Seiji Kaino

Detection of K-ras and p53 gene mutations in pancreatic juice for the diagnosis of intraductal papillary mucinous tumors.

The aim of this study was to investigate mutations of the K-ras oncogene and the p53 tumor suppressor gene in pancreatic juice and to evaluate our method for the diagnosis of intraductal papillary mucinous tumors (IPMT). Pancreatic juice was collected endoscopically from 12 patients with IPMT who underwent surgical resection (eight carcinomas and four adenomas) and eight cases without evident pancreatic diseases. DNA was extracted and both genes were examined by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. In addition, surgically resected specimens were analyzed for both genes by the same methods, and p53 overexpression was investigated immunohistochemically. K-ras point mutations were detected in pancreatic juice from all 12 patients (100%) and p53 mutations were detected in five of 12 (42%). They were detected not only in carcinoma but also in adenoma and there was no difference between the mutations detected in pancreatic juice and surgical specimens. No mutations were found in any cases without pancreatic diseases. These findings suggest that alterations of K-ras and p53 gene are common events in the development of IPMT and that genetic analysis of them in pancreatic juice can be a useful tool for the clinical diagnosis of IPMT before surgery.

Differential diagnosis of benign and malignant branch duct intraductal papillary mucinous neoplasm using contrast-enhanced endoscopic ultrasonography

AIM To elucidate the role of contrast-enhanced endoscopic ultrasonography (CE-EUS) in the diagnosis of branch duct intraductal papillary mucinous neoplasm (BD-IPMN). METHODS A total of 50 patients diagnosed with BD-IPMN by computed tomography (CT) and endoscopic ultrasonography (EUS) at our institute were included in this study. CE-EUS was performed when mural lesions were detected by EUS. The diagnostic accuracy for identifying mural nodules (MNs) was evaluated by CT, EUS, and EUS combined with CE-EUS. In the patients who underwent resection, the accuracy of measuring MN height with each imaging modality was compared. The cut-off values to diagnose malignant BD-IPMNs based on MN height for each imaging modality were determined using receiver operating characteristic curve analysis. RESULTS Fifteen patients were diagnosed with BD-IPMN with MNs and underwent resection. The remaining 35 patients were diagnosed with BD-IPMN without MNs and underwent follow-up monitoring. The pathological findings revealed 14 cases with MNs and one case without. The accuracy for diagnosing MNs was 92% using CT and 72% using EUS; the diagnostic accuracy increased to 98% when EUS and CE-EUS were combined. The accuracy for measuring MN height significantly improved when using CE-EUS compared with using CT or EUS (median measurement error value, CT: 3.3 mm vs CE-EUS: 0.6 mm, P < 0.05; EUS: 2.1 mm vs CE-EUS: 0.6 mm, P < 0.01). A cut-off value of 8.8 mm for MN height as measured by CE-EUS improved the accuracy of diagnosing malignant BD-IPMN to 93%. CONCLUSION Using CE-EUS to measure MN height provides a highly accurate method for differentiating benign from malignant BD-IPMN.

p53 Mutations in Two Patients with Intraductal Papillary Adenoma of the Pancreas

There has been no report on p53 gene mutation in benign human pancreatic intraductal tumors. We examined pancreatic juice and tissue specimens from two patients with intraductal papillary adenoma of the pancreas by polymerase chain reaction‐single‐strand conformation polymorphism analysis and direct sequencing and found point mutations of p53 gene resulting in amino acid substitutions in exons 6 and 8. Thus, p53 gene mutation may be an early event in the neoplastic process of some pancreatic intraductal tumors and may play an important role in tumorigenesis.

Calcium concentration and artificial precipitates in human pancreatic juice.

We studied the role of the increase in the calcium concentration in pure pancreatic juice of alcoholic noncalcified chronic pancreatitis. Pure pancreatic juice was obtained endoscopically. The pancreatic juice from patients with chronic pancreatitis was adjusted to pH 7.5; then the calcium concentration was adjusted to 0.4, 2.9, 5.4, or 10.4 mmol/L. Artificial precipitates were produced by incubation of the samples at 37°C for 6 hours. Proteins in the artificial precipitates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the protein patterns were compared with the patterns of natural protein plugs from patients with chronic pancreatitis. The amount of the precipitate increased as the added calcium increased. The protein patterns of SDS-PAGE of the artificial precipitates were similar to those of protein plugs. Albumin, a-amylase, lipase, trypsinogen, and chymotrypsinogen were identified by immunoblotting both in the precipitate and in the protein plug. The increased calcium concentrations in pancreatic juice induced the formation of precipitates whose protein composition was similar to that of protein plugs. An increased calcium concentration in human pancreatic juice may play an important role in the pathogenesis of protein plugs.

Serum transferrin as a predictor of prognosis for hepatic arterial infusion chemotherapy in advanced hepatocellular carcinoma

We recently reported that the iron chelator deferoxamine (DFO) is efficacious in advanced hepatocellular carcinoma (HCC) patients. Iron regulation may thus have an important impact in HCC therapy. Because transferrin is a native chelator that regulates iron homeostasis, it may act as an anticancer agent in a similar manner as DFO. The objective of this study was to evaluate serum transferrin as a prognostic predictor in advanced HCC patients undergoing hepatic arterial infusion chemotherapy (HAIC).

Utility of the Anterior Oblique-Viewing Endoscope and the Double-Balloon Enteroscope for Endoscopic Retrograde Cholangiopancreatography in Patients with Billroth II Gastrectomy

Background/Purpose. The difficulties of endoscopic retrograde cholangiopancreatography in patients with Billroth II gastrectomy have been reported. We evaluated the usefulness of an anterior oblique-viewing endoscope and a double-balloon enteroscope for endoscopic retrograde cholangiopancreatography in such patients. Methods. From January 2003 to December 2011, 65 patients with Billroth II gastrectomy were enrolled in this study. An anterior oblique-viewing endoscope was used for all patients. From February 2007, a double-balloon enteroscope was used for the failed cases. The success rate of procedures was compared with those in 20 patients with Billroth II gastrectomy using forward-viewing endoscope or side-viewing endoscope from March 1996 to July 2002 as historical controls. Results. In all patients in whom the papilla was reached (60/65), selective cannulation was achieved. The success rate of selective cannulation and accomplishment of planned procedures in the anterior oblique-viewing endoscope group were both significantly higher than that in the control group (100% versus 70.1%, 100 versus 58.8%, resp.). A double-balloon enteroscope was used in 2 patients, and the papilla could be reached and the planned procedures completed. Conclusions. An anterior oblique-viewing endoscope and double-balloon enteroscope appear to be useful in performing endoscopic retrograde cholangiopancreatography in patients with Billroth II gastrectomy.

Efficacy of contrast-enhanced harmonic endoscopic ultrasonography in the diagnosis of pancreatic ductal carcinoma.

Background/Aims: Distinguishing pancreatic ductal carcinoma (DC) from other pancreatic masses remains challenging. This study aims at evaluating the efficacy of contrast-enhanced harmonic endoscopic ultrasonography (CEH-EUS) in the diagnosis of DC. Patients and Methods: Forty-nine patients with solid pancreatic mass lesions underwent CEH-EUS. EUS (B-mode) was used to evaluate the inner echoes, distributions, and borders of the masses. The vascular patterns of the masses were evaluated with CEH-EUS at 30–50 s (early phase) and 70–90 s (late phase) after the administration of Sonazoid®. Results: The final diagnoses included DCs (37), mass-forming pancreatitis (6), endocrine neoplasms (3), a solid pseudopapillary neoplasm (1), a metastatic carcinoma (1), and an acinar cell carcinoma (1). The sensitivity, specificity, and accuracy of the diagnoses of DC in hypoechoic masses using EUS (B-mode) were 89.2%, 16.7%, and 71.4%, respectively. The sensitivity, specificity, and accuracy for the diagnosis of DC in hypovascular masses using CEH-EUS were 73.0%, 91.7%, and 77.6% in the early phase and 83.8%, 91.7%, and 85.7% in the late phase, respectively. Conclusions: CEH-EUS for the diagnosis of DC is superior to EUS. CEH-EUS in the late phase was particularly efficacious in the diagnosis of DC.

Deferasirox, an oral iron chelator, with gemcitabine synergistically inhibits pancreatic cancer cell growth in vitro and in vivo

Objectives Iron is an essential element for cell proliferation and growth processes. We have reported that deferasirox (DFX), an oral iron chelator, showed antiproliferative activity against pancreatic cancer cells. This study aimed to elucidate the effects of combination of gemcitabine (GEM), standard chemotherapy for pancreatic cancer, and DFX in vitro and in vivo. Results GEM+DFX showed antiproliferative activity and induced apoptosis in pancreatic cancer cells in vitro. GEM+DFX suppressed xenograft tumor growth and induced apoptosis without any serious side effects compared with control, GEM, and DFX (average tumor volume: control 697 mm3 vs GEM 372 mm3, p < 0.05; GEM 372 mm3 vs GEM+DFX 234 mm3, p < 0.05). RRM1 and RRM2 protein levels were substantially reduced by DFX in BxPC-3 in vitro. Conclusion GEM+DFX has significant anticancer effects on pancreatic cancer cell through RR activity suppression. Methods BxPC-3, a human pancreatic cancer cell line, was used in all experiments. Cellular proliferation rate was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay. Apoptosis was evaluated by flow cytometry and by measuring caspase 3/7 activity with luminescence assay. In the tumor xenografts in nude mice models, when five weeks after engraftment, drug administration began (day 0). After treatment for 21 days, the mice were sacrificed and the tumors were excised. Apoptotic cells in xenografts were evaluated by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay. Protein levels of ribonucleotide reductase (RR) subunit 1 (RRM1) and RR subunit 2 (RRM2) in BxPC-3 cells were assessed by western blot in vitro.

AL-type amyloidosis with homogeneous bile duct thickening showing biliary sclerosing changes.

A 66-year-old Japanese woman complaining of general fatigue and jaundice (total bilirubin, 3.1 mg/dL) visited our hospital. Serum immunoglobulin G4 (IgG4) was 136 mg/dL. Contrast enhanced computed tomography (CE-CT) and endoscopic ultrasound showed intrahepatic and extrahepatic bile duct dilatation and wall thickening (Fig. 1a,b). Magnetic resonance cholangiopancreatography (MRCP) and endoscopic retrograde cholangiopancreatography (ERCP) showed irregular walls in the main pancreatic duct, and also showed multiple strictures of the hilar and intrahepatic bile ducts, with a ‘pruned-tree’ appearance (Fig. 1c–e). We carried out biopsies from the bile duct and an ultrasound-guided percutaneous liver biopsy. The biopsied specimens showed no evidence of malignancy nor IgG4-positive plasma cells. A steroid trialwas started with 25mg/day prednisolone, andmultiple strictures of the bile ducts were slightly improved. However, unfortunately, she showed repeated cholangitis and pancreatitis and died of the disease 2 years and 8 months later. Autopsy was done and histology of the bile duct showed a massive red amorphous substance by Congo red staining with red-green birefringence under polarized light, and the infiltrating plasma cells showed lambda light-chain restriction. These findings appeared to be compatible with AL amyloidosis, and severe amyloid deposition was observed in the whole body including the bile duct, Vater’s papilla, pancreas, mesenterium and pleura (Fig. 2a–e). To the best of our knowledge, only a few studies have reported amyloidosis showing primary sclerosing cholangitis (PSC)-like or IgG4-related sclerosing cholangitis (IgG4-SC)-like bile duct changes, and this is the first case of AL-type amyloidosis showing homogeneous bile duct wall thickening. Our case suggests that it is important to consider amyloidosis as a differential diagnosis of PSC or IgG4-SC.