KRAS Mutation

Franklin, Wilbur A.; Haney, Jerry; Sugita, Michio; Bemis, Lynne T.; Jimeno, Antonio; Messersmith, Wells A.

doi:10.2353/jmoldx.2010.080131

Cited by 102 publications

(38 citation statements)

References 26 publications

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“…With optimized sample handling and standardized DNA extraction, it is possible to screen paraffin-embedded samples with higher sensitivity [40], [41]. Despite the increased fragmentation of DNA extracted from paraffin-embedded tissue, it is possible to reliably screen shorter amplification products up to 250 bp in length.…”

Section: Discussionmentioning

confidence: 99%

Detection of Somatic Mutations by High-Resolution DNA Melting (HRM) Analysis in Multiple Cancers

et al. 2011

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Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

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Section: Discussionmentioning

confidence: 99%

Detection of Somatic Mutations by High-Resolution DNA Melting (HRM) Analysis in Multiple Cancers

et al. 2011

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“…For first round mutational analysis, DNA samples from selectively microdissected areas were PCR amplified with primers flanking KRAS exon 2 as previously described (35), followed by Sanger DNA sequencing. Positive samples for mutation by Sanger sequencing were not further evaluated, negative samples were evaluated by HRM curve to achieve higher analytical sensitivity.…”

Section: Methodsmentioning

confidence: 99%

ROS1 and ALK Fusions in Colorectal Cancer, with Evidence of Intratumoral Heterogeneity for Molecular Drivers

Aisner

Nguyen

Paskulin

et al. 2014

Molecular Cancer Research

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102

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Activated ALK and ROS1 tyrosine kinases, through gene fusions, has been found in lung adenocarcinomas and are highly sensitive to selective kinase inhibitors. This study aimed at identifying the presence of these rearrangements in human colorectal adenocarcinoma (CRC) specimens using a 4-target, 4-color break-apart fluorescence in situ hybridization (FISH) assay to simultaneously determine the genomic status of ALK and ROS1. Among the clinical CRC specimens analyzed, rearrangement-positive cases for both ALK and ROS1 were observed. The fusion partner for ALK was identified as EML4 and the fusion partner for one of the ROS1-positive cases was SLC34A2, the partner for the other ROS1-positive case remains to be identified. A small fraction of specimens presented duplicated or clustered copies of native ALK and ROS1. In addition, rearrangements were detected in samples that also harbored KRAS and BRAF mutations in two of the three cases. Interestingly, the ALK-positive specimen displayed marked intra-tumoral heterogeneity and rearrangement was also identified in regions of high-grade dysplasia. Despite the additional oncogenic events and tumor heterogeneity observed, elucidation of the first cases of ROS1 rearrangements and confirmation of ALK rearrangements support further evaluation of these genomic fusions as potential therapeutic targets in CRC. Implications ROS1 and ALK fusions occur in colorectal cancer and may have substantial impact in therapy selection.

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“…Several reports have compared the performance of pyrosequencing, real-time PCR, and Sanger sequencing in detecting KRAS mutation for molecular diagnostics 9–11. More recently, the Infiniti assay12,13 and 454 next-generation sequencing14,15 limited to discordant cases have been compared with other techniques.…”

Section: Introductionmentioning

confidence: 99%

454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples

Altimari

Biase²,

Maglio³

et al. 2013

OTT

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Detection of KRAS mutations in archival pathology samples is critical for therapeutic appropriateness of anti-EGFR monoclonal antibodies in colorectal cancer. We compared the sensitivity, specificity, and accuracy of Sanger sequencing, ARMS-Scorpion (TheraScreen®) real-time polymerase chain reaction (PCR), pyrosequencing, chip array hybridization, and 454 next-generation sequencing to assess KRAS codon 12 and 13 mutations in 60 nonconsecutive selected cases of colorectal cancer. Twenty of the 60 cases were detected as wild-type KRAS by all methods with 100% specificity. Among the 40 mutated cases, 13 were discrepant with at least one method. The sensitivity was 85%, 90%, 93%, and 92%, and the accuracy was 90%, 93%, 95%, and 95% for Sanger sequencing, TheraScreen real-time PCR, pyrosequencing, and chip array hybridization, respectively. The main limitation of Sanger sequencing was its low analytical sensitivity, whereas TheraScreen real-time PCR, pyrosequencing, and chip array hybridization showed higher sensitivity but suffered from the limitations of predesigned assays. Concordance between the methods was k = 0.79 for Sanger sequencing and k > 0.85 for the other techniques. Tumor cell enrichment correlated significantly with the abundance of KRAS-mutated deoxyribonucleic acid (DNA), evaluated as ΔCt for TheraScreen real-time PCR (P = 0.03), percentage of mutation for pyrosequencing (P = 0.001), ratio for chip array hybridization (P = 0.003), and percentage of mutation for 454 next-generation sequencing (P = 0.004). Also, 454 next-generation sequencing showed the best cross correlation for quantification of mutation abundance compared with all the other methods (P < 0.001). Our comparison showed the superiority of next-generation sequencing over the other techniques in terms of sensitivity and specificity. Next-generation sequencing will replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues.

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KRAS Mutation

Cited by 102 publications

References 26 publications

Detection of Somatic Mutations by High-Resolution DNA Melting (HRM) Analysis in Multiple Cancers

Detection of Somatic Mutations by High-Resolution DNA Melting (HRM) Analysis in Multiple Cancers

ROS1 and ALK Fusions in Colorectal Cancer, with Evidence of Intratumoral Heterogeneity for Molecular Drivers

454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples

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