EP535 - Phenotyping Circulating Hybrid Cells Across the Colorectal Cancer Continuum

Purpose/Background: Colorectal cancer (CRC) is the second leading cause of death in the United States. Colonoscopy has improved early detection of malignancy and pre-malignant lesions with the highest potential for progression to malignancy (called advanced precursor lesions [APL]); however, compliance is poor, and the procedure itself is costly and resource-intensive with high-risk lesion requiring frequent repeat endoscopies, failing those with limited access to healthcare. Hybrid cells have been identified in CRC and result from the fusion between tumor and immune cells. When detected in blood, hybrid cells are called circulating hybrid cells (CHCs). Hybrid cells have been detected across the cancer continuum in APL, primary, and metastatic tumors as well as in the blood at each stage.

Methods/Interventions: To identify and phenotype hybrid cells across the cancer continuum, we analyzed an outside, publicly available single RNA cell-sequencing (scRNA-seq) dataset that represented N=63 patients for the presence of hybrid cells, deploying stringent doublet discrimination to identify this cell population. To determine if hybrid cells were present in patients at various stages of CRC, we separately collected N=71 colon tissue and blood samples from patients at the time of surgery or endoscopy. Peripheral blood mononuclear cells (PBMCs) were isolated using standard Ficoll gradient centrifugation and processed onto glass slides. We then performed standard immunofluorescent stains for epithelial proteins [E-cadherin (E-cad), epithelial cell adhesion molecule (EpCAM), and pan-cytokeratin (pan-CK)], the pan-leukocyte marker CD45, and proteins that were differentially expressed in normal colon compared to CRC. CHCs were identified as cells co-expressing epithelial proteins and CD45, and were detected in all patients analyzed. We quantified numbers of CHCs in each specimen and analyzed their expression of CRC-associated proteins to determine if levels of CHCs or discriminating CRC-specific phenotypes could differentiate patients with APL versus CRC.

Results/Outcomes: Hybrid cells were detected in all CRC patients within the scRNA-seq datasets and harbored discrete signatures when compared across primary, lymph node and metastatic tumors, indicating that discrete signatures could differentiate hybrid cells from different stages across the cancer continuum. To determine if CHC detection and phenotyping could differentiate patients with APL from those with CRC, we analyzed blood samples from N=71 patients [N=23 healthy, N=26 APL, and N=22 CRC]. We performed further phenotypic analysis on blood and tissue for N=3 CRC, N=6 APL, and N=2 healthy control samples. There were significantly more CHCs in patients with APL compared to healthy controls, and significantly more CHCs in patients with CRC compared to those with APL. CHCs expressing CRC-specific protein expression were found to be present in different numbers in patients with CRC compared to APL.

Conclusion/Discussion: This preliminary study demonstrates that CHCs offer promise as an early biomarker for the development of pre-cancerous and malignant lesions in the colon. Though the generalizability of this data is limited by low sample number, phenotypic expression patterns may be leveraged to improve the specificity of CHCs as a biomarker for early detection of CRC.