- EVALUATION OF ISOLATION AND ANALYSIS SYSTEM FOR CTCS. PREDICTION OF ANTICANCER DRUG RESPONSES USING CIRCULATING TUMOR CELLS (CTCS) ANALYSIS. - EVALUATION OF ISOLATION AND ANALYSIS SYSTEM FOR CTCS. Duyeol HAN1, Dong Seok LEE1, June Sub LEE1, Hyun-Kyung LEE1, Dongin KIM2, Jaehyun EOM2, Dong-Hoon YEOM2, Jin-Hyung AHN2, Weon-Kyoo YOU2, Sang Hoon LEE2, Myoung Shin KIM1, Byung Hee JEON1 1Department of Bio Institute, Cytogen, Inc., Korea, 2Department of R&d Center, Abl Bio, Inc., Korea substitute method for tumor tissue biopsy, and may provide clinical applications. [Background] Delta-like ligand 4 (Dll4) and vascular endothelial growth factor (VEGF) are known as important factors in angiogenesis and cancer cell proliferation. The FDA-approved drugs which inhibit VEGF signaling have been used as anti-cancer drug for various cancer types. ABL001, bi-specific antibody drug, is a novel anti-cancer drug targeting VEGF and Dll4, which will enter phase I clinical trial in next month. In this study, we developed platform tools to isolate circulating tumor cells (CTCs) and to evaluate change of gene expression relating with Dll4/Notch signaling pathway as a pharmacodynamic marker. [Methods] Isolation and analysis system for CTCs were evaluated in a colon cancer cell line, colo205, spiked in blood samples from healthy volunteers. Isolated colo205 cells were immunofluorescent stained for Dll4, NICD, or Notch1. The change of gene expression was evaluated by the expression levels of Dll4/Notch downstream genes. The peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers after ABL001 treatment and then the mRNA levels of the target genes of Dll4/Notch signaling pathway were measured by qRT-PCR. [Results] The recovery rates of colo205 cells from spiked blood samples were determined up to 70% , and the limit of detection (LOD) was 10 cells per reaction. We confirmed that isolated cancer cells expressed CTC marker, Dll4, Notch1 and NICD. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) for target genes of Dll4/Notch signaling pathway was evaluated at 10 copies per reaction of LOD. Expression patterns of Hey1, Hey2, Hes1, Hes5, SFRP2, Notch1 and Dll4 in the isolated cell lines and PBMCs were analyzed and compared. [Conclusion] Various CTC analysis platforms for cancer incidence, prognosis and companion diagnostics (CDx) have been reported and many of them are currently evaluated for clinical sensitivity and specificity. In this study, we showed that our CTC isolation platform has high sensitivity, high recovery rate and effective LOD. In addition, qRT-PCR results revealed that the gene expression analysis was practical in isolated cells via the CTC isolation platform. These results suggest that the CTC isolation platform can be used for the clinical applications as a pharmacodynamic marker. Abstract Materials and Methods Immunofluorescent staining Blood + Colo205 cells CTC enrichment using CytoGen platform Identification of CTCs by IF staining Analysis of Notch1 signaling target gene expression level Blood 6h, 24h, 48, 72h incubation at 37℃ Isolation of PBMC qRT-PCR Blood + ABL001 Results Recovery rate Representative immunofluorescent staining images Recovery rate of spiked cells. Recorvery rate were resulted in up to 70%, LOD was 10 cells/rxn. Spike-in test using Colo205 cell lines. Colo205 cells were enriched by CytoGen platform. Isolated Colo205 cells were immunofluorescent stained for Dll4, NICD, or Notch1. qRT-PCR evaluation Gene Hey1 Hey2 DLL4 SFRP2 Hes5 SPON2 DTX1 FOXO3 BCL2L1 GAPDH LOD (copies/rxn) 1 10 100 1,000 Lower limit of detection for qRT-PCR. qRT-PCR for target genes of DLL4/Notch signaling pathway evaluated at 10 copies per reaction of LOD mRNA expression after ABL001 treatment Blood A Blood B Effect of ABL001 on mRNA expression level of target genes in PBMCs. PBMCs were either untreated or exposed to ABL001 (150ng/ml) for 6, 24, 48, or 72 hr. The mRNA expression level of 12 target genes from healthy donor blood A and B was determined by qRT-PCR.