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Early Symptoms Lung Cancer

Study objective: Epidermal growth factor receptor (EGFR) signaling has been implicated in the pathogenesis of bronchial dysplasia and overt non-small cell lung cancer (NSCLC). We hypothesized that assaying for EGFR activity using an antibody that recognizes phosphorylated EGFR (pEGFR) may identify a subset of patients whose tumor cells are dependent on EGFR signaling. We also hypothesized that EGFR activity may be prognostic for early-stage NSCLC.

Design: We constructed high-density tissue microarrays using tissues from 193 surgically resected stage I NSCLCs. These arrays were immunostained with a pEGFR antibody, and the intensity of staining was correlated with clinicopathologic variables, as well as disease-free and overall survival (OS). Staining was scored by intensity and the percentage of positively stained tumor cells in triplicate.

Measurements and results: We found the expression of pEGFR (with > 50% of tumor cells staining positive) in 51% of tumor tissues. We found an inverse correlation between pEGFR, and both tumor size and the degree of tobacco smoking. In addition, we found a trend in which pEGFR expression was inversely correlated with disease stage (IA higher than IB). There was no correlation with sex, histology, or disease-free or OS.

Conclusions: Our results suggest that pEGFR levels are present in early-stage NSCLC, especially in patients with small tumors and in those with short smoking histories, but there is no prognostic impact on a patient's disease course. Targeting EGFR may therefore have more promise in chemoprevention or in patients with smaller early-stage NSCLCs compared with those with more advanced disease.

Key words: epidermal growth factor; non-small cell lung cancer; prognosis; receptor tyrosine kinase; tobacco smoking

Abbreviations: BAC = bronchioloalveolar cancer; DFS = disease-free survival; EGFR = epidermal growth factor receptor; NSCLC = non-small cell lung cancer; OS = overall survival; PBS = phosphate-buffered saline; pEGFR = phosphorylated epidermal growth factor receptor; TGF = transforming growth factor

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Tumor stage continues to be the most important predictor for outcome following surgical resection for early-stage non-small cell lung cancer (NSCLC). (1) Patients who undergo curative surgical resection for apparently localized disease have 5-year survival rates ranging between 25% and 70%. This implies the need for better systemic treatment to identify and cure occult micrometastatic disease. A better understanding of molecular pathways that impact the lung cancer phenotype may lead to the identification of patients who are at high risk for recurrence and thus of interventions directed at those patients who may derive the maximum benefit. For example, our group recently identified the presence of RRM1 and PTEN as predictors of disease-free and overall survival (OS) in patients undergoing surgical resection for NSCLC. (2)

The epidermal growth factor receptor (EGFR) has been implicated in the molecular pathogenesis of lung cancer through the regulation of cell growth and death, invasion and metastasis, and angiogenesis. The overexpression of EGFR and its natural ligand, transforming growth factor (TGF)-[alpha], is a common event in human lung cancers. (3-6) The overexpression of EGFR occurs in 45 to 70% of NSCLCs. The possibility of EGFR and epidermal growth factor/ TGF-[alpha] acting in an autocrine loop in lung cancer patients has been further supported by work (7) in lung cancer cell lines that produce soluble EGF or TGF-[alpha]. While originally thought of as cytostatic agents, tumor regressions, presumably through enhanced apoptosis, are being seen to occur with agents that inhibit EGFR tyrosine kinase in cases of advanced NSCLC. (8-10) Signaling through EGFR not only regulates events that are important in the cell cycle but also leads to survival signals that down-regulate apoptotic pathways. EGFR signaling can lead to the activation of multiple survival-signaling cascades, including the Ras/mitogen-activated protein kinase pathway, the Janus family of protein-tyrosine kinases, the phosphatidylinositol 3-kinase and Akt pathways, and the signal transducers and activators of transcription pathways. (11) Signaling through the EGFR can affect both the levels and activity of antiapoptotic proteins in the Bcl-2 family. (12) Finally, EGFR signaling has been linked to increased tumor cell invasiveness, suggesting that EGFR signaling may be important for both the local and distant recurrence of disease following surgery. (13)

The role of EGFR in the prognosis of patient outcomes following surgical resection for early-stage NSCLC remains unclear. Some reports (14-17) have indicated a worse prognosis for patients with EGFR overexpression, while still other reports (18-32) have demonstrated no impact on disease-free or OS. The role of EGFR is made even more complex by the wide array of assays used to measure EGFR, including immunohistochemistry for total EGFR receptor protein, quantitative polymerase chain reaction to the assay for messenger RNA copies, enzyme-linked immunosorbent assay, Northern analysis, and gene copy number. The activation of EGFR by natural ligands, such as TGF-[alpha], results in the phosphorylation of tyrosine residues on the cytoplasmic domain of the receptor. This is required for the activation of downstream molecules such as Src and the Janus family of protein-tyrosine kinases, and consequently the biological activity of EGFR signaling. We reasoned that the assessment of phosphorylated EGFR (pEGFR) may be a better indicator of EGFR signaling and may more clearly delineate the impact of EGFR signaling on patient outcome.

MATERIALS AND METHODS

Tissue Arrays

We constructed high-density tissue microarrays using tissues from surgically resected early-stage NSCLCs. These arrays involve arraying up to 1,000 cylindrical tumor cores from individual tumors on a single tissue microarray. This technique is extremely useful for the rapid analysis of a large number of samples under identical technical conditions and for a determination of the statistical relevance of markers in a single experiment. Coupled with complete clinical data on the patient samples, this technology can rapidly assess correlations between markers and output variables such as survival, stage, and histology. We performed immunohistochemistry tests to detect EGFR activation (measured by pEGFR staining in tumor cells) and correlate it with clinicopathologic variables as well as patient outcome following surgical resection for early-stage NSCLCs.

Slides stained with hematoxylin-eosin and paraffin-embedded tissue blocks were examined, and representative tumor areas as well as available normal lung tissues adjacent to the tumor in each sample were marked for the location of tissue microarray punching while reviewing. The donor tissue cores with a diameter of 0.6 mm were punched and arrayed into a recipient paraffin block using a tissue arrayer (Beecher Instrument; Silver Spring, MD). Triplicate recipient blocks were constructed to overcome the problem of tumor heterogeneity in tissue microarray-based analyses and sample bias. (33) Four-micrometer sections of triplicate tissue microarray blocks were cut and transferred to adhesive coated slides (Instrumedics; Hackensack, NJ), and were cured under exposure to ultraviolet light for 30 s to adhere the tissue to the slides.

The sections were deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol, and finally rinsed in deionized water. Endogenous peroxidase was blocked with 3% hydrogen peroxide. For antigen retrieval, the slides were microwaved in an unmasking solution (H-3300; Vector Laboratories Inc; Burlingame, CA) on high power for 7 min. The slides were then allowed to cool in the solution for 20 min at room temperature.

Patient Population

The samples collected for our tissue microarray construction met the following requirements: (1) diagnosis of stage I NSCLC without preoperative radiation or chemotherapy; (2) surgically resected specimens that were fixed in formalin and embedded in a paraffin block; and (3) adequate tumor tissue in size for at least three tissue cores. Adequate tumor samples from 193 patients were collected from patients who had undergone surgical resection for NSCLC between February 1991 and January 2001 at H. Lee Moffitt Cancer Center. During this time interval, the patient's symptoms, medical history, use of tobacco products, and other relevant parameters were prospectively collected on hard copy questionnaires. Tumor staging was based on CT scans of the chest and upper abdomen, mediastinal lymph node dissection, and gross and microscopic evaluation of the resected lung tissue. PET scanning was performed on 24 of 60 patients who had received diagnoses between December 1999 and January 2001. Other staging studies, including brain imaging and bone scans, were performed at the discretion of the surgeon. The date of the first pathologic verification of malignancy was used as date of diagnosis. After surgery, patients were followed up for 3 months by the surgeon. They were then given a choice of follow-up at regular intervals (ie, every 3 to 12 months) by the referring physician or at our institution with standard radiographs and/or CT scans of the chest. The first date of unequivocal clinical evidence of disease or histologic confirmation, in questionable cases, was recorded as the recurrence date. The date of death was obtained through family or care provider contact, and it was verified by a review of public records. OS was defined as the time that elapsed from histologic diagnosis to death. Disease-free survival (DFS) was defined as the time that elapsed from surgery to disease recurrence or death.

Immunohistochemistry

Immunostaining for pEGFR was performed using a rabbit antihuman polyclonal antibody (Phospho-EGFR Tyr 845; Cell Signaling Technology; Beverly, MA). As negative controls, rabbit Igs (Vector Laboratories) were used as a primary antibody. The immunohistochemical stain was performed manually at room temperature using avidin-biotin-peroxidase complex methods (Vectastatin Elite ABC kit; Vector Laboratories). After washing with phosphate-buffered saline (PBS) solution for 5 min, slides were blocked with normal serum with 3% bovine serum albumin for 10 min followed by incubation with the anti-pEGFR primary antibody at a dilution of 1:200 overnight at 4[degrees]C. After rising with PBS for 5 min, slides were incubated with a biotinylated secondary antibody for 60 min and washed again. After washing with PBS for 5 min, slides were incubated with avidin-biotin complex for 1 h, washed again, and developed in chromogen with 3, 3-diaminobenzidine (DAB Substrate Kit for peroxidase; Vector Laboratories).

Immunohistochemical staining of the slides was reviewed and scored. The score system included counting the percentage of positively stained tumor cells and estimating the intensity staining in a semiquantitative manner. Intensity was classified on a scale of 0 to 3 (0, no staining; 1, weak staining; 2, medium; 3, strong staining). For triplicate samples, median values of the percentage of staining, the intensity, and a composite score (percentage multiplied by intensity; range, 0 to 300) were derived.

Statistical Analysis

The expression of pEGFR and demographic data were compared by Spearman correlation coefficient. The Wilcoxon rank sum test was used to test for significant associations between dichotomous variables and pEGFR expression, and the Kruskal-Wallis test was used for variables with more than two categories. Overall and DFS probabilities were estimated using the Kaplan-Meier method, and log-rank testing was used to determine the level of significance between survival curves.

RESULTS

Paraffin-embedded specimens over a 10-year period were collected from pathologically staged patients undergoing complete resection for NSCLC. Table 1 shows the demographic data on the patients whose tumors make up the tissue array. There were 86 women and 107 men. The median age of patients in the study cohort was 69 years, and the median follow-up time for the study cohort was 37 months (range, 0 to 146 months). The histopathology includes adenocarcinoma (81), squamous cell carcinoma (69), bronchioloalveolar carcinoma (BAC) (19), and large cell carcinoma (24). One hundred eleven patients had stage IA disease (tumor size, < 3.0 cm), and 82 patients had stage IB disease (tumor size, [greater than or equal to] 3.0 cm). Eleven patients were lifelong nonsmokers (ie, had smoked < 100 cigarettes), 54 patients were active smokers, 109 patients were former smokers, and 19 patients had unknown smoking histories. At the time of the analysis, 88 patients had died and 105 patients were still alive.

Paraffin-embedded tissues were used to construct the tissue microarray, and immunostaining for pEGFR was performed. Figure 1, top left, A, top middle, B, and top right, C, show a representative collection of tissue cores that were stained for pEGFR. Figure 1, bottom, D, shows the distribution for the percentage of tumor cells staining for pEGFR, the intensity of pEGFR staining, and the composite scores observed in the patient cohort.

[FIGURE 1 OMITTED]

We explored differences in pEGFR expression among women and men, age, smoking status, patients with stage IA and IB disease, performance status 0 and > 0, the absence or presence of weight loss, age, and histopathology. Table 2 shows the correlation between pEGFR and patient characteristics, including sex, histology, tumor size, disease stage (IA vs IB), and smoking history. We found an inverse correlation between pEGFR staining (including the percentage of staining, stain intensity, and composite staining) and smoking history (in pack-years), but not between pEGFR staining and smoking classification (eg, active or former). This is likely due to the inherent inaccuracy of such a classification system. We also detected an inverse relation between pEGFR staining and tumor size as well as disease stage, suggesting higher pEGFR activity in smaller tumors. We found a trend toward a higher percentage of pEGFR-staining cells in BACs compared to other types of tumor histologies, but the finding was not statistically significant. Finally, patients with weight loss had less pEGFR intensity than those without weight loss (p = 0.04 [Wilcoxon rank sum test]), although only 13 patients in our cohort had a history of weight loss.

Kaplan-Meier OS and DFS curves for pEGFR expression, dichotomized by the median level for each gene, are shown Figure 2. No significant relationship was found between DFS or OS and the percentage of pEGFR, the intensity of pEGFR staining, or the composite pEGFR staining.

[FIGURE 2 OMITTED]

DISCUSSION

Our results with pEGFR staining using tissue microarrays demonstrates a spectrum of pEGFR staining in early-stage NSCLC, but no correlation with histology, sex, or patient outcome following surgical resection. Interestingly, pEGFR expression is significantly and inversely correlated with tumor size. In addition, we found a trend in which pEGFR expression is inversely correlated with disease stage (stage IA disease is higher than stage IB disease). Since all of the patients analyzed had stage I disease, it was not possible to determine whether there was a relationship between more advanced tumor stage (ie, stages II to IV) and pEGFR staining. These results suggest that smaller tumors have higher levels of pEGFR, and potentially are more dependent on EGFR signaling than larger and more advanced tumors. These data are consistent with those from other reports demonstrating increased total EGFR expression in the metaplastic bronchial epithelium. (34) There also are reports (35) of differences in the expression of EGFR between high-grade and low-grade dysplasia. The transformation of bronchial epithelial cells is associated with increased EGFR levels, and agents that prevented this increase also prevented the chemical transformation of cells. (36) Similar to our own results, Hirsch and colleagues (21) found a higher expression of total EGFR in earlier stages of disease compared with that in stage III. Collectively, these results and ours may suggest that EGFR signaling is important in the pathogenesis of bronchial dysplasia as well as in early small tumors. Therefore, the targeting of EGFR may have more promise in chemoprevention or in the treatment of smaller early-stage NSCLCs compared with more advanced disease. (37)

One could speculate that increasing tumor size and stage allows for the continued accumulation of oncogenes or the loss of tumor suppressor genes that serve to bypass the requirement of EGFR signaling for tumor growth. It is likely that multiple signaling pathways in lung cancer cells cooperate to ensure tumor growth and survival. In addition to EGFR, NSCLCs can express the receptors for hepatocyte growth factor (c-MET) and insulin-like growth factor-1, as well as receptors for cytokines such as interleukin-6. (10,38-40) Intracellular signaling molecules such as Ras, phosphatidylinositol 3-kinase/Akt, and signal transducers and activators of transcriptions also have been found to be important in NSCLCs. (40-42) It is becoming more apparent that parallel signaling pathways can maintain tumor growth and survival despite the loss of EGFR signaling. Some studies in lung cancer cells have demonstrated that the persistent activity of extracellular signal-regulated kinase or Akt kinase pathways limit the antiproliferative and apoptotic actions of gefitinib, which is a small-molecule inhibitor of EGFR. (43) Other studies (44) have demonstrated that the loss of PTEN or the overexpression of insulin-like growth factor-1 receptor can abrogate the effect of EGFR inhibition. Therefore, a number of signaling pathways may serve as bypass mechanisms for EGFR signaling, and increasing tumor size and/or stage may allow for the accumulation of these parallel signaling pathways.

We found an inverse correlation between smoking history (measured in pack-years) and pEGFR levels. This result is interesting in light of recent studies (45) showing that a limited smoking history is an independent predictor of a response to gefitinib, which is a small-molecule inhibitor of EGFR tyrosine kinase. Additional studies (46) have shown that lung tumors from smokers have widespread chromosomal abnormalities, which are infrequent in nonsmokers. Finally, three groups (47-49) have identified activating mutations in the tyrosine kinase domain of EGFR in patients who responded to gefitinib. Interestingly, the majority of these mutations were identified in patients with minimal or no tobacco smoking history. The antibody used in our studies recognized EGFR phosphotyrosine 845, which is highly phosphorylated in the L848R EGFR mutation, but not in the wild-type or L746-p753 deletion mutant. (50) These results and our own may suggest that EGFR signaling plays an important role in the pathogenesis of lung cancers in patients with limited smoking histories or in lifelong nonsmokers. Since other phosphotyrosines exist on EGFR, additional pEGFR antibodies may identify other relationships that were not found with the pTyr-845 antibody used in our work.

Published reports on EGFR and prognosis vary widely in the assays used, and the impact of EGFR on either DFS or OS following surgery for NSCLCs also vary widely (Table 3). Two reports (15,18) have produced conflicting results about the prognostic role of pEGFR in reseeted NSCLCs. Our analysis with 193 patients does not demonstrate that pEGFR impacts the prognosis for a patient with stage I NSCLC. This is perhaps not surprising since the signaling pathways discussed above, which serve as bypass mechanisms for EGFR signaling, may limit the prognostic importance of EGFR. Bather than examining single molecules or pathways, some investigators have suggested (51,52) using gene expression patterns to predict patient outcome following NSCLC surgery. Finally, although we found no relationship between pEGFR and survival for patients with stage I NSCLC, the same may not be true for patients with more advanced stages of NSCLC.

Two potential confounding effects of studies on signaling pathways in resected NSCLCs are worth mentioning. The first is the effect of ischemia resulting from surgical extraction of the tumor and lung tissues. Studies using gene expression profiling (53) have identified changes in gene expression resulting from ischemia in tumor tissues. Furthermore, published reports (54, 55) have found changes in both EGFR levels and EGFR activity in ischemic tissues. Therefore, it is possible that the levels of total EGFR and pEGFR are being affected by the surgical procedure, which may obscure their true prognostic value. Second, we observed some variability between pEGFR among the three triplicate samples. Despite averaging of the results, our analysis may nonetheless be affected by tumor heterogeneity and variation among the core samples of each tumor.

Our observations about pEGFR staining in patients with early-stage NSCLC may be important for future clinical trials in this patient group with inhibitors of EGFR tyrosine kinase. Some studies (8) in subjects with advanced-stage NSCLC who were treated with small-molecule inhibitors of EGFR tyrosine kinase have shown tumor regression and disease stabilization. The single-agent activity and the identification of patients with activated EGFR may support the incorporation of these agents into the treatment of earlier stage lung cancers or in patients with bronchial dysplasia who are at high risk for the development of lung cancer. This is of importance because of the increasing use of either preoperative or adjuvant cytotoxic chemotherapy for early-stage NSCLC. It is conceivable that pEGFR, along with other markers such as EGFR mutations, could be used to stratify patients whose conditions are appropriate for receiving either adjuvant chemotherapy or adjuvant EGFR-targeted therapy. Finally, our results are consistent with other reported observations, and suggest that the EGFR signaling pathway is an important pathway that drives the lung cancer phenotype, particularly in nonsmokers and those persons who have smoked little during their lifetime.

Table 1--Patient Demographics

     Characteristics           No.        %

Patients
  Total                        193
  Age range, yr               45-82
  Median age, yr                69
  Men                          107       55.4
  Women                         86       44.6
  White                        185       95.9
  Other race                     8        4.1
  Active smoker                 54       28.0
  Former smoker                109       56.5
  Lifelong nonsmoker            11        5.7
  Unknown smoking               19        9.8
Histology
  Adenocarcinoma                81       42.0
  Squamous cell carcinoma       69       35.8
  Large cell carcinoma          24       12.4
  BAC                           19        9.8
Stage
  IA                           111       57.5
  IB                            82       42.5
Follow-up
  Follow-up                  0-146 mo
  Median follow-up            37 mo
  Total alive                  105       54.4
  Total dead                    88       45.6

Table 2--Correlation of pEGFR With Patient
Characteristics *

                                 %         pEGFR        pEGFR
         Variables             pEGFR     Intensity    Composite

Age
  r Value                       0.11        0.09         0.09
  p Value                       0.12        0.24         0.21
Tumor size
  r Value                      -0.20       -0.18        -0.19
  p Value                       0.01        0.01         0.01
Pack-yr of Smoking
  r Value                      -0.22       -0.16        -0.19
  p Value                       0.01        0.04         0.02
Sex
  Male                         49.73        1.17        75.45
  Female                       49.86        1.13        70.74
  p Value ([dagger])            0.99        0.87         0.86
Histology ([double dagger])
  Adenocarcinoma               48.70        1.14        69.30
  BAC                          62.80        1.28        89.89
  Large cell carcinoma         45.30        1.09        75.53
  Squamous cell carcinoma      49.20        1.15        72.96
  p Value ([dagger])            0.24        0.74         0.47
PS
  0 (32)                       51.90        1.19        77.40
  1 (49)                       46.80        1.08        67.27
  p Value ([dagger])            0.38        0.25         0.32
Weight loss
  Yes (13)                     40.00        0.77        51.71
  No (166)                     51.06        1.19        75.98
  p Value ([dagger])            0.22        0.04         0.10
Disease stage
  IA                           54.27        1.25        80.69
  IB                           46.46        1.08        67.93
  p Value ([dagger])            0.07        0.09         0.10

* Values in parentheses signify PS score. PS = Eastern Cooperative
Group Performance Status.

([dagger]) Based on Kruskal-Wallis test.

([double dagger]) Values given as the mean.

Table 3--Published Reports of EGFR and Prognosis *

       Study/Year                  Assay          Patients, No.

Onn et al (18)/2004          IHC EGFR/pEGFR             111
Mukohara et al (20)/2004     IHC                         91
Selvaggi et al (14)/2004     IHC                        130
Hirsch et al (21)/2003       IHC/gene copy No.          183
Mukohara et al (19)/2003     IHC                         60
Kanematsu et al (15)/2003    pEGFR IHC                   36
Meert et al (22)/2002        Metaanalysis             2,185
Cox et al (23)/2001          IHC                        167
Brabender et al (24)/2001    QPCR                        83
Ohsaki et al (16)/2000       IHC                        290
D'Amico et al (25)/1999      IHC                        408
Pfeiffer et al (31)/1998     ELISA/IHC                  190
Fontanini et al (28)/1998    IHC                        195
Volm et al (29)/1998         IHC                        121
Rusch et al (5)/1997         IHC                         96
Pastorino et al (30)/1997    IHC                        515
Pfeiffer et al (26)/1996     IHC                        186
Fontanini et al (27)/1995    IHC                        176
Rusch et al (4)/1993         Northern blot               57
Veale et al (7)/1993         Membrane preps              19
Dazzi et al (32)/1989        IHC                        152

                                                  Prognostic Role of
       Study/Year            Overexpression, %           EGFR

Onn et al (18)/2004                59.5                   Neg
Mukohara et al (20)/2004           79                     Neg
Selvaggi et al (14)/2004           78                     Pos
Hirsch et al (21)/2003             62                     Neg
Mukohara et al (19)/2003           78                     Neg
Kanematsu et al (15)/2003          44.4                   Pos
Meert et al (22)/2002                                     Neg
Cox et al (23)/2001                                       Neg
Brabender et al (24)/2001          33.7                   Neg
Ohsaki et al (16)/2000             42.8                   Pos
D'Amico et al (25)/1999                                   Neg
Pfeiffer et al (31)/1998                                  Neg
Fontanini et al (28)/1998                                 Neg
Volm et al (29)/1998                                      Neg
Rusch et al (5)/1997               70                     Neg
Pastorino et al (30)/1997                                 Neg
Pfeiffer et al (26)/1996           55                     Neg
Fontanini et al (27)/1995                                 Neg
Rusch et al (4)/1993               93                     Neg
Veale et al (7)/1993                                      Pos
Dazzi et al (32)/1989                                     Neg

* IHC = immunohistochemistry, QPCR = quantitative polymerase chain
reaction; ELISA = enzyme-linked immunosorbent assay; Neg = negative;
Pos = positive.

ACKNOWLEDGMENT: The authors thank Rebecca Alexander for administrative assistance.

* From the Thoracic Oncology Program (Drs. Zheng, Bepler, and Haura) and Biostatistics Core (Dr. Cantor), H. Lee Moffitt Cancer Center and Research Institute Tampa FL.

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This work has been supported in part by the Molecular Imaging Core at the H. Lee Moffitt Cancer Center and Research Institute, by the Chiles Endowment Biomedical Research Program of the Florida Department of Health (E.B.H), by National Institutes of Health grants R01 CA 102726 and U01 CA101222 (G.B.), and by the H. Lee Moffitt Cancer Center and Research Institute.

Manuscript received July 21, 2004; revision accepted December 6, 2004.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml).

Correspondence to: Eric B. Haura, MD, Thoracic Oncology Program, The H. Lee Moffitt Cancer Center and Research Institute, MRC3 East, Room 3056, 12902 Magnolia Drive, Tampa, FL 33612-9497; e-mail: hauraeb@moffitt.usf.edu

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