malignant salivary gland neoplasms account for < 0.5% of all malignancies and approximately 35% of all head and neck cancers .
progress in understanding the cell biology of salivary gland carcinomas ( sgcs ) and detecting vulnerable molecular pathways may lead to the development of new targeted therapy options in these rare cancers with   poor prognosis .
anti - egfr agents include ( i ) monoclonal antibodies ( cetuximab or erbitux , panitumumab ) which block the binding of natural egfr ligands like egf or tgf- resulting in inhibition of downstream signal - transduction pathways and ( ii ) small molecule tyrosine kinase inhibitors ( tkis ) which act by binding the atp pocket within the kinase domain of the egfr and impairing its catalytic activity ( gefitinib , erlotinib , lapatinib ) . downstream signaling pathways triggered by egfr include the ras - raf - extracellular signal - regulated kinase / mitogen activated protein kinase ( mek / mapk ) pathway , which is mainly correlated to cell proliferation , and the p13k - pten - akt axis .
recently , we were able to demonstrate that frequent egfr overexpression and the absence of drug - resistance egfr mutations in sgc plead in favor of further therapeutic trials with egfr - targeting monoclonal antibodies .
one of the signaling effectors downstream of egfr , kras , was shown by us to be rarely mutated in sgc [ 2 , 3 ] .
wildtype kras is one of the clinically proven prerequisites for a successful anti - egfr therapy and therefore anti - egfr monoclonal antibodies are approved only for metastatic colorectal cancer patients whose tumors display wildtype kras .   in the absence of kras mutations , resistance to anti - egfr treatments could be caused by alterations of other members of the ras - raf - mapk pathway .
braf ( v - raf murine sarcoma viral oncogene homolog b1 ) , a serine / threonine kinase , is the downstream effector of kras in the ras - raf - mapk signaling pathway . a somatic mutation ( v600e ) in exon 15 of braf has been identified in multiple human cancers with a mutation rate of 66% in malignant melanomas   and at lower frequency in other human carcinomas .
recently , it was demonstrated that wildtype braf is required for the response of patients with metastatic colorectal cancer to cetuximab and panitumumab .
the aim of this study was to determine the braf v600e mutation frequency in a large cohort of sgcs of the main histopathological types and to design an allele - specific pcr as an effective screening method .
surgically removed , formalin - fixed tumor samples were obtained from 65 patients ( 35 males and 30 females with a median age at diagnosis of 55 years ) treated with the histopathological diagnosis of an sgc according to the who classification .
egfr - targeted therapy was not applied . the study cohort consisted of adenoid cystic carcinoma ( n = 25 ) mucoepidermoid carcinoma ( n = 10 ) , myoepithelial carcinoma ( n = 8) , acinic cell carcinoma ( n = 12 ) and adenocarcinoma ex pleomorphic adenoma ( n = 10 ) .   genomic dna was extracted and pooled from oral mucosa samples of five healthy individuals .
this pooled dna was used as a normal dna control for the development of the pcr assay .
heterozygous mutant control dna was extracted from cells of the colorectal cancer cell line ht 29 which contains the heterozygous braf v600e mutation .
the basis for discrimination using allele - specific pcr is that a pcr primer mismatched at its 3 end with the dna template will react less efficiently than one that is entirely complementary .
our allele - specific multiplex pcr was designed with one common forward ( bf ) and two separate reverse primers ( br and bmu ) :   bf : 5-ctcttcataatgcttgctctgatagg-3 , br : 5-agttgagaccttcaatgactttctagt-3 , bmu : 5-cccactccatcgagatttct-3. the forward primer bf and the reverse primer br amplify a 273 bp fragment of both mutant and wildtype alleles and thus serve as amplification control .
the second reverse primer ( bmu ) is specific for the mutated allele at the 3 end .
this primer together with bf generates an 143 bp product only in the presence of the v600e ( gtg > gag ) mutation ( figure 1(a ) ) . a series of annealing temperatures ( 52c60c ) , primer concentrations ( 0.10.4 mol / l ) , and mg concentrations ( 1.53.5
reactions consisted of : 80100 ng genomic dna ; 200 mol / l dntp ; 0.1 mol / l of primers ; 1.5 mmol / l mgcl2 ; 0.5 u taq     polymerase ( roche diagnostics , penzberg , germany ) .
final cycling conditions were as follows : 5 minutes of denaturing at 94c and 30 cycles of 94c for 30 seconds , annealing 55c for 45 seconds and 72c for 60 seconds .
a volume of 10 l of the pcr products was electrophoresed on a standard 2.5% agarose gel stained with sybr - green i for visualization under uv light .
the allele - specific pcr for the detection of the braf v600e mutation demonstrated high specificity ( i.e. ,   detection of only the normal or only the mutant allele ) , high sensitivity ( i.e. , no spurious pcr fragments ) , and acceptable yield . all 65 sgc in this cohort ( 100% ) presented the braf wildtype ( 95% exact confidence limit 00.07 ) .
the 273 bp pcr fragment was always amplified confirming the integrity of the isolated dna from clinical tissue samples ( figure 1(a ) ) . to test the sensitivity of the mutation - specific pcr
, we made a serial dilution of ht 29 dna ( which contains the heterozygous braf v600e mutation ) , in control dna with wildtype braf .
no braf v600e mutation was detected in additionally screened dna samples from microdissected normal tissue adjacent to the tumor cells ( 5 cases ) .
the activation of the egfr - ras - raf signaling cascade is an important pathway in cancer development and is considered a key pathway for therapeutic molecules .
egfr transmits signals to the nuclei instructing cancer cells to proliferate and metastasize , and kras and braf are those downstream signaling molecules .
anti - egfr therapies interrupt the cancer - triggering signaling cascade , however , if the kras or the braf gene is mutated , their proteins are locked into an active conformation , regardless of whether the egfr is therapeutically blocked .  
cetuximab ( erbitux ) has already been tested in two phase ii studies in patients with recurrent and/or metastatic sgc including mainly acc , a cancer with generally poor outcome . in 50% of patients , clinical benefit ( i.e. , response or stable disease for 6 month )
gefitinib was associated with a 53% stable disease rate ( 10/19 ) in acc , but had no effects on patients with salivary duct tumors and mucoepidermoid cancer .
lapatinib , inhibiting erbb1 and erbb2 tyrosine kinases , was studied in a phase ii trial and stabilized disease for greater than 6 months in 47% of acc patients .  
key molecules of the egfr - ras - raf signaling cascade and predictive markers of treatment outcome under anti - egfr therapies have not been comprehensively examined in sgc .
investigations of the mutation status of proteins in the cascade downstream of egfr identified markers for egfr - targeted therapy in colorectal cancer . in recent studies , wildtype braf as well as wildtype kras and intact pten pik3ca
were found to be required for the response of colorectal cancer patients treated with cetuximab or panitumumab [ 5 , 10 , 11 ] .
we were able to demonstrate in this and in a former study that kras and braf mutations seem to be extremely rare in sgc .
these findings imply that salivary gland carcinomas which rarely acquire mutations that result in constitutive activation of the signaling cascade downstream of egfr may be good candidates for anti - egfr therapies .
molecular analyses of alternative members of the egfr signaling cascade , such as akt-1 and mek-1 , may further contribute to elucidating predictive markers of treatment outcome under anti - egfr therapies in sgc . because of its universal availability as a standard methodology in molecular medicine , we designed a braf mutation screening assay based on pcr .
allele - specific pcr , also known as amplification refractory mutation system ( arms ) , is a well - established method for discriminating between different alleles at specific loci resulting from single base mutations [ 12 , 13 ] .
we used the methodology to establish an assay with three pcr primers which allows the discrimination of allele - specific pcr fragments by agarose gel electrophoresis without the need of capillary electrophoresis devices . with our assay
interpretation of the results can be made by simple visual inspection of the stained gel to determine whether or not a specific primer pair amplified a fragment with the template dna .
because microdissected tumor areas were used for the allele - specific pcr , and a control amplification was incorporated into the pcr reaction to ensure dna integrity , we can exclude false negative results .
so far , genomic screening for braf mutations has been based mainly on direct sequencing .
our protocol provides an alternative rapid , sensitive , and cost - effective braf screening method .
 braf is the main effector of kras in the ras - raf - mapk axis , a signaling pathway downstream of egfr . 
 the activation of this cascade is an important pathway in cancer development and is considered a key pathway for therapeutic molecules . 
 recent studies in metastatic colorectal cancer found that an oncogenic activation of braf by a point mutation in exon 15 ( v600e ) could bypass the egfr - initiated signaling cascade with the effect that patients bearing the mutant braf allele are not likely to benefit from egfr - targeted therapies . 
 we designed an allele - specific pcr and screened 65 salivary gland carcinoma ( sgc ) of the main histopathological types for the braf v600e mutation . 
 all 65 sgc in this cohort ( 100% ) presented the braf wildtype . in a previous study 
 , we found a kras wildtype in 98.5% of sgc . 
 
 these findings imply that sgc rarely acquires mutations that result in a constitutive activation of the signaling cascade downstream of egfr and this pleads in favor of further therapeutic trials with egfr - targeting monoclonal 
 antibodies . 