HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Min-Hsiung Huang Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hsieh, C.-C. Articles by Wang, L.-S. Search for Related Content PubMed PubMed Citation Articles by Hsieh, C.-C. Articles by Wang, L.-S.

Ann Thorac Surg 1998;66:1159-1163
© 1998 The Society of Thoracic Surgeons

---

Original articles: general thoracic

Prognostic significance of HER-2/neu overexpression in stage I adenocarcinoma of lung

^a Division of Thoracic Surgery, Department of Surgery, Cancer Center, Section of Thoracic Oncology, Chest Department, and Department of Pathology, Veterans General Hospital-Taipei, Taipei, Taiwan, Republic of China
^b National Yang Ming University, Taipei, Taiwan, Republic of China

Accepted for publication May 5, 1998.

Address reprint requests to Dr Wang, Division of Thoracic Surgery, Department of Surgery, Veterans General Hospital-Taipei, No. 201, Sec 2, Shih-Pai Rd, Taipei, Taiwan, ROC
e-mail: (gcliu{at}www.vghtpe.gov.tw)

Abstract

Background. Even with early diagnosis and adequate resection, the 5-year survival rate for stage I lung cancer patients is around 60% to 70%. Overexpression of HER-2/neu protein is associated with poor prognosis in lung cancers. In this study, we evaluated the expression of HER-2/neu in cancer cells of lung and assessed their clinicopathologic and prognostic significance.

Methods. From 1986 to 1995, clinical data on 42 consecutive patients who underwent complete surgical resection for stage I lung adenocarcinoma were collected. Expression of HER-2/neu in paraffin-embedded tumor samples was determined by immunohistochemistry and scored with a semiquantitative method.

Results. Twenty-one of 42 patients were positive for HER-2/neu overexpression in tumor. Compared with patients with low HER-2/neu expression, patients with HER-2/neu overexpression had a significantly higher incidence of early tumor recurrence (p = 0.014). Survival was also significantly better in patients without HER-2/neu overexpression than in those with HER-2/neu overexpression (p = 0.0047). By univariate analysis, HER-2/neu overexpression and poor cell differentiation are two important factors correlated with poor prognosis.

Conclusions. Expression of HER-2/neu oncoprotein in stage I lung adenocarcinoma can predict the tumor’s aggressiveness. Early tumor recurrence was frequently detected in patients with HER-2/neu overexpression. We recommend an individualized therapeutic strategy based on the level of HER-2/neu oncoprotein in the tumor cells.

Lung cancer is the leading cause of cancer mortality worldwide. In Taiwan, nearly 20% of cancer patients died of this disease and the annual mortality was more than 5,000 patients [1]. Most of the patients who died were at the late stage of the disease when they were diagnosed [2]. Even with early diagnosis and an adequate operation, the 5-year survival rate for stage I patients was only 70% [3]. An intrinsic factor that determines the difference between patients who will have early tumor recurrence and metastasis and those who will not could exist.

Advances in molecular biology and genetics have demonstrated that expressions of several markers are associated with lung cancer, such as p53, c-myc, c-fos, Ki-ras, c-erbB-1 [4, 5], and HER-2/neu [6–10]. HER-2/neu protooncogene, which encodes a transmembrane glycoprotein with a molecular weight of 185,000 (p185^neu), is a receptor with tyrosine kinase activity [11, 12]. Since its first identification [13], HER-2/neu also has been shown to be expressed in a wide variety of human malignancies, including carcinoma of the breast, ovary, gastrointestinal tract, salivary gland, liver, kidney, and bladder [14]. It has been indicated that expression of HER-2/neu could be associated with multidrug resistance, poor prognosis, and short survival [6–7, 9, 15], and measurement of HER-2/neu should be considered as a marker for predicting early recurrence and metastasis.

In this study, we used a semiquantitative method to determine HER-2/neu expression in surgical specimens from patients with stage I lung adenocarcinoma, and to assess the correlation between HER-2/neu expression and the clinicopathologic parameters. The prognostic significance of HER-2/neu overexpression in patients with stage I lung adenocarcinoma also was evaluated.

Material and methods

Materials
From September 1986 to December 1995, samples from 44 consecutive patients who were diagnosed as having stage I lung adenocarcinoma were collected. Stage of the disease was classified according to the new international staging system for lung cancer [16]. In stage I patients, there should be no evidence of metastasis by radionuclide bone scan, brain scan, liver scan, and sonography of the abdomen. All patients had undergone surgical resection and radical ipsilateral mediastinal lymph node dissection. All dissected lymph nodes were proved clear without cancer pathologically. Tumor size, lymph node number, differentiation, vascular invasion, and mitotic number also were evaluated. All patients were routinely followed up every 3 to 6 months in the outpatient department. Tumor recurrence and metastasis were identified when blood examination, biochemical studies, chest radiography, abdominal sonography, whole body bone scan, and computed tomographic scan of the chest showed any suspected evidence of the disease.

Immunohistochemistry
Three paraffin blocks of tumor were prepared for each case. The tissue preparation and staining procedure have been described previously [6, 13]. Briefly, paraffin blocks were sectioned at 4 µm. The wax was melted in a 65°C oven overnight. The slides were deparaffinized in xylene, and xylene was removed subsequently with absolute ethanol before immunostaining. The slides were incubated with monoclonal antibody to HER-2/neu (DAKO A/S, Copenhagen, Denmark) and then with peroxidase-conjugated goat anti-mouse immunoglobulin. Diaminobenzidine was used as chromogenic substrate, and brown precipitate was identified as positive staining. The samples were counterstained with hematoxylin and the slides were mounted with glycerol gelatin. Each batch had a positive and a negative control to ensure the quality.

Slide evaluation
Each slide was evaluated randomly at four areas that contained tumor cells. A photograph was taken at each evaluated area for a record. In each case, normal lung tissue served as internal negative control. Photographs were read by two independent observers without clinicopathologic knowledge of the case. The signal of stain was scored on a 0 to 2+ subjective scale with 0 = no expression, 1+ = intermediate expression, and 2+ = strong expression (Fig 1). Four areas were examined on three slides from each patient, resulting in 12 scores. An average was taken as the final reading. The average score of HER-2/neu in the normal lung tissue is 0.21 ± 0.17. After the specificity was determined from the internal negative control, an average score more than 0.4 was defined as positive for the overexpression of HER-2/neu and less than 0.4 as negative. All patients were then divided into two groups following this guideline.

View larger version (79K): [in this window] [in a new window]   Fig 1. Immunohistochemical staining of HER-2/neu expression in lung adenocarcinoma: (A) strong overexpression, scored as 2+; (B) intermediate expression, scored as 1+; (C) negative for HER-2/neu expression. (x400 before 35% reduction.)

Results

The mean age of the patients was 65.6 years, ranging from 47 to 83 years. Thirty-one patients were male and 13 patients were female. No significant body weight loss was reported by these patients. Eleven patients (25%) were asymptomatic before lung tumors were identified by routine chest radiography. Thirty patients (68.2%) were smokers and 14 patients smoked more than 20 pack-years. Most tumors were found in the left upper lobe (11 cases, 25%), then the right upper lobe (10 cases, 22.7%), right lower lobe (8 cases, 18.2%), left lower lobe (8 cases, 18.2%), and the right middle lobe (7 cases, 15.9%). Among these patients, 37 chose lobectomy, 1 patient chose bilobectomy and 6 patients chose pneumonectomy. Two deaths were related to the operation. One patient died of respiratory failure and the other died of gastric ulcer bleeding and sepsis. The median follow-up time for the remaining 42 patients was 32.2 months, ranging from 3.6 to 102.4 months. Until the date of data analysis, 30 of 42 patients (71.4%) were still well (3 patients had tumor recurrence and 27 patients were tumor-free). Eight patients died of tumor recurrence and 4 patients died of noncancer causes (car accidents and heart attack). Average duration between operation and tumor recurrence was 14.1 ± 6.6 months (ranging from 1.4 to 25.9 months). The cumulative 3-year and 5-year survival rates are 79% and 71%, respectively.

Among these 42 patients, 11 patients had evidence of tumor recurrence. As shown in Table 1, no significant difference was observed between patients with and those without tumor recurrence in the following clinicopathologic parameters: age, sex, tumor location, tumor size (T status), surgical modes, smoking index, and histopathologic characters (mitotic index, evidence of lymphovascular invasion). Nevertheless, a statistical difference was found with respect to the cell differentiation. Patients with poor cell differentiation (n = 10) had a significantly higher incidence of tumor recurrence than those with well and moderate cell differentiation (p = 0.029). Twenty-one patients were negative for HER-2/neu oncoprotein overexpression, and 21 patients were positive for HER-2/neu overexpression. Nevertheless, no significant difference was found between these two groups of patients in terms of the clinicopathologic parameters.

View larger version (15K): [in this window] [in a new window]   Fig 2. (A) Tumor-free survival curves in patients with stage I lung adenocarcinoma (dotted line = negative staining; solid line = positive HER-2/neu staining.) (B) Cumulative survival curves in patients with stage I lung adenocarcinoma (dotted line = negative staining; solid line = positive HER-2/neu staining). Survival curves were plotted with method of Kaplan and Meier. Statistical difference of survivals between different groups was compared by the log rank test.

The results presented here demonstrate that overexpression of HER-2/neu in stage I lung adenocarcinoma correlated with the tumor recurrence and metastasis. Previous reports have described the p185^neu expression in lung cancer. In some cases, overexpression of HER-2/neu also was shown to correlate with survival [7–9]. However, a limited number of cases, no disease staging, lack of a scoring system for HER-2/neu expression, and the conflicting results made interpretation of these studies difficult.

An elegant study by Harpole and associates [10] examined the relationship between disease progression and immunohistopathology along with oncoprotein expression in stage I non–small cell lung cancer. They suggested that male sex, presence of symptoms, tumor size, poor cell differentiation, vascular invasion, HER-2/neu expression, p53 expression, and high Ki-67 index could independently be prognostic factors. They further proposed that the outcome of the disease is the "dose response" of the additive effect of those parameters. It became clear that disease progression is a complex procedure that could be dependent on several clinicopathologic parameters. The cause of early cancer deaths as well as a mechanism for the early relapse and metastasis remains to be determined.

Immunohistochemistry is a frequently used method for evaluating gene expression in tumors. However, a great variation in the interpretation of results has been noted. In the case of lung adenocarcinoma, the positive rate of p185^neu expression ranged from 14% to 87.5%. Variation could be caused by the different methods, materials, or subjective biases. The major cause could be the heterogeneous expression of p185^neu in tumor cells. An objective and quantitative evaluation of immunohistochemical results is required, especially when this immunohistochemically based marker is employed for assessing the prognosis. An insensitive cut-off value used to define high- and low-risk groups of patients can disturb the relationship between variables and outcome.

Osaki and colleagues [17] have shown that patients with stage IIIB or T4 lung adenocarcinoma had an increased serum level of HER-2/neu. The concentration of HER-2/neu in serum was proportional to that in tissue section. When Diez and coworkers [18] examined the expression of HER-2/neu in the fresh samples of non–small cell lung cancer, there was not a significant correlation between p185 level and TNM classification. However, when patients were categorized by p185 levels, the relapse rate, disease-free period, and median time for tumor relapse worsened proportionally with the rise of p185^neu in the tumor tissue. Nevertheless, the results of enzyme-linked immunosorbent assay may not closely correlate with the clinical courses of disease progression. Both techniques can be used complementarily [19].

In patients with early-stage lung adenocarcinoma, lobectomy or pneumonectomy with ipsilateral mediastinal lymph node dissection is considered the best choice of treatment. Postoperative adjuvant therapy is generally not recommended. It is believed that postoperative chemotherapy does not improve the survival rate but increases the risk of chemotherapy complications. From this study, we found that in stage I lung adenocarcinoma, most tumor recurrences developed within 2 years after surgical intervention and were highly associated with overexpression of HER-2/neu oncoprotein. Diez and colleagues [18] have suggested possible individualized patient management based on p185 level and tailoring adjuvant chemotherapy to high-risk patients. In those cases, we would recommend dividing patients into two groups according to the expression of HER-2/neu in the tumor cells. For patients with low HER-2/neu levels, surgical resection may be adequate for cancer control. On the other hand, in patients with high HER-2/neu expression, or with high risk for recurrence and death, adjuvant chemotherapy after the operation would be suggested.

Unfortunately, tumors with HER-2/neu overexpression respond relatively poorly to the standard chemotherapy regimen [20]. Effective chemotherapeutic agents and protocols require further investigation. HER-2/neu protein is homologous to epidermal growth factor receptor with tyrosine kinase activity. Chemoresistance of cells correlated with the activated state of the enzyme. So tyrosine kinase inhibitor could theoretically reverse chemoresistance and increase chemosensitivity [11, 20]. In our preliminary clinical trial, we found that addition of gemcitabine (2,2-difluorodeoxycytidine) or high-dose caffeine could enhance the chemosensitivity of tumors with HER-2/neu overexpression to the standard chemotherapy regimen [21, 22].

In conclusion, our results demonstrated that cell differentiation and HER-2/neu oncoprotein expression in tumor cells are two important parameters to assess the aggressiveness of stage I lung adenocarcinoma. However, use of a similar scoring system in more patients is required to confirm the value of HER-2/neu overexpression and cell differentiation as prognostic factors. Development of prognostic indices based on these general markers will lead to the initiation of adjuvant therapy for patients with high-risk stage I lung adenocarcinoma. The goal of such a regimen is to improve the treatment outcome for these patients.

Acknowledgments

This study was supported, in part, by Lin Rong-San Foundation of Culture and Social Welfare and, in part, by the Department of Health, Executive Yuan, Republic of China (DOH85-HR-524).

We thank Ms Jing-Yi Chao at Cancer Center, Veterans General Hospital-Taipei, for statistical analysis and Ms Li-Ling Yang and Wen-Ser Tseng for their excellent technical assistance.

References

1. Annual reports of the Department of Health, Executive Yuan, Republic of China, 1995.
2. Melamed M.R., Flehinger B.J., Zaman M.B. Impact of early detection on the clinical course of lung cancer. Surg Clin North Am 1987;67:909-924.[Medline]
3. Pairolero P.C., Williams D.E., Bergstralh E.J., Piehler J.M., Bernatz P.E., Payne W.S. Postsurgical stage I bronchogenic carcinoma: morbid implications of recurrent disease. Ann Thorac Surg 1984;38:331-336.[Abstract]
4. Volm M., Effereth T., Mattern J. Oncoprotein (c-myc, c-erbB-1, c-erbB-2, c-fos) and suppressor gene product (p53) expression in squamous cell carcinomas of the lung: Clinical and biological correlations. Anticancer Res 1992;12:11-20.[Medline]
5. Rodenhuis S., van de Wetering M.L., Mooi W.J., Evers S.G., Nico van Zandwijk, Bos J.L. Mutational activity of the K-ras oncogene: a possible pathogenic factor in adenocarcinoma of the lung. N Engl J Med 1987;317:929-935.[Abstract]
6. Schneider P.M., Hung M.C., Chiocca S.M., et al. Differential expression of the c-erbB-2 gene in human small cell and non–small cell lung cancer. Cancer Res 1989;49:4968-4971.[Abstract/Free Full Text]
7. Kern J.A., Schwarts D.A., Nordberg J.E., et al. P185neu expression in human lung adenocarcinoma predicts shortened survival. Cancer Res 1990;50:5184-5191.[Abstract/Free Full Text]
8. Weiner D.B., Nordberg J., Robinson R., et al. Expression of the neu gene-encoded protein (p185neu) in human non–small cell carcinomas of the lung. Cancer Res 1990;50:421-431.[Abstract/Free Full Text]
9. Shi D., He G., Cao S., et al. Overexpression of the c-erbB-2/neu-encoded p185 protein in primary lung cancer. Mol Carcinogen 1992;5:213-218.[Medline]
10. Harpole D.H., Jr, Herndon J.E., II, Wolfe W.G., Iglehart J.D., Marks J.R. A prognostic model of recurrent and death in stage I non–small cell lung cancer utilizing presentation, histopathology, and oncoprotein expression. Cancer Res 1995;55:51-56.[Abstract/Free Full Text]
11. Coussens L., Yang-Feng T.L., Liao Y.C., et al. Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. Science 1985;230:1132-1139.[Abstract/Free Full Text]
12. Akiyama T., Sudo C., Ogawara H., Toyoshima K., Yamamoto T. The product of human c-erbB-2 gene: a 185-kilodalton glycoprotein with tyrosine kinase activity. Science 1986;232:1644-1646.[Abstract/Free Full Text]
13. Slamon D.J., Clark G.M., Wong S.G., Levin W.J., Ullrich A., McGuire W.L. Human breast cancer: correlation of relapse and survival with amplification of the erbB-2/neu oncogene. Science 1987;235:177-182.[Abstract/Free Full Text]
14. Hynes N.E., Stern D.F. The biology of erbB-2/neu/HER-2 and its role in cancer. Biochim Biophys Acta 1994;1198:165-184.[Medline]
15. Tsai C.M., Chang K.T., Perng R.P., et al. Correlation of intrinsic chemoresistance of non–small cell lung cancer lines with HER-2/neu gene expression but not with ras gene mutations. J Natl Cancer Inst 1993;85:897-901.[Abstract/Free Full Text]
16. Mountain C.F. A new international staging system for lung cancer. Chest 1986;89:225S-233S.[Free Full Text]
17. Osaki T., Mitsudomi T., Oyama T., et al. Serum level and tissue expression of c-erbB-2 protein in lung adenocarcinoma. Chest 1995;108:157-162.[Abstract/Free Full Text]
18. Diez M., Pollan M., Maestro M., et al. Prediction of recurrence by quantification of p185^neu protein in non–small-cell lung cancer tissue. Br J Cancer 1997;75:684-689.[Medline]
19. Dittadi R., Donisi P.M., Brazzale A., et al. Immunoenzymatic assay of erbB2 protein in cancer and non-malignant breast tissue: Relationship with clinical and biochemical parameters. Anticancer 1992;12:2005-2010.
20. Tsai C.M., Levitzki A., Wu L.H., et al. Enhancement of chemosensitivity by tyrphostin AG825 in high-p185^neu expressing non–small cell lung cancer cells. Cancer Res 1996;56:1068-1074.[Abstract/Free Full Text]
21. Tsai C.M., Perng R.P., Chen M.M., et al. Greater enhancement of chemosensitivity by caffeine in high p185neu expression human non–small cell lung cancer cell lines. J Natl Cancer Inst 1994;86:1018-1020.[Free Full Text]
22. Tsai C.M., Chang K.T., Chen J.Y., et al. Cytotoxic effects of gemcitabine-containing regimens against human non–small cell lung cancer cell lines which express different levels of p185neu. Cancer Res 1996;56:794-801.[Abstract/Free Full Text]

This article has been cited by other articles:

				L. Thomas, L. A. Doyle, and M. J. Edelman Lung Cancer in Women: Emerging Differences in Epidemiology, Biology, and Therapy Chest, July 1, 2005; 128(1): 370 - 381. [Abstract] [Full Text] [PDF]

				R. J. Korst and R. G. Crystal Active, specific immunotherapy for lung cancer: hurdles and strategies using genetic modification Ann. Thorac. Surg., October 1, 2003; 76(4): 1319 - 1326. [Abstract] [Full Text] [PDF]

				C.-M. Lin, A. F.-Y. Li, L.-H. Wu, Y.-C. Wu, F. C.-F. Lin, and L.-S. Wang Adenoid cystic carcinoma of the trachea and bronchus - a clinicopathologic study with DNA flow cytometric analysis and oncogene expression Eur. J. Cardiothorac. Surg., October 1, 2002; 22(4): 621 - 625. [Abstract] [Full Text] [PDF]

				G. K. Dy and A. A. Adjei Novel Targets for Lung Cancer Therapy: Part I J. Clin. Oncol., June 15, 2002; 20(12): 2881 - 2894. [Abstract] [Full Text] [PDF]

				P. A. Bunn Jr., B. Helfrich, A. F. Soriano, W. A. Franklin, M. Varella-Garcia, F. R. Hirsch, A. Baron, C. Zeng, and D. C. Chan Expression of Her-2/neu in Human Lung Cancer Cell Lines by Immunohistochemistry and Fluorescence in Situ Hybridization and its Relationship to in Vitro Cytotoxicity by Trastuzumab and Chemotherapeutic Agents Clin. Cancer Res., October 1, 2001; 7(10): 3239 - 3250. [Abstract] [Full Text] [PDF]

				D. M. Nguyen, D. Lorang, G. A. Chen, J. H. Stewart IV, E. Tabibi, and D. S. Schrump Enhancement of paclitaxel-mediated cytotoxicity in lung cancer cells by 17-allylamino geldanamycin: in vitro and in vivo analysis Ann. Thorac. Surg., August 1, 2001; 72(2): 371 - 379. [Abstract] [Full Text] [PDF]

				N.-Y. Hsu, H.-C. Ho, K.-C. Chow, T.-Y. Lin, C.-S. Shih, L.-S. Wang, and C.-M. Tsai Overexpression of Dihydrodiol Dehydrogenase as a Prognostic Marker of Non-Small Cell Lung Cancer Cancer Res., March 1, 2001; 61(6): 2727 - 2731. [Abstract] [Full Text]

				D. M. Nguyen, S. Desai, A. Chen, T. S. Weiser, and D. S. Schrump Modulation of metastasis phenotypes of non-small cell lung cancer cells by 17-allylamino 17-demethoxy geldanamycin Ann. Thorac. Surg., December 1, 2000; 70(6): 1853 - 1860. [Abstract] [Full Text] [PDF]

				L.-S. Wang, K.-C. Chow, W.-Y. Li, C.-C. Liu, Y.-C. Wu, and M.-H. Huang Clinical Significance of Serum Soluble Interleukin 2 Receptor-{{alpha}} in Esophageal Squamous Cell Carcinoma Clin. Cancer Res., April 1, 2000; 6(4): 1445 - 1451. [Abstract] [Full Text]

				D. M. Nguyen, A. Chen, A. Mixon, D. S. Schrump, and S. J. A. Roth SEQUENCE-DEPENDENT ENHANCEMENT OF PACLITAXEL TOXICITY IN NON-SMALL CELL LUNG CANCER BY 17-ALLYLAMINO 17-DEMETHOXYGELDANAMYCIN J. Thorac. Cardiovasc. Surg., November 1, 1999; 118(5): 908 - 915. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Min-Hsiung Huang Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hsieh, C.-C. Articles by Wang, L.-S. Search for Related Content PubMed PubMed Citation Articles by Hsieh, C.-C. Articles by Wang, L.-S.