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KRAS, BRAF, and TP53 deep-sequencing for colorectal carcinoma patient diagnostics


Rechsteiner, Markus; von Teichman, Adriana; Rüschoff, Jan H; Fankhauser, Niklaus; Pestalozzi, Bernhard; Schraml, Peter; Weber, Achim; Wild, Peter; Zimmermann, Dieter; Moch, Holger (2013). KRAS, BRAF, and TP53 deep-sequencing for colorectal carcinoma patient diagnostics. Journal of Molecular Diagnostics, 15(3):299-311.

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.

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Institute of Pathology and Molecular Pathology
04 Faculty of Medicine > University Hospital Zurich > Clinic for Oncology
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:2013
Deposited On:09 Apr 2013 10:23
Last Modified:07 Dec 2017 20:58
Publisher:American Society for Investigative Pathology
ISSN:1525-1578
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1016/j.jmoldx.2013.02.001
PubMed ID:23531339

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