Optimization of CTC Recovery Methods and Biomarker Analysis in Hepatocellular Carcinoma Patients

Clinical Application: pharmacodynamic analysis of compound therapy in hepatocellular carcinoma (HCC) patients

Key Words: CTCs, protein expression, pharmacodynamics, head-to-head comparison, ApoStreamTM, HCC

Background: Hepatocellular carcinoma (HCC) is a rare cancer type in the United States; a large number of HCC cases are from distant metastases arising from other cancers, indicating that circulating tumor cells play a role in the development of this disease. This study examines the pharmacodynamic effects of a combination therapy regimen – an HDAC inhibitor coupled with a Raf pathway inhibitor – in isolated CTCs by examining biomarkers that govern a common apoptotic pathway. The biomarkers of interest are direct participants in the pathway under examination and an inhibitory protein that can stop the signaling cascade.

Methods: Blood was drawn from up to ten HCC patients and CTCs enriched using both the CellSearch® CTC kit and the ApoStream™ device. CTCs were enumerated through Laser Scanning Cytometry (LSC) as cells exhibiting a DAPI+/CK+/CD45- phenotype, counts from the two platforms compared, and the ApoStream™ was selected for use in the remainder of the trial. In the second phase, blood from up to an additional ten HCC patients was drawn and enriched using the ApoStream™. Enriched cells were stained for the CTC phenotypic markers and the biomarkers of interest for the trial as discussed above.


Figure 1. CTC enumeration results from ten pilot patients. Alpha fetoprotein (AFP) was used as both a screening method and a downstream biomarker for further cellular classification. Results illustrate ApoStreamTM serves as a capable system for the isolation of circulating tumor cells, recovering many more cells than the CellSearch® CTC kit. ApoStreamTM also isolates potential CTCs of other phenotypes (CK-/CD45-/AFP+ and CK+/CD45+/AFP+). Figure 2. Typical LSC channel images for CTCs. The red arrows on the scatter images represent CTCs (CK+/CD45-/AFP+); the turquoise arrows represent PBMCs. EpCAM was examined to illustrate both tumor heterogeneity and the capability of the ApoStreamTM to isolate both EpCAM+ and EpCAM- CTCs.

ApoCell has illustrated the capabilities of the ApoStreamTM as a means of isolating circulating tumor cells from patients with HCC and incorporated its use into an ongoing clinical

References: Poklepovic, A. et al. “ApoStreamTm, an Antibody Independent Platform, Captures Circulating Tumor Cells in Patients with Hepatocellular Carcinoma.” AACR, 2012.