Cell-Free DNA 5-Hydroxymethylcytosine Signatures for Lung Cancer Prognosis

Abstract

Accurate prognostic markers are essential for guiding effective lung cancer treatment strategies. The level of 5-hydroxymethylcytosine (5hmC) in tissue is independently associated with overall survival (OS) in lung cancer patients. We explored the prognostic value of cell-free DNA (cfDNA) 5hmC through genome-wide analysis of 5hmC in plasma samples from 97 lung cancer patients. In both training and validation sets, we discovered a cfDNA 5hmC signature significantly associated with OS in lung cancer patients. We built a 5hmC prognostic model and calculated the weighted predictive scores (wp-score) for each sample. Low wp-scores were significantly associated with longer OS compared to high wp-scores in the training [median 22.9 versus 8.2 months; p = 1.30 × 10^-10; hazard ratio (HR) 0.04; 95% confidence interval (CI), 0.00-0.16] and validation (median 18.8 versus 5.2 months; p = 0.00059; HR 0.22; 95% CI: 0.09-0.57) sets. The 5hmC signature independently predicted prognosis and outperformed age, sex, smoking, and TNM stage for predicting lung cancer outcomes. Our findings reveal critical genes and signaling pathways with aberrant 5hmC levels, enhancing our understanding of lung cancer pathophysiology. The study underscores the potential of cfDNA 5hmC as a superior prognostic tool for guiding more personalized therapeutic strategies for lung cancer patients.

Keywords: 5-hydroxymethylcytosine; cell-free DNA; lung cancer; prognosis.

Figure 1

The 5hmC prognostic signature is associated with overall survival in lung cancer patients. (A,B) Kaplan–Meier analysis of overall survival (OS) based on weighted prognostic scores in the training set (A) and the validation set (B). A cutoff score of 310.6 was used for different prognostic categories. HR, hazard ratio. CI, confidence interval.

Figure 2

The 5hmC prognostic signature is associated with progression-free survival in lung cancer patients. (A,B) Kaplan–Meier analysis of progression-free survival (PFS) based on prognostic scores in the training set (A) and the validation set (B). A cutoff score of 310.6 was used for different prognostic categories. HR, hazard ratio. CI, confidence interval.

Figure 3

Multivariate Cox regression analysis in lung cancer patients. (A,B) Overall survival multivariate Cox regression analysis, illustrated as a forest plot, considering various clinical parameters in the training set (A) and validation set (B) of lung cancer patients. HR, hazard ratio. CI, confidence interval.

Figure 4

Prognostic value of multiple variables in lung cancer patients. (A,B) Time-dependent receiver operating characteristic (ROC) and corresponding area under the curves (AUCs) for 12-month overall survival predicted by all combined factors, prognostic score, age, sex, smoking history, and TNM stage in the training (A) and validation (B) sets.

Figure 5

Genes and pathways associated with the prognosis of lung cancer. (A) Canonical signaling pathways with genes significantly associated with overall survival (OS) in lung cancer. Pathway analysis was performed using Ingenuity Pathway Analysis. The ratio indicates the number of OS-related genes in each pathway divided by the total number of genes that make up that pathway. (B) Genes appearing in more than 10 canonical pathways are displayed. Hazard ratios for OS in genes significantly enriched in canonical pathways presented by the forest plot.