Study Identifies Prognostic DNA Methylation Signature for Stage I NSCLC
There is an absence of biomarkers to indicate which patients with stage I non–small cell lung cancer (NSCLC) would best benefit from adjuvant chemotherapy. In a study reported in the Journal of Clinical Oncology, Juan Sandoval, PhD, of the Bellvitge Biomedical Research Institute in Barcelona, and colleagues identified a DNA methylation signature that discriminated high- and low-risk patents.
Study Details
The investigators used a DNA methylation microarray analyzing 450,000 CpG sites to study tumor DNA obtained from 444 patients with NSCLC, including 237 stage I tumors. The prognostic DNA methylation markers were then validated by a single-methylation pyrosequencing assay in an independent cohort of 143 patients with stage I NSCLC.
Methylation Microarray in Discovery Cohort
In the discovery cohort, hierarchical clustering distinguished two main types of tumors, accounting for 27% (group 1) and 73% (group 2) of patients. Among the 237 patients with stage I NSCLC, 147 met criteria for inclusion in the relapse-free survival cohort (eg, no receipt of adjuvant chemotherapy before relapse). The ineligible patients did not show a higher recurrence rate.
Group 1 identified patients with high-risk stage I NSCLC that had lower relapse-free survival shown in Kaplan-Meier survival analysis (log-rank test P = .03) and in univariate (hazard ratio [HR] = 1.85, P = .037) and multivariate (HR = 2.35, P = .004) Cox regressions of histology, smoking history, age, and sex. The unsupervised clustering analysis of adenocarcinomas in stage I also identified a group associated with shorter relapse-free survival (HR = 2.94, P = .003), with a trend being observed for squamous cell carcinomas (HR = 2.55, P = .09).
Ranking of CpG sites that, according to methylation status, were best at discriminating the 444 NSCLC samples from 25 histologically normal lung tissue samples ultimately identified 150 sites with hypermethylation significantly enriched in group 1 vs group 2. Testing of the methylation value in the 147 stage I tumors by Kaplan-Meier survival analysis and multivariate Cox regression identified 54 CpGs corresponding to 42 genes significantly associated with shorter relapse-free survival.
Identification of Signature With Pyrosequencing in Validation Cohort
The top 10 genes with a hazard ratio of more than 2 at a 10% false discovery rate were identified for analysis by pyrosequencing in the validation cohort. Four of the top five candidates in the adenocarcinoma set were present in this overall 10-gene candidate list.
Of these 10 genes associated with recurrence in the discovery cohort using the DNA methylation microarray, 5 were significantly associated with recurrence in the validation cohort of 143 stage I NSCLC samples analyzed by pyrosequencing: histone cluster1 H4F (HIST1H4F; HR = 3.55, P < .001), protocadherin gamma subfamily B6 (PCDHGB6; HR = 2.95, P = .002), neuropeptide B/W receptor 1 (NPBWR1; HR = 2.71, P = .004), ALX homeobox protein 1 (ALX1; HR = 2.29, P = .015), and homeobox A9 (HOXA9; HR = 2.03, P = .027).
Signature Identifies High-Risk Group
A greater risk of shorter relapse-free survival was found when stage I tumors harbored a large number of these five hypermethylated markers. To obtain the most useful methylation signature, a cutoff of zero to one vs two or more hypermethylated markers was selected, since it was the best in capturing the percentage of expected recurrences.
This methylation signature divided the patients with stage I tumors into two groups: patients with zero to one methylated markers, who had longer relapse-free survival, and those with two or more hypermethylated genes, who had higher risk of poor relapse-free survival on Kaplan-Meier estimates. The heavily hypermethylated group identified patients with high-risk stage I NSCLC who had shorter relapse-free survival as shown by Kaplan-Meier survival analysis (log-rank test P = .010) and univariate (HR = 2.26, P = .012) and multivariate (HR = 3.24, P = .001) Cox regressions.
The methylation signature remained significantly associated with shorter relapse-free survival in Cox regression multivariate analysis when stage I tumors were divided into IA and IB according to the sixth revision of the TNM classification (HR = 3.09, P = .002), and reclassification of the stage I tumors according to the seventh revision of the TNM classification confirmed the significance of the enriched hypermethylation group for shorter relapse-free survival in 103 original stage I tumors for which all relevant clinicopathologic information was available (HR = 2.89, P = .010). The inclusion of tumor size in the Cox analysis in stage I tumors did not alter the significant association of tumors with two or more methylated markers with shorter relapse-free survival (HR = 2.88, P = .011).
It was calculated that relapse occurred within 3 years of surgery in 48% of patients in the enriched methylated group vs 18% those in the low methylated group. The zero to one vs two or more hypermethylated genes signature obtained by pyrosequencing in the validation cohort was significantly predictive in the 147 stage I tumors in the discovery cohort (HR = 1.95, P = .023).
The investigators concluded, “The DNA methylation signature of NSCLC affects the outcome of stage I patients, and it can be practically determined by user-friendly polymerase chain reaction assays. The analysis of the best DNA methylation biomarkers improved prognostic accuracy beyond standard staging.”
Manel Esteller, MD, PhD, of Bellvitge Biomedical Research Institute in Barcelona, is the corresponding author for the Journal of Clinical Oncology article.
The content in this post has not been reviewed by the American Society of Clinical Oncology, Inc. (ASCO®) and does not necessarily reflect the ideas and opinions of ASCO®.
