Abstract 592: Circulating tumor cells of esophageal and gastric cancers
Authors: Erkizan Hayriye Verda , Wadleigh Robert G. 
Published: July 2021

Abstract
Background: The availability of circulating tumor cells (CTCs) creates a powerful non-invasive approach to monitor tumor progression, therapeutic response, relapse, metastasis, and, in some cases, provide early diagnosis. Esophageal cancer (EC) is the fifth most common cause of cancer-related death in men 40-59 years of age, with a ~90% death rate. Gastric cancers (GC) have a 30% five-year survival rate. Smoking, alcohol, age, H. pylori infection, and specific diets are established risk factors for esophageal and gastric cancers. We aim to isolate, propagate, and identify RNA expression profiles in CTCs from EC and GC patients. This study permits the potential clinical application of identifying markers that may define prognosis, metastatic potential, and therapeutic efficacy.
Materials and methods: Venous blood samples from the esophageal and gastric cancer patients (n=6) were collected before and a week after chemotherapy. A negative enrichment method was performed to isolate CTCs using EasySep™ Direct Human CTC Enrichment Kit (StemCell Technologies). Label-free and viable CTCs were cultured in Epicult-C (StemCell Technologies) containing proliferation supplement and hydrocortisone. After rRNA depletion, strand-specific paired-end sequencing libraries for approximately 100M read-depth will be prepared by SMART-Seq v4 Ultra Low Input RNA Kit (Clontech) for RNA sequencing. Libraries will be constructed using the Illumina Nextera XT kit. The Washington DC VAMC Institutional Review Board approved this study and written informed consent was obtained from patients.
Results: We observed a dynamic range of CTCs in the same patient during chemotherapy treatments. The fluctuation of the number of cells obtained before and a week after infusion may represent the effect of therapy on both primary and circulating tumor cells. Samples with clusters of CTCs preserved their structures during cell culture. Our most extended CTC sample culture approached 55 days, which obtained a patient with intestinal-type gastric cancer.
Conclusion and future directions: We expect to identify differentially expressed genes in CTCs and the primary tumor, along with alternatively spliced isoforms in both primary tumor and CTCs. The fluctuation of the number of cells obtained before and a week after infusion may represent the effect of therapy on circulating tumor cells. RNA sequencing of CTCs will be performed and serve as baseline data for functional studies. This study permits monitoring therapeutic efficacy, identifying therapy-resistant or therapy-sensitive clones/markers that may allow the selection of appropriate therapeutic regimens. We will continue to follow patients and obtain tissues from lymph node or distant metastasis to analyze the evolution of their tumors.
Citation Format: Hayriye Verda Erkizan, Robert G. Wadleigh. Circulating tumor cells of esophageal and gastric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 592.