Masaaki Inoue

Detection of micrometastatic tumor cells in pN0 lymph nodes of patients with completely resected nonsmall cell lung cancer. Impact on recurrence and survival

ObjectiveTo detect occult micrometastatic tumor cells in pN0 lymph nodes of nonsmall cell lung cancer (NSCLC) by a combination of cytokeratin and p53 immunohistochemistry staining, and to evaluate the relation between the micrometastasis in pN0 lymph nodes and the prognosis of patients with completely resected stage 1 NSCLC. Summary Background DataThe average 5-year survival rate for patients with completely resected stage 1 NSCLC is only about 70%; thus, about 30% of these patients have recurrent disease. This suggests that occult micrometastasis may exist at the time of surgery; the rate is clearly underestimated by current clinical staging examinations and conventional histopathologic methods. MethodsA total of 474 hilar and mediastinal lymph nodes were removed during surgery from 49 patients with completely resected stage 1 NSCLC. The lymph nodes analyzed for micrometastasis using immunohistochemical staining with the biclonal anticytokeratin antibody, AE1/AE3. Of these 474 lymph nodes from 49 patients, 263 lymph nodes from 25 patients, whose primary tumors were positive for the p53 protein, were subjected to immunohistochemical staining with the monoclonal anti-p53 protein antibody DO-1. ResultsCells positive for cytokeratin and p53 protein were found in 35 (7.4%) of 474 and 20 (7.6%) of 263 lymph nodes, respectively; 17 (34.7%) of 49 patients had cytokeratin-positive cells and 10 (40.0%) of 25 patients had p53-positive cells in their pN0 lymph nodes. By a combination of cytokeratin and p53 protein immunohistochemical staining, micrometastatic tumor cells were identified in pN0 lymph nodes in 22 (44.9%) of 49 patients. The patients with lymph node micrometastasis identified by a combination of cytokeratin and p53 protein immunohistochemical staining had a poorer prognosis than those without micrometastasis on both univariate and multivariate analyses (overall survival, P = .0003 and 0.013, respectively). ConclusionsThe detection of lymph nodal micrometastasis by cytokeratin and p53 protein immunohistochemical staining will be helpful to predict the recurrence and prognosis of patients with completely resected stage 1 NSCLC.

Induction of chromosomal gene mutations in Escherichia coli by direct incorporation of oxidatively damaged nucleotides. New evaluation method for mutagenesis by damaged DNA precursors in vivo

We have developed a new strategy for the evaluation of the mutagenicity of a damaged DNA precursor (deoxyribonucleoside 5′-triphosphate) in Escherichia coli. 8-Hydroxydeoxyguanosine triphosphate (8-OH-dGTP) and 2-hydroxydeoxyadenosine triphosphate (2-OH-dATP) were chosen for this study because they appear to be formed abundantly by reactive oxygen species in cells. We introduced the oxidatively damaged nucleotides into competent E. coli and selected mutants of the chromosomal lacI gene. Both damaged nucleotides inducedlacI gene mutations in a dose-dependent manner, whereas unmodified dATP and dGTP did not appear to elicit the mutations. The addition of 50 nmol of 8-OH-dGTP and 2-OH-dATP into anE. coli suspension induced 12- and 9-fold more substitution mutations than the spontaneous event, respectively. The 8-OH-dGTP induced A·T → C·G transversions, and the 2-OH-dATP elicited G·C → T·A transversions. These results indicate that the two oxidatively damaged nucleotides are mutagenic in vivo and suggest that 8-OH-dGTP and 2-OH-dATP were incorporated opposite A and G residues, respectively, in the E. coli DNA. This new method enables the evaluation and comparison of the mutagenic potentials of damaged DNA precursors in vivo.

Dietary factors and lung cancer risk in Japanese: with special reference to fish consumption and adenocarcinomas

To investigate risk modification for lung cancer with diet in Japanese, we conducted a hospital-based case–control study and evaluated variation in influence with the histological type. We recruited 367 male and 240 female cases with adenocarcinomas, and 381 male and 57 female cases with squamous cell and small cell carcinomas. Controls comprised 2964 male and 1189 female cancer-free outpatients matched for sex and age with the cases. Odds ratios (ORs) and their 95% confidence intervals (CIs) for lung cancer were calculated with adjustment for potential confounding factors, using an unconditional logistic model. We found decreased ORs for adenocarcinomas in both males (OR = 0.51, 95% CI 0.31–0.84) and females (OR = 0.48, 95% CI 0.24–0.94) who consumed cooked/raw fish, but not dried/salted fish at the highest quartile frequency, compared with the lowest. Soybean curd consumption was associated with a decreased OR for female adenocarcinomas. Decreased ORs for squamous cell and small cell carcinomas were observed in males with frequent consumption of raw and green vegetables, fruit and milk, but consumption of carrot, pumpkin, egg and coffee was associated with increased ORs. This study suggests cooked/raw fish consumption lowers the risk of adenocarcinoma of the lung in Japanese.

Plasma vitamin D and risk of colorectal cancer: the Japan Public Health Center-Based Prospective Study

We investigated the association between plasma 25(OH)D and the subsequent colorectal cancer incidence risk by a nested case–control study in The Japan Public Health Center-based Prospective Study, covering 375 newly diagnosed cases of colorectal cancer from 38 373 study subjects during a 11.5-year follow-up after blood collection. Two controls were matched per case on sex, age, study area, date of blood draw, and fasting time. In a conditional logistic regression model with matched pairs adjusted for smoking, alcohol consumption, body mass index, physical exercise, vitamin supplement use, and family history of colorectal cancer, plasma 25(OH)D was not significantly associated with colorectal cancer in men or in women. However, the lowest category of plasma 25(OH)D was associated with an elevated risk of rectal cancer in both men (odds ratio (OR), 4.6; 95% confidence interval (CI), 1.0–20) and women (OR, 2.7, 95% CI, 0.94–7.6), compared with the combined category of the other quartiles. Our results suggest that a low level of plasma 25(OH)D may increase the risk of rectal cancer.

Lung Cancer Patients Have Increased 8-Hydroxydeoxyguanosine Levels in Peripheral Lung Tissue DNA

The 8‐hydroxydeoxyguanosine (8‐OH‐dG) levels in the peripheral parts of human lung tissues were compared between lung cancer patients (n=70) and non‐cancer patient controls (n=15). An increased level of 8‐OH‐dG was observed in the lung cancer group, in both the adenocarcinoma and non‐adenocarcinoma (mainly squamous cell carcinoma) groups, as compared to the non‐cancer control group. This result suggests that reactive oxygen species are partly involved in the induction of lung carcinomas (both adenocarcinoma and non‐adenocarcinoma).

Fas expression in non-small cell lung cancer: its prognostic effect in completely resected stage III patients

The aim of this study was to examine Fas expression in non-small cell lung cancer (NSCLC) and examine its correlation with clinicopathological features and prognosis. Fas expression was determined by an immunohistochemical analysis using the labelled streptavidin-biotin method from 220 paraffin specimens of completely resected primary stage I-III NSCLC. 80 (36%) of 220 cases were positive for Fas immunostaining. These 80 cases included 44 adenocarcinomas (33%) and 30 squamous cell carcinomas (40%). 33 stage I (33%) 13 (43%) stage II and 34 (37%) stage III tumours were Fas positive. No statistically significant differences were observed regarding the Fas status with respect to age, sex, histological type, or stage of disease. There was no significant difference in survival between early stage (stages I-II) disease patients with positive Fas expression and those with a negative expression (P = 0.719). However, for patients with completely resected stage III tumours, the patients with positive Fas staining were found to survive for a longer period than those with negative staining (P = 0.026).

Association between body mass index and the colorectal cancer risk in Japan: pooled analysis of population-based cohort studies in Japan

BACKGROUND Obesity has been recognized as important risk factors for colorectal cancer. However, limited evidence is available on colorectal cancer and body mass index (BMI) in Asian population. METHODS We conducted a pooled analysis of eight population-based prospective cohorts studies in Japan with more than 300,000 subjects to evaluate an impact of obesity in terms of BMI on colorectal cancer risk with unified categories. We estimated summary hazard ratio (HR) by pooling of study-specific HR for BMI categories with random effect model. RESULTS We found a significant positive association between BMI and colorectal cancer risk in male and female. Adjusted HRs for 1 kg/m(2) increase were 1.03 [95% confidence interval (CI) 1.02-1.04] for males and 1.02 (95% CI 1.00-1.03) for females. The association was stronger in colon, especially in proximal colon, relative to rectum. Males showed a stronger association than females. Population attributable fraction for colorectal cancer by BMI ≥ 25 kg/m(2) was 3.62% (95% CI 1.91-5.30) for males and 2.62% (95% CI 0.74-4.47) for females. CONCLUSIONS We found significant association between BMI and colorectal cancer risk by pooling of data from cohort studies with considerable number of subjects among Japanese population. This information is important in cancer control planning, especially in Asian population.

Comparison of lung cancer cell lines representing four histopathological subtypes with gene expression profiling using quantitative real-time PCR

BackgroundLung cancers are the most common type of human malignancy and are intractable. Lung cancers are generally classified into four histopathological subtypes: adenocarcinoma (AD), squamous cell carcinoma (SQ), large cell carcinoma (LC), and small cell carcinoma (SC). Molecular biological characterization of these subtypes has been performed mainly using DNA microarrays. In this study, we compared the gene expression profiles of these four subtypes using twelve human lung cancer cell lines and the more reliable quantitative real-time PCR (qPCR).ResultsWe selected 100 genes from public DNA microarray data and examined them by DNA microarray analysis in eight test cell lines (A549, ABC-1, EBC-1, LK-2, LU65, LU99, STC 1, RERF-LC-MA) and a normal control lung cell line (MRC-9). From this, we extracted 19 candidate genes. We quantified the expression of the 19 genes and a housekeeping gene, GAPDH, with qPCR, using the same eight cell lines plus four additional validation lung cancer cell lines (RERF-LC-MS, LC-1/sq, 86-2, and MS-1-L). Finally, we characterized the four subtypes of lung cancer cell lines using principal component analysis (PCA) of gene expression profiling for 12 of the 19 genes (AMY2A, CDH1, FOXG1, IGSF3, ISL1, MALL, PLAU, RAB25, S100P, SLCO4A1, STMN1, and TGM2). The combined PCA and gene pathway analyses suggested that these genes were related to cell adhesion, growth, and invasion. S100P in AD cells and CDH1 in AD and SQ cells were identified as candidate markers of these lung cancer subtypes based on their upregulation and the results of PCA analysis. Immunohistochemistry for S100P and RAB25 was closely correlated to gene expression.ConclusionsThese results show that the four subtypes, represented by 12 lung cancer cell lines, were well characterized using qPCR and PCA for the 12 genes examined. Certain genes, in particular S100P and CDH1, may be especially important for distinguishing the different subtypes. Our results confirm that qPCR and PCA analysis provide a useful tool for characterizing cancer cell subtypes, and we discuss the possible clinical applications of this approach.

Successful Induction of Tumor‐specific Cytotoxic T Lymphocytes from Patients with Non‐small Cell Lung Cancer Using CD80‐transfected Autologous Tumor Cells

Cytotoxic T lymphocytes (CTL) against human lung cancer cells are difficult to induce by a conventional method using tumor cell stimulation probably due to an insufficiency of tumor antigens (TA) or costimulatory molecules such as CD80. We, therefore, investigated the potential of CD80‐transfected tumor cells as stimulators of the in vitro induction of autologous tumor‐specific CTL from regional lymph node lymphocytes in patients with lung cancer. Five non‐small cell lung cancer cell lines (two adenocarcinomas, 1 squamous cell carcinoma, 1 large cell carcinoma and 1 adenosquamous cell carcinoma) were established from surgical specimens and were successfully transduced with a plasmid constructed with expression vector pBj and human CD80 cDNA, using a lipofection method. CD80‐transfected tumor cells (CD80‐AT) significantly augmented the proliferation of autologous lymphocytes from all cases as compared with non‐transfected tumor cells (AT). AT‐stimulated lymphocytes from 4 out of 5 cases did not show any cytotoxicity against AT; however, lymphocytes stimulated with CD80‐AT exhibited substantial cytotoxicity against parental AT in all 5 cases tested. AT‐stimulated lymphocytes derived from only one out of 5 cases showed major histocompatibility complex (MHC)‐class I‐restricted cytokine production in response to AT, while the MHC‐class I‐restricted responses were found in CD80‐AT‐stimulated lymphocytes from 4 out of 5 cases. These results indicate that CD80 on tumor cells could be a beneficial costimulatory molecule to elicit CTL against lung cancer, and also show that TA recognized by CTL was frequently expressed on lung cancer cells.

Molecular biological markers and micrometastasis in resected non-small-cell lung cancer

OBJECTIVES Recent advances in molecular biology and genetics have created new diagnostic and treatment possibilities in clinical oncology. We evaluated the usefulness of molecular biological factors in primary tumor and micrometastasis in the bone marrow and pathological negative (pN0) lymph nodes as prognostic parameters in non-small-cell lung cancer (NSCLC) patients. METHODS Pathological specimens were collected from 129 NSCLC patients to analyze molecular biological markers, including K-ras, p53, Rb, p16, loss of heterozygosity (LOH) at 3p, vascular endothelial growth factor (VEGF), and telomerase activity. Bone marrow samples from 250 NSCLC patients and pN0 lymph nodes from 85 of these patients were collected for micrometastasis detection by immunohistochemistry against cytokeratin. RESULTS p53 abnormalities and 3p LOH were significantly associated with reduced patient survival in adenocarcinoma, whereas VEGF expression was significantly associated with reduced survival in a squamous cell carcinoma histological subtype by univariate or multivariate analysis. We identified micrometastatic tumor cells in bone marrow of 78 (31.2%) of 250 patients and in pN0 lymph nodes of 26 (30.6%) of 85 patients. Both bone marrow and lymph nodal micrometastases were associated with decreased survival among patients with stage I, however, only lymph nodal micrometastasis had a significant impact on survival. CONCLUSIONS Molecular biological features of primary tumor and micrometastatic status appear useful in defining groups of patients with a poor prognosis who could benefit from adjuvant systemic treatment.