Abstract 3123: A method for quantifying circulating tumor DNA level and molecular response using methylome sequencing
Authors: Chen Sai , Quinn Katie , Lee Che-Yu , Zhao Jun , Chang Kyle , Jiang Tingting , Zhang Shile , Espenschied Carin , Wienke Sara , Rich Thereasa , Wu Indira , Kim Yvonne , Liu Xianxian , Alla Nageswara , Ma Dustin , Tran Giao , Chuang Han-Yu 
Published: April 2023

Abstract
Background: Circulating tumor DNA (ctDNA) level and the change in level at a subsequent time point (e.g. on-treatment change from baseline or postoperative changes through time) are promising tools for predicting patient prognosis and response to therapy. Existing methods use somatic variant allele frequencies to quantify circulating tumor fractions (cTF). Their performance can be limited by the number of detectable somatic alterations and the associated limit of detection (LoD), as well as interference from copy number variation and non-tumor alterations, such as clonal hematopoiesis. Here, we describe the LoD, precision and limit of quantitation (LoQ) of cTF level and change using GuardantINFINITY, a next generation sequencing panel covering over 800 genes with genome-wide methylation detection.
Method: The cTF of a single sample is estimated from methylation signals across targeted regions of the GuardantINFINITY methylation panel, calibrated using internal training data. cTF change compares two or more samples from the same patient to identify patient-specific methylated regions, and compare the methylation signals of the paired regions. LoQ of cTF level and change were assessed in experimental titrations of advanced colorectal, breast, and lung cancer patient samples and cell line samples into cancer-free backgrounds at different target levels between 0.1%-0.5% cTF. LoD is defined as the lowest cTF level where &gt;95% replicates were detected to have tumor-derived methylation signals. LoQ of cTF level or change is defined as the lowest cTF where the coefficient of variation (CV) across replicates is less than 30%. Accuracy of methylation based cTF compared to cTFs calculated from maximum VAF of somatic mutations was assessed on 1,400 clinical samples of colorectal and lung cancer patients (N=189, 372, 252 and 463 for stage I to IV).
Results: Experimental titrations of cancer samples demonstrated a single-sample LoD of 0.05% cTF (lowest dilution level) and quantitative precision down to a LoQ of below 0.1%, compared to the LoQ of 0.3% estimated by somatic mutations. In paired clinical titration samples, the LoQ of methylation ctDNA level change was also below 0.1%, compared to the LoQ of ctDNA level change estimated by somatic mutations at 0.3-0.5%. In the 1,400 clinical samples, 64% had at least one somatic mutation detected, 90% had ct-DNA detected with methylation and 96% of these ct-DNA detected samples had cTF above the defined methylation LoQ. Among patients with both methylation and genomic signals identified, the methylation method quantified a similar cTF to those that were calculated using maximum somatic driver mutations (Pearson r=0.83).
Conclusion: Methylome sequencing using GuardantINFINITY enables accurate and precise quantification of ctDNA level and change with a liquid-only approach, offering longitudinal ctDNA monitoring for more patients than previous methods.
Citation Format: Sai Chen, Katie Quinn, Che-Yu Lee, Jun Zhao, Kyle Chang, Tingting Jiang, Shile Zhang, Carin Espenschied, Sara Wienke, Thereasa Rich, Indira Wu, Yvonne Kim, Xianxian Liu, Nageswara Alla, Dustin Ma, Giao Tran, Han-Yu Chuang. A method for quantifying circulating tumor DNA level and molecular response using methylome sequencing [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 3123.