Abstract 5588: Methylated ctDNA dynamics correspond with clinical tumor load in metastatic lung cancer patients on therapy
Authors: Ye Patrick , Woodward Brian , Viens Robb , Langpap Sydne , Shelburne Katherine , Zhou Wen , Zhu Joyce , Wignall Jan , Palmer Gary , Tsao David , Atay Oguzhan , Husain Hatim 
Published: June 2023

Treatment monitoring assays are needed to accurately and rapidly assess the efficacy of cancer treatments. While imaging remains the gold standard for monitoring the efficacy of cancer treatment, the use of more sensitive tools, such as liquid biopsy, could be beneficial for the patient’s ultimate treatment outcome. Several liquid biopsy-based assays that measure circulating tumor DNA (ctDNA) have been developed to meet this need. However, approaches that rely on quantifying the variant allele fraction (VAF) of somatic variants may be inaccurate or inconsistent due to a scarcity of detected somatic variants in the ctDNA or may be logistically infeasible if they require a tumor biopsy a priori. Methylated ctDNA has shown promise as a biomarker for treatment monitoring without requiring a tumor biopsy, but current efforts are limited in their ability to precisely quantify the amount of methylation present in the ctDNA. We hypothesize that more precise quantification of methylated ctDNA could enable more accurate correspondence with clinical tumor load and cancer treatment outcomes.

Here, we present a retrospective study characterizing how amounts of methylated ctDNA dynamically change through cancer therapy. We tested 75 patient-treatment events, with each event composed of patient samples from one pre-treatment and two post-treatment time points, all from patients with Stage III-IV metastatic lung cancer receiving targeted therapies or immunotherapy. We employed a novel methylation-based, tumor-naive liquid biopsy assay for treatment monitoring by counting the number of methylated molecules in cell-free DNA. We observed methylation amounts ranging from &lt;50 to &gt;50,000 total methylated tumor molecules per 1000 assayed genomic equivalents at individual time points, indicating that the assay can detect a broad range of tumor load in blood. In addition, we measured changes between time points as large as &gt;96% decrease and &gt;2800% increase in methylation. We found that methylation correlated with maximum variant allele fractions as measured by a treatment selection assay (r2 = 0.74). Moreover, in this tumor-naive assay, we identified a median of 58 informative methylated loci, significantly more than a median of ~1-5 informative SNVs identified in VAF-based tumor-naive assays, and ~4-16 informative SNVs identified in VAF-based tumor-informed assays. This ~6-10x increase in the number of informative loci enables much more precise quantification and underlies the assay’s robustness and precision across patients, time points, and therapies. Finally, these changes in normalized total tumor molecules correlated with radiographic responses on imaging, duration of response, and time on therapy. Overall, our findings support the utility of methylated ctDNA as a biomarker for monitoring tumor load in cancer patients.

Citation Format: Patrick Ye, Brian Woodward, Robb Viens, Sydne Langpap, Katherine Shelburne, Wen Zhou, Joyce Zhu, Jan Wignall, Gary Palmer, David Tsao, Oguzhan Atay, Hatim Husain. Methylated ctDNA dynamics correspond with clinical tumor load in metastatic lung cancer patients on therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5588.