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doi:10.2478/v10019-008-0014-4 research article Biljana Ilievska Poposka1, Snezana Smickova2, Simonida Jovanovska Crvenkovska2, Beti Zafirova Ivanovska3, Tome Stefanovski4, Gordana Petrusevska5 3Ititute for Lung Diseases and Tuberculosis, 2Ititute for Radiology and Oncology, for Epidemiology, 4Clinic for Pneumology and Allergology, 5Ititute for Pathology, 5Medical Faculty, University of "Sv. Kiril and Metdoij", Skopje, Republic of Macedonia 1Ititute Background. The amplification and the overexpression of the Her-2/neu gene have been shown in certain human tumours and are postulated to be important in human carcinogenesis. In this study we evaluated the expression of HER-2/neu gene in patients with lung carcinoma (LC) and assessed its prognostic significance. Patients and methods. HER-2/neu expression was determined in 127 LC patients using immunohistochemistry (IHC) performed on paraffin-embedded section Hercep TestTM (DAKO). Results. The overall HER-2/neu expression was seen in 36 (28.35%) of 127 LC patients. According to the histological type, HER-2/neu overexpression was detected in 12 patients with adenocarcinomas (60%), in 19 patients with squamous cell carcinomas (31.14%), in 4 patients with small cell-lung carcinomas (10%) and in 1 patient with other carcinomas (16.66%). Only in patients with small cell-lung carcinomas HER-2/ neu overexpression was in correlation with the stage of the disease (p<0.001). The patients with HER-2/neu positive expression were associated with a significantly shorter survival compared with those who were HER-2/neu negative (log rank, p<0.002). Conclusio. These observatio suggest that HER-2/neu positivity may serve as a prognostic indicator in patients with LC. HER-2/neu plays a role in identifying patients at risk for the shortened survival, who may benefit from a more aggressive therapy. Key words: HER-2/neu, lung cancer; immunohistochemisty; survival Received 22 July 2008 Accepted 31 July 2008 Correspondence to: Biljana Ilievska Poposka, M.D., M.Sc, Ititute for Lung Disease and Tuberculosis, Vodnjaka, 17, 1000 Skopje, Macedonia. Phone: +38970 234436; 003892 3147510; Fax: +3892 229166; E-mail: firstname.lastname@example.org Introduction Lung cancer is the leading cause of cancer death worldwide with a still increasing incidence.1,2 Despite new chemotherapeutic drugs, improvement in surgical techniques, histological classification, and staging procedures, lung cancer survival has not greatly improved over the past 20 years. The cure rate remai less than 15%.3 Recent advances in biology and molecular biology identified the relatiohip between specific gene alteratio and clinical behaviour of lung cancers. A number of studies have been performed to assess the prognostic role of tumour-suppressor genes and protooncogenes. HER-2/neu (also known as c-erbB2) oncogene is the second member of the epidermal growth factor of the receptor family.4 The HER-2/neu dominant gene is localized in normal human cells as a singular copy on the long arm (q21) of chromosome 17.5 This gene codes for a 185-kDa receptor-type tyrosine protein kinases (p185neu or c-erbB2), similar to EGF-R.6-8 This 1255 aminoacid tramembrane glycoprotein is composed of there domai: extracellular factor-binding domain, tramembrane domain and intracellular domain with a tyrosine kinase activity.9 When an EGF-like ligand (there is no known specific ligand for HER-2/neu) binds to a receptor of the EGF-R family, (HER-1, 3 or 4) there is a heterodimerisation of this receptor with HER2.10 HER-2 is necessary for the regulation of the normal cell growth and differentiation, and can be associated with multiple signal traduction pathways.11 The amplification of the HER-2 gene leads to an overexpression of the receptor and protein product p 185, which could be implicated in the development of many types of tumours. HER-2/neu is expressed in a wide variety of human epithelial malignancies, including breast, ovary, salivary gland, gastrointestinal tract, prostate, lung, kidney, liver.12-18 This suggests that HER-2/neu overexpression probably plays a critical role in the development and progression of human cancers. The aim of this study was to determine the correlation between HER-2/neu expression and clinicopathological features in patients with lung carcinoma and to assess its relation to the survival. Patients and methods Patients and tissue samples Our study population coisted of 127 patients with lung carcinoma (LC) diagnosed between January 2004 and December 2006 in the Ititute for Lung Diseases and Tuberculosis, Skopje, Macedonia. Patients included 115 men and 12 women. Their age ranged from 37 to 87 years old (mean, 58.49 ± 8.15); 80.10% of them were smokers. The diagnosis of LC was established by the histological examination of tissue samples obtained during the bronchoscopy in 120 patients (94.48%) or by surgery in 7 patients (5.51%). All histological analysis of the tumour tissue and immunohistochemistry (ICH) were performed at the Ititute for Pathology, Medical Faculty, Skopje. The histological type and degree of differentiation were assigned according to World Health Organization criteria.19 Tumours included 40 small-cell lung carcinomas (SCLC) and 87 non-small-cell lung carcinomas (CLC) - 20 adenocarcinomas, 61 squamous cell carcinomas and 6 tumours assigned as "others" (bronchoalveolare, large cell carcinomas, nondifferentiated carcinomas). For the histological differentiation, well moderately and poorly differentiated tumours were graded as grade 1, 2 and 3, respectively. According to the International Staging System for Lung Cancer,20 87 CLC patients were divided in: 3 patients with IB stage (3.44%), 1 patient with IIA stage (1.14%), 11 patients with stage IIB (12.64%), 22 patients with stage IIIA (25.28%), 34 patients with stage IIIB (39.08%) and 16 pa- tients with stage IV (18.39%). The patients with SCLC were staging in 23 patients with limited diseases (57.5%) and 17 patients with exteive diseases (42.5%). One hundred and twenty patients were treated with chemotherapy and/or radiotherapy at the Ititute for Radiotherapy and Oncology, Medical Faculty, Skopje. Seven patients underwent surgery first, and after that they were treated with radiotherapy. All patients were followed up regularly in a time frame of 2 to 3 months. The patients were followed up from 1 to 48 months (median 24 months). At the time of the last follow-up 114 patients (89.76%) had died and 13 patients (10.23%) were still alive. The survival time was calculated from the date of histological diagnosis to the date of death or last follow-up. Immunohistochemistry HER-2/neu oncogene expression was determined by immnunohistochemical staining with Hercep Testtm (DAKO, Copenhagen, Denmark). After bronchoscopy or surgery, fresh tumour tissue specime were immediately formalin fixed. Section 4 m thick were cut from tissue blocks, placed on glass slides and exposed to xylene 3 times for 5 min each. Tissues were hydrated in decreasing concentratio of ethanol (100%, 95% and 70% for 2min each) and ried in distilled water. Slides were washed in PBS (Phosphate Buffer Saline) for 20 min. After excess liquid was blotted off, slides were heated at 120°C in 0.01 M citrate buffer (pH 6.0) to expose the HER2 protein antigen. The endogene peroxidase activity was blocked by Peroxidase Blocking Reagent for 5 min. The rabbit antihuman HER2 protein polyclonal antibody was applied for 2 h at room temperature. Slides were ried with PBS, and peroxidase-conjugated antirabbit antibody was added for 30 min at room temperature. Diaminobenzidine solution was added to achieve specific staining. Slides were counterstained with hematoxylin. The evaluation of HER-2/neu immunoreactivity was performed according to the DAKO protocol for the Hercept Testtm, with minor modificatio. Only membrane staining was coidered positive, whereas cytoplasmatic staining was coidered nopecific. Immunostaging was classified as follows: score 0: no staining at all or membrane staining in <10% neoplastic cells; score 1+: a faint/barely appreciable membrane staining is detected in >10% of the tumour cells; the cells are only stained in part of their membrane; score 2+: a with weak to moderate complete membrane staining is observed in >10% of the tumour cells; score 3+: a strong immunoreactivity of the entire membrane is observed in >10% of the tumours cells. Tumours classified as 0 or 1+ were coidered "negative", and those scored 2+ or 3+ were classified as "positive". Statistical Analysis Statistical significances of the relatiohip between clinicopathological data and IHC were assessed by a chi-square test. The Fisher's exact test was used when the frequency of a cell in a 2 x 2 table was <5. The overall survival rate was calculated using the Kaplan-Meier method. The difference was coidered to be statistically significant at p<0.05. Results Figure 1 shows the staining of HER-2/neu in adenocarcinoma using immunohistochemical methods. The dark brown colour denotes positive staining located predominantly at the cell membrane. Quantitative data are shown in Table 1, which summarizes expressio of HER-2/neu Table 1. The relatiohip between the expression of the HER-2/neu and clinicopathological features in patients with lung cancer Features (n) Sex Male (115) Female (12) Age 65 (90) > 65 (37) Smoking history Smoker (103) Nomoker (24) Performa status WHO 0 (27) WHO 1 (79) WHO 2 (11) WHO 3 (9) WHO 4 (1) Tumour type SCLC (40) CLC (87) Differentiation Well (3) Moderate (55) Poorly (69) Stage (CLC) I B (3) II A (1) II B (11) III A (22) III B (34) IV (16) Stage (SCLC) Limited (23) Exteive (17) Total (127) HER-2/neu + n (%) 34 (29.56%) 2 (16.66%) 25 (27.77%) 11 (29.72%) 27 (26.21%) 9 (37.5%) 2 (7.40%) 26 (32.91%) 2 (18.18%) 6 (66.66%) 0 (0%) 4 (10.0 %) 32 (36.78 %) 2 (66.66 %) 14 (25.45 %) 20 (28.98 %) 0 (0 %) 0 (0%) 4 (36.36 %) 7 (31.81 %) 11 (32.35 %) 10 (62.5 %) 0 (0 %) 4 (23.52 %) 36 (28.35%) p Value p<0.006 p<0.001 p<0.001 Note: statistical analysis was performed using x² test and Fisher exact test in 127 LC patients with different clinicopathological features. HER-2/neu immunoreactivity (3+/2+) was detected in 36 of 127 lung cancers cases (28.35%). Fifteen tumours (11.81%) showed strong HER-2/neu immunoreactivity (3+), while twenty one (16.54%) tumours were moderately immunoreactive (2+). Ninety-one cases (71.65 %) were coidered negative; 81 cases were classified as score 0 (63.78 %) and 10 cases as score 1+ (7.87%). As seen in Table 1, no statistically significant correlation was found between the frequency of HER-2/neu protein product expression and some clinicopathological Figure 1. Immunohistochemical staining of HER-2/neu overexpression in lung adenocarcinoma (strong staining 3+). features under the study including sex, age, smoking history and tumour differentiation (well, moderately and poorly). We found the correlation between HER-2/neu expression and performance status (x²=14.1; p<0.006). We also obtained the significant differences in HER-2/neu expression between SCLC and CLC (p<0.001). Among the CLC's HER-2/neu expression was found in 12 of 20 (60%) adenocarcinomas, in 19 of 61 (31.14%) squamous cell carcinomas, and in 1 of 6 (16.66%) "other" carcinomas (Table 2). The difference in the expression rate between the adenocarcinomas and the other groups was highly significant (x²=17.13; p<0.0001). Eight patients (66%) with HER2/neu positive adenocarcinomas showed strong immunoreactivity (3+). In CLC patients HER-2/neu expression was not in correlation with the stage of the diseases. But, there was a significant difference in HER-2/neu expression between SCLC patients with limited and exteive diseases (Fisher exact test p<0.001). We found that the expression of HER-2/ neu protein was associated with a signifi- cant short survival in LC patients. The 24months survival rates of patients with HER2/neu overexpresssion and those without were 21.97% and 2.77%, respectively (Figure 2) with a statistical significant difference (log-rank Test = - 3.06; p=0.002). Discussion Since HER-2/neu proto-oncogene has been originally identified, its overexpression was detected in various types of cancers, first of all in those with epithelial origin.21 HER-2/neu expression is associated mainly with high-grade breast carcinoma and coidered an important prognostic factor for adverse outcome in node positive breast cancer.13 In lung cancer studies, HER-2/neu expression varies depending on the histological classification of the tumours.4,10,22-24 Her-2/neu expression was reported in 1380% in adenocarcinomas,17,22,24 in 2-45% in squamous cell carcinomas,15,16,18 in 0-20% in large-cell carcinomas23 and in 13-31% in small cell lung carcinomas.4,25,26 Table 2. HER-2/neu expression in different histological type of lung cancer Histological type SCLC Squamous cell cacinoma Adenocarcinoma Others No of cases Level of expression* +++ ++ 40 2 2 61 5 14 20 8 4 6 0 1 + 2 3 5 0 - 34 39 3 5 % positive 10.0 31.14 60.0 16.6 * -: no detectable HER-2/neu expression; +, ++, +++: faint, weak and strong expression, respectively We observed HER-2/neu overexpression in 36 of 127 patients with lung carcinoma (28.25%). The overexpression was more frequent in CLC (36.78 %) than in the SCLC (10.0%). The patients with adenocarcinoma showed the highest incidence of positive findings (60%), with the biggest number of strong reactivity patter (53.33%). This observation concurs with the findings of Shi et al., Harpole et al., Pellegriniet et al., Hsieh et al. and others.8,17,27,28 The techniques used to detect HER-2/ neu expression might be one of the potential sources of biases. HER-2/neu protein overexpression is most often measured by IHC using one of several monoclonal antibodies, and the gene expression is measured most often by fluorescence in situ hybridization (FISH) for clinical studies.5 Immunohistochemical results can vary according to the primary used antibody, dilution of the antibody and tissue coervation. Moreover, this technique is semiquantitative and standards for positive or negative specimen vary between studies making difficult the direct comparison of studies. In this study we did not find any correlation between HER-2/neu overexpression and the pathologic stage of disease in patients with CLC. Even our results showed very low HER-2/neu overexpression in patients with SCLC, we found significant differences of HER-2/neu overexpression between SCLC patients with limited and exteive diseases: HER-2/neu overexpression was seen only in patients with the exteive disease (Fisher exact test p<0.05). Tateishi et al.22 reported a higher positivity for HER2/neu in Stage III-IV disease with a poor influence on the prognosis. Similarly, Osaki et al.29 reported increased serum levels of HER-2/neu in Stage IIIB cases. However, the comparison among different studies is difficult due to a coistent lack of balance in the histotype and the stage distribution from one study to another. Although HER-2/neu protein expression is widely studied in lung carcinoma, its prognostic role remai uncertain. Even several investigators reported that HER-2/ neu immunostaining adversely affects the prognosis and the survival in LC patients, especially CLC patients,17,18,22,23,30 some large studies did not find any prognostic implication for HER-2/neu overexpression.31-33 There are many studies showing adverse outcomes among patients with breast and ovarian cancers overexpressing this oncogene. Besides, the overall survival rate and time to relapse for those patients with HER-2/neu overexpression is shorter than those lacking the overexpression.13,21,34 Our results show that patients with HER-2/neu positive tumours had a significantly decreased survival opposite to patients with HER-2/neu negative tumours. After 24 months, only one patient from the group who were HER2/neu positive was still alive, in comparison with 20 patients from the group who were HER-2/neu negative. There are studies which have shown that HER-2/neu overexpression is associated with intriic chemoresistance of CLC.35 Complete 1,0 0,9 0,8 Cumulati ve Proporti on Surviving 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 -0,1 0 5 10 Ceored Group 0, Group 1, 3. Travis WD, Lubin J, Ries L, Devesa S. United States lung carcinoma incidence trends: declining for the most histologic types among males, increasing among females. Cancer 1996; 77: 2464-70. 4. Potti A, Willardson J, Forseen C, Ganti AK, Koch M, Hebert B, et al. Predictive role of HER-2/neu overexpression and clinical features an initial presentation in patients with exteive stage small cell lung carcinoma. Lung Cancer 2002; 36: 257-61. 5. Klijanienko J, Couturier J, Galut M, El-Naggar A, Maciorowski Z, Padoy E, et al. Detection and quantitation by fluorescence in situ hybridization (FISH) and image analysis of Her-2/neu gene amplification in breast cancer fine-needle samples. Cancer 1999; 87: 312-8. 6. Carraway KL 3rd, Cantley LC. A neu acquaintance for ErbB3 and ErbB4: a role for receptor heterodimerization in growth signaling. Cell 1944; 78: 5-8. 7. Fernandes A, Hamburger AW, Gerwin BI. ErbB-2 kinase is required for cotitutive STAT 3 activation in malignant human lung epithelial cells. Int J Cancer 1999; 83: 564-70. 8. Shi D, He G, Cao S, Pan W, Zhang HZ, Yu D, et al. Overexpression of the c-erbB-2/neu-encoded p185 protein in primary lung cancer. Mol Carcinog 1992; 5: 213-8. 9. 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; 42: 1644-6. 10. Meert AP, Martin B, Paesma M, Berghma T, Mascaux C, Verdebout J-M, et al. The role of HER2/neu expression on the survival of patients with lung cancer: a systematic review of the literature. Br J Cancer 2003; 89: 959-65. 11. Hung MC, Lau YK. Basic science of HER-2/neu: a review. Semin Oncol 1999; 26(Suppl 12): 51-9. 12. Brodowicz T, Wiltschke C, Budiky AC, Krainer M, Steger GG, Zieliki CC. Soluble HER-2/neu neutralizes biologic effects of anti-HER/2neu antibody on breast cancer cells in vitro. Int J Cancer 1997; 73: 875-9. 13. Wright C, Angus B, Nisholson S, Saibury JR, Cair JC, Gullick WJ, et al. Expression of the c-erbB-2 oncoprotein: a prognostic indicator in human breast cancer. Cancer Res 1989; 49: 2087-90. 14. Jing X, Kakudo K, Murakami M, Nakamura Y, Nakamura M, Yokoi T, et al. Intraductal spread of invasive breast carcinoma has a positive correlation with c-erb B-2 overexpression and vascular invasion. Cancer 1999; 86: 439-48. 15 Time Figure 2. 24-month survival of a patient with lung cancer as a function of absence (solid line) or presence (dashed line) of HER-2/neu overexpression. Thus, HER-2/neu overexpression may play a role as a predictive factor of respoe to therapy in patients with LC, who may be candidates for adjuvant therapeutic modalities and the gene therapy possibly improving the overall survival. Conclusio In conclusion, HER-2/neu expression detected in LC is a negative predictor of the survival. Immunohistochemical stainig of Her-2/neu may aid in defining a subpopulation of patients with LC whose tumours may behave more aggressively. In addition, focused adjuvant therapeutic modalities and gene therapies might improve the overall survival in these patients expressing the poor prognostic immunohistochemical marker.
Radiology and Oncology – de Gruyter
Published: Sep 1, 2008
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