| Background and Objective Lung cancer continues to be the most common fatal malignancy of men and women. A number of biological and predictive markers of lung cancer have been selected recently. Tumor suppressor gene INK4a/ARF is located on the short arm of chromosome 9p and encodes two proteins, p16INK4a and p14ARF, through the use of independent first exon and shared exon2 and 3. p16INK4a is a cyclin-dependent kinase inhibitor, whereas p14ARF regulates the cell cycle through a p53 and MDM2-dependent pathway. Aberrant expression of p14ARF or p16INK4a protein has been reported in various malignancies. But, their prognostic significance in non-small cell lung cancer (NSCLC) patients remains unclear. The first purpose of this study is to investigate the influence of co-expression states of p14ARF and p16INK4a protein on length of survival period in patients with NSCLC after resection. Secondly, we also pay much attention to the relationship between INK4a/ARF gene inactivation and 5'CpG islands methylation. Methods Sections were obtained from previous 10%-formalin-fixed and paraffin-embedded tissues, including 35 NSCLC samples with p14ARF and p16INK4a negative co-expression and 20 NSCLC samples with p14ARF and p16INK4a positive co-expression. These samples were divided into two groups, named (p14+p16)-group and (p14+p16)+ group respectively. The several clinical and pathological parameters between two groups were analyzed by the χ2 test. The Kaplan-meier method was used to estimate the probability of survival states. The difference of survival rate between two groups was analyzed by log-rank test. Multivariate analysis was performed by Cox regression model with the SPSS11.5 statistical package. 5'CpG islands methylation states of tumor suppressor gene INK4a/ARF was determined by methylation specific polymerase chain reaction (MSP). The values were considered significant at p<0.05. Results 1. The median survival duration, 1-year, 2-year, 3-year and overall survival rate of (p14+p16)-group was 21 months, 72.24%, 42.37%, 22.60% and 38.46%, respectively. Whereas the median survival duration, 1-year, 2-year, 3-year and overall survival rate of (p14+p16)+ group was 42 months, 88.89%, 75.21%, 65.81% and 61.11%, respectively. The survival markers of (p14+p16)-group were significantly worse than that of (p14+p16)+ group (log-rank test, p<0.05). A Cox regression analysis was performed to evaluate prognostic factors for NSCLC, p14ARF and p16INK4a negative co-expression was found to be a significantly adverse factor for the prognosis of NSCLC. 2. The hypermethylation rate of INK4a gene was 51.4%(18/35) in (p14+p16)-group, but 10%(2/20) in (p14+p16)+ group. There was remarkable significance between two groups(p=0.002). 3. The hypermethylation frequency of ARF gene was 22.8%(8/35) in (p14+p16)-group, and 10%(2/20) in (p14+p16)+ group. There was no significance between two groups(p=0.409). 4. Five cases (5/35) showed ARF and INK4a co-hypermethylation in (p14+p16)-group. Three cases (3/35) showed hypermethylation only in ARF gene. Thirteen cases (13/35) showed hypermethylation only in INK4a gene. Besides, hypermethylation of ARF and INK4a gene was not found in 14 cases (14/35). There was no obviously correlation between INK4a gene hypermethylation and ARF gene hypermethylation. However, the hypermethylation of ARF gene was sometimes (5/8) accompanied by the hypermethylation of INK4a gene. Conclusions 1. The 1-year, 2-year, 3-year survival rate and overall survival rate of patients in (p14+p16)+ group was significantly higher than that of patients in (p14+p16)-group, which showed co-expression states of INK4a/ARF gene was intimately relation with the prognosis of NSCLC patients. Patients with negative expression of p14ARF and p16INK4a protein always have a worse prognosis. 2. 5'CpG promoter regions hypermethylation frequency of INK4a gene in (p14+p16)-group was higher than that of (p14+p16)+group, which suggests 5'CpG promoter regions hypermethylation of INK4a gene is one of the important mechanisms that result in p16INK4a proteins negative expression. 3. 5'CpG promoter regions hypermethylation of ARF gene was sometimes accompanied by the hypermethylation of INK4a gene. Howerer, the hypermethylation of ARF and INK4a genes are independent each other.
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