Metastatic colorectal cancer (mCRC) is characterized by its extensive disease heterogeneity, suggesting that individualized analysis could be vital to improving patient outcomes. As a minimally invasive approach, the liquid biopsy has the potential to longitudinally monitor heterogeneous analytes. Current platforms primarily utilize enrichment-based approaches for epithelial-derived circulating tumor cells (CTC), but this subtype is infrequent in the peripheral blood (PB) of mCRC patients, leading to the liquid biopsy’s relative disuse in this cancer type. In this study, we evaluated 18 PB samples from 10 mCRC patients using the unbiased high-definition single-cell assay (HDSCA). We first employed a rare-event (Landscape) immunofluorescence (IF) protocol, which captured a heterogenous CTC and oncosome population, the likes of which was not observed across 50 normal donor (ND) samples. Subsequent analysis was conducted using a colorectal-targeted IF protocol to assess the frequency of CDX2-expressing CTCs and oncosomes. A multi-assay clustering analysis isolated morphologically distinct subtypes across the two IF stains, demonstrating the value of applying an unbiased single-cell approach to multiple assays in tandem. Rare-event enumerations at a single timepoint and the variation of these events over time correlated with progression-free survival. This study supports the clinical utility of an unbiased approach to interrogating the liquid biopsy in mCRC, representing the heterogeneity within the CTC classification and warranting the further molecular characterization of the rare-event analytes with clinical promise.

The liquid biopsy has the potential to improve current clinical practice in oncology by providing real-time personalized information about a patient’s disease status and response to treatment. In this study, we evaluated 161 peripheral blood (PB) samples that were collected around surgical resection from 47 metastatic colorectal cancer (mCRC) patients using the High-Definition Single Cell Assay (HDSCA) workflow. In conjunction with the standard circulating tumor cell (CTC) enumeration, cellular morphology and kinetics between time-points of collection were considered in the survival analysis. CTCs, CTC-Apoptotic, and CTC clusters were found to indicate poor survival with an increase in cell count from pre-resection to post-resection. This study demonstrates that CTC subcategorization based on morphological differences leads to nuanced results between the subtypes, emphasizing the heterogeneity within the CTC classification. Furthermore, we show that factoring in the time-point of each blood collection is critical, both for its static enumeration and for the change in cell populations between draws. By integrating morphology and time-based analysis alongside standard CTC enumeration, liquid biopsy platforms can provide greater insight into the pathophysiology of mCRC by highlighting the complexity of the disease across a patient’s treatment.

As the world’s third most common cancer and second leading cause of oncology-related deaths, colorectal cancer (CRC) poses a variety of clinical challenges. CRC is marked by its extensive solid tumor heterogeneity and the difficulties in longitudinally monitoring a patient’s disease status. Being a minimally invasive prognostic tool, the liquid biopsy has immense potential to improve current clinical practice by providing real-time personalized information over the course of treatment. Utilizing the High-Definition Single Cell Assay (HDSCA) workflow, we employed a “no cell left behind approach” to analyze peripheral blood samples from metastatic CRC (mCRC) patients at various timepoints during treatment. We highlight the circulating rare cell population present in the liquid biopsy of mCRC patients, showing the spatial and temporal heterogeneities of CTCs. We observed a significant association between CTC subcategorization based on morphological differences and survival, emphasizing the heterogeneity within the CTC classification. Moreover, considering the time of sample collection in relation to the clinical scenario is critical in determining the prognostic relevance of static cellular enumerations as well as the change in cell populations over time. Through the integration of cellular morphology in a time-based analysis, the liquid biopsy can provide insight into the pathophysiology of mCRC by highlighting the complexity of the disease to monitor treatment response. Citation Format: Sachin Narayan. Circulating tumor cell kinetics and rare cell morphology monitor disease progression in patients with metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1954.