A CIRCRNA BASED-LIQUID BIOPSY FOR NONINVASIVE AND EARLY DETECTION OF PATIENTS WITH ESOPHAGEAL SQUAMOUS CELL CARCINOMA

Background: Esophageal cancer incidence and mortality are projected to increase by 58.4% and 61.8% by the end of 2040, making esophageal squamous cell carcinoma (ESCC) – the most common subtype a significant public health threat. Serum levels of squamous cell carcinoma antigen (SCC-Ag) are currently used as a common tumor marker; however, it lacks adequate sensitivity and specificity for diagnosing patients with early-stage ESCC. In this study, we developed and validated a circulating circular RNA (circRNA)-based liquid biopsy assay for the early detection of ESCC.
Methods: We conducted a comprehensive genomewide expression profiling analysis of circRNAs to identify biomarkers that are overexpressed both in ESCC vs. adjacent mucosa, as well as in plasma from ESCC patients vs. healthy controls (HC), in two datasets (GSE112496 and GSE131969). Subsequently, we conducted quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays to assess circ-RNA expression levels in two independent clinical cohorts. The training cohort (n=351) from South Africa included 219 ESCC cases and 132 HC subjects, and the validation cohort (n=234) was from a genetically and ethnically different population from Japan and included 132 ESCC cases and 102 HC subjects. We trained our assay using machine-learning algorithms for the early detection of ESCC in the training cohort, which was subsequently confirmed in the independent validation cohort.
Results: The genomewide biomarker discovery phase initially identified a panel of 11 circRNAs that were overexpressed in patients with ESCC. After qRT-PCR analysis and LASSO-based regression, we trained a reduced panel of 5-circRNAs that proved robust in distinguishing patients with ESCC vs. HCs (AUC=0.84). We locked down the assay and subsequently validated the performance of this 5-circRNA panel (AUC=0.81) in an independent validation cohort. Notably, our 5-circRNA panel demonstrated a robust performance in identifying patients with potentially curable, localized stage I/II ESCC (AUC=0.82). Furthermore, our assay was superior to serum SCC expression and, more importantly, performed robustly even in the identification of cancer in otherwise clinically SCC-Ag-negative ESCC patients (<2.7 U/ml; AUC=0.82).
Conclusion: We report a novel 5-circRNA-based liquid biopsy assay for the early detection of patients with early-stage ESCC, which was superior to serum SCC-Ag analysis. The successful validation of our assay in ESCC cohorts from different genetic and ethnic backgrounds (South Africa vs. Japan) highlights the robustness and generalizability of our findings, with a promising potential of this noninvasive assay in clinical practice.