Takanori Morikawa

Intraductal Tubulopapillary Neoplasms of the Pancreas Distinct From Pancreatic Intraepithelial Neoplasia and Intraductal Papillary Mucinous Neoplasms

We have encountered cases of unusual intraductal pancreatic neoplasms with predominant tubulopapillary growth. We collected data on 10 similar cases of “intraductal tubulopapillary neoplasms (ITPNs)” and analyzed their clinicopathologic and molecular features. Tumor specimens were obtained from 5 men and 5 women with a mean age of 58 years. ITPNs were solid and nodular tumors obstructing dilated pancreatic ducts and did not contain any visible mucin. The tumor cells formed tubulopapillae and contained little cytoplasmic mucin. The tumors exhibited uniform high-grade atypia. Necrotic foci were frequently observed, and invasion was observed in some cases. The ITPNs were immunohistochemically positive for cytokeratin 7 and/or cytokeratin 19 and negative for trypsin, MUC2, MUC5AC, and fascin. Molecular studies revealed abnormal expressions of TP53 and SMAD4 in 1 case, but aberrant expression of β-catenin was not observed. No mutations in KRAS and BRAF were observed in the 8 cases that were examined. Eight patients are alive without recurrence, 1 patient died of liver metastases, and 1 patient is alive but had a recurrence and underwent additional pancreatectomy. The mitotic count and Ki-67 labeling index were significantly associated with invasion. All the features of ITPN were distinct from those of other known intraductal pancreatic neoplasms, including pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasm, and the intraductal variant of acinar cell carcinoma. Intraductal tubular carcinomas showed several features that were similar to those of ITPN, except for the tubulopapillary growth pattern. In conclusion, ITPNs can be considered to represent a new disease entity encompassing intraductal tubular carcinoma as a morphologic variant.

Somatic mutations in PIK3CA and activation of AKT in intraductal tubulopapillary neoplasms of the pancreas.

Intraductal tubulopapillary neoplasm (ITPN) is a recently recognized rare variant of intraductal neoplasms of the pancreas. Molecular aberrations underlying the neoplasm remain unknown. We investigated somatic mutations in PIK3CA, PTEN, AKT1, KRAS, and BRAF. We also investigated aberrant expressions of phosphorylated AKT, phosphatase and tensin homolog (PTEN), tumor protein 53 (TP53), SMAD4, and CTNNB1 in 11 cases of ITPNs and compared these data with those of 50 cases of intraductal papillary mucinous neoplasm (IPMN), another distinct variant of pancreatic intraductal neoplasms. Mutations in PIK3CA were found in 3 of 11 ITPNs but not in IPMNs (P=0.005; Fisher exact test). In contrast, mutations in KRAS were found in none of the ITPNs but were found in 26 of the 50 IPMNs (P=0.001; Fisher exact test). PIK3CA mutations were associated with strong expression of phosphorylated AKT (P<0.001; the Mann-Whitney U test). Moreover, the expression of phosphorylated AKT was apparent in most ITPNs but only in a few IPMNs (P<0.001; the Mann-Whitney U test). Aberrant expressions of TP53, SMAD4, and CTNNB1 were not statistically different between these neoplasms. Mutations in PIK3CA and the expression of phosphorylated AKT were not associated with age, sex, tissue invasion, and patients’ prognosis in ITPNs. These results indicate that activation of the phosphatidylinositol 3-kinase pathway may play a crucial role in ITPNs but not in IPMNs. In contrast, the mutation in KRAS seems to play a major role in IPMNs but not in ITPNs. The activated phosphatidylinositol 3-kinase pathway may be a potential target for molecular diagnosis and therapy of ITPNs.

Multicenter comparative study of laparoscopic and open distal pancreatectomy using propensity score-matching

Laparoscopic distal pancreatectomy has been shown to be associated with favorable postoperative outcomes using meta‐analysis. However, there have been no randomized controlled studies yet. This study aimed to compare laparoscopic and open distal pancreatectomy using propensity score‐matching.

A new free radical scavenger, edaravone, ameliorates oxidative liver damage due to ischemia-reperfusion in vitro and in vivo

Ischemia-reperfusion injury causes oxidative stress producing reactive oxygen species, which is a serious problem linked to morbidity and mortality in liver surgery. We investigated the effects of edaravone, a new free radical scavenger, on liver oxidative stress in vitro and in vivo. We employed a hypoxiareoxygenation model of primary cultured hepatocytes using an AnaeroPack (Mitsubishi Gas Chemical Co., Tokyo, Japan). Hepatocytes were exposed to 3 or 4 hours of hypoxia and then returned to oxygenation. We analyzed the time course changes of aspartate aminotransferase (AST), phosphatidylcholine hydroperoxide (PCOOH), and adenosine triphosphate (ATP) content in hepatocytes of edaravone-treated groups or nontreated groups after reoxygenation. Edaravone significantly attenuated the elevation of the AST level of the medium and hepatocellular PCOOH and preserved the hepatocellular ATP level. In vivo, male Sprague-Dawley rats were subjected to 45 minutes of hepatic ischemia and 120 minutes of reperfusion. The rats were intravenously injected with vehicle or edaravone (3 mg/kg or 10 mg/kg) before reperfusion and 1 hour after reperfusion. Serum AST levels and hepatic PCOOH and energy charge were significantly improved in both edaravone groups compared with control. In conclusion, edaravone has the ability to eliminate intra-hepatocellular superoxide species and attenuate oxidative liver damage in liver surgery.

Allelotype analysis of gallbladder carcinoma associated with anomalous junction of pancreaticobiliary duct

Anomalous junction of pancreaticobiliary duct (AJPBD) patients has an increased risk of gallbladder and bile duct carcinomas. However, the relevance of carcinoma with AJPBD is not fully clarified. We performed analysis of loss of heterozygosity (LOH) at p53 locus and immunohistochemistry of p53 and K-ras gene mutation in five cases of gallbladder carcinoma associated with AJPBD. LOH of p53 locus and overexpression of p53 were detected in two out of five (40%) and five out of five (100%), respectively, in the present study. K-ras gene mutation at codon 12 and 13 was not detected (0%, 0/5). These results suggest that aberrations of p53 are involved in carcinogenesis of gallbladder carcinoma associated with AJPBD. Next, in order to find the genetic events besides K-ras mutation and overexpression of mutant p53 in this disease, LOH analysis was performed using 72 microsatellite markers. High frequency of allelic loss (> 50%) was found on 2p (81.8%), 4p (50%), 4q (50%), 8q (60%), 9q (50%), 10p (50%), 14p (60%), 14q (50%), 16p (60%), 19p (50%), 21p (50%) and Xp (66.6%). The highest deletion regions on chromosome 2p24 (3/3, 100%), 14q22 (3/4, 75%) and 21q22 (3/4, 75%) were found. The present study suggests that gallbladder carcinoma associated with AJPBD has high frequent allelic loss and has two new regions which may harbor putative tumor suppressor genes.

Nm23/nucleoside diphosphate kinase-A as a potent prognostic marker in invasive pancreatic ductal carcinoma identified by proteomic analysis of laser micro-dissected formalin-fixed paraffin-embedded tissue.

BackgroundPancreatic cancer is among the most lethal malignancies worldwide. This study aimed to identify a novel prognostic biomarker, facilitating treatment selection, using mass spectrometry (MS)-based proteomic analysis with formalin-fixed paraffin-embedded (FFPE) tissue.ResultsThe two groups with poor prognosis (n = 4) and with better prognosis (n = 4) had been carefully chosen among 96 resected cases of pancreatic cancer during 1998 to 2007 in Tohoku University Hospital. Although those 2 groups had adjusted background (UICC-Stage IIB, Grade2, R0, gemcitabine adjuvant), there was a significant difference in postoperative mean survival time (poor 21.0 months, better 58.1 months, P = 0.0067). Cancerous epithelial cells collected from FFPE tissue sections by laser micro-dissection (LMD) were processed for liquid chromatography-tandem mass spectrometry (LC-MS/MS). In total, 1099 unique proteins were identified and 6 proteins showed different expressions in the 2 groups by semi-quantitative comparison. Among these 6 proteins, we focused on Nm23/Nucleoside Diphosphate Kinase A (NDPK-A) and immunohistochemically confirmed its expression in the cohort of 96 cases. Kaplan-Meier analysis showed high Nm23/NDPK-A expression to correlate with significantly worse overall survival (P = 0.0103). Moreover, in the multivariate Cox regression model, Nm23/NDPK-A over-expression remained an independent predictor of poor survival with a hazard ratio of 1.97 (95% CI 1.16-3.56, P = 0.0110).ConclusionsWe identified 6 candidate prognostic markers for postoperative pancreatic cancer using FFPE tissues and immunohistochemically demonstrated high Nm23/NDPK-A expression to be a useful prognostic marker for pancreatic cancer.

Malignant Pheochromocytoma with Hepatic Metastasis Diagnosed 10 Years After a Resection of the Primary Incidentaloma Adrenal Lesion: Report of a Case

Abstract A 40-year-old woman developed hepatic tumors 10 years after a resection of pheochromocytoma of the left adrenal gland. Computed tomography showed a round tumor measuring about 35 mm in diameter at segment V of the liver (Couinauds classification), and magnetic resonance imaging showed another tumor measuring about 5 mm at segment V–VIII of the liver. The results of the endoscopic examination of her upper gastrointestinal tract and barium enema were normal. Owing to a suspected hepatic metastasis of malignant pheochromocytoma, a right lobectomy of the liver was performed. Postoperatively, [131I]metaiodobenzylguanidine scintigram and computed tomography showed no other residual tumors nor metastasis. The present case suggests that a long-term follow-up only by endocrinological examinations is insufficient to find newly developed metastatic foci, while routine diagnostic imaging at frequent intervals is necessary in cases of pheochromocytoma.

Phase I trial of neoadjuvant chemoradiation with gemcitabine and surgical resection for cholangiocarcinoma patients (NACRAC study).

BACKGROUND/AIMS The feasibility of neoadjuvant chemoradiation therapy for cholangiocarcinoma, followed by conventional resection, has not been determined yet. Here, a phase I study of neoadjuvant chemoradiation therapy, named NACRAC, was performed to determine the maximum tolerated dose (MTD) and the recommended dose (RD) of gemcitabine when combined with external beam radiation therapy for resectable cholangiocarcinoma. METHODOLOGY From August 2007 to June 2008, 12 patients provided informed consent. Preoperative radiation was administered in 1.8Gy daily fractions up to a total dose of 45Gy. Gemcitabine was administered at day 1 and 8 every three weeks. The initial dose of gemcitabine was started from 400mg/m2. RESULTS One patient was not able to start treatment because of bleeding caused by a duodenal ulcer and cholangitis. At 800mg/m2 of gemcitabine, one patient out of three failed to complete the treatment because of Grade 3 hematological toxicity. In another three cases of 800mg/m2, the second case could not complete the treatment because of cholangitis. Then, 600mg/m2 was determined to be the MTD, and the RD dose decided as 600mg/m2. CONCLUSIONS The RD of gemcitabine in NACRAC study was determined to be 600mg/m2. NACRAC study should proceed to a phase II trial to evaluate the effectiveness.

Molecular differences in the microsatellite stable phenotype between left-sided and right-sided colorectal cancer

Differences in the pathogenesis of microsatellite stable (MSS) sporadic colorectal cancers (CRCs) between left‐sided CRC (LC) and right‐sided CRC (RC) have not been clarified. To identify pathogenesis‐related genomic differences between MSS CRCs within the two locations, we performed a comprehensive molecular analysis using crypt isolation with samples from 92 sporadic CRCs. Microsatellite instability (MSI; high and low/negative) and DNA methylation status (low methylation epigenome; intermediate methylation epigenome [IME] or high methylation epigenome [HME]) were determined using polymerase chain reaction (PCR) microsatellite analysis and PCR‐bisulfite pyrosequencing, respectively. Additionally, mutations in the TP53, KRAS, BRAF and PIK3CA genes were examined using PCR‐bisulfite pyrosequencing (for KRAS and BRAF mutations) or PCR‐single conformation polymorphism (for TP53 and PIK3CA mutations), followed by sequencing of aberrant bands. Finally, a genome‐wide study using a copy number alteration (CNA)‐targeted single nucleotide polymorphism array was performed. Ninety‐two CRCs were classified into 71 MSS and 21 MSI phenotypes. We examined 71 CRCs with the MSS phenotype (LC, 56; RC, 15). Mutations in KRAS were associated with RC with the MSS phenotype, whereas mutations in TP53 were more frequently found in LC with the MSS phenotype. There were significant differences in the frequencies of KRAS and TP53 mutations in the IME between LC and RC with the MSS phenotype. Although CNA gains were associated with LC with the MSS phenotype, CNA losses were not major alterations associated with the MSS phenotype. These findings suggested that the molecular pathogenesis of the MSS phenotype in LC was different from that in RC.

Pancreatic intraductal tubulopapillary neoplasm is genetically distinct from intraductal papillary mucinous neoplasm and ductal adenocarcinoma

Intraductal tubulopapillary neoplasm is a relatively recently described member of the pancreatic intraductal neoplasm family. The more common member of this family, intraductal papillary mucinous neoplasm, often carries genetic alterations typical of pancreatic infiltrating ductal adenocarcinoma (KRAS, TP53, and CDKN2A) but additionally has mutations in GNAS and RNF43 genes. However, the genetic characteristics of intraductal tubulopapillary neoplasm have not been well characterized. Twenty-two intraductal tubulopapillary neoplasms were analyzed by either targeted next-generation sequencing, which enabled the identification of sequence mutations, copy number alterations, and selected structural rearrangements involving all targeted (≥300) genes, or whole-exome sequencing. Three of these intraductal tubulopapillary neoplasms were also subjected to whole-genome sequencing. All intraductal tubulopapillary neoplasms revealed the characteristic histologic (cellular intraductal nodules of back-to-back tubular glands lined by predominantly cuboidal cells with atypical nuclei and no obvious intracellular mucin) and immunohistochemical (immunolabeled with MUC1 and MUC6 but were negative for MUC2 and MUC5AC) features. By genomic analyses, there was loss of CDKN2A in 5/20 (25%) of these cases. However, the majority of the previously reported intraductal papillary mucinous neoplasm-related alterations were absent. Moreover, in contrast to most ductal neoplasms of the pancreas, MAP-kinase pathway was not involved. In fact, 2/22 (9%) of intraductal tubulopapillary neoplasms did not reveal any mutations in the tested genes. However, certain chromatin remodeling genes (MLL1, MLL2, MLL3, BAP1, PBRM1, EED, and ATRX) were found to be mutated in 7/22 (32%) of intraductal tubulopapillary neoplasms and 27% harbored phosphatidylinositol 3-kinase (PI3K) pathway (PIK3CA, PIK3CB, INPP4A, and PTEN) mutations. In addition, 4/18 (18%) of intraductal tubulopapillary neoplasms had FGFR2 fusions (FGFR2-CEP55, FGFR2-SASS6, DISP1-FGFR2, FGFR2-TXLNA, and FGFR2-VCL) and 1/18 (5.5%) had STRN-ALK fusion. Intraductal tubulopapillary neoplasm is a distinct clinicopathologic entity in the pancreas. Although its intraductal nature and some clinicopathologic features resemble those of intraductal papillary mucinous neoplasm, our results suggest that intraductal tubulopapillary neoplasm has distinguishing genetic characteristics. Some of these mutated genes are potentially targetable. Future functional studies will be needed to determine the consequences of these gene alterations.