Given the important role of early detection in improving cancer outcomes, epigenetic alterations, particularly DNA methylation, have emerged as powerful biomarkers for cancer screening and diagnosis. However, methylation profiling of cfDNA (circulating free DNA) presents particular technical challenges because cfDNA is typically present at very low concentrations and is highly fragmented. Bisulfite conversion, although essential for distinguishing methylated from unmethylated cytosines, can further compromise DNA integrity through chemical-induced loss of DNA during purification steps. Inadequate conversion or excessive DNA loss may consequently reduce library complexity, increase DNA duplication rates, and negatively affect mapping efficiency and downstream sequencing quality. Therefore, optimizing bisulfite conversion is particularly important for reliable methylation profiling of low-input cfDNA. The effectiveness of these diagnostic approaches relies on the accurate detection of DNA methylation patterns, which is fundamentally dependent on the efficiency of bisulfite conversion of genomic DNA. This pilot study aims to compare the performance of two widely used bisulfite conversion kits, namely the EpiTect Bisulfite (EpiTect) Kit (Qiagen, Hilden, Germany) and the EZ DNA Methylation (Zymo) Kit (Zymo Research, Irvine, CA, USA), in the context of plasma-derived cfDNA methylation profiling using five samples, including two plasma samples from metastatic colorectal cancer (mCRC) patients collected at the Department of Gastrointestinal Oncology at The University of Texas MD Anderson Cancer Center, in Houston, TX, USA, and two plasma samples from healthy donors (controls) purchased from Innovative Research (Novi, MI, USA), along with one control DNA sample (Corriel NA12878-N88). Our objective was to evaluate and compare their conversion efficiency, mapping efficiency (percentage of mapped and unmapped reads), duplication rate, and distribution of methylated cytosines in CpG, CHG, and CHH contexts. Our results indicate that samples processed using the Zymo kit consistently outperformed those treated with the EpiTect kit across multiple performance metrics. The Zymo-treated samples yielded a higher concentration of cfDNA following bisulfite conversion (125.34 nM vs. 45.60 nM in samples processed with the Epitect kit). Additionally, the Zymo kit achieved a higher average bisulfite conversion efficiency (99.3% vs. 95.7%), as well as a significantly higher average mapping rate (94.86% vs. 72.4%), with fewer unmapped reads and lower duplication rates. Head-to-head comparison of Bismark methylation calls across the same reference regions showed high concordance between the two protocols. The SEPT9 region was strongly hypermethylated with both protocols (100% with EpiTect and approximately 94% with Zymo), whereas the BMP3 region was predominantly unmethylated (approximately 4% with EpiTect and 7% with Zymo). These findings suggest that the Zymo kit demonstrated improved performance under the conditions evaluated for cfDNA methylation analysis compared to the EpiTect protocol. Further investigation using a larger number of samples is needed to validate our findings.

Lung cancer (LC) remains the leading cause of cancer-related mortality worldwide, representing a major public health challenge. Although low-dose computed tomography is currently the standard screening method for high-risk individuals to reduce mortality, its clinical performance is limited by false-positive findings and restricted accessibility. Emerging biomarker-based liquid biopsy offers a promising non-invasive approach for cancer detection. This narrative review provides a critical overview and evaluation of clinically endorsed liquid biopsy applications for guiding treatment in advanced-stage lung cancer, as well as investigational advances in liquid biopsy for early cancer detection. We also address challenges of current liquid biopsy technologies, the integration of artificial intelligence, implementation improvements, and future perspectives for their adoption into routine clinical practice. To complement this narrative review, a structured search of the ClinicalTrials.gov registry was performed to identify clinical trials evaluating liquid biopsy in lung cancer. Current evidence supports the clinical use of liquid biopsy for molecular profiling and treatment selection in advanced non-small cell lung cancer (NSCLC), whereas its application in early detection, screening, and minimal residual disease (MRD)-guided management remains investigational and requires further prospective validation. Overall, liquid biopsy represents a promising complementary approach to advance precision oncology and improve lung cancer management.