Abstract
The frequency and poor prognosis of patients with metastatic colorectal cancer (mCRC) emphasizes the requirement for improved biomarkers for use in the treatment and prognosis of mCRC. In the present study, somatic variants in exonic regions of key cancer genes were identified in mCRC patients. Formalin-fixed, paraffin-embedded tissues obtained by biopsy of the metastases of mCRC patients were collected, and the DNA was extracted and sequenced using the Ion Torrent Personal Genome Machine. For the targeted amplification of known cancer genes, the Ion AmpliSeq™ Cancer Panel, which is designed to detect 739 Catalogue of Somatic Mutations in Cancer (COSMIC) mutations in 604 loci from 46 oncogenes and tumor suppressor genes using as little as 10 ng of input DNA, was used. The sequencing results were then analyzed using the Ampliseq™ Variant Caller plug-in within the Ion Torrent Suite software. In addition, Ingenuity Pathway software was used to perform a pathway analysis. The Cox regression analysis was also conducted to investigate the potential correlation between alteration numbers and clinical factors, including response rate, disease-free survival and overall survival. Among 10 specimens, 65 genetic alterations were identified in 24 genes following the exclusion of germline mutations using the SNP database, whereby 41% of the alterations were also present in the COSMIC database. No clinical factors were found to significantly correlate with the alteration numbers in the patients by statistical analysis. However, pathway analysis identified ‘colorectal cancer metastasis signaling’ as the most commonly mutated canonical pathway. This analysis further revealed mutated genes in the Wnt, phosphoinositide 3-kinase (PI3K)/AKT and transforming growth factor (TGF)-β/SMAD signaling pathways. Notably, 11 genes, including the expected APC, BRAF, KRAS, PIK3CA and TP53 genes, were mutated in at least two samples. Notably, 90% (9/10) of mCRC patients harbored at least one ‘druggable’ alteration (range, 1–6 alterations) that has been linked to a clinical treatment option or is currently being investigated in clinical trials of novel targeted therapies. These results indicated that DNA sequencing of key oncogenes and tumor suppressors enables the identification of ‘druggable’ alterations for individual colorectal cancer patients.
Highlights
In 2013, colorectal cancer had the third highest incidence of new cases and the third highest rate of cancer mortality in the USA, with 142,280 and 50,830 individuals, respectively [1]
For the targeted amplification of known cancer genes, the Ion AmpliSeqTM Cancer Panel (Invitrogen Life Technologies), which is designed to detect 739 Catalogue Of Somatic Mutations In Cancer (COSMIC) mutations in 604 loci from 46 oncogenes and tumor suppressor genes using as little as 10 ng of input DNA, was used
These alterations confirmed by COSMIC were all single nucleotide polymorphism (SNP), divided into missense (83%) and nonsense (17%) changes
Summary
In 2013, colorectal cancer had the third highest incidence of new cases and the third highest rate of cancer mortality in the USA, with 142,280 and 50,830 individuals, respectively [1]. The diversity and complexity of somatic mutational processes that underlie carcinogenesis in humans is being revealed through mutational patterns hidden within cancer genomes [2]. Driving the massive data collection is the use of ‐generation sequencing (NGS), which has the ability to probe millions of DNA fragments for mutations and is subsequently enabling clinicians to more accurately gauge the risk of developing cancer and tailor therapies to treat cancers with specific genetic mutations [6]. The Ion Torrent Personal Genome Machine (PGM; Invitrogen Life Technologies, Carlsbad, CA, USA) presents an emerging NGS approach that relies on non‐optical semiconductor sequencing technology with a rapid turnaround time [7]. The deep coverage achieved by the PGM makes it possible to detect somatic mutations in tumor cells with low allele
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