Abstract PR017: A T-cell signature in circulating cell-free DNA at time of diagnosis predicts response to checkpoint inhibition
Authors: Xie Phil , Etzioni Zohar , Song Chun-Xiao , Owen Richard P , Middleton Mark R , Lu Xin , Schuster-Boeckler Benjamin 
Published: November 2024

Abstract
Immune checkpoint inhibitors (ICI) are increasingly used as first line of treatment in a range of cancer types, owing to their efficacy and advantageous toxicological profile. Currently, PD-L1 expression and tumor mutation burden (TMB) are the most common biomarkers to select patients for ICI treatment, yet response to ICI remains highly variable between patients, necessitating more accurate early predictors of treatment response. Circulating cell-free DNA (cfDNA) originates from dying cells throughout the body, each molecule carrying unique epigenetic features of its cell-type of origin. Most studies on cfDNA focus on tumor-derived fragments for diagnostics or minimal residual disease detection. However, variations in the contribution of immune cells and other stromal cell populations could provide important clues to classify a tumor with respect to treatment. To address this, we collected tumor and adjacent normal tissue as well as cfDNA from the blood of 33 patients enrolled in the LUD2015-005 trial in which ICI were administered to patients with non-resectable esophageal adenocarcinoma (EAC) for four weeks, before adding standard-of-care chemotherapy (ICI+CTX). For each patient, we took samples at diagnosis, before ICI+CTX, during and at the end of treatment. Tissue and cfDNA samples were sequenced using TET-Assisted Pyridine-borane Sequencing (TAPS), a method recently developed in Oxford which provides genome-wide high-depth, base- resolution DNA methylation and mutation information from low-input samples. We then selected a representative set of high-purity tumor tissue samples and combined them with whole- genome TAPS data from 9 healthy blood cell types and 5 gastrointestinal tissues to generate a GI-specific atlas of cell-type specific methylation. This atlas allows us to accurately quantify the contribution of tumor and 14 healthy cell types to the cfDNA collected from each patient at each timepoint. Strikingly, the presence of T-cell derived cfDNA at time of diagnosis (tDNA) is a strong positive predictive marker of overall survival (p=0.0024). In contrast, the fraction of tumor-resident T-cells, estimated by either bulk tissue TAPS or RNA sequencing, correlated only weakly with treatment response in this cohort, indicating that T-cell activity is more accurately captured by tDNA. Furthermore, we find that patients with a combination of high TMB and high tDNA have an 80% 2-year survival probability, as opposed to below 25% for others, suggesting that tDNA is derived from T-cells actively engaging tumor cells with high neoantigen load. In summary, we demonstrate that T-cell turnover, as reflected in differential methylation in a set of marker regions spanning only 24kb, is a promising biomarker of treatment response, especially when combined with neoantigen load: in the presented cohort, all but one long-term survivors (alive at last follow up &gt; 3 years after diagnosis) were tDNA positive with high TMB. This demonstrates the power of cfDNA composition analysis for risk stratification in ICI treatment.
Citation Format: Phil Xie, Zohar Etzioni, Chun-Xiao Song, Richard P Owen, Mark R Middleton, Xin Lu, Benjamin Schuster-Boeckler. A T-cell signature in circulating cell-free DNA at time of diagnosis predicts response to checkpoint inhibition [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR017.