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Epidermal Growth Factor Receptor, Cyclooxygenase-2, and BAX Expression in the Primary Non-Small Cell Lung Cancer and Brain Metastases¹

Departments of Radiation Oncology [I. M., R. K., T. H. R. S. B., P. A., J. D. C.], Pathology [J. Y. R., L. L.], Neurosurgery [R. S.], Thoracic and Cardiovascular Surgery [J. B. P., J. A. R.], Molecular Pathology [T. J. M.], Experimental Radiation Oncology [W. B., L. M.], and Cancer Medicine [W. K. H.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

	ABSTRACT

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Purpose: The purpose is to identify biological markers that predict brain metastasis and treatment outcome in non-small cell lung cancer (NSCLC).

Experimental Design: Samples were obtained from the primary tumors, lymph nodes, and brain metastases of 29 patients with NSCLC who had undergone resection of both the pulmonary tumors and the brain lesions. Samples from 29 patients matched for age, sex, and histology whose pulmonary tumors were resected served as controls. Samples were stained with H&E as well as immunohistochemical stains for epidermal growth factor receptor (EGFR), cyclooxygenase 2 (COX-2), and BAX. Comparisons were made between patients with and without brain metastasis. Independent investigators determined the percentage of positive cells.

Results: There was positive correlation in expression of all three biomarkers between primary lung tumors and lymph node metastases. Significantly higher levels of EGFR were found in lymph node metastases in the control group (P = 0.0147). COX-2 expression in brain lesions correlated with expression in primary tumors (P = 0.023). BAX levels were lower in poorly differentiated tumors in lymph node metastases in the control group (P = 0.01) and in brain metastases (P = 0.045). Low EGFR expression and high COX-2 expression in lymph node metastasis were associated with poorer treatment outcome.

Conclusions: Expression of EGFR, COX-2, and BAX in primary lung tumors did not differ between patients with brain metastases from NSCLC and those without brain metastases. These three biomarkers cannot be used to predict brain metastasis. Studies of other biomarkers are under way in an effort to predict brain metastasis among patients with NSCLC.

	INTRODUCTION

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The brain is one of the main metastatic sites in patients with lung cancer. Indeed, brain metastasis is the most common complication of cancers in general but especially patients with primary lung cancer because lung cancer is the leading cause of cancer deaths in the United States (1) . The incidence of brain metastases, based on autopsy findings, can be as high as 70% in patients with SCLC³ and as high as 50% in patients with AC (2, 3, 4) . The risk for developing brain metastasis increases as patients survive longer after aggressive treatment for locoregional disease (5) . Once the patients develop brain metastasis from lung cancer, their median survival is usually 4 months, and whole brain radiation therapy using various fractionation techniques has not demonstrated a survival benefit (6) .

PCI is being investigated as a treatment to prevent additional CNS metastases and improve survival in patients with lung cancer. Already, several studies have shown that PCI decreases the frequency of additional CNS metastases in patients with either SCLC or NSCLC and improved survival in patients with SCLC (7, 8, 9) . Therefore, if we were able to predict which patients with NSCLC will develop brain metastases, we would be able to predict, for example, who would benefit from PCI.

Although clinical and pathologic parameters such as tumor size, the histological type (AC versus squamous cell carcinoma), and weight loss have some predictive value, they are not very precise; there is great variation in the response to the treatment of individual tumors of the same histological types. Recent studies have therefore focused on identifying biological markers that can predict early recurrence and poor prognosis in patients with NSCLC. However, only a few studies have analyzed the biological markers of brain metastasis in the control of lung cancer (10 , 11) .

This study was therefore designed to determine whether the levels of EGFR, COX-2, and BAX in patients with NSCLC were associated with brain metastases and whether the intrapatient expression of these biomarkers in the primary tumors versus brain lesions was similar.

We chose EGFR as an oncoprotein signaling tumor growth because elevated levels of EGFR are a common component of multiple cancer types (12) . We also focused on COX-2 because, recently, increased expression of COX-2 was frequently found in AC of the lung (13 , 14) , indicating that this enzyme may have prognostic significance (15) . BAX has important regulatory roles in apoptosis (16) .

The expression of these three biomarkers was analyzed in resected primary lung tumors, lymph node metastases, and metastatic brain tumor specimens. We selected two groups of patients for comparison: one group comprised of patients with at least one CNS metastasis from a primary NSCLC tumor and one group comprised of patients with primary NSCLC but no CNS metastases during a minimum 2-year follow-up period. These biomarkers were assessed separately and in combination to determine their ability to predict brain metastasis, nonbrain DM and survival characteristics in case and control patients.

	MATERIALS AND METHODS

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Patient Population.
Tumor samples were fixed in formalin and embedded in paraffin blocks. The study population consisted of two groups of patients treated at The University of Texas M. D. Anderson Cancer Center between 1987 and 1999: 29 patients with primary tumors in the lung and metastatic brain tumors (the case group) and 29 patients with primary lung tumors but without brain metastases (the control group). All patients underwent tumor resection at M. D. Anderson Cancer Center. All 29 patients with primary lung tumors who had their brain metastases removed, 27 had solitary lesions and 2 had bilateral solitary lesions. Each control patient was matched by age, sex, and histology to a patient with brain metastasis. Charts were reviewed to collect patient and tumor characteristics, information on treatment, and survival status. Age differences between matched case and control patients were no more than 5 years, and the minimal follow-up in both case and control patients was 2 years after the initial diagnosis of primary lung cancer. The treatment of the patients is listed in Table 2 . Eleven of 29 patients in the case group had surgery only, compared with 18 of 29 patients in the control group, which was not statistically different (P = 0.066). Eight of 29 patients in the case group compared with 6 of 29 patients in the control group had postoperative radiation therapy (P = 0.539). Other treatment combinations were also fairly evenly distributed without statistical difference.

The protocol for staining with the EGFR, COX-2, and BAX antibodies is as follows: the slides were baked at 56°C for 20 min and then cooled for 5 min at room temperature. They were then deparaffinized in xylene and ethanol. The slides were rinsed three times in distilled water before they were steamed for 25 min in a slide holder with a 1x concentration of Dako target retrieval solution (Dako Corp.). After this step, the slides were again rinsed three times with distilled water and then, to block the endogenous peroxidase reaction, soaked in 3% hydrogen peroxide solution in 1x PBS with 0.1% sodium azide for 5 min. Next, the slides were rinsed with a solution of 50 mM TBS with 0.05% Tween 20 at pH 8.0. After this, the sections were exposed to avidin for 15 min to block the reaction, rinsed with TBS, blocked for 15 min more with biotin solution, and rinsed again with TBS. The sections were blocked with Dako serum-free protein block solution for 5 min, and then the excess blocking solution was removed. Incubation of the biomarker with the following antibodies was done for 1 h at room temperature: EGFR clone 3IG7 (1:100 dilution; Zymed Laboratories, Inc., San Francisco, CA); COX-2 clone no. 160112 (1:2000 dilution; Cayman Chemicals), and BAX clone 2D2; (1:80 dilution; Zymed Laboratories, Inc.). The slides were then rinsed with the same 50 mM TBS solution used previously and incubated with the Dako predilute universal secondary link from the linked streptavidin biotin system⁺ kit for 15 min at room temperature. The TBS solution rinse was repeated, and the slides were incubated for 15 min with Dako predilute horseradish peroxidase-labeled streptavidin from the linked streptavidin biotin system⁺ kit at room temperature and then rinsed with the TBS solution. The slides were incubated with diaminobenzidine solution (Biogenex, San Ramon, CA) twice for 8 min each. Before and after counterstaining with Mayer’s hematoxylin (1x concentration; Poly Scientific, Bay Shore, NY) to blue the nuclei of the cells, the slides were rinsed with distilled water. Next, the slides were dehydrated in ethanol, cleared in xylene, and mounted with a xylene-based mounting medium.

Specimen Analysis.
The immunohistochemical scoring for the current study is based on the percentage of positively stained tumor cells. Three independent investigators (R. S. B., T. H., and I. M.) determined the percentage of tumor cells that stained for the biomarkers. Each slide was first reviewed under low magnification to evaluate staining intensity (i.e., strong, intermediate, or weak staining and the distribution of the stained cells (focal versus diffuse). 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 counting purposes, the most prominently stained area of the slide was chosen and marked under x100 magnification. Table 1 gives the criteria for grading biomarker expression. The positive, intermediate, and negative expression of EGFR, COX-2, and BAX were defined in accordance with the definition used in previous studies (17, 18, 19) . In four fields, the number of staining tumor cells was counted under x400 magnification. The counts arrived at by the two investigators were compared, and recounts were done if the percentages of stained cells differed by >10%. If the second recount still did not produce comparable results then a third investigator (J. Y. R.) did a final count.

Survival time was calculated from the date of diagnosis to the first occurrence of the considered event (local-regional recurrence alone, DM alone, or any local-regional or distant recurrence). Overall survival was defined as the time from diagnosis to death from any cause. Disease-specific survival was defined as the time from diagnosis to death because of the lung cancer. Hence, patients who died while in remission were considered dead in the analysis of overall survival but censored from the analysis of disease-specific survival. Local-regional control was defined as the time from diagnosis to the first local-regional failure. DM-free survival was defined as the time from diagnosis to the first DM occurrence. Disease-free survival was defined as the time from diagnosis to the first recurrence (local-regional or distant). When comparing the occurrence of nonbrain metastases in the case and control groups, brain metastases were excluded and only other distant metastases considered.

Cox’s proportional hazard model was used for the multivariate analysis of the effect of patient characteristics and other prognostic factors of significance on the end points. The estimated hazard is reported. The Wald test was used to assess the role of covariates in the model.

	RESULTS

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Patient and Tumor Characteristics.
Details on patients, their tumors and their treatments are given in Table 2 . There were 23 patients with AC in the case group and 24 patients with AC in control group, 5 patients with SCC in each group, and 1 patient with mixed NSCLC in the case group. The tumor grade for each primary lung tumor was determined from pathology results. The median age was 58 years in the case group and 59 years in the control group. There were 11 women and 18 men in each group. The patients were predominantly white (85%), all had a KPS of 80, and 75% had no weight loss before diagnosis of the primary lung tumors. Patients in the case group had a lower mean KPS, 90, than the control group, 94 (P = 0.045). ² analysis showed no differences in the past medical history or symptoms upon diagnosis between the two groups, except for increased sputum production in the case group (n = 4, with no cases in the control group; P = 0.038). Tumor stages were also lower in the control group than the case group: 65% of tumors in the control group were T₁ or T₂, whereas 72% of the case group was staged T₂ or T₃ (P = 0.048). Forty-eight of 58 patients (83%) had a smoking history, with 6 patients still smoking during treatment. There was no significant difference in nodal status or tumor size between the two groups. In addition, the Kaplan-Meier survival curves did not differ between the case and control groups. The median survival time from the date of the diagnosis of the lung primary tumor until death because of lung cancer was 29 months (range, 1–143 months). Control patients survived, on average, longer than case patients (29 versus 25 months). Overall median follow-up from the date of diagnosis of the primary lung tumor was 45 months (range, 12–140 months); this was 45 months (range, 25–115 months) in control patients and 33 months (range, 12–140 months) in case patients. In the case group, 4 of 29 patients had brain metastases at the time of diagnosis of the primary tumor, whereas brain metastases developed in the remaining 25 patients during the observation period. Nine patients in this group had multiple brain metastases. The median time from the diagnosis of NSCLC to the diagnosis of brain metastasis was 12 months (range, <1–133 months). The median survival of these patients after the development of brain metastases was 9 months (range, <1–107 months).

Biomarker Results.
Cells stained for EGFR exhibited cytoplasmic and membrane staining, whereas cells staining for COX-2 and BAX exhibited cytoplasmic staining (Fig. 1) . Table 3 gives the detailed breakdown of results of the IHC staining.

View larger version (116K): [in this window] [in a new window]   Fig. 1. Immunohistochemical staining of AC of the lung. Cells that stain for EGFR (A) demonstrate strong membrane and cytoplasmic staining. Cells that stain for COX-2 (B) and BAX (C) demonstrate strong cytoplasmic staining

² analysis also showed no significant difference in the intensity of staining or the distribution of stained cells for any of the biomarkers between the case and control groups. When the results in both groups were combined, EGFR staining in lung primary tumors was strong in 89% of the cases, COX-2 staining was strong in 63% of the cases, and BAX staining was strong in 59% of the cases. In addition, a distribution of staining for all three biomarkers was more evenly diffuse or focal.

When biomarker staining was analyzed in primary lung tumors from the standpoint of histology (both groups combined), the mean percentage of SCC cells that stained for EGFR was 68% as opposed to 47% of AC cells (P = 0.04). There was no significant difference in the results of COX-2 and BAX stainings, although the mean percentage of cells that stained for COX-2 and BAX was higher in AC than SCC tumors [COX-2: AC (39%) versus SCC (25%), P = 0.16; and BAX: AC (42%) versus SCC (37%), P = 0.55]. Analysis of tumor differentiation showed low levels of BAX in poorly differentiated tumors in lymph node metastases for the control group (P = 0.01) and in brain metastases for the case group (P = 0.045).

Survival.
Analysis of survival in association with biomarker results in primary lung tumors also showed no significant difference between case and control groups. However, both groups combined, t tests showed that overall survival was poor in patients with lower levels of EGFR in lymph node metastases (mean expression of 47%; P = 0.02), and disease-free survival was poor in patients with positive COX-2 levels in lymph node metastases (64%; P = 0.05).

² analysis was performed to assess the correlation of the expression of biomarkers in lung primary tumors, lymph node metastases, and brain metastases. Intermediate and positive COX-2 expression in lung tumors correlated with lymph node metastases values (P = 0.02; r² = 0.2096) and brain metastases values (P = 0.023; r² = 0.1626; Fig. 2, A and B ). EGFR values in lung primary tumors and lymph node metastases also correlated (P = 0.017; r² = 0.2812) and BAX (P = 0.006; r² = 0.3141; Fig. 2, C and D ). Multivariate analysis of hazard ratios showed that neither EGFR, COX-2, and BAX were independent prognostic biomarkers influencing survival.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Linear regression analyses: correlation of expression of COX-2 between (A) primary lung tumors and lymph node metastases and (B) primary lung tumors and brain metastases; correlation of expression for EGFR (C) and BAX (D) between primary lung tumors and lymph node metastases.

	DISCUSSION

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However, some interesting relationships between percentage of stained cells and survival of patients emerged when analysis was performed for both group combined and separately for AC and SCC. Our study did show that COX-2 expression in the case group significantly correlated between primary lung tumor, lymph node metastasis, and brain metastasis. This finding suggested that COX-2 expression is associated with tumor progression. However, although EGFR and BAX expression in the primary lung tumors and lymph node metastases of patients in the case group correlated, it did not correlate with expression in brain metastases.

In this study, EGFR was expressed at higher levels in SCC tumors than in AC tumors as has been found in other studies (17 , 20) . Although EGFR did not show prognostic significance, we did find that low EGFR expression in lymph node metastases (47%) was associated with poor overall survival. In addition, the only statistically significant difference noted between the case and control group was in the expression of EGFR in lymph node metastases (12 cases, 10 control), with the control group showing a higher mean expression than the case group (P = 0.01). These results concur with those from a meta-analysis conducted by Nicholson et al. (12) in which EGFR expression was rarely found to correlate with other clinical and prognostic indicators in NSCLC (only 3 of 10 studies showed an association between EGFR and either relapse-free or overall survival rates).

EGFR has become a focus of research because it is a growth factor receptor tyrosine kinase important in carcinogenesis, and it is commonly elevated in numerous cancer types. However, whether the expression of EGFR has any prognostic importance in NSCLC remains to be established. On the basis of our results, it does not appear to correlate with the risk of brain metastases.

In our study, COX-2 and BAX were both expressed at higher levels in AC than SCC. Recently, increased expression of COX-2 was frequently detected in AC of the lung (13 , 14) and the corresponding lymph nodes (13) . However, in our study, COX-2 expression in primary lung tumor also did not prove to be a prognostic indicator of brain metastases and nonbrain DM. The only notable association was that patients with high COX-2 expression in lymph node metastases had a poor disease-free survival. These findings together with the positive intrapatient correlation in COX-2 expression between lung tumors, lymph nodes, and brain metastases suggest that an increase in expression is associated with AC histology and, therefore, that COX-2 plays a part in the acquisition of an invasive and metastatic phenotype.

In our study, BAX staining was intermediate and positive in 87% of patients, with strong intensity of staining in 59% cases. We therefore considered that our cohort of patients had overexpression of BAX. The study showed a lower level of BAX in poorly differentiated tumors for lymph node metastases and brain metastases. These data are difficult to interpret because the additional statistical analysis did not show any correlation between biomarker expression and prognosis of patients. Similarly, Caputi et al. (21) found that BAX was overexpressed in a cohort of 55 patients with NSCLC, but they found no correlation with any clinicopathological parameters. On the other hand, Apolinario et al. (22) observed that patients with BAX expression alone in their NSCLC tumors did not influence survival, however, patients with expression of BAX in tumors but negative for Bcl-2 biomarker had a poor prognosis. BAX is considered important because of its ability to form heterodimers with Bcl-2, thereby accelerating programmed cell death (23) .

To our knowledge, this is the first study to examine the comparative expression of EGFR, COX-2, and BAX in NSCLC patients with and without brain metastases. In a similar study, Arnold et al. (10) examined the IHC expression of different biomarkers in a series of 35 patients with resected brain metastases tumors and 17 paired primary tumor specimens, but these tumors were from different primary sites and different histological types. In a study, D’Amico et al. (11) demonstrates that molecular markers may predict the site of relapse for NSCLC but without analyzing metastases.

Nonetheless, because the size of the groups was small (n = 29 in each) compared with the patient population in other IHC studies, it is possible that the size was not large enough to reveal significant differences.

In conclusion, although biomarker expression in the case and control groups did not prove to predict brain metastasis or survival, there was a strong correlation in the expression of all three biomarkers in primary lung tumors and lymph node metastases and in the expression of COX-2 in primary lung tumors and brain metastases. Our study also showed the overexpression of BAX, indicating the apoptotic susceptibility of cells during tumor progression.

Univariate analysis showed that low EGFR expression in lymph nodes predicted poor overall survival and high COX-2 expression in lymph nodes predicted poor disease-free survival. Multivariate analysis failed to confirm the independent prognostic significance of the three biomarkers.

It should be noted that our study was performed in patients who had their brain metastases resected and thus had a more favorable prognosis. This limits the extrapolation of our findings to all patients with brain metastases, particularly to those with multiple brain metastases. Our small case-control study can, however, serve as the basis for generating hypotheses to be tested in larger studies.

	ACKNOWLEDGMENTS

 
We thank Hazel Dalton for her technical assistance in cutting tissue blocks and the members of the McDonnell lab at M. D. Anderson Cancer Center, in particular, Shawn Brisbay and Christine Klein. We also thank Dr. Kathrine Mason and Dr. Elizabeth Travis (Department of Experimental Radiation Oncology) for their valued assistance.

	FOOTNOTES

 
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.

¹ This work was supported, in part, by The University of Texas M. D. Anderson Tobacco Research Program (Grant No. 30020026) and the National Cancer Institute, U. S. Department of Health and Human Services (PO1 CA-06294, T32CA77050, and P30CA16672). Presented at the Annual Meeting of American Society of Clinical Oncology, May 18–21, 2002, Orlando, Florida.

² To whom requests for reprints should be addressed, at Department of Radiation Oncology, Box 97, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-3400; Fax: (713) 794-5573; E-mail: rkomaki{at}mdanderson.org

³ The abbreviations used are: SCLC, small cell lung cancer; AC, adenocarcinoma; CNS, central nervous system; NSCLC, non-small cell lung cancer; EGFR, epidermoid growth factor receptor; COX-2, cyclooxygenase-2; PCI, prophylactic cranial irradiation; DM, distant metastasis; KPS, Karnofsky performance status; TBS, Tris-buffered saline; IHC, immunohistochemical.

Received 8/19/02; revised 10/28/02; accepted 10/28/02.

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