Using laser-captured microdissection and a real-time RT -PCR assay, we quantitatively evaluated mRNA levels of the following biomarkers in paraffin-embedded gastric cancer (GC) specimens obtained by surgical resection or biopsy: excision repair crosscomplementing gene 1 (ERCC1), dihydropyrimidine dehydrogenase (DPD), methylenetetrahydrofolate reductase (MTHFR), epidermal growth factor receptor (EGFR), and five other biomarkers related to anticancer drug sensitivity. The study group comprised 140 patients who received first-line chemotherapy for advanced GC. All cancer specimens were obtained before chemotherapy. In patients who received first-line S-1 monotherapy (69 patients), low MTHFR expression correlated with a higher response rate (low: 44.9% vs high: 6.3%; P ¼ 0.006). In patients given first-line cisplatin-based regimens (combined with S-1 or irinotecan) (43 patients), low ERCC1 correlated with a higher response rate (low: 55.6% vs high: 18.8%; P ¼ 0.008). Multivariate survival analysis of all patients demonstrated that high ERCC1 (hazard ratio (HR): 2.38 (95% CI: 1.55 -3.67)), high DPD (HR: 2.04 (1.37 -3.02)), low EGFR (HR: 0.34 (0.20 -0.56)), and an elevated serum alkaline phosphatase level (HR: 1.00 (1.001 -1.002)) were significant predictors of poor survival. Our results suggest that these biomarkers are useful predictors of clinical outcomes in patients with advanced GC.

Promoter methylation of the mismatch repair gene plays a key role in sporadic microsatellite instability (MSI) colorectal cancers. However, promoter methylation often occurs in proximal colon cancers, and molecular phenotypes underlying MSI cancers in distal colon have not been fully clarified. Our goal was to clarify the difference between MSI and microsatellite stability (MSS) cancers and, furthermore, to determine distinct characteristics of proximal and distal MSI cancers. By DNA microarray analysis of 84 cancers (33 MSI and 51 MSS), we identified discriminating genes (177 probe sets), which predicted MSI status with a high accuracy rate (97.6%). These genes were related to phenotypic characteristics of MSI cancers. Next, we identified 24 probe sets that were differentially expressed in proximal and distal MSI cancers. These genes included promoter methylation-mediated genes, whose expression was significantly down-regulated in proximal MSI cancers. Among discriminating genes between MSI and MSS, nine methylation-mediated genes showed down-regulation in MSI cancers. Of these, 7 (77.8%) showed down-regulation in proximal MSI cancers. Furthermore, methylation-specific PCR confirmed that frequency of hMLH1 promoter methylation was significantly higher in proximal MSI cancers (P = 0.0317). These results suggested that there is a difference between proximal and distal MSI cancers in methylation-mediated influence on gene silencing. In conclusion, using DNA microarray, we could distinguish MSI and MSS cancers. We also showed distinct characteristics of proximal and distal MSI cancers. The inactivation form of hMLH, per se, differed between proximal and distal MSI cancers. These results suggested that distal MSI cancers constitute a distinct subgroup of sporadic MSI cancers.

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Purpose: Ulcerative colitis (UC) is associated with a high risk of colorectal cancer. To identify genes that could predict the development of cancer in UC, we conducted a DNA microarray analysis using nonneoplastic rectal mucosa of UC patients. Experimental Design: Gene expression in nonneoplastic mucosa of 53 UC patients were examined. Gene expression profiles were examined using human Genome U133 Plus 2.0 gene chip array (Affymetrix). Among 53 UC patients, 10 had UC-associated cancer (UC-Ca group) whereas 43 did not (UC-NonCa group). Results: By comparing gene expression profiles of nonneoplastic rectal mucosae between the UC-Ca and UC-NonCa groups, we could identify 40 genes that were differentially expressed between two groups. The list of discriminating genes included low-density lipoprotein receptor^related protein (LRP5 and LRP6). Previous studies suggested that LRP5 and LRP6 expression promotes cancer cell proliferation and tumorigenesis and are considered as candidate oncogenes. In the present study, both LRP5 and LRP6 showed significantly higher expression in the UC-Ca group, which suggests the importance of these genes in the development of UC-associated colorectal cancers. With the 40 selected discriminating genes, we did class prediction of the development of colorectal neoplasms in UC patients. Using the k-nearest neighbor method and the support vector machine, we could predict the development of UC-associated neoplasms with an accuracy of 86.8% and 98.1%, respectively. Conclusions: These findings have important implications for the early detection of malignant lesions in UC and may provide directions for future research into the molecular mechanisms of UC-associated cancer.

Aberrant DNA methylation is a commonly observed epigenetic change in lung cancer. Folate has been suggested to play a role in the homeostasis of DNA methylation and has also been implicated in cancer chemotherapy. We investigated a possible role for folate in DNA methylation by measuring folate concentrations in tumors and adjacent normal tissues from 72 non-small cell lung cancer (NSCLC) patients. These were compared to DNA methylation levels and to clinicopathological features. Folate concentrations were determined as the sum of 5,10-methylenetetrahydrofolate and tetrahydrofolate. The MethyLight assay was used to quantitate methylation in promoter regions of P16(CDKN2A), APC, CDH13, RARB, RASSF1, RUNX3, and MYOD1. Methylation of LINE-1 repeats was used as a surrogate for global methylation. Folate levels in tumors correlated positively with LINE-1, CDH13, and RUNX3 methylation. Folate concentrations and methylation of LINE-1, RASSF1, and RUNX3 were significantly higher in adenocarcinoma compared to squamous cell carcinoma (SCC). Two sets of array-based data retrieved from the Gene Expression Omnibus consistently showed that expression of FOLR1, a folate transport enzyme, and GGH, an enzyme that prevents folate retention, were higher and lower, respectively, in adenocarcinomas compared to SCC. This was independently validated by quantitative RT-PCR in 26 adenocarcinomas and 13 SCC. Our results suggest that folate metabolism plays a role in aberrant DNA methylation in NSCLC. The histological subtype differences in folate concentration and DNA methylation observed here were associated with distinct expression patterns for folate metabolizing enzymes. These findings may have clinical applications for histology-directed chemotherapy with fluoropyrimidine and anti-folates in NSCLC. (Cancer Sci 2009; 100: 2325-2330 L ung cancer is a leading cause of cancer death worldwide. Genetic and epigenetic aberrations accumulate throughout lung carcinogenesis. Global hypomethylation of genomic DNA and hypermethylation of gene promoter regions occur simultaneously in a wide variety of malignancies including lung cancer.(1,2) Little is known however about the mechanism leading to these epigenetic alterations in human primary lung cancer.Epidemiological studies have demonstrated that dietary folate supplementation can prevent the development of lung cancer. Folate is an important precursor of one-carbon units required for DNA methylation. Therefore, folate metabolism has been suggested to influence epigenetic alterations in lung cancer and this could provide a mechanism to explain the prevention of lung cancer by folate supplementation. High folate might contribute to the maintenance of global methylation through an adequate supply of one-carbon units for the methylation machinery, thereby stabilizing the genome. Although the status of dietary folate intake has been analyzed in relation to DNA methylation, (4) an association between folate concentration and global methylation in human lung tissue has so far not been reported.In ad...