KRAS Mutation Analysis by PCR: A Comparison of Two Methods

Louise Bolton, Anne Reiman, Katie Lucas, Judith Timms, Ian A Cree

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

BACKGROUND: KRAS mutation assays are important companion diagnostic tests to guide anti-EGFR antibody treatment of metastatic colorectal cancer. Direct comparison of newer diagnostic methods with existing methods is an important part of validation of any new technique. In this this study, we have compared the Therascreen (Qiagen) ARMS assay with Competitive Allele-Specific TaqMan PCR (castPCR, Life Technologies) to determine equivalence for KRAS mutation analysis.

METHODS: DNA was extracted by Maxwell (Promega) from 99 colorectal cancers. The ARMS-based Therascreen and a customized castPCR assay were performed according to the manufacturer's instructions. All assays were performed on either an Applied Biosystems 7500 Fast Dx or a ViiA7 real-time PCR machine (both from Life Technologies). The data were collected and discrepant results re-tested with newly extracted DNA from the same blocks in both assay types.

RESULTS: Of the 99 tumors included, Therascreen showed 62 tumors to be wild-type (WT) for KRAS, while 37 had KRAS mutations on initial testing. CastPCR showed 61 tumors to be wild-type (WT) for KRAS, while 38 had KRAS mutations. Thirteen tumors showed BRAF mutation in castPCR and in one of these there was also a KRAS mutation. The custom castPCR plate included several other KRAS mutations and BRAF V600E, not included in Therascreen, explaining the higher number of mutations detected by castPCR. Re-testing of discrepant results was required in three tumors, all of which then achieved concordance for KRAS. CastPCR assay Ct values were on average 2 cycles lower than Therascreen.

CONCLUSION: There was excellent correlation between the two methods. Although castPCR assay shows lower Ct values than Therascreen, this is unlikely to be clinically significant.

Original languageEnglish
Article numbere0115672
Number of pages13
JournalPLoS ONE
Volume10
Issue number1
DOIs
Publication statusPublished - 8 Jan 2015
Externally publishedYes

Fingerprint

Assays
mutation
Polymerase Chain Reaction
Tumors
Mutation
assays
neoplasms
Neoplasms
colorectal neoplasms
methodology
diagnostic techniques
Colorectal Neoplasms
Technology
DNA
Testing
Routine Diagnostic Tests
Real-Time Polymerase Chain Reaction
Anti-Idiotypic Antibodies
quantitative polymerase chain reaction
Alleles

Keywords

  • Alleles
  • Colorectal Neoplasms
  • DNA
  • DNA Mutational Analysis
  • Humans
  • Mutation
  • Proto-Oncogene Proteins B-raf
  • Reagent Kits, Diagnostic
  • Real-Time Polymerase Chain Reaction
  • ras Proteins
  • Comparative Study
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

KRAS Mutation Analysis by PCR : A Comparison of Two Methods. / Bolton, Louise; Reiman, Anne; Lucas, Katie; Timms, Judith; Cree, Ian A.

In: PLoS ONE, Vol. 10, No. 1, e0115672, 08.01.2015.

Research output: Contribution to journalArticle

Bolton, Louise ; Reiman, Anne ; Lucas, Katie ; Timms, Judith ; Cree, Ian A. / KRAS Mutation Analysis by PCR : A Comparison of Two Methods. In: PLoS ONE. 2015 ; Vol. 10, No. 1.
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N2 - BACKGROUND: KRAS mutation assays are important companion diagnostic tests to guide anti-EGFR antibody treatment of metastatic colorectal cancer. Direct comparison of newer diagnostic methods with existing methods is an important part of validation of any new technique. In this this study, we have compared the Therascreen (Qiagen) ARMS assay with Competitive Allele-Specific TaqMan PCR (castPCR, Life Technologies) to determine equivalence for KRAS mutation analysis.METHODS: DNA was extracted by Maxwell (Promega) from 99 colorectal cancers. The ARMS-based Therascreen and a customized castPCR assay were performed according to the manufacturer's instructions. All assays were performed on either an Applied Biosystems 7500 Fast Dx or a ViiA7 real-time PCR machine (both from Life Technologies). The data were collected and discrepant results re-tested with newly extracted DNA from the same blocks in both assay types.RESULTS: Of the 99 tumors included, Therascreen showed 62 tumors to be wild-type (WT) for KRAS, while 37 had KRAS mutations on initial testing. CastPCR showed 61 tumors to be wild-type (WT) for KRAS, while 38 had KRAS mutations. Thirteen tumors showed BRAF mutation in castPCR and in one of these there was also a KRAS mutation. The custom castPCR plate included several other KRAS mutations and BRAF V600E, not included in Therascreen, explaining the higher number of mutations detected by castPCR. Re-testing of discrepant results was required in three tumors, all of which then achieved concordance for KRAS. CastPCR assay Ct values were on average 2 cycles lower than Therascreen.CONCLUSION: There was excellent correlation between the two methods. Although castPCR assay shows lower Ct values than Therascreen, this is unlikely to be clinically significant.

AB - BACKGROUND: KRAS mutation assays are important companion diagnostic tests to guide anti-EGFR antibody treatment of metastatic colorectal cancer. Direct comparison of newer diagnostic methods with existing methods is an important part of validation of any new technique. In this this study, we have compared the Therascreen (Qiagen) ARMS assay with Competitive Allele-Specific TaqMan PCR (castPCR, Life Technologies) to determine equivalence for KRAS mutation analysis.METHODS: DNA was extracted by Maxwell (Promega) from 99 colorectal cancers. The ARMS-based Therascreen and a customized castPCR assay were performed according to the manufacturer's instructions. All assays were performed on either an Applied Biosystems 7500 Fast Dx or a ViiA7 real-time PCR machine (both from Life Technologies). The data were collected and discrepant results re-tested with newly extracted DNA from the same blocks in both assay types.RESULTS: Of the 99 tumors included, Therascreen showed 62 tumors to be wild-type (WT) for KRAS, while 37 had KRAS mutations on initial testing. CastPCR showed 61 tumors to be wild-type (WT) for KRAS, while 38 had KRAS mutations. Thirteen tumors showed BRAF mutation in castPCR and in one of these there was also a KRAS mutation. The custom castPCR plate included several other KRAS mutations and BRAF V600E, not included in Therascreen, explaining the higher number of mutations detected by castPCR. Re-testing of discrepant results was required in three tumors, all of which then achieved concordance for KRAS. CastPCR assay Ct values were on average 2 cycles lower than Therascreen.CONCLUSION: There was excellent correlation between the two methods. Although castPCR assay shows lower Ct values than Therascreen, this is unlikely to be clinically significant.

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KW - Real-Time Polymerase Chain Reaction

KW - ras Proteins

KW - Comparative Study

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

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