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Original Research: LUNG CANCER |

Clinical Significance of Thyroid Transcription Factor-1 in Advanced Lung Adenocarcinoma Under Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor TreatmentThyroid Transcription Factor-1 and Adenocarcinoma FREE TO VIEW

Kuei-Pin Chung, MD; Yen-Tsung Huang, MD, MPH; Yih-Leong Chang, MD, PhD; Chong-Jen Yu, MD, PhD; Chih-Hsin Yang, MD, PhD; Yeun-Chung Chang, MD; Jin-Yuan Shih, MD, PhD; Pan-Chyr Yang, MD, PhD, FCCP
Author and Funding Information

From the Department of Laboratory Medicine (Dr Chung), the Department of Pathology (Dr Y-L Chang), the Department of Internal Medicine (Drs Yu, Shih, and P-C Yang), and the Department of Medical Imaging (Dr Y-C Chang), National Taiwan University Hospital, College of Medicine, National Taiwan University; Graduate Institute of Oncology (Dr C-H Yang), Cancer Research Center, National Taiwan University, Taipei, Taiwan; and the Departments of Biostatistics and Epidemiology (Dr Huang), School of Public Health, Harvard University, Boston, MA.

Correspondence to: Jin-Yuan Shih, MD, PhD, Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, No. 7, Chung-Shan S Rd, Taipei 100, Taiwan; e-mail: jyshih@ntu.edu.tw


Funding/Support: This work was supported by the National Science Council [Grants 98-2314-B-002-117-MY3 and 98-2628-B-002-087-MY3], Taiwan; and National Taiwan University [Grant 99C101-101], Taiwan.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (http://www.chestpubs.org/site/misc/reprints.xhtml).


© 2012 American College of Chest Physicians


Chest. 2012;141(2):420-428. doi:10.1378/chest.10-3149
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Background:  Thyroid transcription factor 1 (TTF-1) positivity correlates with a higher prevalence of epidermal growth factor receptor (EGFR) mutation in lung adenocarcinoma. It is unknown whether TTF-1 expression affects the clinical outcome of patients with advanced lung adenocarcinoma, who have received EGFR tyrosine kinase inhibitors (TKIs) during the treatment course.

Methods:  This study enrolled patients with advanced lung adenocarcinoma who had results of EGFR mutation analysis and TTF-1 immunostaining. The impact of TTF-1 expression on overall survival (OS) and progression-free survival (PFS) under EGFR TKI treatment was evaluated. Multivariate analyses were done to examine the independent predictors of OS and PFS.

Results:  Of 496 patients with advanced lung adenocarcinoma, 443 had TTF-1-positive adenocarcinoma. Patients with TTF-1-positive lung adenocarcinoma had longer OS than did those with TTF-1-negative lung adenocarcinoma (median survival, 27.4 vs 11.8 months, P = .001). In patients with EGFR TKI treatment, those with TTF-1-positive lung adenocarcinoma and mutant EGFR had longer OS. In patients with EGFR mutation, those with TTF-1-positive lung adenocarcinoma had longer PFS than did those with TTF-1-negative lung adenocarcinoma (median survival, 8.7 vs 5.7 months, P = .043). Multivariate analysis showed that negative TTF-1 expression is a predictor for shorter OS, and a predictor for shorter PFS under EGFR TKI treatment.

Conclusions:  TTF-1 shows independent prognostic significance in advanced lung adenocarcinoma. Patients with TTF-1-negative lung adenocarcinoma have not only shorter OS, but also shorter PFS under EGFR TKI treatment, despite the existence of mutant EGFR. Further studies are needed to investigate the optimal treatment of patients with TTF-1-negative lung adenocarcinoma.

Figures in this Article

Thyroid transcription factor 1 (TTF-1) is an important transcription factor in lung morphogenesis and regulates gene expression of type 2 pneumocytes and Clara cells in the postnatal lung.1 It is an important marker in the immunohistochemistry (IHC) panel for the diagnosis of pulmonary adenocarcinoma.2,3 TTF-1-positive lung adenocarcinoma was found to have a morphologic resemblance to type 2 pneumocytes, Clara cells, and nonciliated bronchiolar cells.4 Previous studies showed that TTF-1 expression is a significant prognostic factor of better survival in lung adenocarcinoma.58 Furthermore, epidermal growth factor receptor (EGFR) mutation is associated with TTF-1 expression in lung adenocarcinoma.9,10 TTF-1-positive adenocarcinoma also shows a higher prevalence of negative p53 staining, less frequent retinoblastoma protein loss, and preserved expression of p27.4 Therefore, the expression of TTF-1 in lung adenocarcinoma is associated with different cancer genetics and clinical prognoses.

EGFR mutation is the most important factor for predicting the response of EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib or erlotinib. Deletions in exon 19 and L858R mutation in exon 21 are the most common mutations associated with the EGFR TKI response, whereas duplication, insertion, or T790M mutation in exon 20 are associated with resistance.1115 In patients with advanced non-small cell lung cancer (NSCLC), EGFR mutation could also confer survival benefits under EGFR TKI treatment.1619 Although a higher EGFR mutation rate was found in TTF-1-positive adenocarcinoma, 17.5% of TTF-1-negative adenocarcinoma still had mutant EGFR.9 For lung adenocarcinoma with mutant EGFR, the best treatment course of EGFR TKI between TTF-1-positive and TTF-1-negative adenocarcinoma is unknown.

Previous studies about the clinical outcome between TTF-1-positive and TTF-1-negative lung adenocarcinoma have enrolled patients mainly in an operable stage.57 In addition, it is unknown whether TTF-1expression affects the treatment efficacy of EGFR TKI. We conducted a study aimed at evaluating the prognostic significance of TTF-1 expression in advanced lung adenocarcinoma and on the efficacy of EGFR TKI treatment.

Study Population

Consecutive patients with lung adenocarcinoma diagnosed between January 2004 and December 2009 were identified using the database of the Cancer Registry, Medical Information Management Office of the National Taiwan University Hospital, a tertiary medical center in northern Taiwan. Lung cancer histology was classified according to World Health Organization (Geneva, Switzerland) pathology classification.20 Patients were included if they had (1) adequate tumor specimens for EGFR sequencing analysis and TTF-1 immunostaining, and (2) advanced disease, which was defined as stage IIIB with malignant pleural effusion and stage IV according to the sixth edition of the American Joint Committee on Cancer, or postoperative recurrence.21

Tumor specimens, including primary lung tumors, malignant pleural effusion cell blocks, and distant metastases, were obtained by surgical or needle biopsy. Patients were excluded if they had combined tumor histology other than adenocarcinoma (such as histologic features suggesting squamous cell carcinoma), or a history of a second malignancy other than lung cancer. The clinical data of enrolled patients retrieved from chart review included age (at diagnosis or recurrence), sex, smoking history, weight loss (≥ 5%) on presentation, Eastern Cooperative Oncology Group (ECOG) performance status, and metastatic sites at diagnosis or recurrence. The metastatic sites were determined by CT scans of the chest (from the neck to the level of the adrenal glands) and brain, and bone scan. When distant metastases were not revealed by CT scans or bone scans, a PET scan was performed. If metastases were suspected by PET, additional imaging studies (such as MRI), biopsies, or both were performed for confirmation. All patients received systemic chemotherapy or EGFR TKIs according to the decision of the attending physicians and the preference of the patients.

For all patients enrolled, we evaluated the factors that affected the overall survival (OS), which was measured from the first day of cancer treatment until death or the last follow-up on July 31, 2010. The study protocol was approved by the institutional review board of the National Taiwan University Hospital, IRB approval #201005008R.

TTF-1, E-cadherin, and Vimentin Expression

TTF-1 expression in cancer cells has been demonstrated with an IHC study, as reported previously.2 From each formalin-fixed, paraffin-embedded tissue block, 4-μm-thick tissue sections were obtained, which were dewaxed with xylene, followed by rehydration with a graded series of ethanol. The sections were then immunostained with a monoclonal antibody to 8G7G3/1 TTF-1 (1:200; Dako Corporation), followed by heat-induced epitope retrieval for 18 min in citrate buffer (pH 6.0). Theavidin-biotin method, with 3,3′-diaminobenzidine as chromogen, was used for antigen localization. A positive external control slide of TTF-1-positive lung adenocarcinoma and an internal control of nonneoplastic type 2 pneumocytes were included in each run. Cases with any definite nuclear staining were considered to be positive TTF-1 expression.

In addition, the membranous expression of E-cadherin and the cytoplasmic expression of vimentin were also determined by IHC studies. Specific monoclonal antibodies to E-cadherin (1:200; TaKaRa Bio Incorporation) and vimentin (1:50; Dako Corporation) were used. Tumor cells were considered positive for E-cadherin or vimentin expression if > 5% of cancer cells had immunoreactivity. Otherwise, negative E-cadherin or vimentin expression in cancer cells was considered.

Mutation Analysis of EGFR and KRAS

Mutation analysis of the EGFR gene was described previously.2224 Briefly, DNA was derived from tumor samples embedded in paraffin blocks using a QIAmp DNA Mini Kit (Qiagen). The tyrosine kinase domain of EGFR (exon 18-21) was amplified by polymerase chain reaction (PCR), and the amplicons were purified and sequenced by an automatic ABI PRISM 3700 DNA analyzer (Applied Biosystems). RNA was extracted from frozen tumor specimens using the RNeasy Mini Kit (Qiagen). Reverse transcription-PCR was used to amplify the four exons (exons 18-21) of the tyrosine kinase domain of the EGFR gene. The amplicons were then purified and sequenced.

KRAS mutation analysis was also performed as described previously.25 DNA from tumor specimens was sequenced for exon 2 of the KRAS gene. Forward and reverse sequencing reactions were performed on an ABI 3700 genetic analyzer (Applied Biosystems). All the sequencing reactions were performed in both forward and reverse directions using tracings from at least two PCRs.

EGFR TKI Treatment

The treatment courses of all study patients were reviewed, and those who had received EGFR TKI were identified. Patients who received TKI as maintenance therapy were excluded. We performed progression-free survival (PFS) and OS analyses in the remaining patients with EGFR TKI treatment. PFS was measured from the first day of TKI treatment until the first objective sign of disease progression or death, whichever occurred first. For evaluation of treatment response and disease progression, a chest radiograph was taken every 2 to 3 weeks and a chest CT scan was taken every 2 to 3 months.

Statistical Analysis

Pearson’s χ2 test or the Fisher exact test was used to compare categorical variables between two groups as appropriate. Kaplan-Meier curves with log-rank tests were used to evaluate the differences of OS and PFS between different stratified patient groups. Multivariate analyses with Cox proportional hazard model were performed to calculate the hazard ratios of death and of disease progression, with adjustment for other potential confounding factors. Details of the statistical method are described in e-Appendix 1. A two-sided P < .05 was considered statistically significant. The Kaplan-Meier curves were plotted with SPSS software (version 17.0 for Windows; SPSS Inc). Other statistical analyses were performed with SAS software (version 9.1; SAS Institute).

Patient Characteristics

From 2004 to 2009, 2,298 patients with lung adenocarcinoma were identified (737 patients with stage IV adenocarcinoma and 159 patients with stage IIIB adenocarcinoma with malignant pleural effusion). Five hundred forty-six patients with advanced-stage adenocarcinoma had tumor specimens adequate for EGFR mutation analysis and TTF-1 immunostaining. None of the patients had a combined tumor histology other than adenocarcinoma. Fifty patients were excluded because of a history of a second malignancy other than lung cancer. The remaining 496 patients constituted the study population. Their clinical characteristics are shown in Table 1. Their mean age was 62 years (range, 24-91 years), and 227 (46%) were men. The histology diagnosis of adenocarcinoma was established by surgical specimens in 136 patients, by small biopsies in 205 patients (156 by fine-needle biopsies and 49 by bronchoscopy-guided biopsies), and by pleural effusion cell blocks in 155 patients.

Table Graphic Jump Location
Table 1 —Clinical Characteristics of the Study Population

ECOG = Eastern Cooperative Oncology Group; EGFR = epidermal growth factor receptor; TKI = tyrosine kinase inhibitor; TTF = thyroid transcription factor 1.

a 

TTF-1(+) vs TTF-1(−).

b 

Includes malignant pleural effusion, malignant pericardial effusion, and metastasis to lung, bone, liver, adrenal gland, soft tissue, brain, peritoneum, spleen, and distant lymph node.

From the study population, 443 (89%) had TTF-1-positive lung adenocarcinoma. The clinical characteristics, including age, sex, smoking history, weight loss at presentation, ECOG performance status, disease stage (stage IV/IIIB or postoperative recurrence), number of metastatic sites, and use of EGFR TKI during the treatment course, were not different between patients with TTF-1-positive and TTF-1-negative tumors. Four hundred two patients received EGFR TKI treatment. Among them, four patients did not receive chemotherapy during the treatment course. The other 94 patients without EGFR TKI treatment received chemotherapy. The median OS of the entire group was 26.7 months (95% CI, 22.8-30.6 months).

EGFR Mutation Pattern

Patients with TTF-1-positive tumors had higher EGFR mutation rates than did those with TTF-1-negative tumors (61.9% vs 32.1%, P < .001) (Table 1). The EGFR mutation patterns are shown in Table 2 (e-Table 1). Of the 291 patients with mutant EGFR, 259 had a single EGFR mutation and the remaining 32 had more than one EGFR mutation. In patients with a single mutation, 117 had deletion in exon 19 and 113 had an L858R mutation. In those with more than one EGFR mutation (double or triple mutations), 14 had mutations involving deletion in exon 19 or L858R. None of these 14 patients had a T790M mutation, or duplication or insertion in exon 20, with the L858R or exon 19 mutations.

Table Graphic Jump Location
Table 2 —EGFR Mutation Patterns in 291 Patients With Mutant EGFR

Data are presented as No. (%). See Table 1 for expansion of abbreviations.

a 

Because of rounding, the total of the percentages does not equal 100.

b 

Includes all EGFR mutations other than deletion in exon 19 and L858R in exon 21. Refer toe-Table 1 for detailed EGFR mutation patterns.

c 

None of these patients had T790M or mutations in exon 20, with deletion in exon 19 or L858R mutation.

d 

Includes all other double or triple EGFR mutations not containing deletion in exon 19 or L858R mutation. Refer to e-Table 1 for detailed EGFR mutation patterns.

New Histology Classification of Lung Adenocarcinoma

During the writing of this article, a new classification scheme of lung adenocarcinoma was reported.26 According to this new classification scheme, 486 patients had a definitive histology diagnosis of adenocarcinoma, established by morphologic features under light microscopy. In the remaining 10 patients, IHC studies of the tumors showed positive TTF-1 expression, and the histology diagnosis was NSCLC, favoring adenocarcinoma. None of these 496 patients had histologic features of squamous cell carcinoma.

Of the 486 patients with a definitive diagnosis of adenocarcinoma, 286 (58.8%) had EGFR mutations. Of the 10 patients with NSCLC favoring adenocarcinoma, five (50%) had EGFR mutations (P = .748 by Fisher exact test, compared with those with a definitive diagnosis of adenocarcinoma). In addition, three patients had a histology of invasive mucinous adenocarcinoma according to the new classification scheme. All these patients had TTF-1-negative tumors, without EGFR mutations.

TTF-1 Expression and OS

Patients with TTF-1-positive lung adenocarcinoma had longer median survival than did those with TTF-1-negative lung adenocarcinoma (median survival, 27.4 vs 11.8 months; P = .001) (Fig 1). To evaluate the prognostic factors in all patients enrolled (N = 496), multivariate analysis for OS was performed with the Cox proportional model. The final Cox proportional model of OS is shown in Table 3. Patients with poor performance status (ECOG ≥ 2) and wide disease extent (number of metastatic sites ≥ 2) had shorter OS. Patients had longer OS if they had mutant EGFR (hazard ratio, 0.448; P < .001) or TTF-1 expression (hazard ratio, 0.452; P < .001). Patients who received EGFR TKI during the treatment course also had longer OS (hazard ratio, 0.257; P < .001). In the entire study population, only four patients did not receive chemotherapy during the treatment course. When chemotherapy treatment was included in the Cox proportional model, the hazard ratio for TTF-1 expression did not change significantly (hazard ratio, 0.456; P = .0001) (e-Table 2).

Figure Jump LinkFigure 1. Kaplan-Meier curves of overall survival (OS) in the entire study population (N = 496). Kaplan-Meier curves of OS were constructed with stratification by TTF-1. Patients with TTF-1-positive lung adenocarcinoma had longer OS than did those with TTF-1-negative lung adenocarcinoma (MST, 27.4 vs 11.8 months, P = .001). MST = median survival time; TTF-1 = thyroid transcription factor-1.Grahic Jump Location
Table Graphic Jump Location
Table 3 —Cox Proportional Hazard Model of Overall Survival in All Patients Enrolled in the Study (N = 496)

Stratified Cox proportional hazard model was performed with stratification factors of sex, smoking history, and weight loss. See Table 1 for expansion of abbreviations.

a 

Refers to postoperative recurrence.

b 

No. metastases > 1; the sites of metastases include malignant pleural effusion, malignant pericardial effusion, and metastasis to the lungs, bone, liver, adrenal gland, soft tissue, brain, peritoneum, spleen, and distant lymph node.

c 

Time-dependent covariates of mutant EGFR and TKI treatment were included in the final model. The estimate of the time-dependent covariate of mutant EGFR (time by mutant EGFR interaction term) was 0.0013 (SE, 0.0004; P = .0012). Two time-dependent covariates of TKI treatment (time by TKI treatment and time2 by TKI treatment interaction terms) were included. The estimate for time by TKI treatment interaction term was 0.0034 (SE, 0.0015; P = .0271), whereas the estimate for time2 by TKI treatment interaction term was −1.9089 × 10 (SE, 1.1195 × 10; P = .0882). The result suggests that the effects of mutant EGFR and TKI treatment decrease with time.

TTF-1 Expression and Survival Under EGFR TKI Treatment

Four hundred two patients received EGFR TKI during treatment. Fourteen patients received EGFR TKI as maintenance therapy (13 patients with TTF-1-positive tumors and one patient with a TTF-1-negative tumor), and they were excluded from further analyses. In the remaining 388 patients, we performed analyses of OS and PFS under EGFR TKI treatment. Two hundred fifty-one patients received EGFR TKI as first-line treatment, 85 as second-line, and 52 as third-line or subsequent lines. Their clinical characteristics are shown in Table 4. Patients were stratified into four groups according to TTF-1 positivity and EGFR mutation. The clinical characteristics were not significantly different among the four groups. Patients with TTF-1-positive adenocarcinoma and mutant EGFR had the longest OS (median survival, 30.1 months), whereas patients with TTF-1-negative adenocarcinoma and mutant EGFR had a median OS of only 10.8 months (P = .004, compared with patients with TTF-1-positive adenocarcinoma and mutant EGFR) (Fig 2A).

Table Graphic Jump Location
Table 4 —Clinical Characteristics of Patients With EGFR TKI Treatment (n = 388)

The table does not include patients who received EGFR TKI as maintenance therapy. See Table 1 for expansion of abbreviations.

a 

TTF-1(+) vs TTF-1(−).

b 

Mutant EGFR vs wild EGFR, in TTF-1(+) and TTF-1(−) groups, respectively

c 

Includes malignant pleural effusion, malignant pericardial effusion, and metastasis to lung, bone, liver, adrenal gland, soft tissue, brain, peritoneum, spleen, and distant lymph node.

Figure Jump LinkFigure 2. Kaplan-Meier curves of OS and progression-free survival in patients who had received EGFR TKI during treatment course (n = 388). A, In 388 patients who received EGFR TKI during treatment course, Kaplan-Meier curves of OS were constructed with stratification by TTF-1 and EGFR mutation. Patients with TTF-1-positive lung adenocarcinoma and mutant EGFR had longer survival. B, Kaplan-Meier curves of PFS were plotted with stratification by TTF-1 and EGFR mutations. In patients with mutations, those with TTF-1-positive adenocarcinoma had longer survival than those with TTF-1-negative adenocarcinoma (MST, 8.7 vs 5.7 months, P = .043). EGFR = epidermal growth factor receptor; TKI = tyrosine kinase inhibitor. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location

Regarding PFS under EGFR TKI treatment, patients with TTF-1-positive lung adenocarcinoma and mutant EGFR had longer PFS than did those with TTF-1-negative lung adenocarcinoma and mutant EGFR (median survival, 8.7 vs 5.7 months; P = .043) (Fig 2B). Multivariate analysis for PFS was performed with Cox proportional model (Table 5). EGFR mutation was the most significant factor associated with longer PFS under TKI treatment (hazard ratio, 0.241; P < .001). Patients with TTF-1-positive lung adenocarcinoma also had longer PFS (hazard ratio, 0.524; P < .001). Patients with poor performance status and wide disease extent (number of metastatic sites ≥ 2) also had shorter PFS. In addition, the clinical settings in which EGFR TKIs were used (first-line, second-line, third-line or more) did not affect PFS significantly.

Table Graphic Jump Location
Table 5 —Cox Proportional Hazard Model of Progression-Free Survival Under TKI Treatment (n = 388)

See Table 1 for expansion of abbreviations.

a 

Reference to postoperative recurrence.

b 

No. metastases > 1; the sites of metastases include malignant pleural effusion, malignant pericardial effusion, and metastasis to the lungs, bone, liver, adrenal gland, soft tissue, brain, peritoneum, spleen, and distant lymph node.

c 

Time-dependent covariate of mutant EGFR (time by mutant EGFR interaction term) was included in the final model, with the estimate as 0.0034 (SE, 0.0010; P = .0006). The result suggests that the effect of mutant EGFR decreases with time.

d 

Reference to first-line EGFR TKI treatment.

E-cadherin and Vimentin Expression and KRAS Mutation in TTF-1-negative Adenocarcinoma

A previous study suggested that TTF-1-positive adenocarcinoma might have a lower KRAS mutation rate than that of TTF-1-negative adenocarcinoma.9 TTF-1 expression was also shown to inhibit the transforming growth factor-β-mediated epithelial-to-mesenchymal transition (EMT) in lung adenocarcinoma cells.27 Moreover, poor clinical outcome under EGFR TKI treatment was found in NSCLCs with KRAS mutation or EMT activation.28,29 For TTF-1-negative lung adenocarcinoma, it is unknown whether the worse clinical outcome is due to EMT activation or KRAS mutation. Therefore, we performed IHC studies for E-cadherin and vimentin and KRAS mutation analysis in TTF-1-negative lung adenocarcinoma. Of 53 patients with TTF-1-negative adenocarcinoma, 37 had tumor specimens adequate for E-cadherin and vimentin IHC studies. Thirty patients had adenocarcinoma with positive E-cadherin and negative vimentin expression. The remaining seven patients had adenocarcinoma with negative E-cadherin (n = 4) or positive vimentin expression (n = 3). Only one patient among these seven had EGFR mutation (L858R). In addition, 35 patients had adequate tumor specimens for KRAS mutation analysis. Only three patients had KRAS mutation, and all of them had wild EGFR.

In patients with advanced lung adenocarcinoma, TTF-1 expression predicted longer OS independently. In patients who had received EGFR TKI during treatment course, those with TTF-1-positive lung adenocarcinoma and mutant EGFR had longer OS than did those with TTF-1-negative lung adenocarcinoma, or TTF-1-positive lung adenocarcinoma but wild EGFR. In patients with mutant EGFR, those with TTF-1-positive lung adenocarcinoma had longer PFS than did those with TTF-1-negative lung adenocarcinoma.

The definition of positive TTF-1 expression under IHC study varies according to the study. Some studies have defined positive TTF-1 expression as any definite nuclear staining, whereas other studies have defined positive TTF-1 expression as tumors with > 5% or > 50% positivity.5,7,9,30 However, one study showed that the clinical outcome between weakly TTF-1-positive and strongly TTF-1-positive adenocarcinoma was not different.7 Therefore, in this study, we defined positive TTF-1 expression as any definite nuclear staining in tumor cells.

In vitro studies show that the expression of TTF-1 in adenocarcinoma is related to proliferation and survival of cancer cells.3133 Furthermore, TTF-1 amplification, examined by fluorescent in situ hybridization, occurs in 6% to 14% of lung adenocarcinoma. However, TTF-1 amplification does not correlate with TTF-1 expression.7,33 In patients with lung adenocarcinoma, those with TTF-1 amplification did not have a different clinical outcome from those without TTF-1 amplification.7,33 The study populations were small in these studies, and further studies are needed to evaluate the clinical significance of TTF-1 amplification in lung adenocarcinoma.

Previous studies discussing TTF-1 expression and clinical outcome have focused mainly on operable stages. In one study, 83% of the study population had advanced lung adenocarcinoma.5 Patients with TTF-1-positive adenocarcinoma had a median survival of 14 months, whereas those with TTF-1-negative adenocarcinoma had a median survival of only 5 months. However, in this study, patients with TTF-1-positive adenocarcinoma had a median survival of 27.4 months, whereas those with TTF-1-negative adenocarcinoma had a median survival of 11.8 months. A high EGFR mutation rate (58.7%) was found in the study population, and most patients (81.0%) received EGFR TKI during the treatment course. This may explain the survival difference between the two studies. Additionally, advancements in chemotherapeutic agents may also account for such differences.

In patients receiving EGFR TKI during the treatment course, this study shows that patients with TTF-1-positive adenocarcinoma and mutant EGFR have a longer survival, compared with those with TTF-1-negative adenocarcinoma, or those with TTF-1-positive but wild EGFR. In patients with mutant EGFR, however, those with TTF-1-positive tumors had a longer PFS (8.7 months) than did those with TTF-1-negative tumors (5.7 months). According to recent phase 3 clinical trials, in patients with advanced NSCLC and sensitive EGFR mutations, PFS under EGFR TKIs is 9.2 to 14 months.18,19,34 In our study, patients with TTF-1-positive lung adenocarcinoma and mutant EGFR had PFS under EGFR TKI similar to that reported by previous clinical trials.

EMT is a critical step in cancer progression, invasion, and metastases.35,36 Lung cancer cells with EMT activation not only lose epithelial markers (such as E-cadherin) but have poor sensitivity to EGFR inhibition.37 Our recent study showed the expression of EMT regulator-Slug correlates with the acquired resistance to EGFR TKI.38 A previous study showed that TTF-1 expression inhibits the transforming growth factor-β-mediated EMT in lung adenocarcinoma cells.27 However, only seven out of 37 patients (18.9%) with TTF-1-negative adenocarcinoma had tumors with negative E-cadherin or positive vimentin expression. Furthermore, KRAS mutation is an important mechanism that causes poor response to EGFR TKI treatment. In our study, only three of 35 patients with TTF-1-negative adenocarcinoma (8.6%) had KRAS mutation. Therefore, the worse clinical outcome in patients with TTF-1-negative adenocarcinoma could not be explained completely by EMT activation or KRAS mutation. One recent study showed that downregulation of TTF-1 in adenocarcinoma was related to loss of differentiation and increased metastatic potential.39 The worse clinical outcome could be caused by the aggressive biologic behavior of TTF-1-negative adenocarcinoma. However, other mechanisms responsible might still exist, which need to be explored in future studies.

There are some limitations to this study. First, there is no report on the response rate to EGFR TKI in TTF-1-positive and TTF-1-negative lung adenocarcinoma. However, PFS may be a more important parameter than response rate in daily practice. Second, in the PFS analyses, EGFR TKIs were used at different times during the treatment course. Although patients might receive EGFR TKIs as first-line, second-line, or third-line treatment, our previous study has suggested that PFS is not affected by the timing of EGFR TKI treatment.23 Moreover, from multivariate analysis, the timing of EGFR TKI treatment (first-line, second-line, third-line or more) was not a significant factor affecting PFS. Therefore, this did not have a significant impact on our results. Finally, because it is a retrospective study, selection bias may exist, and further prospective studies are needed to confirm such results and to investigate whether the expression of TTF-1 affects the clinical outcome in lung adenocarcinoma.

In conclusion, TTF-1 not only is an important cell marker in a routine IHC panel, but also shows independent prognostic significance in advanced lung adenocarcinoma. Under EGFR TKI treatment, patients with TTF-1-positive lung adenocarcinoma and EGFR mutation had longer OS. In patients with EGFR mutation, those with TTF-1-negative adenocarcinoma had shorter PFS than did those with TTF-1-positive adenocarcinoma.

Author contributions:

Dr Chung: contributed to the conception and design of the study, financial support, collection and assembly of data, data analysis and interpretation, manuscript writing, and final approval of the manuscript.

Dr Huang: contributed to the collection and assembly of data, data analysis and interpretation, manuscript writing, and final approval of the manuscript.

Dr Y-L Chang: contributed to the provision of study materials and recruitment of patients, collection and assembly of data, data analysis and interpretation, and final approval of the manuscript.

Dr Yu: contributed to the conception and design of the study, administrative support, provision of study materials and recruitment of patients, and final approval of the manuscript.

Dr C-H Yang: contributed to the conception and design of the study, administrative support, provision of study materials and recruitment of patients, and final approval of the manuscript.

Dr Y-C Chang: contributed to the collection and assembly of data, data analysis and interpretation, and final approval of the manuscript.

Dr Shih: contributed to the conception and design of the study, financial support, provision of study materials and recruitment of patients, collection and assembly of data, data analysis and interpretation, manuscript writing, and final approval of the manuscript.

Dr P-C Yang: contributed to the conception and design of the study, administrative support, provision of study materials and recruitment of patients, manuscript writing, and final approval of the manuscript.

Financial/nonfinancial disclosures: The authors have reported to CHEST the following conflicts of interest: Dr Yu has received honoraria from AstraZeneca and Roche for speeches made. Drs C-H Yang and Shih have received honoraria from AstraZeneca and Roche for speeches and ad hoc advisory committee participation, and also played an unpaid advisory role for Boehringer Ingelheim. Drs Chung, Huang, Y-L Change, Y-C Chang, and P-C Yang have reported that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Role of sponsors: The sponsor had no role in the design of the study, the collection and analysis of the data, or in the preparation of the manuscript.

Additional information: The e-Appendix and e-Tables can be found in the Online Supplement at http://chestjournal.chestpubs.org/content/141/2/420/suppl/DC1.

ECOG

Eastern Cooperative Oncology Group

EGFR

epidermal growth factor receptor

EMT

epithelial-to-mesenchymal transition

IHC

immuno histo chemistry

NSCLC

non-small cell lung cancer

OS

overall survival

PCR

polymerase chain reaction

PFS

progression-free survival

TKI

tyrosine kinase inhibitor

TTF-1

thyroid transcription factor 1

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Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 2004;35021:2129-2139 [PubMed]
 
Pao W, Miller VA, Politi KA, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2005;23:e73 [PubMed]
 
Shih JY, Gow CH, Yang PC. EGFR mutation conferring primary resistance to gefitinib in non-small-cell lung cancer. N Engl J Med. 2005;3532:207-208 [PubMed]
 
Yang CH, Yu CJ, Shih JY, et al. Specific EGFR mutations predict treatment outcome of stage IIIB/IV patients with chemotherapy-naive non-small-cell lung cancer receiving first-line gefitinib monotherapy. J Clin Oncol. 2008;2616:2745-2753 [PubMed]
 
Wu JY, Wu SG, Yang CH, et al. Lung cancer with epidermal growth factor receptor exon 20 mutations is associated with poor gefitinib treatment response. Clin Cancer Res. 2008;1415:4877-4882 [PubMed]
 
Mitsudomi T, Kosaka T, Endoh H, et al. Mutations of the epidermal growth factor receptor gene predict prolonged survival after gefitinib treatment in patients with non-small-cell lung cancer with postoperative recurrence. J Clin Oncol. 2005;2311:2513-2520 [PubMed]
 
Takano T, Fukui T, Ohe Y, et al. EGFR mutations predict survival benefit from gefitinib in patients with advanced lung adenocarcinoma: a historical comparison of patients treated before and after gefitinib approval in Japan. J Clin Oncol. 2008;2634:5589-5595 [PubMed]
 
Mitsudomi T, Morita S, Yatabe Y, et al; West Japan Oncology Group West Japan Oncology Group Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol. 2010;112:121-128 [PubMed]
 
Maemondo M, Inoue A, Kobayashi K, et al; North-East Japan Study Group North-East Japan Study Group Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med. 2010;36225:2380-2388 [PubMed]
 
Travis WD. Pathology and Genetics of Tumours of the Lung, Pleura, Thymus and Heart. 2004; Oxford, England Oxford University Press
 
Greene FL. AJCC Cancer Staging Manual. 2002; New York, NY Springer
 
Shih JY, Gow CH, Yu CJ, et al. Epidermal growth factor receptor mutations in needle biopsy/aspiration samples predict response to gefitinib therapy and survival of patients with advanced nonsmall cell lung cancer. Int J Cancer. 2006;1184:963-969 [PubMed]
 
Wu JY, Yu CJ, Yang CH, et al. First- or second-line therapy with gefitinib produces equal survival in non-small cell lung cancer. Am J Respir Crit Care Med. 2008;1788:847-853 [PubMed]
 
Wu SG, Gow CH, Yu CJ, et al. Frequent epidermal growth factor receptor gene mutations in malignant pleural effusion of lung adenocarcinoma. Eur Respir J. 2008;324:924-930 [PubMed]
 
Wu JY, Yang CH, Hsu YC, et al. Use of cetuximab after failure of gefitinib in patients with advanced non-small-cell lung cancer. Clin Lung Cancer. 2010;114:257-263 [PubMed]
 
Travis WD, Brambilla E, Noguchi M, et al. International association for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol. 2011;62:244-285 [PubMed]
 
Saito RA, Watabe T, Horiguchi K, et al. Thyroid transcription factor-1 inhibits transforming growth factor-beta-mediated epithelial-to-mesenchymal transition in lung adenocarcinoma cells. Cancer Res. 2009;697:2783-2791 [PubMed]
 
Riely GJ, Marks J, Pao W. KRAS mutations in non-small cell lung cancer. Proc Am Thorac Soc. 2009;62:201-205 [PubMed]
 
Yauch RL, Januario T, Eberhard DA, et al. Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients. Clin Cancer Res. 2005;1124 pt 1:8686-8698 [PubMed]
 
Tan D, Li Q, Deeb G, et al. Thyroid transcription factor-1 expression prevalence and its clinical implications in non-small cell lung cancer: a high-throughput tissue microarray and immunohistochemistry study. Hum Pathol. 2003;346:597-604 [PubMed]
 
Tanaka H, Yanagisawa K, Shinjo K, et al. Lineage-specific dependency of lung adenocarcinomas on the lung development regulator TTF-1. Cancer Res. 2007;6713:6007-6011 [PubMed]
 
Kwei KA, Kim YH, Girard L, et al. Genomic profiling identifies TITF1 as a lineage-specific oncogene amplified in lung cancer. Oncogene. 2008;2725:3635-3640 [PubMed]
 
Weir BA, Woo MS, Getz G, et al. Characterizing the cancer genome in lung adenocarcinoma. Nature. 2007;4507171:893-898 [PubMed]
 
Rosell R, Moran T, Queralt C, et al; Spanish Lung Cancer Group Spanish Lung Cancer Group Screening for epidermal growth factor receptor mutations in lung cancer. N Engl J Med. 2009;36110:958-967 [PubMed]
 
Shih JY, Tsai MF, Chang TH, et al. Transcription repressor slug promotes carcinoma invasion and predicts outcome of patients with lung adenocarcinoma. Clin Cancer Res. 2005;1122:8070-8078 [PubMed]
 
Yanagawa J, Walser TC, Zhu LX, et al. Snail promotes CXCR2 ligand-dependent tumor progression in non-small cell lung carcinoma. Clin Cancer Res. 2009;1522:6820-6829 [PubMed]
 
Thomson S, Buck E, Petti F, et al. Epithelial to mesenchymal transition is a determinant of sensitivity of non-small-cell lung carcinoma cell lines and xenografts to epidermal growth factor receptor inhibition. Cancer Res. 2005;6520:9455-9462 [PubMed]
 
Chang TH, Tsai MF, Su KY, et al. Slug confers resistance to the epidermal growth factor receptor tyrosine kinase inhibitor. Am J Respir Crit Care Med. 2011;1838:1071-1079 [PubMed]
 
Winslow MM, Dayton TL, Verhaak RG, et al. Suppression of lung adenocarcinoma progression by Nkx2-1. Nature. 2011;4737345:101-104 [PubMed]
 

Figures

Figure Jump LinkFigure 1. Kaplan-Meier curves of overall survival (OS) in the entire study population (N = 496). Kaplan-Meier curves of OS were constructed with stratification by TTF-1. Patients with TTF-1-positive lung adenocarcinoma had longer OS than did those with TTF-1-negative lung adenocarcinoma (MST, 27.4 vs 11.8 months, P = .001). MST = median survival time; TTF-1 = thyroid transcription factor-1.Grahic Jump Location
Figure Jump LinkFigure 2. Kaplan-Meier curves of OS and progression-free survival in patients who had received EGFR TKI during treatment course (n = 388). A, In 388 patients who received EGFR TKI during treatment course, Kaplan-Meier curves of OS were constructed with stratification by TTF-1 and EGFR mutation. Patients with TTF-1-positive lung adenocarcinoma and mutant EGFR had longer survival. B, Kaplan-Meier curves of PFS were plotted with stratification by TTF-1 and EGFR mutations. In patients with mutations, those with TTF-1-positive adenocarcinoma had longer survival than those with TTF-1-negative adenocarcinoma (MST, 8.7 vs 5.7 months, P = .043). EGFR = epidermal growth factor receptor; TKI = tyrosine kinase inhibitor. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location

Tables

Table Graphic Jump Location
Table 1 —Clinical Characteristics of the Study Population

ECOG = Eastern Cooperative Oncology Group; EGFR = epidermal growth factor receptor; TKI = tyrosine kinase inhibitor; TTF = thyroid transcription factor 1.

a 

TTF-1(+) vs TTF-1(−).

b 

Includes malignant pleural effusion, malignant pericardial effusion, and metastasis to lung, bone, liver, adrenal gland, soft tissue, brain, peritoneum, spleen, and distant lymph node.

Table Graphic Jump Location
Table 2 —EGFR Mutation Patterns in 291 Patients With Mutant EGFR

Data are presented as No. (%). See Table 1 for expansion of abbreviations.

a 

Because of rounding, the total of the percentages does not equal 100.

b 

Includes all EGFR mutations other than deletion in exon 19 and L858R in exon 21. Refer toe-Table 1 for detailed EGFR mutation patterns.

c 

None of these patients had T790M or mutations in exon 20, with deletion in exon 19 or L858R mutation.

d 

Includes all other double or triple EGFR mutations not containing deletion in exon 19 or L858R mutation. Refer to e-Table 1 for detailed EGFR mutation patterns.

Table Graphic Jump Location
Table 3 —Cox Proportional Hazard Model of Overall Survival in All Patients Enrolled in the Study (N = 496)

Stratified Cox proportional hazard model was performed with stratification factors of sex, smoking history, and weight loss. See Table 1 for expansion of abbreviations.

a 

Refers to postoperative recurrence.

b 

No. metastases > 1; the sites of metastases include malignant pleural effusion, malignant pericardial effusion, and metastasis to the lungs, bone, liver, adrenal gland, soft tissue, brain, peritoneum, spleen, and distant lymph node.

c 

Time-dependent covariates of mutant EGFR and TKI treatment were included in the final model. The estimate of the time-dependent covariate of mutant EGFR (time by mutant EGFR interaction term) was 0.0013 (SE, 0.0004; P = .0012). Two time-dependent covariates of TKI treatment (time by TKI treatment and time2 by TKI treatment interaction terms) were included. The estimate for time by TKI treatment interaction term was 0.0034 (SE, 0.0015; P = .0271), whereas the estimate for time2 by TKI treatment interaction term was −1.9089 × 10 (SE, 1.1195 × 10; P = .0882). The result suggests that the effects of mutant EGFR and TKI treatment decrease with time.

Table Graphic Jump Location
Table 4 —Clinical Characteristics of Patients With EGFR TKI Treatment (n = 388)

The table does not include patients who received EGFR TKI as maintenance therapy. See Table 1 for expansion of abbreviations.

a 

TTF-1(+) vs TTF-1(−).

b 

Mutant EGFR vs wild EGFR, in TTF-1(+) and TTF-1(−) groups, respectively

c 

Includes malignant pleural effusion, malignant pericardial effusion, and metastasis to lung, bone, liver, adrenal gland, soft tissue, brain, peritoneum, spleen, and distant lymph node.

Table Graphic Jump Location
Table 5 —Cox Proportional Hazard Model of Progression-Free Survival Under TKI Treatment (n = 388)

See Table 1 for expansion of abbreviations.

a 

Reference to postoperative recurrence.

b 

No. metastases > 1; the sites of metastases include malignant pleural effusion, malignant pericardial effusion, and metastasis to the lungs, bone, liver, adrenal gland, soft tissue, brain, peritoneum, spleen, and distant lymph node.

c 

Time-dependent covariate of mutant EGFR (time by mutant EGFR interaction term) was included in the final model, with the estimate as 0.0034 (SE, 0.0010; P = .0006). The result suggests that the effect of mutant EGFR decreases with time.

d 

Reference to first-line EGFR TKI treatment.

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Anagnostou VK, Syrigos KN, Bepler G, Homer RJ, Rimm DL. Thyroid transcription factor 1 is an independent prognostic factor for patients with stage I lung adenocarcinoma. J Clin Oncol. 2009;272:271-278 [PubMed]
 
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Yatabe Y, Kosaka T, Takahashi T, Mitsudomi T. EGFR mutation is specific for terminal respiratory unit type adenocarcinoma. Am J Surg Pathol. 2005;295:633-639 [PubMed]
 
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Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 2004;35021:2129-2139 [PubMed]
 
Pao W, Miller VA, Politi KA, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2005;23:e73 [PubMed]
 
Shih JY, Gow CH, Yang PC. EGFR mutation conferring primary resistance to gefitinib in non-small-cell lung cancer. N Engl J Med. 2005;3532:207-208 [PubMed]
 
Yang CH, Yu CJ, Shih JY, et al. Specific EGFR mutations predict treatment outcome of stage IIIB/IV patients with chemotherapy-naive non-small-cell lung cancer receiving first-line gefitinib monotherapy. J Clin Oncol. 2008;2616:2745-2753 [PubMed]
 
Wu JY, Wu SG, Yang CH, et al. Lung cancer with epidermal growth factor receptor exon 20 mutations is associated with poor gefitinib treatment response. Clin Cancer Res. 2008;1415:4877-4882 [PubMed]
 
Mitsudomi T, Kosaka T, Endoh H, et al. Mutations of the epidermal growth factor receptor gene predict prolonged survival after gefitinib treatment in patients with non-small-cell lung cancer with postoperative recurrence. J Clin Oncol. 2005;2311:2513-2520 [PubMed]
 
Takano T, Fukui T, Ohe Y, et al. EGFR mutations predict survival benefit from gefitinib in patients with advanced lung adenocarcinoma: a historical comparison of patients treated before and after gefitinib approval in Japan. J Clin Oncol. 2008;2634:5589-5595 [PubMed]
 
Mitsudomi T, Morita S, Yatabe Y, et al; West Japan Oncology Group West Japan Oncology Group Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol. 2010;112:121-128 [PubMed]
 
Maemondo M, Inoue A, Kobayashi K, et al; North-East Japan Study Group North-East Japan Study Group Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med. 2010;36225:2380-2388 [PubMed]
 
Travis WD. Pathology and Genetics of Tumours of the Lung, Pleura, Thymus and Heart. 2004; Oxford, England Oxford University Press
 
Greene FL. AJCC Cancer Staging Manual. 2002; New York, NY Springer
 
Shih JY, Gow CH, Yu CJ, et al. Epidermal growth factor receptor mutations in needle biopsy/aspiration samples predict response to gefitinib therapy and survival of patients with advanced nonsmall cell lung cancer. Int J Cancer. 2006;1184:963-969 [PubMed]
 
Wu JY, Yu CJ, Yang CH, et al. First- or second-line therapy with gefitinib produces equal survival in non-small cell lung cancer. Am J Respir Crit Care Med. 2008;1788:847-853 [PubMed]
 
Wu SG, Gow CH, Yu CJ, et al. Frequent epidermal growth factor receptor gene mutations in malignant pleural effusion of lung adenocarcinoma. Eur Respir J. 2008;324:924-930 [PubMed]
 
Wu JY, Yang CH, Hsu YC, et al. Use of cetuximab after failure of gefitinib in patients with advanced non-small-cell lung cancer. Clin Lung Cancer. 2010;114:257-263 [PubMed]
 
Travis WD, Brambilla E, Noguchi M, et al. International association for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol. 2011;62:244-285 [PubMed]
 
Saito RA, Watabe T, Horiguchi K, et al. Thyroid transcription factor-1 inhibits transforming growth factor-beta-mediated epithelial-to-mesenchymal transition in lung adenocarcinoma cells. Cancer Res. 2009;697:2783-2791 [PubMed]
 
Riely GJ, Marks J, Pao W. KRAS mutations in non-small cell lung cancer. Proc Am Thorac Soc. 2009;62:201-205 [PubMed]
 
Yauch RL, Januario T, Eberhard DA, et al. Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients. Clin Cancer Res. 2005;1124 pt 1:8686-8698 [PubMed]
 
Tan D, Li Q, Deeb G, et al. Thyroid transcription factor-1 expression prevalence and its clinical implications in non-small cell lung cancer: a high-throughput tissue microarray and immunohistochemistry study. Hum Pathol. 2003;346:597-604 [PubMed]
 
Tanaka H, Yanagisawa K, Shinjo K, et al. Lineage-specific dependency of lung adenocarcinomas on the lung development regulator TTF-1. Cancer Res. 2007;6713:6007-6011 [PubMed]
 
Kwei KA, Kim YH, Girard L, et al. Genomic profiling identifies TITF1 as a lineage-specific oncogene amplified in lung cancer. Oncogene. 2008;2725:3635-3640 [PubMed]
 
Weir BA, Woo MS, Getz G, et al. Characterizing the cancer genome in lung adenocarcinoma. Nature. 2007;4507171:893-898 [PubMed]
 
Rosell R, Moran T, Queralt C, et al; Spanish Lung Cancer Group Spanish Lung Cancer Group Screening for epidermal growth factor receptor mutations in lung cancer. N Engl J Med. 2009;36110:958-967 [PubMed]
 
Shih JY, Tsai MF, Chang TH, et al. Transcription repressor slug promotes carcinoma invasion and predicts outcome of patients with lung adenocarcinoma. Clin Cancer Res. 2005;1122:8070-8078 [PubMed]
 
Yanagawa J, Walser TC, Zhu LX, et al. Snail promotes CXCR2 ligand-dependent tumor progression in non-small cell lung carcinoma. Clin Cancer Res. 2009;1522:6820-6829 [PubMed]
 
Thomson S, Buck E, Petti F, et al. Epithelial to mesenchymal transition is a determinant of sensitivity of non-small-cell lung carcinoma cell lines and xenografts to epidermal growth factor receptor inhibition. Cancer Res. 2005;6520:9455-9462 [PubMed]
 
Chang TH, Tsai MF, Su KY, et al. Slug confers resistance to the epidermal growth factor receptor tyrosine kinase inhibitor. Am J Respir Crit Care Med. 2011;1838:1071-1079 [PubMed]
 
Winslow MM, Dayton TL, Verhaak RG, et al. Suppression of lung adenocarcinoma progression by Nkx2-1. Nature. 2011;4737345:101-104 [PubMed]
 
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