Tsunehiro Oyama

EGFR gene alterations as a prognostic biomarker in advanced esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) exhibits abnormalities in epidermal growth factor receptor (EGFR) gene. To identify a prognostic marker, the overexpression of EGFR protein, mutations in EGFR and p53 mutations were analyzed in pretreatment biopsy specimens removed from T3-4 and/or M1 LYM ESCC patients who received chemoradiotherapy. A silent mutation comprised of a single nucleotide polymorphism (SNP) at codon 787 of exon 20 of the EGFR gene was found in 19 patients (33%). In multivariate analysis, a significant difference was seen in the overall survival (odds ratio; 2.347, 95% confidence interval; 1.183-4.656, p = 0.015) between patients with and without the EGFR heterozygous genotype. Among the 57 eligible patients, 3-year survival rates was 21%, while in patients with EGFR heterozygous genotype the rate were 0%. However, neither overexpression of EGFR nor p53 mutations was associated with the overall survival. These results suggest that the EGFR SNP at codon 787 of exon 20 determined in pretreatment biopsy specimens may be a clinically useful biomarker for predicting the prognosis of ESCC patients.

Reduced bone formation in alcohol-induced osteopenia is associated with elevated p21 expression in bone marrow cells in aldehyde dehydrogenase 2-disrupted mice

INTRODUCTIONnHigh consumption of alcohol is one of the risk factors for osteoporosis. Approximately 45% of Chinese and Japanese individuals have the inactive aldehyde dehydrogenase 2 (Aldh2) phenotype. The absence of the ALDH2*2 allele is found to adversely influence the risk of osteoporosis. The aim of this study is to clarify the effects of alcohol consumption on osteoblast differentiation in bone marrow and trabecular bone formation in Aldh2-disrupted mice.nnnMATERIALS AND METHODSnSeven-week-old male Aldh2 knockout mice (Aldh2(-/-)) and wild-type (Aldh2(+/+)) mice were fed with water (groups Aldh2(-/-)/Wa and Aldh2(+/+)/Wa) or with 5% ethanol (groups Aldh2(-/-)/Al and Aldh2(+/+)/Al) for 4 weeks. At the age of 12 weeks, bone histomorphometry was performed at the secondary spongiosa of the tibias. Bone marrow cells from the bilateral femurs and tibias were used for mRNA expression analysis.nnnRESULTSnHistomorphometrical study revealed that trabecular bone was significantly reduced in the Aldh2(-/-)/Al group compared with that in the Aldh2(-/-)/Wa and Aldh2(+/+)/Wa groups. Bone formation rate was significantly decreased in Aldh2(-/-)/Al compared with the other three groups. Quantitative RT-PCR revealed a significant decrease in type I collagen, osterix, osteopontin, and osteocalcin mRNA expressions in Aldh2(-/-)/Al compared with Aldh2(-/-)/Wa. In bone marrow cell cultures, mineralized nodule formation in Aldh2(-/-)/Al was significantly decreased compared with that in Aldh2(+/+)/Wa and Aldh2(-/-)/Wa, while PAK18, a p21-activated kinase inhibitor, recovered the decreased mineralized nodule formation in Aldh2(-/-)/Al.nnnCONCLUSIONnAlcohol consumption suppressed the differentiation and mineralization of osteoblasts and then reduced trabecular bone formation and bone volume in association with the elevated p21 expression in bone marrow cells, especially in aldehyde dehydrogenase 2-disrupted mice.

Effects of 5-week ethanol feeding on the liver of aldehyde dehydrogenase 2 knockout mice.

Objective The polymorphism of aldehyde dehydrogenase 2 (ALDH2), denoted ALDH2*2, is very common in East Asian countries, and the mutated ALDH2 protein derived from ALDH2*2 lacks the ability of acetaldehyde metabolization. Our aim was to determine the consequences of the ALDH2 polymorphism on ethanol-administered liver tissue. Methods Aldh2+/+, +/−, and −/− mice were fed with ethanol solution and standard hard feed for 5 weeks. Results The serum alanine aminotransferase (ALT) level in the Aldh2−/− mice clearly decreased upon ethanol feeding, in contrast to Aldh2+/+ mice, in which the ALT level was unchanged. The levels of malondialdehyde, phospho extracellular signal-regulated kinase 2, and tumor necrosis factor-alpha in the liver tissue all correlated with the ALT level. Conclusion These findings suggest that ethanol intake in the presence of inactive ALDH2 decreases the serum ALT level following a decrease in oxidative stress and tumor necrosis factor-alpha secretion.

The onset of angiogenesis in a multistep process of esophageal squamous cell carcinoma.

Microvessel density (MVD) is an excellent predictive biomarker regarding tumor stage and survival in esophageal squamous cell carcinomas (ESCCs). However, it is obscure when tissues initiate angiogenesis in the malignant transformation of human esophageal squamous epithelium. To investigate the onset of angiogenesis in the multistep progressive process of ESCCs, immunohistochemical staining for CD31, CD105, and vascular endothelial growth factor receptor 2 (VEGFR-2) was performed in normal epithelium, Lugol-unstained lesions with non-dysplastic epithelium (LULs-NDE), low-grade dysplasia (LGD), and high-grade dysplasia (HGD) samples. There were significant differences in the mean MVD for CD31 and CD105 between LULs-NDE and LGD (p less than 0.001, p less than 0.001), and between LGD and HGD (p less than 0.001, p=0.006), respectively. Furthermore, a significant difference in MVD for CD105 was seen in normal controls and LULs-NDE (p=0.002), while thick vessels (less than 10m m) stained with anti-CD105 were not present in normal controls and LULs-NDE despite the presence of these thickened vessels in dysplasia. Our results suggest that CD105 is an efficient marker protein to determine MVD, suggesting that the angiogenic switch occurs at the earliest stage of dysplastic transformation in ESCC.

Expression of aromatase CYP19 and its relationship with parameters in NSCLC.

Human aromatase (CYP19) responsible for the conversion of androgens to estrogens is expressed not only in gonads and adrenals but also in many other tissues, including normal lungs and lung cancers. To investigate the involvement of CYP19 in lung cancer development, purified CYP19 protein and antibody are required. In this study, we have developed an efficient expression method of human aromatase in E. coli (>1000 nmol/L culture). The protein purified from E. coli was used to raise an antibody against the human CYP19 in rabbits. The resulting antibody showed a high titer judged by ELISA, which allowed us to determine the expression of CYP19 in non-small cell lung cancer (NSCLC). Of 78 NSCLC specimens from Japanese patients, 50 (64%) NSCLC aberrantly expressed CYP19. This CYP19 expression in NSCLC was independent of any clinical and pathological parameters as well as the expression of other P450s, except tumor stage. The results suggest that the aromatase inhibitors might be useful for the management of non-small cell lung cancer in postmenopausal women.

Expression of cytochrome P450 in non-small cell lung cancer.

Lung cancer accounts for most of cancer-related deaths in both men and women. Lung cancer is also associated with cigarette smoking that exposes the individual to carcinogenic chemicals. Normally, CYP enzymes (cytochrome P450s) metabolize carcinogens to inactive derivatives, however, occasionally the action of CYP enzymes leads to development of more potent carcinogens. In addition to the metabolism of carcinogenic compounds, CYP enzymes are also involved in the activation and/or inactivation of agents, which are used in the treatment of lung cancer. Therefore, the local level of CYP enzymes in lung cancer and surrounding tissues could be an important determinant in the efficacy of anticancer drugs. Furthermore, the expression of CYP19 (aromatase), estrogen synthesis P450, was found in more than 80 percent of non-small cell lung cancers. Lung cancer was also found to frequently express CYP24A1 that converts 1 alpha, 25-dihydroxyvitamin D3 to its inactive 24-hydroxylated derivatives. The understanding of the local expression of CYP enzymes in tumor tissues is important in the development of better treatment for lung cancer and a standardized treatment, tailor-made, for individual patients.

The effect of ethanol on the formation of N 2-ethylidene-dG adducts in mice: implications for alcohol-related carcinogenicity of the oral cavity and esophagus

The present study aimed to experimentally confirm that long-term alcohol drinking causes a high risk of oral and esophageal cancer in aldehyde dehydrogenase 2 (ALDH2)-deficient individuals. Aldh2 knockout mice, an animal model of ALDH2-deficiency, were treated with 8% ethanol for 14 months. Levels of acetaldehyde-derived DNA adducts were increased in esophagus, tongue and submandibular gland. Our finding that a lack of Aldh2 leads to more DNA damage after chronic ethanol treatment in mice supports epidemiological findings on the carcinogenicity of alcohol in ALDH2-deficient individuals who drink chronically.

TS gene tandem repeats in esophageal cancer patients receiving chemoradiotherapy.

5-Fluorouracil (5-FU) interferes with tumor-cell proliferation by inhibiting thymidylate synthase (TS). We examined the relationship between tandem repeat (TR) variations in the TS gene and survival following concurrent chemoradiotherapy in patients with esophageal squamous cell carcinoma (ESCC). TS-TR variations were analyzed in 57 stage II-IV ESCC patients undergoing chemoradiotherapy combined with 5-FU and cisplatinum (CDDP), and in 106 controls. Pretreatment non-neoplastic biopsy specimens from ESCC patients and lymphocytes from controls were used for analysis. Variations were identified by the size of DNA fragments amplified by polymerase chain reaction. Two to five TRs were found in Japanese individuals. TR3 homozygotes were predominant in 74% of ESCC patients and 61% of controls. Three-year survival rates were significantly longer in patients with TR2/2 or TR2/3 genotypes (38%) than in patients with TR3/3, 3/4, or 3/5 genotypes (9%; p=0.011). In the Cox proportional hazard model, the TR2/2 or TR2/3 genotypes were the only independent predictor for survival (Hazard ratio, 2.647; 95% confidence interval, 1.271-5.513). The TS-TR variations exert an important influence on survival following chemoradiotherapy in ESCC patients.