Tissue and serum EGFR as prognostic factors in malignant pleural mesothelioma

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Lung Cancer 70 (2010) 43–50

Contents lists available at ScienceDirect

Lung Cancer

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issue and serum EGFR as prognostic factors in malignant pleural mesothelioma

abab Gaafara, Abeer Bahnassyb,∗, Ibrahim Abdelsalamc, Mahmoud M. Kameld,mani Helala, Amany Abdel-Hamidb, Nelly Aly Eldine, Nadia Mokhtarb

Medical Oncology Department, National Cancer Institute, Cairo University, EgyptPathology Department, National Cancer Institute, Cairo University, Kaser El-Aini Str., Fom El-Khaleg, Cairo 11967, EgyptCancer Biology Department, National Cancer Institute, Cairo University, EgyptClinical Pathology Department, National Cancer Institute, Cairo University, EgyptEpidemiology and Biostatistics Department, National Cancer Institute, Cairo University, Egypt

r t i c l e i n f o

rticle history:eceived 18 August 2009eceived in revised form5 December 2009ccepted 4 January 2010

eywords:alignant pleural mesothelioma

erum and tissue EGFRrognosis

a b s t r a c t

Background: Malignant pleural mesothelioma (MPM) is an asbestos related aggressive tumor. Asbestoscauses genetic modifications and cell signaling events that favor resistance to chemotherapy. A variety ofreceptor tyrosine kinases have been identified to play a central role in various aspects of tumorigenesis.Epidermal growth factor receptor (EGFR) is overexpressed in a variety of epithelial malignancies includinglung cancer in which EGFR aberrations not only predict response to EGFR tyrosine kinase inhibitors butalso indicate tumor progression. However in MPM, the role of EGFR is less clear. This study was designedto identify serum and tissue EGFR levels in patients with MPM and to evaluate the relationship betweenserum and tissue EGFR levels and clinicao-pathological prognostic factors and survival.Methods: We investigated 71 cases of MPM for EGFR expression in tissue. Serum EGFR was assessed in40 out of those 71 cases and 20 healthy subjects as a control. Pre-treatment serum EGFR levels weremeasured using quantitative enzyme-linked immunosorbent assay. Tissue EGFR protein overexpres-sion was assessed by immunohistochemistry and gene amplification was assessed by the chromogenin situ hybridization (CISH) technique. Results were correlated with the clinical–pathological factors ofthe patients and overall survival (OS).Results: Out of the 71 patients included in the study, 19 had undergone extrapleural pneumonectomy.As for the rest of the patients, 46 received chemotherapy while 6 had only best supportive care. EGFRimmuno-reactivity was detected in 74.6% of the cases, 37 (52.1%) cases were positive for EGFR geneamplification by CISH, 31 of them revealed moderate to high (++, +++) EGFR immuno-reactivity. Elevatedserum EGFR >2.5 ng/ml (the median concentration of EGFR in MPM) was reported in 45% of the cases.

The overall response rate (RR) for the 46 treated patients who received chemotherapy was 24.1%. Aftera median follow up of 29 months, the median overall survival (OS) was 10 months. Elevated serum andtissue EGFR is significantly associated with advanced disease stage. However neither EGFR overexpressionin tissues nor high serum levels were associated with survival rates.Conclusions: EGFR expression is a common feature in MPM patients. High pre-treatment levels of serumEGFR are associated with advanced stage but not with reduced OS. Detailed mutational analysis of EGFR

ients

on a larger number of pat

. Introduction

Malignant pleural mesothelioma (MPM) is an asbestos relatedumor with an increasing incidence, which is difficult to detect early

nd treat effectively. It has been demonstrated that in the next 35ears about one-quarter of a million deaths will occur as a resultf this disease in Western Europe [1,2]. In Egypt, 635 cases of MPMere diagnosed at the National Cancer Institute (NCI), Cairo Uni-

∗ Corresponding author. Tel.: +20 1 01720698/2 23664649; fax: +20 2 23644720.E-mail address: [email protected] (A. Bahnassy).

169-5002/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.lungcan.2010.01.002

is still needed to clarify the exact role of EGFR in MPM patients.© 2010 Elsevier Ireland Ltd. All rights reserved.

versity and in Abassia Chest Hospital in the first four years of thethird millennium (2000–2003). Moreover, NCI hospital-based reg-istry showed an increase in the relative frequency of MPM from0.47 during 2001 to 1.3 during 2003 [3].

Despite recent improvements in the therapeutic managementof MPM through surgery, chemotherapy, radiotherapy, and sup-portive measures, it still has a poor prognosis with a median overall

survival ranging from 6 to 12 months only [4]. Several clinicopatho-logic prognostic scoring systems have been proposed, but they arenot able to predict accurately the individual patient’s outcome, andtherefore specific therapy is possibly deferred. Accordingly, thereis an increasing demand for identifying new biological prognostic

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Table 1Clinical features of the 71 malignant pleural mesothelioma cases studied.

Variable Number of patients (%)

Age≤46 years 36 (51)>46 years 35 (49)

Median age 46

GenderMale 45 (63.4)Female 26 (36.6)

Male:female ratio 1.7:1

Asbestos exposurePositive 50 (70.4)Negative 21 (29.6)

Performance status1 41 (57.8)2 23 (32.4)3 7 (9.8)

Histopatholgical typeEpithelial 39 (54.9)Mixed 19 (26.8)Sarcomatoid 7 (9.8)NOS 6 (8.5)

Tumor stage (IMIG)a

Early (I and II) 19 (26.8)Advanced (III and IV) 52 (73.2)

T-stageEarlyI 7 (9.85)II 27 (38.03)AdvancedIII 20 (28.17)IV 17 (23.9)

Clinical manifestationDyspnea 54 (76.1)Chest pain 48 (67.6)

4 R. Gaafar et al. / Lun

arkers as well as for the development of novel effective therapies5].

The epidermal growth factor receptor (EGFR) is a member of theamily of EGF-related tyrosine kinase (TK) receptors. Upon ligandsinding, the receptors homo- or hetero-dimerize. Subsequently,

t activates receptors’ intrinsic TK activity and broad downstreamignaling cascades, mainly including Ras-Raf-MAP-kinase pathway,I3K-Akt pathway and STAT pathway leading to strong stimulatoryffect on cell proliferation, differentiation, survival, angiogenesis,nd migration [6–8]. The EGFR is expressed in a variety of humanpithelial tumors including lung, head and neck, colorectal, andreast cancers [9]. EGFR has emerged as a critical tumorigenic factor

n the development and progression of non small cell lung can-er (NSCLC). Two specific EGFR tyrosine kinase inhibitors (TKIs),efitinib (ZD, 1839, Iressa) and erlotinib (OSI-774, Tarceva), haveeen developed and used clinically in the treatment of advancedSCLC [10–12]. Both agents can induce dramatic clinical response

n patients who fail chemotherapy by disrupting EGFR signaling viaompeting with ATP for the binding sites at the TK domain, thusnhibiting the phosphorylation and activation of the EGFRs and theirownstream signaling network [10].

On the other hand data regarding EGFR in MPM are still imma-ure and show controversial results. So, EGFR expression levelsanging from 0% to 97% have been reported in MPM patientsith similar variability in patients’ outcome [13–15]. In most of

hese studies EGFR aberrations were evaluated in tissues either bymmunohistochemical staining or by studying genetic mutations.n a recent study, Destro et al. [15] demonstrated a signifi-ant correlation between EGFR immunohistochemical expressionnd the corresponding mRNA levels by real time PCR. In thisork, EGFR was not expressed in any of normal pleural sam-les. This data confirms and enlarges previous observationsuggesting an important role of EGFR overexpression in pleuralarcinogenesis.

EGFR is also readily identifiable and quantifiable in serum andeveral reports have indicated that changes in the serum levels ofGFR are associated with aggressive cancer development, in kid-ey, gastric and lung cancer [11,16,17]. However, the usefulnessf identifying EGFR level in serum from MPM patients is not yetefined.

In this study we investigated the potential prognostic and pre-ictive values of elevated tissue and serum EGFR expression asell as EGFR gene amplification in MPM patients through corre-

ation with the standard clinico-pathological features of patientsnd overall survival rates.

. Patients and methods

.1. Tumor samples

In this prospective study, 71 MPM patients who attended theational Cancer Institute (NCI), Cairo University, Egypt during theeriod from 2004 to 2007 were investigated for EGFR expression

n tissues. Pre-treatment serum samples were available for 40 outf those 71 patients. Paraffin blocks for all patients were recruitedrom the archive of the pathology department following diagno-is whereas pre-treatment serum samples were collected fromatients at presentation and kept at −70 ◦C. In addition, serumamples were obtained from 20 healthy volunteers and used as aontrol for serum EGFR. All cases were diagnosed as MPM and clas-

ified using the WHO criteria on hematoxylin and eosin-stainedections combined with immunohistochemistry, using the routineiagnostic panel for MPM (Table 1) [18]. A written consent shoulde obtained from all patients prior to enrolment in the study, andhe ethical committee of the NCI approved the protocol which was

Pleural effusion 47 (66.2)Cough 33 (46.5)

a MIG: International Mesothelioma Interested Group.

in accordance with the ethical guidelines of the 1975 Declarationof Helsinki.

2.2. Clinical workup and treatment protocols

According to IMIG staging classification [19], patients weredivided into early (stages I and II) and advanced (stages IIIand IV) stages and accordingly, they received treatment in theform of extrapleural pneumonectomy or chemotherapy, respec-tively. The 19 operable cases were pathologically staged whereasthe remaining 52 cases were clinically staged. Advanced casesreceived platinum based chemotherapy with or without radiother-apy according to the treatment protocols of the NCI, Cairo.

2.3. Evaluation of serum EGFR expression

After collection of the venous blood samples from patients (40samples) and control (20 samples) subjects, the samples were cen-

◦

trifuged at 3000 rpm for 10 min and then stored at −70 C untilassay. The human Active EGFR ELISA (Bender Medysystems Diag-nostics GmbH, Rennweg 95b A-1030 Vienna, Austria) kit was usedfor the detection of quantitative level of EGFR in serum accordingto the recommendation of the manufacturer.

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.4. Immunohistochemistry

Immunohistochemistry was performed to confirm the diagno-is of MPM and to assess the expression of EGFR protein using theollowing panel: calretinin, keratin 5/6, epithelial membrane anti-en and/or cytokeratin, CEA, vimentin, and the EGFR pharmDxtmtaining kit (all from Dako, USA) according to manufacturer’snstructions.

The standard streptavidin–biotin–peroxidase detection tech-ique was used as previously described [18]. The antigen retrievalethod was performed by microwave pre-treatment in 0.01 M

itrate buffer. The primary antibody was applied and incubatedvernight at 4 ◦C in a humidified chamber. After 3 washes inBS, the secondary antibody and the avidin–biotin complex (ABC)ere applied to slides. Diaminobenzidine (DAB) was used as a

hromogen and sections were counterstained using Mayer’s hema-oxylin. Manufacturer’s protocol for EGFR staining was strictlyollowed.

.5. Interpretation of immunohistochemical results

Assessment of EGFR was based on a membranous staining pat-ern. Absence of specific membranous staining within tumor cellsas considered negative. In case of positivity (defined as any

mmuno-staining of tumor cell membranes), the intensity of stain-ng, and the percent of positive cells were assessed according to thenstructions of the EGFR pharmDxtm staining kit and reported (1%,%, 10%, etc.).

.6. Chromogen in situ hybridization (CISH)

Paraffin-embedded tissue sections were de-paraffinized in twohanges of xylene for 5 min each; xylene was removed in threeashes of ethanol for 3 min each (100%, 100%, and 95%) and the

lides were washed in distilled running water for 5 min. Slides werelaced in heated (>90 ◦C) CISH Pre-treatment Buffer (Zymed Labo-atories) and microwaved on high power for 30 min, then rinsed inistilled water for 5 min at room temperature (RT). The tissue wasigested for 10 min with pepsin digestion solution (Zymed Lab-ratories) at RT, washed twice in distilled water for 5 min each,ehydrated in 90%, 95%, and 100% alcohol for 2 min each, and thenried in a 37 ◦C oven. Five–seven micro-liters of EGFR Amplificationrobe (Zymed-Invitrogen) was applied to the designated area andcover slip was applied and sealed with rubber cement. Slide wasried at 37 ◦C, followed by probe denaturation at 95 ◦C for 5 minnd hybridization at 37 ◦C overnight. The slides were then washedn 0.5% SSC for 5 min at RT, followed by 0.5% SSC for 5 min at 75 ◦C,nd water for 5 min at RT.

For immuno detection, slides were placed in 3% hydrogen perox-de in absolute methanol for 10 min, washed in phosphate-bufferedaline with 0.025% Tween 20 (PBST) three times for 2 min each,ncubated with nonspecific blocking solution (Zymed-Invitrogen)or 10 min at RT and then with mouse anti-digoxigenin antibodyor 30 min at RT. Slides were then washed in PBST twice for 2 minach, incubated with horseradish peroxidase-conjugated goat anti-ouse antibody for 15 min, and finally washed again with PBST

wice for 2 min each. The slides were then incubated with DABhromagen (Zymed-Invitrogen) for 30 min at room temperaturend washed in distilled water twice for 2 min each. Slides wereounterstained with hematoxylin [20].

The CISH-prepared slides were examined at X400 by bright fieldicroscopy. As in prior studies [20], 200 tumor nuclei were exam-

ned in each case. In the cases where the number of signals perumor nucleus varied between tumor cells, the range of signalsounted was recorded for each case.

er 70 (2010) 43–50 45

2.7. Statistical analysis

Univariate and multivariate survival analyses were done toevaluate the prognostic significance of clinico-pathological fac-tors and EGFR expression on the overall survival. Specifically, theproduct limit or Kaplan-Meier method was applied as univariateanalysis to estimate the survival probabilities, and the log-ranktest (LT) was performed to determine the statistical differencesbetween categories of each prognostic factor. Cox’s proportionalhazards regression model was performed as multivariate analysisto estimate the role of each prognostic factor on survival outcome,adjusted for the possible confounding effect of statistically signif-icant covariates included in the same model. Asymptotic standarderrors of coefficients were used to calculate 95% confidence inter-vals (95% CI) of hazard ratio (HR; relative risk). Chi-square test(�2) was used to test the association variables for categorical data.Survival data were analyzed using the EGRET statistical softwarepackage.

3. Results

The characteristics of the 71 MPM patients enrolled in this areshown in Table 1. The median age for the patients was 46 years(range 26–77 years). Forty-five patients were males and 26 werefemales with a male to female ratio of 1.7:1. A history of asbestosexposure was given in 50 (70.4%) patients. Dyspnea was the com-monest presentation being encountered in 54 patients, followed bychest pain in 48 patients, pleural effusion in 47 patients and coughin 33 patients. The Eastern Cooperative Oncology Group (ECOG) PSwas 1 in 41 (57.7%) patients and >1 in 30 (42.3%) patients. Fifty-two (73.2%) patients were diagnosed as having advanced diseasestage (III and IV) at presentation whereas 19 patients (26.8%) pre-sented at an early stage (I and II). Extrapleural pneumonectomywas performed for the 19 early stage cases.

Out of the 52 patients with advanced disease stage, 46 weretreated with platinum based chemotherapy and/or radiotherapyaccording to NCI, Cairo protocols whereas 6 were treated withpalliative measures only. Two patients out of 46 (4%) achieved com-plete remission (CR) and 9 (20%) had partial remission (PR); theoverall response rate was 24%. On the other hand 22 (48%) patientshad stable disease and 13 (28%) show disease progression. After amedian follow up of 29 months, the median survival for the earlycases 14.3 months, while the median survival for the advancedcases was 10 months.

3.1. Evaluation of serum EGFR

Positive or negative serum EGFR was determined using a cutoffvalue of 0.23 ng/ml, which represents the median concentra-tion of the control group. The median concentration for the 40cases assessed was 2.5 ng/ml. It was significantly higher than themedian concentration for normal control subjects (0.23 ng/ml;p = 0.001). Eighteen patients (45%) had elevated serum EGFR morethan 2.5 ng/ml (Fig. 1). A statistically significant correlation wasfound between a high serum EGFR expression (>2.5 ng/ml) andadvanced disease stage (p = 0.05) as well as between serum EGFRlevel and T-stage (p = 0.001). No relation was found with otherclinico-pathological features of the patients, asbestos exposure, andresponse to treatment or OS (Table 2). However, using 0.23 ng/ml

as a cutoff there was a trend for better response to treatment inpatients with low serum EGFR levels (p = 0.09) as 53% (9/17) of thepatients with good response to treatment have a high serum EGFR>0.23 ng/ml compared to 87.5% (7/8) of the patients with aggressivedisease and a high serum EGFR >0.23 ng/ml.

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Fig. 1. Epidermal growth factor receptor (EGFR) serum levels in malignant pleu-ral mesothelioma cases. The median concentration for the control group was0–23 ng/ml (range 0–0.96) and the median concentration for the tested group was2.5 ng/ml (range 0.5–7.6, p = 0.001).

Table 2Serum EGFR concentration in relation to the clinicopathologic features of the studiedpatients.

Variables Serumconcentration≤ 2.5 (22)

Serumconcentration> 2.5 (18)

p value

Mean age (years)≤46 13 (59.1) 10 (55.6)>46 9 (40.9) 8 (44.4) 0.92

SexMale 14 (63.6) 10 (55.6)Female 8 (36.4) 8 (44.4) 0.6

Performance status<2 14 (63.6) 10 (55.6) 0.6≥2 8 (36.4) 8 (44.4)

Asbestos exposureNegative 9 (40.9) 9 (50)Positive 13 (59.1) 9 (50) 0.56

Histopathologic subtypeEpithelioid 13 (59.1) 13 (72.2)Sarcomatoid 2 (9.1) 1 (5.6)Mixed 4 (18.2) 3 (16.6)NOS 3 (13.6) 1 (5.6) 0.3

Tumor stageEarly (I and II) 10 (45.5) 3 (16.7)Advanced (III and IV) 12 (54.5) 15 (83.3) 0.05

T-stageI and II 18 (81.8) 2 (11.1)III and IV 8 (17.2) 16 (88.9) 0.001

Pleural effusionNegative 13 (59.1) 8 (44.4)Positive 9 (40.9) 10 (55.6) 0.57

Response to CTHClinical benefit 10 (66.7) 7 (70) 0.8a

Progression 5 (33.3) 3 (30)

a Serum samples were available for 25 out of the 46 treated patients.

Fig. 2. A case of epithelioid malignant pleural mesothelioma showing positive mem-brano/cytoplasmic immuno-staining (+++) for EGFR.

3.2. Evaluation of tissue EGFR

EGFR immuno-reactivity was detected in 53/71 cases (74.6%,Fig. 2), 22 (30.98%) were scored (+), 21 (29.6%) were scored (++),10 (14.1%) were scored (+++). No immuno-reactivity was detectedin normal pleural samples. The ranges of CISH signals recorded inthe analyzed cases were as follows: in the majority of the non-amplified cases, the number of CISH signals per cell ranged from2 to 5. In gene-amplified cases, the number of CISH signals percell was estimated at 5–30 with at least focal clustering of signalsconsistent with tandem gene repeats (Fig. 3). Out of the 71 casesexamined, 37 (52.1%) cases were positive for EGFR gene amplifi-cation by CISH, 31 of them revealed moderate to high (++, +++)EGFR immuno-reactivity and 6 revealed mild (+) EGFR immuno-reactivity. The concordance between EGFR protein expression andgene amplification (86%) was statistically significant (p = 0.01).EGFR overexpression and gene amplification were significantlyassociated with T-stage of the tumors (p = 0.021) but not with any ofthe clinico-pathological factors of studied patients, asbestos expo-sure or OS (Table 3 and Fig. 3). Using 0.23 ng/ml as a cutoff point,there was a statistically significant relation between increased EGFRprotein expression and/or gene amplification and elevated EGFRserum levels (p < 0.05). However, there was no statistically sig-

nificant relation between EGFR serum levels and neither proteinoverexpression nor gene amplification (p = 0.0671 and p = 0.884)using 2.5 ng/ml as a cutoff point.

Fig. 3. A case of epithelioid malignant pleural mesothelioma showing multiplebrown nuclear dots and clusters denoting EGFR gene amplification as measuredby chromogen in situ hybridization (CISH) technique.

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Table 3Tissue EGFR expression in relation to the clinico-pathologic features of the 71patients studied.

EGFR negative (18) EGFR positive (53) p value

Mean age (years)≤46 8 (44.5) 28 (52.8)>46 10 (55.5) 25 (47.2) 0.84

SexMale 9 (50) 36 (67.9)Female 9 (50) 17 (32.1) 0.92

Performance status<2 11 (61.1) 30 (56.6) 0.54≥2 7 (38.9) 23 (43.4)

Asbestos exposureNegative 8 (44.5) 13 (24.5)Positive 10 (55.5) 40 (75.5) 0.78

Histopathologic subtypeEpithelioid 8 (44.4) 31 (58.5)Sarcomatoid 2 (11.1) 5 (9.5)Mixed 6 (33.4) 13 (24.5)NOS 2 (11.1) 4 (7.5) 0.77

Tumor stageEarly (I and II) 4 (22.2) 15 (28.3)Late (III and IV) 14 (77.8) 38 (71.7) 0.88

T-stageI and II 14 (77.8) 20 (37.7)III and IV 4 (22.2) 33 (62.3) 0.024

Pleural effusionNegative 6 (33.4) 18 (34) 0.62Positive 12 (66.6) 35 (66)

Response to therapy

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cases included one biphasic type and two epithelial types, andthe high polysomy case was epithelial type. Our study dose

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Clinical benefit 10 (76.9) 23 (70)Progression 3 (23.1) 10 (30) 0.76

.3. Survival analysis

No significant relation was found between EGFR expression inerum or tissues and the OS rate. Similarly, no relation was foundetween EGFR gene amplification by CISH and OS (Table 4). Theedian survival for cases having serum EGFR more than 2.5 ng/ml

nd equal or less than 2.5 ng/ml was 6 months (95% confidencenterval [CI], 2–10 months) and 8 months (95% CI, 5–11 months);espectively (p = 0.44, Fig. 4). Patients with EGFR immunopositiveumors had a median survival of 12 months (95% CI, 9–15 months)ompared to 8 months (95% CI, 3–13 months) for patients withegative tumors (p = 0.27, Fig. 4). Similarly, patients with EGFR gene

mplification had a median survival of 10 months (95% CI, 8–16onths) compared to 7 months (95% CI, 5–13 months) for patientsith negative tumors (p = 0.26, Fig. 4) (Fig. 5).

able 4ox regression analysis results of EGFR in serum and tissue in relation to the overall surv

Median survival B SE

Serum EGFR<2.5 ng/ml 8 3.557 0.996>2.5 ng/ml 6

Tissue EGFR (protein)Negative 8 5.998 1.89Overexpression 12

EGFR gene amplificationNegative 7 2.116 3.974Positive 10

er 70 (2010) 43–50 47

4. Discussion

In lung cancer, EGFR gene mutation at the kinase domain andEGFR gene amplification were reported to be predictors of theresponse to EGFR tyrosine kinase inhibitors [13]. Therefore, someanti temporal drugs have been developed against EGFR. In MPM,data regarding EGFR expression and its prognostic significance arestill scarce and immature.

In the current study, EGFR serum levels were significantly higherin MPM patients than healthy controls (2.5 vs 0.23 ng/ml, p = 0.001).However, this elevated serum level has no prognostic impact sinceno difference in the OS rates was found between patients with lowserum EGFR (<2.5 ng/ml) and those with high serum EGFR levels(≥2.5 ng/ml). However, a high serum EGFR was significantly associ-ated with advanced disease stage since 23.1% (3/13) of patients withearly stage had serum EGFR >2.5 ng/ml compared to 55.6% (15/27)in late stage (p = 0.05). Moreover, there was a significant correla-tion between increased EGFR levels, both in serum and tissues, aswell as EGFR gene amplification and T-stage, which is considereda surrogate prognostic factor in MPM. Our results are comparableto those of Betta et al. [21] and Schneider et al. [22] who foundno differences in serum values of EGFR between MPM patientswith varied clinical outcomes and attributed this to the small num-ber of cases studied. In the present study, there was a trend forpatients with low serum EGFR levels to show better response totreatment compared to those with high serum EGFR >0.23 ng/dlas a cutoff value separating patients from healthy controls thoughthe difference did not reach a statistically significant value(p = 0.09).

We also assessed whether tissue EGFR protein expressionand/or gene amplification have any prognostic impact in ourstudied group with MPM. Seventy-five percent of our casesrevealed EGFR immuno-reactivity and 69% revealed EGFR geneamplification by CISH technique (89% concordance). However, nosignificant difference was reported between high EGFR proteinexpression or EGFR gene amplification and neither patient responsenor OS.

Our reported frequency of tissue EGFR protein expression (intissues) and gene amplification are higher than those of Okudaet al. [13] who studied EGFR gene mutation, amplification andprotein expression in 25 MPM Japanese patients and found thatonly 8/25 (32%) cases were positive for the EGFR protein; all ofthem were epithelioid. Fluorescence in situ hybridization (FISH)analysis revealed three low polysomy and one high polysomycase only; all of them expressed EGFR protein. The low polysomy

not show any significant relation between EGFR gene amplifica-tion or EGFR protein expression and the histopathology type oftumor.

ival of the studied cases.

p value OR 95.0% CI for OR

Lower Upper

0.44 15.864 5 112 10

0.27 24.511 3 139 15

0.026 8.749 5 138 16

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ig. 4. EGFR expression in serum and tissues in relation to the history of asbestos end p = 0.88 respectively) in malignant pleural mesothelioma patients studied.

Our frequency of EGFR protein expression is also higher than,dwards et al. [2], Govindan et al. [23] who reported EGFR proteinxpression in 44% and 58% of their studied cases (21) respectively.n the contrary, Garland et al. [24] reported EGFR protein expres-

ion in 88% of MPM cases and Govindan et al. [25] reported EGFRverexpression in 97% of patients undergoing therapy with theGFR TKI gefitinib, as part of a phase II study.

This heterogeneity in the results of different studies could bexplained by differences in sample size, patients’ characteristics,he detection method, the antibody used to determine tissue EGFRrotein expression, the difference in interpreting the results of IHC

ncluding the intensity of expression and the localization of the

eceptor where some authors reported only membranous stainings opposed to cytoplasmic staining, while others did not report anyreferential localization of the receptor.

No significant relation was found in the current study betweenGFR protein expression or gene amplification and any of the stan-

re (p = 0.56 and p = 0.78 respectively) and disease stage according to IMIG (p = 0.05

dard clinico-pathologic prognostic factors (except the stage of thetumor) or response to therapy.

Prior studies have reached variable conclusions regarding theprognostic significance of EGFR in MPM. Some authors reported thatthe tumor cell expression of the growth factors was associated withfavorable outcome in MPM [2,26]. Others [27] demonstrated thatEGFR is a poor prognostic factor. The inverse correlation betweenEGFR and survival may be explained partly by the differences inexpression between the epithelioid and sarcomatoid cell types,since Dazzi et al. [28] found that, after the cell type was takeninto consideration, lack of EGFR immuno-staining was no longersignificant.

Our findings agree with those of Govindan et al., Okuda et al., andDestro et al. [23,13,15] that EGFR expression and/or gene amplifica-tion in tissue are not related to patients’ survival or response to theavailable therapeutic modalities. Although EGFR status did not cor-relate with overall survival, its frequency in MPM suggests that it

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R. Gaafar et al. / Lung Cancer 70 (2010) 43–50 49

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ig. 5. Kaplan and Mayer curves showing the overall survival rates in patients with

ay be a novel therapeutic target. Confirmation of this hypothesisomes from in vitro studies where treatment of MPM cell lines withhe selective EGFR TKI (Gefitinib) led to significant dose dependenteduction of colony formation of cells grown in soft agarose [29].oreover, the EGFR TKI PD153035 inhibited motility and invasion

f MPM cells in a matrigel assay [30]. However, results from phase IItudies showed that EGFR tyrosine kinase inhibitors did not appearo be effective in unselected patients. The median overall survivalime was 4–10 months and one-year survival was estimated at 43%,lthough some patients had objective response or stable disease14,25,26]. In addition, there is no randomized data from phase IIItudies examining measurable clinical benefits of EGFR pathwaynhibitors in MPM patients. Therefore, further studies are requiredo evaluate the role of targeted therapy in patients with activatedGFR pathways.

We conclude that increased EGFR protein expression (in serumnd tissues) as well as EGFR gene amplification are common ingyptian patients with MPM indicating that EGFR may be a tar-

et for selective therapies in a subset of patients. However, EGFRas not found to be of prognostic value in our cases since no asso-

iation was found between EGFR protein expression in serum andissues or EGFR gene amplification and OS, patients’ response toreatment or the standard clinico-pathological prognostic factors

ted serum and tissue EGFR expression in malignant pleural mesothelioma patients.

except for advanced disease stage. A more extended study includ-ing large number of patients is warranted to define detailed geneticchanges affecting EGFR and other family members.

Conflict of interest

Conflict of interest is declared.

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