Koichi Aiura

Comparison of K-ras point mutation distributions in intraductal papillary-mucinous tumors and ductal adenocarcinoma of the pancreas

Intraductal papillary‐mucinous tumors (IPMT) consist of cells with varying histologic degrees of severity and exhibit multiple tumor loci; however, whether or not these lesions exhibit the same genetic changes has not been clarified. To investigate this point, we analyzed K‐ras mutations in multiple IPMT lesions from each patient enrolled in our study and compared our findings to those for patients with ductal adenocarcinoma of the pancreas (DC). Twenty IPMT specimens and 7 DC specimens were resected, microdissected and analyzed for the presence of K‐ras mutations. The mutated genes were then sequenced using a genetic analyzer. K‐ras mutations were observed in 80% of IPMT and 100% of DC patients. More than 2 types of K‐ras mutation were observed in the main tumors of 43.8% of IPMT and 0% of DC patients. K‐ras mutations in peritumoral and separated lesions were observed in 66.7% and 62.5% of IPMT patients, respectively. At least one identical mutation between the main tumor and the peritumoral or separated lesions was recognized in all of the IPMT patients with those lesions. Different mutations from those in the main tumor were observed in 40% of IPMT patients with separated lesions. The survival curve of IPMT‐carcinoma patients with more than 2 types of K‐ras mutation in the main tumor was better than that with one type of K‐ras mutation. IPMT patients exhibit a remarkably genetic heterogeneity in main tumor and have good prognosis.

Synuclein-γ Is Closely Involved in Perineural Invasion and Distant Metastasis in Mouse Models and Is a Novel Prognostic Factor in Pancreatic Cancer

Purpose: Perineural invasion is associated with the high incidence of local recurrence and a dismal prognosis in pancreatic cancer. We previously reported a novel perineural invasion model and distinguished high– and low–perineural invasion groups in pancreatic cancer cell lines. This study aimed to elucidate the molecular mechanism of perineural invasion. Experimental Design: To identify key biological markers involved in perineural invasion, differentially expressed molecules were investigated by proteomics and transcriptomics. Synuclein-γ emerged as the only up-regulated molecule in high–perineural invasion group by both analyses. The clinical significance and the biological property of synuclein-γ were examined in 62 resected cases of pancreatic cancer and mouse models. Results: Synuclein-γ overexpression was observed in 38 (61%) cases and correlated with major invasive parameters, including perineural invasion and lymph node metastasis (P < 0.05). Multivariate analyses revealed synuclein-γ overexpression as the only independent predictor of diminished overall survival [hazard ratio, 3.4 (95% confidence interval, 1.51-7.51)] and the strongest negative indicator of disease-free survival [2.8 (1.26-6.02)]. In mouse perineural invasion and orthotopic transplantation models, stable synuclein-γ suppression by short hairpin RNA significantly reduced the incidence of perineural invasion (P = 0.009) and liver/lymph node metastasis (P = 0.019 and P = 0.020, respectively) compared with the control. Conclusions: This is the first study to provide in vivo evidence that synuclein-γ is closely involved in perineural invasion/distant metastasis and is a significant prognostic factor in pancreatic cancer. Synuclein-γ may serve as a promising molecular target of early diagnosis and anticancer therapy.

Establishment of Perineural Invasion Models and Analysis of Gene Expression Revealed an Invariant Chain (CD74)as a Possible Molecule Involved in Perineural Invasion in Pancreatic Cancer

Purpose: Perineural invasion causes frequent local recurrence even after resection and a poor prognosis for pancreatic cancer. We established perineural invasion models and analyzed the molecular mechanism of perineural invasion in pancreatic cancer. Experimental Design: Seven pancreatic cancer cell lines with or without human peripheral nerves were s.c. implanted in nonobese diabetes/severe combined immunodeficient mice. We compared expression profiles among high and low perineural invasion cell lines by using an oligonucleotide microarray. We examined up-regulation of the invariant chain (CD74) in high perineural invasion cell lines in mRNA and protein levels and surgical cases immunohistochemically. Results: Four of seven pancreatic cancer cell lines (CaPan1, CaPan2, CFPAC, and MPanc96) showed perineural invasion to s.c. transplanted human peripheral nerves. Moreover, CaPan1 and CaPan2 (high perineural invasion group) also resulted in a high frequency of perineural invasion to mouse s.c. peripheral nerves, whereas three pancreatic cancer cell lines HPAFII, AsPC1, and Panc1 (low perineural invasion group) did not show perineural invasion to either human or mouse nerves. We identified 37 up-regulated genes and 12 down-regulated genes in the high perineural invasion group compared with the low perineural invasion group. Among them, CD74 was up-regulated in the high perineural invasion group in mRNA and protein levels. Furthermore, immunohistochemical expression of CD74 in clinical cases revealed its significant overexpression in pancreatic cancer with perineural invasion (P < 0.008). Conclusions: This is the first report of perineural invasion models using human pancreatic cancer cell lines. In combination with gene expression profiling, it was indicated that CD74 could be a candidate molecule involved in perineural invasion. These models provide new approaches for study of perineural invasion in pancreatic cancer.

Mesenchymal stem cells regulate epithelial–mesenchymal transition and tumor progression of pancreatic cancer cells

Cancer‐associated fibroblasts contribute to cancer progression that is caused by epithelial–mesenchymal transition (EMT). Recently, mesenchymal stem cells (MSCs) were found to be the major candidate involved in the development of tumor‐promoting cancer stroma. Here we report that α‐smooth muscle actin‐positive myofibroblast‐like cells originating from MSCs contribute to inducing EMT in side population cells of pancreatic cancer. More importantly, MSC‐derived myofibroblasts function to maintain tumor‐initiating stem cell‐like characteristics, including augmenting expression levels of various stemness‐associated genes, enhancing sphere‐ forming activity, promoting tumor formation in a mouse xenograft model, and showing resistance to anticancer drugs. Furthermore, both γ‐secretase inhibitor and siRNA directed against Jagged‐1 attenuated MSC‐associated E‐cadherin suppression and sphere formation in pancreatic cancer side population cells. Thus, our results suggest that MSC‐derived myofibroblasts play important roles in regulating EMT and tumor‐initiating stem cell‐like properties of pancreatic cancer cells through an intermediating Notch signal.

Surgery versus radiochemotherapy for resectable locally invasive pancreatic cancer: final results of a randomized multi-institutional trial.

PurposeAlthough the outcome of surgery for locally advanced pancreatic cancer remains poor, it is improving, with 5-year survival up to about 10% in Japan. The preliminary results of our multi-institutional randomized controlled trial revealed better survival after surgery than after radiochemotherapy. We report the final results of this study after 5 years of follow-up.MethodsPatients with preoperative findings of pancreatic cancer invading the pancreatic capsule without involvement of the superior mesenteric or common hepatic arteries, or distant metastasis, were included in this randomized controlled trial, with their consent. If the laparotomy findings were consistent with these criteria, the patient was randomized to a surgery group or a radiochemotherapy group (5-fluorouracil 200 mg/m2/day and 5040 Gy radiotherapy). We compared the mean survival time, 3-and 5-year survival rates, and hazard ratio.ResultsThe surgery and radiochemotherapy groups comprised 20 and 22 patients, respectively. Patients were followed up for 5 years or longer, or until an event occurred to preclude this. The surgery group had significantly better survival than the radiochemotherapy group (P < 0.03). Surgery increased the survival time and 3-year survival rate by an average of 11.8 months and 20%, respectively, and it halved the instantaneous mortality (hazard) rate.ConclusionLocally invasive pancreatic cancer without distant metastases or major arterial invasion is treated most effectively by surgical resection.

Solitary cell infiltration is a novel indicator of poor prognosis and epithelial-mesenchymal transition in pancreatic cancer.

Pancreatic cancer is one of the most aggressive and lethal human malignancies in the Western world. A wide variety of intratumor glandular differentiation, including solitary infiltrating cancer cells, is a prominent microscopic finding in pancreatic cancer. We reviewed 114 resected cases of pancreatic ductal adenocarcinoma to investigate the prognostic impact of the degree of solitary cell infiltration, defined by the number of solitary infiltrating cancer cells. The clinicopathologic correlation of solitary cell infiltration was further evaluated. Seventy-six (67%) cases showed 7 or more solitary infiltrating cancer cells in 10 high-power fields and were labeled as having a high degree of solitary cell infiltration. A high degree of solitary cell infiltration correlated significantly with poor overall survival, the grade, lymphatic invasion, and lymph node metastasis. Multivariate analysis revealed that the degree of solitary cell infiltration, the grade, and the margin status were independent prognostic factors. Grade 1 and 2 tumors with a high degree of solitary cell infiltration, compared with low infiltration, correlated significantly with poor overall survival. Grade 3 tumors showed a worse overall survival than grade 1 and 2 tumors with either a high or a low degree of solitary cell infiltration. Immunohistochemical analysis showed that a high degree of solitary cell infiltration correlated with reduced E-cadherin and increased vimentin expression. In conclusion, solitary cell infiltration is a significant prognostic indicator and serves as a morphological clue to epithelial-mesenchymal transition in pancreatic cancer.

Can Endoscopic Ultrasonography Differentiate Nonneoplastic from Neoplastic Gallbladder Polyps

The present study aimed to clarify the endoscopic ultrasonography (EUS) features of nonneoplastic (cholesterol polyps and adenomyomatosis) and neoplastic (adenoma and adenocarcinoma) gallbladder polyps and to evaluate the effectiveness and limitation of EUS in the differential diagnosis of these lesions. We retrospectively compared EUS images with histologic findings in 29 surgical cases with gallbladder polyps with a diameter of 10 to 20 mm. Those cases were indicated for surgery based on the findings of a sessile appearance, a solitary lesion, low echogenicity, and/or a lobulated surface. Six of 10 cholesterol polyps were atypically seen as partially or completely hypoechoic due to predominant proliferation of glandular epithelia. Nine of 10 cholesterol polyps demonstrated an aggregation of hyperechoic spots, which represented multiple granules of cholesterosis. All adenomyomatoses (n = 10) showed multiple microcysts, which corresponded to proliferated Rokitansky-Aschoff sinuses. However, three of nine neoplastic lesions (three adenomas and six adenocarcinomas) showed one of these signs due to concomitant cholesterosis (n = 2) or proliferated Rokitansky-Aschoff sinuses (n = 1). In conclusion, 69% (20/29) of gallbladder polyps larger than 10 mm that were preoperatively suspected of malignancy were nonneoplastic. An aggregation of hyperechoic spots and multiple microcysts are considered to be important predictive factors for cholesterol polyps and adenomyomatosis, respectively. However, we should caution that these findings can also occur in neoplastic polyps when they contain a concomitant nonneoplastic component (cholesterosis or proliferated Rokitansky-Aschoff sinuses).

The Influence of Platelets on the Promotion of Invasion by Tumor Cells and Inhibition by Antiplatelet Agents

Objective: Using a chemoinvasion assay, we show that platelets promote invasiveness of 5 pancreatic adenocarcinoma cell lines. Methods: Gelatin zymography and Western blot analysis were performed to detect metalloproteinase-9 (MMP-9) secreted from tumor cells in the presence or absence of platelets. The effects of antiplatelet agents on the invasiveness of tumor cells and the secretion level of MMP-9 were evaluated. Results: The number of traversed tumor cells significantly increased when incubated with platelets compared without platelets in all cell lines. The MMP-9 band was detected in all tumor cell lines, and the intensity was obviously greater in conditions of incubation with platelets than without. In the experiment of antiplatelet agents effects, it was confirmed that invasiveness of tumor cells significantly decreased following incubation with cilostazol depending on the concentration in spite of the presence of platelets. The level of MMP-9 also significantly decreased in the ELISA analysis. Conclusions: These data mean platelets activate invasiveness of tumor cells because of enhanced MMP-9 secretion. Furthermore, anti-platelet drugs may inhibit invasiveness of tumor cells due to decreased MMP-9 secretion, and this inhibition may lead to the suppression of tumor cell invasion. We propose that antiplatelet agents are applicable in clinical treatment to inhibit metastasis of malignant tumor cells.

Gallstone Disease after Extended (D2) Lymph Node Dissection for Gastric Cancer

Few studies have reported the incidence and clinical outcomes of gallstone disease after extended (D2) lymph node dissection for gastric cancer. The present study was designed to retrospectively compare limited (D1) and D2 dissections in terms of gallstone formation, presentation of gallstones, and surgery for gallstone disease. A total of 805 Japanese gastric cancer patients (595 male, 210 female) who underwent curative resection with D1 (n = 490) or D2 (n= 315) dissection were retrospectively reviewed. Of those subjects followed for 70.5 ± 44.3 months (range: 2-196 months), 102 (12.7%) developed gallstones. The incidence of gallstone formation was higher in the D2 group than in the D1 group (17.8% vs. 9.4%, p= 0.001). The interval between gastrectomy and detection of gallstones was shorter in the D2 group than in the Dl group (18.8 ± 11.4 months vs. 29.4 ± 18.3 months, p = 0.002). Of those with gallstones followed for 48.0 ± 28.6 months (range: 1-158 months), 74 (72.5%) remained asymptomatic, and 15 (14.7%) experienced mild biliary pain. Thirteen patients (12.7%) developed recurrent biliary pain (n = 3) or biliary complications (n = 10; 6 acute cholecystitis, 3 obstructive jaundice, and 1 cholangitis), and required surgical treatment. Surgery was more frequently sought in the D2 group than in the D1 group (19.5% vs. 4.3%, p = 0.033). In conclusion, patients with D2 dissection developed gallstones more frequently and earlier than patients with D1 dissection. Of those with gallstones, patients with D2 dissection required surgery more often than patients with D1 dissection. A closer follow-up should be mandatory for gallstone disease after D2 dissection, but further studies are needed before generalizations can be made.

Pseudoaneurysm of the cystic artery secondary to cholecystitis as a cause of hemobilia: report of a case.

Spontaneous intracholecystic bleeding is very rare. We report herein a very rare case of a pseudoaneurysm of the cystic artery due to acute cholecystitis. A 58-year-old man presented at the emergency department complaining of colicky pain in the right upper quadrant. Dynamic magnetic resonance imaging demonstrated an early-enhanced pooling of contrast material (suggestive of a pseudoaneurysm of the cystic artery) inside the neck of the gallbladder. After the proximal control of the hepatic artery, the patient underwent a cholecystectomy and a ligation of the cystic artery. The resected specimen of the gallbladder showed evidence of a massive intracholecystic hematoma. Proximal to the impacted gallstone in the neck, a 2-cm diameter saccular-type pseudoaneurysm was identified. Although a pseudoaneurysm of the cystic artery is very rare, it should be included in the differential diagnosis of hemobilia. Once the pseudoaneurysm is confirmed, its embolization before a cholecystectomy (which can be attempted laparoscopically) may be useful to ensure the safety of the patient.