Gastrin-Releasing Peptide Receptor Expression in Lung Cancer Jane Mattei, MD, PhD; Rosane D. Achcar, MD; Carlos H. Cano, MSc; Bruno R. Macedo, MD; Luise Meurer, MD, PhD; Brenda S. Batlle; Steve D. Groshong, MD, PhD; Jane M. Kulczynski, MD, PhD; Rafael Roesler, PhD; Lissandra Dal Lago, MD, PhD; Andre T. Brunetto, MD; Gilberto Schwartsmann, MD, PhD
Context.—Gastrin-releasing peptide receptors (GRPRs) activate mitogen-activated protein kinase signaling pathway primarily through epidermal growth factor receptor activation and are under investigation as a molecular target because they are overexpressed in several solid tumors. Objective.—To determine GRPR expression in both non–small cell lung carcinoma and small cell lung carcinoma, comparing results with clinical stages and demographic data. Design.—We analyzed the immunohistochemical expression of GRPR in 200 non–small cell lung carcinoma and 38 small cell lung carcinoma archival cases from 2004 to 2008. Results.—Non–small cell lung carcinoma cases tended to be higher GRPR expressers at a rate of 62.5% (weak, moderate, and strong expression in 41.5%, 13.5%, and 7.5%, respectively), compared with 52.62% in small cell
lung carcinoma cases (weak, moderate, and strong expression in 34.21%, 15.78%, and 2.63%, respectively; P ¼ .30). In non–small cell lung carcinoma there was a trend for higher percentages of strong expression in adenocarcinoma cases (10%; P ¼ .67), and in patients with advanced stages (III and IV; 9.43% and 6.9%; P ¼ .01). Conclusions.—To the best of our knowledge, this is the first study to demonstrate GRPR tissue expression in a large population of patients with lung cancer. Although GRPR expression was similar in small cell and non–small cell carcinoma, the expression was more pronounced in an advanced-stage lung cancer, particularly in adenocarcinoma cases, and may represent a potential target for the development of new treatment approaches in this population. (Arch Pathol Lab Med. 2014;138:98–104; doi: 10.5858/ arpa.2012-0679-OA)
L
new therapeutic targets are needed to improve survival rates.3,4 Gastrin-releasing peptide (GRP), a bombesin-like peptide growth factor, is expressed by pulmonary neuroendocrine cells and has been shown to stimulate lung development in utero and to increase growth maturation of human fetal lung organ cultures.5,6 It is also a potent mitogen for normal and neoplastic tissues, and it may be involved in growth dysregulation and carcinogenesis.7,8 The effects of GRP are primarily mediated through binding to its receptor, GRP receptor (GRPR), which is a G protein–coupled receptor originally isolated from a small cell lung cancer cell line.6 Upregulation of GRP/GRPR has been reported in several cancers, including prostate, glioma, colon, head and neck, and lung cancer.9–12 It has been proposed that GRP stimulation results in increased release of epidermal growth factor receptor (EGFR) proligands, which is metalloprotease dependent and results in activation of EGFR and mitogen-activated protein kinase downstream pathways13,14 (Figure 1). Moreover, it has been demonstrated that abrogation of EGFR by EGFR-specific tyrosine kinase inhibitors also blocks GRPmediated mitogen-activated protein kinase activation, indicating that GRP activates mitogen-activated protein kinase primarily through EGFR.15 Recent advances using non– small cell lung cancer (NSCLC) cell lines have confirmed that EGF and GRP both stimulate NSCLC proliferation, and
ung cancer is the leading cause of global cancer death and is responsible for more than 1 million deaths per year.1 At diagnosis, approximately 75% of lung cancer patients exhibit locally advanced or metastatic disease.2 Despite recent advances in understanding the biology of lung cancer, the 5-year survival rates of patients suffering from lung cancer remain low at less than 15%; therefore, Accepted for publication April 23, 2014. From the Departments of Internal Medicine (Dr Mattei) and Pathology (Drs Meurer and Kulczynski), School of Medicine, the Laboratory of Molecular Neuropharmacology, Department of Pharmacology, Institute for Basic Health Sciences (Dr Roesler), and the Cancer Research Laboratory, University Hospital Research Center (CPE-HCPA) (Dr Macedo, Roesler, Brunetto, and Schwartsmann), Federal University of Rio Grande do Sul, Porto Alegre, Brazil; the Departments of Urology (Dr Mattei) and Oncology (Mr Cano and Ms Batlle), University of Colorado, Denver; the Pathology Division, Department of Medicine, National Jewish Health, Denver, Colorado (Drs Achcar and Groshong); the National Institutes for Translational Medicine, Porto Alegre, Brazil (Drs Roesler and Schwartsmann); and the Department of Medical Oncology, Institut Jules Bordet, Brussels, Belgium (Dr Dal Lago). The authors have no relevant financial interest in the products or companies described in this article. Presented as a poster at the American Society of Clinical Oncology meeting; June 2011; Chicago, Illinois. Reprints: Jane Mattei, MD, PhD, Denver Health Hospital, 777 Bannock St, Denver, CO 80204 (e-mail: [email protected]). 98 Arch Pathol Lab Med—Vol 138, January 2014
Lung Cancer; Gastrin-Releasing Peptide Receptor—Mattei et al
inhibition of either EGFR or GRPR resulted in cell death. In addition, combining a GRPR antagonist with the EGFR tyrosine kinase inhibitors resulted in additive cytotoxic effects.14,16 To further investigate the possible utility of GRPR as a future therapeutic target in lung cancer, we evaluated the expression of GRPR in small cell lung carcinoma (SCLC) and NSCLC. The results were correlated with sex, clinical stage, smoking status, and survival. To our knowledge, we have the largest number of SCLCs and NSCLCs evaluated for GRPR tissue expression. MATERIALS AND METHODS Population
Figure 1. Gastrin-releasing peptide (GRP)/GRP receptor (GRPR) signaling pathway. When GRP activates GRPR, inactive guanosine diphosphate (GDP)–bound G protein is activated to a guanosine triphosphate (GTP)–bound state, resulting in metalloprotease-dependent increased release of epidermal growth factor receptor (EGFR) proligands, subsequent phosphorylation of EGFR, and activation of RAS/RAF/mitogen-activated protein kinase (MAPK) downstream pathway.
We analyzed the immunohistochemical expression of GRPR in 238 lung cancer archival cases selected from the department of pathology files of Hospital das Clinicas from the University of Rio Grande do Sul (Porto Alegre, Brazil), based on the availability of formalin-fixed, paraffin-embedded tissue blocks from 2004 to 2008. The specimens were obtained from patients with adenocarcinoma (n ¼ 100), squamous cell carcinoma (n ¼ 71), large cell carcinoma (n ¼ 29), and small cell carcinoma (n ¼ 38). Results were correlated with tumor cell type, sex, tumor stage (I–IV in NSCLC cases and limited and extensive disease in SCLC cases), smoking status (smoker versus nonsmoker), and survival (date of diagnosis to date of death or date of last hospital follow-up visit).
Figure 2. Membranous and cytoplasmic immunolocalization of gastrin-releasing peptide receptor in non–small cell lung carcinoma tissue. A, Absent expression. B, Weak expression. C, Moderate expression. D, Strong expression (original magnifications 340). Arch Pathol Lab Med—Vol 138, January 2014
Lung Cancer; Gastrin-Releasing Peptide Receptor—Mattei et al 99
Table 1.
Gastrin-Releasing Peptide Receptor Immunohistochemical Analysis Correlating Percentage and Intensity of Tumor Cell Expressiona % Stained Tumor Cells 1–25
a
Cell Type
Negative
Adenocarcinoma (n ¼ 100) Squamous (n ¼ 71) Large cell (n ¼ 29) Small cell (n ¼ 38)
32 31 12 18
1þ 17 7 6 5
25–50
50–100
2þ
3þ
1þ
2þ
3þ
1þ
2þ
3þ
0 0 0 0
0 0 0 0
12 7 3 4
2 1 1 0
0 0 0 0
13 15 3 5
14 7 2 5
10 3 2 1
Immunostaining and measurement of stain were performed as described in ‘‘Materials and Methods.’’
Immunohistochemistry Technique Immunohistochemical stain for GRPR (rabbit polyclonal antibody, OPA1-15619, 1:200 dilution; Affinity Bioreagents, Golden, Colorado) was performed on formalin-fixed, paraffin-embedded tissue blocks from all cases sectioned at 4-lm thickness. After dewaxing, inactivating endogenous peroxidase activity, and blocking cross-reaction with normal serum, sections were incubated overnight at 48C with the primary antibody diluted solution. Secondary detection was achieved by subsequent application of biotinylated antibody, streptavidin horseradish peroxidase conjugate (LSAB, Dako, Carpinteria, California) and diaminobenzidine tetrahydrochloride/H2O2 (Kit DAB, Dako). Images were obtained with an Aperio scanscope (Scanscope Aperio Technologies, Inc, Vista, California) connected to a personal computer HP xw 4300 Workstation (Hewlett-Packard Development Co, LP, Palo Alto, California). Representative photographs were taken to illustrate the findings.
Immunohistochemistry Evaluation Double-blinded histopathology review was performed in tissue from archival cases of NSCLC (n ¼ 200) and SCLC (n ¼ 38). All tumor cases had one slide of tissue available for review. Five entire high-power fields (340) containing clusters of malignant cells were identified per slide and scored for intensity and percentage of GRPR staining expression. The mean of the 5 intensities and percentages for each power field was recorded. The mean values per case were averaged and calculated for each diagnostic group. Intensity of tumor cells’ positive staining was recorded on a scale of 0 to 3 (0 ¼ absent expression; 1 ¼ mild expression; 2 ¼ moderate expression; and 3 ¼ intense expression; Figure 2, A through D). Percentage of stained tumor cells was recorded on a scale of 0 to 3 (0 ¼ absent expression; 1 ¼ less than
25% of cells; 2 ¼ 25%–50% of cells; 3 ¼ 50%–100% of cells; Table 1). Gastrin-releasing peptide receptor antibody–stained nonneoplastic pancreatic tissue was used as positive control.
Statistical Analysis The significance of differences observed between the groups was calculated using SPSS version 19.0 (SPSS Statistics for Windows, Version 19.0, IBM Corporation, Armonk, New York.) Correlations between GRPR expression and clinicopathologic variables were analyzed by using the Pearson v2 test and Fisher exact test. Overall survival was calculated by the Kaplan-Meier method and differences between groups were assessed by the log-rank test and Fisher exact test. Patients were censored for survival at date of death or date of last hospital follow-up visit. A multivariate analysis using the Cox proportional hazards model was also performed in order to adjust for the effects of age, sex, and stage of disease. Statistical differences were accepted at P , .05 and were 2-sided. The study proposal was reviewed and approved by institutional review board and audit committees from University of Rio Grande do Sul (Porto Alegre, Brazil).
RESULTS Patient Demographics The tumor specimens were obtained from adenocarcinoma (n ¼ 100), squamous cell carcinoma (n ¼ 71), large cell carcinoma (n ¼ 29), and small cell carcinoma (n ¼ 38). The male to female ratio was 1.2:1. Review of the available data showed that 200 patients (84.03%) were smokers, 8 (3.36%) were nonsmokers, and 30 (12.61%) had unknown smoking status. Of NSCLC patients, 37 (15.54%) had stage I or II
Figure 3. Gastrin-releasing peptide receptor (GRPR) expression in non–small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) and clinical stage (I, II, III, and IV for NSCLC and extensive disease [ED] for SCLC). A significant association (P ¼ .01) with more pronounced intensity of GRPR expression and advanced clinical stages was identified.
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Table 2. Non–Small Cell Lung Carcinoma and Small Cell Lung Carcinoma Clinical Data Correlating Sex, Age at Diagnosis, Smoking Status, Cell Type, Stage of Disease, and Survival Clinical Data Sex, No. (%) Female Male Age at diagnosis, mean, y Female Male
106 (44.9) 132 (55.1) 63.08 63.44 61.08
Smoking status Smoker Never smoker Unknown
200 (84.03) 8 (3.36) 30 (12.6)
Cell type Non–small cell lung cancer Adenocarcinoma Squamous Large cell Small cell lung cancer
200 100 71 29 38
(84.03) (42.01) (29.83) (12.18) (15.96)
Disease stage I and II III IV Limited disease Extensive disease Unknown
37 53 110 3 33 2
(15.54) (22.26) (46.21) (1.26) (13.86) (0.84)
Survival, mo Female Male
10.3 10.65 10.02
disease, 53 (22.26%) stage III, and 110 (46.21%) stage IV. Of SCLC patients, 3 (1.26%) had limited disease, 33 (13.86%) had extensive disease, and 2 (0.84%) had unknown disease extension. The average survival was 10.65 months in the female population and 10.02 months in the male popula-
tion, with an overall survival rate of 10.3 months. Clinical data are summarized in Table 2. GRPR Expression and Clinical Stage Absence of GRPR expression was more prominent in early stages I and II (62.16%) compared with patients with advanced disease (34% for clinical stage III, 32.35% for clinical stage IV, and 43.24% for extensive disease). Moderate expression was more conspicuous in clinical stage IV (18.63%) and extensive disease (16.21%). Strong expression was more pronounced in clinical stages III (9.43%), IV (6.9%), and extensive disease (5.4%). There was significant association (P ¼ .01) between strong intensity of GRPR expression and higher clinical stage, independent of tumor cell type (Figure 3). It should be noted that because we had only 3 cases with limited disease, this population may not be representative. GRPR Expression and Cell Type Although the overall GRPR percentage of cell expression was similar between NSCLC (62.51%) and SCLC (52.62%; P ¼ .22), the intensity of GRPR expression tended to be more pronounced in NSCLC (41.5% weak, 13.5% moderate, 7.5% strong) compared with SCLC (34.21% weak, 15.78% moderate, 2.63% strong; P ¼ .30; Figure 4). Similar weak expression of GRPR was seen among all NSCLC cell types, ranging from 40.84% to 42%. Moderate GRPR expression was seen in 16% of adenocarcinomas, 11.26% of squamous cell carcinomas, and 10.34% of large cell carcinomas. Strong GRPR expression was seen in 10% of adenocarcinomas, 4.22% of squamous cell carcinomas, and 6.89% of large cell carcinomas (P ¼ .67). Adenocarcinoma cases tended to be stronger GRPR expressers (Figure 5). GRPR Expression and Sex and Smoking Status Distribution of GRPR expression was comparable between male (weak 41.66%, moderate 12.87%, and strong 6.06%) and female subjects (weak 38.67%, Figure 4. Gastrin-releasing peptide receptor (GRPR) expression in non–small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC). There was no significant difference in GRPR expression between NSCLC and SCLC.
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Figure 5. Gastrin-releasing peptide receptor (GRPR) expression in non–small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC). There was no significant difference in GRPR expression among cases of adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and small cell carcinoma. Adenocarcinoma cases tended to be GRPR overexpressers.
moderate 15.09%, and strong 7.54%). The absence of GRPR was seen in 39.39% of male and 38.67% of female subjects. The differences in GRPR expression between male and female subjects were not statistically significant (P ¼ .91; Figure 6). Among 238 subjects, 200 were smokers, 8 were nonsmokers, and 30 had unknown smoking status. Because of the low number of nonsmoker subjects, it was not possible to correlate GRPR expression with smoking status. GRPR Expression and Survival Two hundred thirty-eight patients were evaluated for overall survival until mortality or the date of the last followup visit if the patients were still alive, with an average follow-up of 15.2 months. The median overall survival time was 8.97 months (95% confidence interval, 5.6–12.3 months) for absent GRPR expression, 11.50 months (95% confidence interval, 3.6–19.3 months) for weak and moderate expression, and 10.6 months (95% confidence interval, 0.4–20.7 months) for strong GRPR expressers (P ¼ .77). The Kaplan-Meier curve was adjusted for clinical stage (Figure 7).
Figure 6. Gastrin-releasing peptide receptor (GRPR) expression in males and females showing no detectable difference in GRPR expression. 102 Arch Pathol Lab Med—Vol 138, January 2014
COMMENT It has been demonstrated that GRPR activates mitogenactivated protein kinase signaling pathway primarily through EGFR activation and that combining GRPR antagonist with EGFR tyrosine kinase inhibitors results in additive cytotoxic effect in tumor cell lines.13 Gastrinreleasing peptide receptor synthetic antagonists, such as RC3095, have been developed as anticancer candidates, and have shown antitumor activity in both in vivo and in vitro murine and human tumor models, producing long-lasting tumor regression.17 In 25 patients with advanced refractory solid malignancies enrolled in a phase I trial of RC-3095 conducted at Hospital das Clinical, in Porto Alegre, Brazil, RC-3095 was well tolerated.18 Gastrin-releasing peptide receptor was originally isolated in SCLC by Cuttitta et al,19 and most previous studies have reported GRPR expression in up to 70% of small cell lung cancer and 10% to 20% in NSCLC.20 Conversely, in our study we noted GRPR expression in NSCLC and SCLC at similar rates of 62.51% and 52.62% respectively (P ¼ .30). Among NSCLC cases, adenocarcinoma subjects tended to be GRPR overexpressers at a rate of 68% (41.5% weak expression, 13.5% moderate expression, and 7.5% strong expression) compared with other NSCLC cases (P ¼ .67). Although we acknowledge that the differences seen in our study were not statistically significant, this pattern of GRPR upregulation may indicate that GRPR represents a future target for adjuvant therapy in lung cancer, particularly in adenocarcinoma cases. The association between GRPR expression, smoking status, and sex is not clear. Some studies suggest that the GRPR gene is expressed more frequently in females than in males in the absence of smoking and that expression is activated earlier in females in response to tobacco exposure.21 On the other hand, GRP expression has also been reported to be increased with tobacco use. Higher levels of GRP protein have been detected in bronchoalveolar lavage and urine specimens of asymptomatic long-standing smokers when compared with nonsmoking control subjects.22 Although our study is limited with respect to smoking status because of a low number of nonsmoker subjects, we noticed no difference in GRPR expression between males and females, suggesting that GRPR plays a similar role in development of lung cancer independent of sex. Gastrin-releasing peptide receptor expression was noted in bronchial epithelium and submucosal gland epithelial cells (not quantified) from small cell and non–small cell Lung Cancer; Gastrin-Releasing Peptide Receptor—Mattei et al
Figure 7. Overall survival of lung cancer and gastrin-releasing peptide receptor (GRPR) expressers calculated using the Kaplan-Meier survival curves. There was no significant correlation with survival and GRPR expression.
carcinoma cases. The role of extratumoral GRPR expression is unclear. However, Egloff and colleagues23 evaluated GRPR mRNA levels in histologically normal bronchial epithelial cells from 224 lung cancer patients and 107 surgical cancer-free controls and demonstrated that GRPR expression in noncancerous bronchial epithelium was significantly associated with lung cancer independent of age, sex, or smoking status. Egloff et al23 showed a 71% rate of GRPR expression in nonneoplastic bronchial cells from lung cancer samples compared with only 38% of cancer-free controls, suggesting that increased GRPR expression in normal epithelial mucosa may be an early event in lung cancer formation. Gastrin-releasing peptide receptor overall percentage and intensity of tumor cell expression staining were more pronounced in clinical stages III and IV and extensive disease compared with GRPR expression in clinical stages I and II. These differences were statistically significant (P ¼ .01) and were independent of tumor cell type, sex, and survival. The impact and implications of increased GRPR expression in patients with advanced lung disease deserve future studies and may indicate that GRPR may be a potential therapeutic target in this population. To our knowledge, this study is the first to correlate GRPR tissue expression and distribution with the clinical stages of lung cancer in a large small cell and non–small cell population. We detected GRPR expression in NSCLC and SCLC at similar rates. Adenocarcinomas and advanced lung cancer disease subjects tended to be GRPR overexpressers, suggesting that GRPR may be a potential future therapeutic target in this population. The GRPR pathway is known to interact with the EGFR pathway in lung cancer cells by increasing the release of EFGR proligands, which could act Arch Pathol Lab Med—Vol 138, January 2014
to further promote cancer in patients who develop EGFR mutations. Molecular profiling of lung cancer patients with GRPR upregulation deserves future study. References 1. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2008. CA Cancer J Clin. 2008;58(2):71–96. 2. Pass HI, Carbone DP, Johnson DH, Minna JD, Scagliotti GV. Principles and Practice of Lung Cancer: The Official Reference Text of the International Association for the Study of Lung Cancer (IASLC). 4th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2010. 3. Sandler A, Gray R, Perry MC, et al. Paclitaxel-carboplatin alone or with bevacizumab for non–small-cell lung cancer. N Engl J Med. 2006;355(24):2542. 4. Pirker R, Pereira JR, von Pawel J, et al. EGFR expression as a predictor of survival for first-line chemotherapy plus cetuximab in patients with advanced non–small-cell lung cancer: analysis of data from the phase 3 FLEX study. Lancet Oncol. 2012;13(1):33–42. 5. Shan L, Emanuel RL, Dewald D, et al. Bombesin-like peptide receptor gene expression, regulation, and function in fetal murine lung. Am J Physiol Lung Cell Mol Physiol. 2004;286(1):L165–L173. 6. Sunday ME, Choi N, Spindel ER, Chin WW, Mark EJ. Gastrin-releasing peptide gene expression in small cell and large cell undifferentiated lung carcinomas. Hum Pathol. 1991;22(10):1030–1039. 7. Patel O, Shulkes A, Baldwin GS. Gastrin-releasing peptide and cancer. Biochim Biophys Acta. 2006;1766(1):23–41. 8. Weber HC. Regulation and signaling of human bombesin receptors and their biological effects. Curr Opin Endocrinol Diabetes Obes. 2009;16(1):66–71. 9. Constantinides C, Lazaris AC, Haritopoulos KN, Pantazopoulos D, Chrisofos M, Giannopoulos A. Immunohistochemical detection of gastrin releasing peptide in patients with prostate cancer. World J Urol. 2003;21(3):183–187. 10. Flores DG, Meurer L, Uberti AF, et al. Gastrin-releasing peptide receptor content in human glioma and normal brain. Brain Res Bull. 2010;82(1–2):95–98. 11. Rivera CA, Ahlberg NC, Taglia L, Kumar M, Blunier A, Benya RV. Expression of GRP and its receptor is associated with improved survival in patients with colon cancer. Clin Exp Metastasis. 2009;26(7):663–671. 12. Lango MN, Dyer KF, Yan VW, et al. Gastrin-releasing peptide receptormediated autocrine growth in squamous cell carcinoma of the head and neck. J Natl Cancer Inst. 2002;94(5):375–383. 13. Thomas SM, Grandis JR, Wentzel AL, Gooding WE, Yan VW, Siegfried JM. Gastrin-releasing peptide receptor mediates activation of the epidermal growth factor receptor in lung cancer cells. Neoplasia. 2005;7(4):426–431.
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14. Zhang Q, Bhola NE, Yan VW, et al. Antitumor mechanisms of combined gastrin-releasing peptide receptor and epidermal growth factor receptor targeting in head and neck cancer. Mol Cancer Ther. 2007;6(4):1414–1424. 15. Liu X, Carlisle DL, Swick MC, Gaither-Davis A, Grandis JR, Siegfried JM. Gastrin-releasing peptide activates Akt through the epidermal growth factor receptor pathway and abrogates the effect of gefitinib. Exp Cell Res. 2007;313(7): 1361–1372. 16. Thatcher N, Chang A, Parikh P, et al. Gefitinib plus best supportive care in previously treated patients with refractory advanced non–small-cell lung cancer: results from a randomised, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer). Lancet. 2005;366(9496):1527–1537. 17. Qin Y, Ertl T, Cai RZ, Halmos G, Sehally AV. Inhibitory effect of bombesin receptor antagonist RC-3095 on the growth of human pancreatic cancer cells in vivo and in vitro. Cancer Res. 54:1035–1041. 18. Schwartsmann G, DiLeone LP, Horowitz M, et al. A phase I trial of the bombesin/gastrin-releasing peptide (BN/GRP) antagonist RC3095 in patients with advanced solid malignancies. Invest New Drugs. 2006;24(5):403–412.
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19. Cuttitta F, Carney DN, Mulshine J, et al. Bombesin-like peptides can function as autocrine growth factors in human small-cell lung cancer. Nature. 1985;316(6031):823–826. 20. Moody TW, Pert CB, Gazdar AF, Carney DN, Minna JD. High levels of intracellular bombesin characterize human small-cell lung carcinoma. Science. 1981;214(4526):1246. 21. Shriver SP, Bourdeau HA, Gubish CT, et al. Sex-specific expression of gastrin-releasing peptide receptor: relationship to smoking history and risk of lung cancer. J Natl Cancer Inst. 2000;92(1):24–33. 22. Aguayo SM, Kane MA, TE King J, Schwarz MI, Grauer L, Miller YE. Increased levels of bombesin-like peptides in the lower respiratory tract of asymptomatic cigarette smokers. J Clin Invest. 1989;84(4):1105. 23. Egloff AM, Davis AG, Shuai Y, et al. Gastrin-releasing peptide receptor expression in non-cancerous bronchial epithelia is associated with lung cancer: a case control study. Respir Res. 2012;13 (1):9.
Lung Cancer; Gastrin-Releasing Peptide Receptor—Mattei et al
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Context.—Gastrin-releasing peptide receptors (GRPRs) activate mitogen-activated protein kinase signaling pathway primarily through epidermal growth factor receptor activation and are under investigation as a molecular target because they are overexpressed in several solid tumors. Objective.—To determine GRPR expression in both non–small cell lung carcinoma and small cell lung carcinoma, comparing results with clinical stages and demographic data. Design.—We analyzed the immunohistochemical expression of GRPR in 200 non–small cell lung carcinoma and 38 small cell lung carcinoma archival cases from 2004 to 2008. Results.—Non–small cell lung carcinoma cases tended to be higher GRPR expressers at a rate of 62.5% (weak, moderate, and strong expression in 41.5%, 13.5%, and 7.5%, respectively), compared with 52.62% in small cell
lung carcinoma cases (weak, moderate, and strong expression in 34.21%, 15.78%, and 2.63%, respectively; P ¼ .30). In non–small cell lung carcinoma there was a trend for higher percentages of strong expression in adenocarcinoma cases (10%; P ¼ .67), and in patients with advanced stages (III and IV; 9.43% and 6.9%; P ¼ .01). Conclusions.—To the best of our knowledge, this is the first study to demonstrate GRPR tissue expression in a large population of patients with lung cancer. Although GRPR expression was similar in small cell and non–small cell carcinoma, the expression was more pronounced in an advanced-stage lung cancer, particularly in adenocarcinoma cases, and may represent a potential target for the development of new treatment approaches in this population. (Arch Pathol Lab Med. 2014;138:98–104; doi: 10.5858/ arpa.2012-0679-OA)
L
new therapeutic targets are needed to improve survival rates.3,4 Gastrin-releasing peptide (GRP), a bombesin-like peptide growth factor, is expressed by pulmonary neuroendocrine cells and has been shown to stimulate lung development in utero and to increase growth maturation of human fetal lung organ cultures.5,6 It is also a potent mitogen for normal and neoplastic tissues, and it may be involved in growth dysregulation and carcinogenesis.7,8 The effects of GRP are primarily mediated through binding to its receptor, GRP receptor (GRPR), which is a G protein–coupled receptor originally isolated from a small cell lung cancer cell line.6 Upregulation of GRP/GRPR has been reported in several cancers, including prostate, glioma, colon, head and neck, and lung cancer.9–12 It has been proposed that GRP stimulation results in increased release of epidermal growth factor receptor (EGFR) proligands, which is metalloprotease dependent and results in activation of EGFR and mitogen-activated protein kinase downstream pathways13,14 (Figure 1). Moreover, it has been demonstrated that abrogation of EGFR by EGFR-specific tyrosine kinase inhibitors also blocks GRPmediated mitogen-activated protein kinase activation, indicating that GRP activates mitogen-activated protein kinase primarily through EGFR.15 Recent advances using non– small cell lung cancer (NSCLC) cell lines have confirmed that EGF and GRP both stimulate NSCLC proliferation, and
ung cancer is the leading cause of global cancer death and is responsible for more than 1 million deaths per year.1 At diagnosis, approximately 75% of lung cancer patients exhibit locally advanced or metastatic disease.2 Despite recent advances in understanding the biology of lung cancer, the 5-year survival rates of patients suffering from lung cancer remain low at less than 15%; therefore, Accepted for publication April 23, 2014. From the Departments of Internal Medicine (Dr Mattei) and Pathology (Drs Meurer and Kulczynski), School of Medicine, the Laboratory of Molecular Neuropharmacology, Department of Pharmacology, Institute for Basic Health Sciences (Dr Roesler), and the Cancer Research Laboratory, University Hospital Research Center (CPE-HCPA) (Dr Macedo, Roesler, Brunetto, and Schwartsmann), Federal University of Rio Grande do Sul, Porto Alegre, Brazil; the Departments of Urology (Dr Mattei) and Oncology (Mr Cano and Ms Batlle), University of Colorado, Denver; the Pathology Division, Department of Medicine, National Jewish Health, Denver, Colorado (Drs Achcar and Groshong); the National Institutes for Translational Medicine, Porto Alegre, Brazil (Drs Roesler and Schwartsmann); and the Department of Medical Oncology, Institut Jules Bordet, Brussels, Belgium (Dr Dal Lago). The authors have no relevant financial interest in the products or companies described in this article. Presented as a poster at the American Society of Clinical Oncology meeting; June 2011; Chicago, Illinois. Reprints: Jane Mattei, MD, PhD, Denver Health Hospital, 777 Bannock St, Denver, CO 80204 (e-mail: [email protected]). 98 Arch Pathol Lab Med—Vol 138, January 2014
Lung Cancer; Gastrin-Releasing Peptide Receptor—Mattei et al
inhibition of either EGFR or GRPR resulted in cell death. In addition, combining a GRPR antagonist with the EGFR tyrosine kinase inhibitors resulted in additive cytotoxic effects.14,16 To further investigate the possible utility of GRPR as a future therapeutic target in lung cancer, we evaluated the expression of GRPR in small cell lung carcinoma (SCLC) and NSCLC. The results were correlated with sex, clinical stage, smoking status, and survival. To our knowledge, we have the largest number of SCLCs and NSCLCs evaluated for GRPR tissue expression. MATERIALS AND METHODS Population
Figure 1. Gastrin-releasing peptide (GRP)/GRP receptor (GRPR) signaling pathway. When GRP activates GRPR, inactive guanosine diphosphate (GDP)–bound G protein is activated to a guanosine triphosphate (GTP)–bound state, resulting in metalloprotease-dependent increased release of epidermal growth factor receptor (EGFR) proligands, subsequent phosphorylation of EGFR, and activation of RAS/RAF/mitogen-activated protein kinase (MAPK) downstream pathway.
We analyzed the immunohistochemical expression of GRPR in 238 lung cancer archival cases selected from the department of pathology files of Hospital das Clinicas from the University of Rio Grande do Sul (Porto Alegre, Brazil), based on the availability of formalin-fixed, paraffin-embedded tissue blocks from 2004 to 2008. The specimens were obtained from patients with adenocarcinoma (n ¼ 100), squamous cell carcinoma (n ¼ 71), large cell carcinoma (n ¼ 29), and small cell carcinoma (n ¼ 38). Results were correlated with tumor cell type, sex, tumor stage (I–IV in NSCLC cases and limited and extensive disease in SCLC cases), smoking status (smoker versus nonsmoker), and survival (date of diagnosis to date of death or date of last hospital follow-up visit).
Figure 2. Membranous and cytoplasmic immunolocalization of gastrin-releasing peptide receptor in non–small cell lung carcinoma tissue. A, Absent expression. B, Weak expression. C, Moderate expression. D, Strong expression (original magnifications 340). Arch Pathol Lab Med—Vol 138, January 2014
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Table 1.
Gastrin-Releasing Peptide Receptor Immunohistochemical Analysis Correlating Percentage and Intensity of Tumor Cell Expressiona % Stained Tumor Cells 1–25
a
Cell Type
Negative
Adenocarcinoma (n ¼ 100) Squamous (n ¼ 71) Large cell (n ¼ 29) Small cell (n ¼ 38)
32 31 12 18
1þ 17 7 6 5
25–50
50–100
2þ
3þ
1þ
2þ
3þ
1þ
2þ
3þ
0 0 0 0
0 0 0 0
12 7 3 4
2 1 1 0
0 0 0 0
13 15 3 5
14 7 2 5
10 3 2 1
Immunostaining and measurement of stain were performed as described in ‘‘Materials and Methods.’’
Immunohistochemistry Technique Immunohistochemical stain for GRPR (rabbit polyclonal antibody, OPA1-15619, 1:200 dilution; Affinity Bioreagents, Golden, Colorado) was performed on formalin-fixed, paraffin-embedded tissue blocks from all cases sectioned at 4-lm thickness. After dewaxing, inactivating endogenous peroxidase activity, and blocking cross-reaction with normal serum, sections were incubated overnight at 48C with the primary antibody diluted solution. Secondary detection was achieved by subsequent application of biotinylated antibody, streptavidin horseradish peroxidase conjugate (LSAB, Dako, Carpinteria, California) and diaminobenzidine tetrahydrochloride/H2O2 (Kit DAB, Dako). Images were obtained with an Aperio scanscope (Scanscope Aperio Technologies, Inc, Vista, California) connected to a personal computer HP xw 4300 Workstation (Hewlett-Packard Development Co, LP, Palo Alto, California). Representative photographs were taken to illustrate the findings.
Immunohistochemistry Evaluation Double-blinded histopathology review was performed in tissue from archival cases of NSCLC (n ¼ 200) and SCLC (n ¼ 38). All tumor cases had one slide of tissue available for review. Five entire high-power fields (340) containing clusters of malignant cells were identified per slide and scored for intensity and percentage of GRPR staining expression. The mean of the 5 intensities and percentages for each power field was recorded. The mean values per case were averaged and calculated for each diagnostic group. Intensity of tumor cells’ positive staining was recorded on a scale of 0 to 3 (0 ¼ absent expression; 1 ¼ mild expression; 2 ¼ moderate expression; and 3 ¼ intense expression; Figure 2, A through D). Percentage of stained tumor cells was recorded on a scale of 0 to 3 (0 ¼ absent expression; 1 ¼ less than
25% of cells; 2 ¼ 25%–50% of cells; 3 ¼ 50%–100% of cells; Table 1). Gastrin-releasing peptide receptor antibody–stained nonneoplastic pancreatic tissue was used as positive control.
Statistical Analysis The significance of differences observed between the groups was calculated using SPSS version 19.0 (SPSS Statistics for Windows, Version 19.0, IBM Corporation, Armonk, New York.) Correlations between GRPR expression and clinicopathologic variables were analyzed by using the Pearson v2 test and Fisher exact test. Overall survival was calculated by the Kaplan-Meier method and differences between groups were assessed by the log-rank test and Fisher exact test. Patients were censored for survival at date of death or date of last hospital follow-up visit. A multivariate analysis using the Cox proportional hazards model was also performed in order to adjust for the effects of age, sex, and stage of disease. Statistical differences were accepted at P , .05 and were 2-sided. The study proposal was reviewed and approved by institutional review board and audit committees from University of Rio Grande do Sul (Porto Alegre, Brazil).
RESULTS Patient Demographics The tumor specimens were obtained from adenocarcinoma (n ¼ 100), squamous cell carcinoma (n ¼ 71), large cell carcinoma (n ¼ 29), and small cell carcinoma (n ¼ 38). The male to female ratio was 1.2:1. Review of the available data showed that 200 patients (84.03%) were smokers, 8 (3.36%) were nonsmokers, and 30 (12.61%) had unknown smoking status. Of NSCLC patients, 37 (15.54%) had stage I or II
Figure 3. Gastrin-releasing peptide receptor (GRPR) expression in non–small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) and clinical stage (I, II, III, and IV for NSCLC and extensive disease [ED] for SCLC). A significant association (P ¼ .01) with more pronounced intensity of GRPR expression and advanced clinical stages was identified.
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Table 2. Non–Small Cell Lung Carcinoma and Small Cell Lung Carcinoma Clinical Data Correlating Sex, Age at Diagnosis, Smoking Status, Cell Type, Stage of Disease, and Survival Clinical Data Sex, No. (%) Female Male Age at diagnosis, mean, y Female Male
106 (44.9) 132 (55.1) 63.08 63.44 61.08
Smoking status Smoker Never smoker Unknown
200 (84.03) 8 (3.36) 30 (12.6)
Cell type Non–small cell lung cancer Adenocarcinoma Squamous Large cell Small cell lung cancer
200 100 71 29 38
(84.03) (42.01) (29.83) (12.18) (15.96)
Disease stage I and II III IV Limited disease Extensive disease Unknown
37 53 110 3 33 2
(15.54) (22.26) (46.21) (1.26) (13.86) (0.84)
Survival, mo Female Male
10.3 10.65 10.02
disease, 53 (22.26%) stage III, and 110 (46.21%) stage IV. Of SCLC patients, 3 (1.26%) had limited disease, 33 (13.86%) had extensive disease, and 2 (0.84%) had unknown disease extension. The average survival was 10.65 months in the female population and 10.02 months in the male popula-
tion, with an overall survival rate of 10.3 months. Clinical data are summarized in Table 2. GRPR Expression and Clinical Stage Absence of GRPR expression was more prominent in early stages I and II (62.16%) compared with patients with advanced disease (34% for clinical stage III, 32.35% for clinical stage IV, and 43.24% for extensive disease). Moderate expression was more conspicuous in clinical stage IV (18.63%) and extensive disease (16.21%). Strong expression was more pronounced in clinical stages III (9.43%), IV (6.9%), and extensive disease (5.4%). There was significant association (P ¼ .01) between strong intensity of GRPR expression and higher clinical stage, independent of tumor cell type (Figure 3). It should be noted that because we had only 3 cases with limited disease, this population may not be representative. GRPR Expression and Cell Type Although the overall GRPR percentage of cell expression was similar between NSCLC (62.51%) and SCLC (52.62%; P ¼ .22), the intensity of GRPR expression tended to be more pronounced in NSCLC (41.5% weak, 13.5% moderate, 7.5% strong) compared with SCLC (34.21% weak, 15.78% moderate, 2.63% strong; P ¼ .30; Figure 4). Similar weak expression of GRPR was seen among all NSCLC cell types, ranging from 40.84% to 42%. Moderate GRPR expression was seen in 16% of adenocarcinomas, 11.26% of squamous cell carcinomas, and 10.34% of large cell carcinomas. Strong GRPR expression was seen in 10% of adenocarcinomas, 4.22% of squamous cell carcinomas, and 6.89% of large cell carcinomas (P ¼ .67). Adenocarcinoma cases tended to be stronger GRPR expressers (Figure 5). GRPR Expression and Sex and Smoking Status Distribution of GRPR expression was comparable between male (weak 41.66%, moderate 12.87%, and strong 6.06%) and female subjects (weak 38.67%, Figure 4. Gastrin-releasing peptide receptor (GRPR) expression in non–small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC). There was no significant difference in GRPR expression between NSCLC and SCLC.
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Figure 5. Gastrin-releasing peptide receptor (GRPR) expression in non–small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC). There was no significant difference in GRPR expression among cases of adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and small cell carcinoma. Adenocarcinoma cases tended to be GRPR overexpressers.
moderate 15.09%, and strong 7.54%). The absence of GRPR was seen in 39.39% of male and 38.67% of female subjects. The differences in GRPR expression between male and female subjects were not statistically significant (P ¼ .91; Figure 6). Among 238 subjects, 200 were smokers, 8 were nonsmokers, and 30 had unknown smoking status. Because of the low number of nonsmoker subjects, it was not possible to correlate GRPR expression with smoking status. GRPR Expression and Survival Two hundred thirty-eight patients were evaluated for overall survival until mortality or the date of the last followup visit if the patients were still alive, with an average follow-up of 15.2 months. The median overall survival time was 8.97 months (95% confidence interval, 5.6–12.3 months) for absent GRPR expression, 11.50 months (95% confidence interval, 3.6–19.3 months) for weak and moderate expression, and 10.6 months (95% confidence interval, 0.4–20.7 months) for strong GRPR expressers (P ¼ .77). The Kaplan-Meier curve was adjusted for clinical stage (Figure 7).
Figure 6. Gastrin-releasing peptide receptor (GRPR) expression in males and females showing no detectable difference in GRPR expression. 102 Arch Pathol Lab Med—Vol 138, January 2014
COMMENT It has been demonstrated that GRPR activates mitogenactivated protein kinase signaling pathway primarily through EGFR activation and that combining GRPR antagonist with EGFR tyrosine kinase inhibitors results in additive cytotoxic effect in tumor cell lines.13 Gastrinreleasing peptide receptor synthetic antagonists, such as RC3095, have been developed as anticancer candidates, and have shown antitumor activity in both in vivo and in vitro murine and human tumor models, producing long-lasting tumor regression.17 In 25 patients with advanced refractory solid malignancies enrolled in a phase I trial of RC-3095 conducted at Hospital das Clinical, in Porto Alegre, Brazil, RC-3095 was well tolerated.18 Gastrin-releasing peptide receptor was originally isolated in SCLC by Cuttitta et al,19 and most previous studies have reported GRPR expression in up to 70% of small cell lung cancer and 10% to 20% in NSCLC.20 Conversely, in our study we noted GRPR expression in NSCLC and SCLC at similar rates of 62.51% and 52.62% respectively (P ¼ .30). Among NSCLC cases, adenocarcinoma subjects tended to be GRPR overexpressers at a rate of 68% (41.5% weak expression, 13.5% moderate expression, and 7.5% strong expression) compared with other NSCLC cases (P ¼ .67). Although we acknowledge that the differences seen in our study were not statistically significant, this pattern of GRPR upregulation may indicate that GRPR represents a future target for adjuvant therapy in lung cancer, particularly in adenocarcinoma cases. The association between GRPR expression, smoking status, and sex is not clear. Some studies suggest that the GRPR gene is expressed more frequently in females than in males in the absence of smoking and that expression is activated earlier in females in response to tobacco exposure.21 On the other hand, GRP expression has also been reported to be increased with tobacco use. Higher levels of GRP protein have been detected in bronchoalveolar lavage and urine specimens of asymptomatic long-standing smokers when compared with nonsmoking control subjects.22 Although our study is limited with respect to smoking status because of a low number of nonsmoker subjects, we noticed no difference in GRPR expression between males and females, suggesting that GRPR plays a similar role in development of lung cancer independent of sex. Gastrin-releasing peptide receptor expression was noted in bronchial epithelium and submucosal gland epithelial cells (not quantified) from small cell and non–small cell Lung Cancer; Gastrin-Releasing Peptide Receptor—Mattei et al
Figure 7. Overall survival of lung cancer and gastrin-releasing peptide receptor (GRPR) expressers calculated using the Kaplan-Meier survival curves. There was no significant correlation with survival and GRPR expression.
carcinoma cases. The role of extratumoral GRPR expression is unclear. However, Egloff and colleagues23 evaluated GRPR mRNA levels in histologically normal bronchial epithelial cells from 224 lung cancer patients and 107 surgical cancer-free controls and demonstrated that GRPR expression in noncancerous bronchial epithelium was significantly associated with lung cancer independent of age, sex, or smoking status. Egloff et al23 showed a 71% rate of GRPR expression in nonneoplastic bronchial cells from lung cancer samples compared with only 38% of cancer-free controls, suggesting that increased GRPR expression in normal epithelial mucosa may be an early event in lung cancer formation. Gastrin-releasing peptide receptor overall percentage and intensity of tumor cell expression staining were more pronounced in clinical stages III and IV and extensive disease compared with GRPR expression in clinical stages I and II. These differences were statistically significant (P ¼ .01) and were independent of tumor cell type, sex, and survival. The impact and implications of increased GRPR expression in patients with advanced lung disease deserve future studies and may indicate that GRPR may be a potential therapeutic target in this population. To our knowledge, this study is the first to correlate GRPR tissue expression and distribution with the clinical stages of lung cancer in a large small cell and non–small cell population. We detected GRPR expression in NSCLC and SCLC at similar rates. Adenocarcinomas and advanced lung cancer disease subjects tended to be GRPR overexpressers, suggesting that GRPR may be a potential future therapeutic target in this population. The GRPR pathway is known to interact with the EGFR pathway in lung cancer cells by increasing the release of EFGR proligands, which could act Arch Pathol Lab Med—Vol 138, January 2014
to further promote cancer in patients who develop EGFR mutations. Molecular profiling of lung cancer patients with GRPR upregulation deserves future study. References 1. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2008. CA Cancer J Clin. 2008;58(2):71–96. 2. Pass HI, Carbone DP, Johnson DH, Minna JD, Scagliotti GV. Principles and Practice of Lung Cancer: The Official Reference Text of the International Association for the Study of Lung Cancer (IASLC). 4th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2010. 3. Sandler A, Gray R, Perry MC, et al. Paclitaxel-carboplatin alone or with bevacizumab for non–small-cell lung cancer. N Engl J Med. 2006;355(24):2542. 4. Pirker R, Pereira JR, von Pawel J, et al. EGFR expression as a predictor of survival for first-line chemotherapy plus cetuximab in patients with advanced non–small-cell lung cancer: analysis of data from the phase 3 FLEX study. Lancet Oncol. 2012;13(1):33–42. 5. Shan L, Emanuel RL, Dewald D, et al. Bombesin-like peptide receptor gene expression, regulation, and function in fetal murine lung. Am J Physiol Lung Cell Mol Physiol. 2004;286(1):L165–L173. 6. Sunday ME, Choi N, Spindel ER, Chin WW, Mark EJ. Gastrin-releasing peptide gene expression in small cell and large cell undifferentiated lung carcinomas. Hum Pathol. 1991;22(10):1030–1039. 7. Patel O, Shulkes A, Baldwin GS. Gastrin-releasing peptide and cancer. Biochim Biophys Acta. 2006;1766(1):23–41. 8. Weber HC. Regulation and signaling of human bombesin receptors and their biological effects. Curr Opin Endocrinol Diabetes Obes. 2009;16(1):66–71. 9. Constantinides C, Lazaris AC, Haritopoulos KN, Pantazopoulos D, Chrisofos M, Giannopoulos A. Immunohistochemical detection of gastrin releasing peptide in patients with prostate cancer. World J Urol. 2003;21(3):183–187. 10. Flores DG, Meurer L, Uberti AF, et al. Gastrin-releasing peptide receptor content in human glioma and normal brain. Brain Res Bull. 2010;82(1–2):95–98. 11. Rivera CA, Ahlberg NC, Taglia L, Kumar M, Blunier A, Benya RV. Expression of GRP and its receptor is associated with improved survival in patients with colon cancer. Clin Exp Metastasis. 2009;26(7):663–671. 12. Lango MN, Dyer KF, Yan VW, et al. Gastrin-releasing peptide receptormediated autocrine growth in squamous cell carcinoma of the head and neck. J Natl Cancer Inst. 2002;94(5):375–383. 13. Thomas SM, Grandis JR, Wentzel AL, Gooding WE, Yan VW, Siegfried JM. Gastrin-releasing peptide receptor mediates activation of the epidermal growth factor receptor in lung cancer cells. Neoplasia. 2005;7(4):426–431.
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14. Zhang Q, Bhola NE, Yan VW, et al. Antitumor mechanisms of combined gastrin-releasing peptide receptor and epidermal growth factor receptor targeting in head and neck cancer. Mol Cancer Ther. 2007;6(4):1414–1424. 15. Liu X, Carlisle DL, Swick MC, Gaither-Davis A, Grandis JR, Siegfried JM. Gastrin-releasing peptide activates Akt through the epidermal growth factor receptor pathway and abrogates the effect of gefitinib. Exp Cell Res. 2007;313(7): 1361–1372. 16. Thatcher N, Chang A, Parikh P, et al. Gefitinib plus best supportive care in previously treated patients with refractory advanced non–small-cell lung cancer: results from a randomised, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer). Lancet. 2005;366(9496):1527–1537. 17. Qin Y, Ertl T, Cai RZ, Halmos G, Sehally AV. Inhibitory effect of bombesin receptor antagonist RC-3095 on the growth of human pancreatic cancer cells in vivo and in vitro. Cancer Res. 54:1035–1041. 18. Schwartsmann G, DiLeone LP, Horowitz M, et al. A phase I trial of the bombesin/gastrin-releasing peptide (BN/GRP) antagonist RC3095 in patients with advanced solid malignancies. Invest New Drugs. 2006;24(5):403–412.
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19. Cuttitta F, Carney DN, Mulshine J, et al. Bombesin-like peptides can function as autocrine growth factors in human small-cell lung cancer. Nature. 1985;316(6031):823–826. 20. Moody TW, Pert CB, Gazdar AF, Carney DN, Minna JD. High levels of intracellular bombesin characterize human small-cell lung carcinoma. Science. 1981;214(4526):1246. 21. Shriver SP, Bourdeau HA, Gubish CT, et al. Sex-specific expression of gastrin-releasing peptide receptor: relationship to smoking history and risk of lung cancer. J Natl Cancer Inst. 2000;92(1):24–33. 22. Aguayo SM, Kane MA, TE King J, Schwarz MI, Grauer L, Miller YE. Increased levels of bombesin-like peptides in the lower respiratory tract of asymptomatic cigarette smokers. J Clin Invest. 1989;84(4):1105. 23. Egloff AM, Davis AG, Shuai Y, et al. Gastrin-releasing peptide receptor expression in non-cancerous bronchial epithelia is associated with lung cancer: a case control study. Respir Res. 2012;13 (1):9.
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