KRAS and BRAF Mutation Analysis in Routine Molecular Diagnostics: Comparison of Three Testing Methods on Formalin-Fixed, Paraffin-Embedded Tumor-Derived DNA

Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands.
The Journal of molecular diagnostics: JMD (Impact Factor: 4.85). 03/2012; 14(3):247-55. DOI: 10.1016/j.jmoldx.2012.01.011
Source: PubMed


Accurate mutation detection assays are strongly needed for use in routine molecular pathology analyses to aid in the selection of patients with cancer for targeted therapy. The high-resolution melting (HRM) assay is an ideal prescreening tool, and SNaPshot analysis offers a straightforward genotyping system. Our present study was determined to compare these mutation testing methods on formalin-fixed, paraffin-embedded (FFPE) tumor-derived DNA. We compared the performance of HRM, followed by cycle sequencing (HRM-sequencing); multiplex PCR assay, followed by SNaPshot analysis (multiplex mutation assay); and a successor assay using HRM, followed by SNaPshot (HRM-SNaPshot) for mutation analysis of both KRAS (codon 12/13/61) and BRAF (codon 600/601). In a series of 195 FFPE-derived DNA specimens, a high genotypic concordance between HRM-sequencing and multiplex mutation assay was found (κ, 0.98; 95% CI, 0.94 to 1), underlining the potential of a combined HRM-SNaPshot approach. In reconstruction experiments, the analytical sensitivity of HRM-SNaPshot was twofold to fourfold higher than HRM-sequencing and multiplex mutation assay, respectively. In addition, HRM-SNaPshot had a good performance rate (99%) on FFPE tumor-derived DNA, and mutation detection was highly concordant with the predecessor assays (κ for both, 0.98). The occurrence of BRAF and KRAS mutations is mutually exclusive. HRM-SNaPshot is an attractive method for mutation analysis in pathology, given its good performance rate on FFPE-derived DNA, high analytical sensitivity, and prescreening approach.

Download full-text


Available from: Ellen C Zwarthoff, Sep 02, 2015
  • Source
    • "Biopsies performed at the VU University Medical Center were assessed for histology with H&E staining and immunohistochemical markers, at the discretion of the pathologist. Mutational analysis was performed on EGFR and KRAS by high-resolution melting (HRM), as described earlier [11] [12] [13]. Sanger sequencing of the HRM PCR products was done to determine the specific sequence alteration when an aberrant melting-out pattern was identified. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Aim Non-small cell lung cancer (NSCLC)-patients with an epidermal growth factor receptor (EGFR)-mutation have median progression-free survival (PFS) of 12 months on tyrosine kinase inhibitors (TKIs). Resistance is mediated by the EGFR T790M-mutation in the majority of patients. Longitudinal follow-up data are lacking. We retrospectively evaluated EGFR-mutated NSCLC-patients who were rebiopsied after TKI-treatment. A subgroup was sequentially rebiopsied along the course of the disease. Patients and methods Advanced EGFR-mutated NSCLC-patients who had both a pre-TKI biopsy and post-TKI biopsy available were included. Information on treatments and (re)biopsies was collected chronologically. Primary endpoint was incidence of T790M-mutation. Results Sixty-six patients fulfilled the inclusion criteria. In first post-TKI biopsies, T790M-mutation was detected in 34 patients (52%) of patients. Twenty-seven patients had subsequent post-TKI rebiopsies with mutation analysis available; in 10 patients (37%) the T790M-status in subsequent post-TKI rebiopsies was not consistent with the T790M-status of the first post-TKI biopsy. Progression free survival (PFS) on TKI-treatment was 12.0 months. Objective response rate on TKI-treatment was 81%. Patients developing T790M-mutation at post-TKI biopsy had longer median PFS compared to T790M-negative patients (14.2 versus 11.1 months respectively (P = 0.034) and longer overall survival (45.9 months versus 29.8 months respectively (P = 0.213). Transformation to SCLC was detected in 1 patient (2%). Conclusion Incidence of T790M-mutation at first post-TKI biopsy in this cohort of EGFR-mutated NSCLC-patients was 50%. Detection of T790M-mutation was not consistent over time; some patients who were T790M-positive at first post-TKI biopsy became T790M-negative in later post-TKI rebiopsies and vice versa. T790M-positive patients showed longer PFS than T790M-negative patients. Whether the low incidence of transformation to SCLC is justifying post-TKI rebiopsy in EGFR-mutated NSCLC-patients with acquired TKI-resistance in regular clinical practice is debatable.
    Full-text · Article · Jul 2014 · Lung cancer (Amsterdam, Netherlands)
  • Source
    • "A variety of methods have been applied for BRAF mutations. PCR-based screening methods such as SNaPshot assays, AS-PCR, COLD-PCR, Taqman® SNP assay, pyrosequencing and HRM analysis have been applied and the commonly used methods in pathology laboratories are summarized in Table 3[13-18]. Although direct sequencing of PCR products is the gold standard for BRAF mutation detection in routine diagnostics, it remains laborious, time consuming and requires rather expensive equipment [5,6]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: BRAF mutation is an important diagnostic and prognostic marker in patient s with papillary thyroid carcinoma (PTC). To be applicable in clinical laboratories with limited equipment, diverse testing methods are required to detect BRAF mutation. A shifted termination assay (STA) fragment analysis was used to detect common V600 BRAF mutations in 159 PTCs with DNAs extracted from formalin-fixed paraffin-embedded tumor tissue. The results of STA fragment analysis were compared to those of direct sequencing. Serial dilutions of BRAF mutant cell line (SNU-790) were used to calculate limit of detection (LOD). BRAF mutations were detected in 119 (74.8 %) PTCs by STA fragment analysis. In direct sequencing, BRAF mutations were observed in 118 (74.2 %) cases. The results of STA fragment analysis had high correlation with those of direct sequencing (p < 0.00001, kappa = 0.98). The LOD of STA fragment analysis and direct sequencing was 6 % and 12.5 %, respectively. In PTCs with pT3/T4 stages, BRAF mutation was observed in 83.8 % of cases. In pT1/T2 carcinomas, BRAF mutation was detected in 65.9 % and this difference was statistically significant (p = 0.007). Moreover, BRAF mutation was more frequent in PTCs with extrathyroidal invasion than tumors without extrathyroidal invasion (84.7 % versus 62.2 %, p = 0.001). To prepare and run the reactions, direct sequencing required 450 minutes while STA fragment analysis needed 290 minutes. STA fragment analysis is a simple and sensitive method to detect BRAF V600 mutations in formalin-fixed paraffin-embedded clinical samples.Virtual Slides: The virtual slide(s) for this article can be found here:
    Full-text · Article · Jul 2013 · Diagnostic Pathology
  • Source
    • "For practical reasons, we have used different methods to evaluate the KRAS mutation status in the different patient cohorts. However, all methods have shown to be able to detect mutations in samples with less than 10% mutated tumour cells (Heideman et al, 2012) and all samples used for extraction had in effect more than 30% of tumour cells. Unfortunately, we do not have access to material from clinical studies investigating the efficacy of EGFR inhibitors. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background: Inhibitors of the epidermal growth factor (EGFR) signaling pathway have a major role in the treatment of KRAS wild-type colorectal cancer patients. The EGFR pathway has been shown to be activated in gastric cancer (GC). However, published data on KRAS and BRAF mutation status is limited in GC and has not been compared between GC from different geographic regions. Methods: The prevalence of KRAS and BRAF mutations was established in 712 GC: 278 GC from the United Kingdom, 230 GC from Japan and 204 GC from Singapore. The relationship between KRAS/BRAF mutation status, DNA mismatch repair (MMR) status, clinicopathological variables and overall survival was analysed. Results: Overall, 30 (4.2%) GC carried a KRAS mutation. In total, 5.8% of the UK GC, 4% of Japan GC and 1.5% of Singapore GC were KRAS mutant. KRAS mutant GC had fewer lymph node metastases in the UK cohort (P=0.005) and were more frequent in elderly patients in the Japan cohort (P=0.034). KRAS mutations were more frequent in MMR-deficient GC in the UK and the Japanese cohort (P<0.05). A BRAF mutation was only detected in a single Japanese GC. Conclusions: This large multicentre study demonstrated that KRAS mutations and DNA MMR deficiency have a role in a small subgroup of GC irrespective of country of origin, suggesting that this subgroup of GC may have developed along a common pathway. Further studies need to establish whether concomitant mutations or amplifications of other EGFR signalling pathway genes may contribute to the activation of this pathway in GC.
    Full-text · Article · Mar 2013 · British Journal of Cancer
Show more