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[Cancer Research 66, 4049-4054, April 15, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Cohypermethylation of p16 and FHIT Promoters as a Prognostic Factor of Recurrence in Surgically Resected Stage I Non–Small Cell Lung Cancer

Jin Seuk Kim1, Jin Wook Kim2, Joungho Han3, Young Mog Shim4, Joobae Park1,5 and Duk-Hwan Kim1,5

1 Center for Genome Research, Samsung Biomedical Research Institute; 2 Seoul Science High School; Departments of 3 Pathology and 4 Thoracic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul; and 5 Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea

Requests for reprints: Duk-Hwan Kim, Center for Genome Research, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Room B155, 50 Ilwon-dong, Kangnam-Ku, Seoul 135-710, Republic of Korea. Phone: 82-23410-3632; Fax: 82-23410-3649; E-mail: dukhwan.kim{at}samsung.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Despite advances in the detection and treatment of lung cancer, the prognosis for patients with lung cancer is poor, partly as a result of recurrences. We retrospectively analyzed the relationship between recurrence and survival in patients with non–small cell lung cancers (NSCLC), and the promoter methylation of p16, GSTP1, FHIT, H-cadherin, and RARß2 genes to identify a prognostic molecular marker associated with the recurrence of NSCLC. Methylation status from 335 paraffin blocks was determined by methylation-specific PCR. Of the 335 NSCLC samples, promoter methylation was detected in 35% for p16, 39% for RARß2, 42% for H-cadherin, 7% for GSTP1, and 21% for FHIT. Recurrence was observed in 39% (132 of 335) of the patients. Recurrence was significantly associated with histology (P = 0.001) and pathologic stage (P = 0.009). Hypermethylation of any single gene was not associated with recurrence in patients. However, cohypermethylation of p16 and FHIT genes in stage I NSCLCs was associated with an increased risk of recurrence [odds ratio, 6.43; 95% confidence interval (CI), 1.04-20.19; P = 0.02] and poor recurrence-free survival after surgery (hazard ratio, 2.03; 95% CI, 1.09-6.23; P = 0.02). In addition, their survival after recurrence was also 4.62 times poorer (95% CI, 1.27-16.48; P = 0.005) than for those without cohypermethylation of both genes. In conclusion, the present study suggests that cohypermethylation of p16 and FHIT genes in patients with stage I NSCLC may be a valuable biomarker for predicting the recurrence-associated prognosis of the disease. (Cancer Res 2006; 66(8): 4049-54)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lung cancer is the most frequent cause of cancer-related death in the world, causing more than one million deaths worldwide each year. Despite advances in the detection and treatment of lung cancer, the overall 5-year survival rate remains <15%. The poor prognosis of patients with lung cancer is partly a result of recurrence, which appears in ~20% to 50% of patients after curative surgical resection with appropriate lymph node dissection (1–3). Thus, the development of biomarkers for predicting recurrence and the implementation of efficient preventive methods to prevent recurrence in these patients is clearly imperative.

The aberrant methylation of normally unmethylated CpG islands is an epigenetic change that induces the transcriptional silencing of tumor suppressor genes. The hypermethylation of CpG islands at the promoter region of >40 tumor suppressor genes has been reported in lung cancer. Among those genes, p16, retinoic acid receptor ß (RARß), H-cadherin (CDH13), and fragile histidine triad (FHIT) genes are important in the pathogenesis of lung cancer and are frequently inactivated by aberrant methylation of CpG islands at their promoter regions (4). Accordingly, epigenetic modification may be a candidate marker for predicting the prognosis of non–small cell lung cancers (NSCLC). In addition, patients with epigenetic modifications could benefit from treatment with demethylating agents after surgery.

Although clinical and histopathologic factors that might assist in the prediction of tumor recurrence after curative resection of NSCLCs have been studied by many groups, epigenetic alterations associated with recurrence after curative resection has been reported by a few groups. Kim et al. (3) studied 61 patients with NSCLC and reported that P2 hypermethylation of RARB2 and unmethylation of DAPK could be used as prognostic markers in predicting the early recurrence of NSCLC. To identify a useful prognostic biomarker for disease recurrence after a curative resection of NSCLC, we investigated the relationship between recurrence and the aberrant methylation of p16, RARß2, H-cadherin (CDH13), GSTP1, and FHIT genes in a large sample.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study population. A total of 335 NSCLC patients, who underwent definitive surgical resection at the Samsung Medical Center in Seoul, Korea, between May 1994 and December 2001, participated in this study. Written informed consent for the use of paraffin-embedded tissues, as approved by the Institutional Review Board at Samsung Medical Center, was obtained from each patient before surgery. All available paraffin blocks were reviewed by a thoracic pathologist (J. Han). Information on sociodemographic characteristics was obtained using an interviewer-administered questionnaire. Postoperative follow-up was scheduled at 1, 2, and every 3 months during the first 2 years after surgery, and every 6 months thereafter, or more frequently if needed. Chest X-ray, chest computed tomography scan, carcinoembryonic antigen, and other serum chemistries were scheduled at every follow-up visit. Whenever patients did not keep the postoperative follow-up schedule, specialized nurses called patients and checked their health status. The median duration of follow-up after a curative resection was 3.7 years.

Recurrence was evaluated from information obtained as of June 30, 2005 from our hospital records and those from other hospitals. The data of patients who died of any causes, whose cancers did not recur before the end of the study, or who were lost to follow-up during the study period, were treated as censored data when calculating recurrence. Second primary tumors were carefully differentiated from recurrence in this study. Recurrence patterns were classified into two categories, locoregional and distant. We defined a locoregional recurrence as evidence of a tumor in the supraclavicular nodes, mediastinal nodes, pleural effusion or seeding, bronchial stump, and other lobes of the ipsilateral lung, whereas distant recurrence was defined as metastasis to the contralateral lung, brain, bone, liver, adrenal, and other organs. Simultaneous locoregional and distant recurrence was considered with the distant recurrence group. Of the 132 patients who had a recurrence, 79 (60%) had undergone radiotherapy, 38 (29%) had received chemotherapy, and 15 (11%) had received surgery.

The 335 patients consisted of 234 men (70%) and 101 women (30%), ranging in age from 15 to 89 years. The mean age at the time of the surgical resection for the first primary NSCLC was 59.8 years. One hundred and ninety-eight of 335 patients with primary NSCLCs had stage I disease, 84 patients had stage II disease, 49 patients had stage III disease, and 4 patients had stage IV disease. The histologic distribution of the first primary cancers at the time of the surgical resection was 36% adenocarcinomas, 53% squamous cell carcinomas, and 11% other cell types.

DNA extraction from paraffin block. Formalin-fixed, paraffin wax–embedded blocks containing at least 75% neoplastic tissues were cut into 10-µm-thick tissue sections. Serial tissue sections from each paraffin block were placed on slides prior to DNA extraction and stained with H&E to evaluate the admixture of tumorous/nontumorous tissues. Areas that corresponded to tumor were carefully microdissected from the surrounding normal stromal tissues, collected in 15 mL centrifuge tubes, and deparaffinized overnight at 63°C in xylene, and then vortexed vigorously. After centrifugation at full speed for 5 minutes, supernatants were removed, ethanol was added to remove residual xylene, and then removed by centrifugation. After ethanol evaporation, tissue pellets were resuspended in lysis buffer ATL (DNeasy Tissue Kit, Qiagen, Valencia, CA) and the genomic DNA was isolated using a DNeasy Tissue kit according to the manufacturer's instruction.

Methylation-specific PCR. The methylation status of the promoter region of the p16, RARß2, GSTP1, H-cadherin, and FHIT genes was determined by methylation-specific PCR, as described by Herman et al. (Fig. 1 ; ref. 5). Two sets of primers were designed for each gene, one specific for DNA methylated at the promoter region and the other specific for unmethylated DNA. Primer sequences and annealing temperatures for the methylation-specific PCR were previously published by our group and others (5–7). Briefly, 1 µg of genomic DNA was denatured by incubation with 0.2 mol/L NaOH for 10 minutes at 37°C. Aliquots of 3 mol/L sodium bisulfite (pH 5.0) and 10 mmol/L of hydroquinone (both from Sigma Chemical, Co., St. Louis, MO) were then added, and the solution was incubated at 50°C for 16 hours. The modified DNA was purified by use of a Wizard DNA purification system (Promega Corp., Madison, WI), followed by ethanol precipitation. Modified DNA was stored in aliquots at –20°C until required.


Figure 1
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  		Figure 1. Methylation analysis of the p16, RARß2, H-cadherin, GSTP1, and FHIT genes in NSCLCs. Methylation-specific PCR for the p16, RARß2, H-cadherin, GSTP1, and FHIT genes were done using unmethylation-specific (U) and methylation-specific (M) primer sets. Twenty microliters of PCR product was run on 2% metaphore agarose gel, stained with ethidium bromide, and visualized under UV illumination. Patient identification numbers indicated (top). DNA from normal lymphocytes served as a negative controls for methylated alleles. Negative control samples without DNA were included for each PCR.

 
The PCR mixture contained 1x PCR buffer [50 mmol/L KCl, 67 mmol/L Tris (pH 8.7), 1.5 mmol/L MgCl2], deoxynucleotide triphosphates (1.25 mmol/L each), primers (300 ng each per reaction), 2.5 units of Taq polymerase, and bisulfite-modified DNA (50 ng). Reactions were hot-started at 94°C before adding 2.5 units of Taq polymerase. Amplification was carried out over 35 cycles (1 minute at 94°C, 1 minute at the annealing temperature, and 1 minute at 72°C), followed by 4 minutes at 72°C. Fifteen microliters of the PCR reaction were loaded on 2% agarose gel, stained with ethidium bromide (Life Technology, Inc., Gaithersburg, MD), and visualized under UV illumination.

DNA from peripheral blood lymphocytes of a healthy individual was treated with SssI methyltransferase (New England Biolabs, Inc., Beverly, MA), subjected to bisulfite modification, and used as a positive control for the methylated alleles. Bisulfite-modified DNA from normal lymphocytes served as a positive control for the unmethylated alleles, and unconverted DNA from normal lymphocytes was used as a negative control for methylated alleles. Negative control samples without DNA were included in each PCR set.

Statistical analysis. The Wilcoxon rank sum test and {chi}2 test (or the Fisher's exact test) were used to analyze continuous and categorical variables by univariate analysis, respectively. Multivariate logistic regression analysis was conducted to estimate the relationship between the development of recurrence and the covariates found to be statistically significant in univariate analysis after controlling for potential confounding factors, and to calculate odds ratios. The effect of promoter methylation on time to death or recurrence was estimated by the Kaplan-Meier method, and the significance of differences in survival between the two groups was evaluated by the log-rank test. The primary end point was recurrence, and the secondary end point was death. Recurrence-free survival was calculated from the date of surgery to the date of recurrence. Overall survival was measured from the date of surgery to the time of last follow-up or death. Cox proportional hazards regression analysis was used to estimate the hazard ratios of independent factors for survival, after controlling for potential confounding factors such as age, sex, histology, and smoking. All statistical analyses were two-sided, with a 5% type I error rate.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Clinicopathologic characteristics and recurrence. The associations between disease recurrence and clinicopathologic features are listed in Table 1 . At a median follow-up of 44 months, 132 patients (39%) had developed recurrence of disease; distant recurrence in 68%, distant and local recurrence in 12%, and local recurrence in 20%. The mean age of patients was similar between those with recurrence and those without (P = 0.86). The recurrence occurred slightly more frequently in males (40%) than in females (39%), but the difference was not statistically significant (P = 0.85). Smoking packyears was not associated with the risk of recurrence (P = 0.36). However, recurrence occurred more frequently in current smokers than those who never smoked or ex-smokers (P = 0.03). The prevalence of recurrence was significantly different according to histologic subtypes (P = 0.001). Recurrence occurred in 65 (54%) of 121 patients with adenocarcinoma and in 56 (31%) of 178 patients with squamous cell carcinoma. A significant relationship was also found between recurrence and pathologic stage (P = 0.009): recurrence occurred in 31% of those with stage I, in 54% of those with stage II, in 45% of those with stage III, and in 75% of those with stage IV. T grade was not associated with recurrence (P = 0.70), but the prevalence of recurrence increased with N grade (P = 0.001): 32% (71 of 223) for N0, 53% (48 of 90) for N1, and 65% (13 of 22) for N2. No relationship was found between recurrence and differentiation (P = 0.15).


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  		Table 1. Clinicopathologic characteristics (n = 335)

 
Recurrence rate and methylation. The relationship between recurrence and the promoter hypermethylation of five genes was investigated to identify biomarkers that would be useful as a risk factor of recurrence. Of the 335 NSCLC samples, promoter methylation was detected in 35% for p16, 39% for RARß, 42% for H-cadherin, 7% for GSTP1, and 21% for FHIT. In univariate analysis, promoter methylation of any single gene was not associated with recurrence (data not shown). Also, the incidence of tumor recurrence was not found to be associated with cohypermethylation of any genes studied by crude data analysis (data not shown). Because tumor recurrence was found to be significantly associated with pathologic stage at the time of curative resection (P = 0.009; Table 1), we stratified the data according to pathologic stage and reanalyzed the relationship between cohypermethylation of genes and the development of recurrence. Using recursive partitioning analysis, we defined recurrently divergent groups on the basis of combinatiorial gene hypermethylation status. Among the five genes studied, cohypermethylation of p16 and FHIT genes was significantly associated with tumor recurrence in stage I cancers (Fig. 2 ).


Figure 2
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  		Figure 2. Prevalence of recurrence according to cohypermethylation of p16 and FHIT genes. Prevalence (56%, 18 of 32) of recurrence in patients with cohypermethylation of p16 and FHIT genes is significantly higher than those without cohypermethylation (27%, 44 of 166) of both genes in only stage I (P = 0.001), but not stage II and stage III.

 
For stage I cancers, recurrence was found to occur in 56% (18 of 32) of patients with cohypermethylation of p16 and FHIT genes and in 27% (44 of 166) of those without, and this difference was statistically significant (P = 0.001). The prevalence of recurrence for stage II and stage III cancers was not significantly different between patients with and without cohypermethylation of p16 and FHIT genes (Fig. 2).

Multivariate logistic regression analysis of recurrence. The data was stratified according to pathologic stage, and stratified multivariate logistic regression analysis was done to control for the potential confounding effects of variables, such as age, sex, smoking status, differentiation, adjuvant therapy and histology, and to calculate the odds ratios (Table 2 ). The coefficient for age variable was not determined to be statistically significant in our univariate analyses (P = 0.86), but age was considered to be a biologically important variable, and it was thus included in the multivariate analysis in order to better construct a parsimonious model. Recurrence occurred at 3.22 times higher prevalence [95% confidence interval (CI), 1.49-4.11; P = 0.001] in adenocarcinomas than in nonadenocarcinomas (squamous cell and other cell types). The risk of recurrence for stage I cases with the cohypermethylation of p16 and FHIT genes was determined to be 6.43 times as high as that of the reference group (95% CI, 1.04-20.19; P = 0.02). There was no relationship between cohypermethylation of p16 and FHIT genes and the risk of recurrence in stage II and stage III (data not shown).


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  		Table 2. Multivariate logistic regression analysis of recurrence in stage I NSCLCs (n = 198)

 
Survival analysis. Overall 5-year survival rates for stage I and stage II were 72% and 42% (P < 0.001), respectively. Five-year recurrence-free survival rates were 57% for stage I and 35% for stage II, and this difference was statistically significant (P < 0.001). The recurrence-free and overall survivals were analyzed between patients with and without cohypermethylation of p16 and FHIT genes. Data was stratified by disease stage because stage is an independent risk factor in NSCLC. Kaplan-Meier survival estimates in stage I are shown in Fig. 3 .


Figure 3
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  		Figure 3. Kaplan-Meier survival curves for p16 and FHIT methylation in stage I NSCLCs. Survival according to cohypermethylation of p16 and FHIT genes in stage I NSCLCs: (A) overall survival, (B) recurrence-free survival, and (C) survival after recurrence were poorer in patients with cohypermethylation of p16 and FHIT genes compared to those without. Black line, group without hypermethylation of p16 and FHIT genes. Green and red lines, unmethylated p16/methylation FHIT and methylated p16/unmethylated FHIT, respectively. Blue line, the group with cohypermethylation of p16 and FHIT genes. P values were calculated for all four groups using the log-rank test.

 
For the 198 stage I cases, the overall 5-year survival rates were 52% and 78% in patients with and without cohypermethylation of p16 and FHIT genes (P = 0.03; Fig. 3A). Recurrence-free 5-year survival rates for the 198 stage I cases with and without cohypermethylation of p16 and FHIT genes were 43% and 69%, respectively. This difference was statistically significant (P = 0.01; Fig. 3B). Survival after recurrence in recurrent cases was finally examined with respect to cohypermethylation of p16 and FHIT genes. Of the 62 patients that did experience recurrence in stage I, survival after recurrence was found to be extremely poor in patients with cohypermethylation of p16 and FHIT genes (Fig. 3C). The median survival after recurrence in recurrent stage I cases with and without cohypermethylation of p16 and FHIT genes was 12 and 26 months, respectively; this difference was statistically significant (P = 0.009). For stage II or stage III cases, overall survival, recurrence-free survival, and survival after recurrence was not significantly different in patients with and without cohypermethylation of p16 and FHIT genes (data not shown).

Cox proportional hazards analysis. The stratified Cox proportional hazards model was created according to pathologic stage to determine whether cohypermethylation of p16 and FHIT genes was an independent prognostic factor of survival associated with recurrence, after controlling for potential confounding factors, including age, sex, smoking, differentiation, histology, adjuvant therapy, and the types of treatment received for recurrent cases (Table 3 ). The present data showed that the cohypermethylation of p16 and FHIT genes was a negative prognostic factor for survival. Patients with cohypermethylation of p16 and FHIT genes in stage I were found to have poorer prognosis than those without cohypermethylation of both genes (hazard ratio, 2.67; 95% CI, 1.21-7.64); this difference was statistically significant (P = 0.02). The recurrence-free survival of stage I cases was also poorer in patients with cohypermethylation of p16 and FHIT genes than those without (hazard ratio, 2.03; 95% CI, 1.09-6.23; P = 0.02). For recurrent stage I cases, the hazard of failure after recurrence was about 4.62 times higher (95% CI, 1.27-16.48; P = 0.005) in patients with cohypermethylation of p16 and FHIT genes than those without. There was no relationship between patient survival and the cohypermethylation of p16 and FHIT genes in stage II and stage III (data not shown).


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  		Table 3. Stratified Cox proportional hazards analysis in stage I NSCLCs

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The long-term survival of patients with NSCLC remains poor because of the high incidence of lymph node metastasis and because of early recurrence after curative surgical resection. Therefore, it is critically important to identify groups at high risk of recurrence, to achieve effective treatment after surgery. In the present study, 39% (132 of 335) of the patients experienced recurrence after curative resection, and the prognosis of these cases was poor compared with those with no recurrences (data not shown). We examined any possible association of methylation status of p16, RARB, FHIT, GSTP1, and ECAD genes with recurrence to identify molecular markers to predict the recurrence of lung cancer after surgery. In the present study, the methylation status of no individual gene had a statistically significant effect on the risk of recurrence in the univariate analysis. These findings are consistent with the findings of Kim et al. (3). Pathologic stage in this study was significantly associated with the risk of tumor recurrence (P = 0.009). Therefore, we stratified the data according to the pathologic stage and reanalyzed the relationship between tumor recurrence and the hypermethylation of those genes. We found that the cohypermethylation of both p16 and FHIT promoters in patients with stage I NSCLC was associated with the poor prognosis of patients and an increased risk of recurrence, suggesting that the cohypermethylation of p16 and FHIT genes may contribute to recurrence after curative resection of stage I NSCLC. The hypermethylation of p16 and FHIT genes in this study were significantly associated with the loss of protein expression (data not shown), suggesting a truly functional inactivation of the two genes. The protein expression was assessed by immunohistochemistry. Second primary lung cancers developed in 18 (5%) of the 335 NSCLCs, but the development of second primary lung cancers was not associated with the hypermethylation of p16 and/or FHIT (data not shown).

It remains unclear how cohypermethylation of p16 and FHIT genes contributes to the recurrence of NSCLC. The p16 gene product is an inhibitor of cyclin-dependent protein kinase 4 (cdk4) which phosphorylates the serine/threonine residues of the retinoblastoma protein (8, 9). The p16 protein inhibits cell cycle progression through G1 into S phase, past the G1 checkpoint by maintaining the retinoblastoma protein in an unphosphorylated state (10, 11). The effect of Fhit on the cell cycle remains the subject of debate (12–14), but reports about the effects of Fhit on apoptosis have produced consistent results. FHIT reexpression in FHIT–/– negative cells that lack Fhit protein expression has been shown to suppress tumor formation by inducing apoptosis in a variety of human cell lines, including lung cancer cell lines (14–20). The induction of apoptosis by FHIT gene transfer in lung cancer cell lines has been associated with the activation of caspase-8 (16). Thus, it is likely that cohypermethylation of p16 and FHIT genes may contribute to the process of multistep carcinogenesis of the lung through different mechanisms, cell cycle and apoptosis control, respectively.

Several reports have shown that the hypermethylation of the p16 gene is detected in the earliest stage of lung cancer and increases with tumor progression. Aberrant methylation of the p16 gene was frequently detected in precursor lesions to lung tumors in rats that were treated with tobacco-specific 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone and increased with disease progression from basal cell hyperplasia (17%) to squamous metaplasia (24%), and then to carcinoma in situ (50%; ref. 21). Aberrant methylation of the p16 gene was also detected in sputum, bronchial lavage, or bronchial brush samples from subjects without any evidence of lung cancer, which suggested that p16 methylation may occur in preneoplastic stage (22–26). The loss of Fhit also occurs very early in lung cancer (27–29), which suggests that Fhit is involved in the initiation of lung cancer tumorigenesis rather than in the progression of lung cancer. Given that hypermethylation of both genes is an early event in the carcinogenesis of lung cancer, the cells that remain after a complete tumor resection in stage I cases with cohypermethylation of p16 and FHIT genes might contribute to the growth advantage and expansion of cells by the failure of both cell cycle and apoptosis control, and eventually increase the risk of recurrence.

After surgery of an initial carcinoma, part of a preneoplastic precursor lesion (i.e., the field) with genetically altered cells may remain in the patient and present a continuous risk for developing a new cancer. However, data about genetic alteration of preneoplastic cells in a field surrounding a tumor were not available in this study. Thus, we could not discriminate a recurrent carcinoma that has developed from minimal residual cancer cells that were left in or near the surgical margins and a second field tumor that developed from preneoplastic precursor cells clonally related to the cells of the excised initial tumor (30), which is in line with the "field cancerization" concept (31). Some of the second field tumors in this study might be misclassified as locally recurrent tumors. Additional studies are needed to differentiate a second field tumor and a recurrent tumor, to validate the usefulness of cohypermethylation of p16 and FHIT genes in identifying a group at high risk of recurrence after surgery.

Fhit-deficient normal cells and cancer cells are more resistant to radiation treatment (UVC and ionizing radiation) and drugs (mitomycin C and cisplatin; ref. 32) and have stronger IR-induced S and G2 checkpoint responses than Fhit+/+ cells (33, 34), suggesting an association between FHIT gene inactivation and increased survival after DNA damage. The overactive checkpoints are known to be regulated by the ATR-CHK1 pathway, which contributes to the radioresistance of Fhit–/– cells (35). Accordingly, the effect of treatment on survival after recurrence in recurrent stage I cases was controlled by stratification and multivariate analysis. However, stratification of data according to the types of treatment for recurrent stage I cases did not show a significant difference in the survival after recurrence between patients with and without cohypermethylation of p16 and/or FHIT genes (data not shown). In contrast, the survival was significantly poorer (hazard ratio, 4.62; 95% CI, 1.27-16.48; P = 0.005) in patients with cohypermethylation of p16 and FHIT genes than those without, after adjusting age, sex, smoking status, histology, and the types of treatment in multivariate analysis (Table 3C). The lack of effect of p16/FHIT methylation on survival after recurrence in the stratified data may have resulted from uncontrolled confounding factors or from the small number of cases in each type of treatment. Among 62 recurrent stage I cases, only 38 patients received radiotherapy and 16 cases received chemotherapy. More studies are needed with a large number of samples to show the effect of Fhit deficiency on survival after recurrence, after controlling the types of treatment received for recurrence.

In the present study, lymph node metastasis, male gender, and adenocarcinoma were found to be significantly associated with an increased risk of recurrence after complete curative resection. The relationship between recurrence and histology is controversial. Our data showed similar results to those of the Lung Cancer Study Group (36) and to recent data reported by Okada et al. (37). In the Lung Cancer Study Group, cancer recurrence was more frequent in non–squamous cell types. Okada et al. (37) also showed that advanced stage, high involvement of lymph nodes, male gender, and non–squamous cell cancer were independent, unfavorable prognostic factors in patients with completely resected lung cancer. In contrast, some groups (2, 38) reported that histologic type did not play a statistically significant role in the incidence of recurrence, and Rena et al. (39) reported that adenocarcinoma has better 5-year survival rates than did squamous cell carcinoma.

Although no significant relationship was observed between cohypermethylation of p16 and FHIT genes and the risk of recurrence in stage II and stage III, this may result from the small number of the sample size. Further study in a larger sample is needed to investigate the significance of cohypermethylation of p16 and FHIT genes in identifying groups at high risk of recurrence in advanced cases. Even though independent studies on a larger scale are required to validate its usefulness before clinical application, the cohypermethylation status of p16 and FHIT genes could be valuable as a molecular biomarker in identifying patients at high risk of recurrence during follow-up treatment after complete tumor resection. These patients might benefit from a more aggressive treatment strategy. The ras gene activation is known to be an early event in lung adenocarcinoma and is a good predictor of poor prognosis. Therefore, the combination effect of ras gene activation with p16/FHIT methylation on prognosis for adenocarcinoma of the lung needs to be investigated. In conclusion, cohypermethylation of p16 and FHIT genes might be a strong indicator of a high risk of recurrence as well as an independent prognostic factor in cases of stage I NSCLC.


   Acknowledgments
 
Grant support: Samsung Biomedical Research Institute (B-A5-102) and the SRC/ERC program of MOST/KOSEF (R11-2005-017).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The authors thank Eun-Kyung Kim for data collection and management, and Hoon Suh for sample collection.

Received 10/21/05. Revised 2/ 1/06. Accepted 2/17/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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