The incidence of lung cancer in women affects an estimated 516,000 women worldwide, of which 100,000 are in the United States and 70,000 in Europe.1 Lung cancer is now the fourth most frequent cause of death from cancer worldwide, and the first cause in the United States and some European countries. These epidemiological data underline that cancer in women deserves specific attention.
Until now, lung cancers occurring in women have been treated similarly to lung cancers in men. However, numerous studies have highlighted different characteristics of lung cancer in women. We along with others have described the specificities of clinical and radiological presentations, pathology types, the response-to-cancer treatments, and patient outcomes in women.2,3 Besides these clinical observations, other research has added new data that reinforce the specificities of lung cancer in women.
Two main mechanisms have emerged from recent findings on lung carcinogenesis in women: the high prevalence of genetic alterations, such as epidermal growth factor receptor (EGFR) mutations,4,5 and the potential involvement of hormonal factors.6EGFR seems to be more frequently mutated in women than in men, leading to a better response rate to EGFR-tyrosine kinase inhibitor (TKI) therapy.7 Recent epidemiological and clinical studies have provided evidence of a role for estrogens in the genesis and progression of lung cancer, especially non–small-cell lung tumors.6,8
Preclinical studies have shown a solid rationale for the crucial involvement of hormones in lung carcinogenesis. Many hormonal receptors, such as ERα, ERβ (estrogen receptors α and β), and PR (progesterone receptors) have been isolated from lung cancer tissues. We have recently reported a higher expression of ERs in women operated on for lung cancer compared with men.9 Moreover, ERs have been shown to be involved in the onset of lung cancer in cells and animal models.10 Last, the interaction of ERs with growth factor receptor signaling, including EGFR, has been demonstrated.11 A direct correlation between both pathways has been also suggested by two recent studies,12,13 indicating that lung cancer treatment could include both EGFR- and hormone-targeting drugs.14
Tobacco smoking is the main cause of lung cancer. However, lung cancer also occurs in people who have never smoked, and it ranks as the seventh most common cause of cancer death worldwide.15,16 Lung cancer in never-smokers is more frequently observed in women, representing 20% to 70% of these cases, according to geographical origin and patient selection.17 Lung cancer in never-smokers is also characterized by a higher rate of gene mutations, involving EGFR, HER2, or PI3K, and of EML4–ALK translocations. We have recently identified, in a national collaborative study, other genetic abnormalities in this population.18 Nevertheless, no clear data are available to improve our understanding of the higher rate of tobacco-independent lung cancer in women.
We hypothesize that genetic events or hormonal factors may be part of this observation. Herein, we have aimed to compare clinical and pathological characteristics of lung cancer in two cohorts of women smokers and women never-smokers, with a specific focus on driver oncogenes and hormonal receptors.
PATIENTS AND METHODS
Patients and Tissues
Tumor specimens were collected from 50 women who underwent surgery at the Thoracic Oncology Department (Toulouse University Hospital, France; this comprised the Ligue collection). A second collection of 90 tumor samples was collected from the Lung Genes (LG-collection) study, which involved 13 centers in France.18 Only women were included in this study, and all patients had been treated with surgery.
We collected specific tobacco-exposure status from all women by checking each patient’s file and subsequently performing a telephone- or mail-based survey. The 63 patients who had never smoked were defined according to current guidelines19 as persons with lifetime exposure of less than 100 cigarettes.
A lung cancer pathologist assessed the diagnoses by applying the latest World Health Organization classification,20 and the clinicopathological stage was assigned according to the tumor, node, metastasis (TNM) classification.21 Patients who were included before the last classification were reclassified, especially for lepidic components. All patients signed an informed consent permitting analysis of tissues. Patients were treated and follow-up was done at our institutions, to ensure collection of clinical data.
We selected the most relevant antibodies8,10,13,22,23 from the literature (Fig. 1 and Table 1). We chose the N-terminal ERα (1D5 clone DAKO) and the PR (PgR 636 clone DAKO) used in breast carcinoma because of their therapeutic implications. A C-terminal α-ER monoclonal antibody (clone sc-8002 F10; Santa-Cruz Biotechnology, Dallas, TX) and an ERβ polyclonal rabbit antibody (Biogenex, Fremont, CA) were also chosen because of their supposed prognostic implications.9
Samples (5-µm sections) of lung tumor tissue were mounted on positively charged slides and dried for 1 hour at 60°C before they underwent immunohistochemical assays for ERα, ERβ, and PR expression. We used the PT link pretreatment module (DAKO, Carpentaria, CA), which allows the entire pretreatment process of deparaffinization, rehydration, and epitope retrieval to be combined in a three-in-one specimen-preparation procedure.
The slides were treated with 3% hydrogen peroxide to block endogenous peroxidase activity. Nonimmune serum was used to block nonspecific staining. Antigen retrieval was achieved by proteinase K digestion for 10 minutes, and the primary antibody was applied at a dilution of 1:50, and then incubated for 2 hours at room temperature. Biotinylated secondary antimouse antibodies (1:100) were applied to the sections for 20 minutes. Visualization was obtained, using the labeled streptavidin–biotin staining method (LSAB kit; DAKO). We then evaluated staining intensity, its nuclear or cytoplasmic localization, and the proportion of positively stained cells. We used semiquantitative scores for nuclear and cytoplasmic staining: that is, mild (+), moderate (++), and high (+++).
For all hormonal receptors, we applied the same score as used in breast cancer, with a 10% positive-cell threshold.24 However, for estrogen,β receptors there is no currently validated score, so we applied a published proportion score.23 For this score, we used a high-score class (6–8) and a low-score class (0–4) by adding the proportion of positively stained cells (five scores from 1–5) and one of the three intensity scores (1–3).
Molecular Analysis of EGFR and K-Ras
For EGFR and K-Ras, direct sequencing was performed after polymerase chain reaction amplification of EGFR exons 18, 19, 20, 21 (NM_005228.3), and K-Ras exons 2 and 3 (NM_033360.2). Purified DNA was sequenced using a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Sequencing reactions were analyzed on a 16-capillary ABI3130 or a 48-capillary 3730 DNA analyzer in both sense and antisense directions, from at least two independent amplifications. Sequence reading and alignment were performed with SeqScape software (Applied Biosystems).
Continuous variables are presented by their means ± standard deviations. We first described and compared the patients’ characteristics between our local series and the national collection. Second, we tested whether the association between smoking status and the patients’ characteristics depended on where the data were collected from; we did not observe any significant interaction between the patients’ characteristics and the place of collection. We subsequently decided to pool data from both these series for the final analysis. The patients’ characteristics were compared between smokers and nonsmokers. The ÷2 or Fisher’s exact test was used to compare categorical variables, and Student’s t test or a nonparametric Wilcoxon test was used for continuous variables. We estimated Spearman’s correlation coefficients (ñ) between each biological characteristic and tested the null hypothesis (ñ = 0) using a Benjamini–Yekutieli correction for multiple tests. All analyses were conducted using Stata SE version 11.2.
Clinical and Pathological Characteristics of Lung Cancer in Women Never-Smokers
A total of 140 women with adenocarcinoma (AC) were included in this study. Of these, 63 were never-smokers and 77 were former or current smokers. We double-checked this status by collecting data from the patients’ files and by sending a specific survey to each patient to ensure that she fulfilled the abovementioned criteria. We compared both groups (Table 2) and did not observe any significant differences regarding either TNM status or tumor stage. In contrast, we observed a higher average age at disease appearance for women who never smoked (67 years) compared with smokers (58.7 years; p < 0.0001). In addition, lepidic features were more frequent in the never-smokers (60.3% versus 37.7%; p = 0.008).
Genetic Characteristics of lung Cancer in Women Never-Smokers
For all patients, we assessed the mutational status of EGFR (exon 19 and 21) and K-Ras (exon 2). Patients with a mutated EGFR represented 28.6% of the overall population in this study. More precisely, 50.8% of never-smokers displayed the mutation compared with 10.4% of former or current smokers (p < 0.001; Table 3). In contrast, the K-Ras mutation was more frequent in smokers (33.8%) compared with never-smokers (9.5%; p = 0.001) (Fig. 2).
Hormone-Receptor Expression in Women Never-Smokers
We analyzed the ERα, ERβ, and the PR for all patients using immunohistochemistry. For all markers, we analyzed the percentage of positive cells and the intensity of staining in both cytoplasm and the nucleus.
We analyzed ERα expression according to the breast cancer testing procedure, as previously described,9 but we also extended our analysis to include other parameters, such as cytoplasm expression and intensity staining. Moreover, we used two different antibodies directed against ERα, as described in the literature9: ERα 1D5 (epitopes NH2 terminal) used in breast cancer diagnosis and ERα F10sc8002 (epitopes COOH terminal)9,13 (Table 1).
For the breast cancer–derived procedure (threshold of 10% nucleus staining with 1D5 antibody), 11.1% of the never-smokers and 3.9% of smokers (p = 0.11) had a positive score (Table 3). We hypothesize that this score might be inappropriate for lung cancer, so we also looked for other parameters. We found that both the 1D5 nucleus and the intensity of cytoplasm staining were significantly higher in never-smokers versus smokers (p = 0.008 and p = 0.03, respectively). Using the ERα F10 antibody, we observed a higher percentage of positive tumor samples (as for the breast cancer score) for never-smokers compared with smokers, 23.8% versus 10.4%, respectively (p = 0.03). In summary, we observed significant differences in nucleus-intensity staining with both antibodies for ERα according to the tobacco status of women with NSCLC (Table 3 and Fig. 2).
For ERβ we used a polyclonal rabbit antibody.23 We observed 100% expression of ERβ in never-smokers compared with 92% in smokers, but ERβ nuclear expression was not significantly different (p = 0.12). Last, we analyzed PR expression and found a very low rate of expression in both populations, with no significant differences (Table 3).
Correlations between Biological Data
We found a negative correlation between the K-Ras and EGFR mutations in this female population. Both mutations remain mutually exclusive for all patients. Interestingly, ERα nucleus-intensity staining (of both antibodies) correlated significantly with the EGFR mutation status of all the women in the study (Table 4). We did not find any correlation between biomarkers according to smoking status (data not shown).
Herein, we have aimed to identify the specificities of lung cancer in women. We first report that lung cancer was similar regarding TNM status at the time of diagnosis. We found that lung cancer appeared later in women who had never smoked than in women who smoked. These data are consistent with a recent study performed in France on primary lung cancer.25 An explanation for this might be that women are more sensitive to tobacco than men, and that lung cancer occurs earlier in this population. The diagnostic age of 58.7 years for women smokers is lower than the median age observed for men smokers. We also observed in our former study that women displayed lung cancer at 58 years (compared with 61.2 years for men).9 In contrast, women not exposed to a carcinogen such as tobacco developed lung cancer later.
We looked at the genetic profiles of both our populations and found that lung cancer had mutated EGFR more frequently and mutated K-Ras less frequently in women who had never smoked. These results are in line with already published data. EGFR is known to be mutated in 14% of women smokers compared with 55% who had never smoked.26 Moreover, EGFR is more frequently mutated in never-smokers.27 Overall, women who had never smoked represented a population characterized by a high probability of EGFR mutation. This finding is associated with a better response to EGFR-TKI as reported in many recent trials.7,28 Interestingly, we observed that EGFR was mutated in 10% of female smokers, suggesting that screening for EGFR mutations should not be limited to never-smokers. K-Ras was mutated in more than 33% of female smokers (whereas only 9.5% women who had never smoked were affected). K-Ras is known to be mutated by tobacco carcinogens, and remains difficult to inhibit by targeted therapy, but drugs such as mitogen-activated ERK kinase inhibitors are currently being tested.29 Overall, these results show that more than 50% of women displayed either EGFR or K-Ras mutations that can be targeted by dedicated drugs. It would be of interest to obtain information on other biomarkers, such as Her2, BRaf, Pi3K, and EML4–ALK, which are known to be deregulated in lung cancer. Unfortunately, we were not able to test these biomarkers at the time of this study.
We found higher expression of both ERα and ERβ in female never-smokers. ERs α and β are expressed in normal lung cells (cultured normal lung fibroblast cell lines and normal bronchial epithelial cells) and in the lung tumors of both men and women.10 Several in vitro and in vivo studies have provided evidence supporting a biological role for estrogens in lung carcinogenesis by directly promoting cell proliferation. Estrogens stimulate the proliferation of NSCLC through ER-mediated signaling, whereas antiestrogens inhibit the growth of NSCLC cells.10,14 We, along with others, have previously reported that Erα, and to a lesser extent, ERβ, was more frequently overexpressed in women than in men, and in never-smokers than in smokers. Nose et al.12 reported a clear correlation between ERβ and the nonsmoking status (p < 0.001) in univariate analysis. Nevertheless, the multivariate analysis failed to show any statistical difference for ERβ expression according to the sex (p = 0.378) and the smoking status (p = 0.294). However, the difference in ER expressions according to the smoking status of women has not been addressed by the authors. we report here for the first time the difference between female never-smokers and smokers.9,13 These data suggest that oncogenesis in never-smokers relies more frequently on hormonal pathways.
There are many reports on the immunohistochemical expression of hormonal receptors. Most data are conflicting because of two ER isoforms (α and β), the presence of truncated forms of ERα, the range of antibodies used, and the absence of a validated threshold or score. Thus, we selected specific antibodies that targeted both COOH-terminal and NH2-terminal ERα and ERβ. We also used previously published scores; for ERα, we applied the same scores used in breast cancer, with a 10% positive-cell threshold and distinguished ER expression in the cytoplasm and the nucleus. Last, we used two antibodies for ERα staining, 1D5, targeting N-terminal part of the receptor, and F10, targeting the COOH part of the receptor. We first applied the breast cancer score with both antibodies and found a statistical difference by using 1D5 antibody and not the F10 antibody. Looking at nuclear intensity staining, we observed a statistical difference for both antibodies. Overall, our data suggest that a different score should be used in lung cancer when comparing it with breast cancer and that an antibody targeting its COOH part is probably more potent in identifying ERα expression in lung cancer. For ERβ, there is currently no validated score, and we used a proportion score.23 Establishment of a dedicated score for ER in lung cancer is warranted to ensure homogeneity between data and studies.
Stabile et al.12 reported that EGFR protein expression was up-regulated in response to antiestrogens in vitro, and that ERβ expression was decreased in response to epidermal growth factor and increased in response to gefitinib. In addition, in line with our results, two previous studies also reported an association between ERα and ERβ expression and EGFR mutations. Raso et al.13 reported the correlation between EGFR mutation and ERα expression in a population of ACs, 27 (in both men and women) of which were found with mutations. Nose et al.12 also reported the correlation between ERβ expression and EGFR mutation in a large population of ACs. To date, our work is the largest study showing this interaction in a specific population. Recently, Garon et al.30 reported the potential synergy of EGFR inhibitors and ER antagonists on preclinical models. Traynor et al.31 reported the result of a pilot study where postmenopausal women diagnosed with NSCLC were treated with gefitinib and fulvestrant. An ongoing clinical trial (NCT01556191) in France is testing the combination of EGFR inhibitors and fulvestrant.
Overall, our study suggests that lung cancer in women who have never smoked is more frequently associated with EGFR mutations and ERα overexpression and that both biomarkers are associated. These findings underline the possibility of treating women who have never smoked with drugs to target hormonal factors, genetic abnormalities, or both.
The authors acknowledge the financial support of Ligue Régionale Contre le Cancer and of Institut National du Cancer for the Programme National d’Excellence Spécialisé (LG; PNES-LungG). LungG participants: Centre Chirurgical Marie-Lannelongue, Le Plessis-Robinson: P. Dartevelle, E. Dulmet, F. Leroy-Ladurie, V. de Montpreville; CHI Créteil: I. Monnet; CHU Dijon: A. Bernard, F. Piard; CHU Hôtel-Dieu, Paris: M. Alifano, S. Camilleri-Broët, J.F. Régnard; CHU Nice: P. Hofman, V. Hofman, J. Mouroux; CHU Saint-Louis, Paris: J. Trédaniel; CHU Strasbourg: M. Beau-Faller, G. Massard, A. Neuville; CHU Tenon, Paris: M. Antoine, J. Cadranel; CHU Toulouse: L. Brouchet, J. Mazières, I. Rouquette; HIA Percy, Clamart: P. Saint-Blancard, F. Vaylet; Institut Gustave-Roussy, Villejuif: A. Berhneim, P. Dessen, F. Dufour, N. Dorvault, P. Fouret, B. Job, L. Lacroix, V. Lazar, C. Richon, V. Roux, P. Saulnier, E. Taranchon, S. Toujani, A. Valent; Institut Mutualiste Montsouris, Paris: P. Girard, D. Gossot, P. Validire; Ligue Nationale contre le Cancer: J. Laffaire.
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