Published papers

Non-muscle invasive (superficial) bladder cancer is a low-grade malignancy with good prognosis, while muscle invasive (invasive) bladder cancer is a high-grade malignancy with poor prognosis. N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) induces superficial bladder cancers with papillary morphology in rats and invasive bladder cancers with infiltrating phenotype in mice. In this study, we analyzed genomic landscapes of rodent BBN-induced bladder cancers using array-based comparative genomic hybridization (array CGH). While no significant copy number alterations were detected in superficial bladder tumors in rats, copy number gains in chromosomal regions 2D-E1, 7qA3, 9F2, and 11C-D were detected in invasive bladder tumors in mice. Amplification of representative genes located on 2D-E1 and 7qA3 chromosomal regions was confirmed by quantitative PCR. Cyp2a22 and Cyp2a5 genes but not Cyp2g1, Cyp2a12, and Rab4b genes on mouse chromosome 7qA3 were amplified in invasive bladder cancers. Although the human ortholog gene of Cyp2a22 has not been confirmed, the mouse Cyp2a5 gene is the ortholog of the human CYP2A6 gene located in chromosomal region 19q13.2, and CYP2A6 was identified by database search as one of the closest human homolog to mouse Cyp2a22. Considering a possibility that this region may be related to mouse 7qA3, we analyzed CYP2A6 copy number and expression in human bladder cancer using cell lines and resected tumor specimens. Although only one of eight cell lines showed more than one copy increase of the CYP2A6 gene, CYP2A6 amplification was detected in six out of 18 primary bladder tumors where it was associated with the invasive phenotype. Immunohistochemical analyses of 118 primary bladder tumors revealed that CYP2A6 protein expression was also higher in invasive tumors, especially in those of the scattered type. Together, these findings indicate that the amplification and overexpression of the CYP2A6 gene are characteristic of human bladder cancers with increased malignancy and that CYP2A6 can be a candidate prognostic biomarker in this type of cancer.

Cholangiocarcinoma is an aggressive malignancy with a poor prognosis, with no effective therapy other than surgical resection. Heat shock protein 90 (HSP90) is a key component of a multi-chaperone complex involved in the posttranslational folding of a number of client proteins, many of which play essential roles in tumorigenesis. Here, we attempted to clarify its prognostic significance and potential utility as a therapeutic target in cholangiocarcinoma. Immunohistochemical expression of HSP90 was assessed retrospectively in 399 cholangiocarcinoma cases and 17 human cholangiocarcinoma cell lines, along with the effect of a small-molecule HSP90 inhibitor (NVP-AUY922) on cholangiocarcinoma tumor growth and angiogenesis in human cholangiocarcinoma cell lines and xenografts. The positivity of HSP90 was 44.6% in intrahepatic cholangiocarcinoma (IHCC) and 32.8% in extrahepatic cholangiocarcinoma (EHCC), respectively. HSP90 expression was significantly associated with the 5-year survival rate for IHCC (P < 0.001) and EHCC (P < 0.001). HSP90 inhibition showed potent antiproliferative activity and reduced growth-associated signaling in human cholangiocarcinoma cells in vitro. Furthermore, treatment of cholangiocarcinoma xenograft-bearingmice with NVP-AUY922 significantly inhibited growth at doses far below the maximum-tolerated dose. HSP90 overexpression is a prognostic marker for cholangiocarcinoma. HSP90-targeted therapy may be an option for a subset of cholangiocarcinoma. (C) 2015 AACR.

Colorectal villous adenoma is thought to be associated with a high risk of progression to adenocarcinoma. To better characterize the genetic alterations involved in colorectal carcinogenesis related to villous adenoma, we analyzed mutations in APC, BRAF, KRAS, TP53, and GNAS in 12 colorectal adenocarcinomas associated with villous adenomas. APC, KRAS, and BRAF mutations were identified in five, 11, and one lesion, respectively, and most of these mutations were shared between the villous adenoma and the adenocarcinoma components in the respective lesions, except in one lesion with APC mutations and in two lesions with KRAS mutations. TP53 mutations were observed exclusively in four adenocarcinoma components, consistent with their role in the progression from adenoma to adenocarcinoma. Activating GNAS mutations were found in nine villous adenomas; however, unexpectedly, these mutations were shared only in three associated adenocarcinomas. Notably, all six adenocarcinomas with discordant GNAS mutation statuses were nonmucinous type, whereas all the other adenocarcinomas, including three adenocarcinomas associated with GNAS wild-type villous adenomas, were mucinous type. The current study suggests that GNAS-mutated villous adenomas may not necessarily be direct precursors of associated adenocarcinomas. At the same time, our observations support the role of activating GNAS mutations in increased mucin production in colorectal neoplasms. © 2014 Wiley Periodicals, Inc.

Gastric and duodenal adenomas exhibit a significant morphological and phenotypical diversity and are classified into intestinal-type, foveolar-type and pyloric gland adenomas. We analysed the mutations in GNAS, KRAS, BRAF and CTNNB1 and the expressions of mismatch repair (MMR) proteins in 80 gastric and 32 duodenal adenomas with histologically distinct subtypes, as well as in 71 gastric adenocarcinomas. Activating GNAS mutations were found in 22 of the 35 pyloric gland adenomas (PGAs; 63%) but in none of the foveolar-type or intestinal-type adenomas or the adenocarcinomas. Fourteen PGAs (41%), two foveolar-type adenomas (9%), five intestinal-type adenomas (9%) and one adenocarcinoma (1%) had KRAS mutations. BRAF mutations were absent in all the adenomas and adenocarcinomas that were examined. CTNNB1 mutations were only found in two intestinal-type adenomas (4%). Notably, 13 of the 14 KRAS-mutated gastric and duodenal PGAs had concurrent GNAS mutations. The loss of the MMR proteins, which is indicative of microsatellite instability, was observed in one PGA (3%), 12 foveolar-type adenomas (52%), one intestinal-type adenoma (2%) and five adenocarcinomas (7%). These observations indicate that each histological subtype of gastric and duodenal adenomas has a distinct genetic background. In particular, the present study identified the frequent presence of activating GNAS mutations, which are often associated with KRAS mutations, as a characteristic genetic feature of PGAs of the stomach and duodenum.

Iron overload has been associated with carcinogenesis in humans. Intraperitoneal administration of ferric nitrilotriacetate initiates a Fenton reaction in renal proximal tubules of rodents that ultimately leads to a high incidence of renal cell carcinoma (RCC) after repeated treatments. We performed high-resolution microarray comparative genomic hybridization to identify characteristics in the genomic profiles of this oxidative stress-induced rat RCCs. The results revealed extensive large-scale genomic alterations with a preference for deletions. Deletions and amplifications were numerous and sometimes fragmented, demonstrating that a Fenton reaction is a cause of such genomic alterations in vivo. Frequency plotting indicated that two of the most commonly altered loci corresponded to a Cdkn2a/2b deletion and a Met amplification. Tumor sizes were proportionally associated with Met expression and/or amplification, and clustering analysis confirmed our results. Furthermore, we developed a procedure to compare whole genomic patterns of the copy number alterations among different species based on chromosomal syntenic relationship. Patterns of the rat RCCs showed the strongest similarity to the human RCCs among five types of human cancers, followed by human malignant mesothelioma, an iron overload-associated cancer. Therefore, an iron-dependent Fenton chemical reaction causes large-scale genomic alterations during carcinogenesis, which may result in distinct genomic profiles. Based on the characteristics of extensive genome alterations in human cancer, our results suggest that this chemical reaction may play a major role during human carcinogenesis.

Recent studies have shown that intratumoral cholestasis is a hallmark of CTNNB1 mutations in hepatocellular carcinomas (HCC). Here, we analyzed the expressions of genes involved in bile acid and bilirubin metabolism and their correlation with the mutational status of CTNNB1 in a series of HCC. The expressions of CYP7A1 and CYP27A1, which encode rate-limiting enzymes in bile acid synthesis, were unaltered or only marginally increased in CTNNB1-mutated HCC compared with those in HCC with wild-type CTNNB1. Among the genes involved in bile acid and bilirubin transport, the expression of SLCO1B3 was significantly elevated in HCC with CTNNB1 mutations, whereas the expression of ABCC4 was elevated in HCC with wild-type CTNNB1. Immunohistochemistry confirmed the frequent expression of SLCO1B3 in CTNNB1-mutated HCC at the protein level, but not in most HCC with wild-type CTNNB1. Immunohistochemistry for MRP4 (encoded by ABCC4) partly agreed with ABCC4 expression, but most cases did not express detectable levels of MRP4. Notably, all HCC with bile accumulation, including those without CTNNB1 mutations, expressed SLCO1B3, suggesting that SLCO1B3 expression, rather than CTNNB1 mutation, is the critical determinant of intratumoral cholestasis. As SLCO1B3 is involved in the uptake of a number of chemotherapeutic and diagnostic agents, SLCO1B3 expression and the status of CTNNB1 mutation might need to be considered in the drug delivery to HCC. (Cancer Sci 2011; 102: 1742-1747)

The aim of this study was to establish new biliary tract carcinoma (BTC) cell lines and identify predictive biomarkers for the potential effectiveness of gemcitabine therapy. Surgical specimens of BTC were transplanted directly into immunodeficient mice to establish xenografts, then subjected to in vitro cell culture. The gemcitabine sensitivity of each cell line was determined and compared with the genome-wide gene expression profile. A new predictive biomarker candidate was validated using an additional cohort of gemcitabine-treated BTC cases. From 55 BTC cases, we established 19 xenografts and six new cell lines. Based on their gemcitabine sensitivity, 10 BTC cell lines (including six new and four publicly available ones) were clearly categorized into two groups, and MAGEH1 mRNA expression in the tumor cells showed a significant negative correlation with their sensitivity to gemcitabine. Immunohistochemically, MAGEH1 protein was detected in three (50%) out of six sensitive cell lines, and four (100%) out of four resistant cell lines. In the validation cohort of gemcitabine-treated recurrence cases, patients were categorized into "effective'' and "non-effective'' groups according to the RECIST guidelines for assessment of chemotherapeutic effects. MAGEH1 protein expression was detected in two (40%) out of five "effective'' cases and all four (100%) "non-effective'' cases. We have established a new BTC bioresource that covers a wide range of biological features, including drug sensitivity, and is linked with clinical information. Negative expression of MAGEH1 protein serves as a potential predictive marker for the effectiveness of gemcitabine therapy in BTC. (Cancer Sci 2010; 101: 882-888)

The fibroblast growth factor receptor (FGFR)-3 gene encodes a receptor tyrosine kinase that is frequently mutated in non-muscle invasive bladder cancer (NMIBC). A sensitive and quantitative assay using peptide nucleic acid-mediated real-time PCR was developed for detecting FGFR3 mutations in the urine samples and evaluated as a molecular marker for detecting intravesical recurrence of NMIBC in patients undergoing transurethral resection of bladder tumor. FGFR3 mutation was examined in tumor tissues and serially taken pre- and postoperative urine sediments in 45 NMIBC patients with a median follow up of 32 months. FGFR3 mutations were detected in 53.3% (24/45) of primary tumor tissues, among which intravesical recurrence developed in 37.5% (9/24) of cases. FGFR3 mutation in the primary tumor was not a significant prognostic indicator for recurrence, while the proportion of FGFR3 mutation (i.e. tumor cellularity was >= 11%) in the preoperative urine sediments was a significant indicator for recurrence in patients with FGFR3 mutations in the primary tumors. FGFR3 mutations were detected in 78% (7/9) of postoperative urine samples from recurrent cases with FGFR3 mutations in the tumor, while no mutations were detected in the urine of 15 non-recurrent cases. Urine cytology was negative in all cases with FGFR3 mutations in the primary tumors, while the sensitivity of cytological examination was as high as 56% (5/9) in cases showing wild-type FGFR3 in the primary tumors. Urine FGFR3 mutation assay and cytological examination may be available in the future as complementary diagnostic modalities in postoperative management of NMIBC. (Cancer Sci 2009).

Mucosal melanomas have genetic alterations distinct from those in cutaneous melanomas. For example, NRAS- and BRAF-activating mutations occur frequently in cutaneous melanomas, but not in mucosal melanomas. We examined 16 esophageal melanomas for genetic alterations in NRAS, BRAF, and KIT to determine whether they exhibit genetic features common to melanomas arising from other mucosal sites. A sequencing analysis identified NRAS mutations in six cases; notably, four of these mutations were located in exon 1, an uncommon mutation site in cutaneous and other mucosal melanomas. BRAF and KIT mutations were found in one case each. Immunohistochemistry showed KIT expression in four cases, including the tumor with a KIT mutation and two other intramucosal tumors. The low frequency of BRAF mutations and the presence of a KIT mutation-positive case are findings similar to those of mucosal melanomas of other sites, but the prevalence of NRAS mutations was even higher than that of cutaneous melanomas. The present study implies that esophageal melanomas have genetic alterations unique from those observed in other mucosal melanomas.

Angiopoietin-like protein 2 (ANGPTL2) is a secreted protein belonging to the angiopoietin family, the members of which are implicated in various biological processes, although its receptor remains unknown. We identified a homozygous loss of ANGPTL2 (9q33.3) in the course of screening a panel of ovarian cancer (OC) cell lines for genomic copy-number aberrations using in-house array-based comparative genomic hybridization. ANGPTL2 mRNA expression was observed in normal ovarian tissue and immortalized normal ovarian epithelial cells, but was reduced in some OC lines without its homozygous deletion (18 of 23 lines) and restored after treatment with 5-aza 2'-deoxycytidine. The methylation status of sequences around the ANGPTL2 CpG-island with clear promoter activity inversely correlated with expression. ANGPTL2 methylation was frequently observed in primary OC tissues as well. In an immunohistochemical analysis of primary OCs, ANGPTL2 expression was frequently reduced (51 of 100 cases), and inversely correlated with methylation status. Patients with OC showing reduced ANGPTL2 immunoreactivity had significantly worse survival in the earlier stages (stages I and II), but better survival in advanced stages (stages III and IV). The restoration of ANGPTL2 expression or treatment with conditioned medium containing ANGPTL2 inhibited the growth of OC cells originally lacking the expression of this gene, whereas the knockdown of endogenous ANGPTL2 accelerated the growth of OC cells with the expression of ANGPTL2. These results suggest that, at least partly, epigenetic silencing by hypermethylation of the ANGPTL2 promoter leads to a loss of ANGPTL2 function, which may be a factor in the carcinogenesis of OC in a stage-dependent manner.

Although Runt-related transcription factors RUNXs (RUNX1-3) have a high similarity in their structure, only RUNX3 is known to be involved in gastric carcinogenesis. First, we examined mRNA expression of these three RUNX genes in the gastric mucosa, and, finding only RUNX2 was not expressed there, we further investigated RUNX1 and RUNX3 expression in three regions including the pit, isthmus/ neck, and gland regions of the human normal stomach and whether RUNX1 is involved in gastric carcinogenesis. The mRNA expression of RUNX1 and RUAX3 was examined by use of the three regions isolated by laser-captured microdissection (LCM) and by use of primary gastric cancer tissues. Furthermore, RUNX1 mutational analysis was performed in the cancer cells, which also were isolated from 44 paraffin-embedded gastric cancer tissues by LCM. RUNX1 was coexpressed with RUNX3 in the pit region, and has cell growth-inhibition activity similar to RUNX3. RUNX3 has been reported to be suppressed by DNA methylation in a subset of gastric cancers; however, the expression of RUNX1 mRNA was observed in all of the gastric cancer cell lines and gastric cancer tissues that we examined. No RUNX1 mutation was found in the 44 gastric cancer patients. Although RUNX1 is similar to RUNX3 in both the expression pattern in the stomach and its cell growth-inhibition activity, RUNX1 is not involved in most cases of gastric cancers. These results suggest that the transcriptional target genes are different between these two family genes.

The aim of this study was to clarify the significance of DNA methylation alterations shared by cancers derived from multiple organs. We analyzed single-institutional methylome data by single-CpG-resolution Infinium assay for 1007 samples of non-cancerous tissue (N) and corresponding cancerous tissue (T) obtained from lung, stomach, kidney, breast and liver. Principal component analysis revealed that N samples of each organ showed distinct DNA methylation profiles, DNA methylation profiles of N samples of each organ being inherited by the corresponding T samples and DNA methylation profiles of T samples being more similar to those of N samples in the same organ than those of T samples in other organs. In contrast to such organ and/or carcinogenetic factor-specificity of DNA methylation profiles, when compared with the corresponding N samples, 231 genes commonly showed DNA hypermethylation in T samples in four or more organs. Gene ontology enrichment analysis showed that such commonly methylated genes were enriched among '' transcriptional factors '' participating in development and/or differentiation, which reportedly show bivalent histone modification in embryonic stem cells. Pyrosequencing and quantitative reverse transcription-PCR revealed an inverse correlation between DNA methylation levels and mRNA expression levels of representative commonly methylated genes, such as ALX1, ATP8A2, CR1 and EFCAB1, in tissue samples. These data suggest that disruption of the differentiated state of precancerous cells via alterations of expression, independent of differences in organs and/or carcinogenetic factors, may be a common feature of DNA methylation alterations during carcinogenesis in multiple organs.

AimsFamilial adenomatous polyposis (FAP) is a hereditary cancer predisposition syndrome caused by a germline APC mutation. A recent study showed the enrichment of pyloric gland adenomas (PGAs) of the stomach, in addition to fundic gland polyps (FGPs) and foveolar-type adenomas (FAs), in patients with FAP. In the present study, we analysed the genetic alterations in these FAP-associated gastric lesions. Methods and resultsMutational statuses of GNAS and KRAS, which are frequently mutated in sporadic PGAs, as well as those of APC, were examined in PGAs, FAs and FGPs in patients with FAP using Sanger sequencing. Our analysis identified GNAS mutations in five of six PGAs (83%), but in none of the three FAs or the 40 FGPs examined. KRAS mutations were identified in four PGAs (67%), one FA (33%) and one FGP (3%). Somatic truncating APC mutations were found in all PGAs (100%), two FAs (67%) and 14 FGPs (47%). We additionally analysed sporadic PGAs of the stomach and duodenum and identified truncating APC mutations in 11 of 25 lesions (44%). ConclusionsFAP-associated and sporadic PGAs not only show similar morphologies, but also share common genetic aberrations, including mutations of GNAS, KRAS and APC.

Background: Phyllodes tumours are rare fibroepithelial tumours of the breast, that include benign, borderline, and malignant lesions. Although the molecular basis of phyllodes tumours largely remains unknown, a recent exome study identified MED12 mutations as a sole recurrent genetic alteration in fibroadenoma, a common benign fibroepithelial tumour that shares some histological features with the phyllodes tumour. Methods: Forty-six phyllodes tumours and 58 fibroadenomas of the breast were analysed for MED12 mutations by using Sanger sequencing. Results: MED12 mutations were identified in 37 out of the 46 phyllodes tumours (80%). The prevalence of MED12 mutations was similar among benign (15/18, 83%), borderline (12/15, 80%), and malignant tumours (10/13, 77%). MED12 mutations were also identified in 36 of the 58 fibroadenomas (62%). The mutations were frequent among intracanalicular-type (24/32, 75%) and complex-type lesions (4/6, 67%), but were significantly less common among the pericanalicular-type lesions (8/20, 40%). A microdissection-based analysis showed that MED12 mutations were confined to the stromal components in both phyllodes tumours and fibroadenomas. Conclusions: MED12 mutations were frequent among the phyllodes tumours of the breast, regardless of the tumour grade. Phyllodes tumours and fibroadenomas share, at least in part, a common genetic background.

To examine the significance of aberrant DNA methylation in hepatocarcinogenesis, the DNA methylation status at the D17S5 locus and mRNA expression of a candidate tumor suppressor gene, HIC-1 (hypermethylated-in- cancer), which was identified at the D17S5 locus, in primary hepatocellular carcinomas (HCCs) and their corresponding noncancerous liver tissues were assessed. DNA hypermethylation at the D17S5 locus was detected in 44% of the noncancerous liver tissues showing chronic hepatitis or cirrhosis, which are widely considered to be precancerous conditions, but was not observed in noncancerous liver tissues showing no remarkable histological findings. The incidence of DNA hypermethylation at this locus was significantly higher in HCCs (90%) than noncancerous liver tissues (P < .001). Loss of heterozygosity at the D17S5 locus, which was preceded by DNA hypermethylation at the same locus, was detected in 54% of HCCs. The HIC-1 mRNA expression level of noncancerous liver tissues showing chronic hepatitis or cirrhosis was significantly lower than that of noncancerous liver tissues showing no remarkable histological findings (P < .01), and that of HCCs was even lower than that of noncancerous liver tissues (P < .05). Poorly differentiated HCCs showed lower expression levels than well- to moderately differentiated HCCs. Mutation of the p53 gene may be involved in HIC-1 inactivation. Moreover, wild-type p53 did not overcome DNA hypermethylation at the D17S5 locus to activate HIC-1 in HCCs. These data suggest that aberrant DNA methylation at this locus and reduced HIC-1 mRNA expression participate in hepatocarcinogenesis during both early developmental stages and malignant progression of HCCs.

The aim of this study was to clarify the significance of DNA methylation alterations during lung carcinogenesis. Infinium assay was performed using 139 paired samples of non-cancerous lung tissue (N) and tumorous tissue (T) from a learning cohort of patients with lung adenocarcinomas (LADCs). Fifty paired N and T samples from a validation cohort were also analyzed. DNA methylation alterations on 1,928 probes occurred in N samples relative to normal lung tissue from patients without primary lung tumors, and were inherited by, or strengthened in, T samples. Unsupervised hierarchical clustering using DNA methylation levels in N samples on all 26,447 probes subclustered patients into Cluster I (n = 32), Cluster II (n = 35) and Cluster III (n = 72). LADCs in Cluster I developed from the inflammatory background in chronic obstructive pulmonary disease (COPD) in heavy smokers and were locally invasive. Most patients in Cluster II were non-smokers and had a favorable outcome. LADCs in Cluster III developed in light smokers were most aggressive (frequently showing lymphatic and blood vessel invasion, lymph node metastasis and an advanced pathological stage), and had a poor outcome. DNA methylation levels of hallmark genes for each cluster, such as IRX2, HOXD8, SPARCL1, RGS5 and EI24, were again correlated with clinicopathological characteristics in the validation cohort. DNA methylation profiles reflecting carcinogenetic factors such as smoking and COPD appear to be established in non-cancerous lung tissue from patients with LADCs and may determine the aggressiveness of tumors developing in individual patients, and thus patient outcome. What's new? While genetic abnormalities are well studied in human cancers, epigenetic changes, especially in the early stages of carcinogenesis, remain largely unknown. Here, the authors perform a genome-wide analysis focusing on DNA methylation profiles in normal lung tissue adjacent to lung adenocarcinomas. Using single-CpG-resolution Infinium assays, they identify distinct DNA methylation profiles clustering with specific risk factors such as cigarette smoking, inflammation and chronic obstructive pulmonary disease. The authors speculate that these epigenetic profiles detected in the neighboring cells may influence the aggressiveness of tumors developing in individual patients and may thus help predict disease outcome.

Early venous return (EVR) is an important radiological feature of hepatic angiomyolipoma (HAML) that can aid in differential diagnosis, but the pathogenic mechanisms of EVR have yet to be elucidated. We present the first HAML case for which a probable mechanism for EVR is described. The patient was a 46-year-old woman, who had a growing 6-cm tumor with EVR in segment 3 of the liver as revealed by dynamic contrast-enhanced computed tomography. Left hepatic lobectomy was performed to prevent tumor rupture. Histopathological and immunohistochemical analyses of the excised tumor indicated HAML. Successive microsections of the tumor were stained with hematoxylin-eosin and Victoria blue to visualize the vascular structure within and around the tumor. These analyses led to three major findings. First, many well-defined thick-walled vessels, such as arteries, were found entering the tumor. Second, many thick-walled vessels within the tumor were connected directly to thin-walled vessels, resembling arteriovenous fistulae. Finally, thin-walled intratumoral vessels were connected directly to the hepatic vein. These histological findings suggested that the rich arterial flow into the tumor was being rapidly drained into the hepatic vein through intratumoral arteriovenous connections. We also detected these same anomalous circulatory pathways in tissue sections from three of four additional HAML cases with EVR. Aberrant arteriovenous fistulae within the tumor may account for many cases of EVR in HAML patients.

The aim of this study was to clarify the significance of DNA methylation alterations at precancerous stages of lung adenocarcinoma. Using single-CpG resolution lnfinium array, genome-wide DNA methylation analysis was performed in 36 samples of normal lung tissue obtained from patients without any primary lung tumor, 145 samples of non-cancerous lung tissue (N) obtained from patients with lung adenocarcinomas, and 145 samples of tumorous tissue (T). Stepwise progression of DNA methylation alterations from normal lung tissue to non-cancerous lung tissue obtained from patients with lung adenocarcinomas, and then tumorous tissue samples, was observed at 3,270 CpG sites, suggesting that non-cancerous lung tissue obtained from patients with lung adenocarcinomas was at precancerous stages with DNA methylation alterations. At CpG sites of 2,083 genes, DNA methylation status in samples of non-cancerous lung tissue obtained from patients with lung adenocarcinomas was significantly correlated with recurrence after establishment of lung adenocarcinomas. Among such recurrence-related genes, 28 genes are normally unmethylated (average beta-values based on lnfinium assay in normal lung tissue samples was less than 0.2) and their DNA hypermethylation at precancerous stages was strengthened during progression to lung adenocarcinomas (Delta beta(T-N)>0.1). Among these 28 genes, we focused on 6 for which implications in transcription regulation, apoptosis or cell adhesion had been reported. DNA hypermethylation of the ADCY5, EVX1, GFRA1, PDE9A, and TBX20 genes resulted in reduced mRNA expression in tumorous tissue samples. 5-Aza-2'-deoxycytidine treatment of lung cancer cell lines restored the mRNA expression levels of these 5 genes. Reduced mRNA expression in tumorous tissue samples was significantly correlated with tumor aggressiveness. These data suggest that DNA methylation alterations at precancerous stages determine tumor aggressiveness and outcome through silencing of specific genes.

Background: Early detection and risk assessment are crucial for treating urothelial cancer (UC), which is characterized by a high recurrence rate, and necessitates frequent and invasive monitoring. We aimed to establish diagnostic markers for UC based on DNA methylation. Methods: In this multi-center study, three independent sample sets were prepared. First, DNA methylation levels at CpG loci were measured in the training sets (tumor samples from 91 UC patients, corresponding normal-appearing tissue from these patients, and 12 normal tissues from age-matched bladder cancer-free patients) using the Illumina Golden Gate methylation assay to identify differentially methylated loci. Next, these methylated loci were validated by quantitative DNA methylation by pyrosequencing, using another cohort of tissue samples (Tissue validation set). Lastly, methylation of these markers was analyzed in the independent urine samples (Urine validation set). ROC analysis was performed to evaluate the diagnostic accuracy of these 12 selected markers. Results: Of the 1303 CpG sites, 158 were hyper ethylated and 356 were hypo ethylated in tumor tissues compared to normal tissues. In the panel analysis, 12 loci showed remarkable alterations between tumor and normal samples, with 94.3% sensitivity and 97.8% specificity. Similarly, corresponding normal tissue could be distinguished from normal tissues with 76.0% sensitivity and 100% specificity. Furthermore, the diagnostic accuracy for UC of these markers determined in urine samples was high, with 100% sensitivity and 100% specificity. Conclusion: Based on these preliminary findings, diagnostic markers based on differential DNA methylation at specific loci can be useful for non-invasive and reliable detection of UC and epigenetic field defect.

Aims: To examine in testicular seminomas the expression of DNA methyltransferase 3B (DNMT3B), which is known to be associated with early embryonic development and carcinogenesis, and to obtain a predictive marker for relapse of stage I seminomas. Methods and results: Immunohistochemical examination of DNMT3B was performed in 88 cases of seminoma, 35 (39.8%) of which showed widely scattered nuclear immunoreactivity for DNMT3B, and 53 (60.2%) of which were completely negative. The incidence of focal DNMT3B expression was higher in stage III seminomas (5/5, 100%) than in stage I (25/70, 35.7%) or stage II (5/13, 38.5%) seminomas (P = 0.011). In stage I seminomas there were no significant correlations between DNMT3B expression and tumour size, invasion of the rete testis, or lymphatic or vascular involvement. Six of 25 cases (24%) showing DNMT3B expression relapsed, whereas only 3/45 cases (6.7%) lacking such expression did so (P = 0.037). Patients with seminomas showing DNMT3B expression had a significantly lower relapse-free survival rate than patients whose tumours lacked this feature (P = 0.0464). Conclusions: Patients with seminomas showing focal DNMT3B expression are at increased risk of relapse, and should be followed up carefully.