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

Figures & data

Table 1 Summary of cases with discrepant results when Sanger sequencing, real-time PCR with the TheraScreen^® kit, pyrosequencing, chip array infiniti^® assay, and 454 next-generation sequencing were used to detect KRAS mutations in formalin-fixed, paraffin-embedded samples

Sample	Tumor cell enrichment	Sanger sequencing	TheraScreen real-time PCR	Pyrosequencing	Chip array assay	NGS 454	Comments
2	50%	Gly13Cys	WT	Gly13Cys	Gly13Cys	Gly13Cys	Mutation not in the TheraScreen kit
17	20%	WT	Gly12Asp	Gly12Asp	Gly12Asp	Gly12Asp	Sanger fails, low enrichment
20	90%	Gly13Cys	WT	Gly13Cys	Gly13Cys	Gly13Cys	Mutation not in the TheraScreen kit
22	20%	WT	Gly12Val	Gly12Val	Gly12Val	Gly12Val	Sanger fails, low enrichment
25	80%	Gly12Cys	Gly12Cys	WT	WT	Gly12Cys	Pyrosequencing and chip array fail, codon 11 mutation
37	30%	WT	Gly13Asp	Gly13Asp	Gly13Asp	Gly13Asp	Sanger fails, low enrichment
39	60%	Gly13Val	WT	Gly13Val	Gly13Val	Gly13Val	Mutation not in the TheraScreen kit
42	5%	WT	Gly12Val	WT	WT	Gly12Val	Detected only by NGS and TheraScreen kit, low enrichment
44	5%	WT	Gly13Asp	Gly13Asp	Gly13Asp	Gly13Asp	Only Sanger fails, low enrichment
45	5%	WT	Gly13Asp	WT	WT	Gly13Asp	Detected only by NGS and TheraScreen kit, low enrichment
52	30%	Gly13Asp	Gly13Asp	Gly13Asp	N/A	Gly13Asp	Chip array fails, low DNA concentration
54	80%	Gly13Cys	WT	Gly13Cys	Gly13Cys	Gly13Cys	Mutation not in the TheraScreen kit
60	60%	Gly12Phe	Gly12Val	Gly12Phe	Gly12Phe	Gly12Phe	Double point mutation in codon 12, detected by all methods; TheraScreen kit fails to detect Phe substitution

Abbreviations: PCR, polymerase chain reaction; WT, wild type; N/A, not amplified; NGS, next-generation sequencing; DNA, deoxyribonucleic acid.

Table 2 Concordance, sensitivity, specificity, and accuracy of Sanger sequencing, real-time polymerase chain reaction with TheraScreen^® kit, pyrosequencing, and chip array infiniti^® assay measured using 454 next-generation sequencing as the reference method

Method	Kappa of concordance	Sensitivity	Specificity	Accuracy
Sanger sequencing	0.791	34/40 (85%)	100%	54/60 (90%)
TheraScreen (ΔCt)	0.857	36/40 (90%)	100%	56/60 (93%)
Pyrosequencing (percent mutated)	0.892	37/40 (93%)	100%	57/60 (95%)
Chip array assay (ratio)*	0.891	36/39 (92%)	100%	56/59 (95%)

Note:

* One sample not assessed, values calculated on 59/60 cases.

Table 3 Statistical correlation between tumor cell enrichment (neoplastic to non-neoplastic cell ratio evaluated on histology sections) and quantification of KRAS mutation abundance according to Sanger sequencing, real-time polymerase chain reaction with TheraScreen^® kit, pyrosequencing, chip array infiniti^® assay, and 454 next-generation sequencing

Method (quantification criteria)	Tumor cell enrichment	TheraScreen	Pyrosequencing	Chip array assay	NGS 454
Sanger sequencing*	N/A	N/A	N/A	N/A	N/A
TheraScreen (ΔCt)	0.030	–	–	–	–
Pyrosequencing (% mutated)	0.007	0.002	–	–	–
Chip array assay (ratio)	0.003	0.004	<0.001	–	–
NGS 454 (% mutated)	0.004	<0.001	<0.001	<0.001	–

Notes:P value < 0.05 considered statistically significant.

* No precise quantification can be achieved with Sanger sequencing.

Abbreviations: N/A, not amplified; NGS, next-generation sequencing.