Abstract
Next generation sequencing (NGS) is an emerging technology becoming relevant for genotyping of clinical samples. Here, we assessed the stability of amplicon sequencing from formalin-fixed paraffin-embedded (FFPE) and paired frozen samples from colorectal cancer metastases with different analysis pipelines. 212 amplicon regions in 48 cancer related genes were sequenced with Illumina MiSeq using DNA isolated from resection specimens from 17 patients with colorectal cancer liver metastases. From ten of these patients, paired fresh frozen and routinely processed FFPE tissue was available for comparative study. Sample quality of FFPE tissues was determined by the amount of amplifiable DNA using qPCR, sequencing libraries were evaluated using Bioanalyzer. Three bioinformatic pipelines were compared for analysis of amplicon sequencing data. Selected hot spot mutations were reviewed using Sanger sequencing. In the sequenced samples from 16 patients, 29 non-synonymous coding mutations were identified in eleven genes. Most frequent were mutations in TP53 (10), APC (7), PIK3CA (3) and KRAS (2). A high concordance of FFPE and paired frozen tissue samples was observed in ten matched samples, revealing 21 identical mutation calls and only two mutations differing. Comparison of these results with two other commonly used variant calling tools, however, showed high discrepancies. Hence, amplicon sequencing can potentially be used to identify hot spot mutations in colorectal cancer metastases in frozen and FFPE tissue. However, remarkable differences exist among results of different variant calling tools, which are not only related to DNA sample quality. Our study highlights the need for standardization and benchmarking of variant calling pipelines, which will be required for translational and clinical applications.
Highlights
Due to recent advances in deep sequencing technologies, remarkable insights have been gained on the alterations acquired by colorectal cancer (CRC) genomes during the carcinogenic process, largely expanding our view on CRC genomic progression [1,2,3]
We sequenced 212 amplicon regions in 48 cancer related genes with Illumina MiSeq using DNA isolated from resection specimens from 17 patients with CRC liver metastases
Mean sequencing quality (Phred score 38 vs. 37) was gradually higher in formalin-fixed paraffin-embedded (FFPE) samples compared to frozen samples; the GC content was higher in FFPE than in frozen tissue (49% vs. 45%)
Summary
Due to recent advances in deep sequencing technologies, remarkable insights have been gained on the alterations acquired by colorectal cancer (CRC) genomes during the carcinogenic process, largely expanding our view on CRC genomic progression [1,2,3]. The development of novel targeted therapeutics highlights the need for reliable and cost effective methods for molecular characterization of cancer genomes to identify patients that respond to treatment on the basis of druggable mutations, predictive alterations or acquired resistance markers. Targeted sequencing based on PCR amplicons represents a feasible approach for evaluation of actionable mutations, mutational hot spots or predictive alterations in cancer genomes for clinical studies. Due to the comparably low number of base pairs to be sequenced per patient, multiple samples, for longitudinal analysis, can be analyzed in parallel on bench-top machines such as Illumina MiSeq, lowering costs and potentially allowing routine clinical application in the near future
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
