Departments of 1 Medicine, 8 Pathology, 3 Biostatistics and 7 CNRS UPR2169, 4 Translational Research Unit, Institut Gustave Roussy, Villejuif; 5 Department of Pathology, Centre Jean Perrin, Clermont-Ferrand; Departments of 9 Thoracique and 6 Pathology, Institut Mutualiste Montsouris, Paris; 2 Laboratoire de Radiobiologie et Oncologie, Commissariat à l'Energie Atomique, Fontenay aux Roses, France
* Correspondence to: Dr J.-C. Soria, Department of Medicine, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, France. Tel: +33-1-42-11-43-01; Fax: +33-1-42-11-52-30; E-mail: soria{at}igr.fr
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Abstract |
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Patients and methods: Using immunohistochemical analysis, we retrospectively analyzed EGFR, pAKT, PTEN, Ki-67, p27 and hTERT expression in specimens from 190 patients with completely resected lung adenocarcinomas (43 never-smokers and 147 smokers). These analyses were performed on tissue microarrays.
Results: EGFR expression was higher in tumors from smokers (P < 0.01), while pAKT was overexpressed mainly in tumors from never-smokers (P = 0.01). As expected, the tumors from smokers presented a higher expression of Ki-67 and a more frequent loss of expression of p27 (P < 0.01). In a multivariate model, two biological factors (p27 and Ki-67) and two clinical factors (age and sex) showed independent significant correlation with never-smoking status.
Conclusions: Lung adenocarcinomas in never-smokers have a very distinct immunohistochemical expression profile of EGFR-related biomarkers as compared with lung adenocarcinomas in smokers. High levels of EGFR and Ki-67 are observed in smokers, while never-smokers are characterized by high levels of pAKT and p27.
Key words: EGFR, immunohistochemistry, lung adenocarcinoma, never-smokers, pAKT
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Introduction |
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EGFR mediates cancer cell growth, proliferation, angiogenesis, invasion and metastasis, and inhibits apoptosis [10]. In NSCLC, overexpression of EGFR has been described in 90% of squamous cell carcinomas and in 3065% of adenocarcinomas [11
], and has been variably correlated with clinical outcome [11
, 12
]. Some authors have stated that EGFR expression is not associated with EGFR TKI sensitivity [13
, 14
]. However, recently, two important studies have suggested that EGFR expression evaluated by immunohistochemistry can be associated with gefitinib [15
] and erlotinib [16
] sensitivity. On the other hand, the response to EGFR TKI (which is noted in up to 20% of treated patients) appears to be more frequent in some clinical groups like Japanese patients [17
], adenocarcinoma subtypes, women and never-smokers [8
, 9
]. High EGFR gene copy number identified by fluorescence in situ hybridization may also be an effective molecular predictor for gefitinib efficacy in advanced NSCLC [15
].
Phosphatidylinositol 3-kinase (PI3K)/AKT is a major signaling pathway that mediates EGFR effects on proliferation and survival. AKT is constitutively active in the majority of NSCLC cell lines [18] and high levels of phosphorylated (activated) AKT have been found in the majority of patients with lung carcinoma [19
]. PTEN is a tumor suppressor gene that physiologically down-regulates the AKT pathway [20
]. PTEN inactivation is found in a great number of cancers and in up to 25% of NSCLC [20
, 21
]. Ki-67 is the most common biomarker used to characterize tumoral proliferation. In NSCLC expression of Ki-67 frequently correlates with poor prognosis [22
, 23
]. p27 plays a crucial role in the regulation of the cellular cycle by inhibiting cdk2cyclin E and thus controlling G1S transition [24
]. Additionally, p27 is a putative tumor-suppressor gene, regulator of drug resistance in solid tumors and promoter of apoptosis [24
]. Loss of expression of p27 has been described in up to 58% of NSCLC [24
26
], especially in smokers [26
], and may correlate with a poor prognosis. Telomerase is a ribonucleoprotein responsible for telomere stabilization and cellular immortality; nucleolar immunohistochemical staining of the catalytic subunit (hTERT) of telomerase is largely restricted to cells with telomerase activity [27
, 28
]. hTERT expression may be stimulated by tobacco exposure, even in the absence of invasive carcinoma [29
, 30
]. Furthermore, telomerase expression is partially under the control of EGFR, while itself modulates epidermal growth factor base level [31
, 32
].
The implication of these biomarkers in NSCLC has been largely described, but limited data exist as for their differential expression in resected lung adenocarcinoma from smokers versus never-smokers.
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Materials and methods |
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Patients' records were retrospectively reviewed for smoking exposure as assessed by trained physicians at diagnosis. A patient was considered as a smoker if he admitted any active smoking history. Thus the smoker category included ex-smokers as well as current smokers. Patients who denied any active smoking exposure were defined as never-smokers. Seven patients were excluded because their smoking history was unavailable. Finally, our study population consisted of 190 patients (147 smokers and 43 never-smokers). The patients' records and biomarker analysis were carried out with ethics committee approval.
Tissue microarrays
A central review by a pathologist (P.V.) confirmed the diagnosis of adenocarcinoma in all hematoxylineosin (HE)-stained sections. One paraffin-embedded adenocarcinoma sample was randomly selected for each case. Three representative tumor areas without necrosis were carefully selected on the corresponding HE-stained slide, and were subsequently punched for tissue microarrays. Blocks were formalin-fixed and paraffin-embedded.
Tissue microarrays production was performed by the Department of Pathology at Institut Gustave Roussy. All 190 tumors were punched in triplicate and cores 0.6 mm in diameter and 5 mm in length were inserted into a recipient paraffin block every 1 mm. Consecutive 5-µm sections were cut from the arrays and mounted onto charged slides. Every five sections, a slide was stained by HE to confirm the presence of the histological features of interest.
Immunohistochemistry
Sections of TMA blocks were stained using monoclonal or polyclonal antibodies against EGFR, pAKT, Ki-67, p27, hTERT and PTEN (Table 1). Deparaffinized sections were either heated in 10 mM citrate buffer (pH 6.0) or partially digested with proteinase K (Table 1). Sections were then immersed in methanol containing 0.3% hydrogen peroxide for 15 min to block the endogenous peroxidase activity and were incubated during 30 min thereafter with 5% blocking horse serum to reduce non-specific binding. Sections were incubated with the primary antibody according to previously validated conditions (Table 1). Following several washes with phosphate-buffered saline, the slides were processed for 30 min with a universal secondary biotinylated antibody, then with avidin-biotinylated horseradish peroxidase H complex (ABC kit; Vector Laboratories, Burlingame, CA, USA). Diaminobenzidine was used as a chromogen (812 min), and hematoxylin was used for counterstaining. Staining was performed simultaneously on all TMA slides. For each reaction, sections of lung cancer previously validated by us to be strongly positive for the assayed antibody were included as positive controls. As negative control, the staining procedure was performed with the primary antibody omitted. For PTEN staining, endothelial cells were also used as an internal control.
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Statistical analysis
Statistical analyses were performed with the SAS system version 8.2 [36]. Data clusters were visualized using Euclidian distance and average linking computed with GENESIS version 1.2.1 [37
].
We used the Fisher's exact, 2 and Wilcoxon two-sample tests to assess differences between the clinicopathological variables of patients who had a smoking history or not. For each semi-quantitative biomarker, univariate analysis of the immunohistochemical expression according to smoking status and other clinical variables was performed using the Wilcoxon two-sample test, comparing the location (median) of the expression values.
Logistic regression models were applied to compare the expression of biomarkers according to smoking history while adjusting for the other clinical variables. For the logistic regression analysis, the expression of each of the five semi-quantitative biomarkers was dichotomized into two classes: expression value below or equal to the median value of the series of 190 patients and expression value above the median value of the series. A multivariate logistic regression model was constructed applying a backwards stepwise selection procedure. Relationships between the expressions of the biomarkers were studied using Spearman's rank order correlation coefficient and tested by Spearman's rank test. The significance level chosen was P < 0.05 and all tests were two-sided.
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Results |
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Among all smokers the median number of pack-years was 40 (range 5120); this value was 40 for men and 35 for female (Wilcoxon test P = 0.06). When testing the correlation between immunohistochemical staining and the degree of smoking as measured by pack-years, only Ki-67 expression was significantly correlated and this correlation was positive (Spearman's rank test P < 0.01).
Relationship between markers and other clinical variables
We investigated the univariate relationship between immunohistochemical expression and clinical variables such as gender, age, TNM stage and tumor differentiation (Table 4). The expression of EGFR, Ki-67 and hTERT was significantly higher in males than in females (P 0.03). Ki-67 was significantly higher expressed in tumors with advanced disease (TNM stage IIIIIa), while p27 was significantly underexpressed (P < 0.01). pAKT and hTERT (P < 0.01) were significantly underexpressed in those patients as compared with patients with limited disease (TNM stage I).
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Unsupervised analysis
A global representation of the immunohistochemical profile of all analyzed biomarkers in the 190 patients studied is displayed in Figure 3. Unsupervised hierarchical clustering of the study series based on the similarities in expression measured over the five semi-quantitative biomarkers revealed two distinct main clusters of patients (vertical axis) with a significantly different proportion of non-smokers (P < 0.001), i.e. a top cluster consisting of only a few non-smokers (seven out of 96) and a bottom cluster with more non-smokers (36 out of 94). The bottom cluster of patients were characterized by a low expression of EGFR and Ki-67 and a high expression for p27, pAKT and hTERT. When taking into account biomarker clusters (horizontal axis), Ki-67 and EGFR were clustered together, as well as p27 and pAKT.
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Discussion |
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We found an enhanced expression of EGFR in tumors from smokers as compared with those from never-smokers. This confirm previous observations suggesting that EGFR expression is enhanced by tobacco exposure [12]. It is largely accepted that the immunohistochemical expression of EGFR in NSCLC does not predict the response to pharmacological agents inhibiting this receptor [13
, 14
]. In line with these findings, high levels of EGFR expression have been reported in squamous cell carcinomas, while response to EGFR inhibitors is higher in lung adenocarcinomas [12
]. Nevertheless, recently, two important studies demonstrated that EGFR expression evaluated by immunohistochemistry associated with gefitinib [15
] and erlotinib [16
] sensitivity. However, more than the degree of EGFR overexpression, the effectiveness of EGFR inhibitors may correlate with the tumor dependency on the EGFR pathway. In this regard, presence of activating mutations of EGFR tyrosine kinase have been reported to correlate with adenocarcinoma subtype [38
], never-smoker status [9
, 38
], and a higher sensitivity to gefitinib or erlotinib [39
41
].
Activation of EGFR subsequently triggers several signal transduction cascades including the PI3K-AKT pathway. Activation of AKT can be induced by tobacco exposure [42] and may be frequent in preneoplasic bronchial lesions in smokers [43
]. A positive correlation has been reported between the expression of EGFR and activated downstream pAKT and pERK expression in NSCLC [19
]. Our results suggest that pAKT might be involved in the development of lung adenocarcinoma in never-smokers, as tumors from such patients have a higher expression of pAKT than those from smokers. In large series of patients with NSCLC treated by gefitinib, other authors have also indicated that strong pAKT expression might correlate with non-smoking status and with an enhanced response to EGFR inhibitors [33
]. Thus, it is possible that the development of lung cancer in never-smokers depends more on the EGFRpAKT pathway than in smokers, in whom the elevated numbers of genomic and molecular abnormalities are responsible for a more complex carcinogenesis. However, in our study, pAKT positivity is not associated with EGFR expression either in smokers or in never-smokers.
PTEN is a negative regulator of the PI3K/AKT pathway [44]. Genomic alterations and loss of expression of the PTEN protein have been found in several malignancies including NSCLC [20
, 21
] but no data exist about PTEN expression according to smoking exposure. Similar to published reports, we found lack of PTEN expression in 10% of analyzed tumors, which is concordant with the frequency previously reported in NSCLC [21
, 45
]. In our study, smoking status did not correlate with the lack of PTEN expression. This suggests that alternative mechanisms (i.e. other than PTEN inactivation) may be responsible for the higher expression of pAKT in tumors from never-smokers.
Our results also suggest that lung adenocarcinoma of smokers tend to be more proliferative than those of never-smokers, as higher levels of expression of Ki-67 and lowers levels of expression of p27 were observed in tumors occurring in patients with a history of active tobacco exposure. The relationship between Ki-67 expression in lung cancers and the cigarette smoking status of patients has been previously reported [23, 46
]. Higher expression levels of Ki-67 correlated with the degree of smoking (measured by the number of pack-years) in our study that included a large number of patients with adenocarcinoma. Several authors have reported that the immunohistochemical expression of p27 may represent a prognostic factor in NSCLC and could be correlated with the tobacco exposure history [25
, 26
]. Compared with our series, these studies had a limited number of patients with adenocarcinomas along with lower frequencies of loss of p27 expression. Our study confirms in a histology-homogenous population that p27 expression is lower in smokers than in never-smokers using a logistic regression analysis adjusted for age, gender and tumor differentiation. Conversely, we have not found any correlation between pack-years and p27 expression nor between the loss of p27 and the expression of Ki-67. The lack of correlation between these two biomarkers may be related to the fact that numerous other abnormalities beyond p27 expression (cyclins, cdk, p53, Rb) do also contribute to tumor proliferation, overall estimated by Ki-67 levels.
Telomerase and its catalytic component hTERT are expressed in the vast majority of NSCLC [35, 47
, 48
]. It has been postulated that tobacco exposure may be responsible for a great number of DNA adducts in the guanine-rich regions [49
], and thus preferentially in the telomeric DNA, which is made by TTAGGG repetitive sequence [29
]. The tobacco-induced alterations of telomeres may be responsible for a frequent activation of telomerase in smokers [29
]. In line with published reports, our study demonstrates that hTERT expression in lung adenocarcinomas is higher in smokers than in never-smokers [29
, 30
, 50
]. However, this difference is mainly related to the higher expression of hTERT among males as compared with females.
Our retrospective analysis raises several questions. First, a potential cause of bias in our analysis relates to the characterization of patients as smokers and never-smokers. Traditionally, never-smokers are defined as individuals having a lifetime exposure of fewer than 100 cigarettes. We consistently assessed the number of pack-years for each patient before surgery and were able to accurately discriminate between smoker and never-smoker individuals. The frequency of never-smokers in our population of patients with adenocarcinomas (23%) is comparable to the range found in other published studies [6]. Secondly, we noted an imbalance between the frequencies of never-smokers in male as compared with female patients. Even if lung adenocarcinoma occurs rarely in male non-smokers subjects, in our study, we decided not to limit ourselves to women with adenocarcinoma. We selected all consecutive intention-to-treat cases from a single institution operated for lung cancer with a final diagnosis of adenocarcinoma. Even though we expected a higher frequency of never-smokers in females, we were nonetheless surprised that in our series only 2% of men were never-smokers compared with 45% in women. However, such an imbalance has been reported previously in patients with lung cancer [6
], and adenocarcinoma subtype in particular. One potential hypothesis for this imbalance is that adenocarcinomas in never-smoker males might be associated with a more advanced stage, thus excluding them from surgical series. Thirdly, we used commercially available antibodies validated for clinical or research applications. We reported median percentages of stained cells in order to discriminate between the different groups. This has allowed us to present a more comprehensive and hierarchical clustering for both biomarkers and tumors using a two-dimensional representation. While our data are not directly linked to any genomic analysis, we believe that clustering of immunohistochemical results according to EGFR mutational status is warranted. In fact, our group is undertaking a collaborative mutational analysis of HER1, HER2, PI3K and RAS in this very same patient population. This should help to elucidate the role of tobacco-exposure in inducing specific gene mutations such as the ones recently described in lung adenocarcinoma.
The development of NSCLC in never-smokers remains largely unknown. Several etiological [2], genetic [4
7
] and molecular [19
, 23
, 33
, 39
] differences have been reported between NSCLC in never-smokers and tobacco-related NSCLC. Our results contribute to improve the description of lung carcinogenesis and highlight that etiology may be important for identifying better prognostic and predictive factors in NSCLC.
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Acknowledgements |
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Received for publication July 1, 2005. Revision received July 28, 2005. Accepted for publication August 2, 2005.
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