Liquid biopsy in colon cancer: comparison of different circulating DNA extraction systems following absolute quantification of KRAS mutations using Intplex allele-specific PCR
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Vera Kloten1, Nadine Rüchel1, Nadina Ortiz Brüchle1, Janina Gasthaus1, Nils Freudenmacher1,2, Florian Steib1, Jolein Mijnes1, Julian Eschenbruch1, Marcel Binnebösel3, Ruth Knüchel1 and Edgar Dahl1,2
1Molecular Oncology Group, Institute of Pathology, University Hospital Aachen, Aachen, Germany
2Centralized Biomaterial Bank of RWTH Aachen University (RWTH cBMB), Institute of Pathology, University Hospital Aachen, Aachen, Germany
3Department of Visceral and Transplantation Surgery, University Hospital Aachen, Aachen, Germany
Vera Kloten, email: [email protected]
Keywords: liquid biopsy, KRAS, cfDNA extraction systems, ctDNA quantification, intplex-allele specific PCR
Received: November 30, 2016 Accepted: August 05, 2017 Published: September 21, 2017
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.
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