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Prognostic significance of matrix metalloproteinase-2, -8, -9, and -13 in oral tongue cancer

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Prognostic significance of matrix metalloproteinase-2, -8, -9, and -13 in oral tongue cancer
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  Prognostic significance of matrix metalloproteinases-1, -2, -7 and -13 and tissueinhibitors of metalloproteinases-1, -2, -3 and -4 in colorectal cancer Marja Hilska 1 *, Peter J. Roberts 1 , Yrj € o U. Collan 2 , Veli Jukka O. Laine 2 , Jyrki K € ossi 3 , Pirkko Hirsim € aki 2 ,Otto Rahkonen 4 and Matti Laato 1,4 1  Department of Surgery, Turku University Central Hospital, Turku, Finland  2  Department of Pathology, University of Turku, Turku, Finland  3  Department of Surgery, P € aij  € at-H  € ame Central Hospital, Lahti, Finland  4  Department of Medical Biochemistry, University of Turku, Turku, Finland  Strong expression of many matrix metalloproteinases (MMPs)has been related to poor survival of colorectal cancer (CRC)patients. The expression of tissue inhibitors of metalloprotei-nases (TIMPs) has been associated with both a beneficial and apoor outcome and there is thus a need to further clarify the sig-nificance of MMPs and TIMPs in CRC. The prognostic signifi-cance of 4 MMPs and TIMPs in CRC was evaluated. Formalin-fixed, paraffin-embedded tissue arrayed samples of 351 patientswith primary colon or rectal cancer of Dukes’ stages A-D wereselected for immunohistochemical staining of MMP-1, -2, -7 and-13, and TIMP-1, -2, -3 and -4. High expression of MMP-2 inthe malignant epithelium as well as in the surrounding stromawas associated with reduced survival of colon cancer patients.Strong epithelial and stromal cytoplasmic staining of TIMP-3was associated with a longer survival in rectal cancer patients,and here the interobserver variation for evaluating the degreeof staining was lower than for epithelial staining. Strong stromalcytoplasmic staining of TIMP-4 predicted longer survival of rec-tal cancer patients. Multivariate analysis showed that stromalcytoplasmic TIMP-3 staining was the only marker of independ-ent prognostic value. MMP-2 might be a useful prognosticmarker in colon cancer, and TIMP-3 and TIMP-4 in rectal can-cer, but the findings associated with stromal staining should beinterpreted with some caution. Different biologic behavior ordifferent genetic development may explain the differences be-tween colon and rectal cancers regarding the expression of MMP-2, TIMP-3 and TIMP-4. '  2007 Wiley-Liss, Inc. Key words:  colorectal cancer; matrix metalloproteinases; tissueinhibitors of metalloproteinases; prognosis; survival Matrix metalloproteinases (MMPs) are a large family of zinc-dependent neutral endopeptidases that play an important role inthe degradation of all matrix components crucial for malignant tu-mor growth, invasion and metastasis. 1,2 Metalloproteinases areinhibited by tissue inhibitors (TIMPs) which are secreted proteins.These bioactive substances are specific inhibitors of MMPs thatbind to enzymatically active MMPs at a 1:1 molar stoichiometrythus inhibiting proteolysis. 3 The role of TIMPs for the homeostasisof the extracellular matrix is critical and may inhibit or stimulatetumsrcenesis. 4 Many studies have shown that the expression of several MMPsis enhanced in a number of malignancies, including colorectalcancer (CRC), but the relation between the expression of MMPsand overall patient survival is not clear. 5,6 Enhanced expression of MMP-1, -7 and -13 has been reported to be associated with metas-tasis and poor survival of patients with CRC. 7–9 Tissue concentra-tions of MMP-1, MMP-2 and TIMP-1 are increased in CRC com-pared to healthy colorectal tissue. This has been related to the roleof these endogenous substances in cancer progression. 2 Healthytissue may contain high levels of TIMP-2 10 and the levels of TIMP-3 mRNA are regionally increased in moderately and poorlydifferentiated CRC. 11 On the other hand, TIMP-3 protein levelsmay decrease progressively as the stage of CRC advances. 12 Theexact role of TIMP-4 in CRC is unknown.In our study, the data of 351 patients were analyzed and cancer tissue specimens from the tumors were examined to clarify theprognostic significance of the expression of MMP-1, -2, -7 and-13 and their tissue inhibitors TIMP-1, -2, -3 and -4 in patientsoperated on for CRC. Any differences between colon and rectalcancers were of special interest. Material and methods Tissue samples The tissue samples were obtained from the archives of theDepartment of Pathology of the Turku University Central Hospi-tal. The data of 351 CRC patients who had undergone bowelresection in 1981–1990 at the Turku University Central Hospitalwere included in the study. Of the patients, 49 had Dukes A, 199Dukes B, 42 Dukes C and 61 Dukes D tumors. All pertinent clini-cal and histopathologic data of the patients were collected fromthe patients’ case records and stored in a computer database. Theclinicopathologic data of the patients are shown in Table I and thesurvival curves according to the modified Dukes’ classification areshown in Figure 1. Tissue microarray Archival paraffin-embedded CRC tissue samples were used toobtain tissue microarray (TMA) blocks for immunohistochemicalstaining. Areas 1 mm in diameter were chosen and marked in he-matoxylin and eosin-stained 4- l m-thin surface sections of theblocks under light microscopy. For carcinomas, invasive areas of malignant tumor of the lowest degree of differentiation or thehighest number of mitoses and of highest cellular atypia were cho-sen. Wide and homogenous areas (compatible with cellular atypiaand poor differentiation) were preferred. To avoid contamination,areas at least 2 mm apart from normal tissue or adenoma werechosen. Necrotic and autolytic areas and areas rich in stromalreaction were avoided. For tumors producing abundant intra- or extracellular mucin, invasive areas with a high number of epithe-lial cells were chosen. Representative areas of cancer mucosawhere marked by a pathologist (VJOL) on slides stained with he-matoxylin and eosin from selected paraffin blocks and a cylinder of tissue 1 mm in diameter was cut with a TMA instrument(Beecher Instruments, Sun Prairie, WI) into a new paraffin block.Serial 4- l m sections were then cut from the TMA paraffin blocks.The sections were mounted on ChemMate TM Capillary Gap PlusSlides (Grey) by DAKO. Normal colorectal mucosa also was Grant sponsor: Special Government Funding (EVO) allocated to TurkuUniversity Central Hospital. * Correspondence to:  Department of Surgery, Turku University CentralHospital, Kiinamyllynkatu 4-8, 20520 Turku, Finland.Fax: 1 358-2-3132284. E-mail: marja.hilska@pp.inet.fiReceived 13 September 2006; Accepted after revision 2 February 2007DOI 10.1002/ijc.22747Published online 23 April 2007 in Wiley InterScience (www.interscience.wiley.com).  Int. J. Cancer:  121,  714–723 (2007) '  2007 Wiley-Liss, Inc. Publicationofthe InternationalUnion AgainstCancer  marked by the pathologist (VJOL) and obtained from tumor blocks adjacent to but at least 2 mm apart from malignant neoplas-tic areas of the section. If available, another sample was obtainedfrom normal colorectal mucosa at either of the resection marginsfrom the surgical specimen. Lymphatic follicles and hyperplasticand inflamed areas were avoided. To obtain enough mucosa for the TMA, tangentially cut areas were discarded.  Immunohistochemical staining Stainings for MMP-1, MMP-2, MMP-7 and TIMP-2 were per-formed using a Techmate 500 immunostaining machine and a per-oxidase/diaminobenzidine (DAB) multilink detection kit (DAKO,Glostrup, Denmark) which is based on an indirect streptavidin-biotin method. Briefly, the sections were first deparaffinized inxylene (3 times for 5 min) and rehydrated through a graded seriesof ethanol after which they were rinsed briefly in Tris-bufferedsaline. For antigen retrieval, the slides were incubated in a micro-wave oven in 10 mmol/L citrate buffer (pH 6.0), but for MMP-7such pretreatment was not required. The dilutions used for MMP-1, MMP-2, MMP-7 and TIMP-2 detection were 1:250, 1:300,1:250 and 1:50, respectively. Sections were counterstained withMayer’s hematoxylin and eosin. After staining, the sections weredehydrated in ethanol, cleared in xylene and covered with Moun-tex and cover-slips.The TMAs for MMP-13, TIMP-1, TIMP-3 and TIMP-4 werehand-stained. For TIMP-3 and TIMP-4 immunostainings, tissuesections were pretreated by boiling in microwave oven in 10mmol/L citrate buffer (pH 6.0) for 10 min as recommended bythe supplier (NeoMarkers, Fremont, CA), and for MMP-13 andTIMP-1 stainings the tissue sections were boiled in microwaveoven for 20 min. The antibody dilutions used for MMP-13,TIMP-1, TIMP-3 and TIMP-4 detection were 1:50, 1:20, 1:400and 1:2000, respectively, and were incubated at 4  C overnight.The reactivity of the antibodies was visualized using the avidin-biotin method as recommended by the supplier (Histostain-PlusKit, Zymed, South San Fransisco, CA). A brown color was devel-oped with diaminobenzidine (DAB-Plus Kit, Zymed, South San TABLE I –  UNIVARIATE ANALYSIS OF VARIABLES RELATED TO TUMOR AND PATIENT: ALL CRC PATIENTSVariable Category/ cutoff value  N  Percentageof patientsin categories5-year survival (%)  p  value Age  < 65 years 155 44 59 0.47  65 years 196 56 58Gender Male 162 46 52 0.033Female 189 54 64Modified Dukes’ stage A 49 14 90  < 0.0001B 199 57 67C 42 12 48D 61 17 13Histologic differentiation Good 4 1 75 0.72 1 Moderate 284 81 58Poor 61 17 59Mucinous 2 1 0Tumor site Colon 233 66 61 0.28Rectum 118 34 53T stage 1 12 3 92  < 0.00012 39 11 903 203 58 594 97 28 40Amount of mucin 0–45% 322 92 59 0.62  50% 29 8 55Urgency of operation Elective 301 86 62 0.0007Emergency 50 14 38MMP-2 epithelial expression  < 0.65 44 13 77 0.011  0.65 307 87 56MMP-2 stromal expression  < 0.65 45 13 76 0.014  0.65 306 87 56TIMP-3 epithelial cytoplasmic expression  < 1.25 291 83 56 0.017  1.25 60 17 72TIMP-3 epithelial nuclear expression  < 1% 106 30 67 0.02  1% 245 70 55TIMP-3 stromal cytoplasmic expression  < 0.1 94 27 47 0.02  0.1 257 73 63TIMP-3 stromal nuclear expression  < 25% 315 90 60 0.047  25% 36 10 44 1 Excluding mucinous tumors due to small sample size. F IGURE  1  – Kaplan–Meier survival curves of all CRC patients ( n 5 351) by modified Dukes’ stage. 715 MMPs AND TIMPs IN COLORECTAL CANCER  Fransisco, CA), and the sections were counterstained with hema-toxylin and mounted (improved Gurr Aquamount, BDH Labora-tory Supplies, Poole, UK). The specificity of the immunoreactionswas controlled by omitting the primary antibody, by using samedilution of nonimmune sera, or—for TIMP-4—by incubating theantibody overnight at 4  C with an excess of blocking peptide(NeoMarkers, Fremont, CA). The specificity of the antibodies hasbeen tested. 13–17 Evaluation of all TMA stainings was done with a Zeiss lightmicroscope at 10 3 objective magnification and 10 3 ocular mag-nification. The one who made these evaluations (MH) wasunaware of tumor grade, stage or clinical outcome. Some stainingswere randomly reevaluated by an independent pathologist (YUC).The following interobserver correlation coefficients were obtainedafter adjustment for the intraclass correlation coefficient (ICC),  i.e. differences in mean level of staining: epithelial cytoplasmic stain-ing of MMP-2 0.88, epithelial cytoplasmic staining of TIMP-30.70 and epithelial nuclear staining of TIMP-3 0.76. The evalua-tion of stromal staining was less reproducible; the ICC for MMP-2was 0.62, for TIMP-3 0.25 and for stromal nuclear TIMP-3 stain-ing 0.50.  Antibodies The monoclonal mouse anti-human antibody used for MMP-1staining was supplied by Oncogene, Boston, MA (code IM35L,clone 41-IE5), and that used for MMP-2 staining by OncogeneResearch, Calbiochem, San Diego, CA (code IM33L, clone 42-5D11). The antibody used for MMP-7 staining was supplied byNeoMarkers, Fremont, CA (code MS-813-P0, clone ID2). Theantibody for MMP-13 detection was a monoclonal mouse anti-body supplied by NeoMarkers, Fremont, CA (code VIIIA2, cloneMS-825-P). The antibodies for TIMP-2 detection (code RB-1488-PO, clone MS-1485), TIMP-3 detection (code RB-1541, Ab-1)and TIMP-4 detection (code RB-1542-PO, LOT 1542P108), wererabbit polyclonal antibodies supplied by NeoMarkers, Fremont,CA. The antibody for TIMP-1 was a mouse anti-human antibody(code MAB 13429, LOT 23061106) supplied by Chemicon Inter-national, Temecula, CA. F IGURE  2  – Staining patterns of MMPs-1, -2, -7 and -13 in normal and cancer epithelium of a Dukes B sigmoid cancer sample. This patient had a poor outcome.Staining of normal colorectal epitheliumfor MMP-1 ( a ), MMP-2 ( c ), MMP-7 ( e )and MMP-13 ( g ). Staining of cancer epi-thelium for MMP-1 ( b ), MMP-2 ( d  ),MMP-7 (  f  ) and MMP-13 ( h ). 716  HILSKA  ET AL .   Evaluation of staining positivity MMPs and TIMPs in cancer cell cytoplasm.  The staining in-tensity of the epithelial cytoplasm was estimated on a scale from 0to 3 1 to a precision of 0.05. The intensity groups were defined asfollows:3 15  50–100% of the cytoplasm of the epithelial cell wasstained with strong intensity,2 15 only part of the cytoplasm was stained with moderate or strong intensity (usually due to mucin goblet) and stainingwith strong intensity was seen in   15–50% of the cyto-plasm of the cell or    15–100% of the cytoplasm wasstained with moderate intensity,1 1 5  5–15% of the cytoplasm of the cell was stained withmoderate or strong intensity or    15–100% of the cyto-plasm was weakly stained,0 5 less than 5% of the cytoplasm was stained.Strong staining intensity was defined as intensive dark browncolor, weak intensity as light brownish staining. Moderate stainingwas the intensity between strong and weak staining intensities.A weighted staining score both for malignant epithelium as wellas for cancerous stromal tissue was counted. The staining scorewas then counted by adding the fraction of cells staining at the in-tensity 1 1 to the fraction of cells staining at the intensity 2 1 mul-tiplied by 2. Finally, the fraction of cells staining at the intensity3 1 multiplied by 3 was added. The fraction of cells at the inten-sity 0 did not influence the staining index. 18 In cases where stain-ing was very weak, half of the area of this just discernible stainingwas scored as intensity 1 1 and half as 0. When the staining inten-sity was intermediate between 2 intensity classes, half of the areawas classified into the higher intensity class and half of the areainto the lower class. TIMP-3 and TIMP-4 in nuclei of malignant epithelial and desmoplasticstromalcells.  Nuclear staining of cancerous epithelial F IGURE  3  – Staining patterns of TIMPs-1, -2, -3 and -4 in normal and cancer epi-thelium of a Dukes B sigmoid cancer sample. This patient had a poor outcome.Staining of normal colorectal epitheliumfor TIMP-1 ( a ), TIMP-2 ( c ), TIMP-3 ( e )and TIMP-4 ( g ). Staining of cancer epi-thelium for TIMP-1 ( b ), TIMP-2 ( d  ),TIMP-3 (  f  ) and TIMP-4 ( h ). 717 MMPs AND TIMPs IN COLORECTAL CANCER  cells was evaluated as the percentage of positive nuclei of the totalnumber of epithelial nuclei observed. Clear brown nuclear stainingwas regarded as positive but nuclear staining intensity was notseparately evaluated. Nuclear staining of stromal cells was eval-uated as in cancer cells.  MMPs and TIMPs in cytoplasm of desmoplastic stromalcells.  The staining intensity of stromal cells was evaluated by thesame principles as epithelial cells on a scale from 0 to 3 1 . Stain-ing of MMP-7 and MMP-13 in stromal cells was considered to beso weak that it was not evaluated.TIMP-1 was scored as negative or positive. Staining wasregarded positive if in at least 1% of the stromal cells moderate or strong TIMP-1 staining could be seen or if at least 5% of the cellshad weak staining. Statistical analyses An optimal cutoff value was identified by Kaplan–Meier’s uni-variate analysis by analyzing the scores of each marker by dividingthe material in 2 parts with every possible cutoff point to find thelowest significant  p  value for the differences between the groupsin survival analyses. The cancer-specific survival curves were esti-mated by the Kaplan–Meier product-limit method. A log-rank testwas used to assess differences between life table data. The Cox’sproportional hazard model was used to determine which factorswere most significantly independently associated with survival. 19 For all statistical analyses, differences between the groups wereconsidered significant at  p  values less than 0.05. All statisticalanalyses were performed using the SAS System for Windows, ver-sion 9.1 (SAS Institute, Cary, NC). Results General observations Normal epithelium.  The staining patterns of all MMPs andTIMPs in the normal colorectal epithelium are shown in Figures 2 a ,2 c , 2 e  and 2 g , and 3 a , 3 c , 3 e  and 3 g , respectively. The epithelialcytoplasm stained occasionally for MMP-1 and MMP-7, but sincethere was strong variation in the level of sectioning no uniformpicture emerged. In the lamina propria, the plasma cells, part of  TABLE II –  UNIVARIATE ANALYSIS OF VARIABLES RELATED TO TUMOR AND PATIENT: COLON CANCER AND RECTAL CANCER PATIENTSVariable Category/ cutoff valueColon cancer Rectal cancer   N   5-year survival (%)  p  value  N   5-year survival (%)  p  value Age  < 65 yrs 107 61 0.94 48 56 0.47  65 yrs 126 61 70 51Gender Male 97 59 0.66 65 42 0.0042Female 136 63 53 68Modified Dukes’ stage A 19 100  < 0.0001 30 83  < 0.0001B 138 73 61 52C 31 52 11 36D 45 13 16 13Histologic differentiation Good 4 75 0.48 1 0 – 0.6 1 Moderate 178 62 106 53Poor 50 58 11 64Mucinous 1 0 1 0T stage 1 2 100  < 0.0001 10 90 0.00022 18 100 21 813 140 64 63 494 73 45 24 25Amount of mucin 0–45% 208 61 0.87 114 54 0.15  50% 25 60 4 25Urgency of operation Elective 184 67 0.0002 117 54 0.29Emergency 49 39 1 0MMP-2 epithelial expression  < 0.65 36 83 0.0049 8 50 0.83  0.65 197 57 110 54MMP-2 stromal expression  < 0.65 31 81 0.023 14 64 0.3  0.65 202 58 104 52TIMP-3 epithelial cytoplasmic expression  < 1.25 191 59 0.17 100 49 0.033  1.25 42 69 18 78TIMP-3 stromal cytoplasmic expression  < 0.1 59 56 0.41 35 31 0.0068  0.1 174 63 83 63TIMP-4 stromal cytoplasmic expression  < 0.05 42 57 0.59 28 36 0.037  0.05 191 62 90 59 1 Excluding mucinous tumors due to small sample size. TABLE III –  CANCER CELL STAINING: MMPS AND TIMPS (351 CRC SAMPLES)Cancer cell cytoplasm Cancer nucleiAntigenNegativesamples (%)MeanscoreMedianscore (range) cutoff   p  value Negativesamples (%)MeanscoreMedianscore (range) cutoff   p  value MMP-1 12 (3.4) 1.28 1.3 (0–2.35) 1.0 0.11 n.d. n.d. n.d.MMP-2 5 (1.4) 1.28 1.3 (0–2.9) 0.65 0.011 n.d. n.d. n.d.MMP-7 57 (16.2) 0.58 0.55 (0–2.6) 0.5 0.4 n.d. n.d. n.d.MMP-13 100 (28.5) 0.53 0.35 (0–2.0) 0.1 0.21 n.d. n.d. n.d.TIMP-1 73 (20.8) 0.51 0.4 (0–1.6) 0.15 0.55 n.d. n.d. n.d.TIMP-2 1 (0.3) 1.71 1.7 (0–2.9) 1.40 0.37 n.d. n.d. n.d.TIMP-3 3 (0.9) 0.94 1 (0–2) 1.25 0.017 106 (30.2) 11% 2% (0–75%) 1% 0.02TIMP-4 28 (8.0) 0.77 0.95 (0–2.0) 0.75 0.3 34 (9.7) 31% 15% (0–95%) 5% 0.27n.d., not defined. 718  HILSKA  ET AL .
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