patients with chemorefractory metastatic colorectal carcinoma were enrolled into one of two panitumumab monotherapy studies ( nct00089635 and nct00083616 ) . the study protocols were approved by the institutional review boards and all patients signed a written consent form . a subset of patients was selected for this analysis from a total of 388 patients . tumor scans were read centrally by a panel of at least two blinded independent radiologists using a modification of the who criteria . assessments were performed at four week intervals through week 28 and every three months thereafter until progression of disease . kras mutational status in the tissue was predetermined using the dxs assay ( qiagen ) . dna was purified from of 0.21ml of banked serum using the qiaamp circulating nucleic acid kit ( qiagen , cat . the ligation assays were performed as previously described using the primers and probes indicated in supplementary table 5 . in brief , kras fragments containing codons 12 and 13 were amplified with primers designed to yield a small pcr product ( 106 bp ) to accommodate the degraded dna found in serum . note that some of the probes contained locked - nucleic acid ( lna ) linkages ( exiqon ) . to confirm and further quantify samples containing kras mutations , beaming assays were employed using the primers and probes listed in supplementary table 5 . the number of mutant fragments per ml serum was determined from the fraction of alleles containing the mutant allele ( determined by beaming ) and the number of alleles assessed per ml of plasma ( determined by qpcr and reported in supplementary table 6 ) . patients with chemorefractory metastatic colorectal carcinoma were enrolled into one of two panitumumab monotherapy studies ( nct00089635 and nct00083616 ) . the study protocols were approved by the institutional review boards and all patients signed a written consent form . a subset of patients was selected for this analysis from a total of 388 patients . tumor scans were read centrally by a panel of at least two blinded independent radiologists using a modification of the who criteria . assessments were performed at four week intervals through week 28 and every three months thereafter until progression of disease . kras mutational status in the tissue was predetermined using the dxs assay ( qiagen ) . dna was purified from of 0.21ml of banked serum using the qiaamp circulating nucleic acid kit ( qiagen , cat . the ligation assays were performed as previously described using the primers and probes indicated in supplementary table 5 . in brief , kras fragments containing codons 12 and 13 were amplified with primers designed to yield a small pcr product ( 106 bp ) to accommodate the degraded dna found in serum . note that some of the probes contained locked - nucleic acid ( lna ) linkages ( exiqon ) . to confirm and further quantify samples containing kras mutations , beaming assays were employed using the primers and probes listed in supplementary table 5 . the number of mutant fragments per ml serum was determined from the fraction of alleles containing the mutant allele ( determined by beaming ) and the number of alleles assessed per ml of plasma ( determined by qpcr and reported in supplementary table 6 ) .
colorectal tumors that are wild - type ( wt ) for kras are often sensitive to egfr blockade , but almost always develop resistance within several months of initiating therapy1,2 . 
 the mechanisms underlying this acquired resistance to anti - egfr antibodies are largely unknown . 
 this situation stands in marked contrast to that of small molecule targeted agents , such as inhibitors of abl , egfr , braf , and mek , in which mutations in the genes encoding the protein targets render the tumors resistant to the effects of the drugs36 . the simplest hypothesis to account for the development of resistance to egfr blockade 
 are that rare cells with kras mutations pre - exist at low levels in tumors with ostensibly wt kras genes . 
 though this hypothesis would seem readily testable , there is no evidence in pre - clinical models to support it , nor is there data from patients . to test this hypothesis 
 , we determined whether mutant kras dna could be detected in the circulation of 28 patients receiving monotherapy with panitumumab , a therapeutic anti - egfr antibody . 
 we found that nine of 24 ( 38% ) patients whose tumors were initially kras wt developed detectable mutations in kras in their sera , three of which developed multiple different kras mutations . 
 the appearance of these mutations was very consistent , generally occurring between five to six months following treatment . 
 mathematical modeling indicated that the mutations were present in expanded subclones prior to the initiation of panitumumab . 
 these results suggest that the emergence of kras mutations is a mediator of acquired resistance to egfr blockade and that these mutations can be detected in a non - invasive manner . 
 moreover , they explain why solid tumors develop resistance to targeted therapies in a highly reproducible fashion .