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From the Department of General and Cardiothoracic Surgery (MT, MO, IM, YT, KK, GW), Kanazawa University School of Medicine; and Department of Thoracic Surgery (YO), Ishikawa Prefectural Central Hospital, Kanazawa, Japan.
ABSTRACT
Background: The aim of the present study was to evaluate the diagnostic utility of levels of circulating vascular endothelial growth factor (VEGF)-C, matrix metalloproteinase-9 (MMP-9), and VEGF and to verify that the combination assay of these circulating factors is a clinically useful indicator to predict the presence of lymph node metastasis in non–small cell lung cancer (NSCLC).
Methods: A series of 78 patients who underwent surgery for NSCLC was used in this study. Serum VEGF-C and VEGF and plasma MMP-9 levels were analyzed with enzyme-linked immunosorbent assay (ELISA) kits. Logistic regression models were used to analyze the influence of VEGF-C, MMP, and VEGF levels on the probability of presence or absence of lymph node metastasis.
Results: Patients with lymph node metastasis had higher serum VEGF- C, VEGF, and plasma MMP-9 concentrations than did those without metastasis (VEGF-C, P = .0004; VEGF, P = .001). Serum VEGF- C reached a sensitivity of 85% and specificity of 68% when a cutoff value of 1762.0 pg/mL was applied, while VEGF reached 80% sensitivity and 59% specificity at 316.8 pg/mL. MMP-9 reached a sensitivity of 63% and specificity of 75% when a cutoff value of 51.4 ng/mL was applied. In the ROC curve analysis, VEGF-C (0.761) had the biggest areas under the ROC curve, followed by MMP-9 (0.723) and VEGF (0.694). Combination assay of three markers had higher sensitivity and specificity for prediction than single-marker assays (AUC = 0.837).
Conclusions: This study has confirmed that combination assay of three markers to determine VEGF-C, MMP-9, and VEGF expression in circulation detects lymph node metastasis in NSCLC with higher accuracy than single-marker assays.
Key Words: Enzyme-linked immunosorbent assay • Lymph node metastasis • Matrix metalloproteinase-9 • Vascular endothelial growth factor • Vascular endothelial growth factor-C
The presence of lymph node metastasis is a very important prognostic factor in patients with potentially resectable non–small cell lung cancer (NSCLC). Preoperative examinations such as computed tomography (CT), radiolabeled 18F-fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET), and mediastinoscopy are the commonly used staging methods for lymph node metastasis. Matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) are two of the most potent factors involved in angiogenesis.1 We have previously reported that VEGF-C expression in NSCLC was significantly associated with lymph node metastasis, lymphatic vessel invasion,2 and nodal microdissemination.3 We have also reported that circulating VEGF- C could give additional information for discriminating between the absence and presence of lymph node metastasis in patients with NSCLC.
Combination assay of circulating VEGF- C with VEGF revealed a high specificity for diagnosing lymph node metastasis preoperatively.4 Assays such as the quantitative measurement of serum protein can be easily and frequently performed because of their minimal invasiveness in comparison with examinations using surgically obtained tissue. However, its diagnostic value is not inferior to that of PET or mediastinoscopy. The aim of the present study was to evaluate the diagnostic utility of levels of circulating VEGF-C, MMP-9, and VEGF and to verify that the combination assay of these circulating factors is a clinically useful indicator to predict the presence of lymph node metastasis in NSCLC.
PATIENTS AND METHODS
Patients
Serum samples were obtained from 78 patients with primary non-small cell lung cancer who underwent curative operations with routine systematic nodal dissemination of the hilar and mediastinal lymph nodes at Kanazawa University Hospital and Ishikawa Prefectural Central Hospital between June 2002 and May 2003. The pathologic stage was classified according to the Japanese Lung Cancer Society classification. The 55 men and 23 women studied had a median age of 67 years (range, 42–79 years). The pathological stage was classified as stage I in 41 patients, stage II in 10, and stage III in 27. The pathological types were 55 adenocarcinomas, 18 squamous cell carcinomas, and 5 adenosquamous cell carcinomas and large cell carcinomas. Patients with malignant pleuritis or a medical condition contraindicating thoracotomy were excluded. No patient had received blood transfusions, radiotherapy, or chemotherapy prior to the study.
Methods
Blood samples were drawn by venous puncture preoperatively and were divided into tubes without anticoagulant (for serum samples) or into heparin-coated tubes (for plasma samples). Within an hour of collection, blood samples were centrifuged at 2000 rpm for 10 minutes, and aliquots were frozen at –80°C for later analysis. Informed consent was obtained from all of the patients.
Serum samples were analyzed for VEGF-C and VEGF with use of commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kits. The VEGF-C kit was kindly provided by the manufacturer (number 17741; IBL Co., Ltd., Fujioka, Gunma, Japan). The sensitivity limit of the VEGF-C assay was 46.9 pg/mL. The VEGF kit was the Human VEGF Immunoassay (Quantiline; R&D Systems, Minneapolis, MN). The sensitivity limit of the VEGF assay was 9.0 pg/mL. The coefficient of variation was <5.0%. Plasma MMP-9 concentrations were measured with a sandwich enzyme immunoassay kit (Fuji Chemical Industries, Toyama, Japan). The minimum detectable concentration of MMP-9 was 0.156 ng/mL.
Statistical Analysis
Because of the skewed distribution, median value (range) is given to describe VEGF-C level. Peripheral levels of angiogenic factors between different groups were compared with the Mann-Whitney U-test. Logistic regression models were used to analyze the influence of VEGF-C, MMP, and VEGF levels on the probability of presence or absence of lymph node metastasis. To calculate the sensitivity and specificity of peripheral levels of each factor at serial cutoff points, a receiver operating characteristics (ROC) curve was plotted with use of the SAS software package (SAS Institute, Cary, NC). The utility of each marker was compared by taking the area under the ROC curve.
RESULTS
Concentrations of VEGF-C, MMP-9, and VEGF and Clinical Features in 78 Primary NSCLC Patients
As shown in Table 1, there were no statistically significant differences in any of the markers in terms of patients’ age, sex, or histologic data. However, the concentrations of all the three markers gradually increased with the progression of pathological stages (VEGF-C: P = .0003; MMP-9: P = .0012; VEGF: P = .0044). Patients with lymph node metastasis had higher serum VEGF- C, VEGF, and plasma MMP-9 concentrations than did those without metastasis (VEGF- C: 2046.7 [1905.1–2398.8 pg/mL] vs. 1419.5 (932.8– 1826.5 pg/mL, P = .0004; VEGF: 362.9 [269.8–519.9 pg/mL] vs. 225.4 [151.8–352.6 pg/mL], P = .0026; MMP-9: 80.3 [32.3–110.5 pg/mL] vs. 31.1 [14.8–65.7 pg/mL], P = .001]) (Figs. 1-3).
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Combination Diagnosis of VEGF-C, MMP-9, and VEGF for Lymph Node Metastasis
The summation of the data for the three markers was calculated, and the ROC curve was reconstructed (Fig. 5). The data from the combination assay were calculated with the following formula:
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The revision coefficient was defined as follows:
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At 4776.2 pg/mL, the combination assay of three markers reached a sensitivity of 83% and specificity of 76%. The combination assay of three markers had higher sensitivity and specificity for prediction (AUC = 0.837) than the single-marker assays (AUC = 0.761, VEGF-C) (confidence interval: –0.065–0.191; P = .048).
DISCUSSION
The involvement of lymph node metastasis is a very important prognostic factor for patients with potentially resectable NSCLC. CT of the chest is the most commonly used noninvasive method for staging the lymph node metastasis, but it is far from satisfying and less accurate than mediastinoscopy. The CT imaging criteria for tumor involvement are morphologic; that is, the criteria rely on the size and shape of the lymph nodes. Evaluation of circulating cytokine concentrations would be accessible at hospitals where there is no access to PET. Furthermore, it would be a noninvasive and inexpensive examination. Our results show that serum VEGF-C level evaluation is superior to CT for nodal staging in primary NSCLC.5
VEGF is the most potent and specific growth factor of endothelial cells.6 It has been reported that strong VEGF expression in primary sites and nodal metastasis are correlated,7 and VEGF-C expression was significantly associated with lymph node metastasis and lymphatic vessel invasion.2 Furthermore, our results suggest that circulating VEGF-C can give additional information for discriminating between the absence or presence of lymph node metastasis in lung cancer patients, and combination assay of circulating VEGF-C with VEGF revealed a high specificity for diagnosing lymph node metastasis preoperatively.4 The mechanism responsible for the elevation of circulating angiogenic factors is an important problem. Regarding this point, we previously reported that circulating VEGF level is correlated with intratumoral VEGF level.8
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.
The gelatinase MMP-9 is one of the 18 members of the MMP family reported so far, and it is known as gelatinase B, or a 92-kDa type IV collagenase. Several steps are required to develop malignant tumor cell metastasis. The first critical phase is the destruction and penetration of the basement membrane, which is part of the extracellular matrix, by the tumor cells.
Hrabec et al.9 reported that circulation levels of MMP-9 in lung cancer patients was 3.6-fold higher than in healthy volunteers. In addition to this, Laack et al.1 reported that the pretreatment MMP-9 serum level is a new powerful prognostic marker in patients with NSCLC. Shou et al.10 reported that MMP-9 expression levels showed a significant correlation to pN-factor. In this study, MMP-9 levels in circulation were higher in patients with NSCLC than in healthy volunteers (data not shown).
The mechanism and function of increasing MMP-9 are unclear at present. Iizasa et al.11 reported that the circulating MMP-9 does not seem to be directly produced by cancer or stromal cells, and there may be other sites responsible for increased circulating levels of MMP-9 that correlate with the existence of tumor tissues.
In this study, VEGF-C and VEGF were assayed in a standard manner with serum samples, but MMP-9 was analyzed with plasma samples. The majority of reported studies used serum samples for analyzing circulating MMP-9 level.1,9,12–14 We could find only two reports analyzing plasma MMP-9 levels.11,15 In a comparison of these studies, higher MMP-9 levels were detected in serum samples than in plasma samples. John et al.16 asserted that increased values in serum are supposedly caused by the release of MMP-9 during platelet activation and the clotting process, and they recommended using heparin plasma for samples to avoid preanalytical mistakes for MMP-9 measurements.
To the best of our knowledge, this is the first report identifying MMP-9 plasma level as a marker for predicting lymph node metastasis in NSCLC. Furthermore, no studies have addressed the usefulness of the combination assay of three markers for detecting lymph node metastasis in NSCLC preoperatively. The way of combining the data for three makers is controversial. In this study, the summation of the data was calculated. All three markers are equally reflected in the data of the combination assay after the data on MMP-9 and VEGF are multiplied by the revision coefficient.
In conclusion, this study has confirmed that combination assay of three markers to determine levels of VEGF-C, MMP-9, and VEGF expression in circulation has greater accuracy than assaying each single marker in detecting lymph node metastasis in NSCLC. Additional expanded studies are necessary to evaluate whether further accuracy can be obtained in detecting lymph node metastasis with use of more than three markers in the circulation of NSCLC patients.
FOOTNOTES
Received January 13, 2004; accepted June 8, 2004.
Address correspondence and reprint requests to: Masaya Tamura, MD, Department of General and Cardiothoracic Surgery, Kanazawa University School of Medicine, Takara-machi 13–1, Kanazawa, 920–8641, Ishikawa, Japan; Fax: 81 076 222 6833; e-mail: m-tamura{at}sf.m.kanazawa-u.ac.jp.
REFERENCES
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