Detection of VAR2CSA‐captured colorectal cancer cells from blood samples by real‐time reverse transcription PCR

Analysis of circulating tumor cells (CTCs) from blood samples provides a non‐invasive approach for early cancer detection. However, the rarity of CTCs makes it challenging to establish assays with the required sensitivity and specificity. We combine a highly sensitive CTC capture assay exploiting the cancer cell binding recombinant malaria VAR2CSA protein (rVAR2) with the detection of colon‐related mRNA transcripts (USH1C and CKMT1A). Cancer cell transcripts are detected by RT‐qPCR using proprietary Target Enrichment Long‐probe Quantitative Amplified Signal (TELQAS) technology. We validate each step of the workflow using colorectal cancer (CRC) cell lines spiked into blood and compare this with antibody‐based cell detection. USH1C and CKMT1A are expressed in healthy colon tissue and CRC cell lines, while only low‐level expression can be detected in healthy white blood cells (WBCs). The qPCR reaction shows a near‐perfect amplification efficiency for all primer targets with minimal interference of WBC cDNA. Spike‐in of 10 cancer cells in 3 mL blood can be detected and statistically separated from control blood using the RT‐qPCR assay after rVAR2 capture (p < 0.01 for both primer targets, Mann‐Whitney test). Our results provide a validated workflow for highly sensitive detection of magnetically enriched cancer cells.