Evaluation of High-Resolution Melting Analysis as a Diagnostic Tool to Detect the BRAF V600E Mutation in Colorectal Tumors

Pichler, Martin; Balić, Marija; Stadelmeyer, Elke; Ausch, Christoph; Wild, Martina; Godballe, Christian; Bauernhofer, Thomas; Samonigg, Hellmut; Höefler, Gerald; Dandachi, Nadia

doi:10.2353/jmoldx.2009.080100

Cited by 83 publications

(93 citation statements)

References 36 publications

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“…Results for dHPLC and HRM suggest that these techniques are adequately sensitive for detecting the mutation when present in ratios as low as 1% to 5% (of V600E allele in the background of WT allele). Pichler et al 6 obtained similar results performing HRM with reagents, real-time PCR equipment, and HRM analysis software (Roche Diagnostics, Vienna, Austria).…”

Section: Discussionmentioning

confidence: 76%

“…A possible explanation for this finding was given by Pichler et al, 6 who also observed a noticeable rate of false-positive cases with HRM and attributed it to the failure of Taq polymerase to recognize modified bases and, therefore, amplify more artifacts existing in DNA extracted from paraffin-embedded tissue than true mutations.…”

Section: Discussionmentioning

confidence: 99%

“…Also, HRM has proved to be a reliable technique for V600E detection when compared with dHPLC, allele-specific PCR, and direct sequencing. 6 The aim of our work was to compare the performance characteristics of BRAF V600E detection by dHPLC and HRM with the previously described TaqMan allelic discrimination method and direct sequencing.…”

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confidence: 99%

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Comparison of Allelic Discrimination by dHPLC, HRM, and TaqMan in the Detection of BRAF Mutation V600E

Carbonell

Turpin

Torres-Moreno

et al. 2011

The Journal of Molecular Diagnostics

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The V600E mutation in the BRAF oncogene is associated with colorectal carcinomas, with mismatch-repair deficiency and, recently, with nonresponse to epidermal growth factor receptor inhibitor therapy. The use of reliable techniques for its detection is important. The aim of our study was to compare the performance characteristics in V600E detection of denaturing high-performance liquid chromatography (dHPLC) and high-resolution melting (HRM) with TaqMan allelic discrimination as well as direct-sequencing methods in a series of 195 colorectal paraffinembedded specimens up to the age of 15 years. The effectiveness for obtaining results on mutation status was best using TaqMan (96.9%), followed by dHPLC (93.3%), HRM (88.7%), and sequencing (88.2%). In general, TaqMan was best for analyzing older tissues, whereas sequencing was the least efficient. Heterozygotic V600E was detected in 11.6%, 9.9%, 11.6%, and 9.9% of tissues using TaqMan, dHPLC, HRM, and sequencing, respectively. Result concordances between dHPLC and TaqMan or sequencing were excellent ( ‫؍‬ 0.9411 and ‫؍‬ 0.8988, respectively); for HRM, the concordances were good ( ‫؍‬ 0.7973 and ‫؍‬ 0.7488, respectively). By using DNA dilutions from tumor tissue, a minimum of 10% of V600E harboring cancer content was required for the analysis by dHPLC and HRM. dHPLC could detect four non-V600E mutations, whereas HRM detected one. Our results indicate that dHPLC and HRM are techniques that can be reliably used for the detection of the BRAFV600E mutation in Recent advances in DNA sequencing technology have made it possible to use high-throughput sequencing on the entire human colorectal cancer (CRC) spectrum, finding 848 genes that may be somatically mutated. 1,2 The subsequent study of these mutations in CRC specimens worldwide would be facilitated by the use of less expensive and more efficient screening methods for mutation detection. Because most of these mutations are found in heterozygosis, screening techniques based on heteroduplex detection, such as denaturing high-performance liquid chromatography (dHPLC) or high-resolution melting (HRM) analysis, are strongly recommended. The V600E mutation in the BRAF oncogene has been found in CRCs with mismatch-repair gene deficiency. Familial CRCs with mismatch-repair gene deficiency do not harbor this mutation; rather, V600E is associated with the sporadic form of CRC associated with mismatch-repair gene deficiency. In addition to the diagnostic value of the BRAF mutation assessment, recent evidence 3,4 has demonstrated that stage IV CRCs with the BRAF V600E mutation do not respond to epidermal growth factor receptor inhibitor therapy, thus extending its value to predictive grounds. In previous articles, 5 V600E detection using TaqMan chemistry (Applied Biosystem, Foster City, CA) saved cost, time, and manual labor over direct sequencing, with the former showing 100% sensitivity and 100% specificity when the latter is considered as the reference method. Also, HRM has proved to be a reliable technique for V600E detection...

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Section: Discussionmentioning

confidence: 76%

Section: Discussionmentioning

confidence: 99%

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Comparison of Allelic Discrimination by dHPLC, HRM, and TaqMan in the Detection of BRAF Mutation V600E

Carbonell

Turpin

Torres-Moreno

et al. 2011

The Journal of Molecular Diagnostics

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“…Whilst this enrichment technique results in high purity leukemia samples (>90%), in the context of a diagnostic molecular laboratory, a more cost-effective, rapid and less labor intensive method that is capable of high throughput is desirable. High resolution melting (HRM) analysis is a DNA mutation screening technique which is fast, relatively simple and has previously been shown to be a sensitive and reliable way of detecting the BRAF V600E mutation in tumor samples from patients with melanoma, 4 colorectal carcinoma 5 and, more recently, HCL. 6 Our aim was to determine the feasibility of using HRM analysis and subsequent SS to detect the BRAF V600E in DNA extracted from unenriched bone marrow aspirate and peripheral blood samples from patients with HCL.…”

Section: Introductionmentioning

confidence: 99%

Detection of BRAF mutations in patients with hairy cell leukemia and related lymphoproliferative disorders

Blombery¹,

Wong²,

Hewitt³

et al. 2011

Haematologica

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“…We request that the authors comment on (1) the different melting behavior of the MCF-7 wildtype DNA and the SKMel28 DNA in the difference plots and (2) the failure to refer to other studies, 2 including their own work, 3 showing a detection limit of 5% mutated BRAF alleles using HRM in combination with conventional PCR.…”

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confidence: 99%

COLD-HRM PCR versus Conventional HRM PCR to Detect the BRAF V600E Mutation

Stadelmeyer

Heitzer

Wolf

et al. 2011

The Journal of Molecular Diagnostics

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In the recent article by Mancini et al, 1 the authors demonstrated the improved sensitivity of the detection of KRAS mutations and the BRAF V600E mutation using coamplification at lower temperature PCR (COLD-PCR) in combination with high-resolution melting (HRM) instead of conventional PCR followed by HRM. They reported an eightfold higher sensitivity for KRAS mutations (from 6.2% to 0.8%) and a debatable fourfold higher sensitivity for the BRAF V600E mutation (from 12.5% to 6.2%, which would seem to be a twofold higher sensitivity), using serial dilutions of DNA from cell lines harboring the mutations (CCRF-CEM and SKMel28) and DNA from MCF-7 as wild-type control.Using conventional PCR and HRM, we 2 and others, including the authors, 3 have previously demonstrated detection limits for the BRAF V600E mutation of less than 5% mutated DNA in a background of wild-type DNA, which is more sensitive than the COLD-PCR assay described in their present article. In addition, our system 2 proved to be applicable to FFPE material. With FFPE samples, the spread of the melting curves increases due to the lower quality of DNA; however, we were able to distinguish samples containing 5% mutated DNA in a background of wild-type DNA.We read with great interest the publications on the advantages of COLD-PCR. Indeed, we have used an extensive optimization process to try to improve the sensitivity of the BRAF assay using COLD-PCR instead of conventional PCR. We designed new primer sets that yield short amplicons to maximize the temperature effect caused by the mismatch in the heteroduplexes during COLD-PCR, taking into account single-nucleotide polymorphisms and the pseudogene described for the BRAF gene. We optimized the reaction composition and the PCR protocol. Unfortunately, we have as yet not been able to improve the sensitivity of 5% mutated DNA (SKMel28) in wild-type DNA (human mononuclear cell DNA) by using COLD-PCR, and we could not observe the expected shift in the quantification cycle values caused by the preferential amplification of the minority alleles (unpublished data). We could, however, confirm the equal melting behavior of 100% homozygously mutated SKMel28 DNA and 100% wild-type DNA in the difference plots (unpublished data). Thus, with regard to Figure 1 (right) in the study by Mancini et al, 1 we do not understand the different melting behavior of curves A (MCF-7 wild-type DNA) and G (100% SKMel28) because no heteroduplexes can be formed in these reactions and the T-A substitution should not cause any alteration in the melting behavior (in consideration of the hydrogen bonds).Thus, although COLD-PCR has been described as a method ideal for the detection of low-level mutations by increasing the sensitivity of PCR-based assays, the application of COLD-PCR for the detection of the BRAF V600E mutation could not improve the sensitivity of our assay (unpublished data). Similar results have been shown by Fadhil and colleagues. 4 In addition, the intensive optimization and increased time necessary for each run (approxi...

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Evaluation of High-Resolution Melting Analysis as a Diagnostic Tool to Detect the BRAF V600E Mutation in Colorectal Tumors

Cited by 83 publications

References 36 publications

Comparison of Allelic Discrimination by dHPLC, HRM, and TaqMan in the Detection of BRAF Mutation V600E

Comparison of Allelic Discrimination by dHPLC, HRM, and TaqMan in the Detection of BRAF Mutation V600E

Detection of BRAF mutations in patients with hairy cell leukemia and related lymphoproliferative disorders

COLD-HRM PCR versus Conventional HRM PCR to Detect the BRAF V600E Mutation

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