Different estrogen receptor β expression in distinct histologic subtypes of lung adenocarcinoma B

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1 Human Pathology (2008) 39, Original contribution Different estrogen receptor β expression in distinct histologic subtypes of lung adenocarcinoma B Greta Alì MD a, Valentina Donati MD a, Barbara Loggini MD a, Adele Servadio MD a, Matteo Dell'Omodarme PhD c, Maria Cristina Prati PhD c, Tiziano Camacci BS a, Marco Lucchi MD b, Franca Melfi MD b, Alfredo Mussi MD b, Gabriella Fontanini MD a, a Department of Surgery, Division of Anatomic Pathology, University of Pisa, Pisa, Italy b Department of Cardio-Thoracic Surgery, University of Pisa, Pisa, Italy c Scuola Normale Superiore and Istituto di Fisica Nucleare, Section of Pisa, Pisa, Italy Received 7 January 2008; revised 18 February 2008; accepted 20 February 2008 Keywords: Lung adenocarcinoma; Histologic subtypes of adenocarcinoma; Estrogen receptor beta; Immunohistochemistry Summary Adenocarcinoma is becoming the most common histologic type of lung cancer in both sex. Although most cases are seen in smokers, it develops more frequently than other histologic types in individuals who have never smoked. This evidence suggests that other putative etiologic factors, such as sex hormones, need to be investigated. Several subtypes of lung adenocarcinoma have been recently described with distinct clinicopathologic features and prognostic implications. The purpose of this study is to investigate the role of estrogen receptor β in lung adenocarcinoma, with particular attention paid to its different histologic subtypes. Nuclear estrogen receptor β expression was evaluated by immunohistochemistry in 112 lung adenocarcinomas, including both single subtype and mixed subtype samples. Using a 2-level (high/low) score system, estrogen receptor β expression was high in most (75%) adenocarcinomas and turned out to be strongly related to the histologic subtypes. In fact, estrogen receptor β expression was low or negative in 68.2% of solid subtypes, whereas it was high in 76.5% of nonmucinous bronchioloalveolar, in 69.4% of acinar, and in 61.2% of papillary patterns (P =.00004). Furthermore, a strong association between estrogen receptor β expression and tumor histologic grade was observed: estrogen receptor β was highly expressed predominantly in well- and moderately differentiated tumors (P =.0014). In conclusion, estrogen receptor β expression has distinct patterns in lung adenocarcinoma, suggesting a specific role for estrogen receptor β in the pathogenesis of different histologic subtypes of this type of cancer. Moreover, loss of estrogen receptor β expression in poorly differentiated (G3) tumors could represent a crucial step in the dedifferentiation process of lung adenocarcinoma Published by Elsevier Inc. Valentina Donati was supported by a fellowship from Fondazione Italiana per la Ricerca sul Cancro. Corresponding author. Department of Surgery, Division of Anatomic Pathology, University of Pisa, Via Roma 57, Pisa, Italy. address: g.fontanini@do.med.unipi.it (G. Fontanini). 1. Introduction Lung cancer is one of the main causes of cancer death in the world, and cigarette smoking is still its principal cause, as 85% to 90% of diagnosed patients have smoked [1] /$ see front matter 2008 Published by Elsevier Inc. doi: /j.humpath

2 1466 G. Alì et al. However, smoking is not the entire story in lung cancer development because 10% of patients are classified as never smokers (b100 cigarettes in lifetime), and 50% of patients are former smokers (at least 100 cigarettes in lifetime but quit smoking more than 12 months before lung cancer diagnosis or before the interview) [1]; therefore, other factors besides cigarette smoking may have a role in the pathogenesis of lung adenocarcinoma. Histologically, lung cancer is classified into small cell lung cancer and non small cell lung cancer, the latter of which includes 3 major histotypes: squamous cell carcinoma, adenocarcinoma, and large cell carcinoma [2]. While squamous cell carcinoma has a very strong association with smoking and is very frequently found in males, adenocarcinoma is the most common form of lung cancer in never smokers, young people, and women of all ages [1]. According to epidemiological data, the incidence of squamous cell carcinoma is decreasing in many countries along with a decline of smoking population, whereas the incidence of adenocarcinoma is rapidly increasing. Adenocarcinoma represents about one third of primary lung cancers in men and about three fourths of primary lung cancers in women [1]. Adenocarcinomas may be classified as single subtype or mixed subtype, with the latter being the most frequent group, representing 80% of resected adenocarcinomas. The major individual histologic patterns/subtypes are acinar, papillary, bronchioalveolar, solid with mucin production, clear cell, mucinous, and signet ring [2]. Adenocarcinoma is characterized by a wide histologic and cytologic heterogeneity, especially in advanced tumors. Recent studies [3,4] focusing on differences in tumor progression observed in distinct subtypes of adenocarcinoma highlighted that different histologic subtypes of lung adenocarcinoma may be considered different entities based on both clinicopathologic and molecular characteristics. Additional studies will be necessary to clarify if the histologic classification of lung adenocarcinoma in different subtypes may add useful information to the current staging system for the management of patients. The increasing incidence of lung cancer in women, with particular reference to adenocarcinoma, has suggested that hormonal status may play an important role in the development of lung cancer [5,6]. Several studies demonstrate that women are more susceptible (dosage-wise) to carcinogenic effects of tobacco smoke [7]. Although the data are still controversial, women with a history of hormone replacement therapy appear to have a reduced risk of lung cancer [8]. The potential of estrogens to initiate and subsequently promote the growth of tumors of female reproductive organs such as the ovary, uterus, and breast, is well established, and their role in other cancer tumorigenesis, such as prostate and colon, has been studied [9]. In 1996, a novel estrogen receptor called estrogen receptor (ER) β was cloned and localized to chromosome 14 [10]. The 2 known wild-type ER forms, ERα and ERβ, share considerable amino acid homology in their sequence and in their ligand-binding regions but differ substantially in tissue distributions [11]. ERβ has been shown to be expressed in both normal lungs as well as in lung tumors [12]. Moreover, it has been demonstrated that ERβ regulates lung development, particularly alveolar formation and surfactant homeostasis in mouse models [13]. Evidence from some studies shows that estrogen-signaling pathways play an important role in normal lung biology and in the control of lung cancer growth [12,14]. Furthermore, in a comparative study, differential expression of both receptor forms (α and β) has been described in both female and male adenocarcinomas of the lung [15]. All these findings suggest the important role of ER, in particular of ERβ, in lung adenocarcinoma, prompting us to investigate their relationship between different histologic aspects to understand the biologic basis of the development of subtypes of lung adenocarcinoma. 2. Materials and methods 2.1. Patients One hundred twelve adenocarcinoma specimens were obtained from 112 patients (71 men and 41 women) who underwent surgical resection at the Department of Cardiothoracic Surgery of the University of Pisa, Pisa, Italy, from January 2002 to March Participation in this study required informed consent. Patients did not receive neoadjuvant chemotherapy or radiation therapy. Clinical information, including patient sex, age, local recurrences, and distant metastasis, were reviewed for each patient. Data concerning smoking status were obtained in 95 of the 112 cases by presurgery or postsurgery personal interviews. An individual who had never smoked or had smoked less than 100 cigarettes in his/her lifetime was defined as a never smoker. An individual who had smoked at least 100 cigarettes in his/her lifetime but quit smoking more than 12 months before lung cancer diagnosis or before the interview was considered to be a former smoker. Current smokers comprised those patients who currently smoke and recent quitters [16] Tissue lung specimens All tumor samples were formalin-fixed and paraffinembedded for microscopic examination. The most representative paraffin block of tumor was selected for analysis for each case. Histologic diagnosis and pathologic features were obtained by 2 pathologists (G Alì and V Donati) according to the World Health Organization (WHO) 2004 histologic criteria [2]. Disagreements were discussed with a third pathologist (G Fontanini), and after a critical discussion, mutual agreement was reached. Special attention was paid to determine the different histologic subtypes/patterns of adenocarcinoma (papillary, acinar, mucinous/nonmucinous/

3 Estrogen receptor β in lung adenocarcinoma mixed bronchioloalveolar, solid, signet ring, mucinous, clear cell). Pathologic grading was determined both for adenocarcinomas single subtype and for each subtype/ pattern composing adenocarcinomas mixed subtype. Pathologic staging was performed according to the TNM classification [17] Immunohistochemistry ERβ immunohistochemical analysis was performed on 3-μm tissue sections. After deparaffinization through serial xylene baths, sections were rehydrated through a graded series of ethanol and water baths (5 minutes each). After a deionized water wash (3 minutes), the sections were heated to 98 C through a bath thermostat for 40 minutes to unmask target antigens then cooled in solution at room temperature and washed with phosphate-buffered saline (PBS) for 3 minutes. After treatment with peroxide block, sections were washed again in PBS and incubated with power block reagent (Biogenex Laboratories, San Ramon, CA) for 30 minutes. Primary mouse antihuman estrogen receptor β1 monoclonal antibody (PPG5/10 diluted 1:50; MCA 1974S; Serotec Ltd, Oxford, UK) was applied overnight at 4 C. After washing with PBS, immunoreactivity was obtained by using the Super Sensitive Polymer-HRP Detection System (Biogenex), following the manufacturer's instructions. After rinsing with PBS for 3 minutes, sections were incubated 20 minutes at room temperature with Super Enhancer Reagent, followed by incubation with Poly-HRP reagent. The reaction was developed using 0.05% 3,3 -diaminobenzidine tetrahydrochloride until adequate color development was seen. A negative control was obtained by substituting primary antibody with PBS Table 1 Clinical and pathologic features of 112 patients with lung adenocarcinoma Clinicopathologic characteristics No. of cases (%) Age Range, y Average, 66 y Sex Male 71 (63.4%) Female 41 (36.6%) Smoking status Current 42 (44.2%) Former 35 (36.8%) Never 18 (19.0%) Tumor size T1 32 (28.6%) T2 53 (47.3%) T3 10 (8.9%) T4 17 (15.2%) Lymph node metastases N0 57 (54.8%) N1 15 (14.4%) N2 32 (30.8%) Stage Ia 15 (14.4%) Ib 25 (24.0%) IIa 3 (2.9%) IIb 12 (11.5%) IIIa 25 (24.0%) IIIb 15 (14.4%) IV 9 (8.8%) Grading G1 (well) 6 (5.4%) G2 (moderate) 81 (72.3%) G3 (poor) 25 (22.3%) Histology Adenocarcinomas with single subtype 55 (49.1%) Adenocarcinomas mixed subtype 57 (50.9%) Fig. 1 Immunohistochemical staining of ERβ in lung adenocarcinoma with mixed subtype showing different ERβ expression between its distinct subtypes: high ERβ expression in the nonmucinous bronchioloalveolar subtype (intensity score 3, proportion score 5) and low ERβ expression in the solid subtype (intensity score 1; proportion score 3) (original magnification 50). Two pathologists (G Alì and V Donati) evaluated and graded immunohistochemical staining by scoring separately the percentage of positive tumor cells (nuclear positivity) and their staining intensity, using previously described scoring systems [18-20]. Disagreements were discussed with a third pathologist (G Fontanini), and mutual agreement was reached in all discordant cases after a critical discussion on the criteria that led to different interpretations. The pathologists were blinded to clinicopathologic characteristics of the patients. Different patterns composing adenocarcinomas mixed subtype were evaluated individually, and the scores for each pattern were determined separately (Fig. 1). A proportion score (PS) was assigned representing the estimated proportion of positive staining cells and was graded in 6 classes: negative (score 0); greater than 0 to 1/ 100 (score 1); greater than 1/100 to 1/10 (score 3); greater than 1/10 to 1/3 (score 3); greater than 1/3 to 2/3 (score 4); and greater than 2/3 to 1 (score 5). An intensity score (IS) was also assigned and graded in 4 classes: none (score 0);

4 1468 G. Alì et al. Table 2 Histologic subtypes of adenocarcinoma of the lung and histologic grade Subtypes Total G1 G2 G3 Acinar Papillary Solid Nonmucinous bronchioloalveolar Mucinous bronchioloalveolar Clear cell Signet ring Mucinous Total subtypes comprised adenocarcinomas of single subtype and each subtype composing adenocarcinomas of mixed subtype. weak (score 1); intermediate (score 2); strong (score 3). Then, a final total score was obtained from the sum of PS and IS and graded in 2 classes: high (5-8) with a higher expression of ERβ and low (0-4) with a lower or negative ERβ expression. In addition, in order to determine the best evaluation system for ERβ expression, total score was also graded in 3 classes: class A (6-8) had a high expression of ERβ; class B (4-5) had a moderate expression of ERβ; and class C (0-3) had a negative or low expression of ERβ Statistical analysis Statistical analyses were performed using R (R Development Core Team, Vienna, Austria) [21]. The correlation between various clinical or pathologic parameters and the expression (score proportion, score intensity, and total score) of ERβ was analyzed using the χ 2 test [22]. Quantitative variables were categorized with respect to their median. Values of P b.05 were considered significant. Fig. 2 Immunohistochemical staining of ERβ in distinct subtypes of adenocarcinomas (A-D). A, ERβ negative staining in a case of lung adenocarcinoma with solid pattern (total score 0 = IS 0 + PS 0). B, An example of high ERβ expression in a lung adenocarcinoma with nonmucinous bronchioloalveolar pattern: an intense (intensity score 3) positive staining is present in 70% of tumor cells (proportion score 5). C, papillary subtype of lung adenocarcinoma with high proportion score (PS 5, 90% of positive staining ERβ tumor cells) and high intensity score (IS 3). D, Lung adenocarcinoma with acinar pattern with total score 7 (intense positive staining in 60% of tumor cells) (original magnification 200).

5 Estrogen receptor β in lung adenocarcinoma 3. Results 3.1. Expression of ERβ in lung adenocarcinomas All analyzed tumors were adenocarcinomas of either mixed subtype (49.6%) or single subtype (50.4%). Clinicopathologic characteristics of patients are summarized in Table 1. Each single histologic pattern of the mixed subtype was evaluated with particular focus on grading (Table 2). Using the 2 levels score system (low and high), ERβ expression was reported as high in 75% (84/112) of the cases and negative or low in the remaining 25% (28/112). With the other scoring system (3 levels: A, B, C), 53.6% (60/112) of adenocarcinomas expressed high ERβ (score A), 36.6% (41/ 112) had moderate expression (score B), and 9.8% (11/112) had negative or low expression of ERβ (score C) Correlation between ERβ expression and different subtypes of adenocarcinomas 1469 A statistically significant association was found between ERβ expression and different subtypes of adenocarcinoma. ERβ expression showed a different trend in solid subtypes compared to nonmucinous bronchioloalveolar, acinar, and papillary patterns (Fig. 2A-D). According to the 2-level scoring system, ERβ expression was low or negative in most (68.2%) of the solid subtypes (Fig. 2A) but only in 23.5% of nonmucinous bronchioloalveolar subtypes (Fig. 2B), in 30.6% of acinar subtypes (Fig. 2D), and in 38.8% of papillary subtypes (Fig. 2C) (P =.00004) (Fig. 3A). Concordant data were obtained using the other score system: 84.1% of solid subtypes had low or moderate expression of ERβ (score B and score C), whereas opposite trends of ERβ Fig. 3 Classification of our study group based on ERβ total scores and histologic subtypes of adenocarcinomas (acinar, nonmucinous bronchioloalveolar, papillary, solid, mucinous, clear cell). A, Different histologic subtypes of lung adenocarcinomas (x-axis) and the number of cases relative to the total scores graded in 2 classes (high/low) (y-axis). B, Different histologic subtypes of lung adenocarcinomas (x-axis) and the number of cases relative to the total scores graded in 3 classes (A/B/C) (y-axis).

6 1470 G. Alì et al. Fig. 4 Classification of our study group based on ERβ total scores and histologic grade of adenocarcinomas with single subtype and for each single subtype composing adenocarcinomas mixed subtype. A, Different tumor histologic grades (G1, well differentiated; G2, moderately differentiated (x-axis; G3, poorly differentiated) and the number of cases relative to the total scores graded in 2 classes (high/low) (y-axis). B, Different tumor histologic grades (x-axis; G1, well-differentiated; G2, moderately differentiated; G3, poorly differentiated) and the number of cases relative to the total scores graded in 3 classes (A/B/C) (y-axis). expression were observed in papillary, acinar, and nonmucinous bronchioloalveolar subtypes (P = ) (Fig. 3B). ERβ expression was reported to be high in all of the 9 cases of mucinous subtype, and in 5 (83.3%) of 6 of clear cell subtype. Signet ring and mucinous bronchioloalveolar subtypes were not included due to the limited number of samples (1 and 2 cases, respectively) Correlation between ERβ expression and histologic grade An interesting association was observed between ERβ expression and the histologic grade of adenocarcinoma subtypes. In the mixed subtype, the histologic grade was analyzed for each single pattern. Using the 2-level scoring system, low or negative expression of ERβ was observed in 59.6% of poorly differentiated tumors (G3), but only in 30.0% of well-differentiated tumors and in 30.4% of moderately differentiated tumors (P =.0014) (Fig. 4A). Similar results were observed using the 3-level scoring system: 50% of poorly differentiated tumors showed low or negative expression of ERβ (score C), whereas only 16.1% of moderate and 17.7% of well-differentiated subtypes had low expression of ERβ (score C) (P = ) (Fig. 4B) Correlation between expression of ERβ and other clinicopathologic features No significant association was found between ERβ expression and other clinicopathologic parameters, such as sex, tumor size, lymph node status, distant metastasis, and stage (data not shown). Smoking status (never, former, current) did not show any statistically significant correlation with ERβ expression,

7 Estrogen receptor β in lung adenocarcinoma Fig. 5 Classification of our study group based on smoking status, histologic grade of adenocarcinomas, and sex. A, Patients' smoking status (C, current smokers; F, former smokers; N, never smokers) (x-axis) and different tumor histologic grades (G1, well-differentiated; G2, moderately differentiated; G3, poorly differentiated) (y-axis). B, Patients' sex (F, females; M, males) (x-axis) and patients' smoking status (C, current smokers; F, former smokers; N, never smokers) (y-axis). although it did show an interesting association with histologic grade. No poorly differentiated adenocarcinoma of the lung was described in never smokers, whereas a progressive increase in its frequency was observed in former (22.8%) and current (33.3%) smokers (P =.034) (Fig. 5A). Moreover, smoking status was significantly associated with sex, with a large disproportion of women versus men in the never smokers group (32.4% versus 10.2%) (Fig. 5B). 4. Discussion Lung carcinoma, such as tumors in other organs, is thought to arise through a stepwise series of molecular and cellular alterations in precursor lung cells. Although many of these genetic changes occur independently from the histologic type, some of these appear to be linked to a specific histology, and increasing knowledge on the different genetic pathways in histologic subtypes of lung cancer has been reported previously [23,24]. There is overwhelming evidence that tobacco smoking is the major cause of lung cancer [1]. Tobacco smoking increases the risk of all major histologic types of lung carcinoma but appears to be strongest for squamous cell carcinoma, followed by small cell carcinoma and adenocarcinoma. Conversely, lung adenocarcinoma is the most frequent histologic type in women, nonsmokers, and young people [1]. This evidence suggests that other etiologic factors 1471 may have an important role in the development of specific type of lung cancer (eg, adenocarcinoma), especially hormonal levels. Several studies indicate that estrogen may play a role in different human cancers [9], including lung cancer [25]. Estrogen receptor expression has been documented in normal lung and tumor tissue, mostly in the β isoforms. Some previous studies [6,12,15] have reported expression of ERβ in lung tumor tissue, particularly in adenocarcinoma, but an absence or very low expression of ERα, whereas another immunohistochemical study evidences a similar expression of ERβ (52%) and ERα (45%) in non small cell lung cancer [26]. Cell lines of lung adenocarcinomas have been reported to mostly express higher levels of the β isoform [27]. In our study, we evaluated the immunohistochemical expression of ERβ in 112 lung adenocarcinomas in order to evaluate whether estrogen levels may represent a biologic basis for this heterogeneous group of lung tumors. In our series of adenocarcinomas, high expression of ERβ was found in most of the cases using both the 2- (75% high versus 25% low) and the 3-level scoring systems (53.6% A; 36.6% B; 9.8% C). Moreover, interesting data were found when the different histologic patterns, both in the mixed subtype and single subtype, were analyzed. The histologic subclassification of lung adenocarcinoma is still a challenge for the heterogeneity of histologic, clinical, and radiologic presentations of such tumors and has evolved considerably since the original WHO classification in The most significant change in histologic subclassification of lung adenocarcinoma was introduced in the 1999 WHO classification with the addition of adenocarcinoma with mixed subtypes. Other attempts of classification, such as the one proposed by Noguchi et al [28] in 1995 and the unique diagnostic criteria proposed by Silver and Askin [29] in 1997 for papillary carcinoma, are noteworthy because of their prognostic implications. Subsequently, other reports have examined solitary, small, peripheral lung adenocarcinomas using different approaches and evaluating distinct features to identify histologic prognostic factors and define a subgroup of mixed-subtype adenocarcinomas with favorable prognosis [30,31]. All these reports are important attempts of classification from the clinical point of view because they suggest that the above cited features should be considered when designing or evaluating new treatment strategies particularly for small adenocarcinoma of the lung. However, in spite of the several attempts of classification, the fourth edition of the WHO classification of lung and pleura tumors in 2004 has become the standard classification system of lung adenocarcinoma [2]. It is readily applicable to research studies and used in almost all routine pathology laboratories. As far as we know, this is the first study in which the relationship between ERβ expression and histologic patterns of adenocarcinomas has been extensively investigated. In fact, according to the 2-level score system, adenocarcinomas with a solid pattern showed a lower ERβ expression in 68.2% of the cases, whereas a high number of papillary (61.2% of

8 1472 G. Alì et al. the cases), acinar (69.4%), and nonmucinous bronchioloalveolar (76.5%) adenocarcinoma patterns expressed high levels of ERβ (P =.00004). Similar results were found using the 3 level score system (A/B/C) (P = ). Our results suggest that ERβ might have a key role in the pathogenesis of adenocarcinomas, specifically driving different differentiative pathways. Adenocarcinomas are a very heterogeneous subgroup of lung cancers, in which oncogenesis is linked to different molecular events. Adenocarcinomas arising in nonsmokers are defined by the presence of tyrosine kinase epidermal growth factor receptor (EGFR) mutations, and various studies [32,33] provide evidence of a functional interaction between the ER and the EGFR pathways. Our results represent the first step for further detailed molecular analysis involving each different histologic pattern of lung adenocarcinoma, starting from the knowledge that different morphological outcomes show different ERβ expression. In addition, we found a high expression of ERβ in all of the 9 cases of the mucinous subtype. These data may be relevant because recent studies [3,4] correlate the mucinous bronchioloalveolar subtype with EGFR and K-ras mutational status. Therefore, ERβ expression in subtypes of lung adenocarcinoma with mucinous differentiation could be interesting to investigate. In our series, adenocarcinoma of mixed subtype showed a different degree of differentiation (well, moderate, poor), with various degrees of cytologic atypia (mild, moderate, marked) among its different patterns. We determined the histologic grade for each subtype identifying well, moderate, and poorly differentiated subgroups. The bronchioloalveolar pattern is generally always moderately or well differentiated, whereas solid adenocarcinomas are poorly differentiated [2]. Using the 2-level score system, a lower or negative expression of ERβ was observed in 59.6% of less differentiated tumors (G3), whereas an opposite trend in well- (30.0%) or moderately (30.4%) differentiated was found. In lung adenocarcinoma, the histologic grade seems to have a prognostic impact. As reported by Chung et al [34], patients with poorly differentiated tumors have generally more local recurrences and lymph node metastases than patients with well or moderately differentiated lesions. Therefore, we hypothesized that ERβ is lost during dedifferentiation of tumor cells, suggesting that the lack of ERβ could be associated with an aggressive biologic behavior. Similar evidence was also observed in breast cancer, as well as other tumors models [9]. The loss of ERβ expression seems to be a critical step in the development and progression of estrogendependent tumor, and this finding could also be true in the progression of lung adenocarcinoma. In our series, smoking status was obtained in 95 of 112 patients. No significant association was found between ERβ expression and smoking status, although tobacco smoking was related to histologic grade. Current and former smokers had a higher frequency of poorly differentiated tumors (33.3% and 22.8%, respectively), compared with never smokers (0.0%) (P =.0034). Moreover, according to data previously reported [35], a correlation was observed between smoking status and sex, as most never smokers patients were women (32.4% versus 10.2%) (P =.027). These findings are consistent with the concept that adenocarcinoma in women may have a different route of development. In this context, our data may offer important details in the understanding of the complex puzzle of adenocarcinoma development, indicating that the loss of ERβ receptor may play a crucial role in the dedifferentiation process. Therefore, ERβ expression could represent a useful marker for a more accurate histopathologic subclassification of adenocarcinomas, with important application in clinical behavior and outcome of patients with distinct histologic subtypes. Further analysis on the ERβ and EGFR or K-ras pathways may provide the basis for a new type of targeted therapy in selected patients with lung cancer. Acknowledgments We thank Dr Laura Boldrini who provided technical support in the data analysis. References [1] Muscat JE, Wynder EL. Lung cancer pathology in smokers, exsmokers, and never smokers. Cancer Lett 1995;88:1-5. [2] Travis WD, Brambilla E, Muller-Hermelink HK, Harris CC. World Health Organization classification of tumours. Pathology and Genetics of tumours of the lung, pleura, thymus and heart. Lyon, France: IARC Press; [3] Sakuma Y, Matsukuma S, Yoshihara M, et al. 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