title: KRAS, BRAF, and TP53 deep sequencing for colorectal carcinoma patient diagnostics.
abstract: In colorectal carcinoma, KRAS (alias Ki-ras) and BRAF mutations have emerged as predictors of resistance to anti-epidermal growth factor receptor antibody treatment and worse patient outcome, respectively. In this study, we aimed to establish a high-throughput deep sequencing workflow according to 454 pyrosequencing technology to cope with the increasing demand for sequence information at medical institutions. A cohort of 81 patients with known KRAS mutation status detected by Sanger sequencing was chosen for deep sequencing. The workflow allowed us to analyze seven amplicons (one BRAF, two KRAS, and four TP53 exons) of nine patients in parallel in one deep sequencing run. Target amplification and variant calling showed reproducible results with input DNA derived from FFPE tissue that ranged from 0.4 to 50 ng with the use of different targets and multiplex identifiers. Equimolar pooling of each amplicon in a deep sequencing run was necessary to counterbalance differences in patient tissue quality. Five BRAF and 49 TP53 mutations with functional consequences were detected. The lowest mutation frequency detected in a patient tumor population was 5% in TP53 exon 5. This low-frequency mutation was successfully verified in a second PCR and deep sequencing run. In summary, our workflow allows us to process 315 targets a week and provides the quality, flexibility, and speed needed to be integrated as standard procedure for mutational analysis in diagnostics.
authors.affiliation: Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland. markus.rechsteiner@usz.ch
authors.forename: Markus
authors.initials: M
authors.lastname: Rechsteiner
authors.forename: Adriana
authors.initials: A
authors.lastname: von Teichman
authors.forename: Jan H
authors.initials: JH
authors.lastname: Rüschoff
authors.forename: Niklaus
authors.initials: N
authors.lastname: Fankhauser
authors.forename: Bernhard
authors.initials: B
authors.lastname: Pestalozzi
authors.forename: Peter
authors.initials: P
authors.lastname: Schraml
authors.forename: Achim
authors.initials: A
authors.lastname: Weber
authors.forename: Peter
authors.initials: P
authors.lastname: Wild
authors.forename: Dieter
authors.initials: D
authors.lastname: Zimmermann
authors.forename: Holger
authors.initials: H
authors.lastname: Moch
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D005091:Exons; D005260:Female; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins