Presented in part at the 2007 Annual Meeting of the United States and Canadian Academy of Pathology (Pulmonary Pathology Society Trainee Award), San Diego, California, March 24-30, 2007.
To the authors' knowledge, there are no reliable markers able to identify patients with nonsmall cell lung cancer (NSCLC) that will develop metastases to the brain. The authors investigated associations between immunohistochemical markers and the development of brain metastases in patients with NSCLC.
This was a hospital-based, case-control study of patients who were newly diagnosed with NSCLC between 1989 and 2003, developed brain metastases, and had pathology material available from both the primary NSCLC and the brain metastases. These patients were compared with a control group of patients who had NSCLC and no evidence of brain metastases. NSCLC was examined for expression levels of Ki-67, caspase-3, vascular endothelial growth factor A (VEGF-A), VEGF-C, E-cadherin, and epidermal growth factor receptor (EGFR) in 54 surgical pathology specimens using immunohistochemistry, and associations were evaluated between those markers and the development of brain metastases.
Brain metastases developed after a median of 12.5 months (range, 1.7-89.4 months) after the diagnosis of NSCLC. A significantly increased risk of developing brain metastases was associated with patients with NSCLC who had primary tumors with high Ki-67 levels (adjusted odds ratio [OR] of 12.2; 95% confidence interval [95% CI], 2.4-70.4 [P < .001]), low caspase-3 expression (adjusted OR of 43; 95% CI, 5.3 to >100 [P < .001]), high VEGF-C expression (adjusted OR of 14.6; 95% CI, 2.0 to >100 [P < .001]), and low E-cadherin (adjusted OR of 3.6; 95% CI, 0.9-16.4 [P = .05]). No significant risk was associated with VEGF-A or EGFR expression. High Ki-67 expression also was associated with a shorter overall survival (P = .04).
Nonsmall cell lung carcinoma (NSCLC) is the leading cause of death from cancer in both men and women.1 Approximately 210,000 cases of NSCLC are diagnosed annually in the US.1 Lung cancer is the malignancy that most commonly gives rise to brain metastases which is a devastating complication and a major cause of morbidity and mortality.2 Approximately 10% of patients have brain metastases at the time of diagnosis, and approximately 40% of all patients with lung cancer will develop brain metastases during the course of the disease.3 Patients with locally advanced NSCLC who are treated with chemotherapy and chest radiotherapy with or without surgery have a high rate of developing brain metastases.2, 4–6 These patients also have a risk that ranges from 15% to 30% of failing first in the brain.4, 7 Brain metastases from NSCLC have received increasing attention because combined-modality therapy has lead to improvements in intrathoracic local control and prolonged overall survival.7–9
It is important to identify patients with NSCLC who are at greater risk of developing brain metastases, because such metastases may exist in the absence of neurologic symptoms.10 Furthermore, recent reports indicate that prophylactic cranial irradiation may be an effective modality for preventing brain metastases in patients with NSCLC who receive adjuvant chemoradiation.5 Despite advances in diagnosis, therapeutic modalities, and clinical practice guidelines, it remains unclear whether patients with early-stage NSCLC should be screened for brain metastases.11–13
Studies have demonstrated that the metastatic cascade is rather complex and involves reciprocal interactions between tumor cells and host tissues, including alterations in tumor cell proliferation, adhesion, proteolysis, invasion, and angiogenesis.8 For the morphologic assessment of the fraction of proliferating cells, Ki-67 staining with MIB-1 is the most commonly used monoclonal antibody, covering late G1-phase, S-phase, G2-phase, and M-phase of the cell cycle.14 Terminal deoxyuridine triphosphate nick-end labeling (TUNEL) assay, and caspase-3 immunohistochemistry are suitable for evaluating apoptosis because these methods cover both the mitochondrial pathway and the death-receptor pathway.15 Studies of both proliferative and proapoptotic factors demonstrated that patients who had tumors with a high proliferative activity and no expression of proapoptotic factors had the shortest survival, and vice versa.16
The vascular endothelial growth factor (VEGF) family of proteins modulates angiogenesis, which is essential for tumor growth and metastasis.17–19 E-cadherin is a calcium-regulated adhesion molecule that is expressed in most normal epithelial tissues. Loss of cell-surface E-cadherin is considered a defining characteristic of epithelial-mesenchymal transition (EMT), a cellular event that occurs during normal embryo development and also may be associated with tumor cell invasion and metastasis.20, 21 Epidermal growth factor receptor (EGFR) is overexpressed in 40% to 80% of NSCLCs and in many other epithelial cancers and is frequently correlated with an adverse prognosis.22, 23
It has been demonstrated previously that the molecular factors we investigated in the current study play a role in tumor invasion and metastasis formation. However, to our knowledge, any association between those markers and the development of brain metastasis from NSCLC has not been established.
In this study, we evaluated patients who had NSCLC with and without brain metastases in a unique and unparalleled series that included tumor material both from the primary lung tumor and from metachronous brain metastases that were obtained from a single institution. These patients typically are treated only for lung cancer, and a brain biopsy is rarely recommended or obtained. This gave us the opportunity to investigate the expression of Ki-67, caspase-3, VEGF-A, VEGF-C, E-cadherin, and EGFR in primary lung tumors using immunohistochemistry and to investigate the risk of developing brain metastasis and death. We compared patients who developed brain metastases with a control group of patients who had NSCLC and no evidence of brain metastases who were followed for a median of 2.5 years.
MATERIALS AND METHODS
We identified patients who were newly diagnosed with NSCLC between 1989 and 2003 who developed brain metastases and had available pathology material from both primary NSCLC and brain metastases (Table 1). The group was identified through a combined search of the registry databases from the Departments of Pathology and Neuropathology maintained by the Department of Pathology and included patients with both confirmed primary NSCLC and metastatic NSCLC to brain who had available pathology material. The patients were reviewed by 2 pathologists and were classified histologically by using World Health Organization criteria for tumors of the lung.24 In each patient, the pathologic and immunophenotypic features of brain metastases were compared with the corresponding NSCLC, and the diagnosis of NSCLC metastatic to brain was confirmed.
Table 1. Clinical and Pathologic Characteristics of the Study Group and Control Group*
No. of Patients (%)
Study Group, N=21
Control Group, N=33
Because of rounding, not all percentages total 100.
Squamous cell carcinoma
Median tumor size [range], cm
Lymph node classification
Pathologic TNM stage
The inclusion criteria for the study were a diagnosis of NSCLC (either lung adenocarcinoma, or squamous cell carcinoma, or NSCLC not otherwise specified); initial treatment by surgery alone, with or without postoperative adjuvant treatment; no other previous or synchronous malignant tumors; histologically confirmed NSCLC metastatic to brain; and no deaths in the perioperative period <30 days after surgery. These criteria resulted in the identification of 21 evaluable patients.
The control group consisted of patients who were seen for follow-up of their NSCLC, who were treated during the same period (between 1989 and 2003) and in the same clinic as the study patients, and who had either no metastases or who developed metastases to distant sites other than brain (Table 1). Only patients with negative neurologic examination and/or negative brain imaging studies were included in the control group, resulting in 33 evaluable patients.
The clinicopathologic features of the 2 subgroups were similar, except that patients in the control group were slightly older than patients in the study group at the time they were diagnosed with primary NSCLC (P = .05) (Table 1). The study protocol was approved by the Institutional Review Board of the Brigham and Women's Hospital.
Immunoperoxidase studies were performed on paraffin-embedded sections from 54 NSCLC specimens after deparaffinization and heat-induced epitope retrieval with buffers (0.01 M citrate [pH 6.0] or 0.001 M ethylenediamine tetraacetic acid [pH 8.0]) and quenching of endogenous peroxidase with 3% aqueous hydrogen peroxide. Tris buffer (pH 7.5) supplemented with 3% porcine serum and Tris buffer alone were used for soaking and rinsing tissues, respectively, during processing. Details of the methodology and sources of the antibodies used in this study are summarized in Table 2.
Table 2. Characteristics of the Antibodies Used in the Study
The immunostained slides were examined by light microscopy. Ki-67 and caspase-3 were recorded as labeling indices (LI) (Fig. 1). The LI was defined as the number of Ki-67-positive or active caspase-3-positive tumor cells per 1000 tumor cells. We selected and evaluated 5 representative high-power fields (1 mm2; Olympus BX41 microscope; ×400 magnification) that had the greatest number of tumor cells stained for Ki-67 or caspase-3 (hot spots). For each patient, we recorded the Ki-67 LI, the caspase-3 LI, and the ratio of Ki-67 LI to caspase-3 LI as a marker of tumor survival (Fig. 1). VEGF-A, VEGF-C, and membranous E-cadherin were recorded as percentages of positive tumor cells in the entire tumor using 5% increments on a representative slide. EGFR was recorded according to published criteria25 as follows: 0 if tumor cells had complete absence of staining or any staining intensity in <10% of tumor cells; 1+ if tumor cells had faint or barely perceptible membrane staining in >10% of tumor cells or weak heterogeneous staining in >50% of positive cells; 2+ if tumor cells had moderate membrane staining in >10% of cells or moderate heterogeneous staining in >50% of positive cells; and 3+ if tumor cells had strong membrane staining in >10% of cells or strong heterogeneous staining in >50% of positive cells. Tumors with 1+, 2+, and 3+ expression were interpreted as positive for EGFR expression, and tumors with no expression (score o) were interpreted as negative for EGFR expression.25
The primary endpoint of this study was the occurrence of brain metastasis. Brain metastasis (present vs absent) was considered as a binary response variable.
The distribution of patient characteristics was compared between the study group and the control group using the Wilcoxon rank-sum test and the Fisher exact test for continuous and discrete variables, respectively. The distribution of protein expression also was compared between the study group and the control groups using the Wilcoxon rank-sum test. In addition, the test was stratified according to age (<60 years, ages 61 to 70 years, and >70 years) to adjust for the age difference between the 2 groups. The association between developing brain metastases and each molecular marker was based on the age-adjusted Mantel-Haenszel estimate of the odds ratio.
Overall survival was calculated from the time of NSCLC diagnosis to the time of death from any cause or to the time of last follow-up, at which point the survival time was censored. Overall survival curves were estimated using the Kaplan-Meier method, and the log-rank test was used to evaluate the survival difference between patient groups. Statistical analyses were computed using SAS statistical software (version 9.1; SAS Institute Inc., Cary, NC), and the plots were produced using S-Plus version 6.2 for Windows (Insightful Corp., Seattle, Wash). All significance tests were based on a 2-sided hypothesis.
Patient and Tumor Characteristics
The patient characteristics are summarized in Table 1. Among the patients with newly diagnosed NSCLC who were included in the study, 21 patients (39%) developed brain metastases after a median of 12.5 months (range, 1.7-89.4 months), and 33 patients (61%) had no evidence of brain metastases according to negative neurologic examination and/or negative brain imaging. The follow-up for the control group (median, 3.5 years; range, 0.8-6.6 years) was at least as long as that for the study group (median, 2.6 years; range, 0.7-8.4 years; P = .54).
Expression of Molecular Markers and the Risk of Developing Brain Metastases
Table 3 shows the distribution of study patients and control patients according to the expression of molecular markers and cutoff values used in the study. The primary analysis in Table 3 is focused on the results for differential expression that is meaningful from a pathology perspective. For each marker, we reported the cutoff point associated with the highest odds ratio in terms of quantifying the risk of developing brain metastases. Nevertheless, the strong association between biomarker expression and development of brain metastases was maintained generally over a broad range of different cutoff values of the semiquantitative score. In general, we explored the range between the 25th and 75th percentiles of marker expression in the combined study and control groups to ensure that there were reasonable patient numbers in the comparison subgroups for the purposes of statistical power and clinical utility.
Table 3. The Risk of Developing Brain Metastases According to the Expression of Molecular Markers in Nonsmall Cell Lung Cancer
Patients in the study group had 15 tumors (71%) with high Ki-67 expression, and patients in the control group had 7 tumors (21%) with high Ki-67 expression (Table 3). A high caspase-3 LI was present in 7 tumors (33%) from the study group, whereas nearly all tumors (97%) in the control group had a high caspase-3 LI (21%) (Table 3).
An increased risk of developing brain metastases was associated with a Ki-67 LI ≥30 (adjusted odds ratio of 12.2; 95% confidence interval [CI], 2.4-70.4 [P < .001]). The caspase-3 LI was also associated significantly with the development of brain metastases (P < .001), but the correlation was negative. Therefore, lower percentages of caspase-3 staining were observed in NSCLC from patients who developed brain metastases (adjusted odds ratio of 43; 95% CI, 5.3 to >100 [P < .001]). The Ki-67 LI:caspase-3 LI ratio was also associated significantly with the development of brain metastases (odds ratio of 68; 95% CI, 7.7 to >100 [P < .001]). The percentages of VEGF-C and E-cadherin staining were also associated significantly with the development of brain metastases (P = .001, and P = .05, respectively) (Table 3) (Fig. 2). No significant risk of developing brain metastases was associated with VEGF-A expression or EGFR expression (Table 3) (Fig. 2).
Outcome of Patients with NSCLC
We observed no significant differences in overall survival between patients in the study group and patients in the control group (P = .46) (Fig. 3). Similar to the results from previous studies,2, 4 brain metastases developed after a median of 12.5 months (range, from 53 days to 89.4 months) after the primary diagnosis of NSCLC. When the study and control groups were analyzed together, a high Ki-67 LI was significantly associated with poor prognosis (P = .04). Patients who had a Ki-67 LI ≥30 had a shorter overall survival than patients who had a Ki-67 LI <30 (median overall survival, 26 months and 47 months, respectively) (Fig. 3). The other molecular markers were not associated with overall survival.
In this study, we evaluated patients who had NSCLC with and without brain metastasis in a unique series that included tumor material from both the primary lung tumor and the matched metachronous brain metastases obtained from a single institution. These patients typically are treated only for lung cancer, and brain biopsy is rarely recommended or obtained. This gave us the opportunity to confirm that the brain tumors were metastases from primary NSCLC and to investigate the expression of a broad panel of biomarkers that reportedly are involved in the pathogenesis of brain metastasis.8, 9, 14–16, 18, 19, 22, 26 Our objective was to determine whether any of these markers or a combination of markers could identify the primary NSCLC tumors that are more likely to metastasize to the brain. We observed that patients with high Ki-67, low caspase-3, high VEGF-C, and low E-cadherin in their primary NSCLC tumors had a greater risk of developing brain metastases compared with a control group of patients with NSCLC who were diagnosed during the same period (Fig. 2). These biomarkers are promising for predicting the development of brain metastases, and additional studies on larger series of patients are needed to validate the findings from our study.
Recent studies of molecular biology in human cancers have demonstrated that numerous factors affect the behavior of malignant neoplasms.27 Tumors with a high Ki-67 LI frequently are more aggressive than tumors with a low Ki-67 LI.28 In addition, factors involved in angiogenesis, such as VEGF, are associated with tumor growth and metastasis.17–19 Furthermore, reduced expression of the molecules involved in EMT, such as E-cadherin, could induce tumor cells with high metastatic potential.21 Although studies have demonstrated an association between biomarker expression and metastatic potential of other cancers,18, 28–30 to our knowledge, the value of these molecular markers in predicting brain metastasis in patients with NSCLC has yet to be explored.
To our knowledge to date, it remains unclear whether patients with early-stage NSCLC should be screened for brain metastases according to published clinical guidelines.11–13 In its latest joint statement, the American Thoracic Society and the European Respiratory Society recommend no preoperative imaging of the brain in patients with NSCLC.11 In fact, it is believed that the routine screening is cost-ineffective because of the low incidence of brain metastasis in asymptomatic patients.31–34 Some reports argue that early detection of occult brain metastases will avoid increased morbidity either by allowing earlier treatment of the brain or by avoiding futile thoracotomies.10, 35–38 However, evidence suggests an increased incidence of cerebral metastases after preoperative radiotherapy and chemotherapy in patients with locally advanced NSCLC.39 In addition, studies have produced contrasting results regarding the correlation between tumor histology and the risk of developing brain metastases.4, 12 Therefore, there is a need to identify biologic markers that can identify patients with NSCLC who are at increased risk of developing brain metastases.
Patients with NSCLC who develop brain metastases are most likely to benefit from therapy addressing the brain that would substantially improve their quality of life.7 An analysis of subgroups of patients with NSCLC demonstrated that certain populations are at particularly high risk of brain metastasis.7
Others have investigated predictors of survival in patients with NSCLC and brain metastases. Similar to our results, Gaspar et al2 observed an increased risk of brain metastases in patients aged <50 years. It also has been reported that performance status, extent of extracranial disease, control of primary cancer, and age are predictors of overall survival in patients with NSCLC.
Immunostaining with the Ki-67 antibody is a widely accepted method for evaluating proliferative activity in a variety of human tumors. The Ki-67 LI correlates well with the predicted growth fraction, although it consistently overestimates the proliferative activity of tumors and, thus, is not an exact reflection of tumor growth.40 Sarbia et al41 observed a significant association between the Ki-67 LI and mitotic activity in tumor tissue. Numerous studies have reported a correlation between the Ki-67 LI and other markers of cell proliferation.42, 43 Our data indicated a strong correlation between a high Ki-67 LI and the development of brain metastases (Fig. 2). Patients who had a Ki-67 LI ≥30% had a 12 times increased risk of developing brain metastases (P < .001) (Table 3) compared with patients who had lower proliferative activity. Furthermore, in our study, a high Ki-67 LI was associated with poor prognosis (Fig. 3).
Abnormalities in the molecular mechanisms of apoptosis are important in the pathogenesis of malignant processes, because they increase the longevity of neoplastic cells, develop resistance to generally harmful stresses, and increase invasiveness, resulting in disease progression and metastasis.29 This process is the result of the interaction between several proteins involved in either inhibition or activation of the apoptotic cascade. Several studies have demonstrated that activation of the caspase system is involved in gefitinib-induced apoptosis in patients with NSCLC.44, 45 Our results indicate that a low caspase-3 LI was associated with a significant increase in the risk of developing brain metastases (Table 3, Fig. 2).
The VEGF family of proteins modulates angiogenesis, which is essential for tumor growth and metastasis.46 Since its identification around 15 years ago, this family has expanded considerably to include VEGF-A, VEGF-B, VEGF-C, and VEGF-D.17, 47, 48 Although VEGF has been linked to angiogenesis in various tumors,49, 50 its role in the metastatic ability of NSCLC has been poorly characterized. VEGF-C production by a tumor is associated with increased incidence of lymphatic metastasis, but the mechanism remains unclear.51 It is believed that tumors with VEGF-C overexpression were surrounded by lymphatic vessels that had abnormal lymphatic flow compared with controls.52, 53 Our results indicated a strong correlation between high expression of VEGF-C and an increased risk of developing brain metastases in patients with NSCLC (Table 3): When >5% of tumor cells expressed VEGF-C, the risk of developing brain metastases increased 15 times (P < .001) (Table 3). There was no association between the expression of VEGF-A and the risk of developing brain metastases (Fig. 2).
E-cadherin is a calcium-regulated adhesion molecule that is expressed in most normal epithelial tissues and plays an important role in cell-sorting mechanisms by offering adhesion specificities that govern tissue integrity. It has been demonstrated that this molecule may regulate the invasiveness of neoplastic cells.54 Our results indicated that loss of E-cadherin is associated with an increased risk of developing brain metastases in patients with NSCLC (P = .05) (Table 3).
EGFR is overexpressed in 40% to 80% of NSCLC and in many other epithelial cancers.22, 23 Although anti-EGFR agents are being used in clinical trials for patients with advanced disseminated disease, the value of EGFR in predicting brain metastases is unknown. In this study, we observed that EGFR was overexpressed in 90% of patients in the study group and 85% of patients in the control group, but the difference was not statistically significant (Table 3, Fig. 2).
The current results indicate that patients with NSCLC and high Ki-67, high VEGF-C, low E-cadherin, and low caspase-3 in their tumors have a greater risk of developing brain metastases (Table 3, Fig. 2). Our study suggests that patients with NSCLC and a specific biomarker expression may benefit from an individualized surveillance regimen and a preventive therapeutic intervention designed to prevent or delay the development of brain metastases.