Genotyping of colorectal cancer for cancer precision medicine: Results from the IPH Center for Molecular Pathology

Abstract

Cancer precision medicine has opened up new avenues for the treatment of colorectal cancer (CRC). To fully realize its potential, high-throughput sequencing platforms that allow genotyping beyond KRAS need to be implemented and require performance assessment. We comprehensively analyzed first-year data of 202 consecutive formalin-fixed paraffin embedded (FFPE) CRC samples for which prospective genotyping at our institution was requested. Deep targeted genotyping was done using a semiconductor-based sequencing platform and a self-designed panel of 30 CRC-related genes. Additionally, microsatellite status (MS) was determined. Ninety-seven percent of tumor samples were suitable for sequencing and in 88% MS could be assessed. The minimal drop-out rates of 6 and 25 cases, respectively were due to too low amounts or heavy degradation of DNA. Of 557 nonsynonymous mutations, 90 (16%) have not been described in COSMIC at the time of data query. Forty-three cases (22%) had double- or triple mutations affecting a single gene. Sixty-four percent had genetic alterations influencing oncological therapy. Eight percent of patients (MSI phenotype: 6%; mutated POLE: 2%) were potentially eligible for treatment with immune checkpoint inhibitors. Of 56% of KRASwt CRC that potentially qualified for anti-EGFR treatment, 30% presented with mutations in BRAF/NRAS. Mutated PIK3CA was detected in 21%. In conclusion, we here present real-life routine diagnostics data that not only demonstrate the robustness and feasibility of deep targeted sequencing and MS-analysis of FFPE CRC samples but also contribute to the understanding of CRC genetics. Most importantly, in more than half of the patients our approach enabled the selection of the best treatment currently available. © 2016 Wiley Periodicals, Inc.

Clinical Application of Targeted Next Generation Sequencing for Colorectal Cancers

“…Regarding the NGS platform, 10 [18,20,21,22,23,24,25,26,27,28] used the Ion Torrent Personal Genome Machine (PGM) sequencer (Life Technologies), three [29,30,31] used the MiSeq sequencer (Illumina) and one [32] used the Illumina Genome Analyser IIx. …”

“…For eight out of 10 studies using the PGM sequencer, the required quantity of DNA was 10 ng [20,21,22,23,24,25,27,28]. Two studies assessed that results were obtained using only 0.1 or 0.8 ng of DNA [18,26].…”

“…It should be noted that for studies using another platform [30,31,32], the required amount of DNA was higher (up to 3 µg of DNA) than for the PGM platform (Table 1), except for the study of Froyen et al [29], in which NGS results were obtained with <50 ng of DNA using a custom panel on the Miseq sequencer. In total, 2480 specimens were analysed across the 14 studies; more than 500 of them were biopsies (this information was reported for eight studies [18,21,22,23,24,26,27,29]).…”

“…Jesinghaus et al [21] reported in their series that 64% of cases have genetic aberrations which potentially influence therapy. However, as already underlined by Meric-Bernstam [33], once the tumour profiling is obtained, one of the hurdles is to perform a clinical trial and to appropriately counsel the patient.…”

“…Moreover, many variants of unknown significance are found with NGS. Nevertheless, correlations between mutational profiles and clinical response will provide in the future a unique framework for assessing the clinical significance of specific variants [21]. …”