Mr 92,000 Type IV Collagenase Is Increased in Plasma of Patients with Colon Cancer and Breast Cancer1

(CANCER RESEARCH 53. 140-146. January I. 1993] Mr 92,000 Type IV Collagenase Is Increased in Plasma of Patients with Colon Cancer and Breast Cancer1

Stanley Zucker,2 Rita M. Lysik, Mohammad H. Zarrabi, and Ute Moll

Departments of Research Â¡S.Z. R. M. LI and Medicine Â¡S.Z., M. H. Z./. Department of Veterans Affairs Medical Center. Nonhpon, New York 11768. and Department of Medicine Â¡S.Z. M. H. Z/ and Pathology Â¡U.M.]. Stale University of New York at Stony Brook. Stony Brook, New York 11794

ABSTRACT Based on the observation that MMP-9 and MMP-2 can be readily identified (19, 20) and quantified in normal human plasma (21, 22), Overproduction of matrix metalloproteinases i MMl'si is a common we hypothesized that the production of these enzymes by aggressive characteristic of metastatic cancer cells. Since MMPs can be identified in cancer cells in the tissues might result in elevated levels of these plasma, we proposed that enhanced MMP-9 secretion by invasive cancer proteinases in the plasma. In contrast to the commonly used serum cells may be detected by plasma assay. To this end, we developed a specific sandwich enzyme-linked immunosorbent assay which uses two mouse tumor markers, such as CEA, which correlate with the body tumor monoclonal antibodies to human I/, 92,000 type IV collagenase (MMP-9). burden, we proposed that MMP markers in plasma might identify the The plasma concentration of MMP-9 (mean Â±SD) in 60 healthy subjects subpopulation of cancer cells destined for metastasis and might ante (9 Â±11 ng/ml), 136 patients without cancer, and 179 patients with cancer date clinically detected cancer dissemination. of the lung, genitourinary tract, or lymphomas-leukemias did not differ In this study, we used a sensitive and specific sandwich-type ELISA significantly. In contrast, plasma MMP-9 was significantly increased (/' < which is able to detect ng/ml concentrations of MMP-9 in human 0.01) in 122 patients with gastrointestinal tract cancer and breast cancer plasma. MMP-9 levels have been measured in >500 patients includ (18 Â±23 and 21 Â±22 ng/ml, respectively). Whereas carcinoembryonic ing those with various types of cancers and other disease categories antigen levels were significantly increased in patients with stage IV gas associated with excessive tissue breakdown. We have determined that trointestinal cancer, MMP-9 concentrations were not significantly in MMP-9 levels are significantly increased in patients with breast can creased in patients with metastatic disease as compared to those with cer and colon cancer. Plasma measurement of MMP-9 may be useful nonmetastatic cancer. Combining both assays improves sensitivity of de tection of colon cancer. MMP-9 was also significantly increased during for early detection of cancer dissemination in a subset of cancer patients and. hence, may supplement data provided by other tumor pregnancy which is consistent with the extensive ongoing tissue remodel ing and the leaching of the tissue proteinase into plasma. markers.

INTRODUCTION MATERIALS AND METHODS The capacity of cancer cells to invade strema and metastasize Commercial Reagents. Biolin-labeled affinity-purified goat antibodies to requires the acquisition of numerous biological properties; central to mouse IgG. IgA. and IgM (H + L) were purchased from Kirkegaard and Perry this process is the capacity to enter and exit blood vessels and lym Laboratories. Inc. (Gaithersburg. MD). Streptavidin-alkaline phosphatase con phatics and to proliferate at distant sites (1, 2). Digestion of blood jugate, 3-bromo-4-(2-h.ydroxymethyl)-l-piperazine ethanesulfonic acid, and vessel basement membrane components, especially type IV collagen, nitroblue tetrazolium chlorine were purchased from Bethesda Research Lab oratories (Gaithersburg. MD). Trypsin. soybean trypsin inhibitor, aminophe appears to be a prerequisite for dissemination of most cancer cells (3,4). Recent studies have implicated MMP-*-2 (A/r 72,000 gelatinase/ nylmercuric acetate. o^M. bovine serum albumin, and />-nitro phenyl phos phate were purchased from Sigma Chemical Co. (St. Louis, MO). Affi-Prep type IV collagenase) (5, 6). MMP-3 (stromelysin, proteoglycanase), Protein A columns were purchased from Bio-Rad Laboratories (Rockville (7) and MMP-9 (M, 92,000 gelatinase/type IV collagenase) (8-10) as Centre. NY). NHS-SS-Biotin was purchased from Pierce (Rockford. IL). playing major roles in the digestion of basement membrane type IV Latent human MMP-9 was purified from phorbol myristate acetate (100 collagen in cancer. A correlation between tumor secretion of MMP-9 ng/ml) stimulated HT 1080 fibrosarcoma cell-conditioned media using sequen (11), as well as MMP-2 (6. 12, 13), and experimental metastasis has tial gelatin-Sepharose affinity chromatography and gel filtration as described been reported. Antibodies to MMP-2 have also been used in clinical previously (23). Human MMP-2 and TIMP-2 (24, 25), purified from A2058 melanoma cells, were provided by Dr. Stetler-Stevenson. Human MMP-1 and immunohistochemical studies to identify human cancer cells with proMMP-3 were provided by Dr. Nagasi. Recombinant fibroblast inhibitor of more invasive and metastatic characteristics (14, 15). Another ap metalloproteinases (TIMP-1) was provided Dr. Carmichael. proach to characterize the role of MMPs in metastasis was to dem Antibodies to MMP-9. Monoclonal antibodies were produced in BALB/c onstrate that inhibition of metalloproteinases by natural TIMPs or mice (6-8 weeks) by injection of MMP-9 purified from HT-1080 human exogenous antibodies to MMPs was accompanied by a loss of inva fibrosarcoma cells (23). Two monoclonal antibodies (6-6B and 7-11C), puri sive and metastatic potential of tumor cells (3. 14, 16-18 ). fied from cell culture supernatants by an Affi-Prep Protein A Sepharose column (Bio-Rad) were selected for use in immunoassays.'* Monoclonal antibody 7-11C was biotinylated twice using biotin-iV-hydroxysuccinimide ester and a total of 10 mg biotin/0.8 mg IgG. Received 2/19/92; accepted 10/20/92. Aliquots of latent and activated MMP-9 were also analyzed by gelatin The costs of publication of this article were defrayed in part by the payment of page zymography and [3H]gelatin substrate degradation assay as previously de charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. scribed (26). 1This research was supported by a grant from the Department of Veterans Affairs, a Sandwich ELISA for Detection of Human MMP-9. Microtiter plates grant from the Center for Biotechnology, State University of New York at Stony Brook (96-welk Dynatech Immulon II, Alexandria, VA) were coated with murine which is sponsored by the New York State Science and Technology Foundation, and the Ann Schermerhom Cancer Foundation. monoclonal antibody 6-6B (400 ng antibody/well in 100 \A) to human MMP-9 2 To whom requests for reprints should be addressed, at Mail code 151. V.A. Medical in 0.1 M NaHCO,. pH 9.6, for 18 h at 4Â°C.The unbound anti-MMP-9 was Center. Nonhpon. NY 11768. ' The abbreviations used are: MMP, matrix metalloproleinase; ELISA, enzyme-linked immunosorbent assay; T1MP, tissue inhibitor of metalloproteinases; CEA. carcinoembry onic antigen; APMA, aminophenylmercuric acetate; oiM. a2-macroglobulin; Gl, gas 4 U. T. Moll, K. B. Rosinski. S. K. Mallya. J. P. Quigley. and D. L. French. A unique trointestinal tract; MMP-1, interstitial collagenase; MMP-3. stromelysin; NHS-SS Biotin. anti-catalytic antibody to the human M, 92.(HX) gelatinase, submitted for publication, sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionale. 1992. 140

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1993 American Association for Cancer Research. ASSAY OF TYPE IV COLLAGENASE IN CANCER removed, and 200 pi of \7c bovine serum albumin in bicarbonate buffer were matological diseases. In our initial study consisting of 43 controls and 206 added twice for 30 min at 37Â°Cto block excess binding sites on the wells. The patients with various types of cancers, we noted significantly elevated levels of plate was then washed 3 times with washing buffer (50 HIMsodium phosphate. MMP-9 in patients with colon cancer and breast cancer and. therefore, chose pH 7.2-0.1 M NaCI-0.05% Tween 20). Human plasma samples and purified to expand these cancer groups by obtaining blood specimens from 75 addi MMP-9 standards were diluted in incubation buffer (50 HIMsodium phosphate. tional patients. These plasma samples were obtained from patients treated in a pH 7.2-0.1 MNaCI-0.02<7r Tween 20-0.1% bovine serum albumin), and 100 pi medical oncology office practice and from stored laboratory specimens previ were added to wells for 2 h at 37Â°C.The wells were then washed 3 times with ously tested for CEA. To avoid the effect of postoperative complications and washing buffer; biotinylated monoclonal 7-11C anti-human MMP-9 antibody healing, specimens were not collected during the month following surgery. (9 ng in 100 ul incubation buffer) was added to each well, and the plates were Laboratory personnel performing the MMP-9 or CEA assays had no knowl incubated at 37Â°Cfor I h. The optimal concentration* of capture antibody edge of the clinical status of patients. Specimens were coded prior to assay. (6-6B) and detecting antibody (7-11C) was determined using checkerboard Each microtiter plate contained at least 4 control specimens, the MMP-9 titration. The plates were then washed 3 times and alkaline phosphatase- reference curve, and a high and low internal plasma standard. Clinical infor conjugated streptavidin ( 1:1000 dilution. 100 ul) was added to each well, and plates were incubated for 30 min at 37Â°C.The wells were then washed 3 times mation was extracted from hospital records. Cancer staging was performed as recommended by the America Joint Commission on Cancer (27). The degree with phosphate-buffered saline-Tween and 3 times with water. The substrate /)-nitrophenyl phosphate (100 ul of I mg/ml) in 0.1 Mglycine-1 HIMMgCU-1 of tumor differentiation was assessed as part of the pathological examination HIMZnCl2. pH 10.4. was added, and after a 20- to 30-min incubation at room of resected tumors. To determine whether the locali/.ed inflammatory response to tumor was a factor contributing to plasma MMP-9 levels, pathological temperature, the plates were read at Am$ in a microplate autoreader (EL 309: BioTek. Winooski, VT). Quantification of MMP-9 was made by extrapolation specimens were graded for inflammatory response as measured by the degree from a log-log linear regression curve using varying concentrations of purified of leukocyte infiltration. Specimens were categorized on a numerical scale of 0-2: 0, normal or marginally increased leukocyte presence; 1, moderately MMP-9 as standard. In preliminary experiments on 5 normal individuals, we noted that serum increased leukocyte infiltrate; 2, substantial leukocyte infiltrate. A Microsoft concentrations (range of 5 specimens. 3.300-31.190 ng/ml) of MMP-9 were Excel program was used. The use of human subjects was approved by our 215- to 3215-fold higher than simultaneously collected plasma concentrations institutional review boards. (range of 5 specimens. 3.0-11.0 ng/ml) of MMP-9 from the same patients. In view of the non-Gaussian distribution of the data, nonparametric tests Blood was. therefore, routinely collected into EDTA (1.5 mg/ml). After blood (Kruskal-Wallis one-way analysis of variance by ranks. Spearman rank corre was collected, cellular elements were removed by centrifugation ( 1000 x i?for lation coefficient, and Kendall rank correlation coefficient) were used to com pare data between different groups using StatView SE software graphics. X2 15 min) to minimi/e contamination by leukocyte proteinases. and plasma was stored at -20Â°C. Plasma MMP-9 assays were performed within 3 months of and contingency tables were used for qualitative data. P < 0.05 was considered blood collection. Plasma specimens for ELISA were noted, however, to be to be statistically significant. Parametric analysis of variance testing yielded stable for at least 6 months when stored at -20Â°C. the same conclusions for each test but will not he described further. The data Intraassay precision of the MMP-9 ELISA was determined by replicate are presented as means Â±SD. testing (10 assays) on the same day of independently diluted plasma samples. Interassay precision was determined by replicate assays of stored plasma samples performed on 8 different days. These values were used to calculate SD RESULTS and coefficient of variation. Blocking Experiments. Because plasma contains high concentrations of Enzyme-linked Immunoassay. The standard curve for the deter metalloproteinase inhibitors (TIMPand osM). experiments were undertaken to mination of MMP-9 in human plasma was established using the determine whether their presence altered the ELISA measurement of MMP-9 optimized reaction parameters (combinations of reagents, concentra in human plasma, purified MMP-9 (530 ng) (23) was added to various dilu tions, and conditions) as described in "Materials and Methods." The tions of native serum or acid-treated human serum as we previously described amount of MMP-9 in a specimen was quantitated by extrapolation (22).5AII samples were then diluted and tested in the MMP-9 ELISA. The from a standard curve of purified human MMP-9 coassayed on each curve for expected MMP-9 recovery was calculated by adding the ng of endogenous MMP-9 in each dilution of serum, as measured in the ELISA, to plate (Fig. 1/4).The sandwich ELISA for MMP-9 is linear on a log-log the actual amount of exogenous MMP-9 added to that sample. scale for protein concentrations between 0.8 and 100.0 ng/ml. When The effect of the addition of an excess of purified a;M or TIMP-1 (>2000 serially diluted patient plasmas were analyzed by MMP-9 ELISA, the M excess) on the immunological reactivity of MMP-9 was analyzed (22). dose-response curve was linear within the concentration range of the MMP-9 (0.63 ng) was incubated for 2 h at 23Â°Cwith either 400 ng TIMP-1 or standard curve (Fig. 1/4). Patient specimens were routinely tested at a 20 ug a2M- Aliquols of these samples were diluted, and immunological activity dilution of 1:4; concentrations falling outside of the curve required was determined in the MMP-9 ELISA. The effect of activation of MMP-9 by APMA (2 min) or trypsin (25 ug) on detection of MMP-9 by ELISA was further dilutions. The intraassay coefficient of variation of the control plasma sample (10 individual tests in duplicate) was 12%. The inter- assessed as previously described (22). CEA was measured by a solid phase radioimmunoassay using beads coated assay coefficient of variation of the control sample assayed weekly with mouse monoclonal anti-CEA according to the instructions of the manu during 8 weeks was 18%. facturer (Abbott Laboratory. Chicago, IL). Blood samples collected at weekly intervals (five times) from two Subjects. Blood was obtained from healthy hospital employees, healthy different individuals showed a coefficient of variation between 8 and pregnant women, patients attending a rheumatology clinic, and patients with 20%. Patients with MMP-9 levels at the upper and lower ranges various types and stages of cancer as part of routine hospital visits. Since we remained in the same category during repetitive testing over 5 months. were trying to identify an early marker of metastasis, we evaluated patients The blocking of immunological activity of MMP-9 by factors in who previously had undergone resection of all clinically detectable cancers, patients with documented mÃ©tastases,and untreated cancer patients. To assess native plasma and serum is illustrated in Fig. IÃŸ.A small (9%) the effect of unrelated acute and chronic illness and organ dysfunction on blocking effect on MMP-9 detection occurred at a relatively high MMP-9 levels, we also tested blood specimens from random hospitali/.ed serum concentration of 25% (diluted with buffer). Acid-treated serum, patients with illnesses other than cancer and patients with nonmalignant he- which is devoid of a2M activity, had no inhibitory effect on MMP-9 detection by ELISA (data not shown). The addition of a large excess (2000-fold) of a2M or TIMP-1 to purified latent MMP-9 resulted in an s In this experiment, serum was used to avoid problems of fibrin formation during 18 and 16% reduction in MMP-9 detection by ELISA, respectively. acidification of plasma. Acid treatment of serum destroys aiM activity and permits an APMA or trypsin activation of latent MMP-9, however, resulted in assessment of Ihe effect of removing a2M from a specimen. After acidification, plasma was neutrali/.ed prior to testing. >85% loss of MMP-9 detection by ELISA. 141

zymography corresponded with those identified in the ELISA for 0.4 MMP-9: the sensitivity of the immunoassay. however, was >10- to 100-fold higher than that of bioassays.6 Concentrations of MMP-9 in Human Plasma. Demographic data concerning patients are provided in Table 1. Plasma MMP-9 concentrations in a healthy population were 9.0 Â±10.7 ng/ml (mean Â±SD). The distribution of MMP-9 values for healthy individuals was skewed to the right and did not fit a Gaussian distribution: 17% of the O O normal group had enzyme levels below the limit of detection (Fig. 2). 'S The mean MMP-9 concentrations for men (10.5 Â±12.1 ng/ml) and O) women (7.0 Â±9.1 ng/ml) were not significantly different (P > O.I). -2 -0.4 Based on the mean Â±2SD calculation, the range of plasma MMP-9 levels in a normal population was determined to be 0-30.4 ng/ml. There was no significant correlation between plasma concentration of MMP-9 and age. There was no correlation between plasma MMP-9 levels and the leukocyte count or neutrophil count of whole blood. As -0.8 a control group, to account for the effects of illnesses other than cancer, we analyzed MMP-9 values in plasma from 136 hospitalized 0.4 0.8 1.2 1.6 patients without evidence of cancer. MMP-9 levels in this group did log of MMP-9 (ng/ml) [â€¢-â€¢] not differ from the normal population: 42% had nonmeasurable levels of MMP-9. Of the hospitalized group without cancer, 11 patients had -2.5 -2 -1.5 -1 -0.5 serious acute infections (pneumonia, abscess, sepsis); the mean log of Plasma Concentration [O -O ] plasma level of MMP-9 (5.9 Â±17.0 ng/ml) was not increased in this group. In addition, MMP-9 levels in patients with rheumatoid arthritis, gout, and systemic lupus erythematosus were not significantly above control values. Plasma samples from healthily pregnant women were analyzed. As illustrated in Table I and Fig. 2, plasma MMP-9 levels of pregnant women were significantly higher (P < 0.05) than nonpregnant women or healthy men. MMP-9 levels in the second and third trimester of pregnancy were higher than in the first trimester of pregnancy, but the difference was not significant. Plasma MMP-9 Concentrations in Patients with Cancer. The results from the initial group and the second group of patients with breast cancer and GI cancer provided similar data showing signifi cantly increased MMP-9 levels in these diseases (P < 0.01). For purposes of clarity, these data have been combined and are presented in Fig. 2. In the GI adenocarcinoma group. 73 patients had colorectal cancer. 16 had gastric cancer. 3 had pancreatic cancer, and 2 had PATIENT SERUM(K! hepatomas. Since the MMP-9 data from these subgroups were similar, Fig. I. A. MMP-9 ELISA. Serial dilutions of purified MMP-9 were assayed for im- the data from these patients were combined into a single category, GI munological reactivity in the sandwich MMP-9 ELISA. Likewise, plasma from a patient with a high concentration of MMP-9 was serially diluted (l:l. l:2. 1:4, 1:8. 1:16, 1:32. and cancer. To minimize interassay variation, patient plasmas with an 1:64) and assayed. Plot of A*Â»versus MMP-9 concentration using a log-log scale. The MMP-9 value >30 ng/ml were rechecked in a single-batch assay; sandwich ELISA for purified human MMP-9 and patient plasma was linear on a log-log 93% of the abnormal values (>30.4 ng/ml) were confirmed, and the scale for protein concentrations from 0.8-100.0 ng/ml. The linear regression for each curve was calculated: the r for the standard curve was 0.9S and for the patient plasma was original data are presented. A third assay was performed when the 0.99. â€¢¿standard curve: O. patient plasma. B. Effect of serum proteins on detection of initial two values differed considerably: the outlier value was elimi MMP-9 in the ELISA. Purified MMP-9 (530 ng) was added to various dilutions of human nated. Specimens from 150 patients were obtained on more than one serum to identify blocking factors which might inhibit the immunological reactivity of endogenous MMP-9. These mixed samples were then assayed in the MMP-9 ELISA. occasion during a 4-month period; results for each individual subject Endogenous levels of MMP-9 in each serum dilution were determined by an independent were averaged, and the mean value is presented. Most of the patients ELISA. The curve for eipected MMP-9 represents the sum of the endogenous MMP-9 in a specific dilution of serum plus the 530 ng of exogenous MMP-9 added to that sample. with stage I-III GI cancer (89%), and stage I-II breast cancer (67%) The curve for actuul MMP-9 represents the concentration of MMP-9 in the mixed sample had undergone complete resection of all macroscopic evidence of (as determined by an independent ELISA). Plasma was not used in this experiment cancer prior to obtaining plasma specimens. Sixty-seven % of the because acid treatment resulted in clotting of the specimen. patients with stage IV colon cancer had undergone surgery prior to specimen collection. The specificity of the MMP-9 ELISA was examined by testing The concentrations of plasma MMP-9 in patients with breast cancer structurally related enzymes of the collagenase family of metallopro- (21.4 Â±22.4 ng/ml) and GI cancer (18.4 Â±23.0 ng/ml) were signif teinases. No cross-reactivity in the MMP-9 ELISA was observed with icantly increased (P < 0.01 by Kruskal Wallis nonparametric analysis proMMP-3 (1.9 ug/ml), MMP-1 (1.0 ug/ml), or MMP-2 (1 ng/ml). and P < 0.05 by analysis of variance) as compared to the healthy The MMP-9 ELISA did not detect free TIMP-1 (0.5 ug/ml) or TIMP-2 controls or to hospitalized patients without cancer. Although the num (0.5 ug/ml). On examination of human plasma enriched for gelati- ber of plasma specimens obtained preoperatively was small, there was nolytic activity by gelatin-Sepharose (eluted with 19!-dimethyl sul- * S. Zucker. P. Mancuso. B. DiMassimo. R. M. Lysik. and C. L. Wu. Comparison of foxide) and gel filtration chromatography (23), active fractions iden techniques for measurement of gelatinase/type IV collagenase in plasma: enzyme-linked tified in the I'Hjgelatin substrate degradation assay and on gelatin immunoassay verana bioassay procedures, submitted for publication. 1992. 142

CONTROL HOSPITALIZED PATIENTS

0 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 70 80 90 MMP-9 (ng/ml) MMP-9(ng/ml)

61 CANCER PREGNANCY 6

5 Fig. 2. Plasma MMP-9 concentrations in healthy subjects, hospitalized patients without cancer, preg 4 nant patients, patients with gastrointestinal cancer, breast cancer, and lung cancer. The data are pre 1 3 sented as the patient count at different intervals of U MMP-9 (width of each column, 0.9 ng/ml). For 2 graphic purposes, patients with plasma MMP-9 lev els >80 ng/ml were reported as 80. lili III I 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90 MMP-9 (nq/ml) MMP-9 (ng/ml)

BREAST CANCER LUN6 CANCER

6'5'4Ihu211li 8 7 6-

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11 Jill 11 0 10 20 30 40 50 60 70 80 90 OL IO 20 30 40 50 60 70 80 90 MMP-9 (ng/ml) MMP-9(ng/ml) no correlation or trend between prior resection of the tumor and Although there was no significant correlation between MMP-9 and MMP-9 levels in patients with breast cancer or GI cancer. CEA levels (Fig. 3), patients with stage IV GI cancer had significantly There was no correlation between breast cancer stage and MMP-9 higher CEA levels. GI cancer patients tended to have either high concentration (Kendall Rank correlation coefficient). The mean plasma MMP-9 (23%) or high CEA (71%); only 6% of GI cancer MMP-9 concentration and the percentage of abnormal MMP-9 values patients had both elevated CEA and MMP-9. However, 4 of 5 patients were not significantly different between stages I, II, III, and IV gas (80%) with both high MMP-9 and high CEA, had stage IV cancer. In trointestinal cancer patients (Kendall Rank correlation coefficient and an exact X2 analysis, categorizing each stage of GI cancer as a X2, respectively). percentage of patients with abnormal versus normal MMP-9 and Evaluation of the degree of tumor differentiation in 11 patients with CEA, it was noted that the distribution of MMP-9/CEA frequencies gastrointestinal cancer and MMP-9 levels >30 ng/ml indicated that was significantly different (P < 0.01) among the stages. During the 9 9 of 11 patients had moderately well-differentiated tumors, while 2 months that patient data were accumulated, 3-10 sequential measure had poorly differentiated tumors. In comparison, in gastrointestinal ments were obtained from 18 breast cancer or colon cancer patients cancer patients with normal levels of MMP-9, 7 of 11 patients had receiving chemotherapy. At present, the correlation between the moderately well-differentiated tumors, 3 had well-differentiated tu clinical response to treatment and changes in MMP-9 concentration is mors, and 1 had a poorly differentiated tumor. inconclusive. The degree of inflammatory infiltrate in resected tumors from 9 of MMP-9 levels were not significantly increased (Table I ) in patients 11 patients with MMP-9 levels >30 ng/ml was normal or marginally with genitourinary tract cancer (males: prostate, bladder, kidney; fe increased, one tumor demonstrated a moderate increase and one tumor males: ovary, uterus, cervix, vagina, bladder), lung cancer, miscella with focal necrosis had a substantial infiltrate. Similar results were neous cancers [thyroid (1 patient), carcinoid (2 patients), adenocarci- obtained when resected tumors from a randomly selected group noma with unknown primary site (6 patients), head and neck cancer of 11 patients with low MMP-9 levels were examined for inflamma (2 patients), Kaposi's sarcoma, (1 patient) malignant melanoma tory infiltrate. (2 patients), squamous cell cancer of skin (4 patients), double primary 143

Table 1 Demographic and experimental dala from patients in this study Data from gastrointestinal cancer and breast cancer patients are presented according to stage of disease. CEA is listed for Gl cancer only. Results are means Â±SD.

ofsubjects603426136351994161922372836415722421162323Age MMP-9 DiagnosisControlMaleFemaleHospitalizedPregnancyRheumatologicGI (yr)41.1 (% subjects)3601222212314321431320335(13311424694CEA(ng/ml)52.7 Â±13.039.5 Â±10.710.5 Â±13.743.1 Â±12.17.0 Â±12.268.0 Â±9.110.2 Â±7.427.8 Â±7.022.4 Â±6.562.5 Â±25.9"15.5 Â±14.868.1 Â±23.218.4 cancerStage Â±7.470.7 Â±23.0"20.4 Â±280.84.1 IStage Â±4.068.4 Â±30.3I7.5Â±21.112.9+16.72 Â±2.44.0 11Stage Â±7.268.7 Â±2.913.1 IIIStage Â±7.166.2 Â±40.8129.8 IVBreast Â±8.258.1 1.2Â±24.42 Â±454.6 cancerStage Â±14.854.7 1.4Â±22.4"6.7 1Stage Â±18.670.0 Â±9.7I9.2Â± IIStage Â±12.060.8 19.230.6 IIIStage Â±11.054.1 Â±29.322.7 IVGenitourinary Â±14.760.3 Â±23.810.4Â± cancerLung tract Â±14.168.1 14.54.5 cancerMiscellaneous Â±6.566.4 Â±7.316.0 cancerLymphomaChronic Â±10.457.8 Â±24.615.5 Â±17.365.7 +39.76.3 leukemiaMyeloproliferativelymphocytic Â±9.969.3 Â±12.996.3 diseasesNo. Â±7.9MMP-9(ng/mll9.0 Â±10.6High " Significant difference when compared to control (P < 0.05).

CANCERo9 1Gl CANCERooOII Gl 1201008060-402On.Oo1ferSTAGE 120100-8Â»W4Â»20*$ortfflSTAGE

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Fig. 3. Correlation between plasma MMP-9 and o CEA levels for patients with gastrointestinal can 20 30 40 20 30 40 cers at different stages. For graphic purposes, pa CEA (ng/ml) CEA (ng/ml) tients with plasma CEA levels >50 ng/ml were expressed as 50 ng/ml. The lines within the graph identify the normal limits of CEA (5 ng/ml) and MMP-9 (30 ng/ml). Patients tended to have either CANCERo(A) III Gl CANCER0oVIV Gl increased CEA or increased MMP-9. Only 5 pa 120100Â»SO4020OO38AtSTAGE 12O100SO4O?rtaO9n tients had both increased CEA and MMP-9. E 1

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cancers (3 patients)], Hodgkin's and non-Hodgkins lymphoma, the measurement in plasma of specific proteins released by cancer chronic lymphocytic leukemia-multiple myeloma, and myeloprolifer- cells during the invasive and metastatic process is a feasible approach ative diseases (chronic leukemias and dysmyelopoietic syndromes). to detect early cancer dissemination. MMP-9 levels (5.0 Â±8.0 ng/ml) in 13 patients with increased gran- Based on the emphasis of the role of type IV collagenases in cancer ulocyte mass secondary to polycythemia rubra vera or chronic myel- metastasis ( 12), we have developed a highly specific and reproducible ogenous leukemia were not increased. Furthermore, the percentage immunoassay for the quantification of MT92,000 type IV collagenase/ of patients with increased plasma MMP-9 levels was not significantly gelatinase (MMP-9) in biological fluids. Using a pair of murine mon elevated in any of these latter cancer groups (X2 test). Analysis of oclonal antibodies to MMP-9 in a sandwich ELISA format, we are clinical status within each of these cancer categories revealed no able to accurately detect concentrations of MMP-9 in human plasma significant differences in MMP-9 between different stages of cancer. as low as 0.8 ng/ml. This ELISA was able to detect free latent MMP-9 and latent MMP-9 complexed to TIMP-1. Trypsin or APMA activa tion of MMP-9 resulted in >85% loss of immunological detection of DISCUSSION antigen by ELISA. The MMP-9 ELISA reported by Bergmann et al. The development of clinical laboratory tests to predict the biolog (21) was able to detect purified activated MMP-9 in the absence of ical behavior of human cancers remains an elusive goal. Conceptually, inhibitors but not after complex formation with a2M in plasma 144

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1993 American Association for Cancer Research. ASSAY OF TYPE IV COLLAGENASE IN CANCER samples (21). MMP-9 levels in 90% of human plasma specimens vergence in MMP-9 and CEA data, we postulate that colon cancer ranged between 27 and 94 ng/ml (log normal distribution) with a cells with a propensity to metastasize secrete increased levels of median of 51 ng/ml in the Bergmann assay, which is approximately MMP-9 early in the course of the disease and CEA at a later stage. 5-fold higher than our results. Discrepancies of this nature are com However, a small number of patients with stage IV GI cancer did mon in ELISAs using different types of reagents (28), especially secrete high levels of both MMP-9 and CEA. considering the absence of a stable well-characterized MMP-9 antigen An important issue raised by this study is the cellular source of standard. The inability of these ELISAs to detect activated MMP-9, MMP-9 in patients with gastrointestinal or breast cancer. In theory, either free or complexed with TIMP or osM, does not appear to be a local macrophages or stromal fibroblasts could be a source of MMP-9 serious problem with clinical specimens, since MMP-9 in plasma is in secretion in GI and breast cancer patients, since macrophage and the latent form and requires activation to demonstrate substrate deg fibroblast MMP-9 are antigenically identical with tumor MMP-9 (9). radation in bioassay. In contrast to the less sensitive substrate degra However, this appears unlikely since elevated MMP-9 levels were dation assay for type IV collagenases/gelatinases in body fluids, the unique to these two cancer types. To determine whether the local MMP-9 ELISA does not cross-react with related metalloproteinases inflammatory response to cancer contributes to the plasma levels of (MMP-1, MMP-2, or MMP-3 ), is not dependent on stability of the MMP-9, we compared the degree of leukocyte infiltration in cancer enzyme, does not require activation of proenzyme forms, and is less tissue from patients with high MMP-9 and normal MMP-9 levels. affected by the formation of a latent complex with TIMP-1 (22, 29). Histological examination revealed no correlation between inflamma In this study we have attempted to determine whether plasma tory cells in the surgical specimens and the plasma MMP-9 levels. MMP-9 measurement could be used as a tumor marker to identify To further address the possibility that MMP-9 originated from a patients with more aggressive tumors in terms of invasive and meta- cellular component of the inflammatory response, we analyzed plasma static potential. We measured MMP-9 levels in 301 patients with a MMP-9 levels in patients with infection or leukocytosis or mono- variety of cancers. The concentration of plasma MMP-9 was signifi cytosis secondary to myeloproliferative disease. Increased MMP-9 cantly increased in patients with breast cancer and cancers originating levels were not found in either of these groups or in patients from the gastrointestinal tract. Setting the range of normal values with rheumatoid arthritis which is associated with an inflammatory (0-30.4 ng/ml) for plasma MMP-9 based on data from 60 healthy response. control subjects, we found that 23% of patients with GI cancer and A practical limitation to widespread use of MMP-9 measurements 32% of patients with breast cancer had increased MMP-9 levels. as a screening test is the bimodal distribution of values found in the Seven of 27 (26%) stage 1-2 GI cancer patients, who had undergone normal population with approximately 60% of normal persons having total resection for clinical tumor at least a month prior to blood values for MMP-9 <6 ng/ml and approximately 40% having values collection, had increased levels of MMP-9. A control group consisting between 7 and 47 ng/ml. This bimodal distribution is maintained in of unselected elderly hospitalized patients without cancer had levels of MMP-9 comparable to healthy subjects, suggesting that the elevated patients with GI cancer and breast cancer as well, but the curves are MMP-9 levels of cancer are not a nonspecific finding related to shifted to the right. For a single healthy individual, however, the level of MMP-9 remains quite constant over time, suggesting that the test general aspects of acute or chronic illness. Patients with cancers might be more useful in following the clinical course of a single originating from the lung and genitourinary system, miscellaneous individual patient. cancers, and lymphomas and leukemias did not have significantly increased levels of plasma MMP-9. In our recent study of many of A correlation between type I collagenolytic activity (substrate deg radation assay for MMP-1) and histolÃ³gica! grade of surgically re these same patients, using an ELISA for MMP-2, we reported no significant increase in MMP-2 levels in any cancer group (30), which sected human colorectal carcinoma specimens has been reported by further supports the specificity of the MMP-9 assay in gastrointestinal van Stappen et al. (35). Hewitt et al. (36) described increased staining of collagenase in the connective tissue stroma of colorectal cancers and breast cancer patients. Contrary to expectations derived from work with other tumor mark but no relationship with tumor differentiation. Ishimura et al. (37), ers, there was no significant correlation between stage of cancer and however, failed to find a significant difference between type I or type levels of MMP-9 in plasma. To explain this discrepancy, it is impor IV collagenolytic activity and clinical stage of cancer on examination tant to recall that most serum tumor marker measurements currently of extracts of colorectal cancer and adjacent normal colonie mucosa. used in clinical medicine generally reflect the tumor burden in the As noted above, substrate degradation assay results are affected not patient and do not assess biological properties of tumor cells except only by the levels of metalloproteinases but also the level of TIMPs in the tissue. Since TIMP-1 levels (measured by ELISA) have been their capacity to secrete or shed specific antigens (31). As a result, tumor markers are most useful for monitoring treatment and deter reported to be increased in human colon cancer tissue extracts as mining prognosis but have not proven useful for cancer screening or compared to adjacent normal mucosa (38), it is understandable that establishing diagnosis, except in selected high-risk populations (32). measurements of metalloproteinase activity by enzymatic assay may Because CEA is a useful tumor marker for advanced gastrointesti not accurately reflect levels of MMPs in tissues (29). nal cancer (31), we compared plasma CEA and MMP-9 levels in Elevated plasma levels of type IV collagenolytic activity (using a patients with GI cancer. Whereas CEA measurements were signifi substrate degradation assay) were reported by Hashimoto et al. (39) in cantly increased in stage IV GI cancer as compared to earlier stages, a selected subset of patients with hepatocellular carcinoma and portal MMP-9 levels were not significantly different between stages. These vein invasion. Distinction between MMP-2 and MMP-9 activity was data indicate that the plasma MMP-9 level is not reflective of tumor not possible in this bioassay. Elevated levels of serum type IV colla burden (as is the case with CEA) but may identify some other bio genase were also reported in an experimental model of cancer (40). logical characteristic of GI cancer cells. Proof of this hypothesis will In view of the extensive amount of uterine and placenta! remodeling have to await long-term observation of stage I and II patients, since that occurs during pregnancy, our detection of increased levels of colon cancer may have a long dormancy state before clinical progres plasma MMP-9 and MMP-2 (22) by ELISA in pregnancy is consistent sion. It is of interest to note that CEA has recently been identified as with the concept that high concentrations of tissue metalloproteinases a member of the cell adhesion family of molecules with linkage to will leach into the blood stream. Using a substrate degradation assay metastatic propensity (33, 34). As a possible explanation for the di for type I collagen, Rajabi et al. (41) reported that plasma levels of 145

MMP-1 were elevated in pregnancy but only at the time of delivery, progression. Mod. Pathol., 4: 239-246, 1991. when enzyme levels reach their maximum. 16. Snultz, R. M., Silberman, S., Persky, B., Bajkowski, A. S., and Carmichael, D. F. Inhibition by human recombinant tissue inhibitor of metalloproteinases of human In summary, the data presented indicate that the detection of in amnion invasion and lung colonization by murine BI6-F10 melanoma cells. Cancer creased MMP-9 levels in the plasma of certain types of cancer patients Res., 48: 5539-5545, 1988. is not related to tumor burden but may reflect biological aspects of 17. DeClerck, Y. E., Yean, T. S., Chan, D., Shimada, H., and Langley, K. E. Inhibition of tumor invasion of smooth muscle cell layers by recombinant human metalloproteinase tumor behavior related to the metastatic process. Long-term follow-up inhibitor. Cancer Res., 51: 2151-2157, 1991. of these patients will be necessary to determine whether increased 18. Albini, A.. Melchiori, A., Santi, L.. Liotta, L., Brown, P. D., and Stetler-Stevenson. W. levels of MMP-9 are predictive of more invasive and metastatic can G. Tumor cell invasion inhibited by TIMP-2. J. Nati. Cancer Inst., 83: 775-779, 1991. 19. Vartio, T.. and Baumann. M. Human gelatinase/type IV collagenase is a regular cers. Since much of the latent MMP-9 that is secreted by cells is plasma component. FEBS Lett., 155: 285-289, 1989. complexed with TIMP-1 (9, 23), it is possible that the measurement of 20. Johansson, S., and Smedsrod, B. Identification of a plasma gelatinase in preparations of fibronectin. J. Biol. Chem., 261: 4363-4366, 1986. this complex in plasma, or other combinations of metalloproteinases 21. Bergmann. U., Michaelis, J.. Oberhoff, R., Knauper, V., Beckmann, R., and and inhibitor complexes, may provide useful information concerning Tschesche, H. 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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1993 American Association for Cancer Research. Mr 92,000 Type IV Collagenase Is Increased in Plasma of Patients with Colon Cancer and Breast Cancer

Stanley Zucker, Rita M. Lysik, Mohammad H. Zarrabi, et al.

Cancer Res 1993;53:140-146.

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