Targeted Methylation Sequencing of Plasma Cell-free DNA for Cancer Detection and Classification

Abstract

Background

Targeted methylation sequencing of plasma cell-free DNA (cfDNA) has a potential to expand liquid biopsies to patients with tumors without detectable oncogenic alterations, which can be potentially useful in early diagnosis.

Patients and methods

We developed a comprehensive methylation sequencing targeting 9,322 CpG sites consistently hypermethylated according to The Cancer Genome Atlas. Next, we performed a clinical validation of our method using plasma cfDNA samples from 78 patients with advanced colorectal cancer, non-small cell lung cancer (NSCLC), breast cancer or melanoma and compared results to patients' outcomes.

Results

Median methylation scores in plasma cfDNA samples from patients on therapy were lower than from patients off therapy (4.74 vs. 85.29; p=0.001). Of 68 plasma samples from patients off therapy, methylation scores detected the presence of cancer in 59 (86.8%), and methylation-based signatures accurately classified the underlying cancer type in 45 (76.3%) of these. Methylation scores were most accurate in detecting colorectal cancer (96.3%), followed by breast cancer (91.7%), melanoma (81.8%) and NSCLC (61.1%), and most accurate in classifying the underlying cancer type in colorectal cancer (88.5%), followed by NSCLC (81.8%), breast cancer (72.7%) and melanoma (55.6%). Low methylation scores versus high were associated with longer survival (10.4 vs. 4.4 months, p<0.001) and longer time to treatment failure (2.8 vs. 1.6 months, p=0.016).

Conclusions

Comprehensive targeted methylation sequencing of 9,322 CpG sites in plasma cfDNA from patients with common advanced cancers detects the presence of cancer and underlying cancer type with high accuracy. Methylation scores in plasma cfDNA correspond with treatment outcomes.