A comparison of Direct sequencing, Pyrosequencing, High resolution melting analysis, TheraScreen DxS, and the K-ras StripAssay for detecting KRAS mutations in non small cell lung carcinomas

Abstract

BackgroundIt is mandatory to confirm the absence of mutations in the KRAS gene before treating metastatic colorectal cancers with epidermal growth factor receptor inhibitors, and similar regulations are being considered for non-small cell lung carcinomas (NSCLC) and other tumor types. Routine diagnosis of KRAS mutations in NSCLC is challenging because of compromised quantity and quality of biological material. Although there are several methods available for detecting mutations in KRAS, there is little comparative data regarding their analytical performance, economic merits, and workflow parameters.MethodsWe compared the specificity, sensitivity, cost, and working time of five methods using 131 frozen NSCLC tissue samples. We extracted genomic DNA from the samples and compared the performance of Sanger cycle sequencing, Pyrosequencing, High-resolution melting analysis (HRM), and the Conformité Européenne (CE)-marked TheraScreen DxS and K-ras StripAssay kits.Results and conclusionsOur results demonstrate that TheraScreen DxS and the StripAssay, in that order, were most effective at diagnosing mutations in KRAS. However, there were still unsatisfactory disagreements between them for 6.1% of all samples tested. Despite this, our findings are likely to assist molecular biologists in making rational decisions when selecting a reliable, efficient, and cost-effective method for detecting KRAS mutations in heterogeneous clinical tumor samples.

Rapid screening for sickle cell disease by polymerase chain reaction-high resolution melting analysis

“…Moreover, RFLP and ARMS assays are required for confirmation testing (14). Although the DNA sequencing technique is widely recognized as the gold standard for screening genomic variations, its widespread use is hampered by its cost-and time-consuming nature (15).…”

KRAS mutations in tumor tissue and plasma by different assays predict survival of patients with metastatic colorectal cancer

“…Over the past five years, several new techniques, including peptide-nucleic-acid-mediated polymerase chain reaction clamping (PNA-PCR), have emerged. These methods have higher sensitivity than direct sequencing, and permit the detection of lower mutation frequencies (1%-5%) [14][15][16].…”

926 KRas Mutation Analysis by PCR – Comparison of Two Methods

“…In tumors with little mutant DNA present, this will lead to differences in mutation detection. Similar small differences between methods using Therascreen as a comparator have been reported by others [18,[24][25][26][27][28][29]. Chang et al [23] conducted a retrospective study of the CRYS-TAL trial samples with Therasceen and LNA, showing excellent concordance, while Gonzalez de Castro et al [18] showed similarly good concordance of 98% between Therascreen and cobas (Roche).…”

“…Altimari et al [27] showed reasonable concordance between Therascreen, pyrosequencing and Roche 454 sequencing, but noted the poor diagnostic sensitivity of Sanger sequencing. Therascreen has also been used to validate other PCR assays [25,28], including high resolution melt analysis [29]. It should be noted that the diagnostic sensitivity of sequencing methods is dependent on the size of PCR products: FFPE tissue samples have fragmentation of their DNA and products > 150 bp suffer loss of diagnostic sensitivity [30].…”