Somatic Mutations of Epidermal Growth Factor Receptor inBile Duct and Gallbladder CarcinomaFrancesco Leone,1Giuliana Cavalloni,1Ymera Pignochino,1Ivana Sarotto,3 Renato Ferraris,1

Wanda Piacibello,1Tiziana Venesio,3 Lorenzo Capussotti,2 Mauro Risio,3 andMassimoAglietta1

Abstract Objective: Conventional therapies are still unsuccessful in patients with carcinoma arising fromthe biliary tract. Somatic mutations of the epidermal growth factor receptor (EGFR) gene andthe activation of its downstream pathways predict the sensitivity to small-molecule inhibitors innon ^ small cell lung carcinoma. Therefore, we analyzed EGFR mutations and related pathwaysin gallbladder and bile duct carcinomas to consider the possible application of these alternativetherapeutic strategies.Experimental Design: Forty paraffin-embedded samples, including intrahepatic or extrahepaticcholangiocarcinoma and gallbladder carcinoma, were studied after tumor cell isolation by lasermicrodissection and sequencing of EGFR tyrosine kinase domain (exons 18-21). Activation ofEGFR pathway was studied by evaluating phosphorylation of mitogen-activated protein kinaseand Akt.Results: None of the 40 specimens hadmutations in exon18; one hadonemissense pointmuta-tion in exon19, two in exon 20, and three in exon 21. In addition, 36 of 40 specimenshad the samesilent mutation at codon 787 in exon 20, which was also found in peripheral blood cells fromhealthy donors. Tumor samples harboring EGFR mutation had phosphorylation of one or bothdownstream transducers analyzed.Conclusions:This is the first evidence of somatic mutations of the EGFR gene in bile duct carci-noma. Our findings suggest that a subgroup of patients with cholangiocarcinoma or gallbladdercarcinoma exhibits somatic mutations of EGFR in the tyrosine kinase domain that can elicit cellsignals sustaining survival and proliferation. These tumors might be further evaluated for theirsusceptibility to small-molecule inhibitor treatment.

Cholangiocarcinoma is a malignant neoplasm originatingfrom the epithelium of the intrahepatic and extrahepatic biliarytree. Usually, it presents itself late in the clinical course and hasa poor prognosis. Most patients are diagnosed when the diseaseis unresectable, and their survival is <12 months followingdiagnosis (1, 2). The results of chemotherapy are disappoint-ing, and no complete remissions have been obtained (3).Moreover, it is not clear if, even in patients with objectiveresponse, the treatment prolongs survival (4, 5). For these

reasons, the search for active agents based on alternativemechanisms of action in bile duct and gallbladder carcinoma ismandatory.

Epidermal growth factor receptor (EGFR) activation triggersmultiple signaling cascades, including the Ras/Raf/mitogen-activated protein kinase (MAPK), Janus-activated kinase/signaltransducers and activators of transcription, and phosphatidyli-nositol 3-kinase/Akt pathways, leading to a multitude of effectsincluding cell proliferation, cell differentiation, angiogenesis,metastasis, and inhibition of apoptosis, participating in thedevelopment of several carcinomas (6, 7). The role of EGFR inhepatic malignancies is not clearly understood. The expressionof EGFR is increased in gallbladder, common bile duct, andampullary carcinomas but not in nonmalignant conditions ofthe gallbladder and biliary tract (8), and it seems related tosome clinical and pathologic features, such as lymph nodemetastasis, aberrant p53 expression, proliferation activity, anddifferentiation (9).

Recently, Yoon et al. showed that in cholangiocarcinoma celllines, EGFR activation was prolonged upon EGF stimulation,and that cell growth was significantly attenuated by EGFRkinase inhibitors (10).

Since the discovery that a subgroup of patients with non–small cell lung cancer with specific mutations in the EGFRgene are responsive to the tyrosine kinase inhibitor gefitinib(11, 12), a large number of studies have focused on EGFR

Human Cancer Biology

Authors’Affiliations: Departments of 1Clinical Oncology, 2Surgical Oncology and3Unit of Pathology, University of Torino Medical School, Institute for CancerResearch andTreatment, Candiolo,Turin, ItalyReceived 8/3/05; revised12/1/05; accepted12/6/05.Grant support: Associazione Italiana per la Ricerca sul Cancro, Milan, Italy;Consiglio Nazionale delle Ricerche/Progetto Strategico Oncologia; and Ministerodell’Istruzione, dell’Universita' e della Ricerca (Ricerca sanitaria finalizzata 2003 andG. Cavalloni,Y. Pignochino,W. Piacibello, andM. Aglietta).The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Requests for reprints: Francesco Leone, Department of Clinical Oncology,Institute for Cancer Research and Treatment, Str. Prov.le 142 Km 3.95, 10060Candiolo,Turin, Italy. Phone: 39-011-993-3628; Fax: 39-011-993-3524; E-mail:francesco.leone@ircc.it.

F2006 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-05-1692

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mutations as a potential target for growth inhibition in solidtumors (13–21). Furthermore, the analysis of EGFR signalingin lung cancer revealed that responsiveness to gefitinib isrelated to the activation of these pathways (22). With thepresent study, we showed that mutations in the EGFR geneare present, and that downstream pathways are activated inbile duct and gallbladder carcinoma, suggesting that small-molecule kinase inhibitors, such as gefitinib or erlotinib,should be evaluated for clinical activity in these tumors.

Materials and Methods

Patients and tissues. The specimens for this study were obtainedfrom paraffin-embedded samples of 40 patients with bile duct andgallbladder carcinomas: 15 of them were intrahepatic cholangiocarci-nomas, 14 were extrahepatic cholangiocarcinomas, and 11 weregallbladder carcinomas. There were 23 males and 17 females with amedian age of 66 years. Patient characteristics, including sex, age, stage,and histologic grading, are summarized in Table 1. The patients were ofItalian origin. Tumor specimens were obtained from the patients beforeany systemic treatment was administered.

Histologic type was determined according to WHO criteria (23).Patient stage at the time of diagnosis was determined according thetumor-node-metastasis staging system (24).

Samples of peripheral blood were obtained from healthy volunteersafter informed consent was given.

Isolation of genomic DNA and mutational analysis of EGFR. GenomicDNA was extracted from deparaffinized samples, with the use of theQIAamp DNA Mini kit (Qiagen, Milan, Italy) following themanufacturer’s instructions. To reduce contamination with normalcells, the tumor portion was obtained by laser microdissection(VSL-337ND-S, Spectra-Physics, Mountain View, CA). The kinasedomain of EGFR coding sequence, from exons 18 to 21, wasamplified by using primers and nested PCR conditions as previouslydescribed by Lynch et al. (12). The PCR products were then purifiedby QIAquick PCR purification kit (Qiagen), and sense and antisensesequences were obtained by using forward and reverse internalprimers, respectively. Each exon was sequenced using the BigDyeTerminator Cycle sequence following the PE Applied Biosystemsstrategy and Applied Biosystems ABI PRISM3100 DNA Sequencer(Applied Biosystems, Foster City, CA). All mutations were confirmed,performing two independent PCR amplifications. In one case inwhich different mutated sequence were found by independent PCRamplifications, the coexistence of several mutants was confirmed bysubcloning PCR products into PCR-4 vector using TOPO TA-cloningkit (Invitrogen, Milan, Italy) and further sequencing of 40 clones foreach PCR product.

EGFR activation analysis. The activation of EGFR downstreamsignaling was evaluated by immunohistochemistry detection ofphosphorylated MAPK and phosphorylated Akt. A rabbit polyclonalantibody against phosphorylated MAPK at Thr202/Tyr204 (Cell SignalingTechnology, Beverly, MA) and a rabbit polyclonal antibody againstphosphorylated Akt at Ser473 (Cell Signaling Technology) were used.Positive immunostaining was attributed to nuclear staining forphosphorylated MAPK and cytoplasmic with a faint membranousstaining for phosphorylated Akt.

Statistical analysis. The variables measured in the study wereinvestigated for association by using the Fisher’s exact test or m2 test asappropriate. P < 0.05 was considered significant.

Results

EGFR mutational analysis. The EGFR genomic sequencecoding for the tyrosine kinase domain was analyzed in a series

of 40 bile duct and gallbladder carcinomas. Exons 18, 19, 20,and 21 were directly sequenced after PCR amplification. Thedetails of the EGFR mutation spectrum are shown in Table 2.Six patients (15%) had point mutations in the EGFR tyrosinekinase domain (Fig. 1): two patients with extrahepatic chol-angiocarcinoma (cases 2 and 6), one patient with gallbladdercarcinoma (case 4), and three patients with intrahepaticcholangiocarcinoma (cases 1, 3, and 5).

Mutations were classified as heterozygous when the wild-typesequence was amplified on the second allele, or homozygous/hemizygous when only the mutated sequence was amplified,

Table 1. Patient characteristics

Case Sex Age Grading Stage Tumor origin

1 M 66 3 T3N1M0 ICC2 F 70 1 TxN1M0 ECC3 F 61 3 TxNxM1 ICC4 F 55 3 T4N2M0 gall5 F 41 2 T2N0M0 ICC6 F 60 3 T3N1M0 ECC7 M 75 2 T2N0M0 gall8 M 72 3 T3N1M0 ICC9 F 48 3 T3N0M0 ICC

10 M 46 3 T3N1M0 ECC11 M 74 2 T3N1M0 ECC12 M 67 1 T3N0M0 ICC13 M 69 2 T2N0M0 ECC14 F 54 3 TxNxM1 gall15 M 69 2 TxN1M1 ICC16 M 47 2 TxNxM1 ICC17 F 50 2 T1N0M0 ICC18 M 64 2 T3N0M0 ECC19 M 53 3 T3N0M0 ECC20 F 57 2 T3N2M0 ECC21 M 63 3 T4N0M0 ECC22 M 70 2 T3N1M0 ECC23 M 50 3 T2NxM0 ICC24 M 74 3 T3N1M0 ECC25 M 73 3 T3N0M0 ECC26 M 67 3 T4N2M0 ECC27 M 66 3 T3NxM0 ICC28 F 53 3 T3N1M0 ICC29 M 53 3 TxNxM1 ICC30 F 69 3 T3N1M0 gall31 F 71 3 T2N1M0 gall32 M 68 2 T2NxM0 gall33 F 75 3 T3NxM0 gall34 F 84 3 T2NxM0 ICC35 M 83 3 T2NxM0 gall36 F 78 3 T2NxM0 gall37 F 51 2 T3N1M0 gall38 M 56 3 T2NxM0 gall39 F 66 3 T4N1M0 ICC40 M 64 3 T2N0M0 ECC

Abbreviations: ICC, intrahepatic cholangiocarcinoma; ECC, extrahepaticcholangiocarcinoma; gall, gallbladder carcinoma.

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suggesting either a biallelic mutation or a monoallelic mutationassociated to the loss of the second allele.

No mutation was detected in exon 18 in all tested tumors.One homozygous/hemizygous point mutation was found in

exon 19; it consisted of a transition from A to G, resulting inamino acid substitution from lysine to arginine at codon 757(K757R).

In exon 20, one silent point mutation or variant, alreadydescribed by Nagahara et al. in colorectal carcinoma (20),was found at codon 787 (Gln; CAG-to-CAA) in 36 of 40(90%) patients. This variant was present both in homozy-gous/hemizygous (78%) and heterozygous status (22%). Inaddition, in two cases missense point mutations of exon 20were found: one patient had one mutation involving thecodon 775 (TGC-to-TAC) with amino acid substitution fromcysteine to tyrosine (C775Y), and the second had onemutation at codon 790 (ACG-to-ATG; Thr-to-Met). This lastmutation was previously described in lung adenocarcinomaby Kobayashi et al. (25). They showed that T790M mutationwas associated to emerging resistance during gefitinibtreatment.

Mutated sequences of exon 21 were found in threesamples. One patient had a homozygous/hemizygous mis-sense point mutation (GCC-to-ACG; Ala-to-Thr) at codon864 (A864T). The second patient had one (GAA-to-AAA; Glu-to-Lys) at codon 872 (E872K). The third patient had differentmutations obtained with independent PCR: a heterozygouspoint mutation with amino acid substitution from glycine toserine (GGC-to-AGC) at codon 882 (G882S), a secondheterozygous point mutation with amino acid substitutionfrom valine to isoleucine (GTA-to-ATA) at codon 843(V843I), and a third heterozygous silent mutation at codon858, which is the most frequently missense mutated codon(L858R) in lung cancer (11–15, 18, 21, 22). The presence ofdifferent mutated sequences was confirmed by PCR productssubcloning into PCR-4 vector and was found not to be dueto microsatellite instability, as shown by the analysis of amarker panel (BAT25, BAT26, D2S123, D2S346, andD17S250) according to Bethesda guidelines (data not shown;ref. 26).

We investigated the somatic origin of the mutations byperforming PCR analysis and sequencing of DNA extractedfrom nontumor tissues of patients. Only exon 20 variant atcodon 787 was also observed in the corresponding nonneo-

plastic tissues of the patients. We confirm the high prevalenceof EGFR exon 20 variant in Italian subjects also in healthydonors, as five of six sequences of exon 20 of EGFR inmononuclear cells obtained from healthy volunteers harboredthe silent G to A. The distribution of EGFR gene mutations wassignificantly different between males and females as alsodescribed in lung adenocarcinoma (27–29). They were presentin 1 of 23 (4.3%) males and in 5 of 17 (29.4%) females (P <0.05). No correlation was found between mutations of theEGFR gene and extrahepatic or intrahepatic origin of the tumornor with other clinical and pathologic findings.

EGFR activation analysis. To evaluate the phosphorylationstatus of EGFR downstream transducers MAPK and Akt,immunohistochemistry was done on all tumor samples. Theresults showed that of the 40 specimens, 16 (40%) show MAPKphosphorylation, and 22 (55%) show Akt phosphorylation(Table 3). Five of six (83.3%) cases with EGFR mutations showMAPK phosphorylation (P < 0.05). The same proportion ofEGFR-mutated cases had Akt phosphorylation without statisti-cal significance (P > 0.05).

Discussion

Chronic biliary inflammation and cholestasis, leading to theproduction of proinflammatory cytokines and reactive oxygenspecies, cause protracted cellular stress and irreversible DNAdamage. This in turn leads to malignant transformation ofcholangiocytes (4). Moreover, the presence of biliary acids inchronic cholestasis functionally transactivates EGFR via trans-forming growth factor-a (10), leading to activation of down-stream pathways that promote cell survival, proliferation,and inhibition of apoptosis, which represent major processescharacteristics of cholangiocarcinogenesis (30). The discoveryof acquired somatic mutations in the EGFR gene that modifythe activity of tyrosine kinase domain could be crucial ingaining a better understanding of molecular pathogenesis ofthis highly aggressive tumor.

EGFR activation regulates a number of cellular functions andhas been strongly implicated in carcinogenesis, thus makingEGFR tyrosine kinase a promising therapeutic target in solidtumors (6, 7). However, it is necessary to carefully evaluatewhether a selective inhibition of pathways involved incarcinogenesis can produce sufficient effect on tumor growth.Previous studies have shown that the activity of small-molecule

Table 2. EGFR mutations in cholangiocarcinoma

Patient Exon Mutation type Mutation status Nucleotide Amino acid

1 19 Substitution Homozygous/hemizygous AAG >AGG K757R2 20 Substitution Homozygous/hemizygous TGC > TAC C775Y3 20 Substitution Heterozygous ACG >ATG T790M4 21 Substitution Homozygous/hemizygous GCG >ACG A864T5 21 Substitution Homozygous/hemizygous GAA >AAA E872K6#1 21 Substitution Heterozygous GGC >AGC G882S6#2 21 Substitution Heterozygous GTA > ATA V843I6#3 21 Silent Heterozygous CTG >CTA L858

Abbreviations: K, lysine; R, arginine; C, cysteine;Y, tyrosine; A, alanine;T, threonine; H, hystidine; E, glutamic acid; G, glycine; S, serine;V, valine; I, isoleucine; L, leucine.

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inhibitors, such as gefitinib or erlotinib, in the treatmentof non–small cell lung carcinoma is associated with theexistence of specific mutations in the tyrosine kinase domain ofEGFR in tumors (11–15, 18, 21). The prevalence of suchmutations is about 20% but is higher in patients of East Asianethnicity (30%) than in other ethnicity (10%). They arestatistically more frequently found in females than males,nonsmokers than smokers, and in adenocarcinoma than otherhistologies. Most tumors that responded to gefitinib orerlotinib displayed mutations in the kinase domain of theEGFR gene (11–13, 29).

In cholangiocarcinoma, somatic mutations of the EGFR genehave not been reported to date. As with lung carcinoma,

mutations in the tyrosine kinase domain of the EGFR gene maybe associated with clinical efficacy of EGFR inhibitors.

In the present study, we found that 6 of 40 (15%) biliarytree and gallbladder carcinoma have EGFR gene mutations inthe sequence coding for the tyrosine kinase domain. All of themutations were somatic acquired point mutations, and mostwere found within exon 21.

Four of eight point mutations found seemed to behomozygous as previously described for other tumor types(14, 19) and might be considered biallelic. Alternatively, theycould be considered hemizygous. In these cases, there may bethe contemporary presence of monoallelic point mutationswith deletions of the second allele involving the intronic

Fig. 1. Electropherograms of tyrosine kinase domain sequence (exons18-21) of the EGFR gene. Mutations found in 6 of 40 patients with bile duct and gallbladdercarcinoma are shown and compared with the correspondingwild-type sequence. Cases1to 5 had single missense point mutations; case 6 hadmultiple mutation detected byindependent PCR (#1 to #3).The exon 20 variant is also shown in a heterozygous status in case 3.

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regions to which primers used for PCR amplification anneal.The technique applied allows the identification of alldeletions previously described (12) in lung tumors; however,other possible deletions not detected by these conditions mayexist.

In one case, different mutations were simultaneously presentin the same tissue suggesting the existence of a heterogeneouspopulation of cancer cells. In our study, tumor cells wereobtained from tissues by laser microdissection, which facilitatesprecise discrimination between tumor cells and analysis groupsof fewer cells. This technique probably contributes to highlighteven minimal differences between tumor clones thus revealingcancer heterogeneity.

In five cases, we detected mutations that have not beenpreviously described in other type of tumors.

No in-frame deletions within the exon 19, frequentlyobserved in non–small cell lung cancer and in head and neckcancer (19), were found. In our study, codon 858, whichrepresents the most common site of mutation in non–smallcell lung carcinoma, showed only one silent nucleotidesubstitution in one case. In two cases, we confirmed previouslydescribed mutations of the tyrosine kinase domain. The V843Imutation determined in case 6 has been detected by Shih et al.(31) in a patient with lung carcinoma that experienced a partialresponse to gefitinib treatment. The T790M mutation deter-mined in case 3 was previously detected in some cases of lungcarcinoma that recur after an initial response to gefitinib (25).These findings suggest that the detection of EGFR mutationsin bile duct carcinoma does not imply that these tumors aresensitive to the clinical available molecular inhibitors. How-ever, functional analysis of the T790M mutation revealed thatother small-molecule inhibitors could actively block EGFRphosphorylation (32).

Previous studies showed the association between EGFR signaltransducers activation and better outcome in lung cancerpatients treated with gefitinib (33). All the mutations that wefound in bile duct carcinoma led to activation of one or both ofthe EGFR signal transduction pathways analyzed. The percent-age of cases with activation of EGFR downstream pathwayswas higher in EGFR-mutated compared with EGFR wild-type tumors. This observation sustains the possibility to obtainobjective response to small-molecule inhibitors in bile ductcarcinoma.

Acknowledgments

We thank Radhika Srinivasan, Ph.D. for her careful editorial assistance.

Table 3. EGFR downstream pathway activation

Case EGFRmutations

PhosphorylatedMAPK

PhosphorylatedAkt

1 Y Y Y2 Y N Y3 Y Y Y4 Y Y Y5 Y Y Y6 Y Y N7 N N N8 N N N9 N N N

10 N N Y11 N N Y12 N Y Y13 N Y Y14 N N N15 N N Y16 N N Y17 N N N18 N Y N19 N N N20 N N N21 N N N22 N N N23 N Y Y24 N N N25 N Y Y26 N Y Y27 N Y Y28 N N Y29 N N N30 N Y N31 N N N32 N N Y33 N Y Y34 N N Y35 N N N36 N N Y37 N Y Y38 N Y Y39 N N N40 N N N

Abbreviations:Y, yes (present); N, not present.

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2006;12:1680-1685. Clin Cancer Res   Francesco Leone, Giuliana Cavalloni, Ymera Pignochino, et al.   Bile Duct and Gallbladder CarcinomaSomatic Mutations of Epidermal Growth Factor Receptor in

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