Background Fast, cheap and reliable methods are needed to identify large populations, which may be at risk in relation to environmental exposure. Polymorphisms in NAT1 (N-acetyl transferase) may be suitable markers to identify individuals at risk.
Background
Fast, cheap and reliable methods are needed to identify large populations, which may be at risk in relation to environmental exposure. Polymorphisms in NAT1 (N-acetyl transferase) may be suitable markers to identify individuals at risk.Results A strategy allowing to address simultaneously 24 various genetic variants in the NAT1 gene using the single sequencing reaction method on the same PCR product is described. A modified automated DNA sequencing using only one of the sequence terminators was used to genotype PCR products in single-track sequencing reactions of NAT1 and was shown to be universal for both DNA sequencing using labeled primers and labeled nucleotides. By this method we detected known SNPs at site T640G, which confers the NAT1*11 allele with frequency of 0.036, further T1088A and C1095A with frequency of 0.172 and 0.188, respectively and a deletion of TAATAATAA in the poly A signal area with a frequency 0.031. All observed frequencies were in Hardy Weinberg equilibrium and comparable to those in Caucasian population. The single-track signatures of the variant genotypes were verified on samples previously genotyped by RLFP.
Results
A strategy allowing to address simultaneously 24 various genetic variants in the NAT1 gene using the single sequencing reaction method on the same PCR product is described. A modified automated DNA sequencing using only one of the sequence terminators was used to genotype PCR products in single-track sequencing reactions of NAT1 and was shown to be universal for both DNA sequencing using labeled primers and labeled nucleotides. By this method we detected known SNPs at site T640G, which confers the NAT1*11 allele with frequency of 0.036, further T1088A and C1095A with frequency of 0.172 and 0.188, respectively and a deletion of TAATAATAA in the poly A signal area with a frequency 0.031. All observed frequencies were in Hardy Weinberg equilibrium and comparable to those in Caucasian population. The single-track signatures of the variant genotypes were verified on samples previously genotyped by RLFP.Conclusions The method could be of great help to scientists in the field of molecular epidemiology of screening of large populations for known informative biomarkers of susceptibility, such as NAT1.
Conclusions
The method could be of great help to scientists in the field of molecular epidemiology of screening of large populations for known informative biomarkers of susceptibility, such as NAT1.

Background We have previously described the single sequencing reaction (SSR) protocol for assessment of a known polymorphism in the 3'utr region of  CYP19  (aromatase) [ 1 , 2 ]. Here we have extended and optimized the use of the method for multiple polymorphisms in the  NAT1  gene. The increasing number of detected mutations in the  NAT1 , makes genotyping using conventional restriction fragment length polymorphism (RFLP) or allele-specific amplification complicated. We developed a rapid and universal strategy based on single-track DNA sequencing analysis of a unique PCR product encompassing the entire  NAT1  coding region (contains no introns) along with the flanking 5' and 3' untranslated regions. It allows a rapid and economic characterization of  NAT1  alleles. Our method brings reproducible results on both Alf Express™ (Pharmacia) and ABI310 PRISM sequencing instruments and may be adopted for majority of epidemiological studies with relevance of  NAT1  in environmentally related diseases. N-acetyltransferases (NAT, EC 2.3.1.5) are implicated in the biotransformation of primary arylamines (e.g. 2-naphthylamine and aminobiphenyls), heterocyclic amines, hydrazines, and their N-hydroxylated metabolites present in tobacco smoke and food [ 3 - 7 ]. An increased activity for p-aminobenzoic acid acetylation (marker for NAT1 activity) was observed in the breast malignant tissues compared to benign and control tissues [ 8 , 9 ]. Human NATs may have adapted a common catalytic mechanism from cysteine proteases for acetyl-transfer reactions [ 10 , 11 ]. The  NAT1  gene is highly polymorphic (for listing of known variant alleles  ). Expression of  NAT1*16  but not  NAT1*10  and  NAT1*11  caused a 2-fold decrease in the amount and catalytic activity of NAT1 in COS-1 cell cytosol [ 12 - 14 ]. All available data suggest that slow NAT1 phenotype results from  NAT1  allelic variants that encode reduced expression of  NAT1  and/or less-stable NAT1 protein [ 15 ]. Epidemiological studies suggest that the  NAT1  and  NAT2  acetylation polymorphisms modify the risk of developing urinary bladder, colorectal, breast, head and neck, lung, and possibly prostate cancers [ 16 - 18 ]. Interactions between  NAT2*4  and  NAT1*10  were suggested by the increased frequency of the  NAT2*4/NAT1*10  haplotype [ 19 - 21 ]. The individual risks associated with NAT1 acetylator phenotypes/genotypes are usually small, but they increase when considered in conjunction with other susceptibility genes and/or aromatic and heterocyclic amine carcinogen exposures. Because of the relatively high frequency of the variant  NAT1  genotypes in the population, the attributable cancer risk may be high. Large-scale molecular epidemiological studies that investigate the role of  NAT1  genotypes and/or phenotypes together with other genetic susceptibility gene polymorphisms and biomarkers of carcinogen exposure are necessary to expand our current understanding of the role of NAT1 acetylation polymorphisms in cancer risk [ 22 ].
Background
We have previously described the single sequencing reaction (SSR) protocol for assessment of a known polymorphism in the 3'utr region of  CYP19  (aromatase) [ 1 , 2 ]. Here we have extended and optimized the use of the method for multiple polymorphisms in the  NAT1  gene. The increasing number of detected mutations in the  NAT1 , makes genotyping using conventional restriction fragment length polymorphism (RFLP) or allele-specific amplification complicated. We developed a rapid and universal strategy based on single-track DNA sequencing analysis of a unique PCR product encompassing the entire  NAT1  coding region (contains no introns) along with the flanking 5' and 3' untranslated regions. It allows a rapid and economic characterization of  NAT1  alleles. Our method brings reproducible results on both Alf Express™ (Pharmacia) and ABI310 PRISM sequencing instruments and may be adopted for majority of epidemiological studies with relevance of  NAT1  in environmentally related diseases.CYP19NAT1NAT1NAT1NAT1NAT1N-acetyltransferases (NAT, EC 2.3.1.5) are implicated in the biotransformation of primary arylamines (e.g. 2-naphthylamine and aminobiphenyls), heterocyclic amines, hydrazines, and their N-hydroxylated metabolites present in tobacco smoke and food [ 3 - 7 ]. An increased activity for p-aminobenzoic acid acetylation (marker for NAT1 activity) was observed in the breast malignant tissues compared to benign and control tissues [ 8 , 9 ]. Human NATs may have adapted a common catalytic mechanism from cysteine proteases for acetyl-transfer reactions [ 10 , 11 ]. The  NAT1  gene is highly polymorphic (for listing of known variant alleles  ). Expression of  NAT1*16  but not  NAT1*10  and  NAT1*11  caused a 2-fold decrease in the amount and catalytic activity of NAT1 in COS-1 cell cytosol [ 12 - 14 ]. All available data suggest that slow NAT1 phenotype results from  NAT1  allelic variants that encode reduced expression of  NAT1  and/or less-stable NAT1 protein [ 15 ].NAT1NAT1*16NAT1*10NAT1*11NAT1NAT1Epidemiological studies suggest that the  NAT1  and  NAT2  acetylation polymorphisms modify the risk of developing urinary bladder, colorectal, breast, head and neck, lung, and possibly prostate cancers [ 16 - 18 ]. Interactions between  NAT2*4  and  NAT1*10  were suggested by the increased frequency of the  NAT2*4/NAT1*10  haplotype [ 19 - 21 ]. The individual risks associated with NAT1 acetylator phenotypes/genotypes are usually small, but they increase when considered in conjunction with other susceptibility genes and/or aromatic and heterocyclic amine carcinogen exposures. Because of the relatively high frequency of the variant  NAT1  genotypes in the population, the attributable cancer risk may be high. Large-scale molecular epidemiological studies that investigate the role of  NAT1  genotypes and/or phenotypes together with other genetic susceptibility gene polymorphisms and biomarkers of carcinogen exposure are necessary to expand our current understanding of the role of NAT1 acetylation polymorphisms in cancer risk [ 22 ].NAT1NAT2NAT2*4NAT1*10NAT2*4/NAT1*10NAT1NAT1Results We outlined a strategy allowing to address simultaneously 13 genetic variants in the coding area of  NAT1  with two sequencing tracks: A and G and 11 variants in the 3' flanking area with two tracks: A and T (Table  1 ). Using single-track sequencing of the  NAT1  coding region, we have detected 11 individuals carrying polymorphisms at site  T640G , which characterizes  NAT1*11  alleles (Figure  1C,1D,1E ). Identical results were obtained when performing the single-track sequencing on an Alf Express™ system as on ABI310 PRISM (Figure  2E,2F ). The frequency of this polymorphism, 0.036 found by single-track sequencing in our study was comparable to the frequency 0.032 found by PCR-RFLP method in 396 German control individuals [ 23 ]. Polymorphisms  T1088A  and  C1095A  (Figure  3A,3B,3C  and Figure  4A,4B,4C , representing each genotype analyzed by both Alf Express™ and ABI310 PRISM) were in perfect linkage disequilibrium. The frequency of these polymorphisms was 0.172 and 0.188 respectively and was comparable to frequency 0.206 reported by Bruhn  et al . [ 24 ]. A-track sequencing of the 3'-untranslated region of  NAT1  revealed a deletion of TAATAATAA (Figure  3D,3E  on Alf Express™ and Figure  4E  on ABI310 PRISM). It was found in 7 individuals accounting for the frequency 0.031, which is comparable to the frequency 0.033 observed in the study on 314 control individuals by Bruhn  et al . [ 24 ]. This change is also unique for the  NAT1*11  alleles and co-segregates with  T640G  polymorphism. Very good co-segregation of  T640G  and  1067-1090delTAATAATAA  polymorphisms in 7 informative samples was observed (4 heterozygotes and 3 homozygotes). Despite the different chemistry, Figures  1 ,  2 ,  3 ,  4  demonstrate perfect concordance between the results obtained by Alf Express™ and ABI310 PRISM analysis, verified on samples with previously characterized genotypes by RFLP.
Results
We outlined a strategy allowing to address simultaneously 13 genetic variants in the coding area of  NAT1  with two sequencing tracks: A and G and 11 variants in the 3' flanking area with two tracks: A and T (Table  1 ). Using single-track sequencing of the  NAT1  coding region, we have detected 11 individuals carrying polymorphisms at site  T640G , which characterizes  NAT1*11  alleles (Figure  1C,1D,1E ). Identical results were obtained when performing the single-track sequencing on an Alf Express™ system as on ABI310 PRISM (Figure  2E,2F ). The frequency of this polymorphism, 0.036 found by single-track sequencing in our study was comparable to the frequency 0.032 found by PCR-RFLP method in 396 German control individuals [ 23 ]. Polymorphisms  T1088A  and  C1095A  (Figure  3A,3B,3C  and Figure  4A,4B,4C , representing each genotype analyzed by both Alf Express™ and ABI310 PRISM) were in perfect linkage disequilibrium. The frequency of these polymorphisms was 0.172 and 0.188 respectively and was comparable to frequency 0.206 reported by Bruhn  et al . [ 24 ].NAT1NAT1T640GNAT1*11T1088AC1095Aet alA-track sequencing of the 3'-untranslated region of  NAT1  revealed a deletion of TAATAATAA (Figure  3D,3E  on Alf Express™ and Figure  4E  on ABI310 PRISM). It was found in 7 individuals accounting for the frequency 0.031, which is comparable to the frequency 0.033 observed in the study on 314 control individuals by Bruhn  et al . [ 24 ]. This change is also unique for the  NAT1*11  alleles and co-segregates with  T640G  polymorphism. Very good co-segregation of  T640G  and  1067-1090delTAATAATAA  polymorphisms in 7 informative samples was observed (4 heterozygotes and 3 homozygotes). Despite the different chemistry, Figures  1 ,  2 ,  3 ,  4  demonstrate perfect concordance between the results obtained by Alf Express™ and ABI310 PRISM analysis, verified on samples with previously characterized genotypes by RFLP.NAT1et alNAT1*11T640GT640G1067-1090delTAATAATAADiscussion A modified automated DNA sequencing with a fluorescent label was used to genotype PCR products spanning through the whole  NAT1  gene by single-track sequencing reactions in 192 control individuals. Previously, we have shown that single-track sequencing reactions performed on PCR products, with subsequent analysis on an Alf Express™ DNA Sequencer, can be as informative, sensitive, and accurate as complete sequencing reactions but more economical option for the genotyping of known polymorphisms in the human aromatase gene [ 1 ]. Compared to analysis by full gel-based sequencing, four times more samples can be analyzed per gel in considerably shorter time. The method could be particularly efficient in cases of high density of polymorphic sites residing in a common area as in the case of  NAT1 .
Discussion
A modified automated DNA sequencing with a fluorescent label was used to genotype PCR products spanning through the whole  NAT1  gene by single-track sequencing reactions in 192 control individuals. Previously, we have shown that single-track sequencing reactions performed on PCR products, with subsequent analysis on an Alf Express™ DNA Sequencer, can be as informative, sensitive, and accurate as complete sequencing reactions but more economical option for the genotyping of known polymorphisms in the human aromatase gene [ 1 ]. Compared to analysis by full gel-based sequencing, four times more samples can be analyzed per gel in considerably shorter time. The method could be particularly efficient in cases of high density of polymorphic sites residing in a common area as in the case of  NAT1 .NAT1NAT1Conclusions This paper shows that single-track sequencing reactions can be used as a tool for screening for all types of known polymorphisms in field laboratories with limited or overloaded sequence capacity. Compared to standard sequencing, this single track sequencing may have better signal to noise ratio. This approach is time and cost effective and can be accommodated for high-throughput analyses in epidemiology studies.
Conclusions
This paper shows that single-track sequencing reactions can be used as a tool for screening for all types of known polymorphisms in field laboratories with limited or overloaded sequence capacity. Compared to standard sequencing, this single track sequencing may have better signal to noise ratio. This approach is time and cost effective and can be accommodated for high-throughput analyses in epidemiology studies.Methods Materials Chemicals for PCR and sequencing were purchased from ABI (Applied Biosystems, Foster City, CA, USA) and Amersham Biosciences (Uppsala, Sweden). Subjects The study included 192 DNA samples from Norwegian healthy individuals obtained through Norwegian Population Registry as a population-based series of residents in the Oslo area. Test samples from healthy Norwegian controls with known genotypes (previously assessed by RFLP) were available. PCR amplification of NAT1 A single PCR amplification of the entire coding region and the 3' flanking area of  NAT1  (923 bp) using forward primer: 5'-tactgggctctgaccactat-3' and reverse primer: 5'-tgctttctagcataaatcacc-3' was performed. PCR mix contained 16.9 μl dH 2 O, 10 × PCR buffer (2.5 μl), 1.25 mM MgCl 2  (4 μl), 2.0 mM dNTP (1.0 μl mixture of each), 10 μM oligonucleotide primer, 0.2 μl Taq DNA polymerase (Perkin Elmer, 5 U/μl) and 1.0 μl of genomic DNA (100 ng/μl) in a final volume of 25 μl. Thermal cycling (GeneAmp 2400, PE, Foster city, CA) included initial denaturation 2 min at 94°C, 10 cycles of 30 sec at 94°C, 30 sec at decreasing annealing temperature 58 to 48°C, 1 min at 72°C, and 25 cycles of 30 sec at 94°C, 30 sec at 50°C and 1 min at 72°C. Quality of PCR products was checked on 6% acrylamide gels. Single-track sequencing The SNPs studied by this assay are summarized in Table  1 . Single A-, G- and T- track sequencing using 1 given terminator at a time was performed using both Alf Express™ system (Pharmacia Biotech, Uppsala, Sweden) and ABI310 PRISM capillary sequencer. Alf Express™ Cy5-labeled primers NAT1A-F-CY5: 5'-gggagggtatgtttacagca-3' and NAT1A-POLYA-CY5: 5'-gcataaatcaccaatttcca-3' were used for genotyping the coding region and 3'untranslated area of  NAT1 , respectively. The single sequencing reactions (7 μl) contained: 1.25 μl of dH 2 O, 0.5 μl of 10-times concentrated FS polymerase buffer (125 mM Tris-HCl, pH 9.5, 50 mM (NH 4 ) 2 SO 4 , 150 mM MgCl 2 ), 2 μl of ddNTP termination mix (containing 10 mM dNTPs and either ddATP or ddTTP (10 μM) for A-track or T-track sequencing respectively, 1 μl of 2 μM primer, 0.25 μl of FS polymerase (5 U/μl), and 2 μl of  NAT1  PCR product. The following conditions were used for thermal cycling: initial denaturation for 5 min at 95°C, 35 cycles of 30 sec at 95°C, 30 sec at 50°C, and 1 min at 68°C. The single-tracks were evaluated using conventional software AlfWin, Fragment Analyzer, v. 1.02 supplied with the sequencer (Alf Express™). For the single-track sequencing by ABI310 PRISM we used the same sequencing primers as for Alf Express™ system however labeled with the fluorophor 6-FAM. Sequencing reactions (10 μl) contained: 5.0 μl dH 2 O, 1.0 μl of 10-times concentrated Thermo Sequenase buffer (260 mM Tris-HCl, pH 9.5, 65 mM MgCl 2 ), 2.0 μl of ddNTP termination mix (containing dNTPs and either ddATP or ddGTP for A-track or G-track sequencing respectively – for composition see Table 3), 0.5 μl of 1 μM primer, 0.5 μl of Thermo Sequenase DNA polymerase (with pyrophosphatase) 3.2 U/μl and 1.0 μl  NAT1  PCR product (described above). Thermal cycling conditions were as follows: initial denaturation 20 sec at 95°C, 25 cycles of 20 sec at 95°C, 20 sec at 55°C and 1 min at 72°C, and then 10 cycles of 20 sec at 95°C, 1 min at 72°C. Results were evaluated using GeneScan Analysis software, v. 3.7.
Methods
Materials Chemicals for PCR and sequencing were purchased from ABI (Applied Biosystems, Foster City, CA, USA) and Amersham Biosciences (Uppsala, Sweden).
Materials
Chemicals for PCR and sequencing were purchased from ABI (Applied Biosystems, Foster City, CA, USA) and Amersham Biosciences (Uppsala, Sweden).Subjects The study included 192 DNA samples from Norwegian healthy individuals obtained through Norwegian Population Registry as a population-based series of residents in the Oslo area. Test samples from healthy Norwegian controls with known genotypes (previously assessed by RFLP) were available.
Subjects
The study included 192 DNA samples from Norwegian healthy individuals obtained through Norwegian Population Registry as a population-based series of residents in the Oslo area. Test samples from healthy Norwegian controls with known genotypes (previously assessed by RFLP) were available.PCR amplification of NAT1 A single PCR amplification of the entire coding region and the 3' flanking area of  NAT1  (923 bp) using forward primer: 5'-tactgggctctgaccactat-3' and reverse primer: 5'-tgctttctagcataaatcacc-3' was performed. PCR mix contained 16.9 μl dH 2 O, 10 × PCR buffer (2.5 μl), 1.25 mM MgCl 2  (4 μl), 2.0 mM dNTP (1.0 μl mixture of each), 10 μM oligonucleotide primer, 0.2 μl Taq DNA polymerase (Perkin Elmer, 5 U/μl) and 1.0 μl of genomic DNA (100 ng/μl) in a final volume of 25 μl. Thermal cycling (GeneAmp 2400, PE, Foster city, CA) included initial denaturation 2 min at 94°C, 10 cycles of 30 sec at 94°C, 30 sec at decreasing annealing temperature 58 to 48°C, 1 min at 72°C, and 25 cycles of 30 sec at 94°C, 30 sec at 50°C and 1 min at 72°C. Quality of PCR products was checked on 6% acrylamide gels.
PCR amplification of NAT1
A single PCR amplification of the entire coding region and the 3' flanking area of  NAT1  (923 bp) using forward primer: 5'-tactgggctctgaccactat-3' and reverse primer: 5'-tgctttctagcataaatcacc-3' was performed. PCR mix contained 16.9 μl dH 2 O, 10 × PCR buffer (2.5 μl), 1.25 mM MgCl 2  (4 μl), 2.0 mM dNTP (1.0 μl mixture of each), 10 μM oligonucleotide primer, 0.2 μl Taq DNA polymerase (Perkin Elmer, 5 U/μl) and 1.0 μl of genomic DNA (100 ng/μl) in a final volume of 25 μl. Thermal cycling (GeneAmp 2400, PE, Foster city, CA) included initial denaturation 2 min at 94°C, 10 cycles of 30 sec at 94°C, 30 sec at decreasing annealing temperature 58 to 48°C, 1 min at 72°C, and 25 cycles of 30 sec at 94°C, 30 sec at 50°C and 1 min at 72°C. Quality of PCR products was checked on 6% acrylamide gels.NAT1Single-track sequencing The SNPs studied by this assay are summarized in Table  1 . Single A-, G- and T- track sequencing using 1 given terminator at a time was performed using both Alf Express™ system (Pharmacia Biotech, Uppsala, Sweden) and ABI310 PRISM capillary sequencer. Alf Express™ Cy5-labeled primers NAT1A-F-CY5: 5'-gggagggtatgtttacagca-3' and NAT1A-POLYA-CY5: 5'-gcataaatcaccaatttcca-3' were used for genotyping the coding region and 3'untranslated area of  NAT1 , respectively. The single sequencing reactions (7 μl) contained: 1.25 μl of dH 2 O, 0.5 μl of 10-times concentrated FS polymerase buffer (125 mM Tris-HCl, pH 9.5, 50 mM (NH 4 ) 2 SO 4 , 150 mM MgCl 2 ), 2 μl of ddNTP termination mix (containing 10 mM dNTPs and either ddATP or ddTTP (10 μM) for A-track or T-track sequencing respectively, 1 μl of 2 μM primer, 0.25 μl of FS polymerase (5 U/μl), and 2 μl of  NAT1  PCR product. The following conditions were used for thermal cycling: initial denaturation for 5 min at 95°C, 35 cycles of 30 sec at 95°C, 30 sec at 50°C, and 1 min at 68°C. The single-tracks were evaluated using conventional software AlfWin, Fragment Analyzer, v. 1.02 supplied with the sequencer (Alf Express™). For the single-track sequencing by ABI310 PRISM we used the same sequencing primers as for Alf Express™ system however labeled with the fluorophor 6-FAM. Sequencing reactions (10 μl) contained: 5.0 μl dH 2 O, 1.0 μl of 10-times concentrated Thermo Sequenase buffer (260 mM Tris-HCl, pH 9.5, 65 mM MgCl 2 ), 2.0 μl of ddNTP termination mix (containing dNTPs and either ddATP or ddGTP for A-track or G-track sequencing respectively – for composition see Table 3), 0.5 μl of 1 μM primer, 0.5 μl of Thermo Sequenase DNA polymerase (with pyrophosphatase) 3.2 U/μl and 1.0 μl  NAT1  PCR product (described above). Thermal cycling conditions were as follows: initial denaturation 20 sec at 95°C, 25 cycles of 20 sec at 95°C, 20 sec at 55°C and 1 min at 72°C, and then 10 cycles of 20 sec at 95°C, 1 min at 72°C. Results were evaluated using GeneScan Analysis software, v. 3.7.
Single-track sequencing
The SNPs studied by this assay are summarized in Table  1 . Single A-, G- and T- track sequencing using 1 given terminator at a time was performed using both Alf Express™ system (Pharmacia Biotech, Uppsala, Sweden) and ABI310 PRISM capillary sequencer. Alf Express™ Cy5-labeled primers NAT1A-F-CY5: 5'-gggagggtatgtttacagca-3' and NAT1A-POLYA-CY5: 5'-gcataaatcaccaatttcca-3' were used for genotyping the coding region and 3'untranslated area of  NAT1 , respectively. The single sequencing reactions (7 μl) contained: 1.25 μl of dH 2 O, 0.5 μl of 10-times concentrated FS polymerase buffer (125 mM Tris-HCl, pH 9.5, 50 mM (NH 4 ) 2 SO 4 , 150 mM MgCl 2 ), 2 μl of ddNTP termination mix (containing 10 mM dNTPs and either ddATP or ddTTP (10 μM) for A-track or T-track sequencing respectively, 1 μl of 2 μM primer, 0.25 μl of FS polymerase (5 U/μl), and 2 μl of  NAT1  PCR product. The following conditions were used for thermal cycling: initial denaturation for 5 min at 95°C, 35 cycles of 30 sec at 95°C, 30 sec at 50°C, and 1 min at 68°C. The single-tracks were evaluated using conventional software AlfWin, Fragment Analyzer, v. 1.02 supplied with the sequencer (Alf Express™). For the single-track sequencing by ABI310 PRISM we used the same sequencing primers as for Alf Express™ system however labeled with the fluorophor 6-FAM. Sequencing reactions (10 μl) contained: 5.0 μl dH 2 O, 1.0 μl of 10-times concentrated Thermo Sequenase buffer (260 mM Tris-HCl, pH 9.5, 65 mM MgCl 2 ), 2.0 μl of ddNTP termination mix (containing dNTPs and either ddATP or ddGTP for A-track or G-track sequencing respectively – for composition see Table 3), 0.5 μl of 1 μM primer, 0.5 μl of Thermo Sequenase DNA polymerase (with pyrophosphatase) 3.2 U/μl and 1.0 μl  NAT1  PCR product (described above). Thermal cycling conditions were as follows: initial denaturation 20 sec at 95°C, 25 cycles of 20 sec at 95°C, 20 sec at 55°C and 1 min at 72°C, and then 10 cycles of 20 sec at 95°C, 1 min at 72°C. Results were evaluated using GeneScan Analysis software, v. 3.7.NAT1NAT1NAT1Abbreviations ABPs – aminobiphenyls, NAT1 – N-acetyltransferase 1, SSR (Single Sequencing Reaction)
Abbreviations
ABPs – aminobiphenyls, NAT1 – N-acetyltransferase 1, SSR (Single Sequencing Reaction)Author's contributions PS performed the SSR analysis on an Alf Express™ and prepared the first draft of the paper, CFS and MS optimized the SSR analysis for an ABI system in the lab of EHK, TK designed primers and contributed with reagents, materials, and advice, VNK brought the idea, organized the study and was responsible for the revisions of the paper.
Author's contributions
PS performed the SSR analysis on an Alf Express™ and prepared the first draft of the paper, CFS and MS optimized the SSR analysis for an ABI system in the lab of EHK, TK designed primers and contributed with reagents, materials, and advice, VNK brought the idea, organized the study and was responsible for the revisions of the paper.


Background Somatic mutations in the gene for the epidermal growth factor receptor (EGFR) are found in adenocarcinomas of the lung and are associated with sensitivity to the kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva). Lung adenocarcinomas also harbor activating mutations in the downstream GTPase, KRAS, and mutations in  EGFR  and  KRAS  appear to be mutually exclusive.
Background
Somatic mutations in the gene for the epidermal growth factor receptor (EGFR) are found in adenocarcinomas of the lung and are associated with sensitivity to the kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva). Lung adenocarcinomas also harbor activating mutations in the downstream GTPase, KRAS, and mutations in  EGFR  and  KRAS  appear to be mutually exclusive.EGFRKRASMethods and Findings We sought to determine whether mutations in  KRAS  could be used to further enhance prediction of response to gefitinib or erlotinib. We screened 60 lung adenocarcinomas defined as sensitive or refractory to gefitinib or erlotinib for mutations in  EGFR  and  KRAS . We show that mutations in  KRAS  are associated with a lack of sensitivity to either drug.
Methods and Findings
We sought to determine whether mutations in  KRAS  could be used to further enhance prediction of response to gefitinib or erlotinib. We screened 60 lung adenocarcinomas defined as sensitive or refractory to gefitinib or erlotinib for mutations in  EGFR  and  KRAS . We show that mutations in  KRAS  are associated with a lack of sensitivity to either drug.KRASEGFRKRASKRASConclusion Our results suggest that treatment decisions regarding use of these kinase inhibitors might be improved by determining the mutational status of both  EGFR  and  KRAS .
Conclusion
Our results suggest that treatment decisions regarding use of these kinase inhibitors might be improved by determining the mutational status of both  EGFR  and  KRAS .EGFRKRAS

Introduction Genes of the  ERBB  family encode receptor tyrosine kinases that mediate cellular responses to growth signals. Somatic mutations in the tyrosine kinase domains of two  ERBB  genes,  epidermal growth factor receptor (EGFR)  and  HER2,  have been found in a proportion of lung adenocarcinomas [ 1 , 2 , 3 , 4 ]. For  EGFR,  mutations are associated with sensitivity to the small-molecule kinase inhibitors gefitinib (Iressa) [ 1 , 2 , 3 ] and erlotinib (Tarceva) [ 3 ]. ERBB signaling pathways include downstream GTPases encoded by  RAS  genes. Some 15%–30% of lung adenocarcinomas contain activating mutations in the  RAS  family member  KRAS . These mutations are most frequently found in codons 12 and 13 in exon 2 [ 5 , 6 ], and may be associated with unfavorable outcomes [ 7 ]. Interestingly,  EGFR  and  KRAS  mutations are rarely found in the same tumors, suggesting that they have functionally equivalent roles in lung tumorigenesis ([ 8 ]; M. Meyerson, personal communication). Furthermore,  EGFR  mutations are common in tumors from patients who have smoked less than 100 cigarettes in their lifetimes (“never smokers”) [ 3 ], while  KRAS  mutations more commonly occur in individuals with a history of substantial cigarette use [ 9 ]. We sought to determine whether  KRAS  mutations could also be used to predict primary sensitivity or resistance to gefitinib or erlotinib. We systematically evaluated 60 lung adenocarcinomas from patients with known responses to either of these drugs for the presence of mutations in  EGFR  (exons 18 through 21) and  KRAS2  (exon 2). Here, we show that mutations in  KRAS  are associated with primary resistance to single-agent gefitinib or erlotinib. Our results suggest that a determination of mutational status for both  EGFR  and  KRAS  may help define which patients are likely to benefit from receiving gefitinib or erlotinib.
Introduction
Genes of the  ERBB  family encode receptor tyrosine kinases that mediate cellular responses to growth signals. Somatic mutations in the tyrosine kinase domains of two  ERBB  genes,  epidermal growth factor receptor (EGFR)  and  HER2,  have been found in a proportion of lung adenocarcinomas [ 1 , 2 , 3 , 4 ]. For  EGFR,  mutations are associated with sensitivity to the small-molecule kinase inhibitors gefitinib (Iressa) [ 1 , 2 , 3 ] and erlotinib (Tarceva) [ 3 ].ERBBERBBepidermal growth factor receptor (EGFR)HER2,EGFR,ERBB signaling pathways include downstream GTPases encoded by  RAS  genes. Some 15%–30% of lung adenocarcinomas contain activating mutations in the  RAS  family member  KRAS . These mutations are most frequently found in codons 12 and 13 in exon 2 [ 5 , 6 ], and may be associated with unfavorable outcomes [ 7 ]. Interestingly,  EGFR  and  KRAS  mutations are rarely found in the same tumors, suggesting that they have functionally equivalent roles in lung tumorigenesis ([ 8 ]; M. Meyerson, personal communication). Furthermore,  EGFR  mutations are common in tumors from patients who have smoked less than 100 cigarettes in their lifetimes (“never smokers”) [ 3 ], while  KRAS  mutations more commonly occur in individuals with a history of substantial cigarette use [ 9 ].RASRASKRASEGFRKRASEGFRKRASWe sought to determine whether  KRAS  mutations could also be used to predict primary sensitivity or resistance to gefitinib or erlotinib. We systematically evaluated 60 lung adenocarcinomas from patients with known responses to either of these drugs for the presence of mutations in  EGFR  (exons 18 through 21) and  KRAS2  (exon 2). Here, we show that mutations in  KRAS  are associated with primary resistance to single-agent gefitinib or erlotinib. Our results suggest that a determination of mutational status for both  EGFR  and  KRAS  may help define which patients are likely to benefit from receiving gefitinib or erlotinib.KRASEGFRKRAS2KRASEGFRKRASMethods Tissue Procurement Tumor specimens were obtained through protocols approved by the institutional review board of Memorial Sloan-Kettering Cancer Center, as previously described [ 3 ] (see  Protocols S1–S3 ). Tumor material, obtained from patients prior to kinase inhibitor treatment for lung cancer, was collected retrospectively for patients on gefitinib, who received 250 mg or 500 mg orally once daily ( n  = 24), and prospectively for patients on erlotinib, who received 150 mg orally once daily ( n  = 36). The latter cohort of patients was part of a clinical trial of erlotinib for patients with bronchioloalveolar carcinoma. The analysis presented here includes specimens we previously reported on ( n  = 17 for gefitinib and  n  = 17 for erlotinib) [ 3 ]. All specimens were reviewed by a single reference pathologist (M. F. Z.). Imaging studies were assessed by a single reference radiologist (R. T. H.), who graded responses according to Response Evaluation Criteria in Solid Tumors (RECIST) [ 10 ]. Both observers were blinded to patient outcomes. Eight of nine patients with tumors sensitive to gefitinib had objective partial responses as defined by RECIST, i.e., at least a 30% decrease in the sum of the longest diameters of target lesions, taking as reference the sum measured at baseline. The ninth patient had marked clinical improvement, as ascertained by two independent reviewing physicians and manifested by lessened dyspnea and cancer-related pain. However, this individual had radiographic lesions (pleural and bone metastases) that were deemed nonmeasurable by RECIST criteria. As erlotinib-treated patients were all in a clinical trial, all had disease measurable using RECIST guidelines. For both drugs in this study, tumors were considered refractory if they did not undergo sufficient shrinkage to qualify for partial response. This definition includes patients whose “best overall response” was either progression of disease ( n  = 26) or stable disease ( n  = 12) as defined by RECIST. No patients had a complete response. Mutational Analyses of  EGFR  and  KRAS  in Lung Tumors Genomic DNA was extracted from tumors embedded in paraffin blocks, except for tumor 109T, which was a fresh-frozen tumor specimen. Primers for  EGFR  analyses (exons 18–21) were as published [ 3 ]. For  KRAS  analyses, the following nested primer sets for exon 2 were used: huKRAS2 ex2F, 5′-
GAATGGTCCTGCACCAGTAA-3′; huKRAS2 ex2R, 5′-
GTGTGACATGTTCTAATATAGTCA-3′; huKRAS2 ex2Fint, 5′-
GTCCTGCACCAGTAATATGC-3′; and huKRAS2 ex2Rint, 5′-
ATGTTCTAATATAGTCACATTTTC-3′.
 For both  EGFR  and  KRAS,  PCR was performed using the HotStarTaq Master Mix Kit (Qiagen, Valencia, California, United States), as per manufacturer's instructions. Use of this method often obviated the need for nested PCR sets. All sequencing reactions were performed in both forward and reverse directions, and all mutations were confirmed by PCR amplification of an independent DNA isolate. In 12 cases, exon 19 deletions were also studied by length analysis of fluorescently labeled PCR products on a capillary electrophoresis device, using the following primers:  EGFR -Ex19-FWD1, 5′-
GCACCATCTCACAATTGCCAGTTA-3′, and  EGFR -Ex19-REV1, 5′-Fam-
AAAAGGTGGGCCTGAGGTTCA-3′. Using serial dilutions of DNA from the H1650 non-small-cell lung cancer cell line (exon 19 deletion-positive [ 11 ]), this assay detects the mutant allele when H1650 DNA comprises 6% or more of the total DNA tested, compared to a sensitivity of 12% for direct sequencing. These same cases were also screened for the exon 21 L858R mutation by a PCR–restriction fragment length polymorphism assay, based on a new Sau96I restriction site created by the L858R mutation (2,573T→G). The Sau96I-digested fluorescently labeled PCR products were analyzed by capillary electrophoresis, and the following primers were used:  EGFR -Ex21-FWD1, 5′-
CCTCACAGCAGGGTCTTCTCTGT-3′, and  EGFR -Ex21-REV1, 5′-Fam-
TCAGGAAAATGCTGGCTGACCTA-3′. Using serial dilutions of DNA from the H1975 cell line (L858R-positive [ 11 ]), this assay detects the mutant allele when H1975 DNA comprises 3% or more of the total DNA tested, compared to a sensitivity of 6% for direct sequencing (Q. Pan, W. Pao, and M. Ladanyi, unpublished data).
 Statistics Fisher's Exact Test was used to calculate  p- values, and confidence intervals were calculated using  Statistics with Confidence  software [ 12 ].
Methods
Tissue Procurement Tumor specimens were obtained through protocols approved by the institutional review board of Memorial Sloan-Kettering Cancer Center, as previously described [ 3 ] (see  Protocols S1–S3 ). Tumor material, obtained from patients prior to kinase inhibitor treatment for lung cancer, was collected retrospectively for patients on gefitinib, who received 250 mg or 500 mg orally once daily ( n  = 24), and prospectively for patients on erlotinib, who received 150 mg orally once daily ( n  = 36). The latter cohort of patients was part of a clinical trial of erlotinib for patients with bronchioloalveolar carcinoma. The analysis presented here includes specimens we previously reported on ( n  = 17 for gefitinib and  n  = 17 for erlotinib) [ 3 ]. All specimens were reviewed by a single reference pathologist (M. F. Z.). Imaging studies were assessed by a single reference radiologist (R. T. H.), who graded responses according to Response Evaluation Criteria in Solid Tumors (RECIST) [ 10 ]. Both observers were blinded to patient outcomes. Eight of nine patients with tumors sensitive to gefitinib had objective partial responses as defined by RECIST, i.e., at least a 30% decrease in the sum of the longest diameters of target lesions, taking as reference the sum measured at baseline. The ninth patient had marked clinical improvement, as ascertained by two independent reviewing physicians and manifested by lessened dyspnea and cancer-related pain. However, this individual had radiographic lesions (pleural and bone metastases) that were deemed nonmeasurable by RECIST criteria. As erlotinib-treated patients were all in a clinical trial, all had disease measurable using RECIST guidelines. For both drugs in this study, tumors were considered refractory if they did not undergo sufficient shrinkage to qualify for partial response. This definition includes patients whose “best overall response” was either progression of disease ( n  = 26) or stable disease ( n  = 12) as defined by RECIST. No patients had a complete response.
Tissue Procurement
Tumor specimens were obtained through protocols approved by the institutional review board of Memorial Sloan-Kettering Cancer Center, as previously described [ 3 ] (see  Protocols S1–S3 ). Tumor material, obtained from patients prior to kinase inhibitor treatment for lung cancer, was collected retrospectively for patients on gefitinib, who received 250 mg or 500 mg orally once daily ( n  = 24), and prospectively for patients on erlotinib, who received 150 mg orally once daily ( n  = 36). The latter cohort of patients was part of a clinical trial of erlotinib for patients with bronchioloalveolar carcinoma. The analysis presented here includes specimens we previously reported on ( n  = 17 for gefitinib and  n  = 17 for erlotinib) [ 3 ].nnnnAll specimens were reviewed by a single reference pathologist (M. F. Z.). Imaging studies were assessed by a single reference radiologist (R. T. H.), who graded responses according to Response Evaluation Criteria in Solid Tumors (RECIST) [ 10 ]. Both observers were blinded to patient outcomes.Eight of nine patients with tumors sensitive to gefitinib had objective partial responses as defined by RECIST, i.e., at least a 30% decrease in the sum of the longest diameters of target lesions, taking as reference the sum measured at baseline. The ninth patient had marked clinical improvement, as ascertained by two independent reviewing physicians and manifested by lessened dyspnea and cancer-related pain. However, this individual had radiographic lesions (pleural and bone metastases) that were deemed nonmeasurable by RECIST criteria. As erlotinib-treated patients were all in a clinical trial, all had disease measurable using RECIST guidelines. For both drugs in this study, tumors were considered refractory if they did not undergo sufficient shrinkage to qualify for partial response. This definition includes patients whose “best overall response” was either progression of disease ( n  = 26) or stable disease ( n  = 12) as defined by RECIST. No patients had a complete response.nnMutational Analyses of  EGFR  and  KRAS  in Lung Tumors Genomic DNA was extracted from tumors embedded in paraffin blocks, except for tumor 109T, which was a fresh-frozen tumor specimen. Primers for  EGFR  analyses (exons 18–21) were as published [ 3 ]. For  KRAS  analyses, the following nested primer sets for exon 2 were used: huKRAS2 ex2F, 5′-
GAATGGTCCTGCACCAGTAA-3′; huKRAS2 ex2R, 5′-
GTGTGACATGTTCTAATATAGTCA-3′; huKRAS2 ex2Fint, 5′-
GTCCTGCACCAGTAATATGC-3′; and huKRAS2 ex2Rint, 5′-
ATGTTCTAATATAGTCACATTTTC-3′.
 For both  EGFR  and  KRAS,  PCR was performed using the HotStarTaq Master Mix Kit (Qiagen, Valencia, California, United States), as per manufacturer's instructions. Use of this method often obviated the need for nested PCR sets. All sequencing reactions were performed in both forward and reverse directions, and all mutations were confirmed by PCR amplification of an independent DNA isolate. In 12 cases, exon 19 deletions were also studied by length analysis of fluorescently labeled PCR products on a capillary electrophoresis device, using the following primers:  EGFR -Ex19-FWD1, 5′-
GCACCATCTCACAATTGCCAGTTA-3′, and  EGFR -Ex19-REV1, 5′-Fam-
AAAAGGTGGGCCTGAGGTTCA-3′. Using serial dilutions of DNA from the H1650 non-small-cell lung cancer cell line (exon 19 deletion-positive [ 11 ]), this assay detects the mutant allele when H1650 DNA comprises 6% or more of the total DNA tested, compared to a sensitivity of 12% for direct sequencing. These same cases were also screened for the exon 21 L858R mutation by a PCR–restriction fragment length polymorphism assay, based on a new Sau96I restriction site created by the L858R mutation (2,573T→G). The Sau96I-digested fluorescently labeled PCR products were analyzed by capillary electrophoresis, and the following primers were used:  EGFR -Ex21-FWD1, 5′-
CCTCACAGCAGGGTCTTCTCTGT-3′, and  EGFR -Ex21-REV1, 5′-Fam-
TCAGGAAAATGCTGGCTGACCTA-3′. Using serial dilutions of DNA from the H1975 cell line (L858R-positive [ 11 ]), this assay detects the mutant allele when H1975 DNA comprises 3% or more of the total DNA tested, compared to a sensitivity of 6% for direct sequencing (Q. Pan, W. Pao, and M. Ladanyi, unpublished data).
Mutational Analyses of  EGFR  and  KRAS  in Lung Tumors
EGFRKRASGenomic DNA was extracted from tumors embedded in paraffin blocks, except for tumor 109T, which was a fresh-frozen tumor specimen. Primers for  EGFR  analyses (exons 18–21) were as published [ 3 ]. For  KRAS  analyses, the following nested primer sets for exon 2 were used: huKRAS2 ex2F, 5′-
GAATGGTCCTGCACCAGTAA-3′; huKRAS2 ex2R, 5′-
GTGTGACATGTTCTAATATAGTCA-3′; huKRAS2 ex2Fint, 5′-
GTCCTGCACCAGTAATATGC-3′; and huKRAS2 ex2Rint, 5′-
ATGTTCTAATATAGTCACATTTTC-3′.EGFRKRASFor both  EGFR  and  KRAS,  PCR was performed using the HotStarTaq Master Mix Kit (Qiagen, Valencia, California, United States), as per manufacturer's instructions. Use of this method often obviated the need for nested PCR sets. All sequencing reactions were performed in both forward and reverse directions, and all mutations were confirmed by PCR amplification of an independent DNA isolate.EGFRKRAS,In 12 cases, exon 19 deletions were also studied by length analysis of fluorescently labeled PCR products on a capillary electrophoresis device, using the following primers:  EGFR -Ex19-FWD1, 5′-
GCACCATCTCACAATTGCCAGTTA-3′, and  EGFR -Ex19-REV1, 5′-Fam-
AAAAGGTGGGCCTGAGGTTCA-3′. Using serial dilutions of DNA from the H1650 non-small-cell lung cancer cell line (exon 19 deletion-positive [ 11 ]), this assay detects the mutant allele when H1650 DNA comprises 6% or more of the total DNA tested, compared to a sensitivity of 12% for direct sequencing. These same cases were also screened for the exon 21 L858R mutation by a PCR–restriction fragment length polymorphism assay, based on a new Sau96I restriction site created by the L858R mutation (2,573T→G). The Sau96I-digested fluorescently labeled PCR products were analyzed by capillary electrophoresis, and the following primers were used:  EGFR -Ex21-FWD1, 5′-
CCTCACAGCAGGGTCTTCTCTGT-3′, and  EGFR -Ex21-REV1, 5′-Fam-
TCAGGAAAATGCTGGCTGACCTA-3′. Using serial dilutions of DNA from the H1975 cell line (L858R-positive [ 11 ]), this assay detects the mutant allele when H1975 DNA comprises 3% or more of the total DNA tested, compared to a sensitivity of 6% for direct sequencing (Q. Pan, W. Pao, and M. Ladanyi, unpublished data).EGFREGFREGFREGFRStatistics Fisher's Exact Test was used to calculate  p- values, and confidence intervals were calculated using  Statistics with Confidence  software [ 12 ].
Statistics
Fisher's Exact Test was used to calculate  p- values, and confidence intervals were calculated using  Statistics with Confidence  software [ 12 ].p-Statistics with ConfidenceResults We identified 60 lung adenocarcinomas from individual patients with tumors shown to be sensitive or refractory to single-agent gefitinib or erlotinib and evaluated these tumors for mutations in  EGFR  and  KRAS . Collectively, nine of 38 (24%) tumors refractory to either kinase inhibitor had  KRAS  mutations, while zero of 21 (0%) drug-sensitive tumors had such mutations ( p  = 0.02) ( Table 1 ). The 95% confidence intervals (CIs) for these observations are 13%–39% and 0%–16%, respectively. Conversely, 17 of 22 (77%) tumors sensitive to either kinase inhibitor had  EGFR  mutations, in contrast to zero of 38 (0%) drug-resistant tumors ( p  = 6.8 × 10 −11 ). The 95% CIs for these observed response rates are 57%–90% and 0%–9%, respectively. All 17 tumors with  EGFR  mutations responded to gefitinib or erlotinib, while all nine tumors with  KRAS  mutations did not ( p  = 3.2 × 10 −7 ). Table 1
 EGFR  and  KRAS  Mutation Status in Lung Adenocarcinomas Sensitive or Refractory to Gefitinib or Erlotinib Lung tumors were examined for mutations in  EGFR  (exons 18–21) and  KRAS  (exon 2). In gefitinib-treated patients, six  EGFR  mutations involved exon 19 deletions that lacked the amino acids Leu-Arg-Glu-Ala, and two were exon 21 amino acid substitutions (L858R). A seventh case with an exon 19 deletion (involving 12 nucleotides) was detected by fluorescent capillary electrophoresis only, so exact sequence deletion information was unavailable (see Methods). In erlotinib-treated patients, three  EGFR  mutations were exon 19 deletions that lacked amino acids Leu-Arg-Glu-Ala, and five were exon 21 amino acid substitutions (L858R)
 a  One erlotinib-sensitive tumor was unavailable for  KRAS  examination
 b  The incidence of  KRAS  mutations in this cohort was low, probably because only cases involving bronchioloalveolar carcinoma were tested (see text) Correlation of  EGFR  and  KRAS  mutational status with drug and treatment response is detailed in  Table 1 . The spectrum of  KRAS  mutations is shown in  Figure 1  and  Table 2 . Results with gefitinib and erlotinib were similar overall. However, the incidence of  KRAS  mutations in the patients treated with erlotinib was low, probably because of the fact that all patients treated with this drug had bronchioloalveolar carcinoma, which rarely has  RAS  mutations [ 13 ]. Alternatively, our analyses involving only exon 2 of  KRAS2  may have missed some  RAS  mutations. However, in our analysis of the exonic regions encoding the first 100 amino acids of  KRAS  in 110 surgically resected early-stage non-small-cell lung cancers, we have found 18 mutations, and all were in either codon 12 or codon 13, encoded by exon 2 (W. Pao, R. Wilson, H. Varmus, unpublished data). Another possibility is that the erlotinib-treated tumors have mutations in other  RAS  genes, since a minority of  RAS  mutations in lung cancer have been reported to occur in  N-  or  HRAS  [ 5 , 6 ]. Figure 1 Sequence Chromatograms Displaying the Types of  KRAS  Mutations Found in This Study Table 2
 KRAS  Exon 2 Mutations Found in Non-Small-Cell Lung Cancers Refractory to Treatment with Gefitinib or Erlotinib
 a  Bold indicates mutations
Results
We identified 60 lung adenocarcinomas from individual patients with tumors shown to be sensitive or refractory to single-agent gefitinib or erlotinib and evaluated these tumors for mutations in  EGFR  and  KRAS . Collectively, nine of 38 (24%) tumors refractory to either kinase inhibitor had  KRAS  mutations, while zero of 21 (0%) drug-sensitive tumors had such mutations ( p  = 0.02) ( Table 1 ). The 95% confidence intervals (CIs) for these observations are 13%–39% and 0%–16%, respectively. Conversely, 17 of 22 (77%) tumors sensitive to either kinase inhibitor had  EGFR  mutations, in contrast to zero of 38 (0%) drug-resistant tumors ( p  = 6.8 × 10 −11 ). The 95% CIs for these observed response rates are 57%–90% and 0%–9%, respectively. All 17 tumors with  EGFR  mutations responded to gefitinib or erlotinib, while all nine tumors with  KRAS  mutations did not ( p  = 3.2 × 10 −7 ).EGFRKRASKRASpEGFRpEGFRKRASpEGFR  and  KRAS  Mutation Status in Lung Adenocarcinomas Sensitive or Refractory to Gefitinib or Erlotinib
EGFRKRASLung tumors were examined for mutations in  EGFR  (exons 18–21) and  KRAS  (exon 2). In gefitinib-treated patients, six  EGFR  mutations involved exon 19 deletions that lacked the amino acids Leu-Arg-Glu-Ala, and two were exon 21 amino acid substitutions (L858R). A seventh case with an exon 19 deletion (involving 12 nucleotides) was detected by fluorescent capillary electrophoresis only, so exact sequence deletion information was unavailable (see Methods). In erlotinib-treated patients, three  EGFR  mutations were exon 19 deletions that lacked amino acids Leu-Arg-Glu-Ala, and five were exon 21 amino acid substitutions (L858R)EGFRKRASEGFREGFRa  One erlotinib-sensitive tumor was unavailable for  KRAS  examinationKRASb  The incidence of  KRAS  mutations in this cohort was low, probably because only cases involving bronchioloalveolar carcinoma were tested (see text)KRASCorrelation of  EGFR  and  KRAS  mutational status with drug and treatment response is detailed in  Table 1 . The spectrum of  KRAS  mutations is shown in  Figure 1  and  Table 2 . Results with gefitinib and erlotinib were similar overall. However, the incidence of  KRAS  mutations in the patients treated with erlotinib was low, probably because of the fact that all patients treated with this drug had bronchioloalveolar carcinoma, which rarely has  RAS  mutations [ 13 ]. Alternatively, our analyses involving only exon 2 of  KRAS2  may have missed some  RAS  mutations. However, in our analysis of the exonic regions encoding the first 100 amino acids of  KRAS  in 110 surgically resected early-stage non-small-cell lung cancers, we have found 18 mutations, and all were in either codon 12 or codon 13, encoded by exon 2 (W. Pao, R. Wilson, H. Varmus, unpublished data). Another possibility is that the erlotinib-treated tumors have mutations in other  RAS  genes, since a minority of  RAS  mutations in lung cancer have been reported to occur in  N-  or  HRAS  [ 5 , 6 ].EGFRKRASKRASKRASRASKRAS2RASKRASRASRASN-HRASKRAS  Exon 2 Mutations Found in Non-Small-Cell Lung Cancers Refractory to Treatment with Gefitinib or Erlotinib
KRASa  Bold indicates mutationsDiscussion These results have important clinical implications. First, they extend previous data from our group and others showing that lung adenocarcinomas containing  EGFR  mutations are associated with sensitivity to gefitinib or erlotinib (17 of 17 in this series; 100% observed response rate; 95% CI, 82%–100%). Second, these data show that tumors with  KRAS  exon 2 mutations ( n  = 9) are associated with a lack of response to these kinase inhibitors (0% observed response rate; 95% CI, 0%–30%). Third, no drug-sensitive tumors had  KRAS  exon 2 mutations ( n  = 21). Whether  KRAS  mutational status can be used to predict responses to gefitinib or erlotinib in patients whose tumors have wild-type  EGFR  sequence is still under investigation: our analysis comparing response rates for tumors with neither  EGFR  nor  KRAS  mutations versus tumors with wild-type  EGFR  but mutated  KRAS  does not reach statistical significance (five of 22 versus zero of nine;  p  = 0.29). Nevertheless, these findings suggest that patients whose lung adenocarcinomas have  KRAS  mutations will not experience significant tumor regression with either drug. The incidence of  EGFR  mutations in tumors responsive to EGFR kinase inhibitors has varied from 71% to 100% ([ 1 , 2 , 3 ] and this paper). Thus, at this point, patients whose tumors test negative for  EGFR  mutations should not necessarily be precluded from treatment with either gefitinib or erlotinib. Data presented here suggest that clinical decisions regarding the use of these agents in patients with lung adenocarcinomas might be improved in the future by pre-treatment mutational profiling of both  EGFR  and  KRAS . These findings warrant validation in large prospective trials using standardized mutation detection techniques. Supporting Information Protocol S1 Preclinical Studies of Blood, Urine, Bone Marrow, and Tissues Collected from Patients with Thoracic Malignancies (32 KB PDF). Click here for additional data file. Protocol S2 Multicenter Phase II Trial of OSI-774 (Erlotinib, Tarceva) in Patients with Advanced Bronchioloalveolar Cell Lung Cancer (1.9 MB PDF). Click here for additional data file. Protocol S3 Protocol Approval Letters (60 KB PDF). Click here for additional data file. Accession Numbers The LocusLink ( http://www.ncbi.nlm.nih.gov/LocusLink/ ) accession number for the  KRAS2  sequence discussed in this paper is 3845; the GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/ ) accession number for the  KRAS2  sequence discussed in this paper is NT_009714.16. Patient Summary Background Two drugs, gefitinib (Iressa) and erlotinib (Tarceva), have been developed that can make lung cancers smaller in some patients. The drugs work by blocking the effect of a molecule called the epidermal growth factor receptor (EGFR), which relays instructions to cells to grow and divide. Recently, researchers found that these drugs most effectively shrink tumors that have acquired abnormal variations (mutations) in the  EGFR  gene. These mutations somehow allow tumor cells to escape normal safety mechanisms that keep cells from growing out of control. Some lung cancers also have mutations in another gene called  KRAS . Interestingly,  KRAS  mutations and  EGFR  mutations are rarely ever found in the same tumor. Why Was This Study Done? Unfortunately,  EGFR  mutations are only found in a minority of patients with lung cancer. This means that gefitinib or erlotinib might be given to a lot of patients who may not benefit from this treatment. Ideally, the drugs would be given only to patients who we know will benefit from them. This study examined whether studying the  KRAS  gene (to see if it had a mutation) could help predict which patients had tumors that would respond well to the drugs. What Did the Researchers Do? They took 60 lung cancer samples from patients who had been treated with one of the drugs and either responded (that is, their tumors shrunk in size) or not, and tested whether the tumors had normal or abnormal  KRAS . What Did They Find? Tumors that got significantly smaller while treated with gefitinib or erlotinib (a total of 22) had a normal  KRAS  gene .  Most of these tumors had  EGFR  mutations. Conversely, tumors that had abnormal  KRAS  (a total of nine) did not shrink while treated with gefitinib or erlotinib. What Does This Mean? Both gefitinib and erlotinib are expensive and have side effects. Testing for  EGFR  and  KRAS  mutations is relatively straightforward, and one could test for abnormalities in both genes first and then decide which patients should be treated with either of the two drugs. What Next? Before doing  EGFR  and  KRAS  tests on a routine basis and taking the results into account when making a decision about who should be treated with gefitinib or erlotinib, larger studies need to be done to see whether the results reported here hold up. More Information Online US Food and Drug Administration information page on Iressa:  http://www.fda.gov/cder/drug/infopage/iressa/iressaQ&A.htm
 Cancer Research UK information page about erlotinib:  http://www.cancerhelp.org.uk/help/default.asp?page=10296
Discussion
These results have important clinical implications. First, they extend previous data from our group and others showing that lung adenocarcinomas containing  EGFR  mutations are associated with sensitivity to gefitinib or erlotinib (17 of 17 in this series; 100% observed response rate; 95% CI, 82%–100%). Second, these data show that tumors with  KRAS  exon 2 mutations ( n  = 9) are associated with a lack of response to these kinase inhibitors (0% observed response rate; 95% CI, 0%–30%). Third, no drug-sensitive tumors had  KRAS  exon 2 mutations ( n  = 21). Whether  KRAS  mutational status can be used to predict responses to gefitinib or erlotinib in patients whose tumors have wild-type  EGFR  sequence is still under investigation: our analysis comparing response rates for tumors with neither  EGFR  nor  KRAS  mutations versus tumors with wild-type  EGFR  but mutated  KRAS  does not reach statistical significance (five of 22 versus zero of nine;  p  = 0.29). Nevertheless, these findings suggest that patients whose lung adenocarcinomas have  KRAS  mutations will not experience significant tumor regression with either drug.EGFRKRASnKRASnKRASEGFREGFRKRASEGFRKRASpKRASThe incidence of  EGFR  mutations in tumors responsive to EGFR kinase inhibitors has varied from 71% to 100% ([ 1 , 2 , 3 ] and this paper). Thus, at this point, patients whose tumors test negative for  EGFR  mutations should not necessarily be precluded from treatment with either gefitinib or erlotinib. Data presented here suggest that clinical decisions regarding the use of these agents in patients with lung adenocarcinomas might be improved in the future by pre-treatment mutational profiling of both  EGFR  and  KRAS . These findings warrant validation in large prospective trials using standardized mutation detection techniques.EGFREGFREGFRKRASSupporting Information Protocol S1 Preclinical Studies of Blood, Urine, Bone Marrow, and Tissues Collected from Patients with Thoracic Malignancies (32 KB PDF). Click here for additional data file. Protocol S2 Multicenter Phase II Trial of OSI-774 (Erlotinib, Tarceva) in Patients with Advanced Bronchioloalveolar Cell Lung Cancer (1.9 MB PDF). Click here for additional data file. Protocol S3 Protocol Approval Letters (60 KB PDF). Click here for additional data file.
Supporting Information
Preclinical Studies of Blood, Urine, Bone Marrow, and Tissues Collected from Patients with Thoracic Malignancies
(32 KB PDF).Click here for additional data file.Multicenter Phase II Trial of OSI-774 (Erlotinib, Tarceva) in Patients with Advanced Bronchioloalveolar Cell Lung Cancer
(1.9 MB PDF).Click here for additional data file.Protocol Approval Letters
(60 KB PDF).Click here for additional data file.Accession Numbers The LocusLink ( http://www.ncbi.nlm.nih.gov/LocusLink/ ) accession number for the  KRAS2  sequence discussed in this paper is 3845; the GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/ ) accession number for the  KRAS2  sequence discussed in this paper is NT_009714.16. Patient Summary Background Two drugs, gefitinib (Iressa) and erlotinib (Tarceva), have been developed that can make lung cancers smaller in some patients. The drugs work by blocking the effect of a molecule called the epidermal growth factor receptor (EGFR), which relays instructions to cells to grow and divide. Recently, researchers found that these drugs most effectively shrink tumors that have acquired abnormal variations (mutations) in the  EGFR  gene. These mutations somehow allow tumor cells to escape normal safety mechanisms that keep cells from growing out of control. Some lung cancers also have mutations in another gene called  KRAS . Interestingly,  KRAS  mutations and  EGFR  mutations are rarely ever found in the same tumor. Why Was This Study Done? Unfortunately,  EGFR  mutations are only found in a minority of patients with lung cancer. This means that gefitinib or erlotinib might be given to a lot of patients who may not benefit from this treatment. Ideally, the drugs would be given only to patients who we know will benefit from them. This study examined whether studying the  KRAS  gene (to see if it had a mutation) could help predict which patients had tumors that would respond well to the drugs. What Did the Researchers Do? They took 60 lung cancer samples from patients who had been treated with one of the drugs and either responded (that is, their tumors shrunk in size) or not, and tested whether the tumors had normal or abnormal  KRAS . What Did They Find? Tumors that got significantly smaller while treated with gefitinib or erlotinib (a total of 22) had a normal  KRAS  gene .  Most of these tumors had  EGFR  mutations. Conversely, tumors that had abnormal  KRAS  (a total of nine) did not shrink while treated with gefitinib or erlotinib. What Does This Mean? Both gefitinib and erlotinib are expensive and have side effects. Testing for  EGFR  and  KRAS  mutations is relatively straightforward, and one could test for abnormalities in both genes first and then decide which patients should be treated with either of the two drugs. What Next? Before doing  EGFR  and  KRAS  tests on a routine basis and taking the results into account when making a decision about who should be treated with gefitinib or erlotinib, larger studies need to be done to see whether the results reported here hold up. More Information Online US Food and Drug Administration information page on Iressa:  http://www.fda.gov/cder/drug/infopage/iressa/iressaQ&A.htm
 Cancer Research UK information page about erlotinib:  http://www.cancerhelp.org.uk/help/default.asp?page=10296
Accession Numbers
The LocusLink ( http://www.ncbi.nlm.nih.gov/LocusLink/ ) accession number for the  KRAS2  sequence discussed in this paper is 3845; the GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/ ) accession number for the  KRAS2  sequence discussed in this paper is NT_009714.16.KRAS2KRAS2Patient Summary Background Two drugs, gefitinib (Iressa) and erlotinib (Tarceva), have been developed that can make lung cancers smaller in some patients. The drugs work by blocking the effect of a molecule called the epidermal growth factor receptor (EGFR), which relays instructions to cells to grow and divide. Recently, researchers found that these drugs most effectively shrink tumors that have acquired abnormal variations (mutations) in the  EGFR  gene. These mutations somehow allow tumor cells to escape normal safety mechanisms that keep cells from growing out of control. Some lung cancers also have mutations in another gene called  KRAS . Interestingly,  KRAS  mutations and  EGFR  mutations are rarely ever found in the same tumor. Why Was This Study Done? Unfortunately,  EGFR  mutations are only found in a minority of patients with lung cancer. This means that gefitinib or erlotinib might be given to a lot of patients who may not benefit from this treatment. Ideally, the drugs would be given only to patients who we know will benefit from them. This study examined whether studying the  KRAS  gene (to see if it had a mutation) could help predict which patients had tumors that would respond well to the drugs. What Did the Researchers Do? They took 60 lung cancer samples from patients who had been treated with one of the drugs and either responded (that is, their tumors shrunk in size) or not, and tested whether the tumors had normal or abnormal  KRAS . What Did They Find? Tumors that got significantly smaller while treated with gefitinib or erlotinib (a total of 22) had a normal  KRAS  gene .  Most of these tumors had  EGFR  mutations. Conversely, tumors that had abnormal  KRAS  (a total of nine) did not shrink while treated with gefitinib or erlotinib. What Does This Mean? Both gefitinib and erlotinib are expensive and have side effects. Testing for  EGFR  and  KRAS  mutations is relatively straightforward, and one could test for abnormalities in both genes first and then decide which patients should be treated with either of the two drugs. What Next? Before doing  EGFR  and  KRAS  tests on a routine basis and taking the results into account when making a decision about who should be treated with gefitinib or erlotinib, larger studies need to be done to see whether the results reported here hold up. More Information Online US Food and Drug Administration information page on Iressa:  http://www.fda.gov/cder/drug/infopage/iressa/iressaQ&A.htm
 Cancer Research UK information page about erlotinib:  http://www.cancerhelp.org.uk/help/default.asp?page=10296
Patient Summary
Background Two drugs, gefitinib (Iressa) and erlotinib (Tarceva), have been developed that can make lung cancers smaller in some patients. The drugs work by blocking the effect of a molecule called the epidermal growth factor receptor (EGFR), which relays instructions to cells to grow and divide. Recently, researchers found that these drugs most effectively shrink tumors that have acquired abnormal variations (mutations) in the  EGFR  gene. These mutations somehow allow tumor cells to escape normal safety mechanisms that keep cells from growing out of control. Some lung cancers also have mutations in another gene called  KRAS . Interestingly,  KRAS  mutations and  EGFR  mutations are rarely ever found in the same tumor. Why Was This Study Done? Unfortunately,  EGFR  mutations are only found in a minority of patients with lung cancer. This means that gefitinib or erlotinib might be given to a lot of patients who may not benefit from this treatment. Ideally, the drugs would be given only to patients who we know will benefit from them. This study examined whether studying the  KRAS  gene (to see if it had a mutation) could help predict which patients had tumors that would respond well to the drugs. What Did the Researchers Do? They took 60 lung cancer samples from patients who had been treated with one of the drugs and either responded (that is, their tumors shrunk in size) or not, and tested whether the tumors had normal or abnormal  KRAS . What Did They Find? Tumors that got significantly smaller while treated with gefitinib or erlotinib (a total of 22) had a normal  KRAS  gene .  Most of these tumors had  EGFR  mutations. Conversely, tumors that had abnormal  KRAS  (a total of nine) did not shrink while treated with gefitinib or erlotinib. What Does This Mean? Both gefitinib and erlotinib are expensive and have side effects. Testing for  EGFR  and  KRAS  mutations is relatively straightforward, and one could test for abnormalities in both genes first and then decide which patients should be treated with either of the two drugs. What Next? Before doing  EGFR  and  KRAS  tests on a routine basis and taking the results into account when making a decision about who should be treated with gefitinib or erlotinib, larger studies need to be done to see whether the results reported here hold up. More Information Online US Food and Drug Administration information page on Iressa:  http://www.fda.gov/cder/drug/infopage/iressa/iressaQ&A.htm
 Cancer Research UK information page about erlotinib:  http://www.cancerhelp.org.uk/help/default.asp?page=10296

Background Two drugs, gefitinib (Iressa) and erlotinib (Tarceva), have been developed that can make lung cancers smaller in some patients. The drugs work by blocking the effect of a molecule called the epidermal growth factor receptor (EGFR), which relays instructions to cells to grow and divide. Recently, researchers found that these drugs most effectively shrink tumors that have acquired abnormal variations (mutations) in the  EGFR  gene. These mutations somehow allow tumor cells to escape normal safety mechanisms that keep cells from growing out of control. Some lung cancers also have mutations in another gene called  KRAS . Interestingly,  KRAS  mutations and  EGFR  mutations are rarely ever found in the same tumor.
Background
Two drugs, gefitinib (Iressa) and erlotinib (Tarceva), have been developed that can make lung cancers smaller in some patients. The drugs work by blocking the effect of a molecule called the epidermal growth factor receptor (EGFR), which relays instructions to cells to grow and divide. Recently, researchers found that these drugs most effectively shrink tumors that have acquired abnormal variations (mutations) in the  EGFR  gene. These mutations somehow allow tumor cells to escape normal safety mechanisms that keep cells from growing out of control. Some lung cancers also have mutations in another gene called  KRAS . Interestingly,  KRAS  mutations and  EGFR  mutations are rarely ever found in the same tumor.EGFRKRASKRASEGFRWhy Was This Study Done? Unfortunately,  EGFR  mutations are only found in a minority of patients with lung cancer. This means that gefitinib or erlotinib might be given to a lot of patients who may not benefit from this treatment. Ideally, the drugs would be given only to patients who we know will benefit from them. This study examined whether studying the  KRAS  gene (to see if it had a mutation) could help predict which patients had tumors that would respond well to the drugs.
Why Was This Study Done?
Unfortunately,  EGFR  mutations are only found in a minority of patients with lung cancer. This means that gefitinib or erlotinib might be given to a lot of patients who may not benefit from this treatment. Ideally, the drugs would be given only to patients who we know will benefit from them. This study examined whether studying the  KRAS  gene (to see if it had a mutation) could help predict which patients had tumors that would respond well to the drugs.EGFRKRASWhat Did the Researchers Do? They took 60 lung cancer samples from patients who had been treated with one of the drugs and either responded (that is, their tumors shrunk in size) or not, and tested whether the tumors had normal or abnormal  KRAS .
What Did the Researchers Do?
They took 60 lung cancer samples from patients who had been treated with one of the drugs and either responded (that is, their tumors shrunk in size) or not, and tested whether the tumors had normal or abnormal  KRAS .KRASWhat Did They Find? Tumors that got significantly smaller while treated with gefitinib or erlotinib (a total of 22) had a normal  KRAS  gene .  Most of these tumors had  EGFR  mutations. Conversely, tumors that had abnormal  KRAS  (a total of nine) did not shrink while treated with gefitinib or erlotinib.
What Did They Find?
Tumors that got significantly smaller while treated with gefitinib or erlotinib (a total of 22) had a normal  KRAS  gene .  Most of these tumors had  EGFR  mutations. Conversely, tumors that had abnormal  KRAS  (a total of nine) did not shrink while treated with gefitinib or erlotinib.KRAS.EGFRKRASWhat Does This Mean? Both gefitinib and erlotinib are expensive and have side effects. Testing for  EGFR  and  KRAS  mutations is relatively straightforward, and one could test for abnormalities in both genes first and then decide which patients should be treated with either of the two drugs.
What Does This Mean?
Both gefitinib and erlotinib are expensive and have side effects. Testing for  EGFR  and  KRAS  mutations is relatively straightforward, and one could test for abnormalities in both genes first and then decide which patients should be treated with either of the two drugs.EGFRKRASWhat Next? Before doing  EGFR  and  KRAS  tests on a routine basis and taking the results into account when making a decision about who should be treated with gefitinib or erlotinib, larger studies need to be done to see whether the results reported here hold up.
What Next?
Before doing  EGFR  and  KRAS  tests on a routine basis and taking the results into account when making a decision about who should be treated with gefitinib or erlotinib, larger studies need to be done to see whether the results reported here hold up.EGFRKRASMore Information Online US Food and Drug Administration information page on Iressa:  http://www.fda.gov/cder/drug/infopage/iressa/iressaQ&A.htm
 Cancer Research UK information page about erlotinib:  http://www.cancerhelp.org.uk/help/default.asp?page=10296
More Information Online
US Food and Drug Administration information page on Iressa:  http://www.fda.gov/cder/drug/infopage/iressa/iressaQ&A.htmCancer Research UK information page about erlotinib:  http://www.cancerhelp.org.uk/help/default.asp?page=10296