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      Serum levels and tissue expression of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinases 2 (TIMP-2) in colorectal cancer patients

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          Abstract

          The objective of the study was the assessment of serum levels and tissue expression of matrix metalloproteinase 2 (MMP-2) and tissue inhibitor of matrix metalloproteinases 2 (TIMP-2) in patients with colorectal cancer (CRC). The study included 72 CRC patients and 68 healthy subjects. The serum levels of MMP-2 and TIMP-2 were measured using enzyme-linked immunosorbent assay (ELISA) method, whereas tissue expression of MMP-2 and TIMP-2 in cancer cells, interstitial inflammatory cells, and adjacent normal colorectal mucosa were examined by immunohistochemical staining of tumor samples. The serum levels of MMP-2 and TIMP-2 in cancer patients were significantly lower than those in control group, but the percentage of positive immunoreactivity of these proteins were higher in malignant and inflammatory cells as compared to normal tissue. There was a significant correlation between MMP-2 immunoreactivity in inflammatory cells and the presence of distant metastases and between TIMP-2 expression in inflammatory cells and tumor size, nodal involvement, and distant metastases. Area under receiver operating characteristic (ROC) curve (AUC) for serum MMP-2 was higher than for serum TIMP-2. Moreover, positive tissue expression of MMP-2 was a significant prognostic factor for CRC patients’ survival. Our findings suggest that MMP-2 and TIMP-2 might play a role in the process of colorectal cancer invasion and metastasis, but the significance of their interactions with tumor stroma and interstitial inflammatory infiltration in colorectal neoplasia require further elucidation.

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          A genetic model for colorectal tumorigenesis.

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            Gelatinase B: a tuner and amplifier of immune functions.

            Gelatinase B (matrix metalloproteinase-9) is a secreted multidomain enzyme that is important for the remodeling of the extracellular matrix and the migration of normal and tumor cells. It cleaves denatured collagens (gelatins) and type IV collagen, which is present in basement membranes. In the immune system, this cleavage helps lymphocytes and other leukocytes to enter and leave the blood and lymph circulations. Gelatinase B also cleaves myelin basic protein and type II gelatins, and this clipping leads to remnant epitopes that generate autoimmunity, the so-called REGA model of autoimmunity. Recently, gelatinase B has been found to process cytokines and chemokines, resulting in skewed immune functions. Therefore, gelatinase B, often considered as a pure effector molecule, acts as a switch and catalyst in both innate and specific immunity, and constitutes a prototypic example of the regulation of immune functions by proteolysis.
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              Matrix metalloproteinase-2 (MMP-2) is associated with survival in breast carcinoma

              Breast carcinoma is the most frequent malignancy among women in Finland as well as in other Western countries (Finnish Cancer Registry, 1995; Parkin et al, 1997). It is a pathologically and clinically heterogenous disease with variable prognosis. Breast carcinomas are potentially highly malignant tumours due to their capacity to invade locally and to metastasise. The traditional prognostic factors of breast carcinoma include the size of the primary tumour, axillary lymph node involvement, tumour grade, oestrogen or progesterone receptor status of the primary tumour and menopausal status of the patients. Tumour invasion and metastasis are the major causes of treatment failure or death for carcinoma patients. The role of matrix metalloproteinases (MMPs) in tumour invasion and metastasis as well as in tumour angiogenesis is important. Matrix metalloproteinase-2 (MMP-2/gelatinase A/72-kDa type IV collagenase) is a member of zinc-dependent endopeptidases that degrade matrix proteins, among other type IV collagens in basement membranes (Liotta and Stetler-Stevenson, 1990; Liotta et al, 1998; Curran and Murray, 1999). The expression of MMP-2 has been strongly associated with the progression of malignancy of several types of carcinoma (D'Errico et al, 1991; Levy et al, 1991; Ellenrieder et al, 2000; Sakata et al, 2000; Giannelli et al, 2002). In primary skin melanoma (Väisänen et al, 1998), lung carcinoma (Kodate et al, 1997), ovarian carcinoma (Davidson et al, 1999) and brain neoplasms (Jäälinoja et al, 2000), the expression of the immunoreactive protein for MMP-2 was associated with a poor prognosis. In several studies, MMP-2 has been shown to be expressed in breast carcinoma (Liotta and Stetler-Stevenson, 1990; Monteagudo et al, 1990; D'Errico et al, 1991; Davies et al, 1993; Tryggvason et al, 1993; Iwata et al, 1996; Garbett et al, 1999, 2000; Jones et al, 1999) and it has been localised in breast carcinoma cells using immunohistochemical methods (Daidone et al, 1991; Höyhtyä et al, 1994). In limited series, MMP-2 positivity is associated with unfavourable prognosis in both premenopausal and postmenopausal node-positive breast carcinoma patients (Talvensaari-Mattila et al, 1998, 1999, 2001). This study is aimed at defining the possible favourable effect of the MMP-2 negativity in primary breast carcinoma in high-risk patient groups while confirming the MMP-2 immunoreactive protein as a prognostic factor also in node-negative breast carcinoma. MATERIALS AND METHODS Breast tissue samples were from the primary tumours of 453 patients operated on during the years 1981–1995 in Northern Finland. The formalin-fixed, paraffin-embedded blocks were obtained from the files of the Departments of Pathology, Oulu University Hospital and the Central Hospitals of Kajaani, Kemi, Kokkola and Rovaniemi. The minimum follow-up time was 60 months (range 60–150 months). Stage, tumour size and axillary node involvement of breast carcinoma were determined according to the UICC TNM classification (Hermanek and Sobin, 1992). The tumours were classified according to the World Health Organization's International Classification of Breast Tumors (Scarff and Torloni, 1986). The ductal carcinomas were graded (I–III) by evaluating tubule formation, nuclear pleomorphism and the mitotic rate according to the criteria of Bloom and Richardson (1957). The patients were 26–85 years of age, the median age being 52 years. Ductal infiltrating carcinoma was the most frequent histological type. In this material, the number of the node-positive cases is over-represented, 302 out of 453 patients, to increase the power to test the effect of MMP-2 negativity in advanced breast carcinoma. A small tumour sample taken during the operation was used for routine steroid receptor assays. Both oestrogen and progesterone receptor charcoal assays were performed in 334 cases. In all, 65 tumours were both oestrogen and progesterone receptor negative. Mastectomy with axillary evacuation was the primary treatment in most of the cases, one case remained inoperable. All patients without distant metastases and with histologically positive axillary lymph nodes, regardless of the number of nodes or size of the primary tumour, received postoperative radiotherapy covering the axillary, supraclavicular and internal mammary lymph nodes and the chest wall around the mastectomy scar. Adjuvant antioestrogen therapy had been used in 138 cases, most of them with a stage II or III disease, and adjuvant cyclophosphamide–methotrexate–fluorouracil (CMF) chemotherapy in 104 cases or FEC (5-fluorouracil-epirubicin-cyclophosphamide) in 96 cases. Patients with metastatic disease (M1) were operated (except one) and local radiotherapy was given. Additionally, the patients received antioestrogen therapy (10 patients) or chemotherapy (two patients). Recurrences in patients with receptor-positive tumour were treated primarily with hormonal therapy. Immunohistochemical staining The histologic material fixed in 10% formalin and embedded in paraffin was cut into 4 μm slides and they were incubated for 12 h at 37°C, dewaxed in a histological clearing agent, Histo-Clear (National Diagnostics, Atlanta, GA, USA), and hydrated. The specimens were treated with 0.4% pepsin (Sigma, St Louis, MO, USA) for 20 min at 37°C. The avidin–biotin–immunoperoxidase technique was used according to Hsu et al, 1981. Mouse monoclonal antibody (CA-4001; Diabor Ltd, Oulu, Finland) against MMP-2 was used as a primary antibody. The antibody has been previously shown to detect the latent (inactive), 70–72 kDa form of MMP-2. The specificity has been confirmed by a Western blot analysis (Höyhtyä et al, 1994). Endogenous peroxidase activity was blocked by incubating the slides in 3% hydrogen peroxide in absolute methanol for 15 min, and nonspecific binding was blocked with 10% goat serum for 15 min. The specimens were incubated for 60 min at room temperature in a humidity chamber, and immunohistological staining was continued using a Histostain-bulk kit (Zymed, San Francisco, CA, USA) according to the manufacturer's instructions. Biotinylated anti-mouse IgG served as a second antibody, and the peroxidase was introduced using a streptavidin conjugate. The slides were washed thoroughly with phosphate-buffered saline between each stage in the procedure. The antibody reaction was visualised using a fresh substrate solution containing an aminoethyl carbazole substrate kit (AEC, Sigma). The sections were counterstained with haematoxylin, dehydrated and mounted in glycerol–vinyl–alcohol (GVA mount, Zymed). For the negative controls, the primary antibody was replaced with mouse nonimmuno IgG. For the positive controls, we used previously known MMP-2-positive specimens of breast carcinoma. Evaluation of the MMP-2 immunostaining A section was considered negative or positive according to the absence or presence of positive staining of the neoplastic cells. The staining was scored as follows: no positive cells, less than 50% of the neoplastic cells staining positive (MMP-2+) and >50% of the neoplastic cells positive (MMP-2++). Immunostaining for MMP-2 was scored by three independent observers. Only cases giving repeatable scores in immunostaining were included in the data. The clinical data were collected and analysed after the evaluation of the immunostaining scores for a given case. The scoring of the immunoreaction and collecting the clinical data were performed independently without knowledge of each other. Statistical analysis The score for MMP-2 immunoreactivity was compared with other prognostic variables by the χ 2 method. P-values 50% of the tumour cells positive; MMP-2++). Negative staining (MMP-2) was found in 22% of the primary tumours of breast carcinoma. The immunoreactive protein in carcinoma cells localised to the cytoplasm (Figure 1A, B Figure 1 Cytoplasmic immunostaining of MMP-2 in primary breast carcinoma. Immunostaining was performed as described in Materials and Methods by using an anti-MMP-2 monoclonal antibody: (A) negative (−), (B) positive (++) case. ). There was no correlation between the MMP-2 protein expression and stage, grade or hormone receptor status (Table 1 Table 1 Expression of MMP-2 immunoreactive protein in breast carcinoma     MMP-2 positive   n n   All patients 453 354 (78%)  Age (years) 26–85      T1–2 404 318 (79%)  T3–4 49 36 (73%)  N0 152 123 (81%)  N1–2 301 231 (77%)  M0 439 341 (78%)  M1 14 13 (93%)         Histology       Ductal infiltrating        Grade I 39 28 (72%)  Grade II–III 302 240 (80%) Oestrogen receptor        Positive 262 199 (76%)  Negative 72 61 (85%)  Unknown 119     Progesterone receptor        Positive 236 181 (77%)  Negative 99 79 (80%)  Unknown 118     Adjuvant treatment        No 187 154 (82%)  Yes 226 200 (88%) ). A statistically significant correlation between MMP-2 positivity and overall survival was found in this study. The 10-year overall survival was 72% in patients with an MMP-2 immunoreactive protein positive (+) and 64% in patients with an MMP-2 immunoreactive protein strongly positive (++) breast carcinoma, compared to 77% in the patient group with an MMP-2-negative primary tumour, the 10-year RFS being 60, 56 and 64%, respectively (Figure 2A, B Figure 2 Survival analysis (Kaplan–Meier) of the breast carcinoma patients according to the MMP-2 immunoreactivity of the primary tumour: (A) overall survival, (B) RFS. MMP-2 negative (−), MMP-2 positivity; weak (+), strong (++). Log-rank analysis of MMP-2 negative vs strong positive: (A) P=0.03, (B) P=0.05. ). In the node-negative patient group, all patients with MMP-2-negative immunostaining were alive in the follow-up time of 10 years. In the patient group with a positive immunohistochemical staining for MMP-2, the overall survival was 87% (P=0.03, Table 2 Table 2 Recurrence-free and overall survival of breast carcinoma patients according to MMP-2 staining in different patient groups   MMP-2 immunoreaction   Recurrence-free survival Overall survival   − +/++ − +/++ All patients 64 59 77 68*** T1–2 66 60 80 70**           Node negative 79 78 100 87**** Node positive 58 48***** 61 56*****           Oestrogen receptor          Negative 90 56***** 90 58*****  Positive 55 55 75 67** Progesterone receptor          Negative 80 56** 95 58*  Positive 55 55 71 67 Grade II–III 57 48***** 64 59**** * P=0.005, ** P=0.02, *** P=0.03, **** P=0.04, ***** P 55 186 0.445 0.183   1.56 1.09–2.23 0.02                 Tumour       0.001        1–2 403       1.00      3–4 49 1.106 0.223   3.02 1.95–4.67 0.001                 Nodes       0.001        0 152       1.00      1–2 300 1.375 0.285   3.95 2.26–6.91 0.001                 Grade       0.01        I 39       1.00      II–III 302 1.923 0.715   6.84 1.68–27.8 0.01 β=coefficient; s.e.=standard error of the mean; OR=odds ratio. ). MMP-2 positivity appeared to increase the risk of death 1.8-fold during the first 10 years of follow-up. DISCUSSION The expression of MMP-2 immunoreactive protein has been associated with invasive and metastatic tumours in previous in vitro studies (Liotta et al, 1980; Garbisa et al, 1987; Nakajima et al, 1987; Bernhard et al, 1990). In this study, intracytoplasmic expression of the protein for MMP-2 was found in 78% of the primary tumours of breast carcinoma. The amount of positive cases is in line with previously published data (Monteagudo et al, 1990; Daidone et al, 1991; Visscher et al, 1994; Talvensaari-Mattila et al, 1998, 1999, 2001; Lee et al, 1996). This study constitutes the largest material of breast carcinoma published showing the prognostic value of MMP-2. In this material consisting of 453 cases, the MMP-2 immunoreactive protein was able to predict a relapse during the 10 years of the follow-up. The 10-year RFS was 60% in patients with a low-grade (+) and 56% in those with a strongly positive (++) tumour, compared to 64% in patients with an MMP-2-negative tumour. Also, the 10-year overall survival rate was significantly inferior among these patients. These differences are high enough to be also clinically significant, and especially the differences between − and ++ groups suggest that the MMP-2 immunoreactive protein is worthy of careful evaluation as a possible marker for biologic aggressiveness in breast carcinoma patients. Further, the previous studies have failed to show any statistically significant differences in survival between the patients with MMP-2-negative vs -positive primary tumours in node-negative patient group. Here the patient group presenting an MMP-2-negative primary breast carcinoma without a lymph node involvement enjoyed an excellent prognosis for survival, 100% of the patients being alive after 10 years of follow-up (Table 2). No difference was, however, found in the RFS suggesting that MMP-2 negativity may be associated with better responses to treatment of the metastatic disease and/or show progression of the disease. The patients with an MMP-2-negative breast carcinoma form a relatively large patient group when the incidence of breast carcinoma is taken into consideration. It is possible that the lack of MMP-2 could become an important factor in certain subgroups of breast carcinoma when selecting the adjuvant therapy. A well-known risk factor in breast carcinoma is hormone receptor negativity. It is interesting that 90% of the patients with an oestrogen receptor negative, MMP-2 negative or 95% of the patients with a progesterone receptor negative, MMP-2 negative primary tumour were alive after the 10 years of the follow-up (Figure 3A, B). On the contrary, only 58% of patients displaying MMP-2 positivity and oestrogen or progesterone receptor negativity were alive at that time. These differences were statistically highly significant, suggesting that these patient groups might need more attention in further studies. The patient group is small in percentages (about 4% of all breast carcinoma patients), but interesting both biologically and clinically. The number of those patients still exceeds the number of patients representing many more uncommon carcinoma types. The regulation of MMP-2 by female sex hormones may be an yet unknown mechanism which could explain this result. This conclusion indicates the need for further studies to explore the value of this enzyme in clinical decision-making. In grade 2 and 3 tumours, MMP-2 correlated significantly with shortened RFS and overall survival (Table 2). It is interesting to note that, MMP-2 negativity in this patient group was a strong marker for a favourable prognosis. Barozzi et al (2002) reported that TGF-α, MMP-2 and IGF-II seem to be suitable candidates for a selective panel of markers designed to provide significant information with respect to the current pathologic staging system for patients with colorectal carcinoma. In conclusion, we show here in a relatively large breast carcinoma patient group that MMP-2 immunoreactive protein is an independent prognostic indicator that might prove valuable in certain subgroups, such as patients with a receptor-negative breast carcinoma. The present data shows for the first time that MMP-2 negativity could serve as a marker for distinctly favourable prognosis in breast carcinoma patients. MMP-2 positivity is also shown to correlate to poor survival in node-negative breast carcinoma.
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                Author and article information

                Contributors
                +48-85-7468587 , +48-85-7468585 , mroczko@umb.edu.pl
                Journal
                Tumour Biol
                Tumour Biol
                Tumour Biology
                Springer Netherlands (Dordrecht )
                1010-4283
                1423-0380
                7 January 2014
                7 January 2014
                April 2014
                : 35
                : 4
                : 3793-3802
                Affiliations
                [ ]Department of Biochemical Diagnostics, University Hospital, Białystok, Poland
                [ ]Department of Biochemical Diagnostics, Medical University, Białystok, Poland
                [ ]Department of Neurodegeneration Diagnostics, Medical University, Białystok, Poland
                [ ]Second Department of General and Gastroenterological Surgery, Medical University, Białystok, Poland
                [ ]Department of General Pathomorphology, Medical University, Białystok, Poland
                Article
                1502
                10.1007/s13277-013-1502-8
                3980035
                24395652
                c31d4b18-1c95-4920-94fc-d9cf7b634b6c
                © The Author(s) 2014

                Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

                History
                : 7 October 2013
                : 29 November 2013
                Categories
                Research Article
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                © International Society of Oncology and BioMarkers (ISOBM) 2014

                Oncology & Radiotherapy
                colorectal cancer,matrix metalloproteinase 2,tissue inhibitor of matrix metalloproteinases 2,immunohistochemical staining

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