Longitudinal monitoring of cell-free DNA methylation in ALK-positive non-small cell lung cancer patients

Abstract

Background DNA methylation (5-mC) signals in cell-free DNA (cfDNA) of cancer patients represent promising biomarkers for minimally invasive tumor detection. The high abundance of cancer-associated 5-mC alterations permits parallel and highly sensitive assessment of multiple 5-mC biomarkers. Here, we performed genome-wide 5-mC profiling in the plasma of metastatic ALK-rearranged non-small cell lung cancer (NSCLC) patients receiving tyrosine kinase inhibitor therapy. We established a strategy to identify ALK-specific 5-mC changes from cfDNA and demonstrated the suitability of the identified markers for cancer detection, prognosis, and therapy monitoring. Methods Longitudinal plasma samples (n = 79) of 21 ALK-positive NSCLC patients and 13 healthy donors were collected alongside 15 ALK-positive tumor tissue and 10 healthy lung tissue specimens. All plasma and tissue samples were analyzed by cell-free DNA methylation immunoprecipitation sequencing to generate genome-wide 5-mC profiles. Information on genomic alterations (i.e., somatic mutations/fusions and copy number alterations) determined in matched plasma samples was available from previous studies. Results We devised a strategy that identified tumor-specific 5-mC biomarkers by reducing 5-mC background signals derived from hematopoietic cells. This was followed by differential methylation analysis (cases vs. controls) and biomarker validation using 5-mC profiles of ALK-positive tumor tissues. The resulting 245 differentially methylated regions were enriched for lung adenocarcinoma-specific 5-mC patterns in TCGA data and indicated transcriptional repression of several genes described to be silenced in NSCLC (e.g., PCDH10, TBX2, CDO1, and HOXA9). Additionally, 5-mC-based tumor DNA (5-mC score) was highly correlated with other genomic alterations in cell-free DNA (Spearman, ρ > 0.6), while samples with high 5-mC scores showed significantly shorter overall survival (log-rank p = 0.025). Longitudinal 5-mC scores reflected radiologic disease assessments and were significantly elevated at disease progression compared to the therapy start (p = 0.0023). In 7 out of 8 instances, rising 5-mC scores preceded imaging-based evaluation of disease progression. Conclusion We demonstrated a strategy to identify 5-mC biomarkers from the plasma of cancer patients and integrated them into a quantitative measure of cancer-associated 5-mC alterations. Using longitudinal plasma samples of ALK-positive NSCLC patients, we highlighted the suitability of cfDNA methylation for prognosis and therapy monitoring.

Cell-Free Methylated PTGER4 and SHOX2 Plasma DNA as a Biomarker for Therapy Monitoring and Prognosis in Advanced Stage NSCLC Patients

“…Since our group and other researchers demonstrated that the detection and quantification of methylated ctDNA is a useful approach for therapy monitoring in lung cancer patients [ 14 , 15 , 16 ], more papers on this subject were published [ 20 , 21 ]. Moreover, the incorporation of liquid profiling as an additional clinical tool for the diagnosis, treatment stratification, detection of resistance mechanisms, and prognostic indication in lung cancer patients has been shown [ 22 , 23 , 24 , 25 ].…”

The role of aberrant DNA methylation in cancer initiation and clinical impacts

“… 180 CtDNA methylation in liquid biopsies can be analysed as either a single region (e.g. a single gene promoter), 181 – 184 as a panel of loci (e.g. multiple gene promoters) 185 – 189 or using genome-wide epigenetic signatures.…”

Liquid Biopsy in Lung Cancer: Biomarkers for the Management of Recurrence and Metastasis

“…Aberrant hypermethylation is a common finding within tumor tissue when compared to generally normal and healthy tissue. Global blood-based methylation biomarkers obtained from liquid biopsy material (cfDNA, ctDNA) have been difficult to determine; however, methylation profiles for lung cancer subtypes have shown robust differences between diseased and control states [133][134][135][136][137], and differences can even be detected between circulating tumor material and direct tumor tissue [138]. These profiles show remarkable differences even in early cancer stages, with certain methylation markers specific to particular cancers, whereas some are detectable pan-cancer [139,140].…”