Ryusei Saito

Association of amino acid substitution polymorphisms in DNA repair genes TP53, POLI, REV1 and LIG4 with lung cancer risk.

Single nucleotide polymorphisms (SNPs) were searched for in 36 genes involved in diverse DNA repair pathways, and 50 nonsynonymous (associated with amino acid changes) SNPs identified were assessed for associations with lung cancer risk by a case‐control study consisting of 752 adenocarcinoma cases, 250 squamous cell carcinoma cases and 685 controls. An SNP, Arg72Pro, of the TP53 gene encoding a DNA damage response protein showed the strongest association with squamous cell carcinoma risk (OR Pro/Pro vs. Arg/Arg = 2.2), while 2 other SNPs, Phe257Ser of the REV gene encoding a translesion DNA polymerase and Ile658Val of the LIG4 gene encoding a DNA double‐strand break repair protein, also showed associations (OR Ser/Ser vs. Phe/Phe = 2.0 and OR Ile/Val vs. Ile/Ile = 0.4, respectively). An SNP, Thr706Ala, in the POLI gene encoding another translesion DNA polymerase was associated with adenocarcinoma and squamous cell carcinoma risk, particularly in individuals of ages < 61 years (OR Ala/Ala + Ala/Thr vs. Thr/Thr = 1.5 and 2.4, respectively). POLI is the human counterpart of PolI, a strong candidate for the Par2 (pulmonary adenoma resistance 2) gene responsible for adenoma/adenocarcinoma susceptibility in mice. The present results suggest that these 4 SNPs function as genetic factors underlying lung cancer susceptibility by modulating activities to maintain the genome integrity of each individual.

Performance Status and Smoking Status Are Independent Favorable Prognostic Factors for Survival in Non-small Cell Lung Cancer: A Comprehensive Analysis of 26,957 Patients with NSCLC

Background: Performance status (PS) is an important factor in determining survival outcome in non-small cell lung cancer (NSCLC) but is generally confounded by stage, age, gender, and smoking status. We investigated the prognostic significance of PS taking into account these important factors. Methods: Retrospective analysis of registry database of the National Hospital Study Group for Lung Cancer (NHSGLC) between1990 and 2005. Univariate analysis was performed using Kaplan-Meier method. Multivariate analysis was performed using Cox proportional hazards model to identify independent prognostic factors. Results: A total of 26,957 patients with NSCLC were analyzed of which 12,613 patients (46.8%) had World Health Organization (WHO) PS = 0, 8,137 patients were never smokers (30.2%), and most of them were females (72.7%). The majority of PS = 0 patients presented with stage I disease (56.9%). Patients with PS = 0 constituted the group with the highest proportion of never smokers (36.7%). There was a significant difference in the median overall survival (OS) between patients with PS = 0 and PS = 1 (51.5 months versus 15.4 months, respectively; p < 0.0001) and among patients with various PS within individual American Joint Committee on Cancer stage (all p values <0.0001). Never smokers had significantly improved median OS than ever smokers (30.0 months versus 19.0 months, respectively; p < 0.0001). Multivariate analysis demonstrated good PS, never smoker (versus ever smoker; hazard ratio = 0.935, 95% confidence interval: 0.884–0.990; p = 0.0205), early stage, female gender, squamous cell carcinoma histology, and treatment were all as independent favorable prognostic factors. Conclusions: PS and smoking status are independent prognostic factors for OS in NSCLC.

Constitutive activation of the Wnt signaling pathway byCTNNB1 (?-catenin) mutations in a subset of human lung adenocarcinoma

Constitutive activation of the Wnt signaling pathway as a result of genetic alterations of APC, AXIN1, and CTNNB1 has been found in various human cancers, including those of the colon, liver, endometrium, ovary, prostate, and stomach. To investigate the pathogenetic significance of constitutive activation of the Wnt signaling pathway in human lung carcinogenesis, CTNNB1 alterations in exon 3, a region known to represent a mutation hot spot, were screened in 46 lung cancer cell lines and 47 primary lung cancers. Missense mutations causing substitutions of Ser/Thr residues critical for regulation by GSK‐3β were detected in one (2%) of the cell lines, A427, and two (4%) of the surgical specimens. The three lung cancers with CTNNB1 mutations were adenocarcinomas. To explore the prevalence of constitutive activation of the Wnt signaling pathway in human lung cancer, we assessed 15 lung cancer cell lines representing major histological subtypes of lung cancers for constitutive Tcf transcriptional activity (CTTA). CTTA was observed only in the A427 adenocarcinoma cell line, but not in the remaining 14 cell lines. The data indicate that constitutive activation of the Wnt signaling pathway caused by CTNNB1 mutation is involved in the development and/or progression of a subset of lung carcinoma, preferentially in adenocarcinoma.

Correlation between the status of the p53 gene and survival in patients with stage I non-small cell lung carcinoma.

The association of p53 abnormalities with the prognosis of patients with non-small cell lung carcinoma (NSCLC) has been extensively investigated to date, however, this association is still controversial. Therefore, we investigated the prognostic significance of p53 mutations through exons 2 to 11 and p53 protein expression in 103 cases of stage I NSCLC. p53 mutations were detected in 49 of 103 (48%) tumors. Two separate mutations were detected in four tumors giving a total of 53 unique mutations in 49 tumors. Ten (19%) of mutations occurred outside exons 5 – 8. Positive immunohistochemical staining of p53 protein was detected in 41 of 103 (40%) tumors. The concordance rate between mutations and protein overexpression was only 69%. p53 mutations, but not expression, were significantly associated with a shortened survival of patients (P<0.001). Furthermore, we investigated the correlation between the types of p53 mutations and prognosis. p53 missense mutations rather than null mutations were associated with poor prognosis (P<0.001 in missense mutations and P=0.243 in null mutations). These results indicated that p53 mutations, in particular missense mutations, rather than p53 expression could be a useful molecular marker for the prognosis of patients with surgically resected stage I NSCLC.

Association of the OGG1-Ser326Cys polymorphism with lung adenocarcinoma risk.

Adenocarcinoma (ADC) is the most frequent histological type of lung cancer and comprises the majority of lung cancers in non‐smokers. Thus, genetic factors responsible for ADC susceptibility need to be determined to establish efficient ways of preventing the disease. The OGG1 gene, encoding a glycosylase for 8‐hydroxyguanine, an oxidatively damaged promutagenic base, has the polymorphism Ser326Cys, and OGG1‐326Cys protein was indicated to have a lower ability to prevent mutagenesis than the OGG1‐326Ser protein. Case‐control studies to date suggest that the OGG1‐326Cys allele is associated with a higher risk for several types of cancers, including overall lung cancer. However, the contribution of this polymorphism to lung ADC risk is unclear. In the present study, the OGG1‐Ser326Cys polymorphism was assessed for association with lung ADC risk using a case‐control study of a Japanese population consisting of 1097 cases and 394 controls. Odds ratios (OR) of the 326Cys allele carriers increased in a dose‐dependent manner with allele number (P for the trend test = 0.04). The OR of homozygotes for the 326Cys allele was increased significantly when homozygotes for the 326Ser allele were used as a reference (OR = 1.5, 95% confidence interval [CI] = 1.0–2.1, P = 0.04). Furthermore, the overall OR for lung ADC of the Cys/Cys homozygotes out of a total of 1925 ADC patients and 3449 controls from six case‐control studies reported up to the present were 1.43 (95% CI = 1.11–1.84, P = 0.0045). These results indicate that OGG1‐326Cys functions as a risk allele for lung ADC development. (Cancer Sci 2006; 97: 724–728)

Gender, Histology, and Time of Diagnosis Are Important Factors for Prognosis: Analysis of 1499 Never-Smokers with Advanced Non-small Cell Lung Cancer in Japan

Background: There has been a growing interest in lung cancer in never-smokers. Methods: Utilizing a database from the National Hospital Study Group for Lung Cancer, information for never-smokers and ever-smokers with advanced non-small cell lung cancer was obtained from 1990 to 2005, including clinicopathologic characteristics, chemotherapy response, and survival data. Time of diagnosis was classified into two periods: 1990–1999 and 2000–2005. Multivariate analysis was performed using the Cox regression and logistic regression method, including gender, age, performance status, histology, stage, and period of diagnosis. Results: There were 1499 never-smokers and 3455 ever-smokers with advanced stage IIIB and IV diseases who received cytotoxic chemotherapy. Never-smokers generally included more females, were younger, with better performance status and more adenocarcinoma diagnosed (p < 0.0001 for all). Smoking status was a significant prognostic factor (never-smoker versus ever-smoker; hazard ratio [HR] = 0.880, 95% confidence interval [CI]: 0.797–0.970; p = 0.0105). In separate multivariate analysis for never-smokers and ever-smokers, female gender and better performance status (p < 0.0001 for both) were both favorable prognostic factors. However, adenocarcinoma histology (versus squamous cell carcinoma; HR = 0.790, 95% CI: 0.630–0.990; p = 0.0403) and the period after 2000 (versus before 2000; HR = 0.846, 95% CI: 0.731–0.980; p = 0.0254) were significant only in the never-smokers, and younger age (HR = 1.007, 95% CI: 1.003–1.011; p = 0.0010) was significant only in the ever-smokers. In an exploratory analysis, different profiles were observed in predictive factors for chemotherapy response between the two groups. Conclusions: Never-smokers with non-small cell lung cancer lived longer than ever-smokers. Gender, histology, and time of diagnosis are important factors for prognosis in these patients.

Over-expression of p27kip1 induces growth arrest and apoptosis mediated by changes of pRb expression in lung cancer cell lines

p27kip1 is a cyclin‐dependent kinase inhibitor which controls the G1 phase of the cell cycle in conjunction with pRb. p27 has been associated with cell‐cycle arrest and apoptosis. In this study, we transferred the full‐length human p27 cDNA using a replication‐deficient recombinant adenoviral vector (Ax‐p27) into lung cancer cell lines and evaluated the potential of this strategy for anti‐cancer gene therapy. After infection with Ax‐p27, the growth of H322, A549 and SQ‐5 cells, which express pRb, was almost completely suppressed, though no such effect was found in H69 and Lu‐135 cells, which do not express pRb. In addition, cell death from day 4 after infection with Ax‐p27 was observed only in H322, A549 and SQ‐5 cells but not in H69 and Lu‐135 cells. The cell cycle of H322 cells treated with Ax‐p27 became arrested at the G1 phase from day 1 to day 3 despite continued over‐expression of p27. When we examined the changes in expression level of pRb and E2F‐1, which play important roles in cell‐cycle progression from G1 to S phase, down‐regulation of pRb expression was detected in H322 cells 3 days after infection with Ax‐p27. These data suggest that (i) the growth‐inhibitory effect and induction of apoptosis by over‐expression of p27 require expression of pRb and (ii) adenovirus‐mediated p27 gene transfer may have promise as a novel strategy in cancer gene therapy. Int. J. Cancer 88:377–383, 2000.

Serum Pro-Gastrin-Releasing Peptide Is a Useful Marker for Treatment Monitoring and Survival in Small-Cell Lung Cancer

We investigated the usefulness of serum pro-gastrin-releasing peptide (Pro-GRP) as a tumor marker for diagnosis, treatment monitoring and the prediction of relapse and prognosis in patients with small-cell lung cancer (SCLC). Serum samples were obtained from 127 patients with primary lung cancer (48 patients with small-cell carcinoma, 31 with adenocarcinoma, 36 with squamous cell carcinoma and 11 with large-cell carcinoma). The cutoff levels of serum Pro-GRP and neuron-specific enolase (NSE) were set at 46 pg/ml and 10 ng/ml, respectively. The specificity of Pro-GRP was significantly higher than that of NSE (Pro-GRP: 93.7%, NSE: 65.8%, p < 0.01). According to the histological type of lung cancer, the positive rates of Pro-GRP were 75% (36/48) in the small-cell carcinomas, 9.7% (3/31) in the adenocarcinomas, 5.6% (2/36) in the squamous cell carcinomas and 0% (0/10) in the large cell carcinomas. The median levels of Pro-GRP in limited disease (LD) and extensive disease (ED) patients were 199 and 295.5 pg/ml, whereas those of NSE were 14.8 and 29.3 ng/ml, respectively. The positive rates of Pro-GRP in LD and ED patients were 80.0% (16/20) and 71.4% (20/28), whereas those of NSE were 70.0% (14/20) and 89.3% (25/28), respectively. The positive rate of NSE tended to elevate with the progression of disease, whereas that of Pro-GRP was already high at an early stage. Among the 29 patients with SCLC who could be followed, the serum Pro-GRP levels of 18 responders were significantly decreased after treatment (p < 0.01), whereas those of the 11 nonresponders were not significantly different between before and after treatment (p = 0.72). In the 9 patients with SCLC who relapsed, the serum Pro-GRP levels were again elevated at the time of relapse. Seventeen patients whose ratio of the Pro-GRP level after treatment to the level before treatment was below 50% (taking the levels before treatment as 100%) survived significantly longer than did the patients whose ratio was over 50% (p < 0.01). The results of the present study suggest that serum Pro-GRP has high specificity and could be a useful marker of SCLC for treatment monitoring and prognosis.

Individuals susceptible to lung adenocarcinoma defined by combined HLA-DQA1 and TERT genotypes.

Adenocarcinoma (ADC) is the commonest histological type of lung cancer, and its weak association with smoking indicates the necessity to identify high-risk individuals for targeted screening and/or prevention. By a genome-wide association study (GWAS), we identified an association of polymorphisms in the 6p21.31 locus containing four human leukocyte antigen (HLA) class II genes with lung ADC risk. DQA1*03 of the HLA-DQA1 gene was defined as a risk allele with odds ratio (OR) of 1.36 [95% confidence interval (CI) = 1.21-1.54, P = 5.3 x 10(-7)] by analysis of 1656 ADC cases and 1173 controls. DQA1*03 and the minor allele for a polymorphism, rs2736100, in TERT, another lung cancer susceptibility locus identified in recent GWASs on Europeans and Americans, were indicated to independently contribute to ADC risk with per allele OR of 1.43 (95% CI = 1.31-1.56, P = 7.8 x 10(-16)). Individuals homozygous both for the DQA1*03 and minor TERT alleles were defined as high-risk individuals with an OR of 4.76 (95% CI = 2.53-9.47, P = 4.2 x 10(-7)). The present results indicated that individuals susceptible to lung ADC can be defined by combined genotypes of HLA-DQA1 and TERT.

A phase II study of amrubicin, a synthetic 9-aminoanthracycline, in patients with previously treated lung cancer.

PURPOSE This study was designed to confirm the efficacy and safety of amrubicin, a new anthracycline agent, in patients with previously treated non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). METHODS Eligible patients were required to have recurrent or refractory NSCLC and SCLC after one or two previous chemotherapy regimens. All patients received intravenous amrubicin 35 mg/m(2) on days 1-3 every 3 weeks. Overall response rate (ORR), progression-free survival (PFS), and overall survival (OS) were evaluated. RESULTS Sixty-six patients (37 NSCLC and 29 SCLC) were assessable for efficacy and safety evaluation. Grade 3 or 4 neutropenia was observed in 39.4% of all patients (NSCLC, 37.8%; SCLC, 41.4%). Nonhematological toxicities were mild. No treatment-related death was observed. The ORRs were 13.5% (95% CI, 4.5-28.8%) in NSCLC and 44.8% (95% CI, 26.4-64.3%) in SCLC. In SCLC, ORRs were 60.0% in the sensitive relapse and 36.8% in the refractory relapse (p=0.2332). In NSCLC, the PFS, OS, and 1-year survival were 3.3 months, 12.0 months, and 35.3%, respectively. In SCLC, the PFS, OS, and 1-year survival were 4.0 months, 12.0 months, and 46.7%, respectively. CONCLUSIONS Amrubicin is an active and well-tolerated regimen in patients with previously treated lung cancer. Amrubicin 35 mg/m(2) seems to achieve similar efficacy with less toxicity than amrubicin 40 mg/m(2) in this patient population. These results warrant further evaluation in previously treated lung cancer.