QIAGEN therascreen KRAS RGQ assay, QIAGEN KRAS pyro assay, and dideoxy sequencing for clinical laboratory analysis of KRAS mutations in tumor Specimens

Qamra M. Alqahtani, Ann Crowley, Sharleen Rapp, Allison M Cushman-Vokoun

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Objective: To compare the performance of assays used to assess KRAS mutations in tumor specimens. Methods: We analyzed DNA extracted from 30 formalin-fixed paraffin-embedded (FFPE) tumor specimens using the QIAGEN Therascreen KRAS RGQ and QIAGEN Pyro reagents, with dideoxy sequencing (colloquially considered to be the gold standard) as the reference method. Results: We detected 22 codon 12 or 13 KRAS mutations using the Pyro assay, whereas the RGQ assay detected 19 mutations. For mutation detection, the clinical sensitivity was 86% for the RGQ assay compared with 100% for the Pyro but 100% for the KRAS mutations that the RGQ was predesigned to detect. The Pyro could detect rare mutations. The RGQ demonstrated a lower limit of detection compared with the Pyro; However, the Pyro required less DNA input than the RGQ. Conclusion: The 2 assays that we tested yielded comparable performance in detecting KRAS mutations, as we had expected based on assay design. Overall, the Pyro assay detects more mutations and requires less DNA input but is less analytically sensitive, compared with the RGQ assay.

Original languageEnglish (US)
Pages (from-to)30-38
Number of pages9
JournalLaboratory Medicine
Volume47
Issue number1
DOIs
StatePublished - 2016

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Clinical laboratories
Tumors
Assays
Mutation
Neoplasms
DNA
Codon
Paraffin
Formaldehyde
Limit of Detection

Keywords

  • Biomarker
  • Colorectal cancer
  • DNA
  • EGFR inhibitor
  • Formalin-fixed paraffin embedded
  • Pyrosequencing
  • Real-time PCR
  • Targeted therapy

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

QIAGEN therascreen KRAS RGQ assay, QIAGEN KRAS pyro assay, and dideoxy sequencing for clinical laboratory analysis of KRAS mutations in tumor Specimens. / Alqahtani, Qamra M.; Crowley, Ann; Rapp, Sharleen; Cushman-Vokoun, Allison M.

In: Laboratory Medicine, Vol. 47, No. 1, 2016, p. 30-38.

Research output: Contribution to journalArticle

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abstract = "Objective: To compare the performance of assays used to assess KRAS mutations in tumor specimens. Methods: We analyzed DNA extracted from 30 formalin-fixed paraffin-embedded (FFPE) tumor specimens using the QIAGEN Therascreen KRAS RGQ and QIAGEN Pyro reagents, with dideoxy sequencing (colloquially considered to be the gold standard) as the reference method. Results: We detected 22 codon 12 or 13 KRAS mutations using the Pyro assay, whereas the RGQ assay detected 19 mutations. For mutation detection, the clinical sensitivity was 86{\%} for the RGQ assay compared with 100{\%} for the Pyro but 100{\%} for the KRAS mutations that the RGQ was predesigned to detect. The Pyro could detect rare mutations. The RGQ demonstrated a lower limit of detection compared with the Pyro; However, the Pyro required less DNA input than the RGQ. Conclusion: The 2 assays that we tested yielded comparable performance in detecting KRAS mutations, as we had expected based on assay design. Overall, the Pyro assay detects more mutations and requires less DNA input but is less analytically sensitive, compared with the RGQ assay.",
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AU - Crowley, Ann

AU - Rapp, Sharleen

AU - Cushman-Vokoun, Allison M

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N2 - Objective: To compare the performance of assays used to assess KRAS mutations in tumor specimens. Methods: We analyzed DNA extracted from 30 formalin-fixed paraffin-embedded (FFPE) tumor specimens using the QIAGEN Therascreen KRAS RGQ and QIAGEN Pyro reagents, with dideoxy sequencing (colloquially considered to be the gold standard) as the reference method. Results: We detected 22 codon 12 or 13 KRAS mutations using the Pyro assay, whereas the RGQ assay detected 19 mutations. For mutation detection, the clinical sensitivity was 86% for the RGQ assay compared with 100% for the Pyro but 100% for the KRAS mutations that the RGQ was predesigned to detect. The Pyro could detect rare mutations. The RGQ demonstrated a lower limit of detection compared with the Pyro; However, the Pyro required less DNA input than the RGQ. Conclusion: The 2 assays that we tested yielded comparable performance in detecting KRAS mutations, as we had expected based on assay design. Overall, the Pyro assay detects more mutations and requires less DNA input but is less analytically sensitive, compared with the RGQ assay.

AB - Objective: To compare the performance of assays used to assess KRAS mutations in tumor specimens. Methods: We analyzed DNA extracted from 30 formalin-fixed paraffin-embedded (FFPE) tumor specimens using the QIAGEN Therascreen KRAS RGQ and QIAGEN Pyro reagents, with dideoxy sequencing (colloquially considered to be the gold standard) as the reference method. Results: We detected 22 codon 12 or 13 KRAS mutations using the Pyro assay, whereas the RGQ assay detected 19 mutations. For mutation detection, the clinical sensitivity was 86% for the RGQ assay compared with 100% for the Pyro but 100% for the KRAS mutations that the RGQ was predesigned to detect. The Pyro could detect rare mutations. The RGQ demonstrated a lower limit of detection compared with the Pyro; However, the Pyro required less DNA input than the RGQ. Conclusion: The 2 assays that we tested yielded comparable performance in detecting KRAS mutations, as we had expected based on assay design. Overall, the Pyro assay detects more mutations and requires less DNA input but is less analytically sensitive, compared with the RGQ assay.

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