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Genes commonly upregulated in HuH7-F6 and -F12 cells compared with HuH7-F0

Clinicopathological characteristics of patients with Tβ4-high and -low HCC

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An 82-year-old woman presented to our hospital with chief complaints of lower abdominal pain and nausea. Contrast- enhanced CT showed ileus of sigmoid colon cancer and a solitary splenic tumor. A metallic stent was placed for the primary lesion. FDG-PET showed high FDG accumulation in the solitary splenic tumor, and synchronous solitary splenic metastasis was diagnosed. Laparoscopic sigmoid colectomy and laparoscopic splenectomy were performed without changing the intraoperative position or port arrangement. Postoperative progress was favorable. The patient was discharged 9 days after surgery, and no sign of recurrence has been observed to date, at 4 months after surgery. Solitary splenic metastasis of colorectal cancer is extremely rare. This is the first case report of synchronous solitary splenic metastasis of colorectal cancer treated with laparoscopic resection in Japan. This procedure is considered effective and minimally invasive. We review and discuss the Japanese literature on this rare disease.

In distal pancreatectomy (DP), the position of the pancreas division line (PDL) changes depending on the location or nature of the tumor. Here, we investigated the relationship between PDL and postoperative complications after DP. We retrospectively analyzed data of 140 patients who underwent DP at Tokyo Medical and Dental University Hospital between January 2011 and September 2018. PDL was defined as the distance from the left margin of the portal vein to the edge of the pancreatic stump on the coronal plane of computed tomography. The mean PDL was 15.1 (range 0–74.3) mm. PDL was significantly longer in patients with portal venous system thrombosis (PVST) than in those without PVST (47.6 vs. 0 mm, p

Because it is unknown whether adjusting the dose of sunitinib can benefit patients with pancreatic neuroendocrine neoplasms (Pan-NENs), this retrospective study examined maximum tumor shrinkage rates and prognoses in patients with and without low doses of sunitinib administration. Eighty-seven patients with metastatic and unresectable neoplasms, treated with sunitinib for > 1 month, were divided into a low-dose (LD) or high-dose (HD) group. The tumor response rates were investigated over time using computed tomography according to the response evaluation criteria in solid tumors criteria. The LD and HD groups included 42 and 45 patients, respectively. There were no differences in baseline characteristics (tumor size, Ki-67 index, mitosis, and differentiation) between the two groups. Progressive disease (PD), stable disease (SD), and partial response (PR) were observed in 16.7, 54.8, and 28.6% of patients in the LD group, respectively, and in 13.3, 60, and 26.7% of patients in the HD group, respectively. There were no differences in tumor shrinkage rates between the two groups (p = 0.87). The 3-year progression-free survival rates for the LD and HD groups were 2.4% and 2.3%, respectively (p = 0.67), and the 3-year overall survival rates were 57.9% and 70.5%, respectively (p = 0.76). The occurrence of adverse events was similar between the two groups (61.9% vs. 60.0%, p > 0.95). Dose reduction of sunitinib did not alter tumor shrinkage rates or prognoses for patients with advanced Pan-NENs.

Pancreatic fistula after distal pancreatectomy (DP) remains an unsolved problem, and postoperative CT imaging often demonstrates fluid collection (FC) around the pancreatic remnant. This study sought to clarify the clinical implications of FC. This study enrolled 146 patients who underwent DP. FC was defined as a cyst-like lesion ≥ 10 mm in diameter on CT imaging at postoperative day (POD) 7. FC size, irregularity of FC margin, and air bubbles in FC were investigated. In addition, clinical data were retrospectively collected, and useful predictive factors for postoperative pancreatic fistula (POPF) were analyzed. Clinically relevant POPF was observed in 26 patients (17.8%), and FC was detected in 136 patients (94.4%). Multivariate analysis identified FC size and drain amylase levels on POD3 as significant risk factors for POPF. Cutoff values were determined by ROC analyses, and the levels of the FC size and drain amylase on POD3 were determined as 41 mm and 1026 IU/L, respectively. The sensitivity and specificity of FC diameters > 41 mm were 76.9% and 75.0%, respectively, while those of drain amylase levels > 1026 IU on POD3 were 73.1% and 75.8%, respectively. While treating some FCs after DP was necessary for the management of POPF, others did not require any intervention since most of them spontaneously disappeared. FC size and drain amylase levels on POD3 were found to be significantly associated with POPF and could potentially help to determine appropriate treatment.

The prognostic importance of the neuroendocrine (NE) markers involving neural cell adhesion molecule (NCAM) has been unclear enough to be adopted for WHO classification in patients with pancreatic neuroendocrine neoplasms (Pan-NENs). This study aimed to elucidate whether the three NE markers such as chromogranin A, synaptophysin, and NCAM decide prognoses for patients with well-differentiated tumors. Between April 2002 and October 2018, 217 patients were included in this study. Tissue samples from tumors of Pan-NENs were immunochemically stained for the aforementioned NE markers. Diffuse and intense staining was defined as positive, while faint or focal staining and non-staining were considered negative. The median age of patients was 55 years. The median observation period was 1415 days. In multivariate analysis of progression-free survival (PFS), liver metastasis, Ki-67 index, and triple-positive staining of NE markers were risk factors. The 5-year PFS rate of patients with and without triple-positive NE markers was 56.3% and 23.8%, respectively (P

Background Hepatocellular carcinoma (HCC) is a heterogeneous disease with various etiological factors, and ranks as the second leading cause of cancer-related mortality worldwide due to multi-focal recurrence. We herein identified three major subtypes of HCC by performing integrative analysis of two omics data sets, and clarified that this classification was closely correlated with clinicopathological factors, immune profiles and recurrence patterns. Methods In the test study, 183 tumor specimens surgically resected from HCC patients were collected for unsupervised clustering analysis of gene expression signatures and comparative analysis of gene mutations. These results were validated by using genome, methylome and transcriptome data of 373 HCC patients provided from the Cancer Genome Atlas Network. In addition, omics data were obtained from pairs of primary and recurrent HCC. Findings Comprehensive molecular evaluation of HCC by multi-platform analysis defined three major subtypes: (1) mitogenic and stem cell-like tumors with chromosomal instability; (2) CTNNB1-mutated tumors displaying immune suppression; and (3) metabolic disease-associated tumors, which included an immunogenic subgroup characterized by macrophage infiltration and favorable prognosis. Although genomic and epigenomic analysis explicitly distinguished between HCC with intrahepatic metastasis (IM) and multi-centric HCC (MC), the phenotypic similarity between the primary and recurrent tumors was not correlated to the IM/MC origin, but to the classification. Interpretation: Identification of these HCC subtypes provides further insights into patient stratification as well as presents opportunities for therapeutic development. Fund Ministry of Education, Culture, Sports, Science and Technology of Japan (16H02670 and 18K19575), Japan Agency for Medical Research and Development (JP15cm0106064, JP17cm0106518, JP18cm0106540 and JP18fk0210040).

INTRODUCTION The use of laparoscopic distal pancreatectomy (LDP) is increasing worldwide. It is important for surgeons to predict preoperatively the difficulty and risks of a surgery. However, very few reports have evaluated the impact of patient or tumor factors on the difficulty of LDP. We aimed to determine the predictors of technical difficulties when performing LDP. METHODS This study included 34 patients who underwent LDP. Patient information was obtained retrospectively and included age, gender, BMI, primary disease, previous abdominal surgery, previous pancreatitis, tumor size, tumor proximity to the splenic arterial origin, type of splenic artery (SpA), operative time, blood loss, postoperative pancreatic fistula, and length of hospital stay. Univariate and multivariate analyses were performed to determine the predictors of a long operative time. SpA anatomy was classified into two types based on the relationship between its origin and the pancreas. Patients whose SpA origin was upward of the pancreatic parenchyma were classified as SpA type 1, whereas patients whose SpA origin was covered by the pancreatic parenchyma were classified as SpA type 2. RESULTS Multivariate analysis revealed SpA type 2 to be an independent risk factor for a long operation (odds ratio = 9.925; 95% confidence interval: 1.461-67.412; P = 0.019). SpA type 2 was related to a longer operative time (P

Recently, several studies have revealed the usefulness of biomarkers to predict the response to chemotherapy for pancreatic ductal adenocarcinoma (PDAC). Among them, human antigen R (HuR) is reported as a powerful marker for response to gemcitabine chemotherapy for PDAC. The present report describes a patient with PDAC who underwent gemcitabine therapy before resection and after recurrence, and HuR expression was examined at multiple stages. A 72-year-old man was diagnosed with locally advanced unresectable PDAC invading the common hepatic artery. After 9 cycles of gemcitabine treatment, a computed tomography (CT) scan demonstrated a partial response. He underwent distal pancreatectomy with portal vein resection. The pathologic assessment for response to the chemotherapy was grade Ib by Evans's criteria, and HuR expression was high. Serum carbohydrate antigen 19-9 (CA19-9) level rose rapidly at 4 months after the first resection. A CT scan and needle biopsy revealed a solitary recurrence in the abdominal wall, and HuR expression remained high. After 4 cycles of gemcitabine and S-1 combination therapy, a CT scan demonstrated a partial response, and serum CA19-9 decreased. However, after 2 additional cycles of the therapy, a CT scan demonstrated progressive disease, and serum CA19-9 increased slightly. By laparotomy, an abdominal wall recurrence and multiple peritoneal dissemination were found. HuR expression in the biopsy specimen obtained during the laparotomy was decreased. Although gemcitabine therapy was reinitiated, the disease progressed rapidly so the treatment was stopped. In this case, a correlation between the acquisition of resistance to gemcitabine therapy and change in HuR expression was demonstrated.

The 2017 revised World Health Organization classification of pancreatic neuroendocrine neoplasms classified conventional G3 tumors into well-differentiated (NET-G3) and poorly differentiated (NEC-G3) tumors. However, guidelines for selection of drug therapy were not established in the 2017 revision. This study aimed to elucidate the rates of maximum tumor reduction of sunitinib, progression-free survival, and overall survival in the new classification. We investigated the reduction rate over time using computed tomography for 60 patients with unresectable or distant metastatic pancreatic neuroendocrine neoplasms who received 37.5 mg of sunitinib in our department from April 2013 to November 2017. Of the 60 cases, 42, 10, and 5 were NET-G1/G2, NET-G3, and NEC-G3, respectively. The prognostic factors were analyzed according to clinicopathological factors using the Cox hazard model. The median observation period was 19 months, and the median duration of sunitinib administration was 7 months. The median maximum reduction rate of sunitinib was 18.3%. Tumor response was classified according to the Response Evaluation Criteria in Solid Tumors: 20 cases (33.3%) showed partial response, 29 cases (48.3%) showed stable disease, and 11 cases (18.3%) showed progressive disease. In a multivariate analysis of factors contributing to progression-free survival from the start of sunitinib administration, only histologically poor differentiation was a significant factor (p = 0.010). Progression-free survival and overall survival were significantly better in patients with NET-G3 than that in patients with NEC-G3 (p = 0.005, p = 0.012), while it was not different between those with NET-G3 and those with NET-G1/2. Our results indicate that sunitinib is as effective for NET-G3 as for NET-G1/2.

Decrease in blood concentration of branched-chain amino acids, especially leucine, is known to promote liver carcinogenesis in patients with chronic liver disease, but the mechanism is unclear. We herein established hepatocellular carcinoma (HCC) cells knocked out for DEPDC5 by using the CRISPR/Cas9 system, and elucidated that cell viability of the DEPDC5 knockout (DEPDC5-KO) cells was higher than that of the DEPDC5 wild-type (DEPDC5-WT) under leucine starvation. Considering that autophagy deficiency might be involved in acquired resistance to leucine deprivation, we observed reduction of LC3-II followed by accumulation of p62 in the DEPDC5-KO, which induced reactive oxygen species (ROS) tolerance. DEPDC5 overexpression suppressed cell proliferation and tumorigenicity in immunocompromised mice, and triggered p62 degradation with increased ROS susceptibility. In clinical specimens of HCC patients, decreased expression of DEPDC5 was positively correlated with p62 overexpression, and the progression-free (PFS) and overall survival (OS) were worse in the DEPDC5-negative cases than in the DEPDC5-positive. Moreover, multivariate analysis demonstrated DEPDC5 was an independent prognostic factor for both PFS and OS. Thus, DEPDC5 inactivation enhanced ROS resistance in HCC under the leucine-depleted conditions of chronic liver disease, contributing to poor patient outcome. It could be a potential target for cancer therapy with oxidative stress control.

BACKGROUND Some patients with pancreatic ductal adenocarcinoma (PDAC) demonstrate a reticular pattern around the superior mesenteric artery (SMA) in computed tomography scans. This study aimed to clarify the clinical significance of the reticular pattern in pancreatic head cancer. METHODS A total of 91 patients with pancreatic head cancer, who underwent upfront pancreaticoduodenectomy between 2004 and 2017, were included. Patients without reticular pattern (Non-group, n = 39); with reticular pattern around SMA (Ret-group, n = 39); and with soft tissue contact (Soft-group, n = 13) were compared. RESULTS Median overall survival (OS) of patients in the Ret-group was significantly worse than that in the Non-group (21.3 vs. 57.0 months; P

Genomic analyses have recently discovered the malignant subtype of human intrahepatic cholangiocarcinoma (ICC) characterized by frequent mutations of chromatin remodeling gene ARID1A; however, the biological and molecular functions still remain obscure. We here examined the clinical and biological significances of ARID1A deficiency in human ICC. Immunohistochemical analysis demonstrated that the loss of ARID1A was an independent prognostic factor for overall survival of ICC patients (P = 0.023). We established ARID1A-knockout (KO) cells by using the CRISPR/Cas9 system from two human cholangiocarcinoma cell lines. ARID1A-KO cells exhibited significantly enhanced migration, invasion, and sphere formation activity. Microarray analysis revealed that ALDH1A1, a stemness gene, was the most significantly elevated genes in ARID1A-KO cells. In addition, ALDH enzymatic activity as a hallmark of cancer stem cells was markedly high in the KO cells. ARID1A and histone deacetylase 1 were directly recruited to the ALDH1A1 promoter region in cholangiocarcinoma cells with undetectable ALDH1A1 expression by chromatin immunoprecipitation assay. The histone H3K27 acetylation level at the ALDH1A1 promoter region was increased in cells when ARID1A was disrupted (P < 0.01). Clinically, inverse correlation between ARID1A and ALDH1A1 expression was also identified in primary ICC (P = 0.018), and ARID1A-negative and ALDH1A1-positve ICCs showed worse prognosis than only ARID1A-negative cases (P = 0.002). In conclusion, ARID1A may function as a tumor suppressor in ICC through transcriptional downregulation of ALDH1A1 expression with decreasing histone H3K27 acetylation. Our studies provide the basis for the development of new epigenetic approaches to ARID1A-negative ICC. Immunohistochemical loss of ARID1A is an independent prognostic factor in intrahepatic cholangiocarcinoma patients. ARID1A recruits HDAC1 to the promoter region of ALDH1A1, a stemness gene, and epigenetically suppresses ALDH1A1 expression with decreasing histone H3K27 acetylation in cholangiocarcinoma cells.

Background The most reliable index to predict the safety of hepatectomy for patients with poor liver function remains unknown. We aimed to construct a novel preoperative index to predict early liver failure (ELF) and mortality due to recurrence-free liver failure (MLF) after hepatectomy. Methods Between 2000 and 2012, 385 consecutive patients with hepatocellular carcinoma undergoing curative minor hepatectomy were divided into 2 sequential cohorts: training set (n = 143) and validation set (n = 242), and observed until 2015. Results Prothrombin time and direct bilirubin were independent predictors of both ELF and MLF in the training set. Thus we devised a novel index, the direct bilirubin to prothrombin time ratio index (DBPTRI). The areas under ROC curves of DBPTRI for predicting ELF and MLF were 0.78 and 0.93, respectively, in the validation set. Using a preoperative DBPTRI cut off of 4.2, we accurately predicted ELF and MLF in 86.8% and 88.4% of patients, respectively. DBPTRI was the best predictor of ELF and MLF when compared with conventional indices such as ICG-R15 and Child-Pugh score. Moreover, the 5-year overall survival rates of the patients with low and high DBPTRI were 59% and 36%, respectively (P

BACKGROUND For the establishment of personalized therapy, we investigated biomarkers that can contribute to the selection of adjuvant therapy for pancreatic ductal adenocarcinoma (PDAC). METHODS Between 2005 and 2014, of 141 consecutive patients with PDAC who underwent R0 or R1 resection, 61 patients given gemcitabine and 31 patients given S-1 as adjuvant therapy were enrolled. We evaluated the correlation between treatment outcomes and the expressions of intratumoral human antigen R (HuR), human equilibrative nucleoside transporter 1 (hENT1), thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD). RESULTS There were no significant differences in clinicopathological features between the gemcitabine and S-1 groups. Among those with high HuR expression and high hENT1 expression, the disease-free survival (DFS) was significantly higher with gemcitabine than with S-1 (MST: 26.2 vs. 11.8 months, P = 0.024; 20.2 vs. 10.2 months, P = 0.029, respectively). Moreover, high HuR/hENT1 (high HuR or high hENT1) was significantly associated with better outcome for gemcitabine (HR for DFS: 0.43, P = 0.027) and low HuR/hENT1 was significantly associated with worse outcome for gemcitabine (HR for DFS: 2.24, P = 0.021). TS and DPD expression levels were not informative in this examination. CONCLUSIONS HuR and hENT1 were good candidates as selective biomarkers and this study's concept could contribute to personalized therapy for PDAC patients.

The incidence of hepatocellular carcinoma (HCC) associated with metabolic risk factors, such as diabetes and obesity, has been increasing. However, the underlying mechanism that links these diseases remains unclear. We performed genome-wide expression analysis of human liver tissues of non-viral HCC patients with or without metabolic risk factors. The upregulated genes that associated with diabetes and obesity were investigated by in vitro and in vivo experiments, and immunohistochemistry of human liver tissues was performed. Among the upregulated genes, connective tissue growth factor (CTGF) expression was induced to a greater extent by combined glucose and insulin administration to human hepatoma cells. Genome-wide expression analysis revealed upregulation of a chemokine network in CTGF-overexpressing hepatoma cells, which displayed an increased ability to induce in vitro activation of macrophages, and in vivo infiltration of liver macrophages. Immunohistochemistry of human liver tissues validated the correlations between CTGF expression and diabetes or obesity as well as activation of liver macrophages in patients with non-viral HCC. Recurrence-free survival was significantly poorer in the CTGF-positive patients compared with the CTGF-negative patients (p = 0.002). Multivariate analysis determined that CTGF expression (HR 2.361; 95 % CI 1.195–4.665; p = 0.013) and vascular invasion (HR 2.367; 95 % CI 1.270–4.410; p = 0.007) were independent prognostic factors for recurrence of non-viral HCC. Our data suggest that CTGF could be involved in oncogenic pathways promoting non-viral HCC associated with metabolic risk factors via induction of liver inflammation and is expected to be a novel HCC risk biomarker and potential therapeutic target.

OBJECTIVE To predict metachronous liver metastasis after pancreatectomy for pancreatic neuroendocrine neoplasms (Pan-NENs). SUMMARY OF BACKGROUND DATA Liver metastasis determines the prognosis of patients with Pan-NENs, but no index exists in the WHO 2017 classification for this prediction. METHODS Between April 2014 and March 2018, resected primary tumors from 20 patients with or without simultaneous liver metastasis were examined using genome-wide gene expression analysis. For validation analysis, resected primary tumors from 62 patients without simultaneous liver metastasis were examined for PAX6 expression. RESULTS Gene expression profiling revealed pancreatic beta cell genes (NES, -2.0; P < 0.001) as the most downregulated set in patients with simultaneous liver metastasis. In the test study, PAX6 was the most valuable index for liver metastasis (log FC, -3.683; P = 0.0096). Multivariate analysis identified PAX6 expression (hazard ratio, 0.2; P = 0.03) as an independent risk factor for metachronous liver metastasis-free survival (mLM-FS). The 5-year mLM-FS of patients with high versus low PAX6 expression was significantly better (95% vs 66%, respectively; P < 0.0001). The 5-year overall survival rate of was also better than in those with high versus low PAX6 expression (100% vs 87%, respectively). Patients with low PAX 6 expression were significantly younger and leaner, had a higher Ki-67 index (P = 0.01, 0.007, 0.008, respectively), and showed a higher mitotic rate than patients with high PAX6 expression. CONCLUSIONS Downregulated pancreatic beta cell genes involving PAX6 in primary tumors may predict mLM and poor overall survival after primary tumor resection in Pan-NEN patients.

Although genomic analysis have recently discovered the malignant subtype of human pancreatic ductal adenocarcinoma (PDAC) characterized by frequent mutations of histone demethylase KDM6A, the biological and molecular roles still remain obscure. We herein elucidated the clinical and biological impacts of KDM6A deficiency on human PDAC and identified the therapeutic potential by pathological and molecular evaluation. Immunohistochemical analysis suggested that loss of KDM6A in cancerous tissues was an independent prognostic factor for both recurrence-free and overall survival in the 103 tumor specimens surgically resected from patients with PDAC. We established KDM6A knocked out cells by using the CRISPR/Cas9 system and KDM6A-expressed cells by doxycycline-inducible system from each two human PDAC cell lines, respectively. KDM6A knockout enhanced aggressive traits of human PDAC cell lines, whereas KDM6A overexpression suppressed them. Microarray analysis revealed reduced expression of 22 genes including five well-known tumor suppressors, such as CDKN1A, and ChIP-PCR analysis displayed depleted enrichment of histone H3 lysine 27 acetylation (H3K27ac) at the promoter regions of the five candidates. The epigenetic alterations were induced by the impaired recruitment of histone acetyltransferase p300, which cooperatively interacted with KDM6A. Consistent with these results, the KDM6A knockout cells demonstrated higher vulnerability to histone deacetylase (HDAC) inhibitors through the reactivation of CDKN1A in vitro and in vivo than the KDM6A wild-type. In conclusion, KDM6A exhibited essential roles in human PDAC as a tumor suppressor and KDM6A deficiency could be a promising biomarker for unfavorable outcome in PDAC patients and a potential surrogate marker for response to HDAC inhibitors.

Pancreatic neuroendocrine tumors (PanNETs) have considerable malignant potential. Frequent somatic mutations and loss of DAXX protein expression have been found in PanNETs. DAXX is known as a transcriptional repressor; however, molecular functions underlying DAXX loss remain unclear in PanNETs. We evaluated DAXX expression by immunohistochemistry in 44 PanNETs.DAXX-knockdown (KD) and -knockout (KO) PanNET cells were analyzed forin vitroandvivo. The target genes were screened by microarray and chromatin immunoprecipitation (ChIP) assays for DAXX, histone H3.3 and H3K9me3 complex. In clinicopathological features, low DAXX expression was significantly correlated with nonfunctional tumors, higher Ki-67 index and WHO grade. Microarray and ChIP assays ofDAXX-KD/KO identified 12 genes as the direct targets of DAXX transcriptional repressor. Among them, expression of five genes including STC2 was suppressed by DAXX/H3.3/H3K9me3 pathway.DAXX-KD/KO cells enhanced sphere forming activity, but its effect was suppressed by knockdown ofSTC2. In xenograft models, tumorigenicity and tumor vessel density were significantly increased inDAXX-KO cells with high expression of STC2. Clinically, higher recurrence rate was recognized in PanNETs with low expression of DAXX and high expression of STC2 than others (P = 0.018). Our data suggest that DAXX plays as a tumor suppressor and DAXX/H3.3 complex suppresses target genes by promoting H3K9me3 in PanNETs. Combination of DAXX loss and its target gene STC2 overexpression might be effective biomarkers and therapeutic candidates.

Background and aim To improve the prognosis of cholangiocarcinoma, we investigated potential biomarkers that may enable the selection of patients for whom postoperative adjuvant chemotherapy is likely effective. Methods The cohort of this retrospective study included 170 surgically resected cholangiocarcinoma patients, 26 with gemcitabine adjuvant chemotherapy (GEM group), 36 with S-1 adjuvant chemotherapy (S-1 group), and 103 receiving no adjuvant chemotherapy (NC group). Propensity score matching was performed to adjust patient backgrounds; 36 patients from the NC group then were selected. Immunohistochemistry of orotate phosphoribosyltransferase (OPRT) and human equilibrative nucleoside transporter 1 (hENT1) was performed to determine the correlation between their expression and disease-free survival (DFS). Results After matching, the backgrounds of these three groups were unbiased. No significant improvement of DFS by adjuvant chemotherapy was observed in the whole cohort. However, among the high-OPRT-expression patients, DFS of GEM, S-1, and NC groups at 5 years was 28.8%, 53.8%, and 25.5%, respectively. The DFS of the S-1 group was significantly longer than that of the NC group (P = 0.034). On the other hand, no significant differences in DFS were observed among the low OPRT expression patients. hENT1 expression was shown to have no predictive value. Multivariate analysis of the high-OPRT-expression patients demonstrated that S-1 adjuvant chemotherapy can reduce tumor recurrence (HR, 0.303; P = 0.013). Conclusion Cholangiocarcinoma patients with high OPRT expression would benefit from postoperative adjuvant S-1 therapy, which increases the DFS. Assessment of OPRT expression may contribute to the optimization of adjuvant chemotherapy for cholangiocarcinoma.

Identification and purification of cancer stem cells (CSCs) lead to the discovery of novel therapeutic targets; however, there has been no study on isolation of the CSC population among pancreatic neuroendocrine tumors (pNETs). This study aimed to identify pNET CSCs and to characterize a therapeutic candidate for pNET CSCs. We identified CSCs by aldehyde dehydrogenase (ALDH) activity in pNET clinical specimens and cell lines. We verified whether or not these cells have the stemness property in vivo and in vitro. ALDHhigh cells, but not control bulk cells, formed spheres, proliferated under hypoxic condition as well as normoxic condition and promoted cell motility, which are features of CSCs. Injection of as few as 10 ALDHhigh cells led to subcutaneous tumor formation, and 105 ALDHhigh cells, but not control bulk cells, established metastases in mice. Comprehensive gene expression analysis revealed that genes associated with mesenchymal stem cells, including CD73, were overexpressed in ALDHhigh cells. Additionally, the in vitro and in vivo effects of an inhibitor of CD73 were investigated. The CD73 inhibitor APCP significantly attenuated in vitro sphere formation and cell motility, as well as in vivo tumor growth observed for ALDHhigh cells. Finally, its expression was evaluated using clinical pNET tissue samples. Immunohistochemical analysis of clinical tissue samples demonstrated CD73 expression was significantly correlated with the invasion into adjacent organs. Since recent studies revealed CD73 as a potential biomarker of anti-PD-1 immune checkpoint therapy, CD73 might be a promising therapeutic target for pNET CSCs.

Background Although hepatocellular carcinoma (HCC) is mostly a lower intensity lesion in the hepatobiliary phase on gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid-enhanced magnetic resonance imaging, some HCCs were shown as a higher intensity lesion (high HCC). This study aimed to reveal the clinicopathological and biological properties of high HCC. Methods Patients who underwent curative hepatectomy as the first treatment for HCC were included. HCC was defined as high HCC if the ratio between the signal intensity of the HCC and the background liver was greater than or equal to 1.0. We retrospectively performed clinicopathological and global gene expression analyses. Results Of the 77 patients, 14 had high HCC. Serum protein induced by vitamin K absence or antagonist II levels in high HCC were lower, and the high HCCs were well differentiated. The 3-year disease-free survival rates in high HCC and low HCC patients were 90% and 54%, respectively (P = .035). Overall survival did not differ significantly. Global gene expression analysis revealed that SLCO1B3 was upregulated in high HCC. Conclusions Clinicopathological analysis revealed low-grade malignancy in high HCCs compared with low HCCs. The expression of SLCO1B3 was key to the hyperintensity in the hepatobiliary phase of ethoxybenzyl-diethylenetriamine pentaacetic acid magnetic resonance imaging.

We report the first case of initially unresectable advanced hepatocellular carcinoma (HCC) with portal vein and hepatic venous tumor thrombosis and multiple lung metastases that allowed for curative hepatectomy after multidisciplinary treatment including sorafenib. A 54-year-old male presented with a large HCC in the right liver with tumor thrombosis of the left portal vein and middle hepatic vein (MHV) as well as multiple lung metastases. His serum alpha-fetoprotein level was elevated at 52,347 ng/mL and palliative treatment with sorafenib was initiated. One month later, a significant reduction in the serum AFP level, decrease in the tumor size with recanalization of the portal vein and the absence of lung metastases were noted. Three months after the start of sorafenib treatment, external-beam radiotherapy was performed to treat enlargement of the area of MHV thrombosis, and the thrombosis regressed. Five months after the initiation of sorafenib treatment, central bisegmentectomy associated with removal of the tumor thrombus in the inferior vena cava was performed. A microscopic examination revealed complete necrosis of the tumor. Sorafenib treatment may be a bridge to curative resection in selected patients with initially unresectable advanced HCC, even in cases involving multiple extrahepatic metastases.

Grading of difficulty is needed for laparoscopic liver resection (LLR). Indications for LLR are expanding worldwide from minor to major resections, particularly in institutions having surgeons with advanced skills. If the degrees of surgical difficulty were defined, it would serve as a useful guide when introducing LLR and stepping up to the more advanced LLR. As no previous study has addressed the degrees of difficulty of various LLR procedures, we devised a practical scoring system for this purpose. We extracted the following five factors from preoperative information to score difficulty levels: (1) tumor location, (2) extent of liver resection, (3) tumor size, (4) proximity to major vessels, and (5) liver function. This difficulty index is comprised of the cumulative score for the five individual factors. There has not yet been a standard definition of difficulty. Our proposed scoring system might be a practical means of assessing the difficulty of LLR procedures. However, this system must be prospectively validated.

Bile duct injury is one of the known serious complications of laparoscopic fenestration for nonparasitic liver cysts. Herein, we report the case of a huge liver cyst for which we performed laparoscopic fenestration using intraoperative fluorescent cholangiography with indocyanine green. A 71-year-old woman with abdominal distention was referred to our hospital. CT demonstrated a 17 × 11.5-cm simple cyst replacing the right lobe of the liver, so laparoscopic fenestration was performed. Although the biliary duct could not be detected because of compression by the huge cyst, fluorescent cholangiography with indocyanine green through endoscopic naso-biliary drainage tube clearly delineated the intrahepatic bile duct in the remaining cystic wall. The patient had no complications at 3 months after surgery. Fluorescent cholangiography using indocyanine green is a safe and effective procedure to avoid bile duct injury during laparoscopic fenestration, especially in patients with a huge liver cyst.

Metabolic syndrome is a newly identified risk factor for hepatocellular carcinoma (HCC); however, tumor-specific biomarkers still remain unclear. We performed cross-species analysis to compare gene signatures of HCC from human patients and melanocortin 4 receptor-knockout mice, which develop HCC with obesity, insulin resistance, and dyslipidemia. Unsupervised hierarchical clustering and principle component analysis of 746 differentially expressed orthologous genes classified HCC of 152 human patients and melanocortin 4 receptor-knockout mice into two distinct subgroups, one of which included mouse HCC and was causatively associated with metabolic risk factors. Nine genes commonly overexpressed in human and mouse metabolic disease-associated HCC were identified; fatty acid binding protein 4 (FABP4) was remarkably enriched in intratumoral activated hepatic stellate cells (HSCs). Subclones constitutively expressing FABP4 were established from a human HSC cell line in which expression levels of inflammatory chemokines, including IL-1A and IL-6, were up-regulated through NF-κB nuclear translocation, resulting in recruitment of macrophages. An immunohistochemical validation study of 106 additional human HCC samples indicated that FABP4-positive HSCs were distributed in tumors of 38 cases, and the FABP4-high group consisted of patients with nonviral and nonalcoholic HCC (P = 0.027) and with multiple metabolic risk factors (P

The use of laparoscopic distal pancreatectomy (LDP) is increasing worldwide. It is important for surgeons to predict preoperatively the difficulty and risks of a surgery. However, very few reports have evaluated the impact of patient or tumor factors on the difficulty of LDP. We aimed to determine the predictors of technical difficulties when performing LDP.This study included 34 patients who underwent LDP. Patient information was obtained retrospectively and included age, gender, BMI, primary disease, previous abdominal surgery, previous pancreatitis, tumor size, tumor proximity to the splenic arterial origin, type of splenic artery (SpA), operative time, blood loss, postoperative pancreatic fistula, and length of hospital stay. Univariate and multivariate analyses were performed to determine the predictors of a long operative time. SpA anatomy was classified into two types based on the relationship between its origin and the pancreas. Patients whose SpA origin was upward of the pancreatic parenchyma were classified as SpA type 1, whereas patients whose SpA origin was covered by the pancreatic parenchyma were classified as SpA type 2.Multivariate analysis revealed SpA type 2 to be an independent risk factor for a long operation (odds ratio = 9.925; 95% confidence interval: 1.461-67.412; P = 0.019). SpA type 2 was related to a longer operative time (P 0.001) and greater intraoperative blood loss (P = 0.001).Classification according to SpA type is simple and useful for predicting technical difficulty when performing LDP.

A novel morphological classification using resected specimens predicted malignant potential and prognosis in patients with pancreatic neuroendocrine tumors (P-NETs). The aim of this study was to examine the predictive ability of morphological diagnoses made using non-invasive multi-detector computed tomography (MDCT) in P-NETs. Between 2002 and 2015, 154 patients were diagnosed with P-NETs at the Tokyo Medical and Dental University, and 82 patients who underwent surgical treatment were enrolled. The primary tumors were classified by MDCT into three types: Type I, simple nodular tumor; Type II, simple nodular tumor with extra-nodular growth; and Type III, confluent multinodular tumor. Patients were stratified by 15 clinical specialists according to classification and without any other clinical or pathological information. Clinicopathological features and patient survival were reviewed retrospectively. The mean observation time was 1004 days. Forty-six, 22, and 14 patients had Type I, II, and III tumors, respectively. Morphological classification was significantly correlated with advanced features such as tumor size, Ki-67 index, and synchronous liver metastasis (p

Abnormal tumor vascularity is one of the typical features of hepatocellular carcinoma (HCC). In this study, the significance of contrast-enhanced intraoperative ultrasonography (CEIOUS) images of HCC vasculature was evaluated by clinicopathological and gene expression analyses. We enrolled 82 patients who underwent curative hepatic resection for HCC with CEIOUS. Clinicopathological and gene expression analyses were performed according to CEIOUS vasculature patterns. CEIOUS images of HCC vasculatures were classified as reticular HCC or thunderbolt HCC. Thunderbolt HCC was significantly correlated with higher alpha-fetoprotein levels, tumor size, histological differentiation, portal vein invasion, and tumor-node-metastasis stage, and these patients demonstrated a significantly poorer prognosis for both recurrence-free survival (P = 0.0193) and overall survival (P = 0.0362) compared with patients who had reticular HCC. Gene expression analysis revealed that a rereplication inhibitor geminin was significantly overexpressed in thunderbolt HCCs (P = 0.00326). In vitro knockdown of geminin gene reduced significantly the proliferation of human HCC cells. Immunohistochemical analysis confirmed overexpression of geminin protein in thunderbolt HCC (P < 0.0001). Multivariate analysis revealed geminin expression to be an independent factor in predicting poor survival in HCC patients (P = 0.0170). Conclusion: CEIOUS vascular patterns were distinctly identifiable by gene expression profiling associated with cellular proliferation of HCC and were significantly related to HCC progression and poor prognosis. These findings might be clinically useful as a determinant factor in the postoperative treatment of HCC. (HEPATOLOGY 2013)

Antiangiogenic therapy is initially effective for several solid tumors including hepatocellular carcinoma; however, they finally relapse and progress, resulting in poor prognosis. We here established in vivo drug-tolerant subclones of human hepatocellular carcinoma cells by long-term treatment with VEGF receptor (VEGFR) inhibitor and serial transplantation in immunocompromised mice (total 12 months), and then compared them with the parental cells in molecular and biological features. Gene expression profiles elucidated a G-actin monomer binding protein thymosin β 4 (Tβ4) as one of the genes enriched in the resistant cancer cells relative to the initially sensitive ones. Highlighting epigenetic alterations involved in drug resistance, we revealed that Tβ4 could be aberrantly expressed following demethylation of DNA and active modification of histone H3 at the promoter region. Ectopic overexpression of Tβ4 in hepatocellular carcinoma cells could significantly enhance sphere-forming capacities and infiltrating phenotypes in vitro, and promote growth of tumors refractory to the VEGFR multikinase inhibitor sorafenib in vivo. Clinically, sorafenib failed to improve the progression-free survival in patients with Tβ4-high hepatocellular carcinoma, indicating that Tβ4 expression could be available as a surrogate marker of susceptibility to this drug. This study suggests that Tβ4 expression triggered by epigenetic alterations could contribute to the development of resistance to antiangiogenic therapy by the acquisition of stemness, and that epigenetic control might be one of the key targets to regulate the resistance in hepatocellular carcinoma. Mol Cancer Ther; 16(6); 1155–65. ©2017 AACR.

The most reliable index to predict the safety of hepatectomy for patients with poor liver function remains unknown. We aimed to construct a novel preoperative index to predict early liver failure (ELF) and mortality due to recurrence-free liver failure (MLF) after hepatectomy.Between 2000 and 2012, 385 consecutive patients with hepatocellular carcinoma undergoing curative minor hepatectomy were divided into two sequential cohorts: training set (n = 143) and validation set (n = 242), and observed until 2015.Prothrombin time and direct bilirubin were independent predictors of both ELF and MLF in the training set. Thus we devised a novel index, the direct bilirubin to prothrombin time ratio index (DBPTRI). The areas under ROC curves of DBPTRI for predicting ELF and MLF were 0.78 and 0.93, respectively, in the validation set. Using a preoperative DBPTRI cut off of 4.2, we accurately predicted ELF and MLF in 86.8% and 88.4% of patients, respectively. DBPTRI was the best predictor of ELF and MLF when compared with conventional indices such as ICG-R15 and Child-Pugh score. Moreover, the 5-year overall survival rates of the patients with low and high DBPTRI were 59% and 36%, respectively (P 0.0001).DBPTRI may serve as a simple, non-invasive index for estimating liver failure after hepatectomy.

For the establishment of personalized therapy, we investigated biomarkers that can contribute to the selection of adjuvant therapy for pancreatic ductal adenocarcinoma (PDAC).Between 2005 and 2014, of 141 consecutive patients with PDAC who underwent R0 or R1 resection, 61 patients given gemcitabine and 31 patients given S-1 as adjuvant therapy were enrolled. We evaluated the correlation between treatment outcomes and the expressions of intratumoral human antigen R (HuR), human equilibrative nucleoside transporter 1 (hENT1), thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD).There were no significant differences in clinicopathological features between the gemcitabine and S-1 groups. Among those with high HuR expression and high hENT1 expression, the disease-free survival (DFS) was significantly higher with gemcitabine than with S-1 (MST: 26.2 vs. 11.8 months, P = 0.024; 20.2 vs. 10.2 months, P = 0.029, respectively). Moreover, high HuR/hENT1 (high HuR or high hENT1) was significantly associated with better outcome for gemcitabine (HR for DFS: 0.43, P = 0.027) and low HuR/hENT1 was significantly associated with worse outcome for gemcitabine (HR for DFS: 2.24, P = 0.021). TS and DPD expression levels were not informative in this examination.HuR and hENT1 were good candidates as selective biomarkers and this study's concept could contribute to personalized therapy for PDAC patients.

Background & Aims Recent genomic studies have identified frequent mutations of AT-rich interactive domain 2 ( ARID2 ) in hepatocellular carcinoma (HCC), but it is not still understood how ARID2 exhibits tumor suppressor activities. Methods We established the ARID2 knockout human HCC cell lines by using CRISPR/Cas9 system, and investigated the gene expression profiles and biological functions. Results Bioinformatic analysis indicated that UV-response genes were negatively regulated in the ARID2 knockout cells, and they were sensitized to UV irradiation. ARID2 depletion attenuated nucleotide excision repair (NER) of DNA damage sites introduced by exposure to UV as well as chemical compounds known as carcinogens for HCC, benzo[a]pyrene and FeCl 3 , since xeroderma pigmentosum complementation group G (XPG) could not accumulate without ARID2. By using large-scale public data sets, we validated that ARID2 knockout could lead to similar molecular changes between in vitro and in vivo settings. A higher number of somatic mutations in the ARID2 -mutated subtypes than that in the ARID2 wild-type across various types of cancers including HCC was observed. Conclusions We provide evidence that ARID2 knockout could contribute to disruption of NER process through inhibiting the recruitment of XPG, resulting in susceptibility to carcinogens and potential hypermutation. These findings have implications for therapeutic targets in cancers harboring ARID2 mutations. Lay summary Recent genomic studies have identified frequent mutations of ARID2, a component of the SWItch/Sucrose Non-Fermentable (SWI/SNF) complex, in hepatocellular carcinoma, but it is not still understood how ARID2 exhibits tumor suppressor activities. In current study, we provided evidence that ARID2 knockout could contribute to disruption of DNA repair process, resulting in susceptibility to carcinogens and potential hypermutation. These findings have far-reaching implications for therapeutic targets in cancers harboring ARID2 mutations.

Proteasome activity is significantly increased in advanced cancers, but its role in cancer initiation is not clear, due to difficulties in monitoring this process in vivo. We established a line of transgenic mice that carried the ZsGreen-degronODC (Gdeg) proteasome reporter to monitor the proteasome activity. In combination with Pdx-1-Cre;LSL-KrasG12D model, proteasome activity was investigated in the initiation of precancerous pancreatic lesions (PanINs). Normal pancreatic acini in Gdeg mice had low proteasome activity. By contrast, proteasome activity was increased in the PanIN lesions that developed in Gdeg;Pdx-1-Cre;LSL-KrasG12D mice. Caerulein administration to Gdeg;Pdx-1-Cre;LSL-KrasG12D mice induced constitutive elevation of proteasome activity in pancreatic tissues and accelerated PanIN formation. The proteasome inhibitor markedly reduced PanIN formation in Gdeg;Pdx-1-Cre;LSL-KrasG12D mice (P = 0.001), whereas it had no effect on PanIN lesions that had already formed. These observations indicated the significance of proteasome activity in the initiation of PanIN but not the maintenance per se. In addition, the expressions of pERK and its downstream factors including cyclin D1, NF-κB, and Cox2 were decreased after proteasome inhibition in PanINs. Our studies showed activation of proteasome is required specifically for the initiation of PanIN. The roles of proteasome in the early stages of pancreatic carcinogenesis warrant further investigation.

Pancreatic neuroendocrine neoplasm (Pan-NEN) representing approximately 1.3 % of pancreatic malignancy cases in incidence has been a so rare disease that it remains major problem to analyze the malignant potential. The aim of this study was to verify whether the macroscopic morphology of Pan-NEN, a novel pathological classification, contributes to malignant potential. From a total of 86 patients with Pan-NEN, 41 surgical sections obtained from the primary site were classified by their morphology into a simple nodular (SN) group and a non-SN group. The non-SN group was further divided into three subtypes: simple nodular with extranodular growth (SNEG), confluent multinodular (CM), and infiltrative (IF). The clinicopathological features of the SN and the non-SN groups were retrospectively compared. Overall 5-year survival rates with and without surgical resection were 94 and 48 %, respectively. SN and non-SN types were identified in 21 and 20 patients, respectively. The non-SN group comprised 14 SNEG type, 2 CM type, and 4 IF type. Synchronous lymph node metastases (p = 0.009), synchronous liver metastases (p = 0.048), microinvasion to an adjacent organ (p

Patients with pancreatic cancer typically develop tumor invasion and metastasis in the early stage. These malignant behaviors might be originated from cancer stem cells (CSCs), but the responsible target is less known about invisible CSCs especially for invasion and metastasis. We previously examined the proteasome activity of CSCs and constructed a real-time visualization system for human pancreatic CSCs. In the present study, we found that CSCs were highly metastatic and dominantly localized at the invading tumor margins in a liver metastasis model. Microarray and siRNA screening assays showed that doublecortin-like kinase 1 (DCLK1) was predominantly expressed with histone modification in pancreatic CSCs with invasive and metastatic potential. Overexpression of DCLK1 led to amoeboid morphology, which promotes the migration of pancreatic cancer cells. Knockdown of DCLK1 profoundly suppressed in vivo liver metastasis of pancreatic CSCs. Clinically, DCLK1 was overexpressed in the metastatic tumors in patients with pancreatic cancer. Our studies revealed that DCLK1 is essential for the invasive and metastatic properties of CSCs and may be a promising epigenetic and therapeutic target in human pancreatic cancer.

Postoperative early recurrence is a crucial issue in the treatment of hepatocellular carcinoma (HCC) patients. Some early recurrences seem to occur from minute tumors which were overlooked during both preoperative and intraoperative investigations. Therefore, it is urgently necessary to increase detectability of minute HCCs during operation. If they could be detected and resected during surgery, the prognosis should be improved. The purpose of this study is to investigate the usefulness of contrast-enhanced intraoperative ultrasound (CEIOUS) for the diagnosis and treatment of HCC. Institutional ethics committee approval and informed consent were obtained. Fifty-two patients (mean age 65 years; 38 males and 14 females) who underwent liver resection with either preoperative computed tomography during angiography (CTA) or CEIOUS with Sonazoid (perflubutane microbubble contrast agent) were studied. We determined the presence of HCC on the basis of the histopathological findings of resected specimens. The sensitivity of CEIOUS [97.6% (95% CI 91.8–99.4)] was higher than that of CTA [89.4% (95% CI 81.1–94.3)]. The positive predictive values of CEIOUS [91.2% (95% CI 83.6–95.5) and CTA [91.6% (95% CI 83.6–95.9)] were similar. Eight new HCCs from 7 patients, which accounted for 9.4% (8/85) of the total HCCs, were correctly detected and diagnosed by CEIOUS, and we performed an additional partial hepatectomy in 3 of these 7 patients. CEIOUS with Sonazoid provided increased sensitivity of detection of small HCCs compared with preoperative CTA, thereby leading to a more appropriate surgical procedure and contributing to complete tumor removal.

症例は34歳，男性．原発性アルドステロン症に対する左副腎摘出時，偶然肝外側区域の腫瘤を指摘され当科を受診．血液検査上，肝炎ウィルスマーカーは陰性，肝機能にも異常を認めなかった．腹部画像所見では，肝外側区域の腫瘤は約6cm大であり，S3のグリソンが腫瘤内を貫通し，それにより腹側部と背側部，2つの領域に分けられる雪だるま状の形態を呈していた．MRI上両者の造影パターンは異なり，腹側部は中心性瘢痕がみられFNHと診断したが，背側部は腹側に比べ，血管造影CT上早期濃染が強く，悪性疾患も考慮され質的診断には至らなかった．手術は肝外側区域切除を行った．切除標本の割面では，腹側部は中心性瘢痕を伴う黄白色調の病変であり組織学的にFNHと診断した．背側部は黄緑色調であり，組織学的には，一層の肝細胞索と拡張した類洞が認められFNHの亜型である血管拡張型FNHと診断した．両者が線維性被膜を介して接するように併存する非常に稀な病変であった．

Background & Aims We previously identified that high Aurora B expression was associated with hepatocellular carcinoma (HCC) recurrence due to tumor dissemination. In this preclinical study, a novel inhibitor of Aurora B kinase was evaluated as a treatment for human HCC. Methods AZD1152 is a selective inhibitor of Aurora B kinase. Twelve human HCC cell lines were analyzed for Aurora B kinase expression and the in vitro effects of AZD1152. The in vivo effects of AZD1152 were analyzed in a subcutaneous xenograft model and a novel orthotopic liver xenograft model. Results Aurora B kinase expression varied among the human HCC cell lines and was found to correlate with inhibition of cell proliferation, accumulation of 4N DNA, and the proportion of polyploid cells following administration of AZD1152-hydroxyquinazoline-pyrazol-anilide (AZD1152-HQPA). AZD1152-HQPA suppressed histone H3 phosphorylation and induced cell death in a dose-dependent manner. Growth of subcutaneous human HCC xenografts was inhibited by AZD1152 administration. In an orthotopic hepatoma model, treatment with AZD1152 significantly decelerated tumor growth and increased survival. Pharmacobiological analysis revealed that AZD1152 induced the rapid suppression of phosphohistone H3, followed by cellular apoptosis in the liver tumors but not in the normal tissues of the orthotopic models. Conclusions Our preclinical studies indicate that AZD1152 is a promising novel therapeutic approach for the treatment of HCC.

Here we described our strategies to attain a better prognosis for hepatocellular carcinoma (HCC) patients by surgery. Among a variety of attempts conducted to date, we focused on anatomical resection and intraoperative contrast-enhanced ultrasonography. There are still controversies with respect to the significance of anatomical resection. We analyzed the significance of this surgical procedure in 207 patients without macrovascular invasion. These patients underwent either anatomical resection or non-anatomical resection. We found that the patients with anatomical resection had higher recurrence-free survival rate than those with non-anatomical resection. Univariable analysis showed that liver damage, the serum level of α-fetoprotein, tumor number, surgical margin, and type of surgery (anatomical or non-anatomical resection) were significant predictive factors for intrahepatic recurrence. Multivariable analysis revealed that multiple tumors, α-fetoprotein, and non-anatomical resection were independent risk factors for recurrence. We conclude that anatomical resection is a recommendable surgical procedure in patients without macrovascular invasion. A recent innovation is the development of contrast-enhanced ultrasonography. Then we have applied this to liver surgery intraoperatively. We confirm that vascular images contribute to a precise diagnosis and the detection of small portal tumor thrombi, and that Kupffer images are useful to discover the minute tumors. In addition, by clarifying the relationship between tumors and the vascular architecture, real-time 3-dimensional images using Kupffer imaging are a promising guide during the surgical procedures, although further development is needed.

Bile duct injury is one of the known serious complications of laparoscopic fenestration for nonparasitic liver cysts. Herein, we report the case of a huge liver cyst for which we performed laparoscopic fenestration using intraoperative fluorescent cholangiography with indocyanine green. A 71-year-old woman with abdominal distention was referred to our hospital. CT demonstrated a 17 × 11.5-cm simple cyst replacing the right lobe of the liver, so laparoscopic fenestration was performed. Although the biliary duct could not be detected because of compression by the huge cyst, fluorescent cholangiography with indocyanine green through endoscopic naso-biliary drainage tube clearly delineated the intrahepatic bile duct in the remaining cystic wall. The patient had no complications at 3 months after surgery. Fluorescent cholangiography using indocyanine green is a safe and effective procedure to avoid bile duct injury during laparoscopic fenestration, especially in patients with a huge liver cyst.