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

Abstract

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.

Molecular Diagnostic of Solid Tumor Using a Next Generation Sequencing Custom-Designed Multi-Gene Panel

“…Our multi-gene custom panel-designed for the NGS MiSeq platform (Illumina)-has an analytical sensitivity of 5%. Sequencing results are concordant with those obtained using a previously validated single-gene targeted approach using the 454 GS-Junior sequencer [26,27,35] and with those obtained with the Gene Studio S5 platform.…”

“…The samples were then sequenced using a MiSeq sequencing platform (Illumina Inc., San Diego, CA, USA), according to manufacturers' instruction. Samples used for panel validation (see "Custom-designed multi-gene panel analytical sensitivity" and "Custom-designed multi-gene panel analytical validation" paragraphs) were also analyzed: (i) following a "single-gene approach" using the 454 GS-Junior sequencer (Roche Diagnostics, Rotkreuz, Switzerland) as previously described [26][27][28][29]; (ii) running the same panel using the GeneStudio S5 sequencing platform with the "Ion Ampliseq Library Kit Plus" (Thermo Fisher Scientific, Waltham, MA, USA), according to manufacturer's instructions.…”

SensiScreen® KRAS exon 2-sensitive simplex and multiplex real-time PCR-based assays for detection of KRAS exon 2 mutations

“…This was especially the case when comparing to DS even though the percentages of mutations detected by DS were in accordance with the literature [37,38]. However, also when comparing to the very sensitive ME-PCR method SensiScreen ® was able to identify an additional mutant case.…”

Comparative examination of various PCR-based methods for DNMT3A and IDH1/2 mutations identification in acute myeloid leukemia

“…This method provides high multiplexing possibilities together with high sensitivity and broad spectrum of detected mutations [38]. However NGS is associated with high costs, high hands-on time and high computational expertise.…”