|Ahead of print publication
Association of cytokines levels with epidermal growth factor receptor mutation in lung cancer patients
Priyanka Gaur1, Sandeep Bhattacharya1, Surya Kant2, R A. S. Kushwaha2, Gaurav Singh3, Sarika Pandey2
1 Department of Physiology, King George's Medical University, Lucknow, Uttar Pradesh, India
2 Department of Respiratory Medicine, King George's Medical University, Lucknow, Uttar Pradesh, India
3 Department of Biotechnology, Kunwar Satya Vira College of Engineering and Management, Bijnor, Uttar Pradesh, India
|Date of Submission||27-Sep-2018|
|Date of Decision||25-Dec-2018|
|Date of Acceptance||11-May-2019|
|Date of Web Publication||13-May-2020|
Department of Physiology, King George's Medical University, Lucknow - 226 010, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Lung cancer is one of the most frequent types of cancer and the leading cause of cancer-related deaths. Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (TK) being highly expressed in lung cancers. Activation of EGFR through oncogenic mutations leads to upregulation of gene expression that may heighten the inflammatory response in certain situations. EGFR acts as a key regulator and a cellular hub for inflammatory cytokine signaling, thereby promoting tumor cell proliferation, invasion, migration, metastases, and survival. The aim of the present study is to determine the serum cytokines levels and EGFR mutation status in lung cancer patients to investigate the association between the EGFR mutation status and cytokines levels with lung cancer patients.
Materials and Methods: Blood and tissue samples of lung cancer patients were collected. The EGFR mutations of lung cancer patients were determined by the immunohistochemistry (IHC) and serum cytokines levels of lung cancer patients were determined using ELISA.
Results: Statistically significant association of EGFR mutations with adenocarcinoma subtypes and non-smokers were found (P < 0.05). Lung cancer patients with EGFR mutations had significantly higher tumor necrosis factor-alpha levels when compared to lung cancer patients without EGFR mutations (P < 0.01), and EGFR mutation status was not significantly associated with interleukin-6 levels (P = 0.24).
Conclusion: EGFR mutation detection by the IHC method is a potentially useful tool to guide clinicians for personalized treatment of lung cancer patients of adenocarcinoma subtype, and cytokines are good biomarkers for the diagnosis, prognosis, and prediction of treatment responses in lung cancer patients as well as act as therapeutic targets. This study will provide biomarkers for lung cancer diagnosis and treatments.
Keywords: Biomarker, cytokines, epidermal growth factor receptor, lung cancer, smoker
|How to cite this URL:|
Gaur P, Bhattacharya S, Kant S, Kushwaha R A, Singh G, Pandey S. Association of cytokines levels with epidermal growth factor receptor mutation in lung cancer patients. J Can Res Ther [Epub ahead of print] [cited 2020 Aug 10]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=284264
| > Introduction|| |
Lung cancer is the most common type of cancer worldwide and is the leading cause of cancer-related deaths. Lung cancers are mainly classified into two major types, including small-cell lung cancer (SCLC) and nonSCLC (NSCLC) and mainly consists of adenocarcinoma and squamous cell carcinoma. It has been shown that approximately 80–85 percent cases are non-small-cell lung cancer (NSCLC). In recent decades, targeted therapies which have been represented by epidermal growth factor receptor (EGFR) inhibitors have gained attention for their ability to significantly improving the clinical outcomes and also reducing the side effects of the chemotherapy. It has been shown that the EGFR mutations were the first targetable mutations to be identified in NSCLC. Lung cancer Patients with these mutations show higher response rates with EGFR TK inhibitors such as gefitinib, erlotinib, and afatinib and thus have a better prognosis than patients without an EGFR mutation.,, The discovery of oncogenic driver mutations in the EGFR gene of exons 18–21 and approval of agents which target against these molecular drivers have been revolutionized the management of NSCLC. Activation of EGFR through oncogenic mutations leads to upregulation of gene expression that may heighten the infiammatory response in certain situations., EGFR signaling also leads to downstream production of interleukin-6 (IL-6) in human lung cancer cells. A substantial portion of the tumor microenvironment is infiammatory, and pro-infiammatory cytokines and growth factors exacerbate cellular processes such as tumor proliferation and inhibit immune responses. In turn, chronic inflammation contributes to the development of malignancies and metastases., Integral in these processes, EGFR acts as a key regulator and a cellular hub for inflammatory cytokine signaling, thereby promoting tumor cell proliferation, invasion, migration, metastases, and survival.,,
Several cytokines have been identified which play an important role in lung cancer. The cytokines are considered as good biomarkers for the diagnosis, prognosis, and prediction of treatment responses in lung cancer patients. Lung cancer is characterized by the poor prognosis with a 5-year survival rate of 15%, which is mainly due to the initial diagnosis of lung cancer is at advanced stages of the disease. For this reason, in addition to advances in treatment, the search for diagnostic strategies of early lung cancer detection is very important. Hence, the use of biomarkers is essential for early detection. The activation of EGFR by its gene mutations such as exon 19 in-frame deletions and exon 21 L858R substitution is currently recognized as the most potent biological predictors of EGFR TKI sensitivity. Surgical intervention, platinum-based chemotherapy and radiotherapy were the principal available therapeutic options for the treatment of lung cancer in the past decade. However, with the description of EGFR mutations in lung adenocarcinoma and the response of these tumors to TKI, a new hope in making a significant difference in the survival of such patients has arisen.,, This study determines the EGFR mutation status and serum cytokines levels in lung cancer patients to investigate the association of EGFR mutation status and cytokines levels with the clinicopathological characters of lung cancer patients.
| > Materials and Methods|| |
Recruitment of subjects
The present study was conducted in the Department of Respiratory King George's Medical University, Lucknow, India. The study was approved by the Ethics Committee of the corresponding institute and all the participants gave their written consent. This study included only lung cancer patients and participants having other disorders such as chronic obstructive pulmonary disease (COPD), asthma, tuberculosis, interstitial lung disease, and other malignancies were excluded from the study.
Epidermal growth factor receptor mutation detection
Tissue samples of lung cancer participants were collected bronchoscopically for the detection of EGFR mutations. The EGFR mutation was detected by immunohistochemistry (IHC) method. Formalin-fixed, paraffin-embedded tissue sections were cut into 4 μm-thick sequential sections. After deparaffinization and rehydration, sections were boiled in citrate buffer (0.01 M, pH 6.0) for antigen retrieval. The sections were incubated with 3% H2O2 and 5% serum to block endogenous peroxidase activity and nonspecific binding. Two primary antibodies (delE746-A750 mutation-specific monoclonal antibody [6B6] and L858R mutation-specific monoclonal antibody [43B2]; Cell Signaling Technology, Danvers, MA, USA) were used for the detection of EGFR mutation. The sections were then incubated with biotinylated secondary antibodies and visualized by 3,3′-Diaminobenzidine (DAB). Counterstaining was carried out with hematoxylin. The sections were then dehydrated in alcohol and mounted with DPX.
The IHC staining score was based on the staining intensity and percentage positivity (0%–100%) of cells in the membrane and/or cytoplasm of tumor cells. Four grades were employed as follows: 0, 1+, 2+, 3+. 0 = no staining; 1+ = faint membrane and/or cytoplasmic staining in <10% positive cells; 2+ = moderate membrane and/or cytoplasmic staining in greater than 10% and <50% cells; 3+ = strong membrane and/or cytoplasmic staining >50% cells positive; 0 and 1 + score was considered as negative; whereas 2+ and 3+ were considered as positive cases.
Estimation of serum cytokines levels
Peripheral blood samples of lung cancer patients and controls were collected into the plain vial by venipuncture on the first admission before chemotherapy. Serum samples were obtained by centrifugation at 3000 rpm for 10 min and stored at − 80°C until use. Serum levels of IL-6 and tumor necrosis factor-alpha (TNF-α) levels were determined using a commercially available ELISA kit according to the manufacturer's instructions.
Data were analyzed using the Graph Pad Prism version 5 (Graph Pad software Inc.; La, Jolla, CA, USA). Descriptive data are presented as mean, standard deviation, or as percentages. The Chi-square test was used for categorical data and groups were compared by unpaired t-test or one-way analysis of variance. P < 0.05 was considered as statistically significant.
| > Results|| |
In this study, 121 lung cancer patients were recruited. The demographic and clinical characteristics of lung cancer patients are shown in [Table 1]. The mean age of lung cancer patients was 53.95 years. The proportion of male patients was higher as compared to females. In the present study, 56.2% of the lung cancer patient was smokers/ex-smokers and 43.8% was nonsmokers. Most of the lung cancer patients were of Stage III/IV, which consists of 93.39%.
EGFR mutations were found in 34.29% of lung cancer patients.
Exon 19 mutation was the most common and seen in 79.17% of the lung cancer patients and L858R point mutations of exon 21 was found in 20.83% of lung cancer patients [Figure 1].
|Figure 1: Exon 21 (L858R) and Exon 19 (E746-A750) EGFR mutation positive lung cancer patients|
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Lung cancer patients with EGFR mutations had significantly higher TNF-α levels when compared to lung cancer patients without EGFR mutations (P < 0.01) and EGFR mutation status was not significantly associated with IL-6 levels (P= 0.24) [Figure 2].
|Figure 2: Levels of IL6 and TNF-α in lung cancer patients with EGFR mutation and without EGFR mutation|
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The level TNF α and IL 6 in lung cancer patients of Stage III/IV was significantly higher than Stage I/II (P< 0.01) [Figure 3].
|Figure 3: Levels of TNF-α and IL-6 in different stages of lung cancer patients|
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Significantly higher levels of TNF-α and IL-6 were observed in smokers/ ex-smokers as compared to non-smokers lung cancer patients (P< 0.001) [Figure 4].
|Figure 4: Levels of TNF-α and IL-6 in Smokers/Exsmokers and Nonsmokers lung cancer patients|
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| > Discussion|| |
EGFR gained pharmaceutical significance with the discovery of its involvement in a number of cancers, including NSCLC and head-and-neck cancers. It has been found that the EGFR is upregulated in several types of cancers. Higher prevalence of EGFR mutations is reported in females, non-smokers and lung cancer patients with adenocarcinoma.
It has been reported that EGFR mutations are found in 40%–55% of adenocarcinoma in various studies., EGFR mutation is found in 34.29% of NSCLC patients in our study. Previous studies shows that in frame deletions mutations in exon 19 were found in 26–79 percent, whereas the point mutations in exon 21 (L858R) was observed in 13–47 percent.,, It has been reported that tumors with exon 19 deletions were more susceptible to gefitinib than exon 21 point mutations. Lung cancer patients showing mutations in the EGFR gene have been associated with better response to TKI such as gefitinib. Although the recent advances are applicable for the management of advanced NSCLC, the cure rate remains still low. Hence, further molecular investigations are required for the development of the new treatment strategies to improve the prognosis of lung cancer patients. It has been shown by several studies that the EGFR mutations are predictive factors of response to EGFR-TKI treatment. Activation of EGFR through oncogenic mutations leads to upregulation of gene expression that may heighten the inflammatory response in certain situations. EGFR acts as a key regulator and a cellular hub for inflammatory cytokine signaling, thereby promoting tumor cell proliferation, invasion, migration, metastases, and survival.
Inflammatory cells may provide growth and survival factors which contribute to various hallmarks of cancer. It has been reported that chronic inflammation due to pulmonary disorders such as COPD significantly increases the patients' risk of developing lung cancer., It has been found that the main mediators of inflammation are cytokines proteins which can be classified as pro-inflammatory and anti-inflammatory molecules such as chemokines and growth factors.,, These proteins can modulate different cellular responses, including inflammation, the immune response, apoptosis as well as chemoattractant processes.,,, Characteristic cytokine patterns have been associated with different cancer patients and are related to their prognosis also. Therefore, some cytokines are the good prognostic biomarkers of cancer.,, The cytokines that are good biomarkers for the diagnosis, prognosis, and the prediction of treatment responses in lung cancer patients. The cytokines could act as therapeutic targets and describe the therapeutic strategies based on these targets. The results of the previous study suggest that therapy with TNF-α would be effective in some cases of nonsmall-cell lung cancer that have acquired resistance to gefitinib. Recent insights into genetic aberrations and the role of the immune system in NSCLC have used in a new era of rapidly evolving targeted therapy and immune-based treatments.,, It has been shown that the blocking of EGFR activation by TKI consequently generates the downregulation of IL-6. The biological rationale for therapeutic anti-IL-6 activity and preliminary clinical evidence which target anti-IL-6 antibodies are well tolerated in cancer patients, these findings provide the insight into a potential strategy for improving gefitinib anticancer efficacy and prolonging PFS by combining the anti-IL-6 agent with gefitinib., It has been reported that the serum TNF-α levels and IL-6 were found increased with the advanced stages of lung cancer suggesting a possible local production of inflammatory cytokines which is also in accordance with some previous studies. The higher levels TNF-α and IL-6 were observed in smokers/ex-smokers as compared with nonsmokers lung cancer patient. Serum TNF-α and IL-6 levels are the promising biomarkers in the identification of lung cancer patients. The study is also supporting the association of inflammatory markers to lung cancer risk. Hence these findings suggest the levels of these biomarkers could be a useful tool for guiding the diagnosis of lung cancer.
| > Conclusion|| |
EGFR mutation detection is a potentially useful tool to guide clinicians for personalized treatment of NSCLC patients. EGFR mutation status is the most valuable indicator for the screening of NSCLC patients for TKI therapy. The detection of EGFR mutation in NSCLC patients is helpful in the selection of targeted therapy. Serum TNF-α and IL-6 levels are the promising biomarkers in the identification of lung cancer patients. The study is also supporting the association of inflammatory markers to lung cancer risk. Hence, these findings suggest the levels of these biomarkers could be a useful tool for guiding the diagnosis of lung cancer.
We would like to thank the faculty and staff of the Department of Respiratory medicine, King George's Medical University, Lucknow for providing samples. The authors also would like to thank all the patients and their families for their cooperation and participation.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, et al
. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11. International Agency for Research on Cancer, Lyon, 2014.
Travis WD, Colby TV, Corrin B, Shimosato Y, Brambilla E. Histological Typing of Lung and Pleural Tumors. Berlin: Springer; 1999. p. 1-156.
Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global Cancer Statistics, 2012, CA Cancer J Clin 2015;65:87-108.
Grigoriu B, Berghmans T, Meert AP. Management of EGFR mutated nonsmall cell lung carcinoma patients, Eur Respir J 2015;45:1132-141.
Zhou C, Wu YL, Chen G, Feng J, Liu XQ, Wang C, et al
. Erlotinib versus chemotherapy asfirst-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): A multicentre, open-label, randomised, phase 3 study, Lancet Oncol 2011;12:735-42.
Scaltriti M, Baselga J. The epidermal growth factor receptor pathway: A model for targeted therapy. Clin Cancer Res 2006;12:5268-72.
Shepard HM, Brdlik CM, Schreiber H. Signal integration: A framework for understanding the efficacy of therapeutics targeting the human EGFR family. J Clin Invest 2008;118:3574-81.
Berasain C, Perugorria MJ, Latasa MU, Castillo J, Goñi S, Santamaría M, et al.
The epidermal growth factor receptor: A link between inflammation and liver cancer. Exp Biol Med (Maywood) 2009;234:713-25.
Singh N, Bal A, Aggarwal AN, Das A, Behera D. Clinical outcomes in non-small-cell lung cancer in relation to expression of predictive and prognostic biomarkers. Future Oncol 2010;6:741-67.
Liu K, Gualano RC, Hibbs ML, Anderson GP, Bozinovski S. Epidermal growth factor receptor signaling to Erk1/2 and STATs control the intensity of the epithelial inflammatory responses to rhinovirus infection. J Biol Chem 2008;283:9977-85.
Liu K, Anderson GP, Bozinovski S. DNA vector augments inflammation in epithelial cells via EGFR-dependent regulation of TLR4 and TLR2. Am J Respir Cell Mol Biol 2008;39:305-11.
Gao SP, Mark KG, Leslie K, Pao W, Motoi N, Gerald WL, et al.
Mutations in the EGFR kinase domain mediate STAT3 activation via IL-6 production in human lung adenocarcinomas. J Clin Invest 2007;117:3846-56.
Balkwill F, Coussens LM. Cancer: An inflammatory link. Nature 2004;431:405-6.
Orozco-Morales M, Soca-Chafre G, Barrios-Bernal P, Hernández-Pedro N, Arrieta O. Interplay between cellular and molecular inflammatory mediators in lung cancer. Mediators Inflamm 2016;2016:3494608.
Dougan M, Li D, Neuberg D, Mihm M, Googe P, Wong KK, et al.
Adual role for the immune response in a mouse model of inflammation-associated lung cancer. J Clin Invest 2011;121:2436-46.
Barrera L, Montes-Servín E, Barrera A, Ramírez-Tirado LA, Salinas-Parra F, Bañales-Méndez JL, et al.
Cytokine profile determined by data-mining analysis set into clusters of non-small-cell lung cancer patients according to prognosis. Ann Oncol 2015;26:428-35.
Paez JG, Jänne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al.
EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science 2004;304:1497-500.
Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, et al.
Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N
Engl J Med 2004;350:2129-39.
Gaur P, Singh G, Bhattacharya S, Kant S, Pandey S, Pandey RK, et al
. EGFR mutation and tyrosine-kinase inhibitors (TKI) in non small cell lung cancer: An overview. Int J Life Sci Sci Res 2018;4:1531-3.
Kosaka T, Yatabe Y, Endoh H, Kuwano H, Takahashi T, Mitsudomi T. Mutations of the epidermal growth factor receptor gene in lung cancer: Biological and clinical implications. Cancer Res 2004;64:8919-23.
Huang SF, Liu HP, Li LH, Ku YC, Fu YN, Tsai HY, et al.
High frequency of epidermal growth factor receptor mutations with complex patterns in non-small cell lung cancers related to gefitinib responsiveness in Taiwan. Clin Cancer Res 2004;10:8195-203.
Shigematsu H, Lin L, Takahashi T, Nomura M, Suzuki M, Wistuba II, et al.
Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst 2005;97:339-46.
Sholl LM, Xiao Y, Joshi V, Yeap BY, Cioffredi LA, Jackman DM, et al.
EGFR mutation is a better predictor of response to tyrosine kinase inhibitors in non-small cell lung carcinoma than FISH, CISH, and immunohistochemistry. Am J Clin Pathol 2010;133:922-34.
Gaur P, Bhattacharya S, Kant S, Kushwaha RA, Singh G, Pandey S. EGFR
mutation detection and its association with clinicopathological characters of lung cancer patients. World J Oncol 2018;9:151-5.
Gaur P, Bhattacharya S, Kant S, Kushwaha RA, Agarwal J, Pathak AK. Epidermal growth factor receptor mutations detection by mutant specific immunohistochemistry in North Indian lung cancer population. Int J Res Dev Pharm L Sci 2017;6:2796-9.
Mitsudomi T, Kosaka T, Endoh H, Horio Y, Hida T, Mori S, et al.
Mutations of the epidermal growth factor receptor gene predict prolonged survival after gefitinib treatment in patients with non-small-cell lung cancer with postoperative recurrence. J Clin Oncol 2005;23:2513-20.
Cappuzzo F, Gregorc V, Rossi E, Cancellieri A, Magrini E, Paties CT, et al.
Gefitinib in pretreated non-small-cell lung cancer (NSCLC): Analysis of efficacy and correlation with HER2 and epidermal growth factor receptor expression in locally advanced or metastatic NSCLC. J Clin Oncol 2003;21:2658-63.
Jiang G, Fan C, Zhang X, Dong Q, Wang L, Liu Y, et al.
Ascertaining an appropriate diagnostic algorithm using EGFR mutation-specific antibodies to detect EGFR status in non-small-cell lung cancer. PLoS One 2013;8:e59183.
Fann JR, Berry DL, Wolpin S, Austin-Seymour M, Bush N, Halpenny B, et al.
Depression screening using the patient health questionnaire-9 administered on a touch screen computer. Psychooncology 2009;18:14-22.
Ell K, Xie B, Quon B, Quinn DI, Dwight-Johnson M, Lee PJ, et al.
Randomized controlled trial of collaborative care management of depression among low-income patients with cancer. J Clin Oncol 2008;26:4488-96.
Dias-Santagata D, Akhavanfard S, David SS, Vernovsky K, Kuhlmann G, Boisvert SL, et al.
Rapid targeted mutational analysis of human tumours: A clinical platform to guide personalized cancer medicine. EMBO Mol Med 2010;2:146-58.
Wilcox RR. Comparing dependent robust correlations. Br J Math Stat Psychol 2016;69:215-24.
Cohen J. Statistical Power Analysis for the Behavioral Sciences. 2nd
. ed. New York: Academic Press; 1998.
Musselman DL, Miller AH, Porter MR, Manatunga A, Gao F, Penna S, et al.
Higher than normal plasma interleukin-6 concentrations in cancer patients with depression: Preliminary findings. Am J Psychiatry 2001;158:1252-7.
Lutgendorf SK, Lamkin DM, DeGeest K, Anderson B, Dao M, McGinn S, et al.
Depressed and anxious mood and T-cell cytokine expressing populations in ovarian cancer patients. Brain Behav Immun 2008;22:890-900.
O'Connor MF, Bower JE, Cho HJ, Creswell JD, Dimitrov S, Hamby ME, et al.
To assess, to control, to exclude: Effects of biobehavioral factors on circulating inflammatory markers. Brain Behav Immun 2009;23:887-97.
Pirl WF, Traeger L, Greer JA, Jackson V, Lennes IT, Gallagher E, et al.
Depression, survival, and epidermal growth factor receptor genotypes in patients with metastatic non-small cell lung cancer. Palliat Support Care 2013;11:223-9.
Liu M, Yang SC, Sharma S, Luo J, Cui X, Peebles KA, et al.
EGFR signaling is required for TGF-beta 1 mediated COX-2 induction in human bronchial epithelial cells. Am J Respir Cell Mol Biol 2007;37:578-88.
Yang JC, Wu YL, Schuler M, Sebastian M, Popat S, Yamamoto N, et al.
Afatinib versus cisplatin-based chemotherapy for EGFR mutation-positive lung adenocarcinoma (LUX-lung 3 and LUX-lung 6): Analysis of overall survival data from two randomised, phase 3 trials. Lancet Oncol 2015;16:141-51.
de Visser KE, Eichten A, Coussens LM. Paradoxical roles of the immune system during cancer development. Nat Rev Cancer 2006;6:24-37.
Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, et al.
Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N
Engl J Med 2012;366:2443-54.
Guo Y, Xu F, Lu T, Duan Z, Zhang Z. Interleukin-6 signaling pathway in targeted therapy for cancer. Cancer Treat Rev 2012;38:904-10.
Trikha M, Corringham R, Klein B, Rossi JF. Targeted anti-interleukin-6 monoclonal antibody therapy for cancer: A review of the rationale and clinical evidence. Clin Cancer Res 2003;9:4653-65.
Boldrini L, Calcinai A, Samaritani E, Pistolesi F, Mussi A, Lucchi M, et al.
Tumour necrosis factor-alpha and transforming growth factor-beta are significantly associated with better prognosis in non-small cell lung carcinoma: Putative relation with BCL-2-mediated neovascularization. Br J Cancer 2000;83:480-6.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]