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Background: MicroRNAs (miRNAs) have been gaining attention as new, key molecules that contribute to carcinogenesis. In pancreatic cancer, previous profiling analyses of miRNA expression have shown that several miRNAs are differently expressed in normal and cancerous tissues. Several pancreatic cancer-specific miRNAs differed, however, in each analysis., Methods: We investigated the miRNA expression profiles of the pancreatic cancer cell lines CAPAN-1 and CFPAC1 and an immortalized human normal pancreatic ductal epithelial cell line (HPDE) using a high-throughput, TaqMan, qRT-PCR array analysis. We also analyzed the expression levels of this miRNA in microdissected (n = 15) and formalin-fixed, paraffin-embedded (FFPE) (n = 115) samples from pancreatic cancers by quantitative RT-PCR. Finally, we investigated the effects of this miRNA on the invasiveness of pancreatic cancer cells., Results: Based on the microarray analysis, miR-372, miR-146a, miR-204, miR-10a, and miR-10b showed particularly large differences (>10-fold changes) between both pancreatic cell lines and HPDE cells. Thirteen of the 15 pancreatic cancer cell lines showed 2.1- to 36.4-fold (median, 15.3-fold) greater levels of miR-10b than HPDE cells. Microdissection analysis revealed that miR-10b exhibited greater expression levels in pancreatic cancer cells (n = 5) than in normal pancreatic ductal cells (n = 10) (P < .020). Analysis of FFPE samples showed that high miR-10b expression was associated with a lesser overall survival (P = .014). Furthermore, miR-10b correlated with the invasiveness of pancreatic cancer cells (P < .01)., Conclusion: miR-10b is overexpressed in pancreatic cancer and may be involved in the invasiveness in pancreatic cancer cells, thereby leading to a poor prognosis., (Copyright © 2011. Published by Mosby, Inc.)

We applied single-incision laparoscopy-assisted surgery for several different types of bowel obstruction in selected patients. Before the operation, a long nasal tube was inserted for intestinal decompression and assessment of a stenotic lesion. A specially-designed instrument for single-incision laparoscopic surgery, the SILS Port, was introduced at the umbilicus or proposed ileostomy site. After intracorporeal procedures, extracorporeal resection and reconstruction of the intestine was performed as needed. Three patients with bowel obstruction due to jejunal carcinoma, colonic stenosis, and adhesion underwent single-incision laparoscopy-assisted surgery. The port site was used for subsequent extracorporeal resection and anastomosis of the jejunum in two patients, and for ileostomy in the remaining patient. All of the procedures were completed safely, and there were no postoperative complications. Single-incision laparoscopy can therefore be applied for selected patients with bowel obstruction. In such cases, the preoperative insertion of a long nasal tube for decompression of intestinal contents and assessment of the stenotic lesion is necessary.

Background: To improve the prognosis of patients after resection of pancreatic cancer, the most appropriate and efficient treatment should be provided to specific subsets of patients. Our aim was to identify promising microRNAs as markers to predict responses to gemcitabine in patients with resected pancreatic cancer., Methods: Two gemcitabine-resistant pancreatic cancer cell lines were established, and global microRNA expression analyses was performed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Eleven miRNAs were selected as putative predictive markers and analyzed by means of macrodissected formalin-fixed, paraffin-embedded samples obtained from 90 patients with or without gemcitabine treatment after resection of pancreatic cancer., Results: We identified 24 microRNAs whose expression was altered in gemcitabine-resistant cells. qRT-PCR analyses showed that patients with high miR-142-5p and miR-204 expression had significantly longer survival times than those with low miR-142-5p (P = 0.0077) and miR-204 (P = 0.0054) expression in the gemcitabine-treated group. This was not seen in the nontreated group. Multivariate analyses showed that miR-142-5p expression was an independent prognostic marker only in patients treated with gemcitabine (P = 0.034)., Conclusions: miR-142-5p is a promising predictive marker for gemcitabine response in patients with resected pancreatic cancer.

Background: Advanced gastric cancers often metastasize to distant organs and the peritoneum, leading to a poor prognosis. Both invasiveness and resistance to anchorage-independent cell death (anoikis) are important factors in the process of metastasis. p600 (600-kDa protein), recently identified from a cervical cancer cell line, plays a role in both anoikis resistance and cell migration. In this study, we examined whether p600 is involved in the progression of gastric cancer., Methods: We used both normal gastric mucosal cells and cancer cells laser-microdissected from 42 gastric cancers and their normal counterparts, and compared their p600 mRNA expression levels with quantitative reverse transcriptase-polymerase chain reaction. We inhibited p600 expression in two gastric cancer cell lines with siRNA and examined its effect on the invasiveness and anoikis resistance both in vitro and in vivo., Results: Expression of p600 mRNA was significantly higher in gastric cancer cells than in normal mucosal cells (P = 0.027). The invasion assay revealed that invasiveness was significantly reduced by inhibition of p600 (P < 0.01). In vitro experiments revealed that cell viability and colony-formation capacity under anchorage-independent conditions were significantly reduced by inhibition of p600 (P < 0.05). In vivo experiments also showed that the establishment of intraperitoneal disseminated tumors was significantly suppressed by transient inhibition of p600 (P < 0.001)., Conclusions: Our results strongly suggest that p600 is involved in gastric cancer progression, and has a potential to be a new molecular target for gastric cancer therapy.

Unlabelled: ETHOPARMACOLOGICAL RELEVANCE: Salacia reticulata, a herbal medicine which has been used for the treatment of early diabetes in Ayurvedic medicine, is reported to have an anti-obesity effect and to be useful in the treatment of diabetes mellitus, insulin resistance and other metabolic diseases., Aim of the Study: The present study was performed to elucidate the mechanism of action of Salacia reticulata with special attention to the adipocytes as the tissue primarily involved in the pathology of metabolic diseases., Materials and Methods: Mouse-derived adipocyte precursor 3T3-L1 cells were treated with differentiation inducers in the presence or absence of Salacia reticulata (SRCD). We determined triacylglycerol accumulations, differentiation makers, released glycerol and adiponectin. Mangiferin, the primary component of SRCD, was also treated to 3T3-L1 cells., Result: Concurrent administration of the extract of SRCD and differentiation inducers resulted in a significant inhibition of differentiation into mature adipocytes. SRCD also exhibited significant inhibitory action on the expression of genes and proteins of peroxisome proliferator-activated receptor (PPAR)γ and CCAAT-enhancer binding protein (C/EBP)α, as well as on the activity of glycerol-3-phosphate dehydrogenase (GPDH), a differentiation marker, and caused a reduction in the concentration of released adiponectin. However, SRCD had no influence on lipolysis as indicated by the release of glycerol into the culture medium. The primary component of SRCD, mangiferin, was investigated for its effect on adipocytes; mangiferin caused no suppression of fat accumulation, suggesting that a component of SRCD other than mangiferin may be involved in the inhibition of adipocyte differentiation., Conclusions: The above results suggest that the inhibitory action of SRCD on adipocyte differentiation, and not the promotion of lipolysis, is involved in the suppression of fat accumulation., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Unlabelled: Aims/Introduction:  When monotherapy with an oral hypoglycemic agent (OHA) is not sufficiently effective for blood glucose control, combination therapy with OHA having different mechanisms of action might be indicated., Materials and Methods:   In the present study, we compared the efficacy of two options in type 2 diabetes mellitus patients whose blood glucose had not been well controlled with mitiglinide (30 mg/day) alone. A total of 20 patients were included in the study and divided into two groups: group A, in which mitiglinide was given concomitantly with the α-glucosidase inhibitor voglibose (0.6 mg/day); and group B, in which a double dose of mitiglinide was given (60 mg/day). Twelve weeks after changing the medication, HbA1c, glycoalbumin and 1,5-anhydroglucitol (1,5-AG) were measured. In addition, at weeks 0 and 12, a meal tolerance test was carried out, and plasma glucose, insulin, glucagon, active glucagon-like peptide-1 (GLP-1) and total glucose-dependent insulinotropic polypeptide levels were measured., Results:   The plasma level of 1,5-AG improved in both groups at week 12. In group A, the plasma insulin level significantly decreased and the plasma active GLP-1 level significantly increased during the meal tolerance test at week 12; thus, bodyweight significantly decreased only in group A., Conclusions:   Our findings suggested that concomitant administration of mitiglinide with voglibose could achieve better glycemic control, particularly in the postprandial period, without bodyweight gain and might have beneficial effects in type 2 diabetic patients at risk of macrovascular complications. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.0082.x, 2011).

Objectives: Although intestinal-type intraductal papillary mucinous carcinoma (IPMC) is reported to have a better prognosis, few studies have addressed its invasive pattern. The meaning of "minimal invasion" (MI) in IPMC also remains unclear. We investigated the prognosis of intraductal papillary mucinous neoplasm (IPMN) focusing on MI and subtypes., Methods: We evaluated 71 patients with IPMC among a total of 179 patients with resected IPMN., Results: Although 2 of 10 MI-IPMC patients had lymph node metastasis, there were no disease-specific deaths among the MI-IPMC patients. Minimally invasive IPMCs were more frequently observed in intestinal-type IPMC (23/33 cases) than in non-intestinal-type IPMCs (16/38 cases; P = 0.019). Among 32 patients with massively invasive IPMC, the prognosis was significantly better for patients with intestinal-type IPMC than for patients with non-intestinal-type IPMC (P = 0.013). When confined to massively invasive IPMC, tubular invasion (P < 0.001) and lymphatic (P = 0.001) or serosal (P = 0.021) invasion were less frequently observed in intestinal-type IPMC than in non-intestinal-type IPMC., Conclusions: Invasive carcinoma derived from intestinal-type IPMN is associated with MI, colloid carcinoma, and less invasive behavior.

Statement of Problem: Magnetic attachments are commonly used for overdentures. However, it can be difficult to identify and provide the same type and size of magnetic assembly and keeper if a repair becomes necessary. Therefore, the size and type may not match., Purpose: This study evaluated the retentive force and magnetic flux strength and leakage of magnetic attachments in different combinations of keepers and magnetic assemblies., Material and Methods: For 6 magnet-keeper combinations using 4 sizes of magnets (GIGAUSS D400, D600, D800, and D1000) (n=5), retentive force was measured 5 times at a crosshead speed of 5 mm/min in a universal testing machine. Magnetic flux strength was measured using a Hall Effect Gaussmeter. Data were statistically analyzed using a 1-way ANOVA, and between-group differences were analyzed with Tukey's HSD post hoc test (α=.05)., Results: The mean retentive force of the same-size magnet-keeper combinations was 3.2 N for GIGAUSS D400 and 5.1 N for GIGAUSS D600, but was significantly reduced when using larger magnets (P<.05). Magnetic flux leakage was significantly lower for corresponding size combinations., Conclusions: Size differences influence the retentive force and magnetic flux strength of magnetic attachments. Retentive force decreased due to the closed field structure becoming incomplete and due to magnetic field leakage., (Copyright © 2011 The Editorial Council of the Journal of Prosthetic Dentistry. Published by Mosby, Inc. All rights reserved.)

Introduction: Recently, incretin-related therapy has been developed for the new treatment of diabetes mellitus; however, incretin response to glucose ingestion in normal glucose tolerant (NGT) subjects has not been clarified in detail with special reference to the role of incretin hormones, glucagon, and a family history of diabetes., Methods: We conducted a 75 g oral glucose tolerance test in 30 NGT subjects., Results: The total glucose-dependent insulinotropic peptide (GIP)-AUC(0-120) (area under the curve over a period of 0-120 minutes) was correlated with immunoreactive insulin (IRI)-AUC(0-120) (P<0.05), insulinogenic index (II; P<0.05), ΔIRI between 0 and 120 minutes (P<0.05). Active glucagon-like peptide-1 (GLP-1) AUC(0-120) was correlated inversely both with Δ glucose between 0 and 30 minutes (P<0.01) and with Δ immunoreactive glucagon between 0 and 30 minutes (P<0.05). Δ Total GIP between 0 and 15 minutes (P<0.01), Δ total GIP between 0 and 30 minutes (P<0.05), and the total GIP-AUC(0-120) (P<0.05) in the subjects with a family history of type 2 diabetes were significantly higher than those in the subjects without a family history., Conclusion: These results suggest that GIP possibly facilitates insulin secretion in response to oral glucose load directly and active GLP-1 may exert the glucoregulatory action via the suppression of glucagon secretion in NGT subjects. Notably, the subjects with a family history of diabetes exert significantly higher GIP response in the early phase of glucose load compared with those without a family history.

The extracts of Salacia reticulata (Salacia extract), a plant that has been used for the treatment of early diabetes, rheumatism and gonorrhea in Ayurveda, have been shown to have an anti-obesity effect and suppress hyperglycemia. In this study, the effects of Salacia extract on various symptoms of metabolic disorder were investigated and compared using these TSOD mice and non-obese TSNO mice. Body weight, food intake, plasma biochemistry, visceral and subcutaneous fat (X-ray and CT), glucose tolerance, blood pressure and pain tolerance were measured, and histopathological examination of the liver was carried out. A significant dose-dependent decline in the gain in body weight, accumulation of visceral and subcutaneous fat and an improvement of abnormal glucose tolerance, hypertension and peripheral neuropathy were noticed in TSOD mice. In addition, hepatocellular swelling, fatty degeneration of hepatocytes, inflammatory cell infiltration and single-cell necrosis were observed on histopathological examination of the liver in TSOD mice. Salacia extract markedly improved these symptoms upon treatment. Based on the above results, it is concluded that Salacia extract has remarkable potential to prevent obesity and associated metabolic disorders including the development of metabolic syndrome.

To determine whether miglitol administration improves glycemic control and reduces the frequency of hypoglycemia in type 1 diabetes mellitus (T1DM) patients treated with intensive insulin therapy, we analyzed the effect of miglitol on daily insulin doses, body weight, hypoglycemia, and incretin hormone responses during meal tolerance tests (MTT). Eleven T1DM subjects (21-77 years) undergoing intensive insulin therapy, took 25 mg (weeks 0-4) and 50 mg miglitol (weeks 4-12) thrice daily, immediately before meals. At weeks 0 and 12, 9 of 11 subjects underwent MTT. In present study, mean HbA1c, glycoalbumin, and 1,5-anhydroglucitol levels were significantly improved. The blood glucose level 1 h after dinner was significantly lower at week 12 than at week 0 (p = 0.008). From week 0 to 12, there was a significant decrease in the body mass index (BMI; p = 0.0051), frequency of preprandial hypoglycemic events (p = 0.012), and daily bolus insulin dosage (p = 0.018). The change in active glucagon-like peptide-1 (GLP-1) at 120 min significantly increased at week 12 (p = 0.015). The change in total glucose-dependent insulinotropic peptide (GIP) significantly decreased in the MTT at week 12. These results demonstrate that addition of miglitol on intensive insulin therapy in T1DM patients has beneficial effects on reducing BMI, bolus and total insulin dosage, and frequency of preprandial hypoglycemic events. MTT findings suggest that this combination therapy improves blood glucose control by delaying carbohydrate absorption and modifying the responses of incretins, GIP, and GLP-1.

Intraductal papillary mucinous neoplasm is characterized by cystically dilated main and/or branch pancreatic duct with mucus. According to the degree of atypia, intraductal papillary mucinous neoplasm is classified into 3 groups: adenoma, borderline, and carcinoma. Furthermore, intraductal papillary mucinous neoplasm is considered to progress through an adenoma-carcinoma sequence like colorectal carcinoma. Programmed cell death 4 is a recently identified tumor suppressor that was found to inhibit translation. Programmed cell death 4 has been reported to inhibit tumorigenesis, tumor progression, proliferation, invasion, and metastasis in several human malignancies. We examined 108 cases of intraductal papillary mucinous neoplasm by immunohistochemistry and revealed that programmed cell death 4 expression was recognized in both the nucleus and cytoplasm in intraductal papillary mucinous neoplasm. The positive rate of programmed cell death 4 was 79%, 43%, and 10% in adenoma, borderline, and carcinoma, respectively. The positive rate of programmed cell death 4 decreased from adenoma to carcinoma (P < .0001, both adenoma versus borderline and borderline versus carcinoma), indicating that programmed cell death 4 might inhibit tumor progression in intraductal papillary mucinous neoplasm. Programmed cell death 4 expression had a strong relationship with p21 expression (P < .0001) and an inverse correlation with Ki-67 labeling index (r = -0.6255, P < .0001). Thus, programmed cell death 4 might inhibit the proliferation of intraductal papillary mucinous neoplasm; and its inhibition might partly result from cell cycle arrest caused by the up-regulation of p21. In conclusion, programmed cell death 4 may inhibit tumor progression in intraductal papillary mucinous neoplasm; and the loss of programmed cell death 4 expression is representative of the malignant potential of intraductal papillary mucinous neoplasm including the proliferative activity. Therefore, programmed cell death 4 can be an important biomarker for intraductal papillary mucinous neoplasm., (Copyright © 2010. Published by Elsevier Inc.)

To overcome the limited clinical efficacy of conditionally replicative adenoviruses (CRAds), we investigated the effects of combination therapy with gemcitabine (GEM) and the hTERT-promoter-dependent CRAd (hTERT-CRAd), Ad5/3hTERTE1. This combination therapy exhibited enhanced cytotoxic effects on pancreatic cancer both in vitro and in vivo. Furthermore, we revealed that this enhancement effect was due to the multiple bidirectional interactions between hTERT-CRAd and GEM. The GEM-sensitizing effect of E1 expression derived from hTERT-CRAd, and the enhancement effect by GEM on hTERT promoter activity which led to the increase of adenovirus E1 and viral infectivity. This combination therapy may be a promising therapeutic approach for pancreatic cancer., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

In Ayurvedic medicine, Salacia reticulata is known to be useful against various metabolic diseases, including diabetes and obesity. In this study, we attempted to clarify the antiobesity mechanism and the safety of S. reticulata in vivo and in vitro. We gave ordinary MF feed, alone or mixed with S. reticulata (0.3 or 1.0%), to Tsumura Suzuki obesity diabetes (TSOD) mice (spontaneous obese type II diabetes model mice) and Tsumura Suzuki non-obese (TSNO) mice (the corresponding reference animals), ad libitum for 2 months. As compared with the TSNO control mice, the TSOD control mice became obese due to fat accumulation and developed various signs of metabolic diseases. The TSOD mouse group receiving S. reticulata showed the following effects: suppression of body weight increase and fat accumulation, alleviation of abnormal lipid metabolism and abnormal glucose tolerance, and suppression of intrahepatic fat accumulation. Also, S. reticulata prevented the mesenteric adipocyte hypertrophy recognized in TSOD mice. In the TSNO controls, the feed containing 1.0% S. reticulata exerted a suppressing effect on body weight increase and fat accumulation, but the feed containing 0.3% S. reticulata did not show any effect at all. In an in vitro experiment using mouse-derived adipocyte precursor 3T3-L1 cells, S. reticulata significantly suppressed fat accumulation in the differentiation induction phase and maturation phase. This suggested that the metabolic disease-preventing effects of S. reticulata, including the antiobesity effect, may involve suppression of differentiation and accumulation in the adipocytes.

Intraductal papillary mucinous neoplasms of the pancreas are subclassified based on morphological features, and different immunohistochemical profiles have been identified in association with the subtypes. We previously reported that S100P was an early developmental marker of pancreatic carcinogenesis and that there was higher S100P expression in intraductal papillary mucinous neoplasms than in normal pancreatic ductal epithelium. However, there have been no reports on novel diagnostic markers to distinguish intraductal papillary mucinous neoplasm from nonneoplastic lesions. Surgical specimens of intraductal papillary mucinous neoplasm obtained from 105 patients were investigated using immunohistochemistry. S100P expression was not detected in normal pancreatic ductal epithelium but was detected in all intraductal papillary mucinous neoplasm cells (100%) with diffuse nuclear or nuclear/cytoplasmic staining. MUC5AC was also expressed in most of the intraductal papillary mucinous neoplasms (102/105; 97%). Furthermore, S100P was clearly expressed in the invasive component of intraductal papillary mucinous neoplasms (32/32; 100%), including perineural and lymphatic and minimal invasion. On the other hand, MUC5AC was expressed in only 23 cases of 32 invasive components (P < .01). These data suggest that the S100P antibody may be a useful marker for detecting all types of intraductal papillary mucinous neoplasms., (Copyright 2010 Elsevier Inc. All rights reserved.)

Extrahepatic bile duct (EBD) carcinoma is a relatively rare neoplasm worldwide, and its prognostic outcome remains unfavorable. Therefore, it is necessary to investigate molecular biologic features of EBD carcinomas. Focal adhesion kinase (FAK) plays a pivotal role in cell adhesion, survival, migration, and signal transduction, but FAK expression in EBD carcinomas has not been evaluated. We measured FAK expression in 76 EBD carcinomas using immunohistochemistry and evaluated its correlation with tumor progression, clinicopathologic factors, and patient outcome. FAK was expressed specifically in the cytoplasm of all normal biliary epithelia (100%). Most dysplastic epithelia also showed positive FAK expression except for 2 cases (92%), whereas EBD carcinomas showed positive FAK expression in 53 (77%) of 76 cases (P < .001, versus normal epithelia). FAK expression tended to be gradually reduced along as dysplasia progressed to carcinoma. Although FAK expression had no association with clinicopathologic factors, the positive FAK expression group showed significantly better survival than the negative FAK expression group (P < .05). However, FAK expression was not an independent prognostic factor by multivariate analysis. In conclusion, FAK expression was significantly lower in EBD carcinomas than in normal biliary epithelia and decreased expression of FAK seemed to be indicative of a poor prognosis, suggesting that FAK might play an inhibitory role for tumor progression in EBD carcinomas. It is important to notice the role of FAK in tumor progression when treatments targeting FAK are performed., (Copyright 2010 Elsevier Inc. All rights reserved.)

Adenovirus-mediated gene therapy shows promise for cancer therapy, but transgene expression of replication-defective adenovirus may be low and transient in clinical settings. Recent reports have shown that the use of a conditionally replication-competent adenovirus (CRAd) enhanced the gene transduction of a replication-defective adenovirus vector. The control of tumor-stromal interactions has also been determined to be important in cancer therapy. In this study, we investigated the effect of the human telomerase reverse transcriptase (hTERT)-CRAd, Ad5/3hTERTE1, which possesses the tumor-specific hTERT promoter with the chimeric fiber 5/3, on the transgene expression and therapeutic efficacy of a replication-defective adenovirus vector expressing NK4 under the control of the CMV promoter, Ad-NK4. In addition, we established a new strategy to target both cancer cells and cancer-stromal interactions. Human pancreatic cancer cells were infected with Ad-NK4 and either Ad5/3hTERTE1 (CRAd-combination group) or Ad5/3hTERTLuc (control-combination group). In the CRAd-combination group, Ad-NK4-delivered transgene expression was increased, leading to an enhanced inhibitory effect on the invasion of cancer cells. In in vivo experiments, NK4 expression within tumors and its inhibitory effect on tumor growth, angiogenesis, and metastasis were enhanced in the CRAd-combination group. These results suggest that hTERT-CRAd enhances the transgene expression and therapeutic efficacies of Ad-NK4, possibly through the in-trans replication of Ad-NK4 induced by adenovirus E1 derived from co-infected hTERT-CRAd. This approach may be a promising combination therapy against advanced pancreatic cancer.

We report the case of a patient demonstrating multiple gastric carcinoids with hypergastrinemia. A 50-year-old Japanese woman was admitted to our hospital for the further examination of multiple carcinoids of the stomach with hypergastrinemia, although she was asymptomatic. However, based on our clinical examination, this case seemed to be neither type I nor II carcinoid. We performed a total gastrectomy with D1 lymph node dissection. A pathological examination showed numerous endocrine micronests, hyperplasia of the parietal cells extending to the foveolar neck region, and numerous dilated oxyntic glands filled with eosinophilic secretions. Many parietal cells exhibited vacuolated cytoplasms and apical snouts. Furthermore, the dilated glands at the base of the mucosa had hyperchromatic nuclei and ciliated surfaces. The postoperative serum gastrin level was soon normalized to 47 pg/ml. This is only the third reported case of multiple gastric carcinoids with hypergastrinemia due to an intrinsic abnormality in the acid secretion of the parietal cells.

Objectives: The purpose of this study was to examine CD44v6 expression in intraductal papillary mucinous neoplasms (IPMNs) and clarify the role of CD44v6 in progression, invasion, metastasis, and morphogenesis of IPMNs., Methods: One hundred fifty-one samples of IPMNs and 30 normal controls were subjected to immunohistochemical analysis for CD44v6. The IPMNs were divided into 4 groups according to the grade of atypia (adenoma, borderline IPMN, noninvasive carcinoma, and invasive carcinoma) and 5 subtypes according to histological phenotype (gastric, intestinal, pancreatobiliary, oncocytic, and unclassified). Correlations were investigated between CD44v6 expression and clinicopathological characteristics including grade of atypia, subtype, lymph node metastasis, and invasion pattern., Results: Whereas normal ductal epithelium did not express CD44v6, CD44v6 expression was observed from the early stage of IPMNs and up-regulated in the progression of IPMNs to invasive carcinoma. CD44v6 expression in intestinal-type IPMNs was significantly lower compared with that in other subtypes. Whereas no correlation was observed between lymph node metastasis and CD44v6 expression in invasive IPM carcinomas, the invasion pattern was significantly correlated to CD44v6 expression., Conclusions: The present data indicate that CD44v6 expression determines the morphology and aggressiveness of IPMNs and is involved in development and invasion of IPMNs.

Objective: The aim of this study is to evaluate the clinical significance of S100A4 mRNA expression in pancreatic cancer., Materials and Methods: We obtained invasive ductal carcinoma (IDC) cells from ten lesions, intraductal papillary mucinous neoplasm (IPMN) cells from 20 lesions, and normal ductal cells from 20 normal pancreatic tissues by laser microdissection of frozen tissues. S100A4 expression was examined in the microdissected cells and in formalin-fixed paraffin-embedded (FFPE) samples of 87 pancreatic cancers by quantitative reverse transcription-polymerase chain reaction., Results: IDC cells expressed higher levels of S100A4 than IPMN cells (P = 0.002) and normal ductal cells (P < 0.001), although the difference between IPMN cells and normal ductal cells was not statistically significant (P = 0.070). Analysis of FFPE samples revealed that high S100A4 expression was significantly associated with a shorter overall survival (P = 0.023). In immunohistochemical analysis, the extent of S100A4 mRNA expression was significantly correlated with the expression of S100A4 protein (P = 0.028)., Conclusion: S100A4 could be a marker for malignancy in pancreatic tumors and for poor prognosis in patients with pancreatic cancer.

Patient: Marginal mandibulectomy was performed on a 30-year-old woman with an ameloblastoma in the left side of the mandible. Subsequently, the patient's chief complaints were in regard to the restoration of masticatory function and esthetics. Realizing that a free iliac crest graft and implant were necessary, we made a diagnostic wax-up and surgical template to match the maxillary and mandibular alveolar bone position. The surgical template was used to determine the position of a free iliac crest in the operating room. When the final restoration was finished, good occlusal balance had been achieved., Discussion: In this case, an oral surgeon as well as a prosthodontist confirmed the graft bone position by using the surgical template in the operating room. Therefore, it was possible to place the graft bone into the preplanned position and carry out the treatment plan smoothly from the first implant operation to the final restoration., Conclusion: This clinical report describes the importance of free iliac crest graft positions, the surgical template and collaboration between a prosthodontist and oral surgeon in forming the proper treatment plan.

Purpose: LIM domain only 2 (LMO2) has been identified as a novel oncogene associated with carcinogenesis and better prognosis in several malignant tumors. We investigate the involvement of LMO2 in pancreatic cancer., Experimental Design: We evaluated LMO2 expression in cultured cells, bulk tissues, and microdissected cells from pancreatic cancers by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry., Results: Of 164 pancreatic cancers, 98 (60%) were positive for LMO2 expression. LMO2 was more frequently detected in high-grade pancreatic intraepithelial neoplasia (PanIN) lesions (PanIN-2 and -3) than in low-grade PanIN lesions (PanIN-1A and -1B; P < .001) and was not detected in normal pancreatic ductal epithelium. The LMO2 messenger RNA levels were significantly higher in invasive ductal carcinoma cells than in normal pancreatic cells as evaluated by quantitative reverse transcription-polymerase chain reaction analyses of microdissected cells (P = .036). We also found higher incidence of LMO2 expression in histologic grade G1/G2 cancers than in grade G3 cancers (P < .001). The median survival time of LMO2-positive patients was significantly longer than that of LMO2-negative patients (P < .001), and multivariate analyses revealed that high LMO2 expression was an independent predictor of longer survival (risk ratio, 0.432, P < .001). Even among patients with a positive operative margin, LMO2-positive patients had a significant survival benefit compared with LMO2-negative patients. We further performed a large cohort study (n = 113) to examine the LMO2 messenger RNA levels in formalin-fixed paraffin-embedded samples and found similar results., Conclusions: LMO2 is a promising marker for predicting a better prognosis in pancreatic cancer.

Due to the poor prognosis of pancreatic cancer, novel diagnostic modalities for early diagnosis and new therapeutic strategy are urgently needed. Recently, microRNA-21 (miR-21) was reported to be strongly overexpressed in pancreatic cancer as well as in other solid cancers. We investigated the functional roles of miR-21, which have not been fully elucidated in pancreatic cancer. miR-21 expression was assessed in pancreatic cancer cell lines (14 cancer cell lines, primary cultures of normal pancreatic epithelial cells and fibroblasts, and a human normal pancreatic ductal epithelial cell line) and pancreatic tissue samples (25 cancer tissues and 25 normal tissues) by quantitative real-time reverse transcription-PCR amplification. Moreover, we investigated the proliferation, invasion, and chemoresistance of pancreatic cancer cells transfected with miR-21 precursor or inhibitor. miR-21 was markedly overexpressed in pancreatic cancer cells compared with nonmalignant cells, and miR-21 in cancer tissues was much higher than in nonmalignant tissues. The cancer cells transfected with the miR-21 precursor showed significantly increased proliferation, Matrigel invasion, and chemoresistance for gemcitabine compared with the control cells. In contrast, inhibition of miR-21 decreased proliferation, Matrigel invasion, and chemoresistance for gemcitabine. Moreover, miR-21 positively correlated with the mRNA expression of invasion-related genes, matrix metalloproteinase-2 and -9, and vascular endothelial growth factor. These data suggest that miR-21 expression is increased in pancreatic cancer cells and that miR-21 contributes to the cell proliferation, invasion, and chemoresistance of pancreatic cancer.

Subclassification of intraductal papillary mucinous neoplasms of the pancreas (IPMNs), based on morphological features and immunohistochemical profiles, has been proposed. Intestinal-type IPMNs frequently show moderate to severe dysplasia. Regenerating islet-derived family, member 4 (REG4) is associated with the adenoma-carcinoma sequence in colon cancer and it is also associated with intestinal phenotype. Therefore, to identify REG4 expression in IPMNs may be helpful to detect high-grade IPMNs. We also investigated REG4 expression and CDX2 expression in IPMNs. To investigate the expressions of REG4 and CDX2 in IPMNs and in invasive ductal adenocarcinoma derived from IPMN, we used immunohistochemical staining and microdissection-based quantitative real-time reverse transcription-polymerase chain reaction. Among 125 IPMNs, 43 (34%) were positive for REG4 and most of the intestinal-type IPMNs showed its expression (35/38). The positive ratio of REG4 expression in colloid carcinoma (5/7) was significantly higher than that in tubular carcinoma (1/17; P=0.003). Most of CDX2-positive cases (31/33) expressed REG4 protein, whereas only 12 of 92 CDX2-negative cases did (P<0.001). The levels of REG4 mRNA in intestinal-type IPMN were significantly higher compared to those in gastric-type IPMN or to normal pancreatic ductal epithelium (P=0.005, P=0.004, respectively). REG4 expression was observed more frequently in borderline lesions (14/28) and carcinoma (21/45) compared to adenoma (8/52). Using the Ki-67 labeling index, REG4 expression was significantly correlated with proliferative activity in borderline lesions. We conclude that REG4 is involved in the 'intestinal' pathway of carcinogenesis in IPMN.

A 68-year-old man underwent esophagogastroduodenoscopy (EGD) in February 2005. A type 2 advanced gastric cancer was observed on the gastric antrum. Abdominal US and CT revealed no distant metastasis. Curative distal gastrectomy with D2 lymph node dissection was therefore performed the next month. Postoperative staging was stage I B. In June 2005, he had symptoms of right hypochondralgia, general fatigue and appetite loss. An abdominal CT the next month revealed multiple liver metastases and so S-1/CDDP combination chemotherapy was initiated. After two courses of chemotherapy, marked decreases in size of the liver metastasis were recognized. However, we had to change the chemotherapy regimen because of adverse effect from the chemotherapy regime after the initial 2 courses. The patient died from tumor progression in May 2006.

Background: The functional roles of deoxycytidine kinase (dCK) in acquired resistance to gemcitabine remain unknown in pancreatic cancer. Here, the functional involvement of dCK in gemcitabine-resistance of pancreatic cancer was investigated., Materials and Methods: The levels of the dCK gene as well as other gemcitabine-related genes (hENT1, RRM1 and RRM2) were analyzed in gemcitabine-resistant pancreatic cancer cells (GR cells) using quantitative real-time reverse transcription polymerase chain reaction. The effects of inhibition of these genes on sensitivity to gemcitabine were evaluated., Results: In GR cells, expression of dCK was significantly reduced compared with that of parental cells (p < 0.05). The dCK-targeting siRNA significantly reduced gemcitabine sensitivity (p < 0.01) without affecting cell proliferation. The RRM1- and RRM2-targeting siRNAs increased gemcitabine sensitivity (p < 0.05) and reduced cell proliferation even without gemcitabine treatment. The hENT-targeting siRNA did not affect gemcitabine sensitivity or cell proliferation., Conclusion: Down-regulation of dCK specifically enhanced acquired resistance to gemcitabine in pancreatic cancer cells without affecting their proliferation.

Morphogenic signals like Hedgehog (Hh) and Wnt are reported to play critical roles in the progression of gastric cancer. We aimed to assess the relationship between Hh and Wnt signaling pathways. In 58 gastric cancer specimens, Wnt pathway activation was inversely correlated with Hh pathway activation. When AGS gastric cancer cells, in which Wnt signaling was constitutively active, were used as a target cell line, Gli1 overexpression suppressed Wnt transcriptional activity, nuclear beta-catenin accumulation and proliferation of AGS cells. Knock-down of beta-catenin by siRNA suppressed Wnt pathway activity and proliferation of AGS cells. Our data may provide some clues for the treatment of gastric cancer associated with Wnt signaling activation.

Purpose: Intraductal papillary mucinous neoplasms (IPMN) are known to show a transition from adenoma to carcinoma accompanied by several molecular abnormalities. ATM-Chk2-p53 DNA damage checkpoint activation, which is involved in prevention of the progression of several tumors, was analyzed to evaluate the role of the DNA damage checkpoint in the progression of IPMNs., Experimental Design: One hundred and twenty-eight IPMNs were classified into four groups (intraductal papillary mucinous adenoma, borderline IPMN, noninvasive intraductal papillary mucinous carcinoma, and invasive intraductal papillary mucinous carcinoma) and stained immunohistochemically using antibody for Thr(68)-phosphorylated Chk2. Expression of ATM, Chk2, and p21(WAF1) and accumulation of p53 were also analyzed., Results: Chk2 phosphorylation was shown in all adenomas and showed a significant decreasing trend with the progression of atypia (P < 0.0001 by the Cochran-Armitage test for trend). Expression of p21(WAF1) also exhibited a decreasing tendency (P < 0.0001), reflecting DNA damage checkpoint inactivation. p53 accumulation was mostly detected in malignant IPMNs. It was suggested that the DNA damage checkpoint provides a selective pressure for p53 mutation., Conclusion: Our findings indicate that DNA damage checkpoint activation occurs in the early stage of IPMNs and prevents their progression. It is suggested that disturbance of the DNA damage checkpoint pathway due to Chk2 inactivation or p53 mutation contributes to the carcinogenesis of IPMNs.

Despite evidence that Twist, a highly conserved basic helix-loop-helix transcription factor, is a novel oncogene, there are no reports describing Twist expression in pancreatic cancer. Intraductal papillary mucinous neoplasm (IPMN) and pancreatic intraepithelial neoplasia (PanIN) are precursor lesions of pancreatic cancer. To clarify involvement of Twist expression in pancreatic cancer, we used quantitative reverse transcription-polymerase chain reaction and examined Twist expression in pancreatic cancer, IPMN, and non-neoplastic pancreas using bulk tissues (11 cancers, 18 IPMNs, and 15 non-neoplastic pancreata), microdissected cells (cancer from 22 sections, IPMN from 19 sections, PanIN from 6 sections, and pancreatitis-affected epithelial cells from 14 sections), and pancreatic juice (16 from cancer, 28 from IPMN, and 17 from pancreatitis). Twist expression differed significantly between cancer and IPMN bulk tissues (p < 0.0001) but not between cancer and non-neoplastic tissues. Twist expressions differed significantly between microdissected cancer cells, IPMN cells, and pancreatitis-affected cells (all comparisons, p < 0.017). PanIN cells expressed significantly lower levels of Twist than did IDC cells (p = 0.016). Twist expression differed significantly between cancer and IPMN juice samples (p = 0.0002) but not between cancer and pancreatitis juice samples. Receiver operation characteristic curve analyses revealed that measurement of Twist was more useful for discriminating cancer from IPMN than from chronic pancreatitis (p = 0.009). Our results suggest that Twist is involved in tumor progression of pancreatic cancer and that measurement of Twist in pancreatic juice may be useful to differentiate pancreatic cancer from nonmalignant neoplasms such as IPMN., ((c) 2007 Wiley-Liss, Inc.)

There are few reports describing the diagnostic significance of S100A6 expression in clinical samples obtained from patients with pancreatic disease. In the present study, we measured S100A6 expression in pancreatic tissues and juice to evaluate its involvement in pancreatic carcinogenesis. We did quantitative real-time reverse transcription-PCR to measure mRNA expression in microdissected cells and pancreatic juice samples. Microdissected invasive ductal carcinoma and intraductal papillary mucinous neoplasm (IPMN) cells expressed significantly higher levels of S100A6 than did microdissected pancreatitis-affected epithelial and normal cells (all comparison; P < 0.008). Median levels of S100A6 in invasive ductal carcinoma were higher than those in IPMN, and those in pancreatitis-affected epithelial cells tended to be higher than those in normal cells, although these differences were not statistically significant. In analyses of pancreatic juice, IPMN and pancreatic cancer samples expressed significantly higher levels of S100A6 than did chronic pancreatitis samples (both; P < 0.017), but levels in pancreatic cancer and IPMN samples did not differ form each other. Receiver operating characteristic (ROC) curve analysis revealed that measurement of S100A6 was useful for discriminating cancer (area under the ROC curve, 0.864) or IPMN (area under the ROC curve, 0.749) from chronic pancreatitis. The present data suggest that expression of S100A6 is increased in a stepwise manner during pancreatic carcinogenesis and may be a biomarker for evaluating malignant potential. Measurement of S100A6 in pancreatic juice may be useful to detect early pancreatic cancer or identify individuals with high-risk lesions that may progress to pancreatic cancer.

Background: A disintegrin and metalloproteases (ADAMs) comprise a multifunctional family of membrane-anchored proteins. ADAM 9 and ADAM 15 are involved in cell migration and invasion. Expression of ADAM 9 and ADAM 15 was reported to be altered in several types of cancer., Materials and Methods: Quantitative real-time reverse transcription-polymerase chain reaction was performed to measure the expression of ADAM 9 mRNA in bulk pancreatic tissues. Results showed no significant difference in the expression of ADAM 9 mRNA between pancreatic cancer and non-neoplastic pancreas. Primary cultured pancreatic fibroblasts also expressed ADAM 9 mRNA. Therefore, a laser microdissection and pressure catapulting technique was employed to isolate cancer cells from tumor tissues. The expression of ADAM 9 and ADAM 15 mRNA was measured in microdissected samples (cancer cells, n = 11; normal epithelial cells, n = 13 for ADAM 9; cancer cells, n = 9; normal epithelial cells, n = 9 for ADAM 15)., Results: Pancreatic cancer cells expressed significantly higher levels of ADAM 9 and ADAM 15 mRNA than did normal pancreatic epithelial cells (p = 0.016 for ADAM 9; p = 0.004 for ADAM 15)., Conclusion: ADAM 9 and ADAM 15 are involved in pancreatic cancer. Microdissection-based analysis appears to be indispensable for the accurate analysis of the expression of certain ADAM family members in pancreatic cancer.

Purpose: Our goal was to clarify the involvement and clinical significance of S100P in pancreatic carcinogenesis., Experimental Design: We examined S100P expression in 45 bulk pancreatic tissues; in microdissected cells, including invasive ductal carcinoma (IDC) cells (20 sections), pancreatic intraepithelial neoplasia (PanIN) cells (12 sections), intraductal papillary mucinous neoplasm (IPMN) cells (19 sections), and normal epithelial cells (11 sections); and in pancreatic juice samples from 99 patients with pancreatic diseases (32 cancer, 35 IPMN, and 32 chronic pancreatitis samples). We used quantitative real-time reverse transcription-PCR with gene-specific priming to measure S100P in these various types of samples., Results: In bulk tissue analyses, pancreatic cancer and IPMN expressed significantly higher levels of S100P than did nonneoplastic pancreas (P<0.017 and P=0.0013, respectively). Microdissection analyses revealed that IPMN expressed significantly higher levels of S100P than did IDC (P<0.0001) and PanIN (P=0.0031), although S100P expression did not differ between IDC and PanIN (P=0.077). In pancreatic juice analyses, cancer and IPMN juice expressed significantly higher levels of S100P than did pancreatitis juice (both P<0.0001). Receiver operating characteristic curve analyses revealed that measurement of S100P in pancreatic juice was useful for discriminating neoplastic disease from chronic pancreatitis (area under the curve=0.837; 95% confidence interval, 0.749-0.903)., Conclusion: S100P may be an early developmental marker of pancreatic carcinogenesis, and measurement of S100P in pancreatic juice may be useful for early detection of pancreatic cancer or screening of early pancreatic carcinogenesis.

Purpose: Recent microarray analyses revealed that expression of S100A11 is up-regulated in pancreatic cancer. The aim of the present study was to evaluate the association of S100A11 with pancreatic carcinogenesis., Experimental Design: We measured S100A11 mRNA expression in various clinical samples related to pancreatic cancer and its precursor lesions, intraductal papillary mucinous neoplasm (IPMN) and pancreatic intraepithelial neoplasia, by quantitative reverse transcription-PCR., Results: Levels of S100A11 were significantly higher in pancreatic cancer (n=22) and IPMN (n=18) bulk tissues than in nonneoplastic bulk tissues (n=22; P<0.0001 for both). Levels of S100A11 did not differ between pancreatic cancer and IPMN bulk tissues. In microdissection analyses, however, IPMN cells (n=21) expressed significantly higher levels of S100A11 than did cancer cells (n=23; P=0.003). The median level of S100A11 expression was higher in pancreatic intraepithelial neoplasia cells (n=6) than in cancer cells. In pancreatic juice analyses, cancer-related (n=24; P=0.004) and IPMN-related (n=18; P=0.001) juice expressed significantly higher levels of S100A11 than did chronic pancreatitis-related juice (n=23)., Conclusions: The present data suggest that expression of S100A11, a putative tumor suppressor gene, is increased in the early stage of pancreatic carcinogenesis and decreased during subsequent progression to cancer. Analysis of the S100A11 level in pancreatic juice may be an effective tool for screening of patients with high-risk lesions that could progress to pancreatic cancer or detecting early-stage pancreatic cancer.

Background: Pancreatic cancer frequently recurs after operative treatment, resulting in a poor prognosis. Inhibition of proliferation of residual cancer cells is important for improved survival of patients with pancreatic cancer. Fibrin glue (FG) is a biocompatible, adherent hemostat that can deliver high concentrations of anticancer drugs to residual cancer cells. The aim of this study was to evaluate the local antitumor effect of a mixture of gemcitabine (GEM) and FG on pancreatic cancer cells implanted orthotopically in nude mice., Methods: SUIT-2 human pancreatic cells were injected into the tail of the pancreas of nude mice. Seven days later, groups of mice were treated with 80 mg/kg GEM mixed with 0.5 mL fibrin glue (GEM + FG), 0.5 mL FG alone (FG), single intraperitoneal (i.p.) injection of 80 mg/kg GEM (GEM1), i.p. injection of 80 mg/kg GEM weekly for 3 weeks (GEM1,2,3), GEM + FG followed by weekly GEM injections for 2 weeks (GEM + FG + GEM2,3), or i.p. injection of PBS weekly for 3 weeks (controls)., Results: Twenty-eight days after cell injections, tumor volumes of groups treated with GEM + FG + GEM2,3, GEM1,2,3, GEM + FG, GEM1, and FG were decreased by 84%, 70%, 62%, 37%, and 10%, respectively, compared to that of control mice. GEM + FG + GEM2,3 had the strongest anticancer effect compared to all other groups (P < .05). Additionally, GEM + FG showed a more potent antitumor effect compared to GEM1 (P < .05). Survival of mice treated with GEM + FG + GEM2,3 was longer than that of mice in all other groups (P < .05)., Conclusions: A mixture of GEM and FG was effective in inhibiting the growth of orthotopically implanted pancreatic neoplasms in nude mice. This procedure may be useful clinically to prevent the local recurrence of pancreatic cancer after pancreatectomy.

Aim: To investigate c-met expression during early pancreatic carcinogenesis., Methods: We used 46 bulk tissues and 36 micro-dissected samples, including normal pancreas, chronic pancreatitis, and pancreatic cancer, for quantitative real-time reverse transcription-polymerase chain reaction., Results: In bulk tissue analyses, pancreatic cancer tissues expressed significantly higher levels of c-met than did chronic pancreatitis and normal pancreas tissues. c-met levels did not differ between chronic pancreatitis and normal pancreas tissues. In microdissection-based analyses, c-met was expressed at higher levels in microdissected pancreatic cancer cells and pancreatitis-affected epithelial cells than in normal ductal epithelial cells (both, P < 0.01). Interestingly, pancreatitis-affected epithelial cells expressed levels of c-met similar to those of pancreatic cancer cells., Conclusion: Overexpression of c-met occurs during the early stage of pancreatic carcinogenesis, and a single alteration of c-met expression is not sufficient for progression of chronic pancreatitis-affected epithelial cells to pancreatic cancer cells.

Although telomerase activity is a promising diagnostic marker, clinical introduction of this marker for cancer diagnosis is still problematic due to the lack of means of evaluating sample quality. Human telomerase reverse transcriptase (hTERT), one of the subunits of telomerase, is also a promising diagnostic marker. In the present study, we did large-scale analysis of 88 pancreatic juice samples to determine the feasibility of quantitative analysis of hTERT mRNA for diagnosis of pancreatic cancer. We found significant differences in hTERT expression among carcinoma-derived, intraductal papillary mucinous neoplasm (IPMN)-derived, and chronic pancreatitis-derived juice samples. Results showed that quantitative analyses of hTERT mRNAs are more useful in discriminating carcinoma from IPMN than from chronic pancreatitis. When the specificity was set at 100%, the sensitivity for differentiation between carcinoma and IPMN was 43.5%, whereas the sensitivity of cytologic examination was 22.0%. There were significant differences in hTERT expression among carcinoma cells, IPMN cells, and normal ductal cells isolated from pancreatic tissues by microdissection. Lymphocytes and hyperplastic epithelial cells isolated from tissues with the histologic appearance of pancreatitis showed various expression levels of hTERT. Our results suggest that quantitative analysis of hTERT mRNA in pancreatic juice is advantageous over cytologic analysis for differentiation between carcinoma and IPMN but probably not for differentiation between carcinoma and chronic pancreatitis.

Pancreatic juice is a promising type of diagnostic sample for pancreatic cancer, and members of the mucin (MUC) family are diagnostic candidates. To evaluate the utility of MUC family members as diagnostic markers, we measured MUC mRNA expression in pancreatic tissues and pancreatic juice obtained from patients with different pancreatic diseases as well as in pancreatic cancer cell lines by real-time PCR. Furthermore, to support the possibility of early diagnosis by quantification of MUC1 and MUC5AC, immunohistochemistry and microdissection-based quantitative analysis of mRNA were carried out. There was no significant correlation between MUC1 and MUC5AC expression in cell lines. When beta-actin was used as a reference gene, median MUC1 and MUC5AC mRNA expression levels were remarkably greater in tumoral tissues than in non-tumoral tissues, but median MUC4 and MUC6 mRNA expression levels were not. Receiver operating characteristic curve analysis showed that quantitative analysis of MUC1 and MUC5AC mRNA in pancreatic juice is better diagnostic modality than that of MUC4 and MUC6 mRNA. Immunohistochemistry showed that MUC1 and MUC5AC were highly expressed in invasive ductal carcinomas (IDC) and moderately expressed in high-grade pancreatic intraepithelial neoplasia (PanIN); no staining was observed in normal ducts. Analysis of cells isolated by microdissection showed stepwise upregulation of MUC1 and MUC5AC in the development of high-grade PanIN to IDC. Our results suggest that MUC1 and MUC5AC are upregulated stepwise in pancreatic carcinogenesis and that quantitative assessment of MUC1 and MUC5AC mRNA in pancreatic juice has high potential for preoperative diagnosis of pancreatic cancer., (Copyright 2005 Wiley-Liss, Inc.)

The DNA chip is a very powerful tool for genetic analysis. Conventional DNA chips that utilize fluorescence detection systems are very complicated, expensive and impractical, but the electrochemical array (ECA) chip is gaining popularity. To investigate the validity of the ECA chip, which utilizes ferrocenyl-naphthalene-diimide (FND), k-ras mutations in 20 pancreatic cancer tissues were examined. DNA was isolated from 20 pancreatic cancer tissues and subjected to a 2-stage polymerase chain reaction (PCR). The k-ras mutations were detected with the ECA chip. To verify the reliability of the ECA chip, the DNA was also analyzed by direct sequencing and the PCR-dependent preferential homoduplex formation assay (PCR-PHFA). The ECA chip could detect one mutation in a background of 1000 wild-type DNAs. K-ras mutations were identified in 17 out of 20 (85%) pancreatic cancer samples. Three mutations of codon 12 of k-ras, GTT, GAT and AGT, were detected. K-ras mutations were detected in 13 out of 20 (65%) samples by sequencing and in 17 out of 20 (85%) samples by PCR-PHFA. These findings were concordant with the ECA chip result. The FND-ECA chip is a sensitive, rapid and reliable method for screening point mutations in a variety of clinical samples., (Copyright© 2006 International Institute of Anticaner Research (Dr. John G. Delinassios), All rights reserved.)

Recent microarray analyses showed that the S100 family contains members that are candidate diagnostic markers or therapeutic targets. In the present study, to evaluate the involvement of S100A6 in pancreatic cancer and its clinical usefulness for diagnosis, we examined S100A6 mRNA expression in pancreatic tissues and pancreatic juice from patients with different pancreatic diseases. To investigate the role of S100A6 in carcinogenesis of pancreatic cancer and the potential of S100A6 as a diagnostic marker for early detection of pancreatic cancer, we did immunohistochemistry and microdissection-based mRNA analysis of pancreatic normal ducts, pancreatic intraepithelial neoplasias, and invasive ductal carcinomas. We also used in vitro experiments and microarray analysis with RNA interference to evaluate the functional role of S100A6 and its potential as a therapeutic target for pancreatic cancer. S100A6 mRNA levels were significantly higher in carcinoma specimens than in nonneoplastic tissues. In pancreatic juice, there was a significant difference in S100A6 expression between patients with carcinoma and those with nonneoplastic disease. Receiver operating characteristic curves revealed that S100A6 might be a useful marker for diagnosis of pancreatic cancer. Immunohistochemistry and microdissection-based analysis showed differential expression of S100A6 among normal ducts, pancreatic intraepithelial neoplasias, and invasive ductal carcinomas. In vitro data showed that inhibition of S100A6 decreased proliferation and invasiveness of cancer cells, and these findings were supported by microarray data. Our present results suggest that quantitation of S100A6 mRNA is a promising tool for diagnosis of pancreatic cancer, and that S100A6 may be a promising therapeutic target for pancreatic cancer.

NK4, a 4-kringle fragment of hepatocyte growth factor (HGF), is an HGF antagonist that also acts as an angiogenesis inhibitor. NK4 strongly inhibits the infiltration, metastasis, and tumor growth of pancreatic cancer. The aim of our study was to evaluate the antitumor effect of adenovirus-mediated NK4 gene transfer to the liver on hepatic metastasis of pancreatic cancer in vivo. We constructed recombinant adenoviral NK4 (Ad-NK4), which encodes a secreted form of human NK4. Intrasplenic injection of Ad-NK4 induced high and relatively maintained expression of NK4 protein in the liver and suppressed the number and growth of metastatic foci in the liver in a nude mouse model. Microscopically, central necrosis was found even in small metastatic foci in Ad-NK4 treated mice. Immunohistochemical analysis of metastatic tumors showed a remarkable decrease in microvessel density and an increase in the number of apoptotic tumor cells after treatment with Ad-NK4. These results indicate that intraportal injection of Ad-NK4 may be a useful therapeutic modality for the clinical control of hepatic metastasis in pancreatic cancer., (Copyright (c) 2005 Wiley-Liss, Inc.)

Background/aims: The differentiation between benign and malignant jaundice is sometimes difficult even with modern diagnostic modalities. With recent advances in molecular biology, Ki-ras point mutation in codon 12 and p53 mutation have been reported as novel biomarkers of hepatobiliary and pancreatic carcinoma. The purpose of this study was to compare exfoliative bile cytology and biomarkers of the bile in patients with hepatobiliary and pancreatic diseases., Methodology: Cytologic examination and novel biomarkers, point mutations of codon 12 of Ki-ras and p53 mutations, were examined in the biliary tract bile aspirated through percutaneous transhepatic biliary drainage (PTBD) tube in 30 Japanese patients with benign and malignant hepatobiliary and pancreatic diseases., Results: Sensitivity of the exfoliative bile cytology for malignant conditions was 31% and specificity 100%, while sensitivity of molecular markers of the bile was 25% and specificity 93%. When the cytologic examination and genetic study were in conjunction, sensitivity increased to 50% and specificity remained 93%., Conclusions: These findings suggest that genetic examinations of the bile, when used in conjunction with cytology, may improve the diagnostic yield for suspected malignant tumors of the hepatobiliary and pancreatic system.

Measurement of telomerase activity is a promising diagnostic tool for pancreatic cancer. Detection of mRNA for human telomerase reverse transcriptase (hTERT), a catalytic subunit of telomerase, is also a diagnostic candidate. In the present study, we developed a telomeric repeat amplification protocol assay with real-time PCR and a protocol for quantification of hTERT mRNA with real-time PCR. To evaluate the feasibility of these methods for diagnosis of pancreatic cancer, we measured telomerase activity and hTERT expression in pancreatic cancer cell lines, pancreatic tissues, and pancreatic juice samples from patients with different pancreatic diseases. There were significant correlations between telomerase activity and hTERT expression in cell lines, tissues, and juice samples. The levels of telomerase activity and hTERT expression were significantly higher in tumoral tissues than in nontumoral tissues. In pancreatic juice specimens, some carcinoma samples showed remarkably high expression of hTERT. However, there were no significant differences in hTERT expression between patients with carcinoma and those with benign diseases, although significant differences in telomerase activity were observed. Our present results suggest that the combined assessment of hTERT and telomerase activities in pancreatic juice provides a potent diagnostic method for pancreatic cancer.

Local recurrence and hepatic metastasis are still the major causes of death of patients who have undergone resection for pancreatic cancer. To decrease the incidence of local recurrence, we have proposed and devised several trans-tissue and local delivery systems, all of which could be applied immediately after surgery at the resected sites: (1) System I: a drug-loaded photocured gelatinous tissue-adhesive gel (bioactive substance reservoir) that enables the sustained release of a drug, protein, or gene-encoding adenovirus, (2) System II: an anti-cytokine antibody-fixed photocured gelatinous, tissue-adhesive gel (cytokine barrier) that prevents cytokine permeation into the resected tissue, (3) System III: a gene-modified cell sheet that enables the sustained release of a very costly protein produced by gene-transduced cells and (4) System IV: a percutaneous drug-delivery device that enables continuous drug infusion and easy removal from the body. This review article is a summary of our several years of efforts and attempts, which are composed of integrated disciplines including active biomaterials and genetic- and tissue-engineerings, to overcome the recurrence of pancreatic cancer. Here, we outline our proposed strategies and therapeutic devices/materials and discuss their potential therapeutic effectiveness, promises and challenges in the clinical settings.

Background: Early detection of pancreatic carcinoma is difficult even with current diagnostic tools. Novel biomarkers and detection techniques are urgently needed. Telomerase activity is a promising diagnostic marker. However, the conventional telomeric repeat amplification protocol (TRAP) assay is not suitable for clinical application because of its complexity, time-consuming nature, and the effects of polymerase chain reaction (PCR) inhibitors in samples leading to difficulties in quantification., Methods: The authors used a hybridization protection assay in combination with TRAP (TRAP/HPA) to investigate the effects of PCR inhibitors in pancreatic juice on quantification of telomerase activity. They analyzed 117 consecutive samples of pancreatic juice to determine the feasibility of TRAP/HPA for diagnosis of pancreatic carcinoma., Results: The authors found that TRAP/HPA was 1000-fold more sensitive than the conventional TRAP assay, and that the effects of PCR inhibitors could be avoided by diluting samples. In a large analysis of pancreatic juice samples with TRAP/HPA, 17 samples were excluded from the final analysis because of insufficient follow-up periods or inadequate treatment of the samples. Relative telomerase activity (RTA) in samples from patients with pancreatic carcinoma was significantly higher in comparison to samples from patients with pancreatitis and 13 (61.9%) of 21 samples from patients with pancreatic carcinoma showed high RTA (> 4 U). Meanwhile, high RTAs were observed in 4 of 35 (11.4%) samples from patients with intraductal papillary mucinous tumor and in 1 of 40 samples (2.5%) fom patients without malignant disease., Conclusions: TRAP/HPA accurately evaluated weak telomerase activity in pancreatic juice samples without the problem due to PCR inhibitors. This large analysis of nonselected pancreatic juice samples suggested that TRAP/HPA is a promising approach for the diagnosis of pancreatic carcinoma., ((c) 2004 American Cancer Society)

Gallbladder and bile duct carcinomas belong to the family of biliary tract tumors, but they demonstrate different clinical behavior. We evaluated a series of biliary tract carcinomas to determine whether they also had genotypic differences by analysis of the tumor suppressor genes DPC4 and p53. Twenty-one gallbladder cancers, 20 intrahepatic bile duct carcinomas, and 10 extrahepatic bile duct carcinomas were retrieved from the surgical pathology files of Kaohsiung Medical University Hospital. Sections were immunostained with monoclonal antibodies to the DPC4 and P53 proteins. Statistical differences between gallbladder cancer and bile duct carcinomas were determined using chi2 analysis or the Fisher's exact test, when appropriate. Two of the 21 gallbladder cancers (9.5%), 7 of the 20 intrahepatic bile duct carcinomas (35%), and five of the 10 extrahepatic bile duct carcinomas (50%) were negatively labeled for DPC4. The differences were significant between gallbladder carcinoma and both intrahepatic bile duct carcinomas (p = 0.023) and extrahepatic bile duct carcinomas (p = 0.012). A higher frequency of P53 overexpression was found in gallbladder cancers (61.9%) than in intrahepatic bile duct carcinomas (26.3%) (p = 0.024). This study suggests that the DPC4 gene may play a limited role in gallbladder carcinoma; however, p53 gene mutation is more frequently found in gallbladder cancers. In contrast, DPC4 deletion may be more common in bile duct carcinomas, especially in those arising from the extrahepatic bile duct. These findings support the concept that gallbladder and bile duct carcinomas are different tumors with differing etiologies and tumorigenesis.

Radiotherapy represents a major treatment option for patients with pancreatic cancer, but recent evidence suggests that radiation can promote invasion and metastasis of cancer cells. Interactions between cancer cells and surrounding stromal cells may play an important role in aggressive tumor progression. In the present study, we investigated the invasive phenotype of pancreatic cancer cells in response to coculture with irradiated fibroblasts. Using in vitro invasion assay, we demonstrated that coculture with nonirradiated fibroblasts significantly increased the invasive ability of pancreatic cancer cells and, surprisingly, the increased invasiveness was further accelerated when they were cocultured with irradiated fibroblasts. The hepatocyte growth factor (HGF) secretion from fibroblasts remained unchanged after irradiation, whereas exposure of pancreatic cancer cells to supernatant from irradiated fibroblasts resulted in increased phosphorylation of c-Met (HGF receptor) and mitogen-activated protein kinase activity, possibly or partially via increased expression of c-Met. We also demonstrated that scattering of pancreatic cancer cells was accelerated by the supernatant from irradiated fibroblasts. The enhanced invasiveness of pancreatic cancer cells induced by coculture with irradiated fibroblasts was completely blocked by NK4, a specific antagonist of HGF. These data suggest that invasive potential of certain pancreatic cancer cells is enhanced by soluble mediator(s) released from irradiated fibroblasts possibly through up-regulation of c-Met expression/phosphorylation and mitogen-activated protein kinase activity in pancreatic cancer cells. Our present findings further support the potential use of NK4 during radiotherapy for patients with pancreatic cancer.

We present 2 cases of extensor pollicis longus tenosynovitis caused by the unusual course of the extensor pollicis longus tendon. The extensor pollicis longus tendon passed through the first extensor compartment and was constricted severely. The symptoms were similar to those of de Quervain's disease. The clinical presentation, diagnosis, and surgical technique of this very rare anatomic condition are discussed.