), and bone marrow-derived fibrocytes (BMDFs). At the primary site, the stroma was strongly positive for α-smooth muscle actin (α-SMA; marker for CAFs), platelet-derived growth factor receptor-β (PDGFR-β) (common marker for HSCs and PFs), fibulin-2, and thymus cell antigen-1 (Thy-1; PF marker), whereas immunoreactivity for fascin (HSC marker) was scarce. Most of the α-SMA-positive cells were found to express PDGFR-β, Thy-1, and fibulin-2 by double immunostaining. A small population of BMDF marker-positive (α-SMA+CD45+CD34+) cells was found by triple immunostaining. In the micro-Met-LNs, α-SMA-positive cells were absent in cancer aggregates of the LN sinus, whereas they were present in the invasion area of cancer cells from the LN sinus to the LN parenchyma. In the macro-Met-LNs, there were abundant α-SMA-positive cells that were also positive for PDGFR-β and Thy-1 but negative for fibulin-2 and fascin. Thus, regarding the expression of molecular markers, CAFs at the primary site of ICC are similar to PFs and different from those of HSCs or CAFs in the Met-LNs. CAFs at the primary sites and in the Met-LN are thought to be derived from PFs/BMDFs and resident cells of LNs, respectively.

A 75-year-old male was admitted to our hospital because of bile duct stenosis. He had no medical history of autoimmune disease. The level of tumor markers, serum IgG, and IgG4 were within normal ranges. Computed tomography showed perihilar and distal bile duct stenosis and wall thickening without swelling or abnormal enhancement of the pancreas. Endoscopic retrograde cholangiopancreatography showed perihilar and distal bile duct stenosis. A biopsy and cytology from the distal bile duct stenosis suggested adenocarcinoma, and cytology from the perihilar bile duct also suggested adenocarcinoma. A preoperative diagnosis of perihilar and distal bile duct cancer was made, and the patient underwent left hepatectomy and pancreaticoduodenectomy. Resected specimens showed wall thickening in the perihilar and distal bile duct; however, tumors were unclear. A histopathological examination revealed lymphoplasmacytic infiltration, storiform fibrosis, and obliterative phlebitis in the perihilar and distal bile ducts. Immunohistochemistry revealed diffuse infiltration of IgG4-positive plasma cells in the perihilar and distal bile ducts. Lymphoplasmacytic infiltration, inflammatory change, storiform fibrosis, and obliterative phlebitis were shown in the pancreas. A final diagnosis of IgG4-related sclerosing cholangitis (IgG4-SC) with autoimmune pancreatitis was made. We herein report a case in which a preoperative diagnosis of IgG4-SC was difficult due to normal serum IgG4 levels and no obvious pancreatic lesion.

Right-sided ligamentum teres (RSLT) is a rare congenital anomaly often accompanied by variation of the hepatic vasculature. We herein report a surgical case of a hilar cholangiocarcinoma with RSLT in whom preoperative hepatectomy simulation proved useful for understanding the anatomical structure of the liver. A 78-year-old male with obstructive jaundice was referred to our department for further examination. The patient was suspected of having a hilar cholangiocarcinoma originating from the left hepatic bile duct by contrast-enhanced computed tomography (CT), and CT also showed right umbilical portion (RUP). Three-dimensional images of the hepatic vasculature and biliary system reconstructed using a hepatectomy simulation system suggested that all portal branches ramified from RUP were right paramedian branches, and three leftward portal branches from these ran parallel to the peripheral bile ducts confluent with the left hepatic bile duct, where the tumor was present. Hepatic resection of part of the ventral area of the right paramedian sector and left hemiliver was performed along the demarcation line drawn after clamping the portal branches; the ratio of estimated liver resection volume was 28.9%. After the operation, bile leakage occurred. However, the leakage was treated with percutaneous drainage alone, and the patient was discharged 77 days after the operation. The patient is doing well without any signs of recurrence 21 months after the operation. The vascular and biliary anatomy in patients with RSLT is complicated and should be evaluated in detail preoperatively using a hepatectomy simulation system.

Background Anatomical hepatectomy aims to eliminate the spread of malignant tumor cells via portal vein systemically. An anatomical concept of the right anterior section (RAS) and preservation of the liver parenchyma within the RAS has been proposed. Methods We focused on the anatomical concept of the RAS based on portal perfusion and described surgical procedures to preserve the ventral or dorsal RAS using preoperative simulation. Results In 370 patients undergoing a preoperative simulation, the ramification of the tertiary portal branches of the RAS could be divided into three types including the cranio-caudal type; Couinaud's classification in 50% of patients, ventro-dorsal type in 26% of patients, and multiple type in 24% of patients. Then in 32 patients of the ventro-dorsal type, curative parenchyma-sparing hepatectomy of the RAS was performed, preserving the ventral and dorsal RAS in 14 and 18 patients, respectively. There were no differences in the postoperative complications and long-term survival compared with the results obtained after segment 5 or 8 resection (n = 33). Conclusion Three-dimensional simulation revealed three types of portal vein ramification of the RAS. Parenchyma-preserving hepatectomy based on the precise portal ramification may contribute to safe and curative hepatectomy in selected cases with liver neoplasm involving the RAS.

Non-parasitic splenic cysts are relatively rare, and the optimal surgical treatment for them remains controversial. Laparoscopic unroofing is a relatively safe and easy technique, but a significant number of recurrences has been reported. Thus, complete cystectomy with partial splenectomy is recommended by several surgeons. However, patients sometimes suffer from intraoperative bleeding. Here, we report a patient with a giant non-parasitic splenic cyst who underwent subtotal cystectomy with partial splenectomy. After the dissection of the vessels circulating the upper pole at the splenic hilum, the resection line of the splenic parenchyma was on the ischemic side of the cyanotic demarcation line. A vessel sealing system and laparoscopic coagulation shears were used for the resection. We intentionally left about 10% of the cyst wall to avoid bleeding from the non-ischemic splenic parenchyma and remaining vessels. No recurrence has been detected after 6 months of observation. We believe this method could be a useful alternative procedure for the treatment of non-parasitic splenic cysts and preservation of the splenic parenchyma.

Fabry disease is caused by deficient activity of α-galactosidase A (GLA) and characterized by systemic accumulation of glycosphingolipids, substrates of the enzyme. To gain insight into the pathogenesis of Fabry disease based on accumulated substrates, we examined the tissue and plasma distributions of globotriaosylceramide (Gb3) isoforms, and globotriaosylsphingosine (lyso-Gb3) and its analogues in a GLA knockout mouse, a model of Fabry disease, by means of liquid chromatography-mass spectrometry and nano-liquid chromatography-tandem mass spectrometry, respectively. The results revealed that the contents of these substrates in the liver, kidneys, heart, and plasma of GLA knockout mice were apparently higher than in those of wild-type ones, and organ specificity in the accumulation of Gb3 isoforms was found. Especially in the kidneys, accumulation of a large amount of Gb3 isoforms including hydroxylated residues was found. In the GLA knockout mice, the proportion of hydrophobic Gb3 isoforms was apparently higher than that in the wild-type mice. On the other hand, hydrophilic residues were abundant in plasma. Unlike that of Gb3, the concentration of lyso-Gb3 was high in the liver, and the lyso-Gb3/Gb3 ratio in plasma was significantly higher than those in the organs. The concentration of lyso-Gb3 was apparently higher than those of its analogues in the organs and plasma from both the GLA knockout and wild-type mice. This information will be useful for elucidating the basis of Fabry disease.

We report an extremely rare case of the development of hepatocellular carcinoma (HCC) in cardiac congestive liver fibrosis. A 62-year-old female presented to our hospital with a complaint of right upper quadrant pain. The patient had undergone cardiac surgery for pulmonary valve insufficiency, pulmonary stenosis and atrial septal defect when she was fifteen years of age. During the subsequent 47 years, she had occasionally suffered from various symptoms associated with right-sided heart failure due to pulmonary stenosis. Computed tomography revealed a liver tumor measuring 63 mm in diameter in segment 5 and other liver tumors in segments 5 (18 mm), 8 (17 mm) and 4 (12 mm), which were diagnosed as HCCs. There was no evidence of stenosis in any hepatic veins or inferior vena cava, and no infectious hepatitis or alcoholic liver damage. Anterior sectionectomy and partial resection of segment 4 was performed, and histological examination showed that these tumors were HCC accompanied by congestive liver fibrosis. Nine months later, multiple recurrent HCCs were detected in segment 6, and transcatheter arterial chemoembolization was employed thereafter. The patient died 40 months after surgery due to advanced recurrence.

Biomarkers useful for diagnosis and evaluation of treatment for patients with Fabry disease are urgently needed. Recently, plasma globotriaosylsphingosine (lyso-Gb3) and lyso-Gb3-related analogues have attracted attention as promising biomarkers of Fabry disease. However, the plasma concentrations of lyso-Gb3 and its analogues are extremely low or below the detection limits in some Fabry patients as well as in healthy subjects. In this paper, we introduce the novel application of a nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) system to the measurement of lyso-Gb3 and its analogues in plasma. Nano-LC-MS/MS requires smaller amounts of samples and is more sensitive than conventional techniques. Using this method, we measured the plasma concentrations of lyso-Gb3 and its analogues in 40 healthy subjects, 5 functional variants (males with E66Q), and various Fabry patients (9 classic Fabry males/9 mutations; 7 later-onset Fabry males/5 mutations; and 10 Fabry females/9 mutations). The results revealed that the mean lyso-Gb3 and lyso-Gb3(-2) concentrations in all the Fabry patient subgroups were statistically higher, especially in the classic Fabry males, than those in the functional variants and healthy subjects. The plasma concentrations of lyso-Gb3 and its analogues in healthy subjects, functional variants, and some Fabry patients with specific mutations (R112H and M296I) that cannot be established by conventional techniques were successfully determined by means of nano-LC-MS/MS. The lyso-Gb3 and lyso-Gb3(-2) concentrations in male patients with these mutations were lower than those in most Fabry patients having other mutations, but higher than those in the functional variants and healthy subjects. This new method is expected to be useful for sensitive determination of the plasma concentrations of lyso-Gb3 and its analogues. This study also revealed that not only lyso-Gb3 but also lyso-Gb3(-2) in plasma is a useful biomarker for the diagnosis of Fabry disease.

Background/Aims Complete operative resection is the only approach to cure for intrahepatic cholangiocellular carcinoma (ICC), but the disease’s prognosis is notably poor. A novel therapeutic approach is urgently required. CXC chemokine receptor 2 (CXCR2) has been associated with tumorigenesis and metastasis in human cancers. In this study, we investigated the suppressive effect of ICC growth by blocking CXCR2. Material and methods The role of CXCR2 was estimated using the human ICC cell lines, RBE and SSP25. CXCR2 small interfering RNA (siRNA) and an antagonist (SB225002) were used to block CXCR2. Proliferation assays, migration assays, and invasion assays were performed to confirm the suppressive effect of blocking CXCR2. Subcutaneous SSP25 tumors were established in athymic nude mice, and the mice were given SB225002. The expression of CXCR2 in ICC was determined by immunohistochemical staining of 34 ICC specimens. We investigated the relationship between CXCR2 expression and prognosis in ICC. Results The prognosis of patients who had higher CXCR2 expression in ICC was significantly poor ( P = .004). CXCR2 siRNA treatment significantly suppressed CXCR2 expression in both RBE and SSP25. Cell proliferation, migration, and invasion were significantly suppressed by both CXCR2 siRNA and SB225002 compared with the control group. SB225002 also suppressed the growth of transplanted subcutaneous tumors ( P = .02) Conclusion Our results demonstrated that blocking CXCR2 clearly suppressed the development of ICC. Blocking CXCR2 may be a promising therapeutic approach for ICC.

Non-parasitic splenic cysts are relatively rare, and the optimal surgical treatment for them remains controversial. Laparoscopic unroofing is a relatively safe and easy technique, but a significant number of recurrences has been reported. Thus, complete cystectomy with partial splenectomy is recommended by several surgeons. However, patients sometimes suffer from intraoperative bleeding. Here, we report a patient with a giant non-parasitic splenic cyst who underwent subtotal cystectomy with partial splenectomy. After the dissection of the vessels circulating the upper pole at the splenic hilum, the resection line of the splenic parenchyma was on the ischemic side of the cyanotic demarcation line. A vessel sealing system and laparoscopic coagulation shears were used for the resection. We intentionally left about 10% of the cyst wall to avoid bleeding from the non-ischemic splenic parenchyma and remaining vessels. No recurrence has been detected after 6 months of observation. We believe this method could be a useful alternative procedure for the treatment of non-parasitic splenic cysts and preservation of the splenic parenchyma.