Development of Circulating Tumor Cell Sequencing as a Biomarker in Gastrointestinal Cancers

For precision oncology to become a reality, clinicians need access to tumor tissue in a real-time, repeatable, and cost-effective manner. Circulating tumor cells (CTCs), cells of tumor origin that circulate in the blood, are one possible solution, with the potential to serve as a “liquid biopsy” for gastrointestinal (GI) tumors. In this thesis, we demonstrate the development and validation of an assay for performing molecular analysis of CTCs as a biomarker for GI cancers. We used a microfluidic platform to develop GI tumor-specific CTC assays for pancreatic and liver cancer through incorporation of tissue-specific markers for CTC capture and identification. We then conducted prospective studies testing the utility of CTC enumeration as a biomarker in pancreatic (n=100) and liver (n=61) cancer. We found that the use of a tissue-specific assay resulted in higher CTC counts than in previous studies, that CTC counts correlated with increasing stage and that CTC enumeration may have diagnostic and prognostic utility as a biomarker in GI cancers. Using a modified assay that allows for the complete isolation of the CTCs identified, we confirmed the tumor origin of the CTCs we identified by comparison of mutational status with the primary tumor using both Sanger and next generation sequencing methods. After confirming their tumor origin, we developed a quality control assay that allowed us to determine how many CTCs we would need to isolate to reliably perform CTC molecular analysis. Using this metric, we modified our CTC sequencing protocol that resulted in reliable sequencing from as few as 3-5 CTCs as opposed to the 10-20 needed using the traditional CTC sequencing protocol. We then used our optimized CTC sequencing protocol in a prospective study to test the feasibility and potential utility of CTC molecular analysis as a biomarker in liver cancer. The research described in this thesis serves two primary goals. The first is as a validated method that allows for the use of CTCs in liquid biopsy-type applications for both HCC and PDAC patients. The second is as a framework for validating CTC assays and definitions to ensure the reliability and reproducibility of CTC sequencing results