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Paper text:
upper aerodigestive cancers including oral cavity , pharynx , and laryngeal malignancies affects an estimated 3 million patients worldwide yearly .
carcinogenesis has been linked to the overuse of tobacco and alcohol ; more recently the human papillomavirus has been implicated .
unfortunately , the cure rates for these malignancies have remained stagnant for several decades , and so new therapies for this malignancy are integral to controlling this disease .   over the past decade ,
the emergence of targeted therapies represents a new development in the treatment of solid tumor malignancies [ 410 ] .
several agent classes have been used to improve survival in a variety of tumor types , and egfr - disruptive therapies have had promising results in lung cancer in specific patient populations [ 1114 ] . in head and neck carcinoma , egfr directed antibody therapy has been approved for simultaneous use with radiation therapy for improved survival of advanced stage disease .
other clinical trials with these agents suggest response rates for recurrent or metastatic head and neck cancer may be similar to studies in lung cancer [ 16 , 17 ] .   in lung cancer , it has recently been reported that tumors harboring mutations of exon 19 and 21 were associated with improved clinical response rates . in the present study ,
we examined 20 head and neck cancer specimens and 4 cell lines for mutations in either exon and report here the high conservation of these sequences of the egfr gene .
tumor specimens were obtained through the university of minnesota tissue procurement facility in compliance under university of minnesota institutional review board approval . to remain within human investigation hipaa compliant guidelines established for the tissue procurement facility ,
the tumors were derived from primary cancers of the upper aerodigestive tact ( oral cavity , oropharynx , larynx , and hypopharynx ) .
head and neck cancer demographics for our university patient population are typical : 2 : 1 male to female preponderance , average age = 60  years .
approximately 5% of the tumors in our tumor bank are from underrepresented minority populations , a reflection of our regional referral base . between 2002 and 2003 , 20 randomly selected frozen specimens from patients with pathologically confirmed head and neck squamous cell carcinoma ( hnscc ) , along with 4 squamous carcinoma cell lines ( ca-9 - 22 , na , um - scc-9 , um - scc-11b ) , were obtained for dna analysis .   for mutational analysis of the egfr coding sequence for exons 19 and 21 , dna extraction was performed using the wizard genomic dna purification kit ( promega , madison , wi ) according to the manufacturer 's instructions . forward and
reverse primers were made for both of the egfr mutational hotspots located on exons 19 and 21 .
previously published primer sequences   were synthesized by integrated dna technologies ( coralville , ia ) .
pcr amplification was carried out using amplitaq gold polymerase ( applied biosystems , foster city , ca ) and an mj research thermocycler ( bio - rad , hercules , ca ) .
pcr cycling parameters were 95c hot start for 9 minutes , then 94c for 30 seconds , 60c for 1  minutes , and 72c for 45 seconds for a total of 25 cycles , followed by a final extension step of 72c for 5 minutes .
pcr product purification was carried out using purelink pcr purification kit ( invitrogen , carlsbad , ca ) .
the resulting dna was then sent for sequencing at the university of minnesota microchemical sequencing facility .
sequencing results were viewed and compared with published sequences using chromas software ( technelysium pty ltd , tewantin qld , australia ) .  
the fisher exact test was used to compare the published frequency of mutations in a recently published lung cancer study using prism graphpad 4 software ( graphpad , san diego , ca ) . to calculate the 95% confidence interval ( ci ) for a proportion
high - quality dna was isolated from all 20 tumor samples and 4 cell lines .
pcr analysis of exons 19 and 21 yielded 100% homology between the nucleotide sequences in question .
no mutations were found in the 20 tumor samples or the four cell lines . there was no statistical difference ( p = .5 ) between published rates of egfr mutations in nsclc   and our data .
next , we calculated a 95% confidence interval based on the presence of 0% mutations in either exon 19 or 21 .
based on a 0% mutation rate , we would predict that no greater than 17% of head and neck tumors would be likely to harbor mutations at exon 19 or 21 .
these data demonstrate that only a small number of head and neck tumors are likely to have mutations of egfr receptor exons 19 and 21
. mutations of exons 19 and 21 are the most common egfr receptor mutations in lung cancer and confer receptor tyrosine kinase activation [ 11 , 18 ] . it is postulated that tumors harboring egfr mutations demonstrate inflexible  tumor addiction  to the high egfr transduction environment since they were born out of this condition .
a number of recent studies in head and neck cancer demonstrate a low , but significant , level of clinical response ( up to 12% ) of single agent egfr tki , in a setting of recurrent or metastatic disease [ 21 , 22 ] .
since activating egfr mutations at exons 19 and 21 is highly associated with a good clinical response to egfr inhibitors in lung cancer , the fact that our tumor specimens do not demonstrate these mutations may support the relatively low levels of clinical response in recent head and neck cancer clinical studies .
since 0% of our specimens and cell lines were associated with mutations , we extrapolated a 95% confidence interval for the likely number of head and neck tumor specimens in a similar large population of u.s .
we would predict that no more than 17% of head and neck tumors would be likely to harbor a mutation at either exon 19 or 21 .
these results are comparable to 3 small studies demonstrating that egfr mutations occur in very few - sampled hnscc in korean , austrian , and spanish   patients .
further , our data also predicts that these mutations should not occur more often than similar mutations in nsclc .  
since tki therapy is expensive and primarily benefits a minority of patients receiving it as a single agent , it would be very useful to predict which patients are most likely to receive benefit . because these agents are most successful in patients whose tumors harbor activating mutations , empiric tki therapy for all patients with recurrent or metastatic head and neck cancer is impractical .
screening patients for tki sensitive mutations may allow for more efficient therapy and improved patient outcomes .  
the tumor patients in our population are fairly typical head and neck cancer patients when segregated for age , gender , and tobacco use . however , the location of our cancer center in the u.s .
midwest has relatively low numbers of non - caucasian patients in our clinical trials and specimen banks pool ( approximately 5% ) , a number that reflects the demographics of our 4 state referral pattern .
another limitation to our study is the fact that the tumor specimens are supplied without identifying data from the patients , per human investigation regulations of the tumor bank . in spite of these limitations , it is helpful to know that since no mutations exist at either locus in our samples , it might suggest that only a minority of such patients will likely respond to current egfr - directed therapies .
further investigations in hnscc correlating exons 19 and 21 mutation status and tki response are required .
objective . 
 epidermal growth factor receptor ( egfr ) tyrosine kinase inhibition ( tki ) is a promising treatment in upper aerodigestive malignancies . 
 egfr inhibitors might be more effective in patients whose tumors harbor specific egfr mutations . 
 the presence of specific efgr mutations is predictive of over a 75% response rate to tki therapies as compared to 10% in wild type cases of non - small cell lung cancer . 
 our objective was to examine whether these mutations might occur in upper aerodigestive cancers .   
 design . 
 dna was extracted from 20 head and neck squamous cell tumors and 4 squamous cell carcinoma cell lines and sequenced the receptor using published primer pairs . 
 we then compared the results against published mutations . 
 results . 
 no exon 19 or 21 mutations were found in any of the 20 tumors and 0 of 4 cell lines . based on the tumor data we would predict that no greater than 8% of head and neck tumors ( ci 97.5% ) would be likely to harbor either of these mutations . conclusions . 
 our findings are comparable to results recently published of korean , austrian , and spanish patient populations and we conclude that exon 19 and 21 egfr mutations are not more common in head and neck cancer than in nonsmall - cell carcinoma .