Therapeutic Target Analysis in CTCs Isolated from NSCLC Patients Using ApoStream®

Clinical Application: Identification of folate receptor alpha (FRA) positive CTCs for patient stratification

Key Words: CTCs, ApoStream®, patient stratification, protein expression, NSCLC

Background: This study was initiated to explore the utility of using biomarker expression levels in CTCs as a means of patient selection for treatment in a clinical trial. The drug being examined is a monoclonal antibody that binds to folate receptor alpha (FRA), which is estimated to be present in up to three-quarters of patients with adenocarcinoma of the lung, a form of non-small cell lung cancer (NSCLC). ApoCell was contacted to develop an assay using our proprietary antibody-independent CTC capture device, ApoStream®, which will be used as a screening mechanism for inclusion into an ongoing trial with this drug – patients who strongly express the drug’s target represent an ideal test group.

Methods: Blood was collected from 14 NSCLC patients and processed for CTC enrichment by ApoStream®. For the first 9 patients, CTCs were also enriched by the CellSearch® CTC kit, and CK+/CD45-/DAPI+ cell counts from the two systems compared. For the remaining five patients and 15 healthy donors, CTCs were enriched by ApoStream® alone. The cells enriched by ApoStream® were placed on glass slides and stained for cytokeratin (CK), CD45, DAPI, and FRA. The stained cells were then analyzed using Laser
Scanning Cytometry (LSC) for the following: total number of CTCs (cells exhibiting a DAPI+/CK+/CD45- phenotype) that are positive for FRA, the total number of CD45+ cells expressing FRA, and the mean fluorescence intensity (MFI) of the biomarker in both CTCs and CD45+ cells.

Results:

Figure 1. Head to Head Comparison. CellSearch® enrichment and identification of CTCs from NSCLC patients was low (mean: 1.56, median: 0); ApoStream enriched significantly higher counts
(mean: 163, median: 81).
Figure 2. FRA Analysis. ApoStream® enriched CTCs from all 5 additional NSCLC patients, and no CTCs from 15 tested healthy donors. FRA was found to be expressed on a subset of CTCs (range: 8.1% – 32.8%) from each patient, but not in the CD45+ leukocyte population, indicating FRA may serve as a suitable cancer specific marker for targeted therapies. Figure 3. Representative LSC Images of CTCs.  ApoStream® enriched CTCs stained with CTC phenotype markers and FRA. Yellow arrows in each channel indicate identified CTCs (cells possessing a CK+CD45-DAPI+ phenotype) that are expressing FRA.

Impact: : ApoCell has demonstrated the feasibility of using folate receptor alpha expression (or the expression of other similar targets) on ApoStream®-enriched CTCs to inform patient selection strategies in a clinical trial.

Reference: Davis DW, et al. “Antibody-Independent ApoStream™ Technology Isolates Folate Receptor Alpha (FRA) ApoCell, Inc.
Positive Circulating Tumor Cells from Blood of Non-Small Cell Lung Adenocarcinoma Patients.” ePub Abstract, ASC0 2012.