epidermal growth factor expression in esophageal adenocarcinoma: a clinically relevant target?

ORIGINAL ARTICLE

Epidermal Growth Factor Expression in EsophagealAdenocarcinoma: A Clinically Relevant Target?

Nicholas Harper & Yan Li & Russell Farmer &

Robert C. G. Martin

Received: 5 October 2011 /Accepted: 31 October 2011 /Published online: 17 November 2011# 2011 The Society for Surgery of the Alimentary Tract

AbstractIntroduction There has been recent widespread enthusiasm in epidermal growth factor (EGFR) as a molecularly activetarget in esophageal adenocarcinoma (EAC). However, there is limited data on the extent of EGFR expression in EAC.Thus, the aim of this study was to evaluated EGFR, pErk1/2, and total Erk1/2 expression in malignant and benignspecimens.Methods Baseline expression of EGFR in the human normal squamous, Barrett’s, and EAC cell lines were determined aswell as after bile acid treatment and curcumin pretreatment. In addition, EGFR expression was also evaluated in 60 matchednormal and malignant EAC resected specimens.Results The in vitro studies in the Het-1a, BarT, and OE19 cell lines failed to show any measurable expression of EGFR viaWestern blot technique. The marker serving as the positive control for the study, MnSOD, showed expression in each cellline for all three treatment regimens at approximately 24 kDa EGFR, showing moderate staining in the malignant tumorspecimens and low staining in the benign tissue specimens. pErk1/2 showed low staining in the malignant tumor specimensand no staining in the benign tissue specimens. Total Erk1/2 showed high staining in both the malignant tumor specimensand benign tissue specimens. The differences in the mean staining scores for the malignant versus benign tissue specimensfor pErk1/2 and total Erk1/2 are not statistically significant (p=0.0726 and p=0.7054, respectively).Conclusion Thus, in conclusion, EGFR expression has been confirmed to be limited to non-existent in EAC and thus its useas a clinically active target is limited at best. Prior to the use of these expensive anti-EGFR therapies, confirmation ofoverexpression should be verified.

Keywords Esophageal . Adenocarcinoma . Epidermalgrowth factor

Introduction

Incidences of esophageal adenocarcinoma, the most prev-alent form of esophageal cancer in the USA, have beensteadily increasing over the past decades. The chroniccondition known as gastroesophageal reflux disease develops

from repeated exposure of the esophagus to bile acids fromthe stomach that can cause significant damage to theesophageal mucosa over time.1 Through this process, bileacids are believed to play a role in the carcinogenictransformation of normal esophageal epithelium to thepremalignant condition known as Barrett’s esophagus andeventually to esophageal adenocarcinoma by causing achronic state of oxidative stress.2

Epidermal growth factor receptor (EGFR) is a trans-membrane, tyrosine kinase receptor that plays a crucial rolein cellular signal transduction to initiate processes such ascellular proliferation, migration, invasion, growth, andsurvival.6Overexpression of EGFR has been linked to awide range of cancers, and some studies have indicated thatEGFR expression may be significantly up-regulated in theprogression of esophageal adenocarcinoma.3

N. Harper :Y. Li : R. Farmer :R. C. G. Martin (*)Division of Surgical Oncology, Department of Surgery,University of Louisville,315 E. Broadway,Louisville, KY 40202, USAe-mail: [email protected]

J Gastrointest Surg (2012) 16:946–955DOI 10.1007/s11605-011-1778-1

The Ras/MAPK pathway has been studied a great dealand the signaling events that occur via the pathway havebeen linked to the development and progression of variousforms of cancer (Fig. 1). The Ras/MAPK pathway isinitiated when the activated EGFR molecule recruits a Ras-activated guanine nucleotide exchange factor called SOS(son of sevenless) through an adaptor protein known asgrowth-factor-receptor-bound-2 (Grb2) to activate Ras bymediating the conversion of GDP to GTP. Activated Ras isthen able to interact with a serine/threonine kinase knownas Raf which is a member of the MAPK kinase kinase(MAPKKK) family. The interaction between Ras and Rafinduces a conformational change in Raf that increases itskinase activity. Therefore, Raf is able to readily phosphor-ylate, and thereby activate, members of the MAPK kinase(MAPKK) and MAPK families. Extracellular signal-regulated kinases 1 and 2 (Erk1/2) are members of theMAPK family that may become activated through thispathway. Phosphorylated Erk1/2 translocate from thecytoplasm to the nucleus of the cell where they inducechanges in the gene expression for cellular activities such asmitosis, embryogenesis, cell differentiation, cell movement,cell metabolism, and apoptosis. A number of studies havereported the overexpression of members of the MAPKfamily, including Erk1/2, as well as abnormal regulation ofRas/MAPK in a wide range of cancers and other diseases.4

Curcumin is an herbal supplement that has been shownto have certain anti-cancer properties in a number of studiesinvolving a wide range of cancers including esophagealadenocarcinoma.5Curcumin has also been shown to have

significant antioxidant and anti-inflammatory effects; there-fore, curcumin has been the focus of many studiesinvolving esophageal adenocarcinoma because oxidativestress has been so heavily implicated in the developmentand progression of that particular form of cancer.6Althoughmany studies have been conducted with curcumin, itseffectiveness at treating cancers such as esophagealadenocarcinoma is still being evaluated; however, curcuminwould certainly be a preferential form of treatmentcompared to chemotherapy regimens because of its abilityto function without causing cytotoxic effects in healthycells.7

The specific aims of this study were to assess the in vitrobaseline expression of EGFR in the Het-1a, BarT, andOE19 cell lines and to examine how bile acid treatment andcurcumin pretreatment affect the expression of thesemarkers in the same human esophageal cell lines. Inaddition, this study aimed to perform an immunohisto-chemical evaluation of EGFR, pErk1/2, and total Erk1/2expression in malignant tumor specimens and benign tissuespecimens resected from the esophagus of human patientsdiagnosed with adenocarcinoma of the esophagus.

Methods and Materials

Cell Culture and Treatment

The human non-tumorigenic esophageal squamous epi-thelial cell line (Het-1a) was purchased from ATCC

Fig. 1 EGFR receptor andactivation of the Ras pathwayfor cell proliferation andcell cycle progression

J Gastrointest Surg (2012) 16:946–955 947

(Manassas, VA, USA). The cell line was maintained inbronchial epithelial cell medium (Clonetics Corp.,Walkersville, MD, USA) that had been supplementedwith 100 U/mL of penicillin and streptomycin. The cellswere grown in standard 75-mL flasks and were stored at37°C in a humidified atmosphere of 5% CO2. Thehuman hTERT-immortalized non-neoplastic Barrett’sesophageal cell line (BarT) was a generous gift fromRhonda Souza, MD, University of Texas Southwestern.The cell line was maintained in keratinocyte-basalmedium-2 (Clonetics), grown in standard 75-mL flasks,and stored at 37°C in a humidified atmosphere of 5% CO2.The human well-differentiated esophageal adenocarcino-ma cell line (OE19) was a generous gift from Gerald C.O’Sullivan, MD. The cell line was maintained in RoswellPark Memorial Institute 1640 (RPMI-1640; ThermoScien-tific; South Logan, UT, USA) medium that had beensupplemented with 2 mML-glutamine and 10% FBS aswell as 100 U/mL of penicillin and streptomycin. The cellline was grown in standard 75-mL flasks and stored at 37°Cin a humidified atmosphere of 5% CO2.

When three flasks containing the BarT cell line hadreached at least 80–90% confluence, this cell line wastreated via the experimental protocol. Of the three flasks,one flask was designated as the “untreated control”. The oldcell medium was removed by vacuum and the untreatedcontrol received 7 mL of new cell medium at the beginningof the experiment. After 1 h, the cell medium was replacedwith 7 mL of new medium pipetted into the flask for theremaining 4 h of the experiment. The second flask wasdesignated as “bile-treated”. The bile Ttreated flaskreceived 7 mL of new cell medium at the beginning ofthe experiment. Our previous research has standardized themixture and dosage of bile acids and was used after the cellmedium was removed by vacuum after 1 h before astandardized bile acid mixture2consisting of 0.4-mMconcentration each of sodium taurocholate, sodium glyco-cholate, sodium choleate, and sodium deoxycholate dis-solved in 7 mL of cell medium was pipetted into the bileTreated flask for the remaining four hours of the experi-ment. The third flask was designated as “curcumin-pre-treated”. As per our previous research, curcumin wasdissolved in 7 μL of dimethyl sulfoxide (DMSO) at aconcentration of 25 mM before the DMSO containing theCurcumin was diluted in 6.993 mL of cell medium (finalcurcumin concentration=25 μM). This 25 μM curcumin incell medium and DMSO was then pipetted into thecurcumin-pretreated flask for 1 h.2,5At the end of the hour,the 25-μM curcumin medium was removed by vacuum andthe standardized bile acid mixture consisting of 0.4 mMeach of sodium taurocholate, sodium glycocholate, sodiumcholeate, and sodium deoxycholate dissolved in 7 mL ofcell medium pipetted into the curcumin-pretreated flask for

the remaining 4 h of the experiment. These procedures werealso used for the treatment of the Het-1a and OE19 celllines.

Protein Extraction

At the end of the treatment protocol, the flasks were washedthree times with 5 mL of ice-cold phosphate-buffered saline(PBS). After the third PBS wash, 0.8 mL of ice-cold lysisbuffer [20 mmol/L HEPES 9 (pH 7.4), 10 mmol/L NaCl,1.5 mmol/L MgCl2, 20% glycerol, 0.1% Triton X-100,1 mmol/L DTT] with 8 μL of a 100× protease inhibitor(P.I.) cocktail was added to each flask and a cell scraperwas used to remove all of the cells from the flask surfaceinto the whole lysate. The whole lysates were transferred toclean, labeled, 1.5-mL microcentrifuge tubes and stored onice for 30 min. The same procedures were used to isolatethe whole lysates for each flask of all three cell lines.

After the whole lysates had been allowed to incubate onice for 30 min, additional P.I. (equal to initial volume addedto lysis buffer) was added to each whole lysate. The wholelysates were allowed to incubate on ice for an additional30 min before the Fisher Sonic Dismembrator Model 150was utilized on each whole lysate to dislodge membrane-bound proteins and free nuclear proteins from within thenucleus by sonicating each sample five times for 3 s. Thewhole lysates were then centrifuged at 14,000 rpm for15 min at 4°C. After centrifugation, the supernatant (celllysate) was transferred into clean, labeled, 1.5-mL micro-centrifuge tubes. The cell lysates were then stored at −80°Cfor future use.

Western Blot Analysis of EGFR

The protein concentration of each of the cell lysate sampleswas determined using the BioRad Protein Assay DyeReagent on 96-well plates. The ThermoScientific MultiskanMCC340 Plate Reader was used to measure absorbance at620 nm so that the concentrations could be calculated.Approximately 40 μg of protein from the BarT and OE19cell lysates and 25 μg of protein from the Het-1a celllysates (with a constant volume of 50 μL per well) were runin triplicate on 10% SDS-PAGE gels at 125 V forapproximately 2 h. After a gel had finished running, theproteins were transferred to a polyvinylidene fluoridemembrane at 15 V overnight. After the transfer wascompleted, the membrane was rinsed with 0.1% Tween 20in Tris-buffered saline (TBST) before being blocked with5% non-fat dry milk in TBST for 1 h at room temperature.After the membrane had been blocked, the membrane wascarefully cut horizontally into three pieces based on thesizes of the proteins being analyzed. Each piece of themembrane was then incubated overnight at 4°C in 3% non-

948 J Gastrointest Surg (2012) 16:946–955

fat dry milk in TBST with one of the three utilized primaryantibodies (1:1,000 concentration). The three primary anti-bodies used for the experiment were the anti-EGFRmonoclonal antibody (175 kDa; Santa Cruz Biotechnology;Santa Cruz, CA, USA), anti-phospho-MAP kinase1/2polyclonal antibody (42 and 44 kDa; Millipore; Bellerica,MA, USA), and anti-MnSOD (manganese superoxidedismutase) polyclonal antibody (24 kDa; Millipore) whichwas used as a positive control. After incubation with theprimary antibody, the membranes were washed three timeswith TBST for 10 min before being incubated for 1 h atroom temperature in 3% non-fat dry milk in TBST with thecorresponding peroxidase-conjugated affinipure donkeyanti-rabbit (ImmunoResearch) or stabilized peroxidase-conjugated goat anti-mouse (ThermoScientific) secondaryantibody (1:2,000 concentration). The membranes wereonce again washed three times with TBST for 10 minbefore the Pierce Fast Western Blot ECL Kit (Thermo-Scientific) was used on the membranes so that interactionsbetween the primary and secondary antibodies could bedetected by developing CL-Xposure Film (ThermoScientific)that had been exposed to the membrane.

Statistical Analyses—Western Blot

Student’s t-tests assuming unequal variance were per-formed. The results are expressed as mean values ±standard deviation. Comparisons were made between thelevels of MnSOD expression for each treatment regimen ofall three human esophageal cell lines by analysis ofvariance. P-values of less than 0.05 were considered asstatistically significant.

Immunohistochemical Assay for EGFR, pErk1/2,and total Erk1/2

Human patients diagnosed with invasive adenocarcino-ma of the esophagus consented to having tissue speci-mens extracted and utilized for histological study.Benign and malignant esophageal tissue specimens weresurgically resected from the esophagus of the patientsand were then fixed in 10% buffered formalin for 24 hand transferred to 80% ethanol. The formalin-fixedesophageal tissues were embedded in paraffin. Serialsections of 5 μm were mounted onto glass slides forhistopathological and immunohistochemical analysis.Hematoxylin and eosin-stained slides were prepared foreach tissue specimen.

EGFR, pErk1/2, and total Erk1/2 protein expressionswere determined using an immunohistochemical assay.Staining was carried out on paraffin-embedded tissuesusing the DAKO EnVision™ + System Kit (DAKOCorporation, Carpinteria, CA, USA). In brief, the sections

were deparaffinized and hydrated. The slides werewashed with TRIS-buffer, and peroxidase blocking wasperformed for 5 min. After rewashing, the anti-phospho-MAP kinase1/2 (pErk1/2) and anti-MAP kinase1/2 (totalErk1/2) rabbit polyclonal antibodies (1:100) (MilliporeInc., Bellerica, MA, USA) and anti-EGFR mousemonoclonal antibody (1:100) (Santa Cruz BiotechnologyInc, CA, USA) were applied for 30 min to separate theslides. The slides were incubated with labeled polymerfor 30 min at room temperature. The substrate,diaminobenzidine, was added as a visualizing reagentbefore the slides were counterstained with hematoxylinor methyl green. A negative control was included whenanalyzing each protein marker.

Cytomorphology

Evaluation of distinct cytomorphological features per-formed microscopically at high-power fields (×40 magnifi-cation) was used to differentiate normal esophagealsquamous epithelial cells from malignant esophagealadenocarcinoma cells. The normal esophageal tissue ischaracterized by non-keratinized stratified squamous epi-thelium with cells arranged in a single layer with their longaxes oriented toward the center of the lumen. Themalignant esophageal tissue is characterized by irregularlyshaped cells with enlarged nuclei arranged in multiple celllayers. The malignant and benign tissue specimens weredistinguished from one for the purposes of staining basedon these cellular classifications.

Immunohistochemistry Scoring

Evaluation of immunohistochemical staining was per-formed microscopically at high-power fields (×40magnification) on the EGFR, pErk1/2, and total Erk1/2staining sections. Membranous immunostaining wasscored for EGFR and nuclear immunostaining wasscored for pErk1/2. Nuclear and cytoplasmic immunos-taining was scored for total Erk1/2. For each marker,eight representative regions of each stained section (atleast 100 cells) were scored. All three markers werescored as 0 (<1% of cells stained), 1 (1–25%), 2 (26–50%), or3 (>50%).

Statistical Analyses—Immunohistochemistry

Mann–Whitney U-test was performed, and the results areexpressed as median values with interquartile ranges.Comparisons were made between the malignant and benignsections stained with the same marker by analysis ofvariance. P-values less than 0.05 were considered asstatistically significant.


Results

In Vitro Analysis of EGFR in the Het-1a, BarT,and OE19 Cell Lines

The in vitro studies in the Het-1a, BarT, and OE19 celllines failed to show any measurable expression ofEGFR via Western blot techniques. Figure 2 shows nobanding patterns in the 175-kDa region for the HET-1Aand BAR-T cells, with minimal banding for the OE-19cells, which is the predicted molecular weight of EGFR.The experiments were repeated five times for each cellline without obtaining any measurable levels of EGFRexpression. The validity of the EGFR primary antibodyused in the Western blot studies was confirmed by testingthe antibody using cell lysates from a high protein-expressing hepatocellular carcinoma cell line known asHepG2. In addition, the marker serving as the positivecontrol for the study, MnSOD, showed expression in eachcell line for all three treatment regimens at approximately24 kDa. Thus, the Western blot techniques used in thisstudy were confirmed. Therefore, this study indicates thatEGFR is not expressed at measurable levels in the Het-1a,BarT, and OE19 cell lines. In addition, the standardizedbile acid treatment and the curcumin pretreatment prior tothe standardized bile acid treatment failed to induce theexpression of EGFR in any of the three human esophagealcell lines as well.

In order to confirm the activity of each cell line, pre-treatment expression evaluation was performed (Fig. 3). Foreach treatment regimen, the OE19 cell line consistently

shows the highest expression of MnSOD while the BarTcell line consistently shows the lowest expression ofMnSOD. For the baseline expression of MnSOD, thereare statistically significant differences between the expres-sion of the protein marker in the Het-1a cell line and theBarT cell line (p=0.05) as well as between the expression inthe OE19 cell line and the BarT cell line (p=0.002). TheBarT and OE19 cell lines treated with a standardized 0.4-mM mixture of bile acids showed significantly differentlevels of MnSOD expression (p=0.001). In addition, therewere statistically significant differences in the expression ofMnSOD in the BarT and OE19 cell lines that underwentpretreatment with 25 μM curcumin prior to the standard-ized bile acid treatment (p=0.01).

Immunohistochemical Evaluation of EGFR, pErk1/2,and Total Erk1/2

Sixty samples matched for benign and malignantadenocarcinoma tumors from the same patients wereevaluated for EGFR expression. Figure 4 shows thehematoxylin and eosin staining of malignant and benignesophageal tissue specimens from two representativehuman patients diagnosed with adenocarcinoma of theesophagus. Figure 5a–c shows the immunohistochemicalstaining of malignant and benign esophageal tissue speci-mens from different human patients diagnosed withadenocarcinoma of the esophagus. The immunohisto-chemical staining of the malignant and benign tissuespecimens was examined microscopically and regions ofthe tissue specimens were scored as 0 (<1% of cells

Fig. 2 Western blot analysis,optical density, and opticaldensity ration of EGFR andErk1/2 expression in theHet-1A, BAR-T, and OE-19 celllines The GAPDH expression isused as a housekeeping geneexpression and the opticaldensity ration is expressed as aratio relative to GAPDHexpression. The results of theWestern blot show minimalEGFR in OE-19 cells withsimilar Erk1/2 expression in allthree cell lines. Optical densityof EGFR and Erk 1/2 ratiosdemonstrating only minimalEGFR expression


stained), 1 (1–25%), 2 (26–50%), or 3 (>50%). Figure 6shows the mean staining scores for the malignant andbenign tissue specimens for each individual proteinmarker of interest. EGFR showed moderate staining inthe malignant tumor specimens and low staining in thebenign tissue specimens. pErk1/2 showed low staining inthe malignant tumor specimens and no staining in thebenign tissue specimens. Total Erk1/2 showed high

staining in both the malignant tumor specimens andbenign tissue specimens. The difference in the meanstaining scores for the malignant versus benign tissuespecimens for EGFR is not statistically significant (p =0.2;Fig. 6). The differences in the mean staining scores for themalignant versus benign tissue specimens for pErk1/2 andtotal Erk1/2 are not statistically significant (p=0.07 andp=0.7, respectively).

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p = 0.0489p = 0.0013

p = 0.0021p = 0.0126

Fig. 3 Mean relative expression of MnSOD in the three humanesophageal cell lines for the three different treatment regimens. Theerror bars indicate the positive/negative standard deviations of themean of the triplicates. Brackets between two columns indicate a

statistically significant difference between the two data sets. The pvalues for significantly different data sets are displayed above thebrackets

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Fig. 4 Hematoxylin andeosin staining of malignant andbenign human esophagealadenocarcinoma tissuespecimens. Malignant tumorspecimens and benign tissuespecimens were surgicallyresected from the esophagusof patients diagnosed withadenocarcinoma of theesophagus at ×20 magnification.a, b Malignant tumor speci-mens. c, b Benign tissuespecimens


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Fig. 5 a Immunohistochemicalevaluation of EGFR expressionin malignant and benign humanesophageal adenocarcinomatissue specimens. Tissues werecounter-stained with hematoxy-lin. Images shown at ×20 mag-nification. Cutout regions at ×40magnification contain arrowsindicating immunohistochemicalstaining. A and B Malignanttumor specimens. C and DBenign tissue specimens.b Immunohistochemical evalua-tion of pErk1/2 expression inmalignant and benign humanesophageal adenocarcinomatissue specimens. Tissues werecounter-stained with methylgreen. Images shown at ×20magnification. Cutout regionsat ×40 magnification containarrows indicating immunohisto-chemical staining. A and BMalignant tumor specimens. Cand D Benign tissue specimens.c Immunohistochemical evalua-tion of total Erk1/2 expressionin malignant and benign humanesophageal adenocarcinoma tis-sue specimens. Tissues werecounter-stained with hematoxy-lin. Images shown at×20magnification. Cutout regionsat×40 magnification containarrows indicating immunohisto-chemical staining. A andB Malignant tumor specimens.C and D Benign tissuespecimens


Discussion

This study sets out to determine the baseline in vitroexpression of EGFR in the Het-1a, BarT, and OE19 celllines as well as to examine how bile acid treatment andcurcumin pretreatment affect the expression of the markerin these same cell lines. The results of the in vitroexperiments performed on the Het-1a, BarT, and OE19 celllines in this study suggest that these cells do not expressEGFR at experimentally measurable levels because nobaseline expression was observed. The standardized bileacid treatment failed to induce the expression of EGFR inany of the human esophageal cell lines as well. In addition,because no measurable expression of EGFR was observedfor any of the cell lines, the effects of curcuminpretreatment on the expression of the receptor could notbe analyzed.

Although this study produced null results for EGFRexpression, the expression of MnSOD, which served as apositive control, in the three cell lines could be analyzedamong the different treatment regimens. As a member ofthe superoxide dismutase family, MnSOD is responsible forthe catalytic conversion of superoxide oxygen radical towater and oxygen.2Therefore, MnSOD is an extremelyimportant enzyme during times of oxidative stress such as

those present in the esophagus during the course of chronicgastroesophageal reflux.2Previous studies have shown thatthe progression of reflux esophagitis to Barrett’s esophagusis characterized by a significant decrease in MnSODexpression.2These studies have also shown that an adaptiveincrease in MnSOD expression occurs in the progression ofBarrett’s esophagus to esophageal adenocarcinoma. Otherstudies that have tested the efficacy of curcumin at treatingBarrett’s esophagus and esophageal adenocarcinoma haveshown that the herbal treatment is able to preserve MnSODexpression in Barrett’s esophagus.5The results from theWestern blots for MnSOD expression in each cell lineappear to be in line with the results from previous studies.Expression of MnSOD for the BarT cell line following bileacid treatment was significantly lower (p=0.044) than theexpression in the OE19 and Het-1a cell lines. Also, theexpression of MnSOD in the OE19 cell line followingcurcumin pretreatment and bile acid treatment was signif-icantly higher (p=0.008) than the expression in the Het-1aand BarT cell lines. Therefore, the results of the MnSODexpression indicate that the bile acid treatment wassuccessful in simulating the effects of gastroesophagealreflux disease that have been previously characterized whilethe curcumin pretreatment was successful in combating theoxidative effects of the bile as would be expected.

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p = 0.2521

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p = 0.7054

Fig. 6 Immunohistochemical evaluation of EGFR, pErk1/2, and totalErk1/2 expression in malignant and benign human esophagealadenocarcinoma tissue specimens. Malignant tumor specimens andbenign tissue specimens were surgically resected from the esophagusof patients diagnosed with adenocarcinoma of the esophagus.

Immunohistochemical staining was scored via the standard 0–3 scaleat ×40 magnification. The error bars indicate the positive/negativestandard deviations of the mean staining scores. The p values for theexpression of each marker in the malignant versus benign tissuespecimens are displayed above the brackets between data sets.


The up-regulation of EGFR has been significantly linkedto various forms of cancer; the lack of EGFR expression inthe OE19 cell line indicates that this protein may not besignificantly involved in the development and progressionof esophageal adenocarcinoma. In the same way, the lack ofobservable EGFR expression in the BarT cell lines suggeststhat EGFR may not be involved in the development of thepremalignant condition of Barrett’s esophagus. In addition,the inability of bile acid exposure to induce EGFRexpression in any of the cell lines would suggest thatEGFR is not an expressible gene and that its use as atreatment marker should be questioned.

In addition to evaluating the in vitro expression of EGFRin the three human esophageal cell lines, this study aimedto examine the immunohistochemical expression of EGFRas well as pErk1/2 and total Erk1/2 in malignant and benigntissue specimens resected from the esophagus of patientsdiagnosed with adenocarcinoma of the esophagus. Immu-nohistochemical staining of the tissue specimens for thethree proteins of interest showed no statistically significantdifferences between the expression of any of the threemarkers among the malignant and benign sections. Al-though the tissue specimens did show some measurableexpression of EGFR, in contrast to the OE19 cell linewhich showed no observable EGFR expression in the invitro experiments, the difference in the expression of thereceptor between the malignant and benign tissue speci-mens was not statistically significant (p=0.2521). pErk1/2was expressed extremely low in both the malignant andbenign specimens while total Erk1/2 was expressed quitehigh in both types of tissue specimen. In addition, neitherpErk1/2 nor total Erk1/2 showed significantly differentlevels of expression in the malignant versus benign speci-mens (p=0.0726 and p=0.7054, respectively). Therefore,the results of the immunohistochemical evaluation appearto be in similar agreement with the results for the OE19 cellline in the in vitro studies that show limited to noexpression of EGFR.

Although EGFR overexpression has been associatedwith malignant transformation in various forms of cancerincluding breast, colon, kidney, lung, and prostate cancers(Amgen Inc., 2010), there is little pre-clinical evidence tosuggest that EGFR may play a significant role in thedevelopment of adenocarcinoma of esophagus. Neverthe-less, there are ongoing clinical trials being conducted toinvestigate the efficacy of anti-EGFR therapeutics incombination with chemoradiotherapy in human patientsdiagnosed with adenocarcinoma of the esophagus. TheRadiation Therapy Oncology Group (RTOG) 0436 phaseIII clinical trial is evaluating the addition of cetuximab to achemoradiotherapy regimen that has been shown to beclinically effective in patients with either adenocarcinomaor squamous cell carcinoma of the esophagus who do not

undergo surgery. This treatment regimen includes cisplatinand paclitaxel in concurrence with daily radiation therapy.The clinical investigators’ rationale for adding an anti-EGFR medication to a chemoradiotherapy regimen forinoperable esophageal cancers is that EGFR signaling hasbeen shown to play a significant role in multiple tumori-genic processes such as cell cycle progression, protectionfrom apoptosis, metastasis, and angiogenesis in variousforms of cancer, and some studies have shown that with theexpression of EGFR is a wide range of 50–80% of cancersof the esophagus.8However, as described by Mukaida et al.,all of the 100 cancers evaluated for EGFR expression weresquamous cell carcinoma.8In esophageal cancer, overex-pression of EGFR by immunohistochemistry has beenwidely reported to occur in up to 80% of patients withesophageal cancer, but again a majority of these weresquamous cell carcinoma.9However, fuorescence in situhybridization analysis shows amplification in only 8–30%of cases.10–12Most importantly, all of these studies reportinghigh EGFR expression were published in 2003 or earlierand no further studies have demonstrated any significantoverexpression of EGFR.

An additional ongoing phase II clinical trial beingconducted is examining the effectiveness of panitumumabin combination with cisplatin and docetaxel in concurrencewith radiation therapy for the treatment of esophagealcarcinomas (Reed et al., 2010). Panitumumab is anotheranti-EGFR monoclonal antibody that is thought to bepotentially more effective than cetuximab at blocking thebinding of EGFR to its ligands (Amgen Inc., 2010). In non-clinical monotherapy studies, panitumumab has beenshown to inhibit tumor growth of breast, renal, pancreatic,head and neck, prostate, ovarian, and non-small cell lungcarcinomas that showed overexpression of EGFR (AmgenInc., 2010) but, again, has not been shown to be aneffective therapy in esophageal adenocarcinoma.

Although both cetuximab and panitumumab have goodtrack records of success in the combination treatment ofcancers that overexpress EGFR, there is currently insuffi-cient pre-clinical or clinical evidence to suggest that EGFRis significantly overexpressed in esophageal adenocarcino-ma to predict the effect these medications will have on thetreatment of the disease. Even though some studies havefound significant in vitro and immunohistochemical ex-pression of the receptor in esophageal cancers, these studiesoften either consider adenocarcinoma and squamous cellcarcinoma together when estimating rates of overexpressionor have not been found to be reproducible in otherlaboratories. Therefore, estimations that 50–80% of esoph-ageal cancers may be skewed by greater incidences ofoverexpression in squamous cell carcinoma of the esoph-agus as opposed to adenocarcinoma of the esophagus or theestimations could be significantly overestimated. Although


cetuximab and panitumumab are biologic agents that arepreferred forms of treatment compared to cytotoxic che-motherapy drugs, these medications carry moderate risks ofseverely adverse reactions. In addition, both cetuximab andpanitumumab are very expensive medications. The treat-ment of a single patient with cetuximab or panitumumab inone of the ongoing clinical trials for a single month coststens of thousands of dollars. For these reasons, it isnecessary to gain a better understanding of the role ofEGFR and its relative expression in esophageal cancers,especially adenocarcinoma of the esophagus, through moreexhaustive pre-clinical studies before spending vastamounts of money and subjecting patients to unnecessaryrisks in clinical trials investigating the efficacy of anti-EGFR medications.

Even though the results of our in vitro and immunohis-tochemical studies suggest that EGFR may not be overex-pressed in esophageal adenocarcinoma, we acknowledgethat the degree of involvement of EGFR in esophagealadenocarcinoma is probably a genetically variable. Thiscould explain the discrepancies between our results andthose from previous studies regarding EGFR expression inhuman esophageal adenocarcinoma cell lines and malignanttumor specimens. Although there are likely to be humanesophageal adenocarcinoma patients for whom EGFR isoverexpressed and thus it plays a significant role incarcinogenesis, the actual percentage of such individualsamong all esophageal adenocarcinoma patients is notknown. Therefore, we propose that genetic testing be usedto evaluate the expression of EGFR at the primary cancersite of patients diagnosed with adenocarcinoma of theesophagus. Once the role of EGFR in the disease of anindividual has been evaluated, it can be determined as towhether the patient should receive the expensive anti-EGFRmedication in combination with traditional chemoradiother-apy treatment. For this to be accomplished, prompt andinexpensive methods of genetic testing for this markerwould need to be developed.

The limitations of this study are the limited number ofcell lines that were evaluated for EGFR expression. Giventhe limited number of cell lines available, these cell lineresults could be biased by the limited number evaluated.

We believe that we have overcome this bias with the large60-patient human esophageal tissue evaluation confirmingthese results of limited EGFR expression.

Thus, in conclusion, EGFR expression has been con-firmed to be limited to non-existent in EAC and thus its useas a clinically active target is limited at best.

References

1. Chen X, Yang CS. Esophageal adenocarcinoma: a review andperspectives on the mechanism of carcinogenesis and chemo-prevention. Carcinogenesis 2001; 22:1119–1129.

2. Schiffman SC, Li Y, Xiao D et al. The resistance ofesophageal adenocarcinoma to bile salt insult is associatedwith manganese superoxide dismutase expression. J Surg Res2010. doi:10.1016/j.jss.2010.04.038.

3. Feagins LA, Souza RF. Molecular targets for treatment of Barrett'sesophagus. Dis Esophagus 2005; 18:75–86.

4. Mebratu Y, Tesfaigzi Y. How ERK1/2 activation controls cellproliferation and cell death: is subcellular localization the answer?Cell Cycle 2009; 8:1168–1175.

5. Li Y, Wo JM, Liu Q et al. Chemoprotective effects of Curcumaaromatica on esophageal carcinogenesis. Ann Surg Oncol 2009;16:515–523.

6. O'Sullivan-Coyne G, O'Sullivan GC, O'Donovan TR et al.Curcumin induces apoptosis-independent death in oesophagealcancer cells. Br J Cancer 2009; 101:1585–1595.

7. Hartojo W, Silvers AL, Thomas DG et al. Curcumin promotesapoptosis, increases chemosensitivity, and inhibits nuclear factorkappaB in esophageal adenocarcinoma. Transl Oncol 2010; 3:99–108.

8. Mukaida H, Toi M, Hirai T et al. Clinical significance of theexpression of epidermal growth factor and its receptor inesophageal cancer. Cancer 1991; 68:142–148.

9. Takaoka M, Harada H, Andl CD et al. Epidermal growth factorreceptor regulates aberrant expression of insulin-like growthfactor-binding protein 3. Cancer Res 2004; 64:7711–7723.

10. al-Kasspooles M, Moore JH, Orringer MB et al. Amplificationand over-expression of the EGFR and erbB-2 genes in humanesophageal adenocarcinomas. Int J Cancer 1993; 54:213–219

11. Rygiel AM, Milano F, Ten Kate FJ et al. Gains and amplificationsof c-myc, EGFR, and 20.q13 loci in the no dysplasia–dysplasia–adenocarcinoma sequence of Barrett's esophagus. Cancer Epide-miol Biomarkers Prev 2008; 17:1380–1385.

12. Miller CT, Moy JR, Lin L et al. Gene amplification in esophagealadenocarcinomas and Barrett's with high-grade dysplasia. ClinCancer Res 2003; 9:4819–4825.


http://dx.doi.org/10.1016/j.jss.2010.04.038

epidermal growth factor expression in esophageal adenocarcinoma: a clinically relevant target?

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