title: Increased detection rates of EGFR and KRAS mutations in NSCLC specimens with low tumour cell content by 454 deep sequencing.
abstract: Detection of activating EGFR mutations in NSCLC is the prerequisite for individualised therapy with receptor tyrosine kinase inhibitors (TKI). In contrast, mutant downstream effector KRAS is associated with TKI resistance. Accordingly, EGFR mutation status is routinely examined in NSCLC specimens, but the employed methods may have a dramatic impact on the interpretation of results and, consequently, therapeutic decisions. Specimens with low tumour cell content are at particular risk for false-negative EGFR mutation reporting by sequencing with Sanger chemistry. To improve reliability of detecting clinically relevant mutant variants of EGFR and KRAS, we took full advantage of 454 deep sequencing and developed a two-step amplification protocol for the analysis of EGFR exons 18-21 and KRAS exons 2 and 3. We systematically addressed the sensitivity, reproducibility and specificity of the developed assay. Mutations could be reliably identified down to an allele frequency of 0.2-1.5 %, as opposed to 10-20 % detection limit of Sanger sequencing. High reproducibility (0-2.1 % variant frequency) and very low background level (0.4-0.8 % frequency) further complement the reliability of this assay. Notably, re-evaluation of 16 NSCLC samples with low tumour cell content ≤40 % and EGFR wild type status according to Sanger sequencing revealed clinically relevant EGFR mutations at allele frequencies of 0.9-10 % in seven cases. In summary, this novel two-step amplification protocol with 454 deep sequencing is superior to Sanger sequencing with significantly increased sensitivity, enabling reliable analysis of EGFR and KRAS in NSCLC samples independent of the tumour cell content.
authors.affiliation: Department of Pathology, University Medical Center, Erlangen, Germany.
authors.forename: Evgeny A
authors.initials: EA
authors.lastname: Moskalev
authors.forename: Robert
authors.initials: R
authors.lastname: Stöhr
authors.forename: Ralf
authors.initials: R
authors.lastname: Rieker
authors.forename: Simone
authors.initials: S
authors.lastname: Hebele
authors.forename: Florian
authors.initials: F
authors.lastname: Fuchs
authors.forename: Horia
authors.initials: H
authors.lastname: Sirbu
authors.forename: Sergey E
authors.initials: SE
authors.lastname: Mastitsky
authors.forename: Carsten
authors.initials: C
authors.lastname: Boltze
authors.forename: Helmut
authors.initials: H
authors.lastname: König
authors.forename: Abbas
authors.initials: A
authors.lastname: Agaimy
authors.forename: Arndt
authors.initials: A
authors.lastname: Hartmann
authors.forename: Florian
authors.initials: F
authors.lastname: Haller
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D009154:Mutation; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D015203:Reproducibility of Results; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D066246:ErbB Receptors; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins