Longitudinal tumor-informed cell-free DNA whole genome sequencing coupled with transcriptomic analysis captures tumor burden dynamics in resectable gastroesophageal cancer

Introduction: Circulating tumor DNA (ctDNA) has been established as a promising biomarker for detecting residual disease and monitoring therapy response across the cancer care continuum, however its utility to assess response to neoadjuvant immune checkpoint inhibition (ICI) is still being investigated. We utilized a tumor-informed cell-free DNA (cfDNA) whole genome sequencing (WGS) approach to molecularly monitor tumor burden dynamics and link clinical outcomes with transcriptomic assessment of baseline tumors based on ctDNA status in gastroesophageal cancer treated with neoadjuvant ICI. Methods: We performed WGS on tumor (n=28), matched white blood cell (WBC, n=28), and serial plasma samples (n=97) from patients with resectable gastroesophageal cancer treated with neoadjuvant ICI and chemoradiation prior to surgical resection (NCT03044613). Tumor and WBC sequence data was used to identify tumor-specific single nucleotide variants (SNVs). High quality SNVs were utilized to determine the presence of ctDNA in plasma through a random forest machine learning model. ctDNA status and tumor fraction (TF) were assessed across four timepoints (baseline, post-ICI cycle 1, post-ICI cycle 2, and pre-operatively). Results were compared with tumor-naïve gene panel ctDNA targeted NGS (n=32 patients, n=152 samples) and correlated with clinical outcomes. Additionally, bulk RNA sequencing (RNAseq) was performed on baseline tumor samples (n=28) and utilized for gene set enrichment analyses (GSEA). Results: Patients with pre-operative ctDNA TF above the median had a significantly shorter overall survival (OS) compared to those with ctDNA TF below the median or undetectable ctDNA (logrank p < 0.0001). Among patients who were undetectable pre-operatively via the tumor-naïve targeted NGS panel, individuals with detectable ctDNA via the tumor-informed WGS approach attained a numerically shorter OS compared to individuals with undetectable ctDNA (logrank p=0.1). Similarly, patients with ≥95% reduction in cfDNA TF pre-operatively had longer recurrence-free (logrank p=0.002) and OS (logrank p= 0.0001). GSEA of RNAseq data from baseline tumors revealed enrichment of G2/M cell cycle checkpoint and E2F targets in tumors from individuals with detectable baseline ctDNA via the tumor-informed WGS approach (FDR-adjusted p=2.31e-12), suggesting that cfDNA TF detection captures cellular turnover in the blood stream. The upregulation of proliferation and cell cycle progression-associated gene sets was not observed in stratified analyses by ctDNA detection utilizing the tumor-naïve approach. Conclusions: Tumor-informed cfDNA WGS analyses accurately capture tumor burden dynamics and cellular turnover during neoadjuvant immunotherapy, opening a window of opportunity for further therapeutic intervention and optimization.