Safety and efficacy of cold snare polypectomy for small colorectal polyps: A prospective randomized control trial and one-year follow-up study.

TRIAL DESIGN: Elimination of small colorectal polyps with cold snare polypectomy (CSP) is reported to be as safe as hot snare polypectomy (HSP). The effectiveness of CSP has not been clearly defined, and the incidence of long-term recurrence has not been determined. We conducted a randomized control study and one-year follow-up study to assess their safety and efficacy. METHODS: Patients with small colorectal polyps were randomized to receive CSP or HSP. Polypectomy was performed to determine the pathological curability, and patients completed a questionnaire about the tolerability of the procedure. Follow-up colonoscopy was performed to determine the local recurrence of adenoma. The major outcome was the non-inferiority of CSP to HSP in the rate of delayed bleeding and minor outcomes, including the incidence of immediate bleeding and perforation, procedural time, and the resection rate. RESULTS: A total of 119 participants were recruited in this randomized study and underwent polypectomy. Among the 458 polyps, 332 eligible polyps were analyzed. The rate of adverse events was 0.6% (1/175) for CSP and 0% (0/157) for HSP, which showed the non-inferiority of CSP. While the complete resection rate of CSP was very high (100%), the R0 rate was not satisfactory (horizontal margin, 65.5%; vertical margin, 89.1%). Two local recurrences (2.5%) were observed in the follow-up of 80 adenomas treated with CSP. No recurrence was found in 79 lesions in the HSP group, which was not significant (P = .06). CONCLUSIONS: Colorectal polyps were safely resected using CSP, similar to HSP. Most would agree to say that CSP is considered safer than HSP. The main question is then related to efficacy. Our results of the present study demonstrate that recurrence after CSP should be carefully managed for curative treatment.

High Response Rate and Prolonged Survival of Unresectable Biliary Tract Cancer Treated With a New Combination Therapy Consisting of Intraarterial Chemotherapy Plus Radiotherapy.

SYNOPSIS: A new combination therapy consisting of intraarterial chemotherapy plus radiotherapy was demonstrated to have the potential to improve the response rate and survival time in patients with unresectable biliary tract cancer. PURPOSE: We retrospectively investigated the effectiveness and safety of a new combination therapy consisting of intraarterial chemotherapy plus radiation therapy (AI+RT), which may have the potential to improve unresectable biliary tract cancer (BTC). METHODS: We retrospectively reviewed 52 BTC cases treated with AI+RT and analyzed the anti-tumor effect, survival time and adverse events. The AI+RT regimen consisted of one-shot intraarterial chemotherapy (AI) at the first angiography session, almost 6 months of reservoir AI (5-FU and cisplatin, q/week) and external radiation with a maximum dose of 50.6 Gy. RESULTS: The response rate and disease control rate were high, at 40.4% and 96.2%, respectively, and the median overall and progression-free survival time were 463 and 431 days; thus, long-term survival was achieved. A univariate analysis identified 12 prognostic factors, and a performance status of 2 (hazard ratio [HR]: 4.82, p=0.02), jaundice (HR: 3.22, p<0.01), peritoneal dissemination (HR: 22.5, p<0.01), number of AI (HR: 0.35, p=0.01) and response to AI+RT (HR: 0.23, p<0.01) were extracted as significant prognostic factors in a multivariate analysis. The following: grade ≥3 adverse events occurred: leucopenia (11.5%), neutropenia (1.9%), anemia (15.4%), thrombocytopenia (11.5%), anorexia (3.8%), gastroduodenal ulcer (25.0%), and cholangitis (23.1%). There were no cases of treatment-related death. CONCLUSIONS: AI+RT was shown to contribute to a high response rate and prolonged survival in patients with unresectable BTC. A sufficient number of AI and the response to this therapy were thought to be significant prognostic factors in patients receiving AI+RT. Advances in multidisciplinary therapies, such as AI+RT, which was described in the present study, are also considered to be important for the future.

Efficacy of Vonoprazan-Based Triple Therapy for Helicobacter pylori Eradication: A Multicenter Study and a Review of the Literature.

BACKGROUND: Eradication therapies for Helicobacter pylori infection are advancing as new acid inhibitory reagents approved. The aim of this study was to assess the efficacy and safety of vonoprazan-based triple treatment. MATERIALS AND METHODS: Triple therapy with vonoprazan and two antibiotics (amoxicillin and clarithromycin or metronidazole) received focus in this analysis. We performed a multicenter retrospective study of patients who received vonoprazan-based eradication therapy between February 2015 and February 2016 and conducted a review of the literature. RESULTS: The eradication rate among the 799 patients in our multicenter study was 94.4% (95% confidence interval [CI] 92.6-96.2%) in the per-protocol analysis for first-line treatment (with vonoprazan 20 mg, amoxicillin 750 mg, and clarithromycin 200 or 400 mg, twice a day for 7 days) and 97.1% (95% CI 93.0-101.1%) for second-line treatment (with vonoprazan 20 mg, amoxicillin 750 mg, and metronidazole 250 mg, twice a day for 7 days). The overall incidence of adverse events was 4.4% in an intention-to-treat analysis with no patients hospitalized. In a literature review, six reports, in which 1380 patients received vonoprazan-based first-line eradication therapy, were included and were all reported by Japanese researchers. The eradication success rates in per-protocol analysis were between 85 and 93%, which was roughly the same among the studies. CONCLUSIONS: Vonoprazan-based triple therapy was effective and safe for Helicobacter pylori eradication in real-world experience, confirmed by a multicenter study and a review of the pertinent literature.

Endoscopic Submucosal Dissection for Depressed-type Early Adenocarcinoma of the Terminal Ileum.

We herein report a rare case of ileal adenocarcinoma that was completely removed by endoscopic submucosal dissection (ESD) without any complications. An 80-year-old man was referred to our hospital to undergo treatment for an ileal tumor. Conventional colonoscopy showed a reddish depressed lesion that was classified as type 0-IIc according to the Paris classification. The ileal tumor was successfully removed en bloc by ESD with a negative surgical margin. The histological findings showed a well-differentiated adenocarcinoma with no submucosal or lymphovascular invasion. Colonoscopy and CT performed one year after ESD showed no local recurrence, stenosis, or lymph node metastasis.

A Rare Case of Epidermoid Cyst in the Pancreatic Tail Invaginated from the Splenic Hilum: The Long-term Changes in the Imaging Findings.

Epidermoid cysts arising from both the pancreas and spleen are rare. We herein report a case of a surgically resected epidermoid cyst in the pancreatic tail invaginated from the spleen. A multi-locular cyst, 2 cm in diameter, without a solid component was discovered incidentally in the pancreatic tail. During the 11-year follow-up, the emergence of satellite cystic lesions with distinct appearances was seen, and surgical resection was selected despite the lack of any associated symptoms or evidence of cytological abnormalities. Histologically, these cysts were lined with benign multi-layered flattened epithelium surrounded by a thin layer consisting of cells positive for CD8 and CD68 and connecting to the spleen.

Primary Poorly Differentiated Squamous Cell Carcinoma of the Extrahepatic Bile Duct.

Squamous cell carcinoma of the extrahepatic bile duct is quite rare. A 77-year-old woman with jaundice and general fatigue was referred to our hospital. Multiphase contrast-enhanced computed tomography visualized a 17-mm solid mass in the junction of the cystic and common bile ducts. The patient underwent pylorus-preserving pancreaticoduodenectomy. The pathological findings demonstrated keratin-positive poorly differentiated squamous cell carcinoma of the extrahepatic bile duct (T3N0M0, stage IIIA). Although adjuvant chemotherapy with gemcitabine was administered, the patient exhibited local recurrence at the site of anastomosis of biliojejunostomy 20 months after resection and died 32 months after resection.

Infection with fully mature Corynosoma cf. validum causes ulcers in the human small intestine.

Corynosoma is a parasite that can normally be found in the intestinal tract of fish-eating mammals, particularly in seals and birds. The present case proposed that Corynosoma could attain full maturity in the human intestine. A 70-year-old female complained of abdominal pain. A computed tomography (CT) scan revealed a swelling of the intraperitoneal lymph nodes with no responsible lesion. Video capsule endoscopy and double-balloon endoscopy detected several ulcerations and one parasite in the ileum, which was tightly attached at the bottom of the ulcerations. The parasite was cylindrical and measured approximately 10 mm (long) x 3 mm (wide). Pathologically, the worm had a four-layered body wall and contained embryonated eggs. The sequences of the parasite-derived nuclear ribosomal DNA fragment and mitochondrial DNA fragment of cox1 were almost identical to those of Corynosoma validum. The patient's abdominal pain immediately improved after the administration of pyrantel pamoate (1,500 mg). Corynosoma was possibly the responsible disease in a patient who complained of abdominal pain and in whom no responsible lesion was detected by CT, gastroduodenoscopy or colonoscopy. Examinations of the small intestines should be aggressively performed in such cases.

Multiple Cancers of the Biliary Tract and Pancreatic Duct after Cholecystectomy for Gallbladder Cancer in a Patient with Pancreaticobiliary Maljunction.

We herein report the rare case of a 76-year-old woman who underwent cholecystectomy with bile duct resection for advanced gallbladder cancer associated with pancreaticobiliary maljunction (PBM) and subsequently developed multiple cancers of the pancreaticobiliary system (the distal bile duct, intrahepatic duct and pancreatic duct) after the operation. We performed conventional endoscopic retrograde cholangiopancreatography (ERCP) using a side-viewing scope to evaluate the masses in the distal bile duct and the pancreatic duct. We also performed ERCP using double-balloon enteroscopy (DBE) to observe the mass in the intrahepatic duct. It was possible to directly observe the lesion using DBE and to perform a biopsy under visual control. All lesions were correctly diagnosed by the combination of ERCP using different endoscopes. The present case suggests that it is necessary to pay close attention (with regard to carcinogenesis) to the whole pancreaticobiliary system in patients with PBM. In addition, the combination of ERCP using DBE and a side-viewing scope may be useful for making a precise diagnosis in patients with altered biliary anatomy who have multiple cancers of the pancreaticobiliary system.

Expression of insulin-like growth factor I receptor as a biomarker for predicting prognosis in biliary tract cancer patients.

Carcinomas of the gallbladder (GBCa) and bile ducts are aggressive tumors with poor survival and it is, therefore, essential to elucidate the molecular mechanisms of the various signaling pathways in order to develop effective therapies. In this study, tumor specimens from 40 GBCa patients, 12 extrahepatic bile duct carcinoma patients and 26 intrahepatic bile duct carcinoma patients from the USA and Japan were investigated for insulin-like growth factor I receptor (IGF-IR), mammalian target of rapamycin (mTOR) and rapidly accelerated fibrosarcoma-1 (Raf-1) expression by immunohistochemistry; in addition, the correlations with histological type, pathological stage and patient outcome were analyzed. Positive expression of IGF-IR, mTOR and Raf-1 were identified in 68, 73 and 85% of the specimens, respectively. There was no association with histological type and pathological stage, although the positive expression rate of Raf-1 was higher in advanced-stage GBCa. Moreover, patients with positive expression of IGF-IR exhibited significantly reduced survival compared to those with negative IGF-IR expression. In conclusion, IGF-IR, mTOR and Raf-1 were highly expressed in biliary tract cancer and targeted therapy against IGF-IR may be an effective strategy. Among these molecules, IGF-IR expression was found to be a useful biomarker for identifying patients who may benefit from additional treatment.

Overexpression and gene amplification of EGFR, HER2, and HER3 in biliary tract carcinomas, and the possibility for therapy with the HER2-targeting antibody pertuzumab.

BACKGROUND: Pertuzumab is a humanized monoclonal antibody that binds to HER2 at an epitope that prevents HER2 from dimerizing with ligand-activated HER-family receptors. To assess the potential of pertuzumab as a new therapy, the expression status of HER family members was determined in biliary tract carcinoma (BTC), and the antitumor activity of pertuzumab was investigated by assessing the inhibition of BTC cell growth. METHODS: The expression status of HER family members in 113 archival specimens of BTC was analyzed by using immunohistochemistry and fluorescence in situ hybridization. Using ten BTC cell lines, heregulin-alpha (HRG-α) stimulated cell proliferation and its inhibition by pertuzumab was tested in vitro. The phosphorylated HER family proteins and their respective downstream molecules were analyzed. In vivo antitumor activity of pertuzumab was evaluated in a xenograft model. RESULTS: Protein overexpression of HER2 and/or HER3 was observed in 23-34 % of the specimens and gene amplification in 17-27 %. Seven of the ten cell lines showed HER2 and/or HER3 protein overexpression and gene amplification, and HRG-α stimulated cell proliferation was observed in four of the ten cell lines. In a BTC cell line co-overexpressing HER2 and HER3, pertuzumab potently inhibited the HRG-α stimulated cell proliferation in a dose-dependent manner, and completely blocked the phosphorylation of HER3. Suppression of downstream pathway molecules including p-AKT was also observed. Pertuzumab inhibited the in vivo growth of subcutaneous tumors, and increased the number of apoptotic cancer cells. CONCLUSIONS: Pertuzumab exerts potent antitumor activity in BTC cells co-overexpressing HER2 and HER3. Pertuzumab provides a new therapeutic option against BTC.

Interaction of Muc4 and ErbB2 in a transgenic mouse model of gallbladder carcinoma: potential pathobiological implications.

The molecular mechanism of gallbladder carcinogenesis and cancer growth remains unknown. BK5.erbB2 transgenic mice in which erbB2 is overexpressed and activated in the biliary epithelia develop adenocarcinoma of the gallbladder at a high incidence. Although it has been reported that erbB2 plays an important role in tumorigenesis, little is known about the involvement of its ligand(s). The expression level of Muc4, a potential functional ligand for erbB2, and its interaction with erbB2 in the gallbladder of BK5.erbB2 mice were determined. By immunohistochemistry and in situ hybridization, both Muc4 mRNA and protein levels were strongly expressed in the cancerous epithelia of gallbladder from BK5.erbB2 mice. Also, in the hyperplastic (precancerous) epithelia, the protein levels were modestly expressed. Immunostaining with Muc4 (ASGP2) Ab overlapped with that with erbB2 Ab in the apical membranous components of the cancerous epithelia, indicating the co-localization of Muc4 and erbB2. Immunoprecipitation experiments revealed an interaction between Muc4 and erbB2 in the gallbladders. The interaction was associated with the hyperphosphorylation of erbB2, MAPK and Akt, and also with the overexpression of cyclooxygenase-2. However, in other organs that overexpressed erbB2 (trachea, esophagus and forestomach), Muc4 was expressed in only trace or modest amounts, and erbB2 was not hyperphosphorylated. Collectively, Muc4 is upregulated and interacts with erbB2 in gallbladders from BK5.erbB2 mice. It is likely that Muc4 plays an important role during gallbladder carcinogenesis and/or cancer growth by potentiating erbB2 signaling.

Cyst infection of intraductal papillary mucinous neoplasms of the pancreas: management of a rare complication: report of 2 cases.

The purpose of this study was to describe the cyst infection of intraductal papillary mucinous neoplasm in 2 patients. The patients were 62- and 74-year-old men. The initial symptom was acute febrile abdominal pain. Laboratory tests revealed severe infection (C-reactive protein concentrations were 23.3 µg/mL in patient 1 and 22.3 µg/mL in patient 2) and multilocular cystic masses (the diameters were 70 mm in patient 1 and 50 mm in patient 2) at the pancreatic head that involved peripancreatic vessels were demonstrated by computed tomography. Laboratory and radiographic findings were markedly improved by endoscopic transpapillary drainage. The enteric bacteria were detected in the drainage specimens. Curative resection was achieved, and histological findings indicated a carcinoma in situ in patient 1 and an invasive carcinoma in patient 2. Neither hyperamylasemia nor histological fat necrosis, frequently observed in acute pancreatitis, was evident. Both patients were free from recurrence after surgery (17 months in patient 1, and 18 months in patient 2). Cyst infection is an unknown complication of intraductal papillary mucinous neoplasm. Transpapillary drainage is highly recommended as an initial intervention. It is difficult to distinguish between cyst infection and unresectable invasive carcinoma with imaging modalities; however, surgical intervention after drainage may contribute to long-term survival.

Expression of N-acetylglucosaminyltransferase V in the subserosal layer correlates with postsurgical survival of pathological tumor stage 2 carcinoma of the gallbladder.

BACKGROUND: N-Acetylglucosaminyltransferase V (GnT-V), an enzyme that catalyzes the ß1-6 branching of N-acetylglucosamine on asparagine-linked oligosaccharides of cellular proteins, enhances the malignant behaviors of carcinoma cells in experimental models. The aim of this study was to determine clinical significance of GnT-V expression in human pT2 gallbladder carcinoma with simple in vitro experiments. METHODS: Ninety patients with pT2 gallbladder carcinoma were included for this study. The in vitro and in vivo biological effects of GnT-V were investigated using gallbladder carcinoma cells with variable GnT-V expression levels induced by a small interfering RNA. RESULTS: Of the 90 cases, 57 showed positive staining and the remaining 33 demonstrated negative staining, the subcellular localization in the 57 cases was classified into the granular-type in 31 cases and the diffuse-type in 26 cases. In 76 cases with curative resection, postsurgical survival was significantly poorer in those showing positive staining than in those showing negative staining (P = 0.028). In all of the 76 cases, postsurgical recurrence was significantly more frequent in those showing diffuse-type localization than in those showing negative staining. Experimental analyses demonstrated that the down-regulation of GnT-V expression in gallbladder carcinoma cells induced suppression of cell growth in vitro. The expression levels of GnT-V in the cells were highly correlated with the rapid in vivo growth coupled with the enhanced angiogenesis, and the tendency to form liver metastasis. CONCLUSIONS: GnT-V expression in the subserosal layer of pT2 gallbladder carcinoma is correlated with the aggressiveness of the disease.

Ex vivo activation of angiogenic property in human peripheral blood-derived monocytes by thrombopoietin.

Human peripheral blood mononuclear cells (PB-MNCs) have angiogenic properties, which make them promising cells for use in angiogenic therapy approaches in regenerative medicine. To explore an efficient method for expanding pro-angiogenic cells from PB-MNCs, we developed a novel serum-free culture system composed of X-VIVO15 medium supplemented with vascular endothelial growth factor, basic fibroblast growth factor, and thrombopoietin (TPO). Using this ex vivo culture, we obtained floating spheres composed mainly of CD11b(+) monocytes expressing c-Mpl (TPO receptor) and which exhibited acetylated low-density lipoprotein uptake and phagocytosis. Expression of IL-8, CXCR4, and vasohibin-2 mRNA was upregulated in these cells. In the presence of TPO, the number and size of the spheres were increased. In a nude mouse hind-limb ischemia model, the intramuscular injection of spheroid cells treated with TPO rescued blood perfusion more effectively than that without TPO. These results indicate that the ex vivo addition of TPO augments the pro-angiogenic activity of peripheral CD11b(+) monocytes, suggesting that this method shows promise for uses in human cell therapy aimed at the induction of vascular regeneration by activating the angiogenic properties of human peripheral blood-derived monocytes.

Glycoproteomics-based cancer marker discovery adopting dual enrichment with Wisteria floribunda agglutinin for high specific glyco-diagnosis of cholangiocarcinoma.

Cholangiocarcinoma (CC) is a lethal malignancy because it exhibits asymptomatic growth infiltrating the surrounding structures and therefore is usually detected at an advanced stage. The mainstay of treatment for CC is complete resection with negative surgical margins. Therefore, its diagnosis at a relatively early stage is demanded for performing relevant surgical resection. Since the definitive CC diagnosis depends on invasive methods such as biliary cytology and biopsy, a noninvasive assay with high diagnostic accuracy is keenly required. We therefore developed a CC marker with high specificity by the Wisteria floribunda agglutinin (WFA)-assisted glycoproteomics approach. WFA-positive glycoproteins were enriched by the direct dissection of the WFA-stained CC tissue region and following WFA-agarose column chromatography. Subsequent analysis by mass spectrometry identified 71 proteins as candidate markers. Screening of these candidates by gene expression profiling and immunohistochemistry resulted in the selection of L1 cell adhesion molecule (L1CAM) as the most specific CC marker. We confirmed the importance of WFA-positivity for L1CAM using both bile and serum of CC and benign bile duct disease patients. Specifically, WFA-positive L1CAM was enriched from serum by the WFA-assisted affinity capturing, with which CC was efficiently distinguished from benign. In the primary verification study using bile from CC patients (n=29) and that of benign bile duct disease (n=29), WFA-positive L1CAM distinguished CC with high specificity (sensitivity=0.66, specificity=0.93, overall accuracy=0.79, area under the receiver operating curve [AUC]=0.82). The combined use of the WFA-positive L1CAM assay with the high sensitive assay detecting WFA-positive sialylated mucin 1 sufficiently improved the diagnostic accuracy of CC (overall accuracy=0.84, AUC=0.93). This combination will possibly be a precise procedure for CC diagnosis compared with conventional diagnostic techniques. BIOLOGICAL SIGNIFICANCE: In this study, we constructed the system for verification of the candidate molecules that exhibit disease specific glyco-alterations and discovered a useful CC marker by the glycoproteomics-assisted strategy for biomarker discovery. Based on the strategy, we previously found that WFA is the best probe to detect CC-specific glycosylation and WFA-positive sialyl MUC1 as a possible biomarker candidate. While the diagnostic specificity of WFA-positive sialyl MUC1 was not superb, we proposed a new biomarker candidate WFA-positive L1CAM with high specificity in bile and serum to complement the previous one. We proved that the novel combination assay of WFA-L1CAM and WFA-sialyl MUC1 selected based on our strategy has the possibility to become a reliable serological test. This study represents application of our strategy, which can be extrapolated to discovery of marker candidates for other diseases.

Biliary tract carcinoma: clinical perspectives on molecular targeting strategies for therapeutic options.

Biliary tract carcinoma (BTC) is a lethal malignancy. This lethality is essentially attributed to both slow carcinogenesis occurring under complex pathological circumstances and to the asymptomatic growth of BTC infiltrating the surrounding structures by varying routes. The disease is therefore usually detected at an advanced stage with a high frequency of distant organ metastasis. To date, conventional chemotherapy and radiation therapy have been notably ineffective against BTC. For an improved treatment outcome of BTC and prolonged survival, there is now a real and urgent need to focus on developing novel and potent therapeutic strategies aimed at exploiting select molecular targets associated with tumor proliferation, invasion, and/or metastasis that would impact in a significant way on clinical outcome. The outcomes of recent studies, by the analysis of BTC cells, BTC animal models, and clinical specimens of BTC patients, have revealed, in detail, the molecular mechanism of carcinogenesis and tumor progression of BTC, and these studies have exploited select molecular targets that could significantly impact the clinical outcome. In the near future, the development of new molecular targeting drugs with potent efficacy against BTC, and the performance of randomized clinical trials of these drugs are urgent and essential for the treatment of patients with BTC.

Transplanting normal vascular proangiogenic cells to tumor-bearing mice triggers vascular remodeling and reduces hypoxia in tumors.

Blood vessels deliver oxygen and nutrients to tissues, and vascular networks are spatially organized to meet the metabolic needs for maintaining homeostasis. In contrast, the vasculature of tumors is immature and leaky, resulting in insufficient delivery of nutrients and oxygen. Vasculogenic processes occur normally in adult tissues to repair "injured" blood vessels, leading us to hypothesize that bone marrow mononuclear cells (BMMNC) may be able to restore appropriate vessel function in the tumor vasculature. Culturing BMMNCs in endothelial growth medium resulted in the early outgrowth of spindle-shaped attached cells expressing CD11b/Flt1/Tie2/c-Kit/CXCR4 with proangiogenic activity. Intravenous administration of these cultured vascular proangiogenic cells (VPC) into nude mice bearing pancreatic cancer xenografts and Pdx1-Cre;LSL-Kras(G12D);p53(lox/+) genetically engineered mice that develop pancreatic ductal adenocarcinoma significantly reduced areas of hypoxia without enhancing tumor growth. The resulting vasculature structurally mimicked normal vessels with intensive pericyte coverage. Increases in vascularized areas within VPC-injected xenografts were visualized with an ultrasound diagnostic system during injection of a microbubble-based contrast agent (Sonazoid), indicating a functional "normalization" of the tumor vasculature. In addition, gene expression profiles in the VPC-transplanted xenografts revealed a marked reduction in major factors involved in drug resistance and "stemness" of cancer cells. Together, our findings identify a novel alternate approach to regulate abnormal tumor vessels, offering the potential to improve the delivery and efficacy of anticancer drugs to hypoxic tumors.

Wisteria floribunda agglutinin-positive mucin 1 is a sensitive biliary marker for human cholangiocarcinoma.

UNLABELLED: Cholangiocarcinoma (CC) is an aggressive malignant tumor for which useful markers are not presently available for early and precise diagnosis. The aim of this study was therefore to identify a high-performance diagnostic marker with a special focus on glyco-alteration of glycoproteins. In the course of study, we found that Wisteria floribunda agglutinin (WFA) is the best probe to differentiate intrahepatic cholangiocarcinoma (ICC) lesions from normal bile duct epithelia (BDE) (P < 0.0001). The subsequent histochemical study confirmed ICC-specific WFA staining on 165 tissue specimens. On the other hand, the WFA staining was shown to be closely associated with that of MY.1E12 established previously against sialylated mucin 1 (MUC1) by double-staining experiments. Moreover, glyco-alteration of MUC1 could be verified by western blotting of WFA-captured bile samples from patients with CC patients. Thus, we attempted to construct an enzyme-linked immunosorbent assay system for more convenient CC diagnosis, where WFA-coated plates, the specific monoclonal antibody MY.1E12, and the bile specimens from CC including ICC (n = 30) and benign diseases (n = 38) were combined. As a result, CC was clearly distinguished from benign diseases with statistical scores (sensitivity = 90.0%, specificity = 76.3%, and area under the curve = 0.85). As a particular note, the obtained sensitivity is the highest score among those having been so far reported. CONCLUSION: Our approach focusing significant glyco-alteration of a particular glycoprotein yielded a novel diagnostic system for CC with satisfactory clinical scores.

Hedgehog promotes neovascularization in pancreatic cancers by regulating Ang-1 and IGF-1 expression in bone-marrow derived pro-angiogenic cells.

BACKGROUND: The hedgehog (Hh) pathway has been implicated in the pathogenesis of cancer including pancreatic ductal adenocarcinoma (PDAC). Recent studies have suggested that the oncogenic function of Hh in PDAC involves signaling in the stromal cells rather than cell autonomous effects on the tumor cells. However, the origin and nature of the stromal cell type(s) that are responsive to Hh signaling remained unknown. Since Hh signaling plays a crucial role during embryonic and postnatal vasculogenesis, we speculated that Hh ligand may act on tumor vasculature specifically focusing on bone marrow (BM)-derived cells. METHODOLOGY/PRINCIPAL FINDINGS: Cyclopamine was utilized to inhibit the Hh pathway in human PDAC cell lines and their xenografts. BM transplants, co-culture systems of tumor cells and BM-derived pro-angiogenic cells (BMPCs) were employed to assess the role of tumor-derived Hh in regulating the BM compartment and the contribution of BM-derived cells to angiogenesis in PDAC. Cyclopamine administration attenuated Hh signaling in the stroma rather than in the cancer cells as reflected by decreased expression of full length Gli2 protein and Gli1 mRNA specifically in the compartment. Cyclopamine inhibited the growth of PDAC xenografts in association with regression of the tumor vasculature and reduced homing of BM-derived cells to the tumor. Host-derived Ang-1 and IGF-1 mRNA levels were downregulated by cyclopamine in the tumor xenografts. In vitro co-culture and matrigel plug assays demonstrated that PDAC cell-derived Shh induced Ang-1 and IGF-1 production in BMPCs, resulting in their enhanced migration and capillary morphogenesis activity. CONCLUSIONS/SIGNIFICANCE: We identified the BMPCs as alternative stromal targets of Hh-ligand in PDAC suggesting that the tumor vasculature is an attractive therapeutic target of Hh blockade. Our data is consistent with the emerging concept that BM-derived cells make important contributions to epithelial tumorigenesis.

Potent in vitro and in vivo antitumor activity of interleukin-4-conjugated Pseudomonas exotoxin against human biliary tract carcinoma.

Targeting cytotoxins or immunotoxins to tumor cell surface receptors represents a new approach for the treatment of cancers. We tested the antitumor activity of a cytotoxin (IL-4-PE) composed of an interleukin-4 (IL-4) targeting moiety and a truncated form of Pseudomonas exotoxin A against human biliary tract carcinoma (BTC). Immunohistochemical analysis showed that cultured BTC cell lines and cancerous epithelia in BTC tissue (e.g., gallbladder carcinoma, extrahepatic cholangiocarcinoma, and intrahepatic cholangiocarcinoma) expressed receptors for IL-4 in situ at high densities. However, normal epithelial cells in gallbladder and bile duct tissues did not express these IL-4 receptors. Eight BTC cell lines expressed IL-4R on the cell surface as determined by radiolabeled ligand binding assays. When these cells were treated with IL-4-PE, significant cytotoxicity was observed as determined by the inhibition of protein synthesis. The concentration of agent causing 50% inhibition of protein synthesis (IC(50)) was found to be less than 10 ng/mL in 4 of the 8 BTC cell lines studied. The antitumor activity of IL-4-PE was assessed for human BTC cells implanted subcutaneously in immunodeficient mice. By intratumoral injection of IL-4-PE, complete disappearance of the established tumors was observed in 40% of animals. Intraperitoneal administration of IL-4-PE at tolerated doses to animals with peritoneally disseminated BTC exhibited significantly prolonged survival compared to untreated animals (>14 weeks vs. 5 weeks in treated and untreated mice, respectively). These results indicate that IL-4 receptor-targeted cytotoxin is a potent agent that may provide a new therapeutic option for BTC.