Clinical Application of Enumeration and Genomic Characterization for Non-invasive Detection and Real-time Monitoring of Circulating Tumor Cells for Esophageal Carcinoma

Abstract

Introduction: Esophageal squamous cell carcinoma (ESCC) is deadly cancer with its highest incidence worldwide amongst Chinese. Despite current upfront chemoradiotherapy (CRT) plus surgical approaches, ESCC patient survival rates are dismal. There is still a need for early predictive markers to track treatment responses. Advances in enrichment and identification of circulating tumor cells (CTCs), which can escape from both primary and metastatic tumors into the blood of cancer patients, allows real-time monitoring of CTC levels at diagnosis/ during/after treatment to assess treatment efficacy. Project Objectives: • Determine the clinical usefulness of noninvasive real-time monitoring of CTCs in ESCC patients during treatment in comparison to current CT/PET imaging • Utilize next-generation sequencing (NGS) targeted gene sequencing to examine genetic changes before/during/after treatment to identify useful biomarkers for metastasis and drug resistance Identification of key driver genes for metastasis and druggable targets from CTC analysis is expected to improve diagnosis and targeted treatment of metastatic ESCC and improve patient precision care. We investigated the prognostication and risk stratification role of liquid biopsy serial monitoring for ESCC. Methods: CTCs and plasma cell-free DNA (cfDNA) were isolated from 199 blood samples of 103 advanced ESCC patients treated by CT or CRT/surgery at serial treatment timepoints. CTCs were isolated using size separation on microfluidic chips and enumeration by immunofluorescent staining with antibody cocktails of CD45/EpCAM/CK/MUC1. Kaplan Meier curve, logrank test, and COX regression analysis were used for statistical analysis of disease relapse and survival. Results: In 57 ESCC patients receiving palliative CT, high CTC counts at CT pre-cycle III is independently associated with response at interim reassessment and progression-free survival (PFS) in multivariate COX analysis. Integration of changes of both baseline pre-cycle III CTC and cfDNA into four risk groups based on the number of favorable/unfavorable changes of CTC/ cfDNA, were independently associated with overall survival (OS) by multivariate COX analysis. In 43 loco-regionally advanced ESCC treated by CRT/surgery, high CTC counts at end of CRT/ pre-operation significantly associated with early-progression at 10-month PFS. Conclusion: CTC counts at pre-cycle III and combined changes of CTC/cfDNA are independent prognostic markers for ESCC patients receiving palliative CT. CTC counts at post CRT is predictive for early disease progression for CRT/surgery-treated ESCC patients. Implications: Longitudinal liquid biopsy serial monitoring provides complementary information for prediction/prognosis for CT responses in advanced ESCC. The CTC/cfDNA bloodbased diagnostics have potential clinical utility for non-invasive monitoring of minimal disease burden to better guide clinical treatment.