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Hindawi Publishing CorporationJournal of Biomedicine and BiotechnologyVolume 2009, Article ID 501736, 3 pagesdoi:10.1155/2009/501736

Research Article

No Incidence of BRAF Mutations in Salivary GlandCarcinomas—Implications for Anti-EGFR Therapies

Regine Dahse,1 Katrin Kromeyer-Hauschild,2 Alexander Berndt,3 and Hartwig Kosmehl1

1 Institute of Pathology, HELIOS Klinikum Erfurt, Nordhauser Str. 74, 99089 Erfurt, Germany2 Institute of Human Genetics and Anthropology, Friedrich Schiller University of Jena, Kollegiengasse 10, 07740 Jena, Germany3 Institute of Pathology, Friedrich Schiller University of Jena, Ziegelmuhlenweg, 07740 Jena, Germany

Correspondence should be addressed to Regine Dahse, [email protected]

Received 7 April 2009; Accepted 7 May 2009

Recommended by Fernando Schmitt

BRAF is the main effector of KRAS in the RAS-RAF-MAPK axis, a signaling pathway downstream of EGFR. The activation ofthis cascade is an important pathway in cancer development and is considered a key pathway for therapeutic molecules. Recentstudies in metastatic colorectal cancer found that an oncogenic activation of BRAF by a point mutation in exon 15 (V600E) couldbypass the EGFR-initiated signaling cascade with the effect that patients bearing the mutant BRAF allele are not likely to benefitfrom EGFR-targeted therapies. We designed an allele-specific PCR and screened 65 salivary gland carcinoma (SGC) of the mainhistopathological types for the BRAF V600E mutation. All 65 SGC in this cohort (100%) presented the BRAF wildtype. In aprevious study, we found a KRAS wildtype in 98.5% of SGC. These findings imply that SGC rarely acquires mutations that resultin a constitutive activation of the signaling cascade downstream of EGFR and this pleads in favor of further therapeutic trials withEGFR-targeting monoclonal antibodies.

Copyright © 2009 Regine Dahse et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Malignant salivary gland neoplasms account for <0.5% of allmalignancies and approximately 3–5% of all head and neckcancers [1]. Progress in understanding the cell biology ofsalivary gland carcinomas (SGCs) and detecting vulnerablemolecular pathways may lead to the development of newtargeted therapy options in these rare cancers with poorprognosis. The EGFR signaling cascade is considered a possi-ble key pathway for therapeutic molecules. Anti-EGFR agentsinclude (I) monoclonal antibodies (cetuximab or erbitux,panitumumab) which block the binding of natural EGFRligands like EGF or TGF-α resulting in inhibition of down-stream signal-transduction pathways and (II) small moleculetyrosine kinase inhibitors (TKIs) which act by binding theATP pocket within the kinase domain of the EGFR andimpairing its catalytic activity (gefitinib, erlotinib, lapatinib).Downstream signaling pathways triggered by EGFR includethe RAS-RAF-extracellular signal-regulated kinase/mitogenactivated protein kinase (MEK/MAPK) pathway, which ismainly correlated to cell proliferation, and the P13K-PTEN-AKT axis.

Recently, we were able to demonstrate that frequentEGFR overexpression and the absence of drug-resistanceEGFR mutations in SGC plead in favor of further therapeutictrials with EGFR-targeting monoclonal antibodies. One ofthe signaling effectors downstream of EGFR, KRAS, wasshown by us to be rarely mutated in SGC [2, 3]. WildtypeKRAS is one of the clinically proven prerequisites fora successful anti-EGFR therapy and therefore anti-EGFRmonoclonal antibodies are approved only for metastaticcolorectal cancer patients whose tumors display wildtypeKRAS.

In the absence of KRAS mutations, resistance to anti-EGFR treatments could be caused by alterations of othermembers of the RAS-RAF-MAPK pathway. BRAF (v-rafmurine sarcoma viral oncogene homolog B1), a serine/threonine kinase, is the downstream effector of KRAS inthe RAS-RAF-MAPK signaling pathway. A somatic mutation(V600E) in exon 15 of BRAF has been identified in multiplehuman cancers with a mutation rate of 66% in malignantmelanomas [4] and at lower frequency in other humancarcinomas. Recently, it was demonstrated that wildtype

2 Journal of Biomedicine and Biotechnology

BRAF is required for the response of patients with metastaticcolorectal cancer to cetuximab and panitumumab [5].

The aim of this study was to determine the BRAF V600Emutation frequency in a large cohort of SGCs of the mainhistopathological types and to design an allele-specific PCRas an effective screening method.

2. Materials and Methods

2.1. Tissue Specimens. Surgically removed, formalin-fixedtumor samples were obtained from 65 patients (35 males and30 females with a median age at diagnosis of 55 years) treatedwith the histopathological diagnosis of an SGC accordingto the WHO classification [6]. All patients received surgeryand postoperative radiation therapy in selected cases. EGFR-targeted therapy was not applied. The study cohort consistedof adenoid cystic carcinoma (n = 25) mucoepidermoidcarcinoma (n = 10), myoepithelial carcinoma (n = 8),acinic cell carcinoma (n = 12) and adenocarcinoma expleomorphic adenoma (n = 10).

2.2. DNA Isolation. Genomic DNA was extracted and pooledfrom oral mucosa samples of five healthy individuals. Thispooled DNA was used as a normal DNA control for thedevelopment of the PCR assay. Heterozygous mutant controlDNA was extracted from cells of the colorectal cancer cellline HT 29 which contains the heterozygous BRAF V600Emutation. DNA from the tumor specimen was isolated aftermicrodissecting apprpriate tumor areas.

2.3. Design of an Allele-Specific PCR for the BRAF V600EMutation. The basis for discrimination using allele-specificPCR is that a PCR primer mismatched at its 3′ end withthe DNA template will react less efficiently than one that isentirely complementary. Our allele-specific multiplex PCRwas designed with one common forward (BF) and twoseparate 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 BRamplify a 273 bp fragment of both mutant and wildtypealleles and thus serve as amplification control. The secondreverse primer (BMu) is specific for the mutated allele at the3′ end. This primer together with BF generates an 143 bpproduct only in the presence of the V600E (GTG>GAG)mutation (Figure 1(a)).

A series of annealing temperatures (52◦C–60◦C), primerconcentrations (0.1–0.4 μmol/L), and Mg2+ concentrations(1.5–3.5 mmol/L) were tested.

2.4. BRAF Mutational Analysis. PCR reactions for screeningSGC were run at a final volume of 25 μL. Reactions consistedof: 80–100 ng genomic DNA; 200 μmol/L dNTP; 0.1 μmol/Lof primers; 1.5 mmol/L MgCl2; 0.5 U Taq polymerase (RocheDiagnostics, Penzberg, Germany). Final cycling conditionswere as follows: 5 minutes of denaturing at 94◦C and 30

1 2 3 4 5 6 7 8 9

(a)

1 2 3 4 5 6 7 8 9

(b)

Figure 1: Allele-specific PCR for the detection of the BRAF V600Emutation (2.5% agarose gel). (a) The 273 bp fragment of bothmutant and wildtype alleles is always amplified and serves asamplification control. The smaller PCR product (143 bp; arrow)is amplified only in the presence of the V600E (GTG>GAG)mutation. Lane 1: Molecular Weight marker; lane 2: heterozygousmutant DNA control (HT 29); lane 3: homozygous wildtype DNAcontrol; lane 4: no template PCR control (water). Lanes 5–9:analysis of tumor samples. The SGC samples in lanes 5–9 havethe BRAF V600E wildtype sequence. (b) Serial dilution of HT 29DNA in normal wildtype DNA. The allele-specific PCR is ableto detect the 143 bp mutation specific fragment in an 15-foldexcess of normal DNA. Lane 1: Molecular Weight marker; lane 2:heterozygous mutant DNA control [HT 29] undiluted; lanes 3–9:serial dilutions HT 29/WT DNA (starting from 1:2 up to1:64).

cycles of 94◦C for 30 seconds, annealing 55◦C for 45 secondsand 72◦C for 60 seconds. A volume of 10 μL of the PCRproducts was electrophoresed on a standard 2.5% agarose gelstained with SYBR-Green I for visualization under UV light.

3. Results

The allele-specific PCR for the detection of the BRAF V600Emutation demonstrated high specificity (i.e., detection ofonly the normal or only the mutant allele), high sensitivity(i.e., no spurious PCR fragments), and acceptable yield. All65 SGC in this cohort (100%) presented the BRAF wildtype(95% exact confidence limit 0–0.07). The 273 bp PCRfragment was always amplified confirming the integrity ofthe isolated DNA from clinical tissue samples (Figure 1(a)).

To test the sensitivity of the mutation-specific PCR, wemade a serial dilution of HT 29 DNA (which containsthe heterozygous BRAF V600E mutation), in control DNAwith wildtype BRAF. Less than 6% of mutant DNA wasreproducibly detected (Figure 1(b)).

No BRAF V600E mutation was detected in additionallyscreened DNA samples from microdissected normal tissueadjacent to the tumor cells (5 cases).

Journal of Biomedicine and Biotechnology 3

4. Discussion and Conclusions

The activation of the EGFR-RAS-RAF signaling cascade is animportant pathway in cancer development and is considereda key pathway for therapeutic molecules. EGFR transmitssignals to the nuclei instructing cancer cells to proliferateand metastasize, and KRAS and BRAF are those downstreamsignaling molecules. Anti-EGFR therapies interrupt thecancer-triggering signaling cascade, however, if the KRAS orthe BRAF gene is mutated, their proteins are locked intoan active conformation, regardless of whether the EGFR istherapeutically blocked.

Cetuximab (erbitux) has already been tested in twophase II studies in patients with recurrent and/or metastaticSGC including mainly ACC, a cancer with generally pooroutcome. In 50% of patients, clinical benefit (i.e., response orstable disease for 6 month) could be achieved [1, 7]. Gefitinibwas associated with a 53% stable disease rate (10/19) in ACC,but had no effects on patients with salivary duct tumors andmucoepidermoid cancer [8]. Lapatinib, inhibiting ErbB1 andErbB2 tyrosine kinases, was studied in a phase II trial andstabilized disease for greater than 6 months in 47% of ACCpatients [9].

Key molecules of the EGFR-RAS-RAF signaling cascadeand predictive markers of treatment outcome under anti-EGFR therapies have not been comprehensively examinedin SGC. Investigations of the mutation status of proteinsin the cascade downstream of EGFR identified markersfor EGFR-targeted therapy in colorectal cancer. In recentstudies, wildtype BRAF as well as wildtype KRAS and intactPTEN PIK3CA were found to be required for the responseof colorectal cancer patients treated with cetuximab orpanitumumab [5, 10, 11].

We were able to demonstrate in this and in a former studythat KRAS and BRAF mutations seem to be extremely rarein SGC. These findings imply that salivary gland carcinomaswhich rarely acquire mutations that result in constitutiveactivation of the signaling cascade downstream of EGFRmay be good candidates for anti-EGFR therapies. Molecularanalyses of alternative members of the EGFR signalingcascade, such as Akt-1 and MEK-1, may further contribute toelucidating predictive markers of treatment outcome underanti-EGFR therapies in SGC.

Because of its universal availability as a standard method-ology in molecular medicine, we designed a BRAF muta-tion screening assay based on PCR. Allele-specific PCR,also known as Amplification Refractory Mutation System(ARMS), is a well-established method for discriminatingbetween different alleles at specific loci resulting from singlebase mutations [12, 13]. We used the methodology to estab-lish an assay with three PCR primers which allows the dis-crimination of allele-specific PCR fragments by agarose gelelectrophoresis without the need of capillary electrophoresisdevices. With our assay, the specificity was incorporatedinto the amplification reaction itself. Interpretation of theresults can be made by simple visual inspection of thestained gel to determine whether or not a specific primerpair amplified a fragment with the template DNA. Becausemicrodissected tumor areas were used for the allele-specific

PCR, and a control amplification was incorporated intothe PCR reaction to ensure DNA integrity, we can excludefalse negative results. So far, genomic screening for BRAFmutations has been based mainly on direct sequencing. Ourprotocol provides an alternative rapid, sensitive, and cost-effective BRAF screening method.

References

[1] A. Milano, F. Longo, M. Basile, R. V. Iaffaioli, and F.Caponigro, “Recent advances in the treatment of salivarygland cancers: emphasis on molecular targeted therapy,” OralOncology, vol. 43, no. 8, pp. 729–734, 2007.

[2] R. Dahse, O. Driemel, S. Schwarz, et al., “Epidermal growthfactor receptor kinase domain mutations are rare in salivarygland carcinomas,” British Journal of Cancer, vol. 100, no. 4,pp. 623–625, 2009.

[3] R. Dahse, O. Driemel, S. Schwarz, K. Kromeyer-Hauschild,A. Berndt, and H. Kosmehl, “KRAS status and epidermalgrowth factor receptor expression as determinants for anti-EGFR therapies in salivary gland carcinomas,” Oral Oncology.In press.

[4] H. Davies, G. R. Bignell, C. Cox, et al., “Mutations of the BRAFgene in human cancer,” Nature, vol. 417, no. 6892, pp. 949–954, 2002.

[5] F. Di Nicolantonio, M. Martini, F. Molinari, et al., “WildtypeBRAF is required for response to panitumumab or cetuximabin metastatic colorectal cancer,” Journal of Clinical Oncology,vol. 26, no. 35, pp. 5705–5712, 2008.

[6] L. Barnes, W. Eveson, P. Reichart, and W. Sidransky, “Pathol-ogy and genetics of head and neck tumours,” in World HealthOrganization Classification of Tumours, pp. 210–242, IARCPress, Lyon, France, 2005.

[7] L. D. Locati, P. Bossi, F. Perrone, et al., “Cetuximab inrecurrent and/or metastatic salivary gland carcinomas: a phaseII study,” Oral Oncology. In press.

[8] B. S. Glisson, G. Blumenschein, M. Francisco, J. Erasmus, R.Zinner, and M. Kies, “Phase II trial of gefitinib in patients withincurable salivary gland cancer,” Journal of Clinical Oncology,vol. 23, no. 16S, p. 5532, 2005.

[9] M. Agulnik, E. W. E. Cohen, R. B. Cohen, et al., “Phase II studyof lapatinib in recurrent or metastatic epidermal growth factorreceptor and/or erbB2 expressing adenoid cystic carcinomaand non-adenoid cystic carcinoma malignant tumors of thesalivary glands,” Journal of Clinical Oncology, vol. 25, no. 25,pp. 3978–3984, 2007.

[10] M. Jhawer, S. Goel, A. J. Wilson, et al., “PIK3CA muta-tion/PTEN expression status predicts response of colon cancercells to the epidermal growth factor receptor inhibitor cetux-imab,” Cancer Research, vol. 68, no. 6, pp. 1953–1961, 2008.

[11] A. Sartore-Bianchi, M. Martini, F. Molinari, et al., “PIK3CAmutations in colorectal cancer are associated with clinicalresistance to EGFR-targeted monoclonal antibodies,” CancerResearch, vol. 69, no. 5, pp. 1851–1857, 2009.

[12] G. Sarkar, J. Cassady, C. D. K. Bottema, and S. S. Sommer,“Characterization of polymerase chain reaction amplificationof specific alleles,” Analytical Biochemistry, vol. 186, no. 1, pp.64–68, 1990.

[13] C. R. Newton, A. Graham, L. E. Heptinstall, et al., “Analysisof any point mutation in DNA. The amplification refractorymutation system [ARMS],” Nucleic Acids Research, vol. 17, pp.2503–2516, 1989.

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