ar in cholesteatoma

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The LaryngoscopeVC 2010 The American Laryngological,Rhinological and Otological Society, Inc.

Increased Amphiregulin Expression as a

Biomarker of Cholesteatoma Activity

MiMi P. Macias, PhD; Richard D. Gerkin, MD, MS; John D. Macias, MD

Objectives/Hypothesis: The purpose of thisstudy was to evaluate human surgical specimens forcholesteatoma-associated changes in amphiregulinexpression and determine potential relations to clini-cal disease variables. Amphiregulin, an epidermalgrowth factor receptor ligand, has functions in normal

epithelial proliferation and aberrant neoplastic cellgrowth and is proinflammatory (e.g., rheumatoid ar-thritis, fibrosis) and active in hyperproliferative cuta-neous conditions including psoriasis and wound heal-ing. These known amphiregulin activities and thecharacteristic epithelial expansion and bone erosionof cholesteatoma pathophysiology prompted testing ofthe hypothesis that amphiregulin expression levelsare altered in cholesteatoma and correlate to the dis-ease state.

Study Design: Prospective experimental study,cross-sectional analysis.

Methods: Relative changes in amphiregulingene expression were quantitated by real-timereverse-transcription polymerase chain reaction anal-

yses of cholesteatoma epithelium compared to unin-volved control tissues from patients postauricularand external auditory canal regions. Western immu-noblot assays were performed for qualitative evalua-tion of amphiregulin protein expression. The t testand Fisher exact test were used for analysis.

Results: A statistically significant increase inamphiregulin gene expression was associated withcholesteatoma specimens compared to uninvolvedpostauricular skin (PAS) and external auditory canal(EAC) skin, P .004 and P .002, respectively. Fromcomparisons of 60 sets of skin pairs, the mean ratio ofamphiregulin RNA expression for cholesteatoma/

PAS is 4.94 (standard error of the mean [SEM] 1.53, n 30) and for cholesteatoma/EAC is 7.70(SEM 1.57, n 30).

Conclusions: Amphiregulin is overexpressed inepithelial tissues of human cholesteatoma. Significantrelationships were identified between increased

amphiregulin expression levels and the extent of cho-lesteatoma migration and bone erosion. Our studyresults indicate amphiregulin is a potential biomarkerof early cholesteatoma disease processes.

Key Words: Cholesteatoma, hyperproliferative,migration, biomarker, epidermal growth factorreceptor, amphiregulin, real-time reverse-transcriptionpolymerase chain reaction.

Level of Evidence: 1b.Laryngoscope, 120:22582263, 2010

INTRODUCTIONCholesteatoma (CHLST) is a common, progressive

disease of the temporal bone that affects both childrenand adults. The invasive properties of the CHLST sac

(which is composed of hyperproliferative desquamating,

keratinizing squamous epithelium), frequent bone ero-

sion of the ossicular chain, and high rate of recurrence

following surgical removal are responsible for associated

hearing loss and other serious morbidity.1 However, clin-

ical progression varies greatly among individuals, with

some CHLSTs showing rapid growth and bone erosion

and others remaining indolent for years. Surgery repre-

sents the only interventional option, and multiple

operations are often required for eradication.2 Identify-

ing stimulatory signals that functionally drive the

expansion of human CHLST epithelium will help distin-

guish potential molecular targets for developingpreventive and nonsurgical intervention strategies.

Amphiregulin (AR) is a major autocrine growth fac-

tor for normal proliferation of human keratinocytes and

fibroblasts in many tissues including skin, colon, spleen,

breast, ovary, and T lymphocytes.3 However, AR also

increases the proliferation and invasiveness of various

neoplastic cell types4 and is upregulated in inflammatory

and hyperproliferative skin lesions such as psoriasis, ac-

tinic keratoses, and verrucae.5,6 The experimental

overexpression of AR in keratinocytes of transgenic mice

is sufficient to induce epidermal hyperproliferation and

From the Biomedical Research Program, The EAR Foundation ofArizona (M.P.M.); Scientific Services, Banner Health Research Institute(R.D.G.); and Macias Otology, P.C. (J.D.M.), Phoenix, Arizona, U.S.A.

Editors Note: This Manuscript was accepted for publication June29, 2010.

This research was supported by Banner Good Samaritan MedicalCenter, The Nathan Cummings Foundation with the support of Sheilaand Michael Zuieback, The Arizona Community Foundation, TheNational Organization for Hearing Research Foundation, and The EARFoundation of Arizona. The authors have no other funding, financialrelationships, or conflicts of interest to disclose.

Send correspondence to MiMi P. Macias, PhD, BiomedicalResearch Program, The EAR Foundation of Arizona, 668 North 44th St,Ste 300, Phoenix, AZ 85008. E-mail: [email protected]

DOI: 10.1002/lary.21142

Laryngoscope 120: November 2010 Macias et al.: Amphiregulin Expression in Cholesteatoma2258


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promote inflammatory skin responses.7 AR is a ligand of

the epidermal growth factor receptor (EGFR), a trans-

membrane signal transducing receptor that is a crucial

regulator of cellular proliferative, migratory, and differen-

tiation responses. Previous reports have shown human

CHLSTs express elevated levels of EGFR and family

member ErbB28 in the basal and suprabasal layers of the

diseased epithelium.9,10 The striking functional capacity

of AR to stimulate proliferation and invasiveness in othercutaneous systems, along with reports that the EGFR

receptors for AR11 are expressed in human CHLST, led us

to investigate whether AR expression is dysregulated in

CHLST epithelial lesions and potentially contributes to

the etiology and pathologic changes in the temporal bone.

MATERIALS AND METHODS

PatientsThirty-three patients undergoing surgery for CHLST

removal between May 2003 and September 2008 were

prospectively enrolled in this study after providing

informed consent for participation. Simple retractionpockets were excluded from the study. Protocols were

approved by Banner Health Institutional Review Board.

For the study, a skin specimen of approximately 15 mm3

was collected intraoperatively from the patients CHLST

and the remainder was sent to pathology for routine ex-

amination. In addition, two sources of normal epithelia,

postauricular skin (PAS) and external auditory canal

(EAC) from the vascular strip, were collected from the

subject to serve as uninvolved skin controls. Upon surgi-

cal removal, coded specimen sets were immediately

transferred to separate 1.5-mL tubes containing stabiliz-

ing solution (RNAlater; Ambion, Austin, TX). Before

processing, specimens were weighed and documented by

digital imaging using a KODAK EDAS 290 System (East-man Kodak Co., Rochester, NY). Prospectively, individual

clinical histories were compiled and coded for subsequent

correlative analysis with experimental gene expression

data. Catalogued data included patient age, sex, family

history of skin conditions, CHLST etiology (congenital,

acquired, initial, recurrent), migration landmarks, extent

of bone erosion, and presence or absence of infection.

RNA Preparation and Real-time QuantitativePolymerase Chain Reaction

Total RNA from each surgical specimen was iso-

lated and DNase-treated by using the Absolutely RNA

Miniprep Kit (Stratagene, La Jolla, CA). Each set ofpatient CHLST and control tissue specimens was batch

processed to maximize consistency in handling. Tissues

were homogenized on ice by using a rotor/stator 7-mm

rotating-blade homogenizer; concentration and purity of

isolated total RNA preparations were determined by

spectrophotometry (BioMate3; Thermo Spectronic, Roch-

ester, NY) measurements at 260 nm and 280 nm.

Quantitation and integrity were visually confirmed

when possible by 0.7% agarose/1X 3-(N-morpholino) pro-

panesulfonic acid gel electrophoretic separation and

ethidium bromide or SYBR Gold (Molecular Probes,

Eugene, OR) staining. cDNA was reverse transcribed

from 2 lg aliquots of total RNA in a 20 lL reaction vol-

ume at 42C for 90 minutes by using SuperScript II

(Invitrogen, Carlsbad, CA) according to manufacturers

conditions, 500 lM dNTP mix (Eppendorf, Hamburg,

Germany), 1 U/lL RNase inhibitor (Invitrogen, Carls-

bad, CA), and 1 lM modified oligo-dT primer, dT-ACP1

primer: 50-CTGTGAATGCTGCGACTACGATXXXXX(T)18-

30 (Seegene, Rockville, MD).Real-time quantitative reverse-transcriptionpoly-

merase chain reaction (QRT-PCR) was run for each set

of patient cDNA samples PAS, EAC, and CHLST, con-

currently and in triplicate in a Stratagene Mx3000P

with analysis software MxPro Version 4.0.1 (Stratagene,

La Jolla, CA), for both AR and the internal normalizer

control, glyceraldehyde-3-phosphate dehydrogenase

(GAPDH). GAPDH demonstrated the most stable expres-

sion in these tissues when tested against alternative

control gene options including beta-actin, 18S RNA, and

cyclophilin (data not shown). Each 25 lL reaction

included 30 ng cDNA, Brilliant SYBR Green QPCR Mas-

ter Mix (Stratagene, La Jolla, CA), 30 nM ROX dye

(United States Biochemical, Cleveland, OH), and the AR

or GAPDH primer pair at 150 nM each primer:

upstream 50-GCTTAGAA GACAATACGTCAGG-30, down-

stream 50-GGGTCCATTG TCTTATGATCCAC-30 (AR); or

upstream 50-TCTGACTTCAACAGCGACAC-3 0, down-

stream 50-TGTTGCTGTAGC CAAATTCG-30 (GAPDH).

Primers were designed using Amplify software (W.

Engels, Madison, WI) to anneal to exon sequences and

span genomic intron sequences. Relative amplification

efficiencies of the primer pairs were optimized to be sim-

ilar, and dissociation curves and no template control

reactions confirmed specificity of the predicted QRT-PCR

products and absence of DNA contamination. An addi-

tional level of correction was introduced by includingpassive fluorescent reference dye, 30 nM ROX, to nor-

malize potential well-to-well differences. Cycling

conditions were as follows: 94C, 10 minutes; (94C, 30

seconds; 60C, 1 minute; 72C, 1 minute) 40 cycles.

GAPDH-normalized AR expression is presented as a

fold-change by the ratios CHLST/PAS or CHLST/EAC,

and calibrator AR ratio values for PAS or EAC are

defined as 1.0. Error bars are derived from threshold

cycles, fluorescence measurements at each cycle, and

software-based estimates of imprecision of the normal-

izer. Twenty-seven patients had values for CHLST AR

expression relative to both PAS and EAC; three had val-

ues for only CHLST/PAS, and three had values for only

CHLST/EAC.

Protein Preparation and Anti-AR Western BlotsWhen both RNA and protein were isolated from a

set of surgical specimens, each biopsy was first divided

into two portions, the RNA portion extracted as

described previously and the protein portion processed

by using the PARIS lysate preparation system (Ambion,

Austin, TX). Concentrations were determined by using

Bradford reagent (Sigma-Aldrich, St. Louis, MO), 595

nm absorbance readings and comparison to a standard



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curve prepared in parallel. Protein samples (15lg/lane)

were loaded in duplicate and electrophoretically sepa-

rated through discontinuous Laemmli 4%/12% sodium

dodecyl sulfate polyacrylamide gels for transfer to What-

man Protran BA85, 0.45 lm, nitrocellulose membrane

(Sigma-Aldrich, St. Louis, MO). Endogenous cellular AR

is synthesized as a 252 amino acid transmembrane pre-

cursor protein that is glycosylated and cleaved by matrix

metalloproteinase to release a mature 18 kDa polypep-tide.6,12 Positive control sample lanes of recombinant

human AR (rhAR) contained 2.5 ng/lane of a 98 amino

acid, mature form AR (R&D Systems, Minneapolis, MN),

and a 50-fold excess (125 ng/lane) of carrier protein, bo-

vine serum albumin (BSA). Two different protein

molecular weight marker mixes were used for more

accurate sizing: Pre-stained Blue Ranger (Pierce/Thermo

Scientific, Rockford, IL) and Sigma Wide (Sigma-Aldrich,

St. Louis, MO). Protein gels were electrotransferred by

using the Genie Blotter (Idea Scientific, Minneapolis,

MN) at 6 V for 3 hours with transfer buffer 25 mM Tris/

192 mM Glycine/20% methanol (pH 8.3). Duplicate blots

were stained with MemCode Reversible (MCR) (Pierce/

Thermo Scientific, Rockford, IL) to assess transfer effi-

ciency and were documented by using the KODAK

EDAS 290 System and were then destained before West-

ern blotting steps. Blocking and antibody incubation

buffer was Starting Block T20 (Pierce/Thermo Scientific,

Rockford, IL). Incubations and washes were performed

at room temperature with constant agitation on a rotat-

ing platform (Davis R&D, Carlsbad, NM). Two sources of

primary antibodies were pooled for anti-AR Western

blots: AF262 goat antihuman AR polyclonal sera (R&D

Systems, Minneapolis, MN), 1:1,000 working dilution,

and N20 goat antihuman AR polyclonal sera (Santa

Cruz Biotechnology, Santa Cruz, CA), 1:200 working

dilution. Negative control primary antibody was normalgoat serum (R&D Systems, Minneapolis, MN). Second-

ary antibody-enzyme conjugate was donkey antigoat

IgG-alkaline phosphatase, 1:5,000 working dilution

(Santa Cruz Biotechnology, Santa Cruz, CA). Color sub-

strate for detection was 1-Step NBT/BCIP (Pierce/

Thermo Scientific, Rockford, IL) with 1 mM levamisole

added fresh to eliminate endogenous phosphatase activ-

ity. Antibody incubation steps were for 1 hour, and color

substrate development was for 20 minutes. Western

results were recorded with the KODAK EDAS 290.

Statistical AnalysisDescriptive statistics were used. Continuous varia-

bles were reported as means and the standard error of

the mean (SEM). Medians were also reported. Categori-

cal variables were reported as percentages. Continuous

variables were analyzed using t tests, or Mann-Whitney

tests if the data were not normal. The Fisher exact test

was used to analyze categorical variables. All specimens

were run in triplicate. Standard deviations (SD) and

coefficients of variation (SD/mean) were calculated for

each sample. A two-tailed P < .05 was considered

significant.

RESULTSOf the 33 patients enrolled in the study, 20 (60.6%)

were males and 13 (39.4%) were females. The mean age

was 39.1 years, and ages ranged from 7 to 72 years. At

the time of surgery, 16 of the 33 patients (48.5%) were

presenting with recurrent disease. Five of these 16 had

also undergone contralateral procedures. Of the other 17

patients with initial, acquired CHLST, three had previ-

ously undergone surgical removal of CHLST in their

contralateral ear. Nineteen of the 33 enrolled patients

had infections at the time of surgery. Thirty of the 33

patients had PAS measured, and 27 of these 30 as well

as the remaining three subjects had EAC measured.

CHLST/PAS comparisons showed 22 of 30 CHLST

samples (73.3%) had AR ratios greater than 1.0 (P

.004), and the mean AR expression ratio for CHLST/PAS

was 4.94 (SEM 1.53, n 30, median 2.87). Twenty-

four of the 30 paired CHLST/EAC samples (80.0%) had

AR ratios greater than 1.0 (P .002), and the mean AR

expression ratio was 7.70 (SEM 1.57, n 30, median

5.08). Interassay variation and reproducibility of tech-

nical replicates was measured for all 60 sample pairs,and the mean coefficient of variation was 6.94%. These

data are displayed in the comparative quantity charts in

Figure 1.

Protein lysates were prepared from patients PAS,

EAC, and CHLST specimens for anti-AR Western immu-

noblots to evaluate endogenous AR expression at the

protein level. Representative duplicate membranes pre-

pared in parallel are shown with lysate patterns

visualized by transient, nonspecific stain (left images in

Fig. 2A and 2B). The membranes were then destained

and incubated with primary antibodies, either goat anti-

ARspecific antibodies or normal goat serumnegative

control. Colorimetric detection reveals AR-specific anti-

body staining of the 18 kDa AR product in CHLST

lysate only (Fig. 2A, right image). Of the six patients

tested with Western blots, four patients specimens

exhibited AR-specific protein staining identical to the

pattern shown in Fig. 2A. Two patients tested did not

show endogenous AR staining of any tissue. The positive

control lane containing 2.5 ng rhAR protein was as

expected, only stained by anti-AR specific polyclonal

sera (Fig. 2A) and not normal goat serum (Fig. 2B). Neg-

ative control carrier protein BSA (66 kDa), in 50-fold

excess of rhAR, was weakly visible by MCR staining but

was not recognized by antibodies on either Western blot

(Fig. 2A or 2B), further confirming anti-AR antibody

specificity. The negative control normal goat serumappeared to react weakly with a few unknown human

products, one of approximately 25 kDa in PAS and EAC

lysates (Fig. 2B) that were not consistently seen in other

patient lysates and one comigrating with BSA at 66

kDa, which may be reactivity or passive reagent trap-

ping (see staining intensity at 66 kDa in all lysate lanes,

left images in Fig. 2A and 2B).

Classification of the clinical severity of patient pa-

thology was based on evaluation of both the extent of

CHLST epithelia migration and the extent of CHLST-

associated bone erosion. For both classifications,



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category 2 CHLSTs are defined to be more pathologically

aggressive than category 1. Migration of eight of the 33

CHLSTs was classified as category 1 and limited to the

middle ear and/or attic. Migration of the other 25 speci-

mens was classified as category 2 and extended into the

antrum and/or mastoid. For bone erosion, eight of 33

CHLSTs were classified as category 1 and exhibited no

bone destruction or erosion of one or more ossicles,

whereas the 25 category 2 CHLSTs were classified by

erosion of the scutum and/or mastoid.

AR overexpression is clearly associated with diseased

CHLST epithelia. Additional tests were performed to

examine relationships between levels of AR gene expres-

sion and patients clinical information. Analyses

demonstrated an inverse relationship in which higher lev-

els of AR expression were found early in CHLST

development (category 1 CHLSTs). The CHLST/EAC AR

ratio had a statistically significant, inverse relationship to

both extent of CHLST migration (P .007) and degree ofCHLST-associated bone erosion (P .011) (Table I). The

CHLST/PAS AR ratio also demonstrated significant,

inverse relation to CHLST-associated bone erosion (P

.032) but not to migration, as mean CHLST/PAS AR ratios

were elevated similarly in both migration category 1 and

2 lesions (Table I). Neither CHLST/PAS nor CHLST/EAC

ratio was related to infection or recurrent disease.

Recurrence of disease correlated significantly with

the presence of infection (P .006), but not with the

extent of migration or bone erosion. Recurrence was also

related to age, with younger patients more likely to ex-

perience recurrence. The mean age in those with

recurrence was 31.3 years, and the mean age in those

without recurrence was 45.8 years (P .034). Gender

was significantly associated with migration in 18 of 20

males in our study showing more aggressive disease (P

.009) as compared with seven of 13 females.

DISCUSSIONCHLST has long been recognized as a serious condi-

tion of the temporal bone, with hearing loss the frequent

outcome of its propensity for rapid growth and bone

destruction. Better understanding of molecular factors

regulating disease progression and the biomarkers indic-

ative of CHLST activity can help identify potential

targets for the long-term objectives of developing nonsur-gical and preventive treatment strategies for CHLST.

Our study is the first to report the statistically signifi-

cant upregulation of AR in human middle ear CHLST

and the correlation to disease state.

Several technical and biological limitations were

identified and overcome to perform this study. Chal-

lenges to studies of human CHLST include 1) limited

quantity of clinical biopsy material available for experi-

mental manipulations and 2) inherent biological

heterogeneity of human clinical samples. To address

logistical constraints, we first optimized protocols for col-

lecting and processing patient specimen sets. Working at

this time with only one otologic surgeon (j.d.m.) ensured

crucial technical confidence that specimen sets were col-lected and stabilized in a well-defined manner. Next,

sensitive processing methods combined with real-time

PCR technology maximized RNA and protein purifica-

tion yields and the quantitative information obtained

from limited starting materials.

Intraindividual controls were used for experimental

comparisons in our study design. Specimens collected

from each patients nondiseased skin regions served as

personalized normal controls to minimize interindividual

biological differences that potentially complicate data

interpretation of heterogeneous clinical isolates.

Fig. 1. Comparative quantitation charts. Relative amphiregulin (AR)values with upper and lower error bars are shown for each pair ofpatient cholesteatoma (CHLST) and postauricular skin (PAS) orexternal auditory canal (EAC) calibrator reference specimens.Quantity of AR mRNA in PAS or EAC was defined as 1.0 (solidblack bars). CHLST AR values (striped bars) are expressed asglyceraldehyde-3-phosphate dehydrogenase (GAPDH)normalizedrelative ratios of CHLST/PAS or CHLST/EAC. In both graphs, off-scale CHLST AR ratio values are indicated next to the truncatedstriped bar(s). (A) CHLST/PAS AR graph. The mean ratio of ARRNA expressed in CHLST relative to PAS is 4.94 (standard error ofthe mean [SEM] 1.53, n 30). (B) CHLST/EAC AR graph. Themean ratio of AR mRNA expressed in CHLST relative to EAC is7.70 (SEM 1.57, n 30). Specimen 22 was measured usingendpoint real-time quantitative reverse-transcription polymerasechain reaction and KODAK EDAS 290 digital image analysis sys-tem; CHLST/PAS AR ratio 3.8 (standard deviation [SD] 0.2),CHLST/EAC AR ratio 10.6 (SD 0.1).



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Previous publications investigating pathophysiology of

human CHLSTs generally used either PAS or EAC as

appropriate normal skin controls, PAS being a distal

ear-associated epithelium and EAC a proximal, migra-

tory epithelial reference. Incorporating both in this

current study allowed simultaneous comparisons of

CHLST to two reference skin sources for better under-

standing of gene expression patterns in these pertinent

yet distinct epithelial regions of the ear. Despite differ-ences in the magnitude of AR upregulation, mean

expression levels of AR in CHLST are increased as com-

pared with both PAS and EAC reference controls.

AR gene expression levels correspond inversely to

the clinical stage of disease based on migration and tem-

poral bone erosion. Elevated AR expression in early

stage clinical specimens was independent of relation to

age, recurrence, or infection and suggests AR stimulation

may contribute to progressive growth during CHLST de-

velopment. These findings are even more striking when

considering the clinically diverse backgrounds under

which the samples were collected. If AR plays an impor-

tant functional role during a specific point in CHLST

development, reporting averaged values from clinical

specimens at different stages of progression potentially

underestimates the relative magnitude of AR overexpres-

sion. Understandably, CHLST is often identified later in

disease when epithelial expansion has caused notable

sequelae such as hearing loss or infection. And at the

time of surgery, the metabolic status of specimens is var-

ied and generally unknown. Specimens collected during

a metabolically active phase of disease (e.g., response toproinflammatory signaling molecules) or an indolent or

latent period (e.g., following antibiotic resolution of a sec-

ondary infection) may present correspondingly up- and

down-regulated levels of AR. To address the relationship

of metabolic status to AR expression, we used QRT-PCR

assays to measure the expression of proliferating cell nu-

clear antigen (PCNA) in several of our paired specimen

sets. Our results are consistent with those of previous

reports identifying CHLST as hyperproliferative tissue

and having upregulated proliferation markers PCNA13

and Ki-67 nuclear antigen.14 Our CHLST study speci-

mens with higher AR expression also had higher PCNA

expression (data not shown).

Fig. 2. Representative Western blot detection of amphiregulin (AR) protein in a patients set of epithelial lysates (15 lg/lane). Duplicate blots(left images in panels A and B) are transiently stained with MemCode Reversible to visualize protein transfer and pattern and thendestained for immunoblotting. (A) Anti-AR Western immunoblot. Human AR protein (18 kDa) detected with anti-AR specific antibodies is

indicated with the AR-labeled arrow. Positions of recombinant human AR (rhAR) positive () control at 2.5 ng, bovine serum albumin (BSA)negative (-) control at 125 ng per lane, and Sigma Wide molecular weight standards are indicated. (B) Negative control Western blot assayusing normal goat serum. Prestained Blue Ranger molecular weight standards and sizes are indicated to the left. PAS postauricular skin;EAC external auditory canal; CHLST cholesteatoma; con control.

TABLE I.

Cholesteatoma Amphiregulin Expression Levels and Relation to Clinical Variables.

MigrationCategory 1(n 8)

MigrationCategory 2(n 25) P Value*

ErosionCategory 1(n 8)

ErosionCategory 2(n 25) P Value*

CHLST/PAS AR mean (SEM) 5.50 (3.22) 4.77 (1.78) .845 10.30 (2.77) 2.99 (1.67) .032

CHLST/EAC AR mean (SEM) 15.08 (2.89) 5.45 (1.59) .007 14.73 (2.93) 5.56 (1.62) .011

Migration category 1 extension to middle ear and/or attic; migration category 2 extension to antrum and/or mastoid; erosion category 1 none orerosion of one or more ossicles; erosion category 2 erosion of scutum and/or mastoid.

*Statistical significance is indicated by P .05.CHLST cholesteatoma; PAS postauricular skin; AR amphiregulin; SEM standard error of the mean; EAC external auditory canal.



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This study demonstrates AR is overexpressed in

CHLST; in the presence of its cell-surface receptor

EGFR, AR has the potential to play an important role in

driving dynamic processes of the disease state. Future

studies of the function of AR in human CHLST disease

progression will likely require synchronized, manipulat-

able model systems such as the gerbil animal model,

which has been carefully defined,15 or development of a

short-term human skin organ culture system to testeffects of growth factor and signaling inhibitors on mo-

lecular responses in human CHLST tissues. In both

normal and tumor cells, activation of EGFR-dependent

signaling cascades by binding of specific ligand such as

AR induces an intrinsic tyrosine kinase and mediates

complex proliferative, invasive, angiogenic, and antia-

poptotic responses. Recent advances in U.S. Food and

Drug Administrationapproved inhibitors of EGFR and

overexpression of components of the EGFR signaling

pathway in CHLST make this molecular network a com-

pelling target for future studies of treatment options for

CHLST. Development of topical delivery systems for

anti-EGFR therapeutics16 (e.g., monoclonal antibodies,

tyrosine kinase inhibitors, ligand-toxin conjugates, or

antisense oligonucleotides) may be efficacious for inhibi-

ting CHLST progression in nonsurgical candidates or

preventing recurrence of disease.

CONCLUSIONOur study results show the growth factor AR is over-

expressed in the epithelium of aural CHLST compared to

nondiseased epithelial sites of the ear. Demographic and

clinical variables were prospectively evaluated to investi-

gate correlations between AR levels and CHLST and to

provide evidence that increased AR expression is a poten-

tial biomarker of CHLST activity. Significant inverse

relationships were identified between AR gene expressionand both the extent of expansion into the temporal bone

and the severity of erosion. AR levels were significantly

higher when CHLST migration was in the earliest stages

of temporal bone invasion, before the development of sig-

nificant tissue destruction. The significantly increased

expression of AR detected at the onset of CHLST pathol-

ogy suggests a functional role during developmental

stages of disease progression and makes AR a promising

target for pharmacologic medical intervention.

AcknowledgmentThe authors gratefully acknowledge the support of

Victor Hsu, Jamie Lee, Tanya Thal, John Pepper, Jerry

and Amy Hugo, Lylis Olsen, Linda Mardel, Kim Greenlief,

and ShayneDavis.

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