Published papers

Biliary tract cancer occurs frequently in patients with pancreaticobiliary maljunction (PBM), although no details of its clinicopathological characteristics have been reported. Here we describe a case of bile duct (BD) cancer that developed in association with PBM. This BD cancer involved the common channel, extended to the main pancreatic duct (MPD) via the common channel, and invaded the pancreatic parenchyma, where its growth and spread, and features of its recurrence, were similar to those of ductal carcinoma of the pancreas. We assumed that MPD extension of BD cancer via the common channel was the reason for its deep spread to the pancreas, since BD cancer usually spreads along the BD and rarely reaches the common channel of the ampulla of Vater. During the follow-up of patients with PBM, attention should be paid to involvement of the common channel by BD cancer and also to cancer developing silently in the pancreas after extrahepatic BD resection.

Background & Aims Bile reflux contributes to development of Barrett's esophagus (BE) and could be involved in its progression to esophageal adenocarcinoma (EAC). We investigated whether bile acids affect levels or functions of microRNAs (MIRs) 221 and 222, which bind to the 3′-UTR of p27Kip1 messenger RNA to inhibit its translation. Reduced p27Kip1 increases degradation of the transcription factor CDX2 levels of CDX2 have been reported to decrease during progression of BE to EAC. Methods We used quantitative reverse transcriptase polymerase chain reaction to compare levels of MIRs 221 and 222 and immunohistochemistry to compare levels of p27Kip1 and CDX2 proteins in areas of BE and EAC from each of 11 patients. We examined the effects of bile acid exposure on levels of MIRs 221 and 222 and CDX2 in EAC cells. We investigated the effects of inhibitors of MIRs 221 and 222 on growth of human EAC xenograft tumors in NOD/SCID/IL-2Rγnull mice. Results Levels of MIRs 221 and 222 increased and levels of p27Kip1 and CDX2 decreased in areas of EAC vs BE. Levels of MIRs 221 and 222 increased, along with activity of nuclear bile acid receptor/farnesoid X receptor (FXR), when cultured cells were exposed to bile acids. Incubation of cells with bile acids increased degradation of CDX2 this process was reduced when cells were also incubated with proteasome inhibitors. Overexpression of MIRs 221 and 222 reduced levels of p27Kip1 and CDX2, and knockdown of these MIRs increased levels of these proteins in cultured cells. Inhibitors of MIRs 221 and 222 increased levels of p27Kip1 and CDX2 in EAC cells and reduced growth of xenograft tumors in NOD/SCID/IL-2Rγnull mice. Conclusions We observed increased levels of MIRs 221 and 222 in human EAC tissues, compared with areas of BE from the same patient. We found that exposure of esophageal cells to bile acids activates FXR and increases levels of MIRs 221 and 222, reducing levels of p27Kip1 and promoting degradation of CDX2 by the proteasome. Our work opened the perspective of therapeutically targeting this pathway either via FXR antagonists or inhibitors of MIRs as a treatment option for BE and EAC. © 2013 by the AGA Institute.

The aim of this study was to identify clinicopathological variables associated with the clinical outcomes of patients with lymph node metastasis-positive urothelial bladder carcinoma after radical cystectomy. Forty-six patients who underwent radical cystectomy without preoperative chemotherapy and had histologically proven nodal metastasis were included in the study. The status of lymph nodes and primary lesion was analyzed in terms of disease-specific survival and recurrence-free survival. The 5-year disease-specific survival and recurrence-free survival for the 46 patients overall were 41.3 and 32.2, respectively. Univariate analysis showed that pN status, the total number of involved lymph nodes, lymph node density and extranodal invasion were statistically significant variables predictive of disease-specific survival. Multivariate analysis revealed that the total number of involved lymph nodes, extranodal invasion and diameter of the metastatic lesion were statistically significant variables predictive of disease-specific survival. Interestingly, the diameter of metastatic lesions was inversely correlated with poorer survival. Patients with large (epsilon 10 mm) metastatic lesions and no extranodal invasion (expansive growth) had significantly better disease-specific survival than those with multiple small (10 mm) metastatic lesions and no extranodal invasion (highly spreading) (P 0.0156) or those with extranodal invasion (infiltrative growth) (P 0.0181). Our data indicate that the clinical outcome of node-positive patients is not only stratified according to the tumor burden reflected in the total number of involved lymph nodes, but also affected by tumor biology including invasiveness and potential for metastasis, which is reflected in pathological characteristics such as extranodal invasion and the diameter of metastatic lesions.

For appropriate surveillance of patients at the precancerous stage for hepatocellular carcinomas (HCCs), carcinogenetic risk estimation is advantageous. The aim of our study was to establish criteria for such estimation based on DNA methylation profiling. The DNA methylation status of 203 CpG sites on 25 bacterial artificial chromosome (BAC) clones, whose DNA methylation status had been proven to discriminate samples of noncancerous liver tissue obtained from patients with HCC (N) from normal liver tissue (C) samples by BAC array-based methylated CpG island amplification, was evaluated quantitatively using pyrosequencing. The 45 CpG sites whose DNA methylation levels differed significantly between C and N in the learning cohort (n = 22) were identified. The criteria combining DNA methylation status for the 30 regions including the 45 CpG sites were able to diagnose N as being at high risk of carcinogenesis with 100% sensitivity and specificity in the learning cohort and 95.6% sensitivity and 100% specificity in the validation (n = 90) cohort. DNA methylation status for the 30 regions in N samples was significantly correlated with the outcome of patients with HCCs, indicating that clinicopathologically valid DNA methylation alterations have already accumulated at the precancerous stage. The DNA methylation status of the 30 regions did not depend on the presence or absence of hepatitis virus infection, or the status of noncancerous liver tissue (chronic hepatitis or cirrhosis). These criteria may be applicable for carcinogenetic risk estimation using liver biopsy specimens obtained from patients who are followed up because of chronic liver diseases.

During the analysis of phosphotyrosine-containing proteins in scirrhous gastric carcinoma cell lines, we observed an unusual expression of Arf-GAP with Rho-GAP domain, ankyrin repeat and PH domain 3 (ARAP3), a multimodular signaling protein that is a substrate of Src family kinases. Unlike other phosphotyrosine proteins, such as CUB domain-containing protein 1 (CDCP1) and Homo sapiens chromosome 9 open reading frame 10/oxidative stress-associated Src activator (C9orf10/Ossa), which are overexpressed and hyperphosphorylated in scirrhous gastric carcinoma cell lines, ARAP3 was underexpressed in cancerous human gastric tissues. In this study, we found that overexpression of ARAP3 in the scirrhous gastric carcinoma cell lines significantly reduced peritoneal dissemination. In vitro studies also showed that ARAP3 regulated cell attachment to the extracellular matrix, as well as invasive activities. These effects were suppressed by mutations in the Rho-GTPase-activating protein (GAP) domain or in the C-terminal two tyrosine residues that are phosphorylated by Src. Thus, the expression and phosphorylation state of ARAP3 may affect the invasiveness of cancer by modulating cell adhesion and motility. Our results suggest that ARAP3 is a unique Src substrate that suppresses peritoneal dissemination of scirrhous gastric carcinoma cells. Oncogene (2011) 30, 1413-1421; doi:10.1038/onc.2010.522; published online 15 November 2010

Estrogen has been indicated to play an etiological role in the development of lung adenocarcinoma (ADC), particularly bronchioloalveolar carcinoma (BAC), a type of ADC that develops from a benign adenomatous lesion, atypical adenomatous hyperplasia (AAH). Polymorphisms in the CYP19A1 gene cause interindividual differences in estrogen levels. Here, 13 CYP19A1 single-nucleotide polymorphisms (SNPs) were examined for associations with lung AAH risk. AAH is detected as ground-glass opacity (GGO) by computed tomography (CT) examination, and this study consisted of 100 individuals diagnosed with GGO in their lungs among 3088 CT-based cancer screening examinees and 424 without. Minor allele carriers for the rs3764221 SNP showed an elevated risk for GGO [odds ratio (OR) = 1.72, P = 0.017]. Associations of this SNP with risks for lung AAH and BAC in the lungs were next examined using 359 ADC cases whose resected lung lobes were subjected to a histological examination for AAH accompaniment and the presence of BAC components and 330 controls without cancer. The ORs were also increased for lung ADC accompanied by AAH (OR = 1.74, P = 0.029) as well as lung ADC with BAC components (OR = 1.41, P = 0.091). The minor allele was associated with an increased circulating estradiol level (P = 0.079) in a population of 363 postmenopausal women without cancer. These results indicate that CYP19A1 polymorphisms are involved in the risk for lung AAH and BAC in the lungs by causing differences in estrogen levels.

© 2016 American Society for Clinical Investigation. All rights reserved. Tumor cells are thought to evade immune surveillance through interaction with immune cells. Much recent attention has focused on the modification of immune responses as a basis for new cancer treatments. SIRPα is an Ig superfamily protein that inhibits phagocytosis in macrophages upon interaction with its ligand CD47 expressed on the surface of target cells. Here, we show that SIRPα is highly expressed in human renal cell carcinoma and melanoma. Furthermore, an anti-SIRPα Ab that blocks the interaction with CD47 markedly suppressed tumor formation by renal cell carcinoma or melanoma cells in immunocompetent syngeneic mice. This inhibitory effect of the Ab appeared to be mediated by dual mechanisms: direct induction of Ab-dependent cellular phagocytosis of tumor cells by macrophages and blockade of CD47-SIRPα signaling that negatively regulates such phagocytosis. The antitumor effect of the Ab was greatly attenuated by selective depletion not only of macrophages but also of NK cells or CD8+ T cells. In addition, the anti-SIRPα Ab also enhances the inhibitory effects of Abs against CD20 and programmed cell death 1 (PD-1) on tumor formation in mice injected with SIRPα-nonexpressing tumor cells. Anti-SIRPα Abs thus warrant further study as a potential new therapy for a broad range of cancers.

CpG-island methylator phenotype (CIMP)-positive clear cell renal cell carcinomas (RCCs) are characterized by accumulation of DNA hypermethylation of CpG islands, clinicopathological aggressiveness and poor patient outcome. The aim of this study was to clarify the molecular pathways participating in CIMP-positive renal carcinogenesis. Genome (whole-exome and copy number), transcriptome and proteome (two-dimensional image converted analysis of liquid chromatography-mass spectrometry) analyses were performed using tissue specimens of 87 CIMP-negative and 14 CIMP-positive clear cell RCCs and corresponding specimens of non-cancerous renal cortex. Genes encoding microtubule-associated proteins, such as DNAH2, DNAH5, DNAH10, RP1 and HAUS8, showed a 10% or higher incidence of genetic aberrations (non-synonymous single-nucleotide mutations and insertions/deletions) in CIMP-positive RCCs, whereas CIMP-negative RCCs lacked distinct genetic characteristics. MetaCore pathway analysis of CIMP-positive RCCs revealed that alterations of mRNA or protein expression were significantly accumulated in six pathways, all participating in the spindle checkpoint, including the "The metaphase checkpoint (p=1.427 x 10(-6))," "Role of Anaphase Promoting Complex in cell cycle regulation (p=7.444 x 10(-6))" and " Spindle assembly and chromosome separation (p=9.260 x 10(-6))" pathways. Quantitative RT-PCR analysis revealed that mRNA expression levels for genes included in such pathways, i.e., AURKA, AURKB, BIRC5, BUB1, CDC20, NEK2 and SPC25, were significantly higher in CIMP-positive than in CIMP-negative RCCs. All CIMP-positive RCCs showed overexpression of Aurora kinases, AURKA and AURKB, and this overexpression was mainly attributable to increased copy number. These data suggest that abnormalities of the spindle checkpoint pathway participate in CIMP-positive renal carcinogenesis, and that AURKA and AURKB may be potential therapeutic targets in more aggressive CIMP-positive RCCs.

Introduction: Up to 30% stage I lung cancer patients suffer recurrence within 5 years of curative surgery. We sought to improve existing protein-coding gene and microRNA expression prognostic classifiers by incorporating epigenetic biomarkers. Methods: Genome-wide screening of DNA methylation and pyrosequencing analysis of HOXA9 promoter methylation were performed in two independently collected cohorts of stage I lung adenocarcinoma. The prognostic value of HOXA9 promoter methylation alone and in combination with mRNA and miRNA biomarkers was assessed by Cox regression and Kaplan-Meier survival analysis in both cohorts. Results: Promoters of genes marked by polycomb in embryonic stem cells were methylated de novo in tumors and identified patients with poor prognosis. The HOXA9 locus was methylated de novo in stage I tumors (p < 0.0005). High HOXA9 promoter methylation was associated with worse cancer-specific survival (hazard ratio [HR], 2.6; p = 0.02) and recurrence-free survival (HR, 3.0; p = 0.01), and identified high-risk patients in stratified analysis of stages IA and IB. Four protein-coding gene (XPO1, BRCA1, HIF1, and DLC1), miR-21 expression, and HOXA9 promoter methylation were each independently associated with outcome (HR, 2.8; p = 0.002; HR, 2.3; p = 0.01; and HR, 2.4; p = 0.005, respectively), and when combined, identified high-risk, therapy naive, stage I patients (HR, 10.2; p = 3x10(-5)). All associations were confirmed in two independently collected cohorts. Conclusion: A prognostic classifier comprising three types of genomic and epigenomic data may help guide the postoperative management of stage I lung cancer patients at high risk of recurrence.

ObjectiveRecent studies have shown an improvement in prostate cancer diagnosis with the use of 3.0-Tesla magnetic resonance imaging. We retrospectively assessed the ability of this imaging technique to predict side-specific extracapsular extension of prostate cancer. MethodsFrom October 2007 to August 2011, prostatectomy was carried out in 396 patients after preoperative 3.0-Tesla magnetic resonance imaging. Among these, 132 (primary sample) and 134 patients (validation sample) underwent 12-core prostate biopsy at the National Cancer Center Hospital of Tokyo, Japan, and at other institutions, respectively. In the primary dataset, univariate and multivariate analyses were carried out to predict side-specific extracapsular extension using variables determined preoperatively, including 3.0-Tesla magnetic resonance imaging findings (T2-weighted and diffusion-weighted imaging). A prediction model was then constructed and applied to the validation study sample. ResultsMultivariate analysis identified four significant independent predictors (P<0.05), including a biopsy Gleason score of 8, positive 3.0-Tesla diffusion-weighted magnetic resonance imaging findings, 2 positive biopsy cores on each side and a maximum percentage of positive cores 31% on each side. The negative predictive value was 93.9% in the combination model with these four predictors, meanwhile the positive predictive value was 33.8%. Good reproducibility of these four significant predictors and the combination model was observed in the validation study sample. ConclusionsThe side-specific extracapsular extension prediction by the biopsy Gleason score and factors associated with tumor location, including a positive 3.0-Tesla diffusion-weighted magnetic resonance imaging finding, have a high negative predictive value, but a low positive predictive value.

Alterations of DNA methylation can account for the histological heterogeneity, reflected in the stepwise progression and complex biological characteristics of human cancers, that genetic alterations alone cannot explain. Analysis of DNA methylation status in tissue samples can be an aid to understanding the molecular mechanisms of multistage carcinogenesis. Human cancer cells show a drastic change in DNA methylation status, that is, overall DNA hypomethylation and regional DNA hypermethylation, which results in chromosomal instability and silencing of tumor-suppressor genes. Overexpression of DNA methyltransferase (DNMT) 1 is not a secondary result of increased cell proliferative activity but may underline the CpG island methylator phenotype of cancers. Splicing alteration of DNMT3B may result in chromosomal instability through DNA hypomethylation of pericentromeric satellite regions. Alterations of DNA methylation are observed even in the precancerous stage frequently associated with chronic inflammation and/or persistent viral infection or with cigarette smoking. Precancerous conditions showing alterations of DNA methylation may generate more malignant cancers. Aberrant DNA methylation is significantly associated with aggressiveness of cancers and poorer outcome of cancer patients. Genome-wide analysis of DNA methylation status based on array-based technology may identify DNA methylation profiles that can be used as appropriate indicators for carcinogenetic risk estimation and prognostication.

Alterations of DNA methylation are one of the most consistent epigenetic changes in human cancers. Human cancers generally show global DNA hypomethylation accompanied by region-specific hypermethylation. Alterations of DNA methylation may result in chromosomal instability as a result of changes in chromatin structure. DNA hypermethylation of CpG islands silences various tumor-related genes. Alterations of DNA methylation are frequently observed in cancers associated with chronic inflammation and/or persistent infection with viruses or other pathogenic microorganisms, such as hepatitis B or C viruses, Epstein-Barr virus, human papillomavirus and Helicobacter pylori, or with cigarette smoking. Accumulating evidence suggests that alterations of DNA methylation are involved even in the early and precancerous stages. On the other hand, in patients with cancers, aberrant DNA methylation is significantly associated with poorer tumor differentiation, tumor aggressiveness and poor prognosis. Precancerous conditions showing alterations of DNA methylation may progress rapidly and generate more malignant cancers. DNA methyltransferase (DNMT) 1 over-expression is not a secondary result of increased cell proliferative activity but is significantly correlated with the CpG island methylator phenotype, which is defined as frequent DNA hypermethylation of C-type CpG islands that are usually methylated in a cancer-specific (not age-dependent) manner. Splicing alteration of DNMT3b may result in chromosomal instability through DNA hypomethylation of pericentromeric satellite regions. Alteration of DNA methylation may become an indicator for carcinogenetic risk estimation and early diagnosis of cancers and a biological predictor of poor prognosis in patients with cancers. Correction of DNA methylation status may offer a new strategy for prevention and therapy of cancers.

© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. ADAMs (a disintegrin and metalloproteinases) are involved in various biological events such as cell adhesion, migration and invasion, membrane protein shedding and proteolysis. However, there have been no systematic studies on the expression of ADAMs in human ovarian carcinomas. We therefore examined mRNA expression of all the proteolytic ADAM species including ADAM8, 9, 10, 12, 15, 17, 19, 20, 21, 28, 30, 33 and ADAMDEC1 in human ovarian carcinomas, and found that prototype membrane-anchored ADAM9m, but not secreted isoform ADAM9s, is significantly over-expressed in carcinomas than in control non-neoplastic ovarian tissue. Among the histological subtypes of serous, endometrioid, mucinous and clear cell carcinomas, ADAM9m expression was highest in clear cell carcinomas. Immunohistochemistry showed that all the clear cell carcinoma samples displayed ADAM9m primarily on the carcinoma cell membrane. By immunoblotting, ADAM9m was detected mainly in an active form in the clear cell carcinoma tissues. When two clear cell carcinoma cell lines (RMG-I and TOV21G cell

Background: Heterotopic gastric-type epithelium, including gastric foveolar metaplasia (GFM) and gastric heterotopia (GH), is a common finding in duodenal biopsy specimens; however, there is still controversy regarding their histogenetic backgrounds. Methods: We analysed a total of 177 duodenal lesions, including 66 GFM lesions, 81 GH lesions, and 30 adenocarcinomas, for the presence of GNAS, KRAS, and BRAF mutations. Results: Activating GNAS mutations were identified in 27 GFM lesions (41%) and 23 GH lesions (28%). The KRAS mutations were found in 17 GFM lesions (26%) and 2 GH lesions (2%). A BRAF mutation was found in only one GFM lesion (2%). These mutations were absent in all 32 normal duodenal mucosa specimens that were examined, suggesting a somatic nature. Among the GFM lesions, GNAS mutations were more common in lesions without active inflammation. Analyses of adenocarcinomas identified GNAS and KRAS mutations in 5 (17%) and 11 lesions (37%), respectively. Immunohistochemically, all the GNAS-mutated adenocarcinomas diffusely expressed MUC5AC, indicating gastric epithelial differentiation. Conclusions: A significant proportion of GFM and GH harbours GNAS and/or KRAS mutations. The common presence of these mutations in duodenal adenoma and adenocarcinoma with a gastric epithelial phenotype implies that GFM and GH might be precursors of these tumours.

Objective: The purpose of this study was to establish a new formula predicting liver metastasis in patients with colorectal cancer (CRC). Methods: Nine previously reported predictive markers for liver metastasis and/or prognosis (COX-2, dysadherin, E-cadherin, beta-catenin, Ki-67, p53, laminin5 gamma 2, matrilysin and MUC-1) were immunohistochemically investigated in 439 consecutive patients with CRC. We tried to determine the combination of molecules which best predicted liver metastasis. A formula for predicting liver metastasis was constructed using a training cohort comprising 150 cases, and applied to a validation cohort comprising 190 cases and another comprising 99 cases from an outside hospital. Results: A combination of dysadherin, E-cadherin and matrilysin was identified to be best for predicting liver metastasis (area under the curve value, 0.807). The predictive formula: 3 x dysadherin score [0 for low expression (<= 50% of tumor cells positive) or 1 for high expression (> 50%)] + 4 x E-cadherin score [0 for preserved (> 80% of tumor cells positive) or 1 for reduced (<= 80%)] + 2 x matrilysin score [0 for low expression (<= 30% of tumor cells positive) or 1 for high expression (> 30%)] was able to discriminate patients with liver metastasis in the training cohort with a sensitivity of 85.7% and a specificity of 58.9%. The discriminative capacity of the formula was validated in the first cohort with a sensitivity of 87.0% and a specificity of 66.5%, and in the second cohort with a sensitivity of 80% and a specificity of 60.0%. Conclusions: We have established a formula for predicting liver metastasis in patients with CRC, and confirmed that it has a high sensitivity potentially useful for clinical application. Copyright (C) 2008 S. Karger AG, Basel.

To evaluate the significance of alterations in DNA methylation during multistage carcinogenesis of the pancreas, tissue samples of 13 peripheral pancreatic duct epithelia showing no remarkable histological changes without inflammatory background (DE), 20 peripheral pancreatic duct epithelia showing no remarkable histological changes with inflammatory background (DEI), 40 pancreatic intraepithelial neoplasias (PanIN) and 147 areas of ductal carcinoma were microdissected from surgically resected specimens from 58 patients and were embedded into agarose beads. The embedded tissue samples were subjected to methylation-specific PCR (MSP) to evaluate the DNA methylation status of the p14, p15, p16, p73, APC, hMLH1, MGMT, BRCA1, GSTPI, TIMP-3, CDH1 and DAPK-1 genes. The prevalence of DNA methylation of at least one of the 12 genes and the average number of methylated genes were significantly higher in both DEI (60% and 0.85 +/- 0.88, P = 0.0151 and P = 0.0224, respectively) and PanIN (67.5% and 0.95 +/- 0.85, P = 0.0014 and P = 0.0028, respectively) than in DE (15.4% and 0.15 +/- 0.38), and were further increased in ductal carcinoma (98.3% and 2.50 +/- 1.35, P < 0.0001 and P < 0.0001, respectively). The BRCA1, APC, p16 and TIMP-3 genes were frequently methylated in ductal carcinoma (60.3, 58.6, 39.3 and 30.9%, respectively). Considerable heterogeneity of DNA methylation status was observed among multiple microdissected areas from individual ductal carcinomas, and the number of methylated genes per area was significantly correlated with poorer tumor differentiation (P = 0.0249). The average number of methylated genes in ductal carcinomas was significantly correlated with DNMT1 protein expression level (P = 0.0093). These data suggest that accumulation of DNA methylation of multiple tumor-related genes is involved in multistage carcinogenesis of the pancreas from early precancerous stages to malignant progression and that DNMT1 protein overexpression may be responsible for this aberrant DNA methylation.

Solid-pseudopapillary neoplasms (SPNs) of the pancreas are uncommon and occur preferentially in young women. The question whether the features of SPNs occurring in men differ from those in women has not yet been studied. For a better understanding of the clinicopathological features of SPNs of both sexes, we studied a series of 14 tumors surgically resected at a Japanese hospital within a period of 14 years. This series was composed of seven men and seven women. All these SPNs demonstrated nuclear and cytoplasmic accumulation of beta-catenin protein in immunohistochemistry and 86% of them had activating mutations of beta-catenin gene. No pancreatic neuroendocrine tumors showed such immunohistochemical findings and genetic alterations. In our series, most SPNs in women showed encapsulation by thick fibrous tissue and massive degenerative changes. Most SPNs in men exhibited solid components without prominent degenerative changes, even though they were of a similar size to those in women. These findings suggest that SPNs in men tend to be a solid mass with slower progression of degenerative changes during their growth compared to that in women. Nuclear accumulation of beta-catenin appears to be a useful marker of SPN, which allows male SPNs to be correctly diagnosed despite their less typical features.

We report a case of leiomyosarcoma of the renal vein, which is a rare tumor with no more than 30 cases found in the published English language literature. This case demonstrates encasement of the renal artery by the tumor mass, a previously unreported manifestation. The present study could be useful in considering this rare tumor in the differential diagnosis of renal hilar tumors.