Published papers

Background: The CpG island methylator phenotype (CIMP) of clear cell renal cell carcinomas (ccRCCs) is characterized by accumulation of DNA methylation at CpG islands and poorer patient outcome. The aim of this study was to establish criteria for prognostication of patients with ccRCCs using the ccRCC-specific CIMP marker genes. Methods: DNA methylation levels at 299 CpG sites in the 14 CIMP marker genes were evaluated quantitatively in tissue specimens of 88 CIMP-negative and 14 CIMP-positive ccRCCs in a learning cohort using the MassARRAY system. An additional 100 ccRCCs were also analyzed as a validation cohort. Results: Receiver operating characteristic curve analysis showed that area under the curve values for the 23 CpG units including the 32 CpG sites in the 7 CIMP-marker genes, i.e. FAM150A, ZNF540, ZNF671, ZNF154, PRAC, TRH and SLC13A5, for discrimination of CIMP-positive from CIMP-negative ccRCCs were larger than 0.95. Criteria combining the 23 CpG units discriminated CIMP-positive from CIMP-negative ccRCCs with 100% sensitivity and specificity in the learning cohort. Cancer-free and overall survival rates of patients with CIMP-positive ccRCCs diagnosed using the criteria combining the 23 CpG units in a validation cohort were significantly lower than those of patients with CIMP-negative ccRCCs (P = 1.41 x 10(-5) and 2.43 x 10(-13), respectively). Patients with CIMP-positive ccRCCs in the validation cohort had a higher likelihood of disease-related death (hazard ratio, 75.8; 95% confidence interval, 7.81 to 735; P = 1.89 x 10(-4)) than those with CIMP-negative ccRCCs. Conclusions: The established criteria are able to reproducibly diagnose CIMP-positive ccRCCs and may be useful for personalized medicine for patients with ccRCCs.

Gallbladder cancer (GBC) is relatively rare but has a high mortality rate. One candidate molecule which might be involved in GBC development is prostate stem cell antigen (PSCA), a glycosylphosphatidylinositol- anchored cell surface antigen with a tissue- specific pattern of expression in the epithelium of several organs, such as the prostate, stomach, bladder, and gallbladder. It is up- regulated in a number of cancers including prostate, urinary bladder, and pancreatic cancers, while it is down- regulated in esophageal and gastric cancers, suggesting that PSCA has an oncogenic activity in the former but a tumor suppressor activity in the latter. However, the precise function of PSCA and the regulatory mechanism for its expression in normal and cancer cells are yet to be determined. In this study, immunohistochemical analyses with a specific antibody revealed that PSCA is down- regulated in non- neoplastic gallbladder lesions such as cholesterolosis, cholecystolithiasis, and cholecystitis (9/17; 53%), and also in adenocarcinoma (40/44; 91%), a common neoplasm in gallbladder. Analyses of the DNA methylation status in the GBC cell lines by bisulfite- Pyrosequencing and a reporter assay for the PSCA promoter activity suggested that the down- regulation is explained, at least partly, by DNA methylation. Moreover, colony formation assay revealed that PSCA has cell- proliferation inhibition activity in the GBC cell lines, which was also observed in vivo. These lines of in vivo and in vitro evidence suggest that PSCA is acting as a tumor suppressor in GBC development. (C) 2011 Wiley Periodicals, Inc.

Connective tissue growth factor (CTGF) is a secreted protein belonging to the CCN family, members of which are implicated in various biological processes. We identified a homozygous loss of CTGF (6q23.2) in the course of screening a panel of ovarian cancer cell lines for genomic copy number aberrations using in-house array-based comparative genomic hybridization. CTGF mRNA expression was observed in normal ovarian tissue and immortalized ovarian epithelial cells but was reduced in many ovarian cancer cell lines without its homozygous deletion (12 of 23 lines) and restored after treatment with 5-aza 2 '-deoxycytidine. The methylation status around the CTGF CpG island correlated inversely with the expression, and a putative target region for methylation showed promoter activity. CTGF methylation was frequently observed in primary ovarian cancer tissues (39 of 66, 59%) and inversely correlated with CTGF mRNA expression. In an immunohistochemical analysis of primary ovarian cancers, CTGF protein expression was frequently reduced (84 of 103 cases, 82%). Ovarian cancer tended to lack CTGF expression more frequently in the earlier stages (stages I and II) than the advanced stages (stages III and IV). CTGF protein was also differentially expressed among histologic subtypes. Exogenous restoration of CTGF expression or treatment with recombinant CTGF inhibited the growth of ovarian cancer cells lacking its expression, whereas knockdown of endogenous CTGF accelerated growth of ovarian cancer cells with expression of this gene. These results suggest that epigenetic silencing by hypermethylation of the CTGF promoter leads to a loss of CTGF function, which may be a factor in the carcinogenesis of ovarian cancer in a stage-dependent and/or histologic subtype-dependent manner.

The molecular mechanisms underlying the development of pancreatic neuroendocrine tumors (PanNETs) have not been well defined. We report here that the genomic region of the PHLDA3 gene undergoes loss of heterozygosity (LOH) at a remarkably high frequency in human PanNETs, and this genetic change is correlated with disease progression and poor prognosis. We also show that the PHLDA3 locus undergoes methylation in addition to LOH, suggesting that a two-hit inactivation of the PHLDA3 gene is required for PanNET development. We demonstrate that PHLDA3 represses Akt activity and Akt-regulated biological processes in pancreatic endocrine tissues, and that PHLDA3-deficient mice develop islet hyperplasia. In addition, we show that the tumor-suppressing pathway mediated by MEN1, a well-known tumor suppressor of PanNETs, is dependent on the pathway mediated by PHLDA3, and inactivation of PHLDA3 and MEN1 cooperatively contribute to PanNET development. Collectively, these results indicate the existence of a novel PHLDA3-mediated pathway of tumor suppression that is important in the development of PanNETs.

Flat epithelial lesions of the biliary tract cannot be detected by the image analysis, and the diagnosis entirely depends on pathological examination. The biliary tract is often affected by inflammatory conditions, and the resultant changes of the biliary epithelium make it difficult to differentiate them from neoplasia. Thus, the pathological diagnosis of biliary flat epithelial lesions can be challenging. In the biliary tract, there are several forms of intraepithelial neoplasia of the flat type, and biliary intraepithelial neoplasia (BilIN) is known as one of such lesions that represent the multistep cholangiocarcinogenesis. In this article, the diagnostic criteria and the differential diagnosis of biliary flat epithelial lesions, particularly focusing on BilIN, were presented and discussed to provide help to advance clinical and research applications of the BilIN system.

Aims: To determine the prevalence of MED12 mutations in smooth muscle tumours of different organs. Methods and results: A total of 142 smooth muscle tumours of the uterus, gastrointestinal tract, retroperitoneum and soft tissue were analysed for MED12 mutations using Sanger sequencing. Among the uterine tumours that were examined, MED12 mutations were identified in 36 of 45 conventional leiomyomas (80%), two of six cellular leiomyomas (33%), one of four bizarre leiomyomas (25%), none of four lipoleiomyomas (0%), and two of 12 leiomyosarcomas (17%). The two MED12-mutated leiomyosarcomas were associated with benign leiomyomatous components that also harboured MED12 mutations identical to those in the respective leiomyosarcomatous components. None of the extrauterine smooth muscle tumours, including the leiomyomas, leiomyosarcomas, and angioleiomyomas, had MED12 mutations. Conclusions: Among uterine smooth muscle tumours, MED12 mutations are frequently present in conventional leiomyomas, but are significantly less common in histological variants of leiomyoma and leiomyosarcoma. In contrast to uterine lesions, none of the extrauterine smooth muscle tumours had MED12 mutations. © 2012 Blackwell Publishing Ltd.

Purpose: We identify clinicopathological variables predicting overall survival in patients with recurrent bladder urothelial carcinoma after radical cystectomy. Materials and Methods: We retrospectively collected data on 114 patients treated with radical cystectomy for bladder urothelial carcinoma who subsequently had remote metastasis and/or local recurrence. The Kaplan-Meier method with the log rank test and multivariate Cox regression models were used to address overall survival after recurrence. Results: During followup 99 of the 114 patients died. Median survival in the 114 patients was 11.2 months. One and 3-year overall survival rates were 48.0% and 12.1%, respectively. On multivariate analysis independent predictors of poorer overall survival included less than 1 year to recurrence, symptoms at recurrence, 2 or more metastatic organs at recurrence, high serum C-reactive protein, high lactate dehydrogenase, no post-recurrence platinum based chemotherapy and no metastasectomy. Based on the 4 variables (time to recurrence, symptoms, number of metastatic organs and C-reactive protein), we constructed a risk model predicting post-recurrence overall survival that classified patients into 3 groups with significantly different overall survival (p < 0.0001). Conclusions: Our data confirm that recurrent urothelial carcinoma after radical cystectomy is a highly aggressive, lethal disease. Seven clinicopathological factors were identified that predicted post-recurrence overall survival. Our risk model based on the 4 variables could be useful to provide relevant prognostic information to patients and physicians, and better stratify patients in clinical trials. © 2013 American Urological Association Education and Research, Inc.

Intraorgan metastasis of a primary cancer within the organ of origin, such as intrahepatic metastasis of hepatocellular carcinoma, is one of the key features for clinicopathologic staging of the cancer. Pancreatic intraglandular metastasis (P-IM) of pancreatic ductal carcinoma (PDC) is encountered occasionally but has not yet been evaluated. The aim of this study was to investigate the clinicopathologic characteristics and prognostic value of P-IM in patients with PDC. The histopathologic features of 393 consecutive patients with PDC who had undergone pancreatic resection at the National Cancer Center Hospital, Tokyo, between 2003 and 2010 were reviewed. For the purposes of the study, P-IM was defined as an independent tumor showing histopathologic features similar to those of the primary one. Twenty-six cases of P-IM were identified in 21 (5.3%) of the reviewed patients. The incidence of P-IM at each stage of the TNM classification was 0% (0/7) at stage IA, 17% (1/6) at stage IB, 5% (5/92) at stage IIA, 4% (11/252) at stage IIB, 0% (0/1) at stage III, and 11% (4/35) at stage IV. Univariate survival analysis showed that both overall survival and disease-free survival for patients with P-IM were significantly shorter than for those without P-IM (P < 0.001 and P = 0.019, respectively). Multivariate survival analysis showed that P-IM was significantly correlated with shorter overall survival (P = 0.002; hazard ratio = 2.239; 95% confidence interval: 1.328-3.773). Our findings suggest that the presence of P-IM in patients with PDC is an independent prognosticator and may represent aggressive tumor behavior.

Gastric cancer is one of the most common malignancies worldwide and patients with advanced gastric cancer still have poor clinical outcomes. The overexpression of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) mRNA in colorectal cancer and its correlation with clinicopathological factors were recently reported by us. In this study, we show LGR5 mRNA overexpression in human gastric cancer specimens by quantitative RT-PCR and in situ hybridization and assess a correlation with clinicopathological factors. The mean expression of LGR5 mRNA in cancerous tissues was five times higher than that in normal tissue (P=0.0002). Furthermore, LGR5 mRNA expression show marked variation among cases and significantly increased in cases where lymphatic invasion was present compared with those where it was absent (P=0.0056). Although the mean expression level of LGR5 was observed to be higher in nodal metastasis and venous invasion positive cases compared to negative cases, a significant difference was not observed. These results suggest that LGR5 can be a biomarker for malignancy in gastric cancer. © 2012 The Authors. Pathology International © 2012 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.

Uterine leiomyosarcoma (LMS) is a highly metastatic smooth muscle neoplasm for which calponin h1 is suspected to have a biological role as a tumor-suppressor. We earlier reported that LMP2-null mice spontaneously develop uterine LMS through malignant transformation of the myometrium, thus implicating this protein as an anti-tumorigenic candidate as well. In the present study, we show that LMP2 may negatively regulate LMS independently of its role in the proteasome. Moreover, several lines of evidence indicate that although calponin h1 does not directly influence tumorigenesis, it clearly affects LMP2-induced cellular morphological changes. Modulation of LMP2 may lead to new therapeutic approaches in human uterine LMS. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Aims: alpha-Methylacyl-CoA racemase (AMACR) is expressed in the majority of hepatocellular carcinomas (HCCs) at variable levels, but the significance of AMACR overexpression remains elusive. The aim of this study was to investigate the relationship between AMACR expression and the presence of CTNNB1 mutations in HCCs. Methods and results: The expression of AMACR and GLUL, an established downstream target of beta-catenin was examined in HCCs, by quantitative reverse transcription polymerase chain reaction (PCR), and the expression of their protein products by immunohistochemistry. The quantitative reverse transcription PCR analysis showed that the expression of AMACR was significantly higher in HCCs with CTNNB1 mutations than in mutation-negative HCCs or normal livers, like the expression of GLUL. Immunohistochemistry also showed that strong AMACR protein expression was closely correlated with the presence of CTNNB1 mutations. HCCs with CTNNB1 mutations and those with AMACR overexpression frequently exhibited bile production. Conclusions: The overexpression of AMACR was closely correlated with the presence of CTNNB1 mutations in HCCs. AMACR is a putative target of beta-catenin as well as an excellent immunohistochemically detectable marker of HCCs with CTNNB1 mutations. As AMACR is physiologically involved in bile acid synthesis, the current observation implies a regulatory role of beta-catenin in bile acid metabolism.

Although the majority of smooth muscle neoplasms found in the uterus are benign, uterine leiomyosarcoma (LMS) is extremely malignant, with high rates of recurrence and metastasis. We earlier reported that mice with a homozygous deficiency for LMP2, an interferon (IFN)-gamma-inducible factor, spontaneously develop uterine LMS. The IFN-gamma pathway is important for control of tumor growth and invasion and has been implicated in several cancers. In this study, experiments with human and mouse uterine tissues revealed a defective LMP2 expression in human uterine LMS that was traced to the IFN-gamma pathway and the specific effect of JAK-1 somatic mutations on the LMP2 transcriptional activation. Furthermore, analysis of a human uterine LMS cell line clarified the biological significance of LMP2 in malignant myometrium transformation and cell cycle, thus implicating LMP2 as an anti-tumorigenic candidate. This role of LMP2 as a tumor suppressor may lead to new therapeutic targets in human uterine LMS.

Acinar cell carcinoma (ACC) of the pancreas is very rare, which usually grows expansively. Recently, a variant of ACC with predominant growth in the pancreatic ducts has been proposed, and is speculated to have potentially less aggressive behavior. The aim of this study was to investigate how the pancreatic duct system is related to the growth and extension of ACC. We reviewed the detailed gross and histologic features of 13 cases of ACC, of which 7 (54%) showed intraductal polypoid growth (IPG) of the tumor in the large pancreatic ducts with a mean IPG length of 24.8 mm. Tumors with IPG were found to spread characteristically along the pancreatic ducts as extending polypoid projections, filling the ducts and destroying the duct walls, although tumors did not tend to extend beyond the pancreatic parenchyma. Comparison of the clinicopathologic characteristics showed that ACC with IPG had less infiltrative features including lymphatic, venous, and neural invasion, formation of tumor thrombus in the portal vein, nodal metastasis, and invasion beyond the pancreas to the surrounding organs; death in only 1 case (14%) of ACC with IPG was the result of ACC itself. In contrast, ACC without IPG frequently showed more infiltrative growth, and was the cause of death in 50% of patients with this type of tumor. Intraductal dissemination of ACC in pancreatic ducts was proven in 1 case of ACC with IPG. These findings suggest that a significant proportion of ACC shows IPG, which is potentially linked to less aggressive clinicopathologic characteristics.

The present study investigated the protein expression level of CXCL12 in colorectal cancer and aimed to elucidate its association with prognosis. CXCL 12 positivity in 50% or more of tumor cells was defined as high expression and that in less than 50% of the tumor cells as low expression. CXCL12+ tumor budding at the invasive from was divided into 2 grades: high with 10 or more budding foci per x 200 field of view and low grade with fewer than 10 budding foci. Patients with high expression (72.7%) and high grade CXCL12+ tumor budding (43.0%) had significantly shorter survival than patients with low expression (P = .014) and low grade (P = .003), respectively. Patients with a combination of high expression and high grade had the worst outcome (P < .001). Our study demonstrated that CXCL12 expression in colorectal cancer cells and at sites of budding were significant prognostic factors. Furthermore, together with lymph node metastasis, a combination of both expression patterns was a more powerful independent prognostic factor.

Objective: The microenvironment of cancer plays a critical role in its progression. However, the molecular features of cancer-associated fibroblasts (CAFs) are less well understood than those of cancer cells. We investigated the clinicopathological significance of podoplanin expression in stromal fibroblasts in patients with colorectal cancer (CRC). Methods: We selected podoplanin as an upregulated marker in CAF from a DNA microarray experiment. Consequently, podoplanin was identified as an upregulated gene. Immunohistochemical podoplanin expression was investigated at the National Cancer Center Hospital, Tokyo, Japan, in 120 patients with advanced CRC, and its clinicopathological significance was examined. The biological function of podoplanin expression was also assessed by a coculture invasion assay with CRC cell lines such as HCT116 and HCT15. Results: Podoplanin expression was exclusively confined to stromal fibroblasts and absent in tumor cells. Podoplanin is absent in normal stroma except for lymphatic vessels. Staining was considered positive when over 30% of the cancer stroma was stained. Positive podoplanin expression was significantly correlated with a more distal tumor localization (p = 0.013) and a shallower depth of tumor invasion (p = 0.011). Univariate analysis revealed that negative podoplanin expression in stromal fibroblasts was significantly associated with reduced disease-specific survival (p = 0.0017) and disease-free survival (p < 0.0001). Multivariate analysis revealed that negative podoplanin expression (p = 0.016) and lymph node metastasis (p = 0.027) were significantly associated with disease-free survival. CRC cell invasion was augmented by coculture with CAFs that were treated with siRNA for podoplanin. Conclusions: Our results suggest that a positive podoplanin expression in stromal fibroblasts could have a protective role against CRC cell invasion and is a significant indicator of a good prognosis in patients with advanced CRC, supported by biological analysis showing that podoplanin expression in CAFs is associated with decreased CRC cell invasion. Copyright (C) 2009 S. Karger AG, Basel

PTPRH is a receptor-type protein tyrosine phosphatase thought to be a potential regulator of tumorigenesis. The aim of the present study was to clarify the significance of PTPRH expression and its regulation by DNA methylation in non-small cell lung cancer (NSCLC), especially in lung adenocarcinoma (LADC). PTPRH mRNA expression was examined in 89 NSCLC and corresponding non-cancerous tissues. The correlation between DNA methylation and PTPRH gene expression was investigated in another cohort that consisted of 145 patients with LADC, a major NSCLC subtype. Gene regulation by DNA methylation was assessed using a DNA methylation inhibitor. PTPRH mRNA expression was significantly upregulated in NSCLC. PTPRH DNA methylation was reduced in LADC samples and inversely correlated with mRNA expression. 5-Aza-2'-deoxycytidine treatment of lung cancer cell lines with low PTPRH expression, restored mRNA PTPRH expression levels. Furthermore, low PTPRH methylation was associated with shorter recurrence-free survival (P=1.64x10(-4)) and overall survival (P=5.54x10(-5)). Multivariate analysis revealed that PTPRH DNA methylation was an independent prognostic factor (P=6.88x10(-3)). It was confirmed that PTPRH is overexpressed in NSCLC. Furthermore, we determined that PTPRH is epigenetically regulated by DNA hypomethylation, with prognostic implications for LADC.

The aim of this study was to identify pathways that have a significant impact during renal carcinogenesis. Sixty-seven paired samples of both noncancerous renal cortex tissue and cancerous tissue from patients with clear cell renal cell carcinomas (RCCs) were subjected to whole-exome, methylome and transcriptome analyses using Agilent SureSelect All Exon capture followed by sequencing on an Illumina HiSeq 2000 platform, Illumina Infinium HumanMethylation27 BeadArray and Agilent Sure-Print Human Gene Expression microarray, respectively. Sanger sequencing and quantitative reverse transcription-PCR were performed for technical verification. MetaCore software was used for pathway analysis. Somatic nonsynonymous single-nucleotide mutations, insertions/deletions and intragenic breaks of 2,153, 359 and 8 genes were detected, respectively. Mutations of GCN1L1, MED12 and CCNC, which are members of CDK8 mediator complex directly regulating beta-catenin-driven transcription, were identified in 16% of the RCCs. Mutations of MACF1, which functions in the Wnt/beta-catenin signaling pathway, were identified in 4% of the RCCs. A combination of methylome and transcriptome analyses further highlighted the significant role of the Wnt/beta-catenin signaling pathway in renal carcinogenesis. Genetic aberrations and reduced expression of ERC2 and ABCA13 were frequent in RCCs, and MTOR mutations were identified as one of the major disrupters of cell signaling during renal carcinogenesis. Our results confirm that multilayer-omics analysis can be a powerful tool for revealing pathways that play a significant role in carcinogenesis.

Uterine leiomyosarcoma (LMS) develops more often in the muscle tissue layer of the uterine body than in the uterine cervix. The development of gynecologic tumors is often correlated with female hormone secretion however, the development of uterine LMS is not substantially correlated with hormonal conditions, and the risk factors are not yet known. Importantly, a diagnostic-biomarker which distinguishes malignant LMS from benign tumor leiomyoma (LMA) is yet to be established. Accordingly, it is necessary to analyze risk factors associated with uterine LMS, in order to establish a treatment method. LMP2-deficient mice spontaneously develop uterine LMS, with a disease prevalence of ~40 by 14 months of age. We found LMP2 expression to be absent in human LMS, but present in human LMA. Therefore, defective LMP2 expression may be one of the risk factors for LMS. LMP2 is a potential diagnostic-biomarker for uterine LMS, and may be targeted-molecule for a new therapeutic approach. Copyright © 2011 Takuma Hayashi et al.

The purposes of this study were to investigate whether the biological characteristics or outcomes of patients with metaplastic carcinoma, invasive ductal carcinoma, or invasive lobular carcinoma of the breast differ; to determine whether the metaplastic carcinoma subtypes have similar malignant potentials; and to identify accurate predictors of outcome in patients with metaplastic carcinoma. The subject comprised 6137 invasive ductal carcinoma patients, 301 invasive lobular carcinoma patients, and 46 metaplastic carcinoma patients of the breast. The metaplastic carcinomas were classified according to the World Health Organization classification. Multivariate analyses clearly demonstrated that the metaplastic carcinoma patients had a significantly poorer outcome than the invasive ductal carcinoma patients or the invasive lobular carcinoma patients independent of the nodal status or age not exceeding 39 years, whereas patients with triple-negative metaplastic carcinomas or triple-negative invasive lobular carcinomas had a poorer outcome than those with triple-negative invasive ductal carcinomas. Although no significant differences in clinical outcome were observed among the metaplastic carcinoma subtypes in multivariate analyses, an age not exceeding 39 years, the presence of skin invasion, and the presence of a squamous cell carcinoma component in nodal tumors were significant outcome predictors for metaplastic carcinoma patients. In conclusion, the results of this study clearly demonstrated that metaplastic carcinoma is more aggressive than invasive ductal carcinoma or invasive lobular carcinoma. Although the metaplastic carcinoma subtypes had no prognostic significance, an age not exceeding 39 years, the presence of skin invasion, and the presence of a squamous cell carcinoma component in nodal tumors were significant predictors of outcome among metaplastic carcinoma patients. (C) 2010 Elsevier Inc. All rights reserved.