1 Department of Oncology and Haematology, University Hospital Hamburg-Eppendorf; 2 Department of Surgery, 3 Department of Laboratory Medicine and 4 Department of Pathology, General Hospital Hamburg-Harburg; 5 Institute of Mathematics and Computer Science in Medicine, University Hospital Hamburg-Eppendorf, Germany
Received 17 December 2001; revised 5 April 2002; accepted 25 April 2002
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Abstract |
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Matrix metalloproteinase (MMP)-9 and vascular endothelial growth factor (VEGF) are two proteins involved in angiogenesis. In the present study we investigated the association of pretreatment MMP-9 and VEGF serum levels with clinicopathological parameters and outcome in patients with non-small-cell lung cancer (NSCLC).
Patients and methods:
From February 1998 to October 1999, pretreatment serum levels of MMP-9 and VEGF were analysed in 118 patients with enzyme-linked immunoassays. At diagnosis 50 patients (42%) were staged as early disease (I/II), 27 patients (23%) as locally advanced (IIIA/IIIB), and 41 patients (35%) had metastatic disease (IV). In 72 of the 118 patients tumours were resected and 46 patients received combination chemotherapy with gemcitabine and vinorelbine.
Results:
The median survival of all 118 patients was 602 days. The 72 patients who had undergone surgery had a median survival of 972 days and the 46 patients who were treated with chemotherapy had a median survival of 298 days (P <0.001). Resected patients with stage I/II disease and an MMP-9 serum level 1293 ng/ml or a VEGF serum level
630 pg/ml had a significantly longer survival (median survival longer than 1218 days) than patients with higher serum levels (median survival 421 days) (P = 0.001 for MMP-9; P = 0.04 for VEGF). No significant difference in survival was observed in patients with resected stage III disease. Besides tumour stage, Karnofsky performance status and gender, the pretreatment serum level of MMP-9 was identified as an independent prognostic factor in a multivariate Cox regression analysis.
Conclusions:
Future studies may support our hypothesis that the pretreatment serum level of MMP-9 is a new powerful prognostic marker and can help to stratify NSCLC patients with stage I/II disease into low- and high-risk groups.
Key words: angiogenic factors, matrix metalloproteinase-9, non-small-cell lung cancer, prognostic factors, vascular endothelial growth factor
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Introduction |
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Angiogenesis represents the formation of new blood vessels from existing vasculature. Neovascularisation is a requirement for solid tumour growth beyond 12 mm in diameter [4, 5]. The angiogenic process is a balance between stimulatory and inhibitory factors. The pro-angiogenic stimuli may be released by the tumour, stromal and inflammatory cells, by the extracellular matrix, or by the endothelial cells themselves. Tumour cells secrete or induce the release of growth factors which stimulate migration and proliferation of endothelial cells. Furthermore these factors may be involved in capillary morphogenesis or release of proteolytic enzymes [68].
Matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor (VEGF) are two of the most potent factors involved in angiogenesis. The MMPs belong to a family of zinc-dependent neutral endopeptidases. Under physiological conditions they are capable of degrading extracellular matrix and basement membrane components. Increased MMP activity has been implicated in tumour invasion and the formation of metastases. Although 18 members of the MMP family have been described [9], the gelatinases MMP-9, formerly known as gelatinase B or 92 kDa type IV collagenase, and MMP-2 have been most consistently detected in malignant tissues and are associated with tumour aggressiveness and metastatic potential [1012].
VEGF is the most potent and specific growth factor (e.g. proliferation and migration) for endothelial cells. High levels of expression of VEGF are found in many solid tumour types including colon [13], breast [14], gastric [15] and lung [16] cancer. It has been reported that overexpression of VEGF in tumour tissue represents an independent prognostic factor in patients with breast [17], gastric [18] and squamous-cell lung [19] cancer. Four isoforms of VEGF (VEGF121, VEGF165, VEGF189, VEGF206) exist in human tissues [20]. VEGF189 and VEGF206 are cell-associated and have strong affinity to cell-surface proteoglycans [20] whereas VEGF121 and VEGF165 are secretory forms.
The prognostic value of pretreatment serum levels of MMP-9 and VEGF in patients with NSCLC is still unclear.
The aim of the present study was to analyse the association of pretreatment serum levels of MMP-9 and VEGF with clinicopathological parameters and outcome, and to evaluate their prognostic relevance in patients with NSCLC.
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Patients and methods |
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Pretreatment evaluation included a complete history and physical examination with evaluation of the Karnofsky performance status score, chest X-rays in posterioanterior and lateral views, a computed tomography (CT) scan of the chest, sonography or CT of the upper abdomen, radionuclide bone scan, fiberoptic bronchoscopy with bronchoaspirate and/or brushing and/or bronchial biopsy, complete blood cell count, and serum chemistry analysis. Brain CT was only performed if clinically indicated. Patients who had signs of metastasis in mediastinal lymph nodes in the CT scan of the chest received a mediastinoscopy before surgery. All patients gave informed consent before entering this study.
Twenty millilitres of venous blood were taken before surgery or chemotherapy from each non-fasting patient, and was subsequently centrifuged at 2100 g for 7 min at 4°C. The supernatant was transferred into microtubes and stored at 70°C until use.
Serum samples were analysed for MMP-9 and VEGF with Human MMP-9 (total) Immunoassay QuantikineTM and Human VEGF Immunoassay QuantikineTM (R&D Systems, Inc., Minneapolis, MN, USA). The principle of these assays employs a quantitative sandwich enzyme immunoassay technique. The minimum detectable concentration of MMP-9 is <0.156 ng/ml and of VEGF<9.0 pg/ml. Each serum sample was determined twice. The mean intrabatch coefficients of variation range between 1.9% and 2.9% for the MMP-9 immunoassay and between 4.5% and 6.7% for the VEGF immunoassay. MMP-9 and VEGF concentrations were calculated using Delta SOFT 3 computer software (Bio Metallics, Inc., Princeton, NJ, USA).
Statistical analysis
The median follow up of all patients was 945 days (minimum 154 days, maximum 1218 days). Dates of diagnosis, relapse and death were recorded. From these data, survival curves were prepared according to the KaplanMeier method for time to death. Survival curves were compared with a log-rank test. The serum concentrations of MMP-9 and VEGF in our study population were compared with data from healthy controls (37 persons) provided by R&D Systems with a two-tailed Students t-test. The KruskalWallis test was used to compare serum levels of MMP-9 or VEGF with tumour stage.
All factors were investigated for their prognostic value in a univariate analysis using the Cox regression model. Variables with sufficient statistical prognostic power (P <0.1) were investigated, then subjected to a multivariate analysis and to a model selection approach in order to determine a final parsimonious prognostic model for patient survival based on this data set. Forward and backward variable selection and likelihood-ratio statistic were calculated with SPSS software (Version 10.0; SPSS, Inc., Chicago, IL, USA). The outcome of the Cox regression was described quantitatively by the statistical estimate of the regression parameter ß and its standard error, its risk ratio exp (ß), the 95% confidence interval, and the respective P value obtained from the Wald test statistic. MMP-9 and VEGF were categorised in quartiles (MMP-9: 806 ng/ml, 807 to
1293 ng/ml, 1294 to
1912 ng/ml, >1912 ng/ml; VEGF:
417 pg/ml, 418 to
630 pg/ml, 631 to
1205 pg/ml, >1205 pg/ml) for survival and Cox regression analysis.
All statistical tests were performed using SPSS software.
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Results |
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Serum levels of MMP-9/VEGF and patient characteristics
A significant relationship between serum levels of MMP-9 and tumour stage was observed (P = 0.01). Patients with early disease had a median serum level of 910 ng/ml (range 1563055), those with locally advanced disease 1340 ng/ml (range 2592892), and those with metastatic disease 1796 ng/ml (range 1612897) (Figure 1). The relationship between serum levels of VEGF and tumour stage was also highly significant (P <0.0005). The median serum levels in patients with stage I/II disease was 506 pg/ml (range 692087), with stage IIIA/B disease 630 pg/ml (range 802708), and with stage IV disease 890 pg/ml (range 3156241) (Figure 2).
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Correlation between serum levels of MMP-9/VEGF and outcome
The median survival of all 118 patients was 602 days [range 291218+ (+ indicates that the patient with the longest follow up is still alive)]. The 72 patients who had undergone surgery had a median survival of 972 days (range 351218+), whereas the 46 patients who were treated with chemotherapy had a median survival of 298 days (range 29989) (P <0.00005) (Figure 3).
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Patients with resected disease and an MMP-9 serum level of 1293 ng/ml or lower (i.e. cutoff level between the second and third quartile) had a significantly longer survival [median survival longer than 1218 days (range 421218+)] than those with a serum level of higher than 1293 ng/ml [median survival 540 days (range 351168+)] (P = 0.002).
Subanalysis of resected patients with stage I/II and stage IIIA/B disease has demonstrated that only patients with stage I/II disease and an MMP-9 serum level of 1293 ng/ml or lower had a significantly longer survival [median survival longer than 1218 days (range 461218+)] than those with a serum level of higher than 1293 ng/ml [median survival 421 days (range 351168+)] (P = 0.001) (Figure 5), whereas no significant difference was found in patients with resected stage IIIA/B disease (P = 0.49) nor in patients who were treated with chemotherapy (P = 0.06).
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An analysis including all resected patients has also demonstrated no significant difference in survival for patients with a VEGF serum level of 630 pg/ml or lower (cutoff level between the second and third quartile) versus higher than 630 pg/ml (P = 0.20). On the other hand, subanalysis showed a significant survival benefit for patients with stage I/II disease and a VEGF serum level of 630 pg/ml or lower [median survival longer than 1218 days (range 1031218+) versus 421 days (range 351125+)] (P = 0.04) (Figure 6). No significant difference in survival was observed for patients with resected stage IIIA/B disease (P = 0.38). In contrast, patients treated with chemotherapy who had a VEGF serum level of 630 pg/ml or lower did survive significantly longer than those with serum levels higher than 630 pg/ml [median survival 630 days (range 29989) versus 290 days (range 46587)] (P = 0.02).
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Discussion |
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We observed a significant elevation of serum levels of MMP-9 and VEGF in our patient population in comparison with healthy controls. Similar results were reported for patients with various types of cancer, including lung cancer [2232].
In our investigation we found a significant positive correlation between serum levels of both MMP-9 and VEGF and tumour stage. So far, only one group [33] has described a significant elevation of serum VEGF in a small patient population with advanced NSCLC in comparison with those with early disease; no significant difference was detected between locally advanced and metastatic disease. Furthermore a positive correlation between VEGF serum level and size of the primary tumour has been reported [34]. In contrast, other authors did not find a significant correlation between VEGF serum level and tumour stage or the presence of distant metastases in 70 patients with small-cell or non-small-cell lung cancer [25].
To the best of our knowledge, our findings are the first report of a significant association between MMP-9 serum level and tumour stage in NSCLC. So far, two groups could not identify a relationship between MMP-9 serum or plasma levels and tumour stage in NSCLC in small patient populations [29, 30].
So far, no association has been reported between serum levels of MMP-9 or VEGF and the different histological types of NSCLC (squamous-cell carcinoma, adenocarcinoma, large-cell carcinoma) [25, 29, 33], supporting our findings.
We observed a significant correlation between serum levels of MMP-9 as well as VEGF and survival in resected patients with stage I/II disease. Patients with an MMP-9 serum level of 1293 ng/ml or lower or a VEGF serum level of 630 pg/ml or lower had a significantly longer survival than patients with higher serum concentrations. No significant difference was found in patients with stage IIIA/B disease, whereas a significant correlation between VEGF serum level and survival was observed in the patients with metastatic disease who were treated with chemotherapy.
In the present study, tumour stage, histological type, serum level of MMP-9 as well as VEGF, Karnofsky performance status, haemoglobin concentration, lactate dehydrogenase concentration and platelet count had prognostic significance in the univariate Cox regression analysis. However, in a multivariate analysis only tumour stage, the Karnofsky performance status, gender and pretreatment MMP-9 serum level were independent prognostic factors.
The prognostic impact of MMP-9 and VEGF serum or plasma levels still remains unclear. In most published studies, serum VEGF has no prognostic influence on survival [24, 25]. The prognostic role of VEGF expression in tumour tissue is discussed controversially by several study groups [3541]. In an immunohistochemical study of 69 stage I/II NSCLC patients no prognostic influence of VEGF expression has been found [36]. Only in the context of microvessel density did VEGF expression yield prognostic information in 81 operable NSCLC patients [37]. In contrast, VEGF expression was found to be a significant prognostic factor in a univariate analysis in 223 patients with operable NSCLC [35]. Other study groups identified VEGF expression in tumour tissue as an independent prognostic factor in NSCLC [3840].
To the best of our knowledge, this is the first report identifying MMP-9 serum level as an independent prognostic factor in NSCLC in a multivariate analysis. A formerly described significant correlation between MMP-9 plasma level and 1-year survival in 79 NSCLC patients by Ylisirnio et al. [42] could not be confirmed in a multivariate analysis. The same authors could not show prognostic significance for serum MMP-9 in a univariate analysis in another comparable patient population [43].
Similar to VEGF, contradictory results exist concerning the prognostic impact of MMP-9 expression in lung cancer tissue detected by immunhistochemistry methods. In one investigation the expression of MMP-9 in tumour tissue did not correlate with survival in 90 patients with stage IIV adenocarcinomas of the lung [44], whereas a significant association was observed in 79 patients with adenocarcinomas staged as T1 disease [45]. In an additional further study with 169 NSCLC patients with stage IIIIA the expression of MMP-9 in tumour tissue was identified as an independent prognostic factor [46]. In this investigation a significant proportion of tumours co-expressed MMP-9 and epidermal growth factor receptor (EGFR). The co-expression of both markers was significantly associated with a poor prognosis. The authors discussed that the EGFR signalling pathway may play an important role in the invasive behaviour of NSCLC via specific up-regulation of MMP-9 [46].
In conclusion, our results demonstrate a correlation between tumour stage and pretreatment serum levels of both MMP-9 and VEGF. However, in our patient population only the pretreatment serum level of MMP-9 was identified as an independent prognostic factor and had a higher prognostic relevance than those of VEGF. Future studies should be conducted and may support our hypothesis that the pretreatment MMP-9 serum level is a new powerful prognostic marker and may help to assess an individual risk profile for NSCLC patients with stage I/II diseasetogether with classical prognostic markers like TNM stage and performance statusin order to stratify these patients into low- and high-risk groups. Based on the data of this investigation, we plan a large study where patients with stage I/II disease are stratified for adjuvant chemotherapy according to their serum level of MMP-9 before surgery. Furthermore, 200 patients with metastatic disease have currently been enrolled in an ongoing study to investigate the prognostic value of pretreatment MMP-9 and VEGF serum levels in patients who received palliative chemotherapy.
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Acknowledgements |
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Footnotes |
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