Japanese Journal of Clinical Oncology 33:186-191 (2003)
© 2003 Foundation for Promotion of Cancer Research
Determining the Levels of Matrix Metalloproteinase-9 in Portal and Peripheral Blood is Useful for Predicting Liver Metastasis of Colorectal Cancer
1 Department of Surgery, Saitama Medical Center, Saitama Medical School, Kawagoe, Saitama, 2 Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, University of Tokyo, Tokyo and 3 Pharmacology Division, National Cancer Center Research Institute, Tokyo, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Background: Matrix metalloproteinase-9 (MMP-9) is one of the MMPs that play an important role in cancer invasion and metastasis. Increased levels of MMP-9 in tumor tissue have been found to correlate with advanced stages of colorectal cancer. However, the clinical significance of determining the levels of MMP-9 in blood samples from patients with colorectal cancer has not yet been clarified. The purpose of this study was to clarify the relationship between the clinicopathological variables of colorectal cancer and MMP-9 levels of drainage (portal) or peripheral venous blood and to examine whether this assay would be useful for predicting liver metastasis.
Methods: Blood samples were obtained from peripheral and drainage veins of 102 patients with colorectal cancer during surgery and the plasma levels of MMP-9 were determined by a one-step sandwich enzyme immunoassay.
Results: The levels of portal MMP-9 were significantly higher than those of peripheral blood (P < 0.01, n = 102). The levels of MMP-9 in peripheral venous blood did not correlate with any of the 12 clinicopathological variables examined, while the levels of MMP-9 in portal blood correlated with macroscopic type of the primary tumor (P = 0.02), Dukes’ stage (P = 0.03), liver metastasis (P < 0.01) and lymph node metastasis (P = 0.02). By setting the cutoff ratio of portal to peripheral MMP-9 levels at 1.6 in patients with curative resection (n = 73), elevated ratios predicted subsequent emergence of liver metastases with 77.8% sensitivity, 81.3% specificity and 80.8% accuracy.
Conclusion: The results suggest that synchronous determination of the levels of MMP-9 in portal and peripheral blood would be useful for selecting colorectal cancer patients at high risk of hepatic recurrence.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Matrix metalloproteinase-9 (MMP-9) is one of the MMPs that play an important role in cancer invasion and metastasis by degrading extracellular matrix components and basement membrane (1,2). Increased levels of MMP-9 in the tumor tissue, identified by mRNA measurement (3,4,5) or zymography (6,7) have been found to correlate with advanced stages of colorectal cancer. Specifically, Zeng and Guillem (8) suggested that activation of MMP-9 may be a pivotal event during the formation of colorectal liver metastasis. The blood levels of MMP-9 in patients with colorectal cancer also have been confirmed to be elevated compared with those of normal subjects (9,10). However, correlations between the peripheral blood level of MMP-9 and clinicopathological variables including liver metastasis have not yet been clarified. In addition, to the best of our knowledge, correlations between the level of MMP-9 of drainage venous (portal) blood from colorectal cancer lesions and the clinicopathological variables have not yet been examined, even though the level of MMP-9 in portal blood is thought to be related more closely to the quantity of this enzyme released from cancer lesions than those of the peripheral venous blood.
The purpose of this study was to clarify the relationship between the clinicopathological variables of colorectal cancer and the levels of MMP-9 in portal and peripheral blood and to examine whether determining the levels of MMP-9 in portal and peripheral blood would be useful for selecting patients at high risk of developing liver metastasis. Since we found in this study that the levels of MMP-9 in portal blood were significantly higher than those of peripheral blood, the ratios of portal to peripheral MMP-9 were also determined. We examined the relationship between the ratios of portal to peripheral MMP-9 and clinicopathological factors and found important results.
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PATIENTS AND METHODS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Patients
A total of 102 patients who underwent colorectal resection for histologically verified adenocarcinoma between November 1996 and April 1999 were included in this study. Patients’ ages ranged from 37 to 87 years (median, 64.8 years) and the male to female ratio was 1.8. Radical surgery for cure was performed in 82 patients (Dukes’ A, 15; Dukes’ B, 33; Dukes’ C, 34) and a palliative resection was selected in 20 Dukes’ D patients with massive peritoneal dissemination, lymph node involvement or hematogenous metastases. The description of clinicopathological variables was undertaken according to the rules adopted by the Japanese Research Society for Cancer of the Colon and Rectum (11) or the TNM classification (12).
Collection of Blood Samples
Blood samples were obtained during laparotomy. Before mobilization of the tumor, peripheral venous and portal blood specimens were collected from all the patients. Peripheral blood was taken from an antecubital venepuncture. The portal blood was collected from the main drainage vein closest to the tumor by inserting a venous catheter. The plasma samples were stored at –40°C until the measurement of MMP-9 concentration. Informed consent was obtained from all the patients.
Measurement of MMP-9 Level
Plasma MMP-9 concentrations were determined using a previously reported one-step enzyme immunoassay (EIA) employing monoclonal antibodies (Fuji Chemical, Takaoka, Japan) (13,14). This assay detects the precursor form proMMP-9, the 83 kDa intermediate form of MMP-9 and their complex forms with tissue inhibitor of metalloproteinase-1 (TIMP-1) instead of the active 67 kDa form. The upper limit of the normal range of plasma MMP-9 was determined to be 64.0 ng/ml, corresponding to the mean + 2SD (standard deviation of the mean) from normal healthy volunteers (n = 20).
Follow-Up
The patients undergoing curative colorectal resection for Dukes’ A, B or C tumor were followed-up periodically with clinical and laboratory examinations including ultrasonography and computed tomography and chest X-P performed at intervals of 3–6 months. Of these, four patients developed extrahepatic lesions (lung, 3; local, 1) as the initial site of recurrence 13–24 months postoperatively. Four patients developed extracolonic malignancies (multiple myeloma, 1; primary lung cancer, 2; hepatocellular carcinoma, 1). Death unrelated to colorectal cancer was confirmed in one patient. The other 73 patients remained at risk of development of liver metastasis as the first recurrence site. The median follow-up period of these patients was 41 months (range, 31–60 months) or until death.
Statistical Methods
Statistical analysis was performed using the software StatFlex (version 5.0; Artech, Osaka, Japan). Data were expressed as means ± standard error (SE) of means. Comparisons of continuous variables between groups were performed by one-way analysis of variance (ANOVA) or Student’s t-test. Determining the optimal cutoff value was performed by evaluating receiver operating characteristics (ROC) plots. P-values <0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasma Levels of MMP-9 in Portal and Peripheral Blood
For all patients (n = 102), the plasma levels of peripheral blood were within the range 43.9 ± 4.5 ng/ml. Eighteen out of 102 patients (17.6%) showed plasma level of MMP-9 in peripheral blood greater than the cutoff value (64.0 ng/ml) determined from the healthy volunteers. The plasma levels of MMP-9 in portal blood (66.3 ± 8.6 ng/ml) were significantly higher than those of peripheral blood (P < 0.01, n = 102) (Table 1).
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Clinicopathological Variables and MMP-9 Level
The level of MMP-9 in peripheral blood did not significantly correlate with the 12 clinicopathological variables age, gender, site of tumor, size of tumor, histological differentiation, depth of tumor invasion, macroscopic type, Dukes’ staging, liver metastasis, lymph node metastasis, lymphatic invasion and venous invasion. The plasma level of MMP-9 in portal blood was significantly elevated in patients with type 3 or 4 tumors (P = 0.02), advanced Dukes’ stage (P = 0.03), liver metastasis (P < 0.01) and lymph node metastasis (P = 0.02) and tended to be higher in patients with moderately or poorly differentiated adenocarcinoma (P = 0.09) (Table 2). The ratio of portal to peripheral MMP-9 levels was higher in patients with moderately or poorly differentiated adenocarcinoma (P = 0.045), liver metastasis (P < 0.01), lymph node metastasis (P = 0.04) and excessive venous invasion (P = 0.02) and tended to be higher in patients with advanced Dukes’ stage (P = 0.08) (Table 3).
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Ratio of Portal to Peripheral MMP-9, MMP-9 in Portal and Peripheral Blood According to Development of Liver Metastasis
Of the 73 patients who were evaluable for recurrence, nine developed liver metastasis as the first recurrence site. The plasma levels of MMP-9 in peripheral blood were not significantly different between patients without recurrence (46.8 ± 5.6 ng/ml, n = 64) and those who developed liver metastasis (20.4 ± 5.1 ng/ml, n = 9) (P = 0.12). The levels in portal blood also were not significantly different between patients without recurrence (57.6 ± 9.0 ng/ml, n = 64) and those who developed liver metastasis (56.8 ± 13.9 ng/ml, n = 9) (P = 0.94). However, the ratio of MMP-9 was significantly higher in patients who developed liver metastasis at the first recurrence site (3.6 ± 1.0, n = 9) than those without recurrence (1.6 ± 0.2, n = 64) (P < 0. 01) (Fig. 1).
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The optimal cutoff value of the ratio of portal to peripheral MMP-9 levels for predicting the development of liver metastasis was set at 1.6 with 77.8% sensitivity, 81.3% specificity and 80.8% accuracy by evaluating ROC plots (Table 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several researchers (15,16) have demonstrated that MMP-9 mRNA and protein were localized in colon cancer cells in addition to macrophages and neutrophils. Others (3,17–19) reported that MMP-9 mRNA and protein were not found in colon cancer cells but only in macrophages and neutrophils and in the surrounding stromal cells. Despite the conflicting results of the cellular source of MMP-9, elevated blood levels of this protein have been assumed to be a reflection of the enhanced expression associated with colorectal cancer.
With regard to the release of MMP-9 from cancerous tissue into the peripheral blood, it has not yet been clarified whether MMP-9 is mainly released into the portal system or the lymphatic system, or both. In the present study, the mean plasma level of the portal blood was significantly higher than that of the peripheral blood. This result indicates that MMP-9 enters the portal blood at least partially via the drainage vein, because theoretically MMP-9 in the portal blood should be the same as in the peripheral blood, if MMP-9 does not enter the portal blood and is mainly drained into the thoracic duct via lymphatics. The other possible explanations for the higher level of MMP-9 in the portal blood are that this protein in the drainage venous blood has not yet been diluted by the whole blood and has not yet been degraded by the liver or by the systemic circulation.
We did not find any correlations between the plasma level of MMP-9 in the peripheral blood and the clinicopathological variables. Although the levels of MMP-9 in the peripheral blood have been known to be elevated in patients with colorectal cancer compared with those of normal subjects (9,10), only a few studies investigated the correlation between the blood level of MMP-9 and the clinicopathological variables. According to Öberg et al. (20), serum levels of MMP-9 did not differ according to Dukes’ stage and determining the levels was not useful for predicting the prognosis of patients with colorectal cancer. Sonnante et al. (21) reported that the plasma MMP-9 levels in peripheral blood were not influenced by different histological grades of tumors or by different Dukes’ stages. Our results are in agreement with their results, suggesting that the measurement of plasma MMP-9 levels in peripheral venous blood has little importance in the management of colorectal cancer.
In contrast to the plasma levels in peripheral blood, the elevated plasma levels of MMP-9 in portal blood correlated with several pathological factors including infiltrative features of the primary tumor (type 3 or 4), advanced Dukes’ stage, presence of liver metastasis and lymph node metastasis. These results suggest that the plasma MMP-9 levels of the portal blood reflect the metastatic status or potential of colorectal cancer more accurately than those of the peripheral venous blood. The exact reasons for the close relationship between the elevated levels of this protein in the portal blood and the several clinicopathological factors associated with metastasis or prognosis are unclear. Perhaps these clinicopathological factors may be associated with the increased quantity of this protein released directly into the liver from cancer lesions. However, in cases when the level of portal MMP-9 is high and the peripheral MMP-9 is similarly increased, we must inevitably conclude that the direct release of the protein into the portal system is not reflected.
Elevated ratios of portal to peripheral MMP-9 levels also correlated with several pathological factors associated with metastatic status or potential when all patients were analyzed. Importantly, when analysis was restricted to patients who had undergone curative surgery, the elevated ratios could be a marker for predicting the emergence of metachronous liver metastasis, but the underlying mechanism is unclear. Although the concept may be much more complex, the following explanations can be proposed. First, the ratio may represent the tendency of MMP-9 to be transported from the primary lesion to the liver via the portal system. Furthermore, the ratio may correlate positively with the number of colorectal cancer cells, which secrete MMP-9 into the portal system and consequently form the liver metastasis. Another explanation is that the MMP-9 production from cancer cells and/or non-malignant cells exceeds the deactivating capability of tissue inhibitor of metalloproteinase-1 (TIMP-1), leading to the formation of liver metastasis. The relationship between the ratio of portal to peripheral MMP-9 levels and the imbalance of MMP-9 to TIMP-1 in the primary lesions deserves further investigation.
The method for determining the optimal cutoff ratio of portal to peripheral MMP-9 levels may be controversial. However, the cutoff ratio determined based on ROC plotting is one of the standard methods and this value subsequently discriminated patients with metachronous liver metastasis from those without recurrence with satisfactory sensitivity, specificity and accuracy. Further collection of cases may be needed to draw conclusions about the optimal cutoff value for predicting the emergence of liver metastasis.
In conclusion, we found that elevated plasma MMP-9 levels of the portal blood reflect the presence of metastatic lesions of colorectal cancer, while no relationship was found between the plasma MMP-9 levels of peripheral blood and various clinicopathological factors. In addition, elevated ratios of portal to peripheral MMP-9 levels predicted the subsequent emergence of liver metastasis. Synchronous determination of the plasma MMP-9 levels of the portal and peripheral blood at curative resection would be useful for selecting colorectal cancer patients at high risk of hepatic recurrence, who may be good candidates for postoperative adjuvant chemotherapy.
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FOOTNOTES |
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+ For reprints and all correspondence: Hideyuki Ishida, Department of Surgery, Saitama Medical Center, Saitama Medical School, 1981, Kamoda, Kawagoe, Saitama 350-8550, Japan. E-mail: ishida{at}zb3.so-net.ne.jp
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REFERENCES |
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1 Liotta LA, Stetler-Stevenson WG. Metalloproteinases and cancer invasion. Semin Cancer Biol 1990;1:99–106.[Medline]
2 De Clerk YA, Shimada H, Taylor SM, Langley KE. Matrix metalloproteinases and their inhibitors in tumor progression. Ann N Y Acad Aci 1994;732:222–32.[Web of Science][Medline]
3 Zeng ZS, Guillem JG. Colocalisation of matrix metalloproteinase-9-mRNA and protein in human colorectal cancer stromal cells. Br J Cancer 1996;74:1161–7.[Web of Science][Medline]
4 Zeng ZS, Guillem JG. Distinct pattern of matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1 mRNA expression in human colorectal cancer and liver metastasis. Br J Cancer 1995;72:575–82.[Web of Science][Medline]
5 Zeng ZS, Huang Y, Cohen AM, Guillem JG. Prediction of colorectal cancer relapse and survival via tissue RNA levels of matrix metalloproteinase-9. J Clin Oncol 1996;14:3133–40.[Abstract]
6 Linbakk NB, Talbot I, Smith RA, Wilkinson K, Balkwill F. Matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) type IV collagenases in colorectal cancer. Cancer Res 1996;56:190–6.
7 Baker EA, Bergin FG, Leaper DJ. Matrix metalloproteinases, their tissue inhibitors and colorectal cancer staging. Br J Surg 2000;87:1215–21.[CrossRef][Web of Science][Medline]
8 Zeng ZS, Guillem JG. Unique activation of matrix metalloproteinase-9 within human liver metastasis from colorectal cancer. Br J Cancer 1998;78:349–53.[Web of Science][Medline]
9 Zucker S, Lysik RM, Zarrabi MH, Moll U. Mr 92 000 type IV collagenase is increased in plasma of patients with colon cancer and breast cancer. Cancer Res 1993;53:140–6.
10 Zucker S, Lysik RM, Dimassoni BS, Zarrabi H, Moll U, Grimson R, et al. Plasma assay of gelatinase B: tissue inhibitor of metalloproteinase complexes in cancer. Cancer 1995;76:700–8.[CrossRef][Web of Science][Medline]
11 Japanese Research Society for Cancer of the Colon and Rectum. General Rules for Clinical and Pathological Studies on Cancer of Colon, Rectum and Anus in Japan. Tokyo: Kanehara 1998.
12 Sobin LH, Wittekind C, editors. TNM Classification of Malignant Tumors, 5th ed. New York: Wiley-Liss 1997.
13 Shinya T, Okada Y, Hayakawa T. A one-step sandwich enzyme immunoassay for inactive precursor and complex form of human matrix metalloproteinases 9 (92 kDa gelatinase/type type IV collagenase, gelatinage B) using monoclonal antibodies. Clin Chim Acta 1994;231:79–88.[CrossRef][Web of Science][Medline]
14 Torii A, Kodera Y, Uesaka K, Hirai T, Yasui K, Morimoto T, et al. Plasma concentration of matrix metalloproteinase 9 in gastric cancer. Br J Surg 1997;84:133–6.[CrossRef][Web of Science][Medline]
15 Ogata Y, Miura K, Ohkita A, Nagase H, Shirouzu K. Imbalance between matrix metalloproteinase 9 and tissue inhibitor of metalloproteinases 1 expression by tumor cells implicated in liver metastasis from colorectal carcinoma. Kurume Med J 2001;48:211–8.[Medline]
16 Jeriorska M, Haboubi NY, Schofield PF, Ogata Y, Nagase H, Woolley DE. Distribution of gelatinase B (MMP-9) and type IV collagen in colorectal carcinoma. Int J Colorectal Dis 1994;9:141–8.[CrossRef][Web of Science][Medline]
17 Nielsen BS, Timshel S, Kjeldsen L, Sehested M, Pyke C, Borregaard N, et al. 92 kDa type IV collagenase (MMP-9) is expressed in neutrophils and macrophages but not in malignant epithelial cells in human colon cancer. Int J Cancer 1996;65:57–62.[CrossRef][Web of Science][Medline]
18 Gallegos NC, Smales C, Savage FJ, Hembry RM, Boulos PB. The distribution of matrix metalloproteinases and tissue inhibitor of metalloproteinases in colorectal cancer. Surg Oncol 1995;4:21–9.[Web of Science][Medline]
19 Pyke C, Ralkiaer E, Tryggvason K, Dano K. Messenger RNA for two type IV collagenases is located in stromal cells in human colorectal cancer. Am J Pathol 1993;142:349–63.
20 Öberg Å, Höyhyä M, Tavelin B, Stenling R, Lindmark G. Limited value of preoperative serum analyses of matrix metalloproteinases (MMP-2, MMP-9) and tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2) in colorectal cancer. Anticancer Res 2000;20:1085–92.[Web of Science][Medline]
21 Sonnante AM, Correale M, Linsalata M, DiLeo A, Guerra V. Circulating levels of matrix metalloproteinase-9 in patients with colorectal cancer. Scand J Gastroenterol 2000;35:671–2.[CrossRef][Web of Science][Medline]
Received October 22, 2002; accepted March 11, 2003
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