Real-time serial monitoring of liquid biopsies allows earlier detection of treatment responses and relapse for metastatic esophageal squamous cell carcinoma

Abstract

Purpose: Esophageal squamous cell carcinoma (ESCC) is a deadly cancer, frequently diagnosed at advanced stages, when it has already metastasized, making treatment difficult and survival low. We utilized liquid biopsies from ESCC patients for real-time monitoring of treatment responses and detection of relapse and metastasis. We aimed to determine the usefulness of circulating tumor biomarkers for monitoring disease burden and provide real-time assessment of the evolving tumor under treatment pressure. Experimental methods: Blood was collected from metastatic ESCC patients before, during, and at the end of treatment. It was processed to lyse red blood cells and enrich for circulating tumor cells (CTCs). Spiral microfluidics chips for size separation enriched for larger CTCs and removed smaller white blood cells. CTC enumeration was performed by immunofluorescence staining using antibody cocktails [pan-cytokeratin (CK), epithelial cell adhesion molecule (EpCAM), and clusters of differentiation 45 (CD-45)]. We performed next-generation sequencing (NGS) target capture analysis for a customized 300-gene panel of selected cancer-relevant and druggable genes. cfDNAs extracted from plasma were utilized to determine mutation profiles for circulating tumor DNA (ctDNA) with the Roche AVENIO ctDNA 77-gene pan-cancer kit. Mutations or amplifications detected were validated by Sanger sequencing or digital PCR. Summary: The sensitivity and specificity of the cfDNA platform was determined using in-house controls with known mutations, which were validated by digital PCR. At the baseline timepoint, genetic alterations were detected in 90.3% of 31 ESCC patients. TP53 gene was the most frequently mutated with point mutations detected in 77% of ESCC patients and EGFR gains in 19% of patients. Longitudinal cfDNA analysis showed good concordance between mutant allele frequency and tumor mutation burden, suggesting the usefulness of real-time non-invasive monitoring to detect minimal residual disease. Analysis of cfDNAs also provides the opportunity to determine the extent of inter-tumoral heterogeneity between primary and metastatic tumors. Our preliminary data show CTC enumeration at ~6 weeks pre-cycle III for patients treated with palliative CT and at end of chemo-radiation therapy (CRT) for locally advanced ESCC patients, may be useful prognostic and predictive biomarkers for treatment efficacy and to gauge risk relapse and survival. Ongoing studies will increase sample sizes. The clinical relevance of serial CTC enumeration data and the cfDNA and CTC mutational load detected will be correlated with disease progression to determine their usefulness for prognostication. Conclusions: Serial analysis of CTCs and ctDNAs of metastatic ESCC patients indicates the usefulness of real-time monitoring using liquid biopsies to assess tumor mutation burden and to predict treatment efficacy, disease relapse, and patient survival. Acknowledgements: TBRS grant T12-701/17R and HMRF grant 05160926 to MLL