Journal of Immunoassay and Immunochemistry
ISSN: 1532-1819 (Print) 1532-4230 (Online) Journal homepage: http://www.tandfonline.com/loi/ljii20
Over-Expression of Egfr is Closely Correlated to Poor Prognosis in Tunisian Patients with NonSmall Cell Lung Adenocarcinoma Amira Arfaoui, Lilia Kriaa, Nadia Znaidi, Sami Gritli, Hend Bouacha, Rachida Zermani & Soumaya Rammeh To cite this article: Amira Arfaoui, Lilia Kriaa, Nadia Znaidi, Sami Gritli, Hend Bouacha, Rachida Zermani & Soumaya Rammeh (2014) Over-Expression of Egfr is Closely Correlated to Poor Prognosis in Tunisian Patients with Non-Small Cell Lung Adenocarcinoma, Journal of Immunoassay and Immunochemistry, 35:3, 256-268, DOI: 10.1080/15321819.2013.848813 To link to this article: https://doi.org/10.1080/15321819.2013.848813
Accepted author version posted online: 25 Oct 2013. Published online: 25 Oct 2013. Submit your article to this journal
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ljii20
Journal of Immunoassay and Immunochemistry, 35:256–268, 2014 Copyright © Taylor & Francis Group, LLC ISSN: 1532-1819 print/1532-4230 online DOI: 10.1080/15321819.2013.848813
OVER-EXPRESSION OF EGFR IS CLOSELY CORRELATED TO POOR PROGNOSIS IN TUNISIAN PATIENTS WITH NON-SMALL CELL LUNG ADENOCARCINOMA
Amira Arfaoui,1 Lilia Kriaa,2 Nadia Znaidi,1 Sami Gritli,1 Hend Bouacha,3 Rachida Zermani,1 and Soumaya Rammeh1 1 Department of Pathology, Charles Nicolle Hospital, Tunis, Tunisia 2 Department of Biology, Central University, Tunis, Tunisia 3 Department of Pneumology, Charles Nicolle Hospital, Tunis, Tunisia
We studied epidermal growth factor receptor (EGFR) expression profile with the aim of an individualized therapy for patients with non-small cell lung cancer (NSCLC) from whom tumor materials are not sufficient for molecular investigations. Using immunohistochemistry, we found a markedly increased EGFR expression with significant difference in term of intensity and distribution from normal mucosa to primary tumors (p < 0.05). Furthermore, patients with EGFR positive tumors had significantly shorter survival than those with EGFR negative tumors (p = 0.0001). Thus, EGFR over-expression is a valuable prognostic marker to predict poor outcome in Tunisian patients with NSCLC. Keywords EGFR expression, lung cancer, immunohistochemistry, marker, prognosis
INTRODUCTION Lung cancer is the most prevalent of all cancers also known to have a very poor prognosis. Lung tumorigenesis is a multistep process that involves genetic and epigenetic events.[1] Non-small cell lung cancer (NSCLC) is the major type of lung cancer and is classified into three histological types: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma.[2,3] The majority of patients with NSCLC are in an advanced stage at the time of clinical and/or histological diagnosis, and less than 30% of patients undergo surgical resection.[4] Despite great achievements in molecular studies, which focus on finding markers of early diagnosis and prognosis useful for targeted Address correspondence to Amira Arfaoui, 2, Rue des Narcisses les Jardins de L’Aouina, 2045 la Soukra, Tunisia. E-mail: [email protected] Color versions of one or more of the figures in this article can be found online at www.tandfonline. com/ljii.
Prognostic Value of EGFR Over-Expression in Lung Cancer
257
therapies, survival rate in NSCLC remains very low.[5] This poor outcome could be caused by a lack of efficient therapeutic strategies, mainly hampered by drug resistance.[6,7] The discovery of novel targeted treatments for NSCLC has led to a search for new genetic and epigenetic markers able to selectively predict response to these new drugs.[8] Somatic alterations in the epidermal growth factor receptor (EGFR) gene are analyzed to predict response to tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib used in the treatment of NSCLC, whose efficacy depends on the presence or the absence of specific molecular alterations.[9−11] The protein encoded by the EGFR gene, EGFR, is a transmembrane glycoprotein that belongs to the protein kinase superfamily.[12] EGFR is a member of the epidermal growth factor family and is a cell surface protein that binds to epidermal growth factor.[12] Binding of the protein to its ligand induces receptor dimerization and tyrosine autophosphorylation, which leads to cell proliferation.[13] Mutations in the EGFR gene are associated with lung cancer.[4–10] Mutations in the tyrosine kinase (TK) domain of the EGFR gene in NSCLC patients are associated with clinical response to the TKIs gefitinib and erlotinib.[14−17] These mutations are somatic and more common in patients with clinical features known to be associated with sensitivity to TKIs, such as female gender, type of cancer (adenocarcinoma), Asian ethnicity, and lack of smoking history.[14] The discovery of these mutations and their clinical significance became a major step toward the development of targeted therapies with EGFR TKIs.[14−17] Activating mutations within the TK domain of EGFR are found in approximately 10–20% of NSCLC and are associated with response to anti-EGFR therapy.[18] Furthermore, it has been well established that EGFR expression profile could guide therapy choice for patients with NSCLC.[18] To the best of our knowledge, there is no information on EGFR mutation or EGFR expression profile in Tunisian patients with NSCLC. Herein, we describe for the first time the EGFR immunohistochemical profile in a series of Tunisian patients with NSCLC and evaluate the involvement of this protein in lung cancer pathogenesis and prognosis. MATERIAL AND METHODS Sample Selection Between January 2004 and June 2011, a total of 65 patients were diagnosed with NSCLC at Charles Nicolle’s Hospital (Tunis, Tunisia). All patients signed an informed consent, which permitted the use of their specimens for our present work.
258
A. Arfaoui et al.
Immunohistochemical Analyses For immunohistochemical (IHC) purposes, normal or tumor lung tissue samples were obtained on biopsy. These samples were fixed in 10% formaldehyde solution, dehydrated, and placed in paraffin for histological analyses. Formalin-fixed 5 µm-thick sections were stained with haematoxylin and eosin for the evaluation of tumor type. The histological analyses were performed by two independent observers. Five µm-thick sections were prepared and mounted on poly-L-lysinecoated slides. IHC analysis was performed using the following monoclonal antibodies: anti-cytokeratin 7, anti-cytokeratin 20, anti-TTF1, anti-napsin, and anti-EGFR (NovoCastra, New Castle Upon Tyne, UK). All antibodies used in this work are listed in Table 1. The staining procedure was conducted using an automated immunostainer BOND-MAX Leica (Leica Biosystems, Milton Keynes, UK) and a three-step indirect procedure based on the biotin-streptavidin-peroxidase method. Briefly, after proteinase pretreatment (Ventana Medical Systems Inc., Tucson, AZ, USA), tissue sections were incubated with fresh 3% hydrogen peroxide in methanol to block endogenous peroxidase. The sections were then sequentially incubated with the diluted mouse monoclonal antibody, a biotinylated rabbit anti-mouse antibody and streptavidine-peroxidase conjugate. The sections were then developed with diaminobenzidine and counterstained with hematoxylin.[18] Specimens were examined for the distribution of EGFR immunostaining. Only cells showing a membranous staining alone or in association with a cytoplasmic staining were considered as positive and scored. EGFR expression was assessed according to the percentage of positive cells using the “0 to 3+” scale as follows: a score 0 is an absence of positive cells; a score of 1+ is > 0–1%; a score of 2+ is 2–50%; and a score of 3+ is > 50%. A section of esophagus tissue known to express EGFR was used as a positive control. Statistical Analysis Statistical analyses were performed using SPSS software (IBM, Armonk, NY, USA). X 2 test and McNemar’s method were used to test significance TABLE 1 Antibodies used in immunohistochemistry Antibodies TTF1 Napsin Cytokeratin 7 Cytokeratin 20 EGFR
Source
Clone
Dilution
NovoCastra NovoCastra NovoCastra NovoCastra NovoCastra
Spt24 − LP5k Ks20.8 EGFR.113
1:50 1:50 1:5–1:10 1:25–1:50 1:50
Prognostic Value of EGFR Over-Expression in Lung Cancer
259
with regard to the difference in frequency and intensity of EGFR expression between normal tissue and primary tumor and the association of EGFR expression with clinical and pathologic variables. Additionally, we estimated the relationship between EGFR expression and survival by Cox’s proportional hazards model. The curves describing survival were computerized according to Kaplan and Meier. Tests were 2-sided and p < 0.05 was considered as statistically significant. RESULTS We have recruited nine males and three females. The mean age was 64 years (range: 52–76 years). On histologic examination, the tumors consisted of NSCLC adenocarcinoma. Five cases were moderately differentiated and seven were poorly differentiated. All cases were pT4 and accompanied by lymphatic and/or visceral metastases according to TNM classification. TTF1, Napsin, Cytokeratin7, and Cytokeratin20 Expression Figures 1A and 1B show representative positive immunostaining images of NSCLC for TTF1 and napsin, respectively. Our results showed that primary lung adenocarcinoma was TTF1+, napsin+, CK7+, and CK20-, while secondary lung adenocarcinoma was TTF1-, napsin-, CK7-/+, and CK20-/+. Among 65 cases, we selected 12 patients who had primary lung cancer.
FIGURE 1 TTF1 and napsin IHC staining in NSCLC. (A) TTF1 shows nuclear positivity in NSCLC (original magnification 40X); (B) napsin shows cytoplasmic positivity in NSCLC (original magnification 40X).
260
A. Arfaoui et al.
EGFR Expression in Normal Mucosa and Primary Tumors In matched samples, the frequency and intensity of EGFR immunostaining were markedly increased in normal tissue compared to those in primary tumors (p < 0.05). We found that only two tumors revealed a completely negative reaction with anti-EGFR. Pattern Distribution of EGFR Immunostaining As shown in Table 3, 7 cases are positive (64%), while 5 cases turned out to be negative (36%; Figure 2A). Among the positive cases, 4 specimens exhibited intense and diffuse membranous expression with (3+, 80–90%; Figure 2B), 2 cases with discontinuous and moderate intensity (2+, 80–90%; Figure 2C), and one case showed a discontinuous expression and weak intensity (1+, 20%). All results are presented in Table 2. On the other hand, as shown in Table 3, 10 cases were positive (84%) and 2 were negative (16%). Among the specimens with positive cytoplasmic EGFR immunostaining, we observed that 5 tumors exhibited an intense and diffuse signal (3+, 90%; Figure 3), 4 cases showed a moderate signal (2+, 80%), and one case showed a weak and discontinuous immunostaining (1+, 40%; Table 2). Moreover, specimens were examined for the double distribution of the immunostaining: membranous and cytoplasmic. We observed that seven tumors exhibited this mixed expression pattern (Figure 4). However, we were unable to find any significant association between the EGFR positive expression and the distribution of immunostaining (membranous vs. cytoplasmic; p = 0.28).
FIGURE 2 Membranous EGFR IHC staining in NSCLC. (A) Negative immunostaining of EGFR (case no. 12; original magnification 100X); (B) intense and diffuse membranous positivity of EGFR [3+, 90%], (case no. 5; original magnification 250X); (C) discontinuous expression and moderate intensity of EGFR [2+, 80%], (case no. 4; original magnification 250X).
261
Prognostic Value of EGFR Over-Expression in Lung Cancer
TABLE 2 Expression of EGFR in relation to sex, age, TNM stage, differentiation, and smoking in patients with NSCLC EGFR Positive (%)
Negative (%)
Sex - Male - Female
9 (75) 1 (8)
0 (0) 2 (17)
Age (years) -
ISSN: 1532-1819 (Print) 1532-4230 (Online) Journal homepage: http://www.tandfonline.com/loi/ljii20
Over-Expression of Egfr is Closely Correlated to Poor Prognosis in Tunisian Patients with NonSmall Cell Lung Adenocarcinoma Amira Arfaoui, Lilia Kriaa, Nadia Znaidi, Sami Gritli, Hend Bouacha, Rachida Zermani & Soumaya Rammeh To cite this article: Amira Arfaoui, Lilia Kriaa, Nadia Znaidi, Sami Gritli, Hend Bouacha, Rachida Zermani & Soumaya Rammeh (2014) Over-Expression of Egfr is Closely Correlated to Poor Prognosis in Tunisian Patients with Non-Small Cell Lung Adenocarcinoma, Journal of Immunoassay and Immunochemistry, 35:3, 256-268, DOI: 10.1080/15321819.2013.848813 To link to this article: https://doi.org/10.1080/15321819.2013.848813
Accepted author version posted online: 25 Oct 2013. Published online: 25 Oct 2013. Submit your article to this journal
Article views: 64
View related articles
View Crossmark data
Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ljii20
Journal of Immunoassay and Immunochemistry, 35:256–268, 2014 Copyright © Taylor & Francis Group, LLC ISSN: 1532-1819 print/1532-4230 online DOI: 10.1080/15321819.2013.848813
OVER-EXPRESSION OF EGFR IS CLOSELY CORRELATED TO POOR PROGNOSIS IN TUNISIAN PATIENTS WITH NON-SMALL CELL LUNG ADENOCARCINOMA
Amira Arfaoui,1 Lilia Kriaa,2 Nadia Znaidi,1 Sami Gritli,1 Hend Bouacha,3 Rachida Zermani,1 and Soumaya Rammeh1 1 Department of Pathology, Charles Nicolle Hospital, Tunis, Tunisia 2 Department of Biology, Central University, Tunis, Tunisia 3 Department of Pneumology, Charles Nicolle Hospital, Tunis, Tunisia
We studied epidermal growth factor receptor (EGFR) expression profile with the aim of an individualized therapy for patients with non-small cell lung cancer (NSCLC) from whom tumor materials are not sufficient for molecular investigations. Using immunohistochemistry, we found a markedly increased EGFR expression with significant difference in term of intensity and distribution from normal mucosa to primary tumors (p < 0.05). Furthermore, patients with EGFR positive tumors had significantly shorter survival than those with EGFR negative tumors (p = 0.0001). Thus, EGFR over-expression is a valuable prognostic marker to predict poor outcome in Tunisian patients with NSCLC. Keywords EGFR expression, lung cancer, immunohistochemistry, marker, prognosis
INTRODUCTION Lung cancer is the most prevalent of all cancers also known to have a very poor prognosis. Lung tumorigenesis is a multistep process that involves genetic and epigenetic events.[1] Non-small cell lung cancer (NSCLC) is the major type of lung cancer and is classified into three histological types: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma.[2,3] The majority of patients with NSCLC are in an advanced stage at the time of clinical and/or histological diagnosis, and less than 30% of patients undergo surgical resection.[4] Despite great achievements in molecular studies, which focus on finding markers of early diagnosis and prognosis useful for targeted Address correspondence to Amira Arfaoui, 2, Rue des Narcisses les Jardins de L’Aouina, 2045 la Soukra, Tunisia. E-mail: [email protected] Color versions of one or more of the figures in this article can be found online at www.tandfonline. com/ljii.
Prognostic Value of EGFR Over-Expression in Lung Cancer
257
therapies, survival rate in NSCLC remains very low.[5] This poor outcome could be caused by a lack of efficient therapeutic strategies, mainly hampered by drug resistance.[6,7] The discovery of novel targeted treatments for NSCLC has led to a search for new genetic and epigenetic markers able to selectively predict response to these new drugs.[8] Somatic alterations in the epidermal growth factor receptor (EGFR) gene are analyzed to predict response to tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib used in the treatment of NSCLC, whose efficacy depends on the presence or the absence of specific molecular alterations.[9−11] The protein encoded by the EGFR gene, EGFR, is a transmembrane glycoprotein that belongs to the protein kinase superfamily.[12] EGFR is a member of the epidermal growth factor family and is a cell surface protein that binds to epidermal growth factor.[12] Binding of the protein to its ligand induces receptor dimerization and tyrosine autophosphorylation, which leads to cell proliferation.[13] Mutations in the EGFR gene are associated with lung cancer.[4–10] Mutations in the tyrosine kinase (TK) domain of the EGFR gene in NSCLC patients are associated with clinical response to the TKIs gefitinib and erlotinib.[14−17] These mutations are somatic and more common in patients with clinical features known to be associated with sensitivity to TKIs, such as female gender, type of cancer (adenocarcinoma), Asian ethnicity, and lack of smoking history.[14] The discovery of these mutations and their clinical significance became a major step toward the development of targeted therapies with EGFR TKIs.[14−17] Activating mutations within the TK domain of EGFR are found in approximately 10–20% of NSCLC and are associated with response to anti-EGFR therapy.[18] Furthermore, it has been well established that EGFR expression profile could guide therapy choice for patients with NSCLC.[18] To the best of our knowledge, there is no information on EGFR mutation or EGFR expression profile in Tunisian patients with NSCLC. Herein, we describe for the first time the EGFR immunohistochemical profile in a series of Tunisian patients with NSCLC and evaluate the involvement of this protein in lung cancer pathogenesis and prognosis. MATERIAL AND METHODS Sample Selection Between January 2004 and June 2011, a total of 65 patients were diagnosed with NSCLC at Charles Nicolle’s Hospital (Tunis, Tunisia). All patients signed an informed consent, which permitted the use of their specimens for our present work.
258
A. Arfaoui et al.
Immunohistochemical Analyses For immunohistochemical (IHC) purposes, normal or tumor lung tissue samples were obtained on biopsy. These samples were fixed in 10% formaldehyde solution, dehydrated, and placed in paraffin for histological analyses. Formalin-fixed 5 µm-thick sections were stained with haematoxylin and eosin for the evaluation of tumor type. The histological analyses were performed by two independent observers. Five µm-thick sections were prepared and mounted on poly-L-lysinecoated slides. IHC analysis was performed using the following monoclonal antibodies: anti-cytokeratin 7, anti-cytokeratin 20, anti-TTF1, anti-napsin, and anti-EGFR (NovoCastra, New Castle Upon Tyne, UK). All antibodies used in this work are listed in Table 1. The staining procedure was conducted using an automated immunostainer BOND-MAX Leica (Leica Biosystems, Milton Keynes, UK) and a three-step indirect procedure based on the biotin-streptavidin-peroxidase method. Briefly, after proteinase pretreatment (Ventana Medical Systems Inc., Tucson, AZ, USA), tissue sections were incubated with fresh 3% hydrogen peroxide in methanol to block endogenous peroxidase. The sections were then sequentially incubated with the diluted mouse monoclonal antibody, a biotinylated rabbit anti-mouse antibody and streptavidine-peroxidase conjugate. The sections were then developed with diaminobenzidine and counterstained with hematoxylin.[18] Specimens were examined for the distribution of EGFR immunostaining. Only cells showing a membranous staining alone or in association with a cytoplasmic staining were considered as positive and scored. EGFR expression was assessed according to the percentage of positive cells using the “0 to 3+” scale as follows: a score 0 is an absence of positive cells; a score of 1+ is > 0–1%; a score of 2+ is 2–50%; and a score of 3+ is > 50%. A section of esophagus tissue known to express EGFR was used as a positive control. Statistical Analysis Statistical analyses were performed using SPSS software (IBM, Armonk, NY, USA). X 2 test and McNemar’s method were used to test significance TABLE 1 Antibodies used in immunohistochemistry Antibodies TTF1 Napsin Cytokeratin 7 Cytokeratin 20 EGFR
Source
Clone
Dilution
NovoCastra NovoCastra NovoCastra NovoCastra NovoCastra
Spt24 − LP5k Ks20.8 EGFR.113
1:50 1:50 1:5–1:10 1:25–1:50 1:50
Prognostic Value of EGFR Over-Expression in Lung Cancer
259
with regard to the difference in frequency and intensity of EGFR expression between normal tissue and primary tumor and the association of EGFR expression with clinical and pathologic variables. Additionally, we estimated the relationship between EGFR expression and survival by Cox’s proportional hazards model. The curves describing survival were computerized according to Kaplan and Meier. Tests were 2-sided and p < 0.05 was considered as statistically significant. RESULTS We have recruited nine males and three females. The mean age was 64 years (range: 52–76 years). On histologic examination, the tumors consisted of NSCLC adenocarcinoma. Five cases were moderately differentiated and seven were poorly differentiated. All cases were pT4 and accompanied by lymphatic and/or visceral metastases according to TNM classification. TTF1, Napsin, Cytokeratin7, and Cytokeratin20 Expression Figures 1A and 1B show representative positive immunostaining images of NSCLC for TTF1 and napsin, respectively. Our results showed that primary lung adenocarcinoma was TTF1+, napsin+, CK7+, and CK20-, while secondary lung adenocarcinoma was TTF1-, napsin-, CK7-/+, and CK20-/+. Among 65 cases, we selected 12 patients who had primary lung cancer.
FIGURE 1 TTF1 and napsin IHC staining in NSCLC. (A) TTF1 shows nuclear positivity in NSCLC (original magnification 40X); (B) napsin shows cytoplasmic positivity in NSCLC (original magnification 40X).
260
A. Arfaoui et al.
EGFR Expression in Normal Mucosa and Primary Tumors In matched samples, the frequency and intensity of EGFR immunostaining were markedly increased in normal tissue compared to those in primary tumors (p < 0.05). We found that only two tumors revealed a completely negative reaction with anti-EGFR. Pattern Distribution of EGFR Immunostaining As shown in Table 3, 7 cases are positive (64%), while 5 cases turned out to be negative (36%; Figure 2A). Among the positive cases, 4 specimens exhibited intense and diffuse membranous expression with (3+, 80–90%; Figure 2B), 2 cases with discontinuous and moderate intensity (2+, 80–90%; Figure 2C), and one case showed a discontinuous expression and weak intensity (1+, 20%). All results are presented in Table 2. On the other hand, as shown in Table 3, 10 cases were positive (84%) and 2 were negative (16%). Among the specimens with positive cytoplasmic EGFR immunostaining, we observed that 5 tumors exhibited an intense and diffuse signal (3+, 90%; Figure 3), 4 cases showed a moderate signal (2+, 80%), and one case showed a weak and discontinuous immunostaining (1+, 40%; Table 2). Moreover, specimens were examined for the double distribution of the immunostaining: membranous and cytoplasmic. We observed that seven tumors exhibited this mixed expression pattern (Figure 4). However, we were unable to find any significant association between the EGFR positive expression and the distribution of immunostaining (membranous vs. cytoplasmic; p = 0.28).
FIGURE 2 Membranous EGFR IHC staining in NSCLC. (A) Negative immunostaining of EGFR (case no. 12; original magnification 100X); (B) intense and diffuse membranous positivity of EGFR [3+, 90%], (case no. 5; original magnification 250X); (C) discontinuous expression and moderate intensity of EGFR [2+, 80%], (case no. 4; original magnification 250X).
261
Prognostic Value of EGFR Over-Expression in Lung Cancer
TABLE 2 Expression of EGFR in relation to sex, age, TNM stage, differentiation, and smoking in patients with NSCLC EGFR Positive (%)
Negative (%)
Sex - Male - Female
9 (75) 1 (8)
0 (0) 2 (17)
Age (years) -
