Abstract
Molecular diagnostics of human cancers may increase accuracy in prognosis, facilitate the selection of the optimal therapeutic regimen, improve patient outcome, reduce costs of treatment and favour development of personalized approaches to patient care. Moreover sensitivity and specificity are fundamental characteristics of any diagnostic method. We developed a highly sensitive microarray for the detection of common KRAS and BRAF oncogenic mutations. In colorectal cancer, KRAS and BRAF mutations have been shown to identify a cluster of patients that does not respond to anti-EGFR therapies; the identification of these mutations is therefore clinically extremely important. To verify the technical characteristics of the microarray system for the correct identification of the KRAS mutational status at the two hotspot codons 12 and 13 and of the BRAFV600E mutation in colorectal tumor, we selected 75 samples previously characterized by conventional and CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) followed by High Resolution Melting analysis and direct sequencing. Among these samples, 60 were collected during surgery and immediately steeped in RNAlater while the 15 remainders were formalin-fixed and paraffin-embedded (FFPE) tissues. The detection limit of the proposed method was different for the 7 KRAS mutations tested and for the V600E BRAF mutation. In particular, the microarray system has been able to detect a minimum of about 0.01% of mutated alleles in a background of wild-type DNA. A blind validation displayed complete concordance of results. The excellent agreement of the results showed that the new microarray substrate is highly specific in assigning the correct genotype without any enrichment strategy.
Highlights
Defining the molecular signature of human cancers could be central to the development of a personalized approach to patient care
We used two cell lines provided by ATCC-LGC Standards Partnership: the CCRF-CEM cell line as reference for KRAS mutation p.G12D and the human melanoma cell line A375 as the source of homozygous BRAFV600E DNA
All of the 75 samples were screened for the research of KRAS mutations and BRAFV600E by High Resolution Melting (HRM) and sequencing
Summary
Defining the molecular signature of human cancers could be central to the development of a personalized approach to patient care. Therapeutic agents targeting specific genetic variants and well characterized molecular pathways have been developed. This is the case of the oncogene KRAS which is part of the signaling pathway of several different molecules. Gain-of-function missense mutations are often somatically acquired in colorectal cancer, prevalently at three hot spots represented by codons 12, 13, and 61. At these levels the number of substitutions is high, making their detection more complex with allele specific techniques. Europa.eu/pdfs/human/press/pr/ 27923508en.pdf.) as well as the American Society for Clinical Oncology [7] and the National Comprehensive Cancer Network (NCCN, http://www.nccn.org/ professionals/physician_gls/PDF/colon.pdf.) require KRAS mutational analysis on colorectal cancer prior to anti-EGFR therapy The European health authority (http://www.emea. europa.eu/pdfs/human/press/pr/ 27923508en.pdf.) as well as the American Society for Clinical Oncology [7] and the National Comprehensive Cancer Network (NCCN, http://www.nccn.org/ professionals/physician_gls/PDF/colon.pdf.) require KRAS mutational analysis on colorectal cancer prior to anti-EGFR therapy
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