Identification and Enumeration of Circulating Tumour Cells in Dogs with Naturally - Occurring Appendicular Osteosarcoma Using Flow Cytometry

Abstract

Osteosarcoma (OSA) is a highly locally aggressive and metastatic neoplasm of dogs. Despite gold standard therapy (amputation or limb-sparing tumour resection followed by chemotherapy) the reported median survival time (MST) is 235-540 days. Detection of early tumour dissemination and metastasis using current image-based staging modalities represents a major challenge in veterinary and human cancer patients and affects prognostication. Circulating tumour cells (CTC) are released from the primary tumour, and have the potential to form distant metastases. Enumeration of CTCs is used for outcome prediction, monitoring disease progression, and evaluating therapy effectiveness in various human cancers. A flow cytometric assay was developed to detect and enumerate CTCs in dogs with OSA. The optimized protocol was applied to enumerate CTCs at standardized time points in a clinical cohort of 21 dogs with naturally occurring OSA and no evidence of metastasis, and findings were correlated with patient outcome and disease progression. At the end of the study period, 19 (90%) dogs had been euthanized, with two (10%) alive and free of detectable metastasis. Fifteen (79%) of 19 dogs were euthanized because of metastatic OSA-related disease. Overall survival times (ST) ranged from 88 to 1,058 days. Median ST was 374 days (lower quartile 242 days, upper quartile 687 days). The 1-, 2-, and 2.5-year survival rates were 52% (11/21), 24% (5/21), and 14% (3/21), respectively. CTCs were common and highly variable in dogs with OSA (mean 356 CTCs/1,000,000 leukocytes; range 0-4443 CTSs/1,000,000 leukocytes). In 12 of 15 (80%) dogs that developed metastases, a pre-metastatic spike in CTC number occurred within 100 days prior to the development of clinically detectable metastasis. This pre-metastatic CTC spike was detected on average 36.5 days (range: 1-100 days) prior to the development of clinical OSA metastasis, and was significantly associated with a shorter ST (301±64days (spike) vs. 626 (±55) days (no spike); p=0.0107). Dogs with a CTC pre-metastatic spike were 10x more likely to die compared to those who did not experience a spike. The results suggest that a pre-metastatic spike in CTC number may be a precursor to clinically apparent metastasis and could serve to guide clinicians in therapeutic decision-making in dogs with OSA.