Low-dose computed tomography (LDCT) is recommended for early lung cancer screening in high-risk populations. Small lung nodules are identified by these CT scans in up to 50% of high-risk patients. While over 50% of lung nodules less than 1 cm are benign, discriminating benign nodules from malignant nodules remains extremely challenging without an invasive lung biopsy. Taking advantage of methylation signatures in circulating tumor DNA (ctDNA), we developed a non-invasive screening assay to identify patients with malignant lung nodules from those with benign lung nodules.

Methods: We first adopted Singlera's MONOD+ assay as a marker screening technology to interrogate the methylation state of over 4,000,000 CpG sites across more than 200,000 methylation haplotype blocks in the human genome from as little as 10 ng of cell-free or tissue DNA. We first processed 129 fresh frozen and FFPE tissue samples from healthy lung, benign lung nodules, and lung cancer at various stages of malignancy. We identified a preliminary set of methylation markers capable of segregating lung nodules by malignancy and invasiveness. To examine the reproducibility of these markers in plasma, we applied the same screening assay to plasma cell-free DNA from 46 healthy patients and 175 lung nodule patients with either benign or malignant nodules (most at an early stage of Ia1/Ia2). We refined the marker set by removing methylation markers that were unstable in plasma samples. We next applied Singlera's MethylTitan assay to deeply interrogate approximately 1,000 methylation haplotype blocks enriched in malignant lung nodules in plasma samples, as MethylTitan's higher conversion rate allowed us to better handle the limited amount of DNA in plasma samples. We ran the MethylTitan assay on both healthy and lung nodule plasma samples; using this method, we confirmed that tissue-derived methylation signatures could be rapidly screened in plasma samples in a high-throughput and highly sensitive manner.

Results: Using the MONOD+ marker screening assay on plasma samples, we were able to utilize cell-free DNA methylation to identify early-stage malignant lung nodule patients with a sensitivity of 80.25% and a specificity of 83.08%. We were able to further improve upon these results using the MethylTitan assay, as we could more efficiently and more accurately screen the methylation markers in a large number of plasma samples.

Conclusion: In summary, we have applied Singlera's MONOD+ and MethylTitan methylation assays to non-invasively identify early stage lung cancer in patients with small lung nodules. We are currently expanding this study to a much larger cohort of lung nodule patients in order to clinically validate our early lung cancer screening assay. The approach of using MONOD+ to identify markers and MethylTitan to analyze plasma samples will allow us to noninvasively screen for additional cancer types in a high-throughput clinical setting.

Citation Format: Ruijun Liu, Xiaojie Li, Athurva Gore, Zi Qin, Jeff Gole, Qiye He, Rui Liu, Shun Lu. Detection of early-stage malignant lung cancer using methylation signatures in circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3249.