Purpose: The primary objective of this study was to determine whether markers of differentiation and activation of the Akt pathway are associated with metastasis in adenocarcinoma of the lung.

Experimental Design: Paired primary and metastatic tumor samples were obtained from 41 patients who had undergone resection of both primary lung adenocarcinoma and brain metastatic lesions. Paired samples were compared for relative expression of thyroid transcription factor 1 (TTF-1) and E-cadherin as potential markers of differentiation. Activation of the Akt pathway was assessed by expression of p-Akt and p-S6. Biomarkers that showed relative discordance in expression between the matched pairs were then assessed in a cohort of 77 primary lung adenocarcinomas. Validation was done in an independent cohort of 82 primary lung adenocarcinomas.

Results: Among the 41 matched pairs, E-cadherin (23 discordant pairs) and TTF-1 (18 discordant pairs) were overexpressed in primary tumors (20 of 23 and 15 of 18, respectively). In contrast, p-S6 overexpression was significantly associated with metastatic tumors (20 of 21 discordant pairs). The expression of E-cadherin, p-S6, and TTF-1 was evaluated in 77 primary lung adenocarcinomas, in which high p-S6 expression was associated with shorter time to metastasis. The association of p-S6 with metastasis was then validated in an independent set of 82 tumors. In multivariable analysis, p-S6 expression was a negative independent predictor of metastasis-free survival after adjustment for tumor stage.

Conclusions: The biomarker p-S6 is overexpressed in metastatic tumors. In primary tumors, higher p-S6 expression is associated with shorter metastatic-free survival. This biomarker has the potential for risk stratification in future clinical trials.

Translational Relevance

This retrospective study shows the potential utility for using p-S6, identified by immunohistochemistry in tumor specimen thin sections, as a predictive biomarker of a more rapid time to the development of distant metastasis in early-stage adenocarcinoma of the lung. Obtaining the status of this biomarker may guide adjuvant therapy decisions in these patients after the initial, definitive surgery has been done.

The leading cause of cancer death in the United States is lung cancer. It has been estimated that there were more than 160,000 deaths from lung cancer in the United States during 2007, with more than 213,000 new cases diagnosed (1). About 80% of these cases were classified as one of the three major types of non–small cell lung carcinoma (NSCLC): adenocarcinoma, squamous cell carcinoma, or large-cell anaplastic carcinoma. The most important factor for survival in NSCLC patients is tumor stage, in part due to the potential for complete resection (2). Only patients who undergo complete tumor resection have a significant chance for cure (3).

Even with complete surgical resection, survival rates for NSCLC are disappointing. Depending on the pathologic stage, 5-year survival rates range from 23% to 67% (2). Thus, it is likely that at the time of diagnosis many cancers have already spread or metastasized at the microscopic level. About half of locally advanced lung adenocarcinoma patients will develop brain metastatic disease (4, 5). Because resection of these lesions is the cornerstone of therapy, examining the differences between primary and metastatic tissue is possible.

Currently there are no diagnostic tests to determine which patients will likely relapse after potentially curative surgery. Previous studies have been conducted to identify molecular markers with the potential for prognostic/diagnostic use. Thyroid transcription factor 1 (TTF-1) expression has been found predominately in lung adenocarcinoma among the different types of NSCLC (6, 7), and has been shown to be a favorable, independent predictor of survival in lung adenocarcinoma patients (8). The phosphatidylinositol 3-kinase/Akt pathway is frequently deregulated in cancer (9). Overexpression of activated or phosphorylated AKT (p-AKT) in preinvasive lesions has been associated with severe dysplasia, suggesting early involvement of this pathway in lung cancer (10, 11). Overexpression of p-AKT has been shown to be a poor prognostic factor for NSCLC patients with lymph node involvement (12, 13). AKT is a known repressor of E-cadherin; reduced E-cadherin expression, along with epidermal growth factor receptor expression, has also been associated with poorer survival in NSCLC (14).

These reports show the possibility that biomarkers associated with these pathways may be used to stratify lung adenocarcinoma patients. Unfortunately, few studies have included analyses of both primary tissue and corresponding metastatic tissue to further our knowledge of the development of lung tumor metastasis. To address issues related to metastasis in lung adenocarcinoma, studies using matched primary-metastatic pairs have been examined. With the use of a variety of markers (p53, bcl-2, Ki-67, epidermal growth factor receptor, cyclooxygenase-2, and bax), differences were not detected between the expression of these markers between the paired samples (15, 16). Since the publications of those studies, additional potential markers have been identified that may elucidate important factors regarding metastatic behavior.

In this study, we sought to determine whether markers of differentiation and activation of the Akt pathway are associated with metastasis in adenocarcinoma of the lung. Matched primary tumors and brain metastases from 41 patients were used to identify differentially expressed markers. We chose brain metastasis for this purpose because of all potential metastatic sites, those occurring in the brain are the most frequently surgically excised and provide the best quality of tissue for molecular evaluation. Further investigation and validation of these markers was done in an additional 159 primary lung adenocarcinoma samples.

Patient population. This study was conducted under an Institutional Review Board–approved, retrospective laboratory protocol that was compliant under the Health Insurance Portability and Accountability Act. Patients that had primary thoracic tumor resection at the University of Texas M. D. Anderson Cancer Center from 1992 to 2004 for primary adenocarcinoma of the lung were considered for inclusion in this study. Forty-one patients were first ascertained to have additionally undergone metastatic neurosurgery at the University of Texas M. D. Anderson Cancer Center; these 41 patients also had sufficient samples of tissue for whole section assessment from both surgeries. The study and validation cohorts comprised an additional 159 patients that (a) had a positive history of smoking and (b) did not receive induction chemotherapy. The difference between the study and the validation cohorts was based solely on tissue availability. Cases included in the test cohort had enough sample for whole section assessment, whereas cases included in the validation cohort were available for assessment only in tissue microarray format.

The study cohort, in which the results of the matched-pair analysis were tested, comprised 41 women and 36 men diagnosed with primary lung adenocarcinomas (n = 77). The staging of these patients at initial diagnosis were as follows: 40 stage I, 15 stage II, 21 stage III, and 1 stage IV. The median age at primary tumor diagnosis was 66.3 y (range, 40.1-83.9 y), and the median clinical follow-up was 104.4 wk. Twenty-eight patients had suffered metastatic lesions at the time of this study, with 10 involving brain metastatic disease. The validation cohort (n = 82), in which findings from the study cohort were tested, consisted of 43 women and 39 men. The staging of these patients at initial diagnosis were as follows: 61 stage I, 12 stage II, 8 stage III, and 1 stage IV. The median age at primary tumor diagnosis was 66.0 y (range, 33.5-84.3 y), and the median clinical follow-up was 98.0 wk. Twenty-five patients had suffered metastatic lesions by the point of study, with eight involving the brain.

All 241 tumor samples (200 primaries, 41 metastases) were obtained from formalin-fixed, paraffin-embedded tissue blocks collected by The University of Texas M. D. Anderson Cancer Center Department of Pathology, and each sample was assessed histologically for tumor tissue by one or more pathologists (M.S., G.R., I.I.W., K.A.). A Beecher tissue arrayer was used to manufacture tissue arrays that comprised our validation set of 82 primary lung adenocarcinomas.

Immunohistochemistry. Immunohistochemistry was done as described previously (17). Briefly, 5-μm sections were deparaffinized, followed by microwave antigen retrieval in 10 mmol/L sodium citrate (pH 6.0; except for TTF-1, which was retrieved in 10 mmol/L Tris-EDTA). For the matched primary/brain metastatic pair cohort, the antibodies against E-cadherin (1:100 of Ab-3, Lab Vision, Inc.), p-AKT (1:250 of Thr308, New England BioLabs, Inc.), p-S6 (1:1,000 of Ser235/6, Cell Signaling Technology, Inc.), and TTF-1 (1:50 of 8G7G3/1, Lab Vision, Inc.; 1:100 of NCL-L-TTF-1, Novocastra Laboratories) were applied and incubated overnight at 4°C. The DAKO EnVision kit was used to detect staining. The p-S6 antibody was tested for specificity (18), and the 1:1,000 dilution worked well when comparing primary tumor with metastatic tumor for differential expression. However, the amount of p-S6 antibody was increased 4-fold to enhance the detection of signal for the two cohorts of primary tumor samples. The increased sensitivity provided by the lower dilution allowed for better assessment of the primary cohorts. Negative control experiments were conducted by excluding primary antibodies from the protocol described above.

Scoring for expression was based on the percentage of positively stained tumor cells (Fig. 1). Each slide was reviewed by one of us (K.A) under low magnification to evaluate staining intensity (e.g., positive and negative). Staining was considered diffuse if >50% of the tumor cells stained for the biomarker and focal if <50% of the tumor cells stained for the biomarker. For scoring purposes, the most prominently stained area of the slide was chosen. The positive and negative expression of E-cadherin, p-AKT, p-S6, and TTF-1 were defined in accordance with the definition used in previous studies (1214). Immunoreactivity for TTF-1 was detected as nuclear staining and scored as 0 to 1 based on the proportion of positively stained tumor nuclei (0 if ≤5% positive tumor nuclei, 1 if >5% positive tumor nuclei). Cytoplasmic staining was observed but disregarded, in accordance with previous studies (19). E-cadherin was scored as 0 to 1 based on positive membrane staining (0 if ≤75% positive cells, 1 if >75% positive cells). The immunoreactivity of p-S6 and p-AKT was scored as 0 to 2 based on positive cytoplasmic staining (0 if <5% positive cells, 1 if 5-30% positive cells, 2 if >30% positive cells).

Fig. 1.

Differential expression in primary-metastatic pairs. Loss of E-cadherin and TTF-1 expression was seen in these metastatic tumors (B and D, respectively), compared with their corresponding primary tumors (A and C, respectively). In contrast, acquisition of p-S6 was observed for another metastatic tumor (F), compared with its corresponding primary tumor (E).

Fig. 1.

Differential expression in primary-metastatic pairs. Loss of E-cadherin and TTF-1 expression was seen in these metastatic tumors (B and D, respectively), compared with their corresponding primary tumors (A and C, respectively). In contrast, acquisition of p-S6 was observed for another metastatic tumor (F), compared with its corresponding primary tumor (E).

Close modal

Statistical analysis. A paired two-sided t test was done to determine the differences of marker scores between the matched primary and metastatic tumor specimens. Log-rank and Kaplan-Meier analyses were done to determine the correlations of variables with time-to-metastasis in univariate analyses. Cox multivariate analysis was used to determine independent associations with time-to-metastasis. Correlations of independent variables with one another were determined using Spearman's rank. Time-to-metastasis was defined as the time from initial diagnosis to the first clinically evident metastasis.

Markers in matched primary and brain-metastatic pairs. All 41 patients with matched primary/metastatic pairs in the current study were diagnosed with primary adenocarcinoma of the lung. The median age at initial diagnosis was 57.5 years (range, 35.6-85.1 years). There were 16 women and 25 men in this test group, and the majority of patients were Caucasian (88%). Four patients presented with brain metastases at initial diagnosis. For the 37 remaining patients, brain metastases became clinically evident after surgical resection of the primary tumor at a median rate of 54.4 weeks. As described in the Methods section, these paired samples were evaluated for expression of four markers. In instances of immunohistochemistry staining intensity discordance (e.g., the primary tumor was high whereas the metastatic tumor was low), the direction of the discrepancy was noted and a paired sample t test was done to determine significance. A summary of these results is given in Table 1. For TTF-1 there were 18 instances of discordant expression, of which 15 (83%) pairs had less TTF-1 expression in the metastatic tumor compared with the corresponding primary tumor. There were 23 discordant pairs for E-cadherin, of which 20 (87%) pairs showed loss of expression in the metastatic lesion. There were 21 discordant examples of positive p-S6 staining, of which 20 (95%) showed higher expression in the metastatic lesion. The distribution of the differences in expression among the discordant pairs for these three markers (TTF-1, E-cadherin, and p-S6) were all statistically significant (Table 1). In contrast, among the 15 discordant p-AKT cases 5 were higher in the primary whereas 10 were higher in the metastasis, which was not a statistically significant difference.

Table 1.

Comparison of biomarkers in paired primary and metastatic pairs

MarkerConcordant (positive)Concordant (negative)Discordant
Two-sided t test
Higher in primaryHigher in metastasisP
E-cadherin 18 20 <0.01 
TTF-1 13 10 15 <0.01 
p-AKT 26 10 0.28 
p-S6 20 20 <0.01 
MarkerConcordant (positive)Concordant (negative)Discordant
Two-sided t test
Higher in primaryHigher in metastasisP
E-cadherin 18 20 <0.01 
TTF-1 13 10 15 <0.01 
p-AKT 26 10 0.28 
p-S6 20 20 <0.01 

NOTE: Markers were determined to be either concordant or discordant, and if discordant, the direction of discordance is indicated. The significance of these differences, based on a two-sided t test, is shown.

Association of biomarkers and metastasis in primary lung tumors. The finding of higher p-S6 expression and decreased TTF-1 and E-cadherin expression in metastatic lesions among matched primary-metastatic pairs suggested that these biomarkers may be predictive of metastasis in primary tumors. Specifically, these data generated the hypothesis that increased p-S6 expression and decreased TTF-1 and E-cadherin in primary lung tumors were associated with increased risk of metastasis. To test this, 77 primary lung adenocarcinoma cases were tested. Positive staining was detected in 61 (79%), 65 (84%), and 64 (83%) cases for E-cadherin, p-S6, and TTF-1, respectively. The proportion of tumors that were positive for p-S6 was higher in the primary tumors in this sample set compared with the 41 tumors in the matched pairs, as the antibody concentration for the immunohistochemistry was raised to increase detection. The median time-to-metastasis for the p-S6–negative cases could not be ascertained because 11 of the 12 cases did not have metastatic disease at last follow-up. In the cases staining positive for p-S6, the median time-to-metastasis was 64.1 weeks. Kaplan-Meier survival curves and log-rank testing showed a statistically significant correlation between p-S6 expression (negative versus positive) and time-to-metastasis (Fig. 2A). The difference in time-to-metastasis was significant between p-S6–negative cases and positive cases (P = 0.02). Significant differences were not observed in time-to-metastasis when stratified by TTF-1 (P = 0.73) or E-cadherin (P = 0.49; Fig. 2B and C). Interestingly, we did not observe a correlation between TNM stage and any of the three markers tested. Along with the p-S6 status, T-stage (P = 0.02; data not shown), but not N-stage, was predictive in univariate analysis. Cox proportional hazards analysis showed only positive p-S6 staining to be an independent predictive factor for shortened time-to-metastasis, after accounting for T-stage and patient age (Table 2).

Fig. 2.

Kaplan-Meier time-to-metastasis curves of the study primary tumor set, when stratified by p-S6 status. For p-S6–negative cases (dashed line; n = 12), a median survival could not be ascertained as all but one case were censored. For p-S6–positive (solid line; n = 65), the median time-to-metastasis was 199 wk. The difference in time-to-metastasis was significant between p-S6–negative cases and positive cases (P = 0.02, log-rank). B, there was no significant difference in time-to-metastasis when stratified by TTF1 (P = 0.73) or C, E-cadherin (P = 0.49).

Fig. 2.

Kaplan-Meier time-to-metastasis curves of the study primary tumor set, when stratified by p-S6 status. For p-S6–negative cases (dashed line; n = 12), a median survival could not be ascertained as all but one case were censored. For p-S6–positive (solid line; n = 65), the median time-to-metastasis was 199 wk. The difference in time-to-metastasis was significant between p-S6–negative cases and positive cases (P = 0.02, log-rank). B, there was no significant difference in time-to-metastasis when stratified by TTF1 (P = 0.73) or C, E-cadherin (P = 0.49).

Close modal
Table 2.

Cox proportional hazards analysis of marker expression and T-stage in study and validation cohorts of primary lung tumors

FactorStudy set (n = 77)
Validation set (n = 82)
Combined set (n = 159)
HRPHRPHRP
p-S6 7.6 0.05 2.6 0.04 3.2 <0.01 
T2-4 NS NS 2.0 0.09 2.0 0.02 
FactorStudy set (n = 77)
Validation set (n = 82)
Combined set (n = 159)
HRPHRPHRP
p-S6 7.6 0.05 2.6 0.04 3.2 <0.01 
T2-4 NS NS 2.0 0.09 2.0 0.02 

Abbreviations: HR, hazard ratio; NS, not significant.

Validation of p-S6 as a marker of metastasis. To validate the finding that p-S6 was a biomarker of metastasis, p-S6 expression was examined in an independent set of tumors on tissue arrays. We found 50 of 82 cases positive for p-S6 (61%). The Kaplan-Meier time-to-metastasis curves of the validation set are shown in Fig. 3. The difference in time-to-metastasis was significant between the cases with positive p-S6 staining and those with no staining (P = 0.04, log-rank). As with the study set, a correlation between TNM stage and positive p-S6 staining was not observed. T-stage was also predictive of a shortened time-to-distant metastasis in univariate analysis (P = 0.03; data not shown). Multivariable analysis of this set showed that positive p-S6 staining and a T-stage >1 were independent predictive factors for shortened time-to-metastasis (Table 2). Lastly, when both the study and validation datasets from the lung primary tumors are combined, positive p-S6 staining (hazard ratio, 3.2; 95% confidence interval, 1.5-6.8; P < 0.01) and a T-stage >1 (hazard ratio, 2.0; 95% confidence interval, 1.1-3.7; P < 0.01) were independent, adverse predictive factors for time-to-metastasis (Table 2).

Fig. 3.

Kaplan-Meier time-to-metastasis curves of the validation set when stratified by p-S6 status. For p-S6–negative cases (dashed line; n = 32), a median survival could not be ascertained as 25 cases were censored. For p-S6–positive (solid line; n = 50), the median time-to-metastasis was 215 wk. The difference in time-to-metastasis was significant between p-S6–negative cases and positive cases (P = 0.04, log-rank).

Fig. 3.

Kaplan-Meier time-to-metastasis curves of the validation set when stratified by p-S6 status. For p-S6–negative cases (dashed line; n = 32), a median survival could not be ascertained as 25 cases were censored. For p-S6–positive (solid line; n = 50), the median time-to-metastasis was 215 wk. The difference in time-to-metastasis was significant between p-S6–negative cases and positive cases (P = 0.04, log-rank).

Close modal

Although not the primary end point of our study, the relationship of p-S6 status with overall survival was also examined. Analysis of the 159 combined cases (study and validation primary adenocarcinoma sets) showed that the 44 cases with a negative p-S6 status had a 420-week median overall survival, compared with a 300-week median survival for the 115 p-S6–positive cases (P = 0.04, log-rank).

The phosphatidylinositol 3-kinase/AKT pathway is critical for the regulation of cell proliferation, growth, differentiation, migration, and survival in many human cancers. Activation of this pathway seems to occur early in NSCLC development, because overexpression of p-AKT in preinvasive NSCLC lesions have been detected (10, 11). The overexpression of p-AKT has been seen as a poor prognostic factor for NSCLC patients with lymph node involvement (12, 13). It has been shown that AKT transmits some of its downstream effects through activation of the serine/threonine protein kinase mammalian target of rapamycin (mTOR; ref. 20). The current model has p-AKT directly activating mTOR through phosphorylation of its Ser2448 residue (21, 22), which, in turn, activates ribosomal S6K. S6K affects mRNA translation indirectly through intermediates such as the 40S ribosomal protein S6 (23). In fact, immunohistochemical testing for p-S6 has recently been used to detect S6K and mTOR activity in lung adenocarcinomas (24).

The major finding of this study is that high p-S6 expression was a negative prognostic factor for lung adenocarcinoma, as it was associated with time-to-metastasis in patients with early-stage lung adenocarcinoma. We first tested p-S6 for concordant expression in 41 primary lung adenocarcinoma-brain metastatic pairs. Half of these cases had discordant expression, with a vast majority of the higher p-S6 expression in the metastatic tissues (20 of 21). This led to the hypothesis that high p-S6 expression in the primary tumor might be a predictor of metastatic behavior. We tested this by examining p-S6 expression levels in a set of 77 primary lung adenocarcinoma tissues. We found that high expression of p-S6 was associated with a shorter average time-to-metastasis (200 weeks versus >400 weeks, postthoracic surgery). This finding was confirmed in a validation group (120 weeks versus 400 weeks, postthoracic surgery), a tumor set consisting of 82 primary lung adenocarcinoma cases. Although the finding remained robust, the time-to-metastasis curves seem somewhat different in this set compared with the study set of primary tumors. We believe that this is largely due to technical reasons, in that the immunohistochemistry on the study set was done on whole sections, whereas the validation set was done on tissue arrays, where one to three cores of tumor were evaluable for scoring. Whole-sectioning allowed more of the tumor to be assessed, and the area with the highest amount of staining was used. For this reason the undersampling of tumor inherent to the tissue array format likely led to false negatives (no staining observed in a tumor which actually expressed p-S6 if interrogated elsewhere). In support of this explanation, we found that the proportion of cases positive for p-S6 was higher when whole sections were used, rather than tissue cores (84% versus 61%, respectively), indicating that some of the cores where no expression was detected likely represented false negatives. Despite these technical differences, p-S6 remained associated with time-to-metastasis in both sets. In addition, the results indicate that the increased p-S6 seen in the brain-metastatic tissue samples (Table 1) could be due to an acquisition of phosphatidylinositol 3-kinase/Akt signaling in the course of metastasis, but further study will be required to verify this.

Although reports describing the relationship between clinical outcome and p-S6 status in lung cancer were lacking before this study, there are several studies that suggest that activation of the mTOR pathway, and hence, S6 activation, are indeed adverse prognostic characteristics in a variety of other malignancies. Activation of the mTOR pathway has been shown to be prognostic in renal cell carcinoma (25). Immunohistochemical analysis of tumor specimens of 375 renal cell carcinoma patients showed a correlation between p-S6 positivity and the clear cell histologic subtype, higher histologic grade, and other adverse pathologic features. Further, it was shown that p-S6 was an independent, adverse prognostic factor when accounting for patient performance status, tumor TNM stage, and the status of the biomarkers p-Akt and PTEN. One report indicated the presence of p-S6K (an upstream regulator of S6) to be much higher in 101 cases that presented with disseminated, stage IV (70%) compared with 51 cases with local-regional stage II-III disease (39%; ref. 26). Lastly, results from our laboratory indicated that p-S6K was an adverse prognostic marker in glioblastoma (27). Collectively, these data suggest that pathways which lead to S6 activation confer clinical aggressiveness in solid tumors.

There is considerable evidence that loss of differentiation and unchecked proliferation are two mechanisms by which primary neoplastic lesions metastasize to distant organ sites. We chose two well-known differentiation markers for lung tissue as well as two well-characterized signal transduction markers to test for differential expression in a set of primary lung adenocarcinomas and their subsequent metastatic brain lesions. The basis for choosing these markers for study was taken from various reports. Increased E-cadherin expression has been associated with favorable survival in NSCLC (14). Further, as stated previously, adenocarcinoma of the lung is known for its relatively high TTF-1 expression in comparison with the other types of NSCLC (6, 7), and increased expression has been reported as a favorable independent predictor of survival in lung adenocarcinoma patients (8). A recent meta-analysis of 10 studies examining the prognostic relevance of TTF-1 in NSCLC (28) found that, indeed, this is a favorable biomarker in this disease. Although our observations do not support this conclusion, there may be several factors for this discrepancy. Because TTF-1 is a favorable, independent prognostic factor, the benefit of TTF-1 would be more pronounced in later-staged disease. In our study, only 31 of 159 (19%) were stage III-IV. Because the majority of the cases within our study were otherwise favorable (clinical stages I-II and medically operable), a greater number of cases would be needed to show the benefit of TTF-1 positivity. Also, our primary clinical measure was time-to-metastasis. Although loosely correlated with overall survival time, these are different biological end points. Lastly, we only included patients with adenocarcinoma and a smoking history. This excludes other histologies, including bronchioalveolar carcinoma, which commonly affects nonsmokers (29). Out of the 10 reports that were included in this meta-analysis, only four were adenocarcinoma-specific, and only two showed a statistically significant survival advantage. One of these included bronchioalveolar carcinoma in their analysis.

In summary, analyses of matched primary-metastatic paired samples, as well as two cohorts of lung primary samples, suggest that activation of S6 protein is involved in metastasis of lung adenocarcinoma. Our findings further suggest that evaluation of p-S6 expression could contribute to the stratification of lung adenocarcinoma patients for potential treatment regimens. In addition, those found to have increased risk of early metastasis, by the use of p-S6 as well as other clinical and molecular factors, may benefit from particularly close clinical follow-up.

No potential conflicts of interest were disclosed.

Grant support: American Cancer Society Postdoctoral Fellowship grant PF-06-272-01-CCE (McDonald) and Specialized Program of Research Excellence in Lung Cancer grant P50CA70907, National Cancer Institute, Bethesda, MD (I.I. Wistuba and K. Aldape).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: J.M. McDonald and C.E. Pelloski contributed equally to this study.

1
American Cancer Society. Cancer facts & figures 2007. Atlanta: American Cancer Society; 2007.
2
Mountain CF. Revisions in the International System for Staging Lung Cancer.
Chest
1997
;
111
:
1710
–7.
3
Flehinger BJ, Kimmel M, Melamed MR. The effect of surgical treatment on survival from early lung cancer. Implications for screening.
Chest
1992
;
101
:
1013
–8.
4
Stuschke M, Eberhardt W, Pottgen C, et al. Prophylactic cranial irradiation in locally advanced non-small-cell lung cancer after multimodality treatment: long-term follow-up and investigations of late neuropsychologic effects.
J Clin Oncol
1999
;
17
:
2700
–9.
5
Mamon HJ, Yeap BY, Janne PA, et al. High risk of brain metastases in surgically staged IIIA non-small-cell lung cancer patients treated with surgery, chemotherapy, and radiation.
J Clin Oncol
2005
;
23
:
1530
–7.
6
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
;
34
:
597
–604.
7
Myong NH. Thyroid transcription factor-1 (TTF-1) expression in human lung carcinomas: its prognostic implication and relationship with expressions of p53 and Ki-67 proteins.
J Korean Med Sci
2003
;
18
:
494
–500.
8
Saad RS, Liu YL, Han H, Landreneau RJ, Silverman JF. Prognostic significance of thyroid transcription factor-1 expression in both early-stage conventional adenocarcinoma and bronchioloalveolar carcinoma of the lung.
Hum Pathol
2004
;
35
:
3
–7.
9
Miakotina OL, Goss KL, Snyder JM. Insulin utilizes the PI 3-kinase pathway to inhibit SP-A gene expression in lung epithelial cells.
Respir Res
2002
;
3
:
27
.
10
Massion PP, Taflan PM, Shyr Y, et al. Early involvement of the phosphatidylinositol 3-kinase/Akt pathway in lung cancer progression.
Am J Respir Crit Care Med
2004
;
170
:
1088
–94.
11
Liu LZ, Zhou XD, Qian G, Shi X, Fang J, Jiang BH. AKT1 amplification regulates cisplatin resistance in human lung cancer cells through the mammalian target of rapamycin/p70S6K1 pathway.
Cancer Res
2007
;
67
:
6325
–32.
12
Hirami Y, Aoe M, Tsukuda K, et al. Relation of epidermal growth factor receptor, phosphorylated-Akt, and hypoxia-inducible factor-1α in non-small cell lung cancers.
Cancer Lett
2004
;
214
:
157
–64.
13
David O, Jett J, LeBeau H, et al. Phospho-Akt overexpression in non-small cell lung cancer confers significant stage-independent survival disadvantage.
Clin Cancer Res
2004
;
10
:
6865
–71.
14
Deeb G, Wang J, Ramnath N, et al. Altered E-cadherin and epidermal growth factor receptor expressions are associated with patient survival in lung cancer: a study utilizing high-density tissue microarray and immunohistochemistry.
Mod Pathol
2004
;
17
:
430
–9.
15
Bubb RS, Komaki R, Hachiya T, et al. Association of Ki-67, p53, and bcl-2 expression of the primary non-small-cell lung cancer lesion with brain metastatic lesion.
Int J Radiat Oncol Biol Phys
2002
;
53
:
1216
–24.
16
Milas I, Komaki R, Hachiya T, et al. Epidermal growth factor receptor, cyclooxygenase-2, and BAX expression in the primary non-small cell lung cancer and brain metastases.
Clin Cancer Res
2003
;
9
:
1070
–6.
17
Simmons ML, Lamborn KR, Takahashi M, et al. Analysis of complex relationships between age, p53, epidermal growth factor receptor, and survival in glioblastoma patients.
Cancer Res
2001
;
61
:
1122
–8.
18
Cell Signaling Technology, Inc. [homepage on the Internet]. Danvers (MA); c1999-2008 [updated 2008 Jun 12; cited 2008 Jul 16]. Product Pathways—Translational Control; [about 2 screens]. Available from: http://www.cellsignal.com/products/4857.html.
19
Bejarano PA, Mousavi F. Incidence and significance of cytoplasmic thyroid transcription factor-1 immunoreactivity.
Arch Pathol Lab Med
2003
;
127
:
193
–5.
20
Bjornsti MA, Houghton PJ. The TOR pathway: a target for cancer therapy.
Nat Rev Cancer
2004
;
4
:
335
–48.
21
Abraham RT. Identification of TOR signaling complexes: more TORC for the cell growth engine.
Cell
2002
;
111
:
9
–12.
22
Jacinto E, Hall MN. Tor signaling in bugs, brain and brawn.
Nat Rev Mol Cell Biol
2003
;
4
:
117
–26.
23
Volarevic S, Thomas G. Role of S6 phosphorylation and S6 kinase in cell growth.
Prog Nucleic Acid Res Mol Biol
2001
;
65
:
101
–27.
24
Conde E, Angulo B, Tang M, et al. Molecular context of the EGFR mutations: evidence for the activation of mTOR/S6K signaling.
Clin Cancer Res
2006
;
12
:
710
–7.
25
Pantuck AJ, Seligson DB, Klatte T, et al. Prognostic relevance of the mTOR pathway in renal cell carcinoma: implications for molecular patient selection for targeted therapy.
Cancer
2007
;
109
:
2257
–67.
26
Tampellini M, Longo M, Cappia S, et al. Co-expression of EGF receptor, TGFα and S6 kinase is significantly associated with colorectal carcinomas with distant metastases at diagnosis.
Virchows Arch
2007
;
450
:
321
–8.
27
Pelloski CE, Lin E, Zhang L, et al. Prognostic associations of activated mitogen-activated protein kinase and Akt pathways in glioblastoma.
Clin Cancer Res
2006
;
12
:
3935
–41.
28
Steels E, Paesmans M, Berghmans T, et al. Role of p53 as a prognostic factor for survival in lung cancer: a systematic review of the literature with a meta-analysis.
Eur Respir J
2001
;
18
:
705
–19.
29
Morabia A, Wynder EL Relation of bronchioloalveolar carcinoma to tobacco.
BMJ
1992
;
304
:
541
–3.