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EGFR overexpression in malignant pleural mesothelioma
Lung Cancer (2006) 51, 207—215
EGFR overexpression in malignant pleural mesothelioma An immunohistochemical and molecular study with clinico-pathological correlations夽 A. Destro a, G.L. Ceresoli b, M. Falleni c, P.A. Zucali b, E. Morenghi d, P. Bianchi a, C. Pellegrini c, N. Cordani c, V. Vaira c, M. Alloisio e, A. Rizzi f, S. Bosari c, M. Roncalli g,∗ a
Molecular Genetics Laboratory, IRCCS Istituto Clinico Humanitas, Via Manzoni 56, 20089 Rozzano, Milano, Italy b Department of Oncology, IRCCS Istituto Clinico Humanitas, Via Manzoni 56, 20089 Rozzano, Milano, Italy c Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, via A. di Rudin´ ı 8, 20142 Milan, Italy d Clinical Trial Office, IRCCS Istituto Clinico Humanitas, Via Manzoni 56, 20089 Rozzano, Milano, Italy e Department of Thoracic Surgery, IRCCS Istituto Clinico Humanitas, Via Manzoni 56, 20089 Rozzano, Milano, Italy f Department of Thoracic Surgery, Humanitas Gavazzeni, Via Mauro Gavazzeni, 21-24125 Bergamo, Italy g Department of Pathology, University of Milan, IRCCS Istituto Clinico Humanitas, Via Manzoni 56, 20089 Rozzano, Milano, Italy Received 24 June 2005 ; received in revised form 22 September 2005; accepted 4 October 2005 KEYWORDS Mesothelioma; EGFR; Immunohistochemistry; Real-time PCR; Prognostic factors
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Summary The epidermal growth factor receptor (EGFR) is overexpressed in many epithelial malignancies, against which some antitumoral drugs have been developed. There is a lack of information as to EGFR expression in malignant pleural mesothelioma (MPM), an aggressive and fatal cancer poorly responsive to current oncological treatments. Our aim was to: (a) compare EGFR immunohistochemical expression with mRNA levels measured by real time PCR; (b) assess the relationships between EGFR expression and clinico-pathological data including survival; (c) analyze the EGFR mutations. We developed an immunohistochemical method of EGFR evaluation based on the number of immunoreactive cells and staining intensity in 61 MPMs.
This work was partly supported by MURST (COFIN 2003). Corresponding author. Tel.: +39 02 82244714; fax: +39 02 82244791. E-mail address: massimo [email protected] (M. Roncalli).
0169-5002/$ — see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2005.10.016
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A. Destro et al. EGFR immunoreactivity was documented in 34/61 (55.7%) cases. A significant correlation between EGFR protein and mRNA levels (p = 0.0077) was found, demonstrating the reliability of our quantification method of EGFR membrane expression. Radically resected patients (p = 0.005) and those with epithelial histotype (p = 0.048) showed an increased survival. No statistical correlation between EGFR immunoreactivity and patients survival was observed. No EGFR mutation was documented. This study documents EGFR overexpression in MPM at the protein and the transcriptional levels; it proposes a reliable method for EGFR expression evaluation in MPM. EGFR levels are not associated with clinico-pathological features of patients, including survival. © 2005 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Malignant pleural mesothelioma (MPM) is an aggressive cancer, with predicted increasing incidence in the next 10—20 years [1]. MPM is poorly responsive to current oncological therapies and is rarely amenable to radical surgical resection [2—7]. Epidermal growth factor receptor (EGFR) is a tyrosine-kinase receptor involved in cell death and proliferation, cell motility, angiogenesis and extracellular matrix composition [8]. EGFR gene alterations are thought to be important in neoplastic cell trasformation, tumor growth and cancer progression. EGFR is overexpressed in many human malignancies including lung, head and neck, colorectal and breast cancers [9] where it is variably associated with patients’ prognosis [10—12]. Several inhibitors of EGFR have been recently developed, including monoclonal antibodies (cetuximab) and small molecule inhibitors (gefitinib, erlotinib), which have been shown to be effective in animal models, in preclinical and clinical studies [13]. A correlation between EGFR expression and response to therapy has been reported in some human cancers (breast, lung and prostate) [14—18]. Furthermore, somatically acquired hot-spot gene activating mutations of the EGFR tyrosine-kinase domain have been recently documented to predict increased sensitivity to gefitinib in NSCLC [19,20]. EGFR expression in MPM was previously investigated mainly at protein level. EGFR immunoreactivity was detected in non-neoplastic/reactive pleural mesothelium and in variable percentages of MPM [21—25] with higher expression in the epithelial subtype [22,24]. Although EGFR protein overexpression did not seem to be an independent prognostic factor for survival of MPM patients [22,23], recent data suggest that EGFR could play a crucial role in biology and aggressiveness of this disease [8]. Furthermore, EGFR tyrosine-kinase inhibitors have been reported to inhibit MPM cell growth and migration, as well as matrix metalloprotease production [26]. The lack of a standardised methodol-
ogy of EGFR expression evaluation and quantification is thought to be one of the factors responsible for these discrepancies. Given the potential clinical significance of EGFR expression, there is a need to optimise and standardise the method of detection and quantification of EGFR expression in individual types of tumors, similarly to Her-2 immunohistochemical evaluation in breast cancer [27]. In this study, we evaluated EGFR protein expression and mRNA levels in MPM and non-neoplastic mesothelium; furthermore, gene activating mutations of EGFR tyrosine-kinase domain previously detected in NSCLC were investigated in a subset of patients. Immunohistochemical data were finally correlated to patients characteristics (gender and age, tumor histological type, proliferation index) and survival.
2. Materials and methods 2.1. Patients and samples Sixty-one consecutive MPM patients who underwent surgical procedures at Istituto Clinico Humanitas of Milan from 1997 to 2003 have been selected for this study. Twenty-two patients (36%) were radically resected with extrapleural pneumonectomy (EPP), 35 patients underwent partial pleurectomy (PP) (57%); in four cases (7%), a diagnostic biopsy only was performed. Informed consent was obtained from all patients under study. Tissue samples representative of the tumors were routinely fixed in 10% buffered neutral formalin and processed for conventional histological examination. In 17 cases, immediately adjacent tumor samples were snapfrozen in liquid nitrogen within 10 min from excision and stored at −80 ◦ C. Five and seven tissue samples obtained from macroscopically normal pleura were used as controls for immunohistochemical and molecular analysis, respectively. Standard 2 m-thick sections from all routinely processed tissue samples were stained with hematoxylin and eosin, examined by light microscopy. MPM histo-
EGFR overexpression in malignant pleural mesothelioma Table 1 patients
Clinico-pathological features of 61 MPM Cases
Mean age Gender Female Male
61.7 ± 11 19 (31%) 42 (69%)
Histology Epithelial Mixed Sarcomatous
50 (82%) 9 (15%) 2 (3%)
Surgery Radical (EPP) Palliative (PP) None
22 (36%) 35 (57%) 4 (7%)
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staining intensity. We obtained the following three categories of EGFR protein membrane expression. (a) Negative: score 0 (0—10% IR cells regardless the membrane staining intensity). (b) Low expression: score 1, 2 (more than 10% IR cells and weak membrane staining intensity or 11—50% IR cells and strong membrane staining intensity). (c) High expression: score 4 (more than 50% IR and strong membrane staining intensity). Examples of EGFR membrane immunoreactivity evaluation in MPM are represented in Fig. 1.
EPP: extrapleural pneumonectomy; PP: partial pleurectomy.
type was established according to the WHO classification of lung and pleural tumors [28]. All the macroscopically non-neoplastic pleural samples were judged to be benign. The amount of tumor cells in tissue samples for molecular analysis was equal or exceeded 80% of the sample. The clinicopathological features of these patients are reported in Table 1.
2.2. Immunohistochemistry Standard 2-m thick sections after deparaffination and rehydration were submitted to antigen retrieval (10 min at 40 ◦ C in 1 mg/ml Protease, Sigma Aldrich). As previously described [29], slides were incubated 1 h with mouse monoclonal antibody anti-human EGFR antigen (Neomarkers, Union City, CA) diluted to a final concentration of 4 g/ml. Slides were then incubated with the secondary antibody using the DAKO EnVision Universal kit (DAKO Corporation, Carpinteria, CA). Staining was performed with 3,3 -diaminobenzidine (DAB) as a chromogen and sections were then counterstained with hematoxylin. 2.2.1. EGFR protein evaluation To evaluate EGFR membrane staining were considered the percentage of positive neoplastic cells and the intensity of immunoreactivity. According to the percentage of immunoreactive (IR) tumor cells, cases were scored as follows: 0 (0—10% IR cells); 1 (11—50% IR cells); 2 (>50% IR cells); the intensity of EGFR immunoreactivity was scored as follows: 0 (negative); 1 (weak); 2 (strong). A final score was assigned to each case by multiplying the percentage of tumor positive cells score and the score
Fig. 1 Examples of EGFR membrane immunoreactivity: (A) high expression; (B) low expression; (C) negative.
210 2.2.2. Proliferation index evaluation To evaluate the proliferation index, we used a mouse monoclonal antibody anti-human Ki-67 antigen, clone MIB-1 (DAKO Corporation, Carpinteria, CA), at final concentration of 4 g/ml, after antigen retrieval in 10 nmol/l citrate buffer pH 6.0, 15 min at 98 ◦ C. The proliferation index was evaluated as percentage of nuclear immunoreactivity/10 highpower field. For each batch negative control slides with absence of the primary antibody were included as negative control.
2.3. EGFR mRNA quantification by real-time RT-PCR EGFR mRNA levels in non-neoplastic pleura and in MPMs were measured by real-time quantitative RT-PCR based on TaqMan methodology, using ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Real-time RT-PCR data analysis was performed with geNorm software (http://medgen. ugent.be/∼jvdesomp/genorm/). This software determines the most stable housekeeping genes from a set of tested genes in a given cDNA sample panel and calculates a gene expression normalization factor for each sample based on the geometric mean of a user-defined number of housekeeping genes [30]. With this approach, we selected the following three internal control genes: -2 microglobulin (B2M), -actin (ACTB) and succinate dehydrogenase complex, subunit A (SDHA). Our results are expressed as relative levels of EGFR mRNA referred to a calibrator, which is a sample with the lowest EGFR mRNA level normalized to internal control genes. All of the analyzed samples express n-fold EGFR mRNA relative to the calibrator, normalized to internal control genes. The amount of target, normalized to an internal control gene and relative to calibrator is definite by the Ct method as described by Livak K (Sequence Detector User Bulletin 2, Applied Biosystems). Specifically, the formula is: Target Amount = 2−Ct , where Ct = {[Ct (EGFR sample) − Ct (housekeeping gene of sample)] − [Ct(EGFR calibrator) − Ct (housekeeping gene of calibrator)]}. 2.3.1. RNA extraction and cDNA synthesis Total RNA was extracted from 17 frozen tumor and 7 non-neoplastic pleural specimens using a commercial kit, Trizol (Invitrogen, Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Total RNA was quantified spectrophotometrically, and 500 ng was reverse transcribed in a final
A. Destro et al. volume of 100 l, using High-Capacity cDNA Archive kit (Applied Biosystems), as described by the manufacturer. The samples were incubated at 25 ◦ C for 10 min, 37 ◦ C for 120 min. 2.3.2. Primers and probes For the quantification of three internal controls, we used a ready-to-use assay (Assay-on-DemandTM Gene Expression Products), purchased from Applied Biosystems. It consists of a 20× mix of unlabeled PCR primers and TaqMan MGB probe (6-FAM at 5 -end and a no fluorescent quencher at 3 end). The assay identification numbers of selected genes are: Hs99999903 m1 (ACTB), Hs99999907 m1 (B2M), Hs00188166 m1 (SDHA). Primers and probe for EGFR mRNA were chosen using Primer Express computer program. Primer and probe nucleotide sequences for EGFR were: forward primer 5 -CCC AGT ACC TGC TCA ACT GGT-3 , reverse primer 5 -TGC CAG GTC GCG GTG-3 and TaqMan probe 5 -6(FAM)-TGC AGA TCG CAA AGG GCA TGA ACT AC-3 (TAMRA). 2.3.3. Real-time PCR conditions Amplification reactions were performed with TaqMan Universal PCR master mix (Applied Biosystems), using 5 l of cDNA in a final volume of 25 l. ACTB, B2M, SDHA primers and probes were added to the reaction mixture according to the manufacture’s directions, while EGFR primers and probes were present at 0.9 and 0.3 M, respectively. All reactions were performed in duplicate. The thermal cycling conditions included 2 min at 50 ◦ C and 10 min at 95 ◦ C, followed by 40 cycles of 95 ◦ C for 15 s and 60 ◦ C for 1 min.
2.4. Mutational analysis of EGFR by DHPLC DNA was isolated from three section (5 m thick) paraffin-embedded tumor samples of 16 MPM patients. After xylene deparaffinization, all samples were treated by digestion with proteinase K (Finzyme), followed by standard phenol—chloroform extraction and ethanol precipitation. PCR was used to amplify exon 18—21 of EGFR from genomics DNAs using standard methods with primers previously described [19]. PCR amplification was performed in 25 l final volume mix containing 10—50 ng of genomic DNA, 200 M dNTPs, 1.5 mM MgCl2 , 10 pmol of each primer and 1.25 U AmpliTaq-Gold (Applied Biosystem). All EGFR amplimers were amplified successfully at an annealing temperature of 55 ◦ C. Products were subjected to mutational analysis by denaturation high performance liquid chromatography (DHPLC WATEM system Transgenomic Inc., Omaha,
EGFR overexpression in malignant pleural mesothelioma NE). To ensure equimolar amounts of the heteroduplex, amplicons were denatured at 95 ◦ C for 5 min and allowed to reanneal gradually (45 min) to 30 ◦ C. PCR products (10 l) diluted with 10 l MilliQ H2 O was loaded on the DHPLC column and eluted with a linear acetonitrile gradient calculate by the WAVEMaker(tm) software at a flow rate of 1.5 ml/min. The WAVE DNA Fragment Analysis System (Transgenomic) and associated WAVE-Maker(tm) software were used. Preparation and analysis of the PCR products was carried out as previously described [31]. The temperature of heteroduplex analysis was primarily established by using the DHPLC melting algorithm WAVE MakerTM of the WAVETM instrument. The final temperature for optimal resolution of homoduplexes and heteroduplexes of each fragment were: 60.9—62.9 ◦ C exon 18, 60.1—60.5 ◦ C exon 19, 61.2—61.8 ◦ C exon 20, 60.8—61.3 ◦ C exon 21. Sequences obtained were aligned with normal sequences and examined for the presence of mutations. Wild-type EGFR sequence was obtained from Genbank number AY588246. Samples with an altered DHPLC profiles were purified with DyeEx 2.0 Spin kit (Qiagen, Hilden, Germany) and then sequenced. The bi-directional sequencing amplification was performed with primers used in DHPLC and with BigDye(tm) Terminator v1.1 Cycle Sequencing Kits (Applied Biosystems) in accordance with the manufacturer’s instructions and sequenced by ABIPRISM 310(r) Genetic Analyzer (Applied Biosystem), using sequence analysis 3.4.1 software (Applied Biosystem).
2.5. Statistical analysis The Fisher exact test was used to analyze the correlation between EGFR immunohistochemical expression and MPM histotype, or gender of patients. Correlation between immunohistochemical expression of EGFR and EGFR mRNA levels, proliferation index and age of patients were analyzed with ANOVA. A p-value less than 0.05 was considered significant. Survival curves were generated with Kaplan—Meier method. Overall survival was calculated starting from the date of surgical resection to death or last follow-up visit. The possible prognostic factors were subjected to a univariate analysis using the Cox regression analysis. The following variables were analyzed: gender, age, tumor histotype (epithelial versus non-epithelial), therapy (radical versus palliative or no surgery), EGFR score (0 versus 1—2 versus 4). All variables found to have a p ≤ 0.1 in the univariate evaluation
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were considered to be candidates for a subsequent stepwise Cox regression analysis. All calculations were performed with Stata 6 (http://www.stata.com/).
3. Results 3.1. EGFR immunoreactivity EGFR membrane immunoreactivity was documented in 34/61 (55.7%) cases: 9/61 (14.8%) cases showed high expression levels (strong membrane staining in more than 50% IR cells) and 25/61 (41.0%) cases showed low expression levels (weak membrane staining in more than 10% IR cells and strong membrane staining in 11—50% IR cells). No immunoreactivity was detected in the five normal pleural samples. No statistical association was found between EGFR expression and gender, age, histotype and proliferation index. Specifically, proliferation index evaluated by Ki-67 was 20.9% in cases with high EGFR expression, 19.7% in cases with low expression and 19.8% in negative cases. Fifty-six patients were valuable for analysis of prognostic factors. Five were excluded: three were lost to follow-up and two died post-operatively. No other patients died of cancer unrelated causes. Cox regression analysis (Table 2) showed that an increased survival correlated with the extension of the surgical resection (p = 0.005) (Fig. 2A) and with the epithelial histotype (p = 0.048) (Fig. 2B). Survival curves failed to document a significant association between patients’ outcome and EGFR expression (Fig. 2C).
3.2. Correlation between immunohistochemical expression of EGFR and mRNA levels Seven specimens of non-neoplastic pleura were used as control to determine EGFR mRNA basal levels. All samples expressed detectable levels of EGFR mRNA, ranging from 3 to 15 (mean value: 8.3). EGFR mRNA levels were evaluated in 17 MPM and in all cases EGFR mRNA could be documented. Four of the 17 cases (23.5%) showed very low levels of EGFR transcript, comparable to normal counterparts, whereas 13 (76.5%) tumors displayed mRNA levels higher than normal samples ranging from 22 to 110 (Table 3). A significant correlation was detected between EGFR immunohystochemical expression and mRNA levels (p = 0.0077). Specifically, higher mRNA levels
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Table 2 Correlation between survival and radical surgery, tumor histology and EGFR score, by univariate and multivariate analysis Univariate
Surgery Histology EGFR score
Multivariate
Haz. ratio
(95% conf. interval)
P > |z|
Haz. ratio
(95% conf. interval)
P > |z|
0.3440 0.496 1.108
0.153 0.221 0.980
0.010 0.089 0.100
0.306 0.433 1.112
0.135 0.189 0.985
0.005 0.048 0.086
0.772 1.113 1.253
0.695 0.991 1.255
Table 3 EGFR immunohistochemical expression and mRNA levels in 17 MPM cases Case
Immunoreactivity
Score
mRNA levels
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Negative Negative Low expression Low expression Negative Negative Low expression Negative Low expression High expression High expression Low expression Low expression High expression Low expression High expression High expression
0 0 1 1 0 0 1 0 2 4 4 1 1 4 2 4 4
1 6 10 13 22 24 24 28 29 42 46 48 49 50 66 99 110
(mean value 69.4) were seen in cases showing high EGFR immunohystochemical expression, intermediate mRNA levels (mean value 34.1) in cases showing lower EGFR expression; the lowest mRNA levels (mean value 16.2) were seen in EGFR immunonegative cases (Fig. 3).
3.3. EGFR mutational analysis by DHPLC Mutations in exons 18—21 of EGFR were investigated by DHPLC in 16 cases analyzed with realtime PCR. No mutations of the EGFR gene were found.
4. Discussion
Fig. 2 Survival curves related to radical surgery (A), histotype (B) and EGFR score (C). For each of the curves, the number of patients is shown.
EGFR activation is undergoing intensive research in human tumors, given the current availability of an increasing number of drugs with antireceptor activity. EGFR immunohistochemical expression in malignant mesothelioma has been previously reported, with controversial results, possibly due to
EGFR overexpression in malignant pleural mesothelioma
Fig. 3 Correlation of mRNA levels and protein immunoreactivity. mRNA levels are expressed as relative levels referred to a calibrator and normalized to internal control genes (see Section 2). (A) Non-neoplastic pleura; (B) immunonegative MPM; (C) low immunoexpressor MPM; (D) high immunoexpressors MPM. The lowest, the median and the highest values, the first and third quartile values for each category are shown.
the lack of a standardized method of EGFR detection and quantification [21—25]. To document EGFR overexpression, we compared immunohistochemical expression in tissue with mRNA levels detected by real-time PCR. To the best of our knowledge, this is the first study in which EGFR mRNA levels have been quantified in MPM and in which EGFR expression, both at protein and at transcript levels, has been analyzed. In this study, we have developed a scoring system of EGFR membrane staining quantification. We preliminarily defined three categories of immunohistochemical expression (negative, low and high expression) based on both the staining intensity and the number of immunoreactive malignant cells. We found that more than 50% of patients showed EGFR membrane immunoreactivity. Interestingly, we documented a statistically significant correlation between the immunohistochemical expression of EGFR and corresponding mRNA levels (p = 0.0077). Therefore, our quantification method of EGFR membrane expression in the three staining categories (i.e. negative, low and high expression) reflected the actual synthesis of EGFR in malignant mesothelial cells. In spite of the reduced number of non-neoplastic samples analyzed, EGFR mRNA levels were higher in tumor specimens than nonneoplastic pleura. This data confirms and enlarges previous observations suggesting an important role of EGFR overexpression in pleural carcinogenesis [8]. The second aim of the present study was to correlate EGFR immunohistochemical data with common clinico-pathological parameters (patients
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age and gender, tumor histotype and proliferating index), including follow-up informations. We have not documented any significant association between EGFR expression levels and age, gender or proliferating index. As recently reported by Govindan et al., we have not observed a statistical correlation between EGFR expression and patients survival [23]. Patients’ outcome was dependent on the histological diagnosis as already reported [32]. A recent report of Phase II study on MPM patients concludes that the effectiveness of Gefitinib is not dependent on EGFR overexpression [23]. In order to select potentially sensitive patients, it could be useful to perform immunohistochemical studies of phosphorylated EGFR and downstream molecules [33]. Recently, no objective response was observed in MPM patients treated with erlotinib in SWOG S01218 trial; in this study, lack of Akt phosphorilation and lack of PTEN expression in the Akt pathway downstream of EGFR was postulated as a mechanism of MPM’s clinical resistance to erlotinib [34]. A significant association between clinical response to gefitinib and mutations in exons 18—21 of EGFR has been reported in lung cancer patients [19,20,35]. Lynch et al. documented these mutations in 8% of NSCLC patients, proposing these genetic alterations as potential markers of sensitivity to antireceptor therapy [19]. In another study on 860 NSCLC patients, Marchetti et al. [36] reported a 10% incidence of mutations in adenocarcinoma with higher rates in bronchiolaralveolar carcinoma (26%). Therefore, screening for these mutations in NSCLC may identify responsive patients to gefitinib. Consequently, a third target of our study was to investigate whether EGFR mutations (exons 18—21) could also be a feature of MPM. We did not find any mutations in the tested exons suggesting that in MPM EGFR mutations do not contribute to receptor activation. However, in NSCLC, the frequency of this mutations is very low, and given that we could conduct our genomic analysis in 16 cases only, we cannot exclude the possible presence of a EGFR mutation in a small subset of MPM patients. In conclusion, in this study, we documented EGFR overexpression, both at protein and at transcriptional level, in human MPM. We have validated an immunohistochemical method for the semiquantitative evaluation of EGFR expression levels in MPM. However, no correlation between EGFR expression levels and clinico-pathological data, including survival, was found. Whether EGFR mutation could be a marker of sensitivity to antireceptor therapy in MPM, remains to be established in specific clinical trials.
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References [18] [1] Peto J, Decarli A, La Vecchia C, Levi F, Negri E. The European mesothelioma epidemic. Br J Cancer 1999;79:666—72. [2] Treasure T, Sedrakyan A. Pleural mesothelioma: little evidence, still time to do trials. Lancet 2004;364:1183—5. [3] Adjei AA. Pemetrexed (ALIMTA), a novel multitargeted antineoplastic agent. Clin Cancer Res 2004;10:4276S—80S. [4] Vogelzang NJ, Rusthoven JJ, Symanowski J, Denham C, Kaukel E, Ruffie P, et al. Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma. J Clin Oncol 2003;21:2636—44. [5] Baldini E. External beam radiation therapy for the treatment of pleural mesothelioma. Thorac Surg Clin 2004;14:543—8. [6] Sugarbaker DJ, Flores RM, Jaklitsch MT, Richards WG, Strauss GM, Corson JM, et al. Resection margins, extrapleural nodal status, and cell type determine postoperative long-term survival in trimodality therapy of malignant pleural mesothelioma: results in 183 patients. J Thorac Cardiovasc Surg 1999;117:54—65. [7] Sugarbaker DJ, Jaklitsch MT, Bueno R, Richards W, Lukanich J, Mentzer SJ, et al. Prevention, early detection, and management of complications after 328 consecutive extrapleural pneumonectomies. J Thorac Cardiovasc Surg 2004;128:138—46. [8] Janne PA, Taffaro ML, Salgia R, Johnson BE. Inhibition of epidermal growth factor receptor signaling in malignant pleural mesothelioma. Cancer Res 2002;62:5242—7. [9] Arteaga CL. Epidermal growth factor receptor dependence in human tumors: more than just expression? Oncologist 2002;7:31—9. [10] Brabender J, Danenberg KD, Metzger R, Schneider PM, Park J, Salonga D, et al. Epidermal growth factor receptor and HER2-neu mRNA expression in non-small cell lung cancer is correlated with survival. Clin Cancer Res 2001;7:1850—5. [11] Fox S, Smith K, Hollyer J, Greenall M, Hastrich D, Harris A. The epidermal growth factor receptor as a prognostic marker: results of 370 patients and review of 3009 patients. Breast Cancer Res Treat 1994;29:41—9. [12] Gasparini G, Gullick W, Bevilacqua P, Sainsbury J, Meli S, Boracchi P, et al. Human breast cancer: prognostic significance of the c-erbB-2 oncoprotein compared with epidermal growth factor receptor, DNA ploidy, and conventional pathologic features. J Clin Oncol 1992;10:686—95. [13] Mendelsohn J, Baselga J. Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer. J Clin Oncol 2003;21:2787—99. [14] Janmaat ML, Giaccone G. Small-molecule epidermal growth factor receptor tyrosine-kinase inhibitors. Oncologist 2003;8:576—86. [15] Magne N, Fischel J, Dubreuil A, Formento P, Poupon M, Laurent-Puig P, et al. Influence of epidermal growth factor receptor (EGFR), p53 and intrinsic MAP kinase pathway status of tumor cells on the antiproliferative effect of ZD1839 (‘‘Iressa’’). Br J Cancer 2002;86:1518—23. [16] Cappuzzo F, Gregorc V, Rossi E, Cancellieri A, Magrini E, Paties CT, et al. Gefitinib in pretreated non-small-cell lung cancer (NSCLC): analysis of efficacy and correlation with HER2 and epidermal growth factor receptor expression in locally advanced or metastatic NSCLC. J Clin Oncol 2003;21:2658—63. [17] Santoro A, Cavina R, Latteri F, Zucali PA, Ginanni V, Campagnoli E, et al. Activity of a specific inhibitor, gefitinib (IressaTM, ZD1839), of epidermal growth factor recep-
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28] [29]
[30]
[31]
[32]
[33]
tor in refractory non-small-cell lung cancer. Ann Oncol 2004;15:33—7. Kakiuchi S, Daigo Y, Ishikawa N, Furukawa C, Tsunoda T, Yano S, et al. Prediction of sensitivity of advanced non-small cell lung cancers to gefitinib (Iressa, ZD1839). Hum Mol Genet 2004;13:3029—43. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129—39. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304:1497—500. Cai Y, Roggli V, Mark E, Cagle P, Fraire A. Transforming growth factor alpha and epidermal growth factor receptor in reactive and malignant mesothelial proliferations. Arch Pathol Lab Med 2004;128:68—70. Dazzi H, Hasleton P, Thatcher N, Wilkes S, Swindell R, Chatterjee A. Malignant pleural mesothelioma and epidermal growth factor receptor (EGF-R). Relationship of EGFR with histology and survival using fixed paraffin embedded tissue and the F4, monoclonal antibody. Br J Cancer 1990;61:924—6. Govindan R, Kratzke RA, Herndon II JE, Niehans GA, Vollmer R, Watson HL, et al., On behalf of the Cancer and Leukemia Group B. Gefitinib in patients with malignant mesothelioma: a phase ii study by the Cancer and Leukemia Group B. Clin Cancer Res 2005;11:2300—4. Ramael M, Segers K, Buysse C, Van den Bossche J, Van Marck E. Immunohistochemical distribution patterns of epidermal growth factor receptor in malignant mesothelioma and non-neoplastic mesothelium. Virch Arch A Pathol Anat Histopathol 1991;419:171—5. Trupiano J, Geisinger K, Willingham M, Manders P, Zbieranski N, Case D, et al. Diffuse malignant mesothelioma of the peritoneum and pleura, analysis of markers. Mod Pathol 2004;17:476—81. Liu Z, Klominek J. Inhibition of proliferation, migration, and matrix metalloprotease production in malignant mesothelioma cells by tyrosine-kinase inhibitors. Neoplasia 2004;6:705—12. Lewis F, Jackson P, Lane S, Coast G, Hanby A. Testing for HER2 in breast cancer. Histopathology 2004;45: 207—17. Travis WD, Colby TV, Corrin B, Shimosato Y, Brambilla E. Histological typing of lung and pleural tumors. Springer; 1999. Parra H, Cavina R, Latteri F, Zucali P, Campagnoli E, Morenghi E, et al. Analysis of epidermal growth factor receptor expression as a predictive factor for response to gefitinib (‘Iressa’, ZD1839) in non-small-cell lung cancer. Br J Cancer 2004;91:208—12. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of realtime quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002;3. RESEARCH0034. Mihalatos M, Apessos A, Triantafillidis J, Kosmidis P, Fountzilas G, Agnantis N, et al. Evaluation of dHPLC in mutation screening of the APC gene in a Greek FAP cohort. Anticancer Res 2003;23:2691—5. Ceresoli GL, Locati L, Ferreri A, Cozzarini C, Passoni P, Melloni G, et al. Therapeutic outcome according to histologic subtype in 121 patients with malignant pleural mesothelioma. Lung Cancer 2001;34:279—87. Cappuzzo F, Magrini E, Ceresoli GL, Bartolini S, Rossi E, Ludovini V, et al. Akt phosphorylation and gefitinib efficacy
EGFR overexpression in malignant pleural mesothelioma in patients with advanced non-small-cell lung cancer. J Natl Cancer Inst 2004;96:1133—41. [34] Garland L, Rankin C, Scott K, Nagle R, Lobell M, Gandara D, et al. Molecular correlates of the EGFR signaling pathway in association with SWOG S0218: a phase II study of oral EGFR tyrosine-kinase inhibitor OSI-774 (NSC-718781) in patients with malignant pleural mesothelioma (MPM). J Clin Oncol, 2004 ASCO Annual Meeting, Vol. 22, 145, 2004:3007. [35] Pao W, Miller V, Zakowski M, Doherty J, Politi K, Sarkaria I, et al. EGF receptor gene mutations are common in
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lung cancers from ‘‘never smokers’’ and are associated with sensitivity of tumors to gefitinib and erlotinib. PNAS 2004;101:13306—11. [36] Marchetti A, Martella C, Felicioni L, Barassi F, Salvatore S, Chella A, et al. EGFR mutations in non-small-cell lung cancer: analysis of a large series of cases and development of a rapid and sensitive method for diagnostic screening with potential implications on pharmacologic treatment. J Clin Oncol 2005;23:857—65.
EGFR overexpression in malignant pleural mesothelioma An immunohistochemical and molecular study with clinico-pathological correlations夽 A. Destro a, G.L. Ceresoli b, M. Falleni c, P.A. Zucali b, E. Morenghi d, P. Bianchi a, C. Pellegrini c, N. Cordani c, V. Vaira c, M. Alloisio e, A. Rizzi f, S. Bosari c, M. Roncalli g,∗ a
Molecular Genetics Laboratory, IRCCS Istituto Clinico Humanitas, Via Manzoni 56, 20089 Rozzano, Milano, Italy b Department of Oncology, IRCCS Istituto Clinico Humanitas, Via Manzoni 56, 20089 Rozzano, Milano, Italy c Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, via A. di Rudin´ ı 8, 20142 Milan, Italy d Clinical Trial Office, IRCCS Istituto Clinico Humanitas, Via Manzoni 56, 20089 Rozzano, Milano, Italy e Department of Thoracic Surgery, IRCCS Istituto Clinico Humanitas, Via Manzoni 56, 20089 Rozzano, Milano, Italy f Department of Thoracic Surgery, Humanitas Gavazzeni, Via Mauro Gavazzeni, 21-24125 Bergamo, Italy g Department of Pathology, University of Milan, IRCCS Istituto Clinico Humanitas, Via Manzoni 56, 20089 Rozzano, Milano, Italy Received 24 June 2005 ; received in revised form 22 September 2005; accepted 4 October 2005 KEYWORDS Mesothelioma; EGFR; Immunohistochemistry; Real-time PCR; Prognostic factors
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Summary The epidermal growth factor receptor (EGFR) is overexpressed in many epithelial malignancies, against which some antitumoral drugs have been developed. There is a lack of information as to EGFR expression in malignant pleural mesothelioma (MPM), an aggressive and fatal cancer poorly responsive to current oncological treatments. Our aim was to: (a) compare EGFR immunohistochemical expression with mRNA levels measured by real time PCR; (b) assess the relationships between EGFR expression and clinico-pathological data including survival; (c) analyze the EGFR mutations. We developed an immunohistochemical method of EGFR evaluation based on the number of immunoreactive cells and staining intensity in 61 MPMs.
This work was partly supported by MURST (COFIN 2003). Corresponding author. Tel.: +39 02 82244714; fax: +39 02 82244791. E-mail address: massimo [email protected] (M. Roncalli).
0169-5002/$ — see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2005.10.016
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A. Destro et al. EGFR immunoreactivity was documented in 34/61 (55.7%) cases. A significant correlation between EGFR protein and mRNA levels (p = 0.0077) was found, demonstrating the reliability of our quantification method of EGFR membrane expression. Radically resected patients (p = 0.005) and those with epithelial histotype (p = 0.048) showed an increased survival. No statistical correlation between EGFR immunoreactivity and patients survival was observed. No EGFR mutation was documented. This study documents EGFR overexpression in MPM at the protein and the transcriptional levels; it proposes a reliable method for EGFR expression evaluation in MPM. EGFR levels are not associated with clinico-pathological features of patients, including survival. © 2005 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Malignant pleural mesothelioma (MPM) is an aggressive cancer, with predicted increasing incidence in the next 10—20 years [1]. MPM is poorly responsive to current oncological therapies and is rarely amenable to radical surgical resection [2—7]. Epidermal growth factor receptor (EGFR) is a tyrosine-kinase receptor involved in cell death and proliferation, cell motility, angiogenesis and extracellular matrix composition [8]. EGFR gene alterations are thought to be important in neoplastic cell trasformation, tumor growth and cancer progression. EGFR is overexpressed in many human malignancies including lung, head and neck, colorectal and breast cancers [9] where it is variably associated with patients’ prognosis [10—12]. Several inhibitors of EGFR have been recently developed, including monoclonal antibodies (cetuximab) and small molecule inhibitors (gefitinib, erlotinib), which have been shown to be effective in animal models, in preclinical and clinical studies [13]. A correlation between EGFR expression and response to therapy has been reported in some human cancers (breast, lung and prostate) [14—18]. Furthermore, somatically acquired hot-spot gene activating mutations of the EGFR tyrosine-kinase domain have been recently documented to predict increased sensitivity to gefitinib in NSCLC [19,20]. EGFR expression in MPM was previously investigated mainly at protein level. EGFR immunoreactivity was detected in non-neoplastic/reactive pleural mesothelium and in variable percentages of MPM [21—25] with higher expression in the epithelial subtype [22,24]. Although EGFR protein overexpression did not seem to be an independent prognostic factor for survival of MPM patients [22,23], recent data suggest that EGFR could play a crucial role in biology and aggressiveness of this disease [8]. Furthermore, EGFR tyrosine-kinase inhibitors have been reported to inhibit MPM cell growth and migration, as well as matrix metalloprotease production [26]. The lack of a standardised methodol-
ogy of EGFR expression evaluation and quantification is thought to be one of the factors responsible for these discrepancies. Given the potential clinical significance of EGFR expression, there is a need to optimise and standardise the method of detection and quantification of EGFR expression in individual types of tumors, similarly to Her-2 immunohistochemical evaluation in breast cancer [27]. In this study, we evaluated EGFR protein expression and mRNA levels in MPM and non-neoplastic mesothelium; furthermore, gene activating mutations of EGFR tyrosine-kinase domain previously detected in NSCLC were investigated in a subset of patients. Immunohistochemical data were finally correlated to patients characteristics (gender and age, tumor histological type, proliferation index) and survival.
2. Materials and methods 2.1. Patients and samples Sixty-one consecutive MPM patients who underwent surgical procedures at Istituto Clinico Humanitas of Milan from 1997 to 2003 have been selected for this study. Twenty-two patients (36%) were radically resected with extrapleural pneumonectomy (EPP), 35 patients underwent partial pleurectomy (PP) (57%); in four cases (7%), a diagnostic biopsy only was performed. Informed consent was obtained from all patients under study. Tissue samples representative of the tumors were routinely fixed in 10% buffered neutral formalin and processed for conventional histological examination. In 17 cases, immediately adjacent tumor samples were snapfrozen in liquid nitrogen within 10 min from excision and stored at −80 ◦ C. Five and seven tissue samples obtained from macroscopically normal pleura were used as controls for immunohistochemical and molecular analysis, respectively. Standard 2 m-thick sections from all routinely processed tissue samples were stained with hematoxylin and eosin, examined by light microscopy. MPM histo-
EGFR overexpression in malignant pleural mesothelioma Table 1 patients
Clinico-pathological features of 61 MPM Cases
Mean age Gender Female Male
61.7 ± 11 19 (31%) 42 (69%)
Histology Epithelial Mixed Sarcomatous
50 (82%) 9 (15%) 2 (3%)
Surgery Radical (EPP) Palliative (PP) None
22 (36%) 35 (57%) 4 (7%)
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staining intensity. We obtained the following three categories of EGFR protein membrane expression. (a) Negative: score 0 (0—10% IR cells regardless the membrane staining intensity). (b) Low expression: score 1, 2 (more than 10% IR cells and weak membrane staining intensity or 11—50% IR cells and strong membrane staining intensity). (c) High expression: score 4 (more than 50% IR and strong membrane staining intensity). Examples of EGFR membrane immunoreactivity evaluation in MPM are represented in Fig. 1.
EPP: extrapleural pneumonectomy; PP: partial pleurectomy.
type was established according to the WHO classification of lung and pleural tumors [28]. All the macroscopically non-neoplastic pleural samples were judged to be benign. The amount of tumor cells in tissue samples for molecular analysis was equal or exceeded 80% of the sample. The clinicopathological features of these patients are reported in Table 1.
2.2. Immunohistochemistry Standard 2-m thick sections after deparaffination and rehydration were submitted to antigen retrieval (10 min at 40 ◦ C in 1 mg/ml Protease, Sigma Aldrich). As previously described [29], slides were incubated 1 h with mouse monoclonal antibody anti-human EGFR antigen (Neomarkers, Union City, CA) diluted to a final concentration of 4 g/ml. Slides were then incubated with the secondary antibody using the DAKO EnVision Universal kit (DAKO Corporation, Carpinteria, CA). Staining was performed with 3,3 -diaminobenzidine (DAB) as a chromogen and sections were then counterstained with hematoxylin. 2.2.1. EGFR protein evaluation To evaluate EGFR membrane staining were considered the percentage of positive neoplastic cells and the intensity of immunoreactivity. According to the percentage of immunoreactive (IR) tumor cells, cases were scored as follows: 0 (0—10% IR cells); 1 (11—50% IR cells); 2 (>50% IR cells); the intensity of EGFR immunoreactivity was scored as follows: 0 (negative); 1 (weak); 2 (strong). A final score was assigned to each case by multiplying the percentage of tumor positive cells score and the score
Fig. 1 Examples of EGFR membrane immunoreactivity: (A) high expression; (B) low expression; (C) negative.
210 2.2.2. Proliferation index evaluation To evaluate the proliferation index, we used a mouse monoclonal antibody anti-human Ki-67 antigen, clone MIB-1 (DAKO Corporation, Carpinteria, CA), at final concentration of 4 g/ml, after antigen retrieval in 10 nmol/l citrate buffer pH 6.0, 15 min at 98 ◦ C. The proliferation index was evaluated as percentage of nuclear immunoreactivity/10 highpower field. For each batch negative control slides with absence of the primary antibody were included as negative control.
2.3. EGFR mRNA quantification by real-time RT-PCR EGFR mRNA levels in non-neoplastic pleura and in MPMs were measured by real-time quantitative RT-PCR based on TaqMan methodology, using ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Real-time RT-PCR data analysis was performed with geNorm software (http://medgen. ugent.be/∼jvdesomp/genorm/). This software determines the most stable housekeeping genes from a set of tested genes in a given cDNA sample panel and calculates a gene expression normalization factor for each sample based on the geometric mean of a user-defined number of housekeeping genes [30]. With this approach, we selected the following three internal control genes: -2 microglobulin (B2M), -actin (ACTB) and succinate dehydrogenase complex, subunit A (SDHA). Our results are expressed as relative levels of EGFR mRNA referred to a calibrator, which is a sample with the lowest EGFR mRNA level normalized to internal control genes. All of the analyzed samples express n-fold EGFR mRNA relative to the calibrator, normalized to internal control genes. The amount of target, normalized to an internal control gene and relative to calibrator is definite by the Ct method as described by Livak K (Sequence Detector User Bulletin 2, Applied Biosystems). Specifically, the formula is: Target Amount = 2−Ct , where Ct = {[Ct (EGFR sample) − Ct (housekeeping gene of sample)] − [Ct(EGFR calibrator) − Ct (housekeeping gene of calibrator)]}. 2.3.1. RNA extraction and cDNA synthesis Total RNA was extracted from 17 frozen tumor and 7 non-neoplastic pleural specimens using a commercial kit, Trizol (Invitrogen, Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Total RNA was quantified spectrophotometrically, and 500 ng was reverse transcribed in a final
A. Destro et al. volume of 100 l, using High-Capacity cDNA Archive kit (Applied Biosystems), as described by the manufacturer. The samples were incubated at 25 ◦ C for 10 min, 37 ◦ C for 120 min. 2.3.2. Primers and probes For the quantification of three internal controls, we used a ready-to-use assay (Assay-on-DemandTM Gene Expression Products), purchased from Applied Biosystems. It consists of a 20× mix of unlabeled PCR primers and TaqMan MGB probe (6-FAM at 5 -end and a no fluorescent quencher at 3 end). The assay identification numbers of selected genes are: Hs99999903 m1 (ACTB), Hs99999907 m1 (B2M), Hs00188166 m1 (SDHA). Primers and probe for EGFR mRNA were chosen using Primer Express computer program. Primer and probe nucleotide sequences for EGFR were: forward primer 5 -CCC AGT ACC TGC TCA ACT GGT-3 , reverse primer 5 -TGC CAG GTC GCG GTG-3 and TaqMan probe 5 -6(FAM)-TGC AGA TCG CAA AGG GCA TGA ACT AC-3 (TAMRA). 2.3.3. Real-time PCR conditions Amplification reactions were performed with TaqMan Universal PCR master mix (Applied Biosystems), using 5 l of cDNA in a final volume of 25 l. ACTB, B2M, SDHA primers and probes were added to the reaction mixture according to the manufacture’s directions, while EGFR primers and probes were present at 0.9 and 0.3 M, respectively. All reactions were performed in duplicate. The thermal cycling conditions included 2 min at 50 ◦ C and 10 min at 95 ◦ C, followed by 40 cycles of 95 ◦ C for 15 s and 60 ◦ C for 1 min.
2.4. Mutational analysis of EGFR by DHPLC DNA was isolated from three section (5 m thick) paraffin-embedded tumor samples of 16 MPM patients. After xylene deparaffinization, all samples were treated by digestion with proteinase K (Finzyme), followed by standard phenol—chloroform extraction and ethanol precipitation. PCR was used to amplify exon 18—21 of EGFR from genomics DNAs using standard methods with primers previously described [19]. PCR amplification was performed in 25 l final volume mix containing 10—50 ng of genomic DNA, 200 M dNTPs, 1.5 mM MgCl2 , 10 pmol of each primer and 1.25 U AmpliTaq-Gold (Applied Biosystem). All EGFR amplimers were amplified successfully at an annealing temperature of 55 ◦ C. Products were subjected to mutational analysis by denaturation high performance liquid chromatography (DHPLC WATEM system Transgenomic Inc., Omaha,
EGFR overexpression in malignant pleural mesothelioma NE). To ensure equimolar amounts of the heteroduplex, amplicons were denatured at 95 ◦ C for 5 min and allowed to reanneal gradually (45 min) to 30 ◦ C. PCR products (10 l) diluted with 10 l MilliQ H2 O was loaded on the DHPLC column and eluted with a linear acetonitrile gradient calculate by the WAVEMaker(tm) software at a flow rate of 1.5 ml/min. The WAVE DNA Fragment Analysis System (Transgenomic) and associated WAVE-Maker(tm) software were used. Preparation and analysis of the PCR products was carried out as previously described [31]. The temperature of heteroduplex analysis was primarily established by using the DHPLC melting algorithm WAVE MakerTM of the WAVETM instrument. The final temperature for optimal resolution of homoduplexes and heteroduplexes of each fragment were: 60.9—62.9 ◦ C exon 18, 60.1—60.5 ◦ C exon 19, 61.2—61.8 ◦ C exon 20, 60.8—61.3 ◦ C exon 21. Sequences obtained were aligned with normal sequences and examined for the presence of mutations. Wild-type EGFR sequence was obtained from Genbank number AY588246. Samples with an altered DHPLC profiles were purified with DyeEx 2.0 Spin kit (Qiagen, Hilden, Germany) and then sequenced. The bi-directional sequencing amplification was performed with primers used in DHPLC and with BigDye(tm) Terminator v1.1 Cycle Sequencing Kits (Applied Biosystems) in accordance with the manufacturer’s instructions and sequenced by ABIPRISM 310(r) Genetic Analyzer (Applied Biosystem), using sequence analysis 3.4.1 software (Applied Biosystem).
2.5. Statistical analysis The Fisher exact test was used to analyze the correlation between EGFR immunohistochemical expression and MPM histotype, or gender of patients. Correlation between immunohistochemical expression of EGFR and EGFR mRNA levels, proliferation index and age of patients were analyzed with ANOVA. A p-value less than 0.05 was considered significant. Survival curves were generated with Kaplan—Meier method. Overall survival was calculated starting from the date of surgical resection to death or last follow-up visit. The possible prognostic factors were subjected to a univariate analysis using the Cox regression analysis. The following variables were analyzed: gender, age, tumor histotype (epithelial versus non-epithelial), therapy (radical versus palliative or no surgery), EGFR score (0 versus 1—2 versus 4). All variables found to have a p ≤ 0.1 in the univariate evaluation
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were considered to be candidates for a subsequent stepwise Cox regression analysis. All calculations were performed with Stata 6 (http://www.stata.com/).
3. Results 3.1. EGFR immunoreactivity EGFR membrane immunoreactivity was documented in 34/61 (55.7%) cases: 9/61 (14.8%) cases showed high expression levels (strong membrane staining in more than 50% IR cells) and 25/61 (41.0%) cases showed low expression levels (weak membrane staining in more than 10% IR cells and strong membrane staining in 11—50% IR cells). No immunoreactivity was detected in the five normal pleural samples. No statistical association was found between EGFR expression and gender, age, histotype and proliferation index. Specifically, proliferation index evaluated by Ki-67 was 20.9% in cases with high EGFR expression, 19.7% in cases with low expression and 19.8% in negative cases. Fifty-six patients were valuable for analysis of prognostic factors. Five were excluded: three were lost to follow-up and two died post-operatively. No other patients died of cancer unrelated causes. Cox regression analysis (Table 2) showed that an increased survival correlated with the extension of the surgical resection (p = 0.005) (Fig. 2A) and with the epithelial histotype (p = 0.048) (Fig. 2B). Survival curves failed to document a significant association between patients’ outcome and EGFR expression (Fig. 2C).
3.2. Correlation between immunohistochemical expression of EGFR and mRNA levels Seven specimens of non-neoplastic pleura were used as control to determine EGFR mRNA basal levels. All samples expressed detectable levels of EGFR mRNA, ranging from 3 to 15 (mean value: 8.3). EGFR mRNA levels were evaluated in 17 MPM and in all cases EGFR mRNA could be documented. Four of the 17 cases (23.5%) showed very low levels of EGFR transcript, comparable to normal counterparts, whereas 13 (76.5%) tumors displayed mRNA levels higher than normal samples ranging from 22 to 110 (Table 3). A significant correlation was detected between EGFR immunohystochemical expression and mRNA levels (p = 0.0077). Specifically, higher mRNA levels
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Table 2 Correlation between survival and radical surgery, tumor histology and EGFR score, by univariate and multivariate analysis Univariate
Surgery Histology EGFR score
Multivariate
Haz. ratio
(95% conf. interval)
P > |z|
Haz. ratio
(95% conf. interval)
P > |z|
0.3440 0.496 1.108
0.153 0.221 0.980
0.010 0.089 0.100
0.306 0.433 1.112
0.135 0.189 0.985
0.005 0.048 0.086
0.772 1.113 1.253
0.695 0.991 1.255
Table 3 EGFR immunohistochemical expression and mRNA levels in 17 MPM cases Case
Immunoreactivity
Score
mRNA levels
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Negative Negative Low expression Low expression Negative Negative Low expression Negative Low expression High expression High expression Low expression Low expression High expression Low expression High expression High expression
0 0 1 1 0 0 1 0 2 4 4 1 1 4 2 4 4
1 6 10 13 22 24 24 28 29 42 46 48 49 50 66 99 110
(mean value 69.4) were seen in cases showing high EGFR immunohystochemical expression, intermediate mRNA levels (mean value 34.1) in cases showing lower EGFR expression; the lowest mRNA levels (mean value 16.2) were seen in EGFR immunonegative cases (Fig. 3).
3.3. EGFR mutational analysis by DHPLC Mutations in exons 18—21 of EGFR were investigated by DHPLC in 16 cases analyzed with realtime PCR. No mutations of the EGFR gene were found.
4. Discussion
Fig. 2 Survival curves related to radical surgery (A), histotype (B) and EGFR score (C). For each of the curves, the number of patients is shown.
EGFR activation is undergoing intensive research in human tumors, given the current availability of an increasing number of drugs with antireceptor activity. EGFR immunohistochemical expression in malignant mesothelioma has been previously reported, with controversial results, possibly due to
EGFR overexpression in malignant pleural mesothelioma
Fig. 3 Correlation of mRNA levels and protein immunoreactivity. mRNA levels are expressed as relative levels referred to a calibrator and normalized to internal control genes (see Section 2). (A) Non-neoplastic pleura; (B) immunonegative MPM; (C) low immunoexpressor MPM; (D) high immunoexpressors MPM. The lowest, the median and the highest values, the first and third quartile values for each category are shown.
the lack of a standardized method of EGFR detection and quantification [21—25]. To document EGFR overexpression, we compared immunohistochemical expression in tissue with mRNA levels detected by real-time PCR. To the best of our knowledge, this is the first study in which EGFR mRNA levels have been quantified in MPM and in which EGFR expression, both at protein and at transcript levels, has been analyzed. In this study, we have developed a scoring system of EGFR membrane staining quantification. We preliminarily defined three categories of immunohistochemical expression (negative, low and high expression) based on both the staining intensity and the number of immunoreactive malignant cells. We found that more than 50% of patients showed EGFR membrane immunoreactivity. Interestingly, we documented a statistically significant correlation between the immunohistochemical expression of EGFR and corresponding mRNA levels (p = 0.0077). Therefore, our quantification method of EGFR membrane expression in the three staining categories (i.e. negative, low and high expression) reflected the actual synthesis of EGFR in malignant mesothelial cells. In spite of the reduced number of non-neoplastic samples analyzed, EGFR mRNA levels were higher in tumor specimens than nonneoplastic pleura. This data confirms and enlarges previous observations suggesting an important role of EGFR overexpression in pleural carcinogenesis [8]. The second aim of the present study was to correlate EGFR immunohistochemical data with common clinico-pathological parameters (patients
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age and gender, tumor histotype and proliferating index), including follow-up informations. We have not documented any significant association between EGFR expression levels and age, gender or proliferating index. As recently reported by Govindan et al., we have not observed a statistical correlation between EGFR expression and patients survival [23]. Patients’ outcome was dependent on the histological diagnosis as already reported [32]. A recent report of Phase II study on MPM patients concludes that the effectiveness of Gefitinib is not dependent on EGFR overexpression [23]. In order to select potentially sensitive patients, it could be useful to perform immunohistochemical studies of phosphorylated EGFR and downstream molecules [33]. Recently, no objective response was observed in MPM patients treated with erlotinib in SWOG S01218 trial; in this study, lack of Akt phosphorilation and lack of PTEN expression in the Akt pathway downstream of EGFR was postulated as a mechanism of MPM’s clinical resistance to erlotinib [34]. A significant association between clinical response to gefitinib and mutations in exons 18—21 of EGFR has been reported in lung cancer patients [19,20,35]. Lynch et al. documented these mutations in 8% of NSCLC patients, proposing these genetic alterations as potential markers of sensitivity to antireceptor therapy [19]. In another study on 860 NSCLC patients, Marchetti et al. [36] reported a 10% incidence of mutations in adenocarcinoma with higher rates in bronchiolaralveolar carcinoma (26%). Therefore, screening for these mutations in NSCLC may identify responsive patients to gefitinib. Consequently, a third target of our study was to investigate whether EGFR mutations (exons 18—21) could also be a feature of MPM. We did not find any mutations in the tested exons suggesting that in MPM EGFR mutations do not contribute to receptor activation. However, in NSCLC, the frequency of this mutations is very low, and given that we could conduct our genomic analysis in 16 cases only, we cannot exclude the possible presence of a EGFR mutation in a small subset of MPM patients. In conclusion, in this study, we documented EGFR overexpression, both at protein and at transcriptional level, in human MPM. We have validated an immunohistochemical method for the semiquantitative evaluation of EGFR expression levels in MPM. However, no correlation between EGFR expression levels and clinico-pathological data, including survival, was found. Whether EGFR mutation could be a marker of sensitivity to antireceptor therapy in MPM, remains to be established in specific clinical trials.
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References [18] [1] Peto J, Decarli A, La Vecchia C, Levi F, Negri E. The European mesothelioma epidemic. Br J Cancer 1999;79:666—72. [2] Treasure T, Sedrakyan A. Pleural mesothelioma: little evidence, still time to do trials. Lancet 2004;364:1183—5. [3] Adjei AA. Pemetrexed (ALIMTA), a novel multitargeted antineoplastic agent. Clin Cancer Res 2004;10:4276S—80S. [4] Vogelzang NJ, Rusthoven JJ, Symanowski J, Denham C, Kaukel E, Ruffie P, et al. Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma. J Clin Oncol 2003;21:2636—44. [5] Baldini E. External beam radiation therapy for the treatment of pleural mesothelioma. Thorac Surg Clin 2004;14:543—8. [6] Sugarbaker DJ, Flores RM, Jaklitsch MT, Richards WG, Strauss GM, Corson JM, et al. Resection margins, extrapleural nodal status, and cell type determine postoperative long-term survival in trimodality therapy of malignant pleural mesothelioma: results in 183 patients. J Thorac Cardiovasc Surg 1999;117:54—65. [7] Sugarbaker DJ, Jaklitsch MT, Bueno R, Richards W, Lukanich J, Mentzer SJ, et al. Prevention, early detection, and management of complications after 328 consecutive extrapleural pneumonectomies. J Thorac Cardiovasc Surg 2004;128:138—46. [8] Janne PA, Taffaro ML, Salgia R, Johnson BE. Inhibition of epidermal growth factor receptor signaling in malignant pleural mesothelioma. Cancer Res 2002;62:5242—7. [9] Arteaga CL. Epidermal growth factor receptor dependence in human tumors: more than just expression? Oncologist 2002;7:31—9. [10] Brabender J, Danenberg KD, Metzger R, Schneider PM, Park J, Salonga D, et al. Epidermal growth factor receptor and HER2-neu mRNA expression in non-small cell lung cancer is correlated with survival. Clin Cancer Res 2001;7:1850—5. [11] Fox S, Smith K, Hollyer J, Greenall M, Hastrich D, Harris A. The epidermal growth factor receptor as a prognostic marker: results of 370 patients and review of 3009 patients. Breast Cancer Res Treat 1994;29:41—9. [12] Gasparini G, Gullick W, Bevilacqua P, Sainsbury J, Meli S, Boracchi P, et al. Human breast cancer: prognostic significance of the c-erbB-2 oncoprotein compared with epidermal growth factor receptor, DNA ploidy, and conventional pathologic features. J Clin Oncol 1992;10:686—95. [13] Mendelsohn J, Baselga J. Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer. J Clin Oncol 2003;21:2787—99. [14] Janmaat ML, Giaccone G. Small-molecule epidermal growth factor receptor tyrosine-kinase inhibitors. Oncologist 2003;8:576—86. [15] Magne N, Fischel J, Dubreuil A, Formento P, Poupon M, Laurent-Puig P, et al. Influence of epidermal growth factor receptor (EGFR), p53 and intrinsic MAP kinase pathway status of tumor cells on the antiproliferative effect of ZD1839 (‘‘Iressa’’). Br J Cancer 2002;86:1518—23. [16] Cappuzzo F, Gregorc V, Rossi E, Cancellieri A, Magrini E, Paties CT, et al. Gefitinib in pretreated non-small-cell lung cancer (NSCLC): analysis of efficacy and correlation with HER2 and epidermal growth factor receptor expression in locally advanced or metastatic NSCLC. J Clin Oncol 2003;21:2658—63. [17] Santoro A, Cavina R, Latteri F, Zucali PA, Ginanni V, Campagnoli E, et al. Activity of a specific inhibitor, gefitinib (IressaTM, ZD1839), of epidermal growth factor recep-
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28] [29]
[30]
[31]
[32]
[33]
tor in refractory non-small-cell lung cancer. Ann Oncol 2004;15:33—7. Kakiuchi S, Daigo Y, Ishikawa N, Furukawa C, Tsunoda T, Yano S, et al. Prediction of sensitivity of advanced non-small cell lung cancers to gefitinib (Iressa, ZD1839). Hum Mol Genet 2004;13:3029—43. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129—39. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304:1497—500. Cai Y, Roggli V, Mark E, Cagle P, Fraire A. Transforming growth factor alpha and epidermal growth factor receptor in reactive and malignant mesothelial proliferations. Arch Pathol Lab Med 2004;128:68—70. Dazzi H, Hasleton P, Thatcher N, Wilkes S, Swindell R, Chatterjee A. Malignant pleural mesothelioma and epidermal growth factor receptor (EGF-R). Relationship of EGFR with histology and survival using fixed paraffin embedded tissue and the F4, monoclonal antibody. Br J Cancer 1990;61:924—6. Govindan R, Kratzke RA, Herndon II JE, Niehans GA, Vollmer R, Watson HL, et al., On behalf of the Cancer and Leukemia Group B. Gefitinib in patients with malignant mesothelioma: a phase ii study by the Cancer and Leukemia Group B. Clin Cancer Res 2005;11:2300—4. Ramael M, Segers K, Buysse C, Van den Bossche J, Van Marck E. Immunohistochemical distribution patterns of epidermal growth factor receptor in malignant mesothelioma and non-neoplastic mesothelium. Virch Arch A Pathol Anat Histopathol 1991;419:171—5. Trupiano J, Geisinger K, Willingham M, Manders P, Zbieranski N, Case D, et al. Diffuse malignant mesothelioma of the peritoneum and pleura, analysis of markers. Mod Pathol 2004;17:476—81. Liu Z, Klominek J. Inhibition of proliferation, migration, and matrix metalloprotease production in malignant mesothelioma cells by tyrosine-kinase inhibitors. Neoplasia 2004;6:705—12. Lewis F, Jackson P, Lane S, Coast G, Hanby A. Testing for HER2 in breast cancer. Histopathology 2004;45: 207—17. Travis WD, Colby TV, Corrin B, Shimosato Y, Brambilla E. Histological typing of lung and pleural tumors. Springer; 1999. Parra H, Cavina R, Latteri F, Zucali P, Campagnoli E, Morenghi E, et al. Analysis of epidermal growth factor receptor expression as a predictive factor for response to gefitinib (‘Iressa’, ZD1839) in non-small-cell lung cancer. Br J Cancer 2004;91:208—12. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of realtime quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002;3. RESEARCH0034. Mihalatos M, Apessos A, Triantafillidis J, Kosmidis P, Fountzilas G, Agnantis N, et al. Evaluation of dHPLC in mutation screening of the APC gene in a Greek FAP cohort. Anticancer Res 2003;23:2691—5. Ceresoli GL, Locati L, Ferreri A, Cozzarini C, Passoni P, Melloni G, et al. Therapeutic outcome according to histologic subtype in 121 patients with malignant pleural mesothelioma. Lung Cancer 2001;34:279—87. Cappuzzo F, Magrini E, Ceresoli GL, Bartolini S, Rossi E, Ludovini V, et al. Akt phosphorylation and gefitinib efficacy
EGFR overexpression in malignant pleural mesothelioma in patients with advanced non-small-cell lung cancer. J Natl Cancer Inst 2004;96:1133—41. [34] Garland L, Rankin C, Scott K, Nagle R, Lobell M, Gandara D, et al. Molecular correlates of the EGFR signaling pathway in association with SWOG S0218: a phase II study of oral EGFR tyrosine-kinase inhibitor OSI-774 (NSC-718781) in patients with malignant pleural mesothelioma (MPM). J Clin Oncol, 2004 ASCO Annual Meeting, Vol. 22, 145, 2004:3007. [35] Pao W, Miller V, Zakowski M, Doherty J, Politi K, Sarkaria I, et al. EGF receptor gene mutations are common in
215
lung cancers from ‘‘never smokers’’ and are associated with sensitivity of tumors to gefitinib and erlotinib. PNAS 2004;101:13306—11. [36] Marchetti A, Martella C, Felicioni L, Barassi F, Salvatore S, Chella A, et al. EGFR mutations in non-small-cell lung cancer: analysis of a large series of cases and development of a rapid and sensitive method for diagnostic screening with potential implications on pharmacologic treatment. J Clin Oncol 2005;23:857—65.