BACKGROUND: Following the discovery that mutant KRAS is associated with 
resistance to anti-epidermal growth factor receptor (EGFR) antibodies, the 
tumours of patients with metastatic colorectal cancer are now profiled for seven 
KRAS mutations before receiving cetuximab or panitumumab. However, most patients 
with KRAS wild-type tumours still do not respond. We studied the effect of other 
downstream mutations on the efficacy of cetuximab in, to our knowledge, the 
largest cohort to date of patients with chemotherapy-refractory metastatic 
colorectal cancer treated with cetuximab plus chemotherapy in the pre-KRAS 
selection era.
METHODS: 1022 tumour DNA samples (73 from fresh-frozen and 949 from 
formalin-fixed, paraffin-embedded tissue) from patients treated with cetuximab 
between 2001 and 2008 were gathered from 11 centres in seven European countries. 
773 primary tumour samples had sufficient quality DNA and were included in 
mutation frequency analyses; mass spectrometry genotyping of tumour samples for 
KRAS, BRAF, NRAS, and PIK3CA was done centrally. We analysed objective response, 
progression-free survival (PFS), and overall survival in molecularly defined 
subgroups of the 649 chemotherapy-refractory patients treated with cetuximab 
plus chemotherapy.
FINDINGS: 40.0% (299/747) of the tumours harboured a KRAS mutation, 14.5% 
(108/743) harboured a PIK3CA mutation (of which 68.5% [74/108] were located in 
exon 9 and 20.4% [22/108] in exon 20), 4.7% (36/761) harboured a BRAF mutation, 
and 2.6% (17/644) harboured an NRAS mutation. KRAS mutants did not derive 
benefit compared with wild types, with a response rate of 6.7% (17/253) versus 
35.8% (126/352; odds ratio [OR] 0.13, 95% CI 0.07-0.22; p<0.0001), a median PFS 
of 12 weeks versus 24 weeks (hazard ratio [HR] 1.98, 1.66-2.36; p<0.0001), and a 
median overall survival of 32 weeks versus 50 weeks (1.75, 1.47-2.09; p<0.0001). 
In KRAS wild types, carriers of BRAF and NRAS mutations had a significantly 
lower response rate than did BRAF and NRAS wild types, with a response rate of 
8.3% (2/24) in carriers of BRAF mutations versus 38.0% in BRAF wild types 
(124/326; OR 0.15, 95% CI 0.02-0.51; p=0.0012); and 7.7% (1/13) in carriers of 
NRAS mutations versus 38.1% in NRAS wild types (110/289; OR 0.14, 0.007-0.70; 
p=0.013). PIK3CA exon 9 mutations had no effect, whereas exon 20 mutations were 
associated with a worse outcome compared with wild types, with a response rate 
of 0.0% (0/9) versus 36.8% (121/329; OR 0.00, 0.00-0.89; p=0.029), a median PFS 
of 11.5 weeks versus 24 weeks (HR 2.52, 1.33-4.78; p=0.013), and a median 
overall survival of 34 weeks versus 51 weeks (3.29, 1.60-6.74; p=0.0057). 
Multivariate analysis and conditional inference trees confirmed that, if KRAS is 
not mutated, assessing BRAF, NRAS, and PIK3CA exon 20 mutations (in that order) 
gives additional information about outcome. Objective response rates in our 
series were 24.4% in the unselected population, 36.3% in the KRAS wild-type 
selected population, and 41.2% in the KRAS, BRAF, NRAS, and PIK3CA exon 20 
wild-type population.
INTERPRETATION: While confirming the negative effect of KRAS mutations on 
outcome after cetuximab, we show that BRAF, NRAS, and PIK3CA exon 20 mutations 
are significantly associated with a low response rate. Objective response rates 
could be improved by additional genotyping of BRAF, NRAS, and PIK3CA exon 20 
mutations in a KRAS wild-type population.
FUNDING: Belgian Federation against Cancer (Stichting tegen Kanker).