KRAS mutation testing in metastatic colorectal cancer

Table 1 Predictive value of KRAS for anti-epidermal growth factor receptor therapy in metastatic colorectal cancer

Reference	Regimen	Treatment line	Phase	n	Mutation status (%)	Method	Remarkable results
Monotherapy
Karapetis et al[9], 2008	Cetuximab vs BSC	Chemotherapy refractory	III	394	42.3	Sequencing	Cetuximab alone works on patient with WT KRAS tumors
Amado et al[10], 2008	Panitumumab vs BSC	Chemotherapy refractory	III	427	43	Allele-specific PCR (DxS, United Kingdom)	Panitumumab alone works on patient with WT KRAS tumors
Combined with chemotherapy
Van Cutsem et al[11], 2009	Cetuximab + FOLFIRI, FOLFIRI	First-line CRYSTAL trial	III	540	35.6	PCR clamping and HRM (TIB MolBioL, Germany)	Cetuximab plus FOLFIRI, reduced the risk of progression of metastatic colorectal cancer
Bokemeyer et al[12], 2009	Cetuximab + FOLFOX, FOLFOX	First-line, OPUS trial	II	233	42	PCR clamping and HRM (TIB MolBioL, Germany)	Significantly increased ORR in patients with WT KRAS tumors
Peeters et al[13], 2010	Panitumumab + FOLFIRI FOLFIRI	Second-line	III	1083	45	Allele-specific PCR (DxS, United Kingdom)	Significantly improved PFS in patients with WT KRAS tumors
Douillard et al[14], 2010	Panitumumab + FOLFOX FOLFOX	First-line	III	1096	40	Allele-specific PCR (DxS, United Kingdom)	Significantly improved PFS in patients with WT KRAS tumors
Van Cutsem et al[15], 2011	Cetuximab + FOLFIRI, FOLFIRI	First-line	III	1063	37	PCR clamping and HRM (TIB MolBioL, Germany)	Significantly improved OS in patients with WT KRAS tumors

BSC: Best supportive care; WT: Wild type; ORR: Overall response rate; FOLFIRI: 5-fluorouracil, leucovorin and irinotecan; FOLFOX: 5-fluorouracil, leucovorin and oxaliplatin; PFS: Progression-free survival; OS: Overall survival; PCR: Polymerase chain reaction; HRM: High-resolution melting.

Table 2 Methods used for KRAS mutation testing[45,55,60-62]

Method	Sensitivity (mutant/wild-type) (%)	Turnaround time	Main advantages	Main disadvantages
Sanger sequencing	20–30	Slow (4 d to 2 wk)	Detects all possible mutations, cost-effective	Insensitive, time consuming, open PCR system is easily contaminated
Pyrosequencing	5	Rapid	Detects all possible mutations, sensitive	Open PCR system is easily contaminated
Real-time PCR with HRMA	5	Rapid	Rapid, closed PCR system, detects all possible mutations (heterozygous and homozygous)	Occasionally difficult to distinguish between mutation types
Allele-specific real-time PCR	10	Rapid	Rapid, closed PCR system	Detects only the 7 most common mutations, requires more tissue for analysis compared with other methods
RFLP with sequencing	0.1	Slow (4 d to 2 wk)	Sensitive	Requires confirmation by sequencing, complicated
DxS (ARMS/S)	1	Rapid	Sensitive, time-saving	Expensive, detects specific mutations targeted by the designed primers
COLD-PCR with sequencing	1-2.5	Rapid	Sensitive, cost-effective, detects all possible mutations	-

ARMS: Amplification refractory mutation system; RFLP: Restriction fragment length polymorphism; PCR: Polymerase chain reaction; COLD-PCR: Coamplification at lower denaturation temperature PCR.