Analysis of KRAS, NRAS and BRAF mutational profile by combination of in-tube hybridization and universal tag-microarray in tumor tissue and plasma of colorectal cancer patients

RESEARCH ARTICLE Analysis of KRAS, NRAS and BRAF mutational profile by combination of in-tube hybridization and universal tag-microarray in tumor tissue and plasma of colorectal cancer patients a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Francesco Damin ID1☯*, Silvia Galbiati2☯, Nadia Soriani2, Valentina Burgio3, Monica Ronzoni3, Maurizio Ferrari2,4,5, Marcella Chiari1 1 Istituto di Chimica del Riconoscimento Molecolare, CNR, Milano, Italy, 2 Unit of Genomic for the Diagnosis of Human Pathologies, Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy, 3 Dipartimento di Oncologia Medica, IRCCS Ospedale San Raffaele, Milan, Italy, 4 Laboratory of Clinical Molecular Biology, IRCCS Ospedale San Raffaele, Milan, Italy, 5 Università Vita-Salute San Raffaele, Milan, Italy ☯ These authors contributed equally to this work. * OPEN ACCESS Citation: Damin F, Galbiati S, Soriani N, Burgio V, Ronzoni M, Ferrari M, et al. (2018) Analysis of KRAS, NRAS and BRAF mutational profile by combination of in-tube hybridization and universal tag-microarray in tumor tissue and plasma of colorectal cancer patients. PLoS ONE 13(12): e0207876. https://doi.org/10.1371/journal. pone.0207876 Editor: Surinder K. Batra, University of Nebraska Medical Center, UNITED STATES Received: July 11, 2018 Accepted: November 6, 2018 Published: December 18, 2018 Copyright: © 2018 Damin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 766466. Abstract Microarray technology fails in detecting point mutations present in a small fraction of cells from heterogeneous tissue samples or in plasma in a background of wild-type cell-free circulating tumor DNA (ctDNA). The aim of this study is to overcome the lack of sensitivity and specificity of current microarray approaches introducing a rapid and sensitive microarraybased assay for the multiplex detection of minority mutations of oncogenes (KRAS, NRAS and BRAF) with relevant diagnostics implications in tissue biopsies and plasma samples in metastatic colorectal cancer patients. In our approach, either wild-type or mutated PCR fragments are hybridized in solution, in a temperature gradient, with a set of reporters with a 5’ domain, complementary to the target sequences and a 3’ domain complementary to a surface immobilized probe. Upon specific hybridization in solution, which occurs specifically thanks to the temperature gradients, wild-type and mutated samples are captured at specific location on the surface by hybridization of the 3’ reporter domain with its complementary immobilized probe sequence. The most common mutations in KRAS, NRAS and BRAF genes were detected in less than 90 minutes in tissue biopsies and plasma samples of metastatic colorectal cancer patients. Moreover, the method was able to reveal mutant alleles representing less than 0,3% of total DNA. We demonstrated detection limits superior to those provided by many current technologies in the detection of RAS and BRAF gene superfamily mutations, a level of sensitivity compatible with the analysis of cell free circulating tumor DNA in liquid biopsy. Competing interests: I have read the journal’s policy and the authors (Francesco Damin, Silvia PLOS ONE | https://doi.org/10.1371/journal.pone.0207876 December 18, 2018 1 / 15 Tissue and ctDNA genotyping by combination of in-tube hybridization and universal tag-microarray Galbiati, Maurizio Ferrari and Marcella Chiari) of this manuscript have the following competing interests: they have filed one patent application (PCT/IB2018/052219 Title: Genotyping of mutations by combination of in-tube hybridization and universal tag-microarray) on the method described in this paper. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Introduction The identification of DNA variants that can cause diseases is a central aim in human genetics. In particular, the ability to detect mutations in oncogenes facilitates early diagnosis, monitoring and treatment [1,2] of cancer. The discovery that tumor cells release DNA fragments (circulating tumor DNA -ctDNA-) in blood, urine or other body fluid samples, paves the way to a paradigm shift in cancer diagnostics introducing the concept of liquid biopsy: a term that refers to a novel, non-invasive technique used for detecting cancer biomarkers [3,4]. ctDNA belongs to the pool of the total circulating cell free-DNA in blood. The mechanisms of its release are not completely disclosed; probably it derives from apoptotic or necrotic cells as well as from living cells through a mechanism of active secretion. ctDNA provides real-time molecular information allowing monitoring treatment response and relapsing as it contains genetic alteration of both primary and metastatic lesions, such as point mutations, copy number variations and insertions/deletions [5,6]. Detecting mutation in ctDNA is challenging since the lower number of mutant copies of cancer origin are masked by the large amount of wild-type DNA mostly from contaminant leukocytes [7]. Liquid biopsy is still in its infancy and efforts will be required before the field can mature and achieve widespread routine use in oncology clinical practice. The analysis of low-abundance mutations requires cfDNA isolation and amplification followed by mutations detection either in disease specific genes (PCR based sequencing) [8–12] or in multiple genes simultaneously (next generation sequencing -NGS- multiplex testing) [13]. Droplet digital PCR (ddPCR) is one of newly developed methods that allow for enumeration of rare mutant variants. Based on water-emulsion droplet technology, ddPCR fractionates a DNA sample in 20.000 droplets [14]. Mutation-specific amplification of the template subsequently occurs in each individual droplet, and counting the positive droplets gives precise, absolute target quantification as copies per milliliter of plasma. It was reported that ddPCR can detect mutant alleles with high sensitivity (0.01–0.001%) [15]. However, with ddPCR only the genes that are the most susceptible to mutations are analyzed, at first, giving the patient the choice of whether to pursue additional tests based on the results. The downside of this approach to testing is it is time and cost consuming. Another sophisticated ctDNA based cancer test is the targeted amplicon sequencing [16,17]. NGS in particular conditions, can reach the high sensitivity required for the analysis of ctDNA. This technique has the potential to uncover additional actionable findings that cou (...truncated)