Ion Torrent next-generation sequencing for routine identification of clinically relevant mutations in colorectal cancer patients

Abstract

AimsTo evaluate the accuracy, consumable cost and time around testing (TAT) of a next-generation sequencing (NGS) assay, the Ion Torrent AmpliSeq Colon and Lung Cancer Panel, as an alternative to Sanger sequencing to genotype KRAS, NRAS and BRAF in colorectal cancer patients.MethodsThe Ion Torrent panel was first verified on cell lines and on control samples and then prospectively applied to routine specimens (n=114), with Sanger sequencing as reference.ResultsThe Ion Torrent panel detected mutant alleles at the 5% level on cell lines and correctly classified all control tissues. The Ion Torrent assay was successfully carried out on most (95.6%) routine diagnostic samples. Of these, 12 (11%) harboured mutations in the BRAF gene and 47 (43%) in either of the two RAS genes, in two cases with a low abundance of RAS mutant allele which was missed by Sanger sequencing. The mean TAT, from sample receipt to reporting, was 10.4 (Sanger) and 13.0 (Ion Torrent) working days. The consumable cost for genotyping KRAS, NRAS and BRAF was €196 (Sanger) and €187 (Ion Torrent).ConclusionsIon Torrent AmpliSeq Colon and Lung Cancer Panel sequencing is as robust as Sanger sequencing in routine diagnostics to select patients for anti-epidermal growth factor receptor (EGFR) therapy for metastatic colorectal cancer.

EGFRmutation detection on lung cancer cytological specimens by the novel fully automated PCR-based IdyllaEGFRMutation Assay

“…Then, Idylla EGFR Mutation Assay was carried out on DNA preparations previously extracted from routine cytological samples (n=76) quantified (ng/µL) and tested for clinical reporting by in-house PCR-based methods (fragment length and TaqMan assays) as previously described 11. In cases of discrepancies, next generation sequencing was used as an orthogonal technique, following our previously validated protocol 12. In 17 cases, an additional direct smear was available and Idylla EGFR Mutation Test was also carried out directly on cytological material exploiting the whole Idylla system workflow, as explained below.…”

Current practices and guidelines for clinical next-generation sequencing oncology testing

“…The most prevalent current implementation of NGS for oncology is mutation detection via targeted panels [1][2][3][4][5] . These assays use molecular methods such as multiplex polymerase chain reactions (PCR) to isolate clinically relevant segments of the genome, such as mutation hotspots or coding exons of entire genes.…”

Liquid biopsy - emergence of a new era in personalized cancer care

“…Though, it has been optimized to work with highly fragmented and degraded FFPE DNA, recent studies have shown that it can be used to analyze mutations in cfDNA (https://www.illumina.com/). The Ion Ampliseq Colon and Lung cancer panel on the Ion Torrent Personal Genome Machine selectively amplifies 90 amplicons that encompass 1825 mutational hotspots of 22 genes related to colon and lung cancer [86][87][88]. It has been clinically validated in a retrospective study of 39 NSCLC samples and 51 colorectal cancer samples [84].…”