title: Deep ion sequencing of amplicon adapter ligated libraries: a novel tool in molecular diagnostics of formalin fixed and paraffin embedded tissues.
abstract: Due to the advanced progress in personalised therapy concepts for non-small cell lung cancer (NSCLC), we applied the ion semiconductor sequencing (ISS) approach to molecular diagnosis of NSCLC, analysing a set of therapy relevant gene loci. DNA from macrodissected tumour samples of formalin fixed biopsies was used for PCR amplification of EGFR exons 18, 19, 21 and KRAS exon 1. A total of 128 PCR products were analysed by conventional termination sequencing as well as by ISS. Sensitivity of ISS was additionally determined using 100-10 000 copies of reference mutants. All somatic mutations detected by direct Sanger sequencing were also identified by ISS. No additional mutants were detected. Running samples with limited copies of mutated alleles revealed high sensitivity, detecting less than 10% (2500 copies) mutants in a human wild type background. In conclusion, multiplexed mutation analyses by ISS is an efficient technology that can easily be linked to existing PCR approaches in molecular pathology.
authors.affiliation: Cologne Center for Genomics, University of Cologne, Cologne, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Becker
authors.forename: Claudia
authors.initials: C
authors.lastname: Vollbrecht
authors.forename: Ulrike
authors.initials: U
authors.lastname: Koitzsch
authors.forename: Katharina
authors.initials: K
authors.lastname: Koenig
authors.forename: Jana
authors.initials: J
authors.lastname: Fassunke
authors.forename: Sebastian
authors.initials: S
authors.lastname: Huss
authors.forename: Peter
authors.initials: P
authors.lastname: Nuernberg
authors.forename: Lukas C
authors.initials: LC
authors.lastname: Heukamp
authors.forename: Reinhard
authors.initials: R
authors.lastname: Buettner
authors.forename: Margarete
authors.initials: M
authors.lastname: Odenthal
authors.forename: Janine
authors.initials: J
authors.lastname: Altmueller
authors.forename: Sabine
authors.initials: S
authors.lastname: Merkelbach-Bruse
mesh_terms: D000483:Alleles; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D005557:Formaldehyde; D006801:Humans; D008168:Lung / Q000378:metabolism* / Q000473:pathology; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009154:Mutation; D016612:Paraffin Embedding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D005557:Formaldehyde; C512478:EGFR protein, human; D066246:ErbB Receptors; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
keywords: CANCER; DIAGNOSTICS; LUNG CANCER; MOLECULAR PATHOLOGY; TUMOUR MARKERS