An Ultrasensitive Molecular Diagnostic Method for Blood Biopsy in Personalized Treatment of Colorectal Cancer

Abstract

ABSTRACT Aim: KRAS status is given as a predictive molecular biomarker of responsiveness to targeted therapy in metastatic colorectal cancer (mCRC). Previous evidence shows that blood-based testing can track the emergence of drug resistant and provide an early warning of treatment failure. However existing methods have complicated protocols and insufficient sensitivity for clinical liquid biopsies. For the analysis of circulating cell free DNA (ccfDNA), we have constructed a simple and ultrasensitive genotyping method which is based on Invader Plus technology. Here we report the clinical validation study of the method, comparing it to droplet digital PCR (ddPCR) system on the detection of KRAS and BRAF mutations. Methods: Assays were set up using plasmids containing hot spots KRAS (G12A, G12C, G12D, G12R, G12S, G12V, G13D, G61H, Q61L and Q61R) and BRAF (V600E) mutations. ccfDNA samples were purified from serum samples (n = 74) of mCRC patients (n = 50). Analysis of KRAS seven point mutations in codon 12 and 13 was compared from one tumor-tissue FFPE block versus one blood sampling. Mutations of KRAS in codon 61 and BRAF in codon 600 were additionally determined from residual DNA samples. Droplet digital PCR system was performed by QX-100 (BioRad). Results: Invader Plus method clearly detected KRAS mutations at a 0.2% level (max. 0.01%) in the plasmid titration study. In the clinical performance study, as a result (Table), the method showed 81.8% specificity and 91.7% sensitivity in comparison with ddPCR. Invader Plus could detect highly variable mutation load which is the ratio as the proportion of mutant alleles in ccfDNA (0.3–48.1%, median 2.6%). Besides, numbers of ccfDNA fragments in the sera were quite broad (1.3 x 103 copies/ml–6.4 x 105 copies/ml, median 8.1 x 103). 2 x 2 comparison of ddPCR and Invader Plus for the KRAS mutation detection in the ccfDNA ddPCR Mt Wt total Invader Plus Mt 11 2 13 Wt 1 9 10 total 12 11 23 Conclusions: In the real-time monitoring using blood based testing, this method possesses several other advantages including its all-in-one chip reaction, simple procedure and excellent sensitivity. This method is a promising clinical tool for noninvasive assessment of emerging resistance and early relapse. Disclosure: All authors have declared no conflicts of interest.