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. 2025 Apr 25;17(1):63.
doi: 10.1186/s13148-025-01870-8.

Plasma cell-free DNA methylation profile before afatinib treatment is associated with progression-free and overall survival of patients with epidermal growth factor receptor gene mutation-positive non-small cell lung cancer

Mao Fujimoto #  1 , Hiroyuki Yasuda #  2 , Eri Arai  3 , Makoto Nakajima  1 , Saori Takata  4 , Kei Morikawa  5 , Hisashi Tanaka  6 , Hidetoshi Itani  7 , Takeshi Honda  8 , Kazuya Horiuchi  9 , Kageaki Watanabe  10 , Hideyuki Nakagawa  11 , Yoshiro Nakahara  12 , Yoshitaka Seki  13 , Akihiro Bessho  14 , Nobumasa Takahashi  15 , Kentaro Hayashi  16 , Takeo Endo  17 , Kiyoshi Takeyama  18 , Toshiya Maekura  19 , Nagio Takigawa  20 , Akikazu Kawase  21 , Makoto Endoh  22 , Kenji Nemoto  23 , Kazuma Kishi  24 , Kenzo Soejima  25 , Yusuke Okuma  10 , Akira Togashi  26 , Noriyuki Matsutani  27 , Nobuhiko Seki  8 , Yae Kanai  28
Affiliations

Plasma cell-free DNA methylation profile before afatinib treatment is associated with progression-free and overall survival of patients with epidermal growth factor receptor gene mutation-positive non-small cell lung cancer

Mao Fujimoto et al. Clin Epigenetics. .

Abstract

Background: The present study aimed to clarify the clinical significance of the cell-free DNA (cfDNA) methylation profile of patients with non-small cell lung cancer (NSCLC) showing the epidermal growth factor receptor (EGFR) gene mutation.

Methods: In 103 patients, genome-wide DNA methylation analysis using Infinium Methylation EPIC array was performed using samples of pre-tyrosine kinase inhibitor afatinib-treatment plasma cfDNA (n = 101) and post-afatinib cfDNA (n = 84).

Results: Principal component analysis indicated that the cfDNA methylation profile was altered after afatinib treatment. Hierarchical clustering using the pre-afatinib cfDNA methylation profile revealed that cases with a fatal outcome were accumulated in specific clusters. Moreover, Kaplan-Meier analysis showed that the pre-afatinib cfDNA methylation profile was significantly associated with both progression-free survival (PFS) and overall survival (OS), whereas the post-afatinib profile was not. The genes for which pre-afatinib cfDNA methylation levels were associated with PFS were accumulated in the cadherin, Wnt, and EGFR signaling pathways. Activation of EGFR-related signaling due to DNA methylation alterations might overturn the effect of afatinib. Pre-afatinib levels of CEP170 and CHCHD6 cfDNA methylation were associated with both PFS and OS. Both pre- and post-afatinib cfDNA methylation levels of SLC9A3R2 and INTS1 were associated with bone metastasis. Using the cfDNA methylation levels at two CpG sites, cg12721600 and cg05905155, patients showing an overall response were predicted with a sensitivity of 96% or more.

Conclusions: The non-invasively measurable cfDNA methylation profile may reflect the corresponding profile in cancer cells, and that pre-treatment measurement may provide clinically useful information on EGFR mutation-positive NSCLC.

Keywords: Afatinib; Cell-free DNA; DNA methylation; Epidermal growth factor receptor; Lung adenocarcinoma.

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Conflict of interest statement

Declarations. Ethics approval and consent to participate: This study complied with the Declaration of Helsinki and was approved by the Ethics Committee at Keio University School of Medicine (2021–0147) and the Ethical Review Board for Medical and Health Research Involving Human Subjects at Teikyo University (16–066). All participants provided written informed consent. Competing interests: AT is an employee of Nippon Boehringer Ingelheim Co., Ltd., Tokyo, Japan.

Figures

Fig. 1
Fig. 1
Cell-free DNA (cfDNA) methylation profiles of non-small cell lung cancer patients. A Principal component (PC) analysis of 101 pre-afatinib cfDNA samples (black circles) and 84 post-afatinib cfDNA samples (blue triangles) from among 103 patients using the DNA methylation levels of all of the 815,746 probe CpG sites. The pre-afatinib samples are scattered over a wide area on the scatterplot, whereas the post-afatinib samples appear clustered in a smaller area, suggesting that the cfDNA methylation profile tended to be altered by afatinib treatment. B Hierarchical clustering (Manhattan distances, Ward’s method) in the 101 patients from whom pre-afatinib blood samples were obtained. Patients who survived and who died are shown in blue and red, respectively, at the bottom of the dendrogram. Fatal cases are accumulated in Clusters I and IIA (n = 43; 62.8%) relative to Cluster IIB (n = 58; 24.1%) (P = 1.85 × 10–4, Fisher's exact test)
Fig. 2
Fig. 2
Correlations between pre-afatinib cell-free DNA (cfDNA) methylation profile and progression-free survival (PFS) in patients with non-small cell lung cancer. Patients belonging to the initial cohort (n = 60) A and the validation cohort (n = 41) B are divided into a DNA hypermethylation group (Hyper) and a DNA hypomethylation group (Hypo) using the median values of the pre-afatinib cfDNA methylation levels in the initial cohort as the cutoff values (CV). Kaplan–Meier curves for PFS at 5 CpG sites showing the smallest P-values in the initial cohort using Log-rank test are shown. The Infinium assay probe ID for each CpG site and gene symbol are shown on the left side of each Kaplan–Meier curve
Fig. 3
Fig. 3
Correlation between the pre-afatinib cell-free DNA (cfDNA) methylation profile and overall survival (OS) in patients with non-small cell lung cancer. Patients belonging to the initial cohort (n = 60) A and the validation cohort (n = 41) B are divided into a DNA hypermethylation group (Hyper) and a DNA hypomethylation group (Hypo) using the median values of the pre-afatinib cfDNA methylation levels in the initial cohort as the cutoff values (CV). Kaplan–Meier curves for OS at 5 CpG sites showing the smallest P-values in the initial cohort using Log-rank test are shown. The Infinium assay probe ID for each CpG site and gene symbol are shown on the left side of each Kaplan–Meier curve
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