KRAS and BRAF mutation analysis in routine molecular diagnostics: comparison of three testing methods on formalin-fixed, paraffin-embedded tumor-derived DNA.
ABSTRACT 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.
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ABSTRACT: Next generation DNA sequencing (NGS) holds promise for diagnostic applications, yet implementation in routine molecular pathology practice requires performance evaluation on DNA derived from routine formalin-fixed paraffin-embedded (FFPE) tissue specimens. The current study presents a comprehensive analysis of TruSeq Amplicon Cancer Panel-based NGS using a MiSeq Personal sequencer (TSACP-MiSeq-NGS) for somatic mutation profiling.Cellular oncology (Dordrecht). 09/2014;
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ABSTRACT: Non-small cell lung cancer (NSCLC) is a heterogeneous disease, with a burden of genomic alterations exceeding most other tumours. The goal of this study was to evaluate the frequencies of co-occurring mutations and copy-number aberrations (CNAs) within the same tumour and to evaluate their potential clinical impact. Mass-spectrometry based mutation profiling using a customized lung cancer panel evaluating 214 mutations across 26 key NSCLC genes was performed on 230 non-squamous NSCLC and integrated with genome-wide CNAs and clinical variables. Among the 138 cases having at least one mutation, one third (41, 29.7%) showed two or more mutations, either in the same gene (double mutation) or in different genes (co-mutations). In EGFR mutant cancers, there was a double mutation in 18% and co-mutations in the following genes: TP53 (10%), PIK3CA (8%), STK11 (6%) and MET (4%). Significant relationships were detected between EGFR mutation and 1p, 7p copy gains (harbouring the EGFR gene), as well as 13q copy loss. KRAS mutation was significantly related with 1q gain and 3q loss. For stage I, tumours harbouring at least one mutation or PIK3CA mutation were significantly correlated with poor prognosis (p-value=0.02). When combining CNAs and mutational status, patients having both KRAS mutation and the highest related CNA (3q22.3 copy loss) showed a significant poorer prognosis (p-value=0.03). Our study highlights the clinical relevance of studying the tumour complexity by integrative genomic analysis and the need for developing assays that broadly screen for both "actionable" mutations and copy-number alterations to improve precision of stratified treatment approaches. © 2014 Wiley Periodicals, Inc.International Journal of Cancer 01/2014; · 6.20 Impact Factor
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ABSTRACT: v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) genotyping is required prior to anti-epidermal growth factor receptor monoclonal antibody therapy administered in cases of metastatic colorectal carcinoma (CRC). Thus, KRAS mutation screening is required for patient management. The present study reported the experience of KRAS/v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutational screening on synchronous CRC pairs from 26 patients, which were defined as index lesions (ILs) and concurrent lesions (CLs) on the basis of tumor grade and dimension and their respective lymph node and distant metastases. Overall, KRAS mutations were present in 38.4% of patients, whereas BRAF mutations were present at a frequency of 11.5%. The genotyping of paired synchronous carcinomas indicated that 11 patients (42.3%) exhibited discordant KRAS mutational statuses in terms of the presence of a mutation in only one lesion of the pair or of two different mutations harbored by each lesion. BRAF mutations were present in the synchronous tumors of two cases, whereas in two other cases, only the IL or CL harbored mutant BRAF. Overall, the mutational statuses of distant and lymph node metastases confirm the genetic heterogeneity of synchronous primary tumors. These results highlighted the fact that adequate sampling and comprehensive testing, when feasible, is likely to optimize the decision-making process for treatment approaches, even in the relatively rare event of multiple synchronous lesions.Oncology letters 05/2014; 7(5):1532-1536. · 0.24 Impact Factor