Abstract
Non-invasive molecular analysis of circulating tumor DNA (ctDNA) is a promising application in personalized cancer management, although there is still much to learn about the biological characteristics of ctDNA. The present study compared absolute amounts of KRAS mutated ctDNA and total circulating cell-free DNA (cfDNA) in colorectal cancer (CRC) patients (n=50) from various stages and healthy controls (n=8) by Intplex allele-specific and digital droplet PCR. In addition, the impact of two prominent extraction techniques (silica-based membrane vs. magnetic beads) on cfDNA and ctDNA recovery was analyzed in 38 paired samples from CRC patients and specific spike-in DNA controls. CfDNA fragment size was assessed using the Agilent 2100 Bioanalyzer. Relative quantities of total cfDNA quantities were measured using the Qubit fluorometer. Statistical analysis on total cfDNA yield revealed a strong correlation (r=0.976) between Qubit and absolute Intplex allele-specific PCR measurements in cancer patients and healthy controls. Total cfDNA was significantly increased in cancer patients compared to healthy controls, with the highest yield in distant metastatic disease. In line, the highest amount of ctDNA (1.35 ng/μL) was found in patients with distant organ metastasis. Of great interest, the silica-based membrane method significantly promoted extraction of long cfDNA fragments. In contrast, the magnetic bead system more efficiently recovered short cfDNA fragments in serum of cancer patients. Further, a decreased KRAS allele frequency was observed in serum compared to plasma. This study suggests that the source of cfDNA and choice of pre-analytical extraction systems needs to be more carefully validated in routine clinical practice.
Highlights
Blood-based molecular analysis of tumor-derived circulating DNA is becoming an established tool for monitoring tumor burden and detection of resistance early during targeted cancer therapies [1,2,3]
Keeping in mind that clinically highly valuable patient collectives [18,19,20,21,22] are based on serum samples, we wondered if innovative Intplex polymerase chain reaction (PCR), first published by Thierry et al [6], could be a reliable technology for sensitive analysis of oncological Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in colorectal cancer (CRC) patients’ serum samples as well
Digital PCR methods revealed a maximal technical detection limit of 0.01%, sensitivity of the mentioned mutations in circulating tumor DNA (ctDNA) was unexpectedly low in some studies [7, 23]
Summary
Blood-based molecular analysis of tumor-derived circulating DNA (ctDNA) is becoming an established tool for monitoring tumor burden and detection of resistance early during targeted cancer therapies [1,2,3]. Bettegowda and colleagues already revealed an extreme variability of mutated ctDNA fragments in e.g. colon cancer patients (between 1 and 100.000 mutated fragments) illustrating www.impactjournals.com/oncotarget the need for highly sensitive detection techniques. Recent studies investigating the secondary EGFR T790M resistance mutation in advanced non-small cell lung cancer (NSCLC) patients showed that about 2030% of tissue T790M mutations were not detectable in plasma-ctDNA, even with highly sensitive digital PCR technologies [7]. While ctDNA-based detection of the EGFR T790M mutation is already an approved diagnostic application in those cases where no tissue biopsy is available, it becomes clear that recent developments in liquid biopsy are rather based on innovative technologies than on an established knowledge about the analytes themselves
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