Lack of MMP-9 expression is a marker for poor prognosis in Dukes’ B colorectal cancer

  • Selja Koskensalo1,

    Affiliated with

    • Jaana Hagström2,

      Affiliated with

      • Nina Linder3,

        Affiliated with

        • Mikael Lundin3,

          Affiliated with

          • Timo Sorsa4,

            Affiliated with

            • Johanna Louhimo1 and

              Affiliated with

              • Caj Haglund1, 2Email author

                Affiliated with

                BMC Clinical Pathology201212:24

                DOI: 10.1186/1472-6890-12-24

                Received: 7 June 2012

                Accepted: 29 November 2012

                Published: 7 December 2012

                Abstract

                Background

                Matrix metalloproteinases (MMPs) play a role in cancer progression by degrading extracellular matrix and basement membranes, assisting in tumour neovascularization and in supporting immune response in cancer.

                Methods

                We studied the prognostic value of immunohistochemical expression of MMP-2, MMP-8, and MMP-9 in a series of 619 colorectal cancer patients using tissue microarray specimens.

                Results

                Of the samples, 56% were positive for MMP-2, 78% for MMP-8, and 60% for MMP-9. MMP-9 associated with low WHO grade (p < 0.001). In univariate analysis of Dukes’ B tumours, MMP-9 negativity associated with poor survival (p = 0.018), and MMP-9 positivity was an independent prognostic marker in multivariate analysis of these tumours (p = 0.034).

                Conclusion

                Negative MMP-9 expression can predict poor prognosis in Dukes’ B colorectal tumours and may prove useful for identifying patients, who should be offered adjuvant treatment.

                Keywords

                MMP-2 MMP-8 MMP-9 Colorectal cancer Prognosis Immunohistochemistry

                Background

                Colorectal cancer (CRC) is the third most common malignancy in the world [1]. The most important prognostic factor in colorectal cancer is stage of disease at diagnosis. Other factors for poor prognosis are venous and lymphatic invasion, deficient healthy tissue margins at surgery, obstruction or perforation of the bowel, and poor differentiation at histology [25]. In addition to tumour-specific factors, also host responses, such as intra- or peritumoral inflammation and desmoplasia or reactive lymph nodes can suppress tumour spread and predict better outcome [68]. In cancer progression and spread, tumours must invade their surrounding tissues, basement membranes (BM), and extracellular matrix (ECM), must avoid the host’s immunoresponse, and must ascertain their circulation by neovascularization. In all these phenomena, matrix metalloproteinases (MMPs) play an important role [9].

                MMPs are zinc-dependent endopeptidases capable of degrading both BM and ECM proteins and extracellular adhesion molecules. MMP-9, also known as gelatinase B, is able to degrade collagens IV (the main component of BM) and V, gelatins, and elastin. MMP-2, also known as gelatinase A, is able to degrade the same substrates as MMP-9, but also collagens I, VII, X, fibronectin and procollagenase-3 [9]. MMP-8, also called collagenase-2, is able to degrade collagens I, II and III [10].

                In CRC, MMP-2 immunoexpression associates with advanced disease [11, 12], and high MMP-2 expression in cancer cells and the stroma associates with poor prognosis [11, 13]. MMP-9 correlates with metastatic disease [11, 14, 15] and poor prognosis [14], although conflicting findings exist [16]. MMP-8 is expressed in many cancer types [17, 18] and may protect against cancer spread by regulating tumour metastasis [18].

                In this study, we studied the prognostic value of MMP-2, MMP-8 and MMP-9 immunoexpression in colorectal cancer.

                Methods

                Patients

                Sample material came from 643 consecutive patients who underwent surgery for CRC at the Department of Surgery, Meilahti Hospital, Helsinki University Central Hospital, between 1982 and 1998. Of these, 18 patients were excluded on account of incorrect final diagnosis (9 patients) or synchronous multiple tumours (9 patients). Six cases were excluded because of insufficient archival surgical tissue specimens. Finally, 619 cases remained, 330 of them male. Tumour staging was performed according to the modified Dukes’ classification [19], with 91 tumours classified as Dukes’ stage A, 226 as stage B, 161 as stage C, and 145 as stage D. For patients’ clinicopathological characteristics, see Table 1.
                Table 1

                Patient clinicopathological characteristics and their correlation with MMP-2, MMP-8, and MMP-9 immunoreactivity in colorectal cancer patients assessed with chi-square test

                 

                MMP-2

                MMP-8

                MMP-9

                Clinicopathological

                Patients

                positive

                p-value

                Patients

                positive

                p-value

                Patients

                positive

                p-value

                variable

                (n=)

                (n)

                %

                 

                (n=)

                (n)

                %

                 

                (n=)

                (n)

                %

                 

                Gender

                   

                0.887

                   

                0.496

                   

                0.574

                Female

                272

                153

                56.3

                 

                257

                194

                75.5

                 

                257

                158

                61.5

                 

                Male

                309

                172

                55.7

                 

                301

                235

                78.1

                 

                301

                192

                63.8

                 

                Age

                   

                0.107

                   

                0.343

                   

                0.197

                < 65 years

                244

                146

                59.8

                 

                228

                183

                80.3

                 

                238

                142

                59.7

                 

                ≥ 65 years

                337

                179

                53.1

                 

                320

                246

                76.9

                 

                320

                208

                65.0

                 

                Dukes’ stage

                   

                0.317

                   

                0.148

                   

                0.100

                A

                87

                44

                50.6

                 

                82

                63

                76.8

                 

                83

                57

                68.7

                 

                B

                208

                110

                52.9

                 

                197

                163

                82.7

                 

                206

                138

                67.0

                 

                C

                155

                93

                60.0

                 

                137

                99

                72.3

                 

                138

                77

                55.8

                 

                D

                131

                78

                59.5

                 

                132

                104

                78.8

                 

                131

                78

                59.5

                 

                Dukes’ stage

                   

                0.066

                   

                0.116

                   

                0.016*

                A and B

                295

                154

                52.2

                 

                279

                226

                81.0

                 

                289

                195

                67.5

                 

                C and D

                286

                171

                59.8

                 

                269

                203

                75.5

                 

                269

                155

                57.6

                 

                Differentiation (WHO grade)

                   

                0.116

                   

                0.326

                   

                <0.001*

                1

                19

                9

                47.4

                 

                19

                14

                73.7

                 

                18

                12

                66.7

                 

                2

                382

                211

                55.2

                 

                368

                290

                78.8

                 

                374

                254

                67.9

                 

                3

                155

                86

                55.5

                 

                138

                111

                80.4

                 

                142

                77

                54.2

                 

                4

                24

                19

                79.2

                 

                22

                14

                63.6

                 

                23

                7

                30.4

                 

                missing

                1

                   

                1

                   

                1

                   

                Histologic type

                   

                0.680

                   

                0.085

                   

                0.053

                Adenocarcinoma

                512

                288

                56.3

                 

                481

                382

                79.4

                 

                489

                314

                64.2

                 

                Mucinous carcinoma

                69

                37

                53.6

                 

                67

                47

                70.1

                 

                69

                36

                52.2

                 

                Tumour location

                   

                0.202

                   

                0.073

                   

                0.419

                Colon

                315

                169

                53.7

                 

                305

                247

                81.0

                 

                311

                191

                61.4

                 

                Rectum

                263

                155

                58.9

                 

                240

                179

                74.6

                 

                244

                158

                64.8

                 

                missing

                3

                   

                3

                   

                3

                   

                * = p-value significant.

                Clinical data were retrieved from patient records, and survival and cause of death data until March 2011 from the Population Register Centre of Finland, and Statistics Finland.

                For MMP-9, we also stained tissue microarray (TMA) specimens from a validation series of 213 CRC patients treated between 1998–2001, 82 of them male; 31 tumours were classified as Dukes’ stage A, 70 stage B, 69 stage C, and 41 stage D, with 201 of tumours being adenocarcinomas; with 7 being grade I, 161 grade II, 37 grade III, and 4 grade IV. Clinical data was retrieved, as for the main cohort, until October 2011.

                Tissue samples and preparation of TMA blocks

                Formalin-fixed and paraffin-embedded surgical tissue samples were collected from the archives of the Department of Pathology, University of Helsinki. Histopathologically representative regions of tumour specimens were defined and marked on H&E slides. Three cores from each tumour block were sampled with 1.0 mm punchers with a manual tissue microarrayer (Tissue Arrayer 1, Beecher Instruments Inc., Silver Spring, MD, USA). Three series of blocks were constructed, all including one sample from each patient. From each block, 4-μm slides were cut and processed for immunohistochemistry.

                Immunohistochemistry of MMP-2, MMP-8, and MMP-9

                The Lab Vision Autostainer TM 480 (LabVision, Fremont, CA, USA) served for immunohistochemistry. Specimens were deparaffinized in xylene and rehydrated through graded alcohol series. To retrieve antigen, samples were heated in the pretreatment module of the autostainer in Tris–HCl pH 8.5 buffer for 20 minutes at 98°C. The samples were incubated for 5 min in DAKO REAL Peroxidase-Blocking Solution (DAKO, Glostrup, Denmark) for inactivation of endogenous peroxidases. The sections were incubated for one hour with a monoclonal primary MMP-2 antibody MS-806-PO (NeoMarkers) diluted 1:700, with a polyclonal MMP-8 antibody 1:100 [20] or with a polyclonal MMP-9 antibody RB-1539-R7 (NeoMarkers) 1:1500 and reacted for 30 min with the Dako REAL EnVision™/HRP detection system, Rabbit/Mouse (ENV) reagent. Between each pair of steps the sections were rinsed with Tween-20/PBS. The peroxidase staining was visualized with 3-amino-9-ethylcarbatzole (Sigma-Aldrich, Inc., St. Louis, MO, USA). Slides were counterstained with Meyer’s haematoxylin, washed in tap water for 10 min, and mounted in aqueous mounting medium (Aquamount, BDH, Poole, UK). Stainings of specimens without primary MMP-antibody were used as negative controls. Gingival tissue was used as positive control in MMP-8 stainings, and gastric tissue in MMP-2 and MMP-9 stainings.

                Scoring of immunostainings

                MMP-2, MMP-8, and MMP-9 immunostainings were scored by two independent investigators (S.K. and J.H.) without knowledge of clinical outcome. In case of different scores, the consensus score was determined. The interobserver variation was low, below 5%. The intensity of cytoplasmic staining in cancer cells was evaluated: strong intensity, scored as 3, moderate as 2, and weak as 1. Absence of positivity in all spots was scored as 0 (Figure 1). Spots without cancer cells were excluded. For further analysis, the patients were divided into two groups, negative (0) versus positive (score 1–3).
                http://static-content.springer.com/image/art%3A10.1186%2F1472-6890-12-24/MediaObjects/12907_2012_113_Fig1_HTML.jpg
                Figure 1

                Immunohistochemical scoring pattern of MMP-9 in colorectal cancer. A no expression, B mild, C moderate, D strong.

                Statistical analysis

                Correlations between stainings and clinicopathological variables were assessed with the χ2 test or Fisher’s exact test when applicable. The Mann–Whitney U-test was applied to determine the correlation between age and the immunoreactivity. Life-tables were calculated with the Kaplan-Meier method. The significance of the difference between groups was assessed with the log-rank test or log-rank test for trend. Patients alive at the end of the follow-up (March 2011) and patients who died from unrelated causes or postoperatively (within 30 days from surgery) were censored. The Cox proportional hazards model served for multivariate survival analysis. Clinical variables included in the model as covariates were age, Dukes’ stage, differentiation (WHO grade), tumour location (colon or rectum), and tumour histology (adenocarcinoma or mucinous carcinoma).

                The likelihood ratio test was applied for exclusion or inclusion of significant variables. A p-value of 0.05 was considered significant. Statistical analyses were performed with SPSS 17.0 software.

                Results

                Tissue expression of MMP-2, MMP-8, and MMP-9

                Immunostaining for MMP-2

                Of 581 cases, MMP-2 immunoreactivity was observed in 325 (55.9%). In 56 patients (9.6%), MMP-2 immunopositivity was scored as strong, in 98 (16.9%) as moderate, and in 171 (29.4%) as weak, whereas 256 (44.1%) lacked any MMP-2 immunopositivity. MMP-2 positivity did not correlate with clinicopathological variables (Table 1).

                Immunostaining for MMP-8

                Of 548 cases, MMP-8 immunoreactivity was noticed in 429 (78.3%). In 39 patients (7.1%), MMP-8 immunopositivity was scored as strong, in 153 (27.9%) as moderate, and in 237 (43.2%) as weak, whereas 119 (21.7%) lacked any MMP-8 immunopositivity. MMP-8 positivity showed no correlation with clinicopathological variables (Table 1).

                Immunostaining for MMP-9

                Of the 581 cases, MMP-9 immunoreactivity was observed in 350 (60.2%). In 50 patients (8.6%), MMP-9 immunopositivity was scored as strong, in 108 (18.6%) as moderate, and in 192 (33.0%) as weak, whereas 208 (35.8%) lacked any MMP-9 immunopositivity. MMP-9 correlated with WHO grade (p < 0.001); it was more often positive in high to moderately differentiated tumours. MMP-9 immunopositivity did not correlate with Dukes’ stage, but was more often positive in local (Dukes’ A-B) tumours than in advanced disease (p = 0.016 ) (Table 1).

                In the validation series of 213 cases MMP-9 was positive in 167 (78.4%). In positive patients, in 12 (7.2%), the immunopositivity was scored as strong, in 41 (24.6%) as moderate, and in 114 (68.3%) as weak. No correlation appeared between clinicopathological variables and MMP-9 immunoexpression.

                Prognostic roles of MMP-2, MMP-8, and MMP-9

                In univariate analysis, 5-year survival was 62.5% in MMP-9-positive, and 52.2% in MMP-9-negative patients, a difference that was significant (p = 0.015). Advanced Dukes’ stage (p < 0.001), old age (p = 0.005), and higher WHO grade (p < 0.001) associated with poor prognosis (Table 2). The association between elevated MMP-9 immunoexpression and improved prognosis was evident only in Dukes’ B tumours, but it was such a strong prognostic factor that the association was significant in analysis of the whole cohort (p = 0.018). In Dukes’ A stage 5-year survival was 92.3% in MMP-9 positive and 87.1% in MMP-9 negative patients (p = 0.417), in Dukes’ C patients 56.6% versus 50.3% (p = 0.618), and in Dukes’ D patients 6.8% versus 9.6% (p = 0.992).
                Table 2

                Univariate analysis of correlations between preoperative characteristics and survival with Kaplan-Meier life-table and logrank test analyses

                Clinicopathological

                Patients

                Cumulative 5-year

                χ2

                p-value

                variable

                (n)

                %

                survival %

                statistic

                 

                MMP2 immunoreactivity

                581

                  

                0.507

                0.477

                   Negative

                256

                44.1

                58.8

                  

                   Positive

                325

                55.9

                56.0

                  

                MMP8 immunoreactivity

                548

                  

                0.129

                0.719

                   Negative

                119

                21.7

                56.6

                  

                   Positive

                429

                78.3

                58.8

                  

                MMP9 immunoreactivity

                558

                  

                5.923

                0.015*

                   Negative

                208

                44.9

                52.2

                  

                   Positive

                350

                55.1

                62.5

                  

                Gender

                619

                  

                0.000

                0.992

                   Female

                289

                46.7

                56.1

                  

                   Male

                330

                53.7

                57.5

                  

                Age

                619

                  

                7.777

                0.005*

                   < 65 years

                259

                41.8

                61.6

                  

                   ≥ 65 years

                360

                58.2

                53.4

                  

                Dukes’ stage

                619

                  

                293.83

                <0.001*

                   A

                91

                14.7

                90.3

                  

                   B

                222

                35.9

                77.7

                  

                   C

                161

                26.0

                51.1

                  

                   D

                145

                23.4

                8.7

                  

                Differentiation (WHO grade)

                618

                  

                18.982

                <0.001*

                   1

                19

                3.1

                83.1

                  

                   2

                406

                65.7

                60.9

                  

                   3

                165

                26.7

                46.3

                  

                   4

                28

                4.5

                41.3

                  

                Histologic type

                619

                  

                1.311

                0.252

                   adenocarcinoma

                539

                87.1

                58.1

                  

                   mucinous carcinoma

                80

                12.9

                48.6

                  

                Tumour location

                616

                  

                2.126

                0.145

                   Colon

                339

                55.0

                58.3

                  

                   Rectum

                277

                45.0

                55.4

                  

                   missing

                3

                    

                * = p-value significant.

                In Cox stepwise multivariate analysis, age (p < 0.001), Dukes’ stage (p < 0.001), location (p = 0.016), and differentiation (p = 0.005) were all independent prognostic factors (Table 3). In Dukes’ B tumours, MMP-9 positivity was an independent prognostic factor (p = 0.034), as was tumour location (p = 0.041).
                Table 3

                Cox stepwise multivariate regression analysis of prognostic factors in 558 colorectal cancer patients (MMP-9)

                Covariate

                Wald statistic

                p-value

                RH

                95% CI

                Age

                31.209

                <0.001

                1.031

                1.020-1.043

                Dukes’ stage

                299.615

                <0.001

                  

                A

                    

                B

                2.397

                 

                1.664

                0.874-3.166

                C

                27.913

                 

                5.274

                2.846-9.773

                D

                117.503

                 

                30.446

                16.417-56.464

                Differentiation (WHO Grade)

                12.502

                0.005

                  

                1

                    

                2

                2.246

                 

                2.143

                0.791-5.806

                3

                4.577

                 

                3.031

                1.097-8.373

                4

                6.353

                 

                4.293

                1.383-13.330

                Tumour location in rectum

                5.815

                0.016

                1.373

                1.061-1.776

                Histologic type

                 

                NS

                  

                MMP9

                 

                NS

                  

                NS = not significant, RH = relative hazard, CI = confidence interval at 95% level.

                In the validation series, 5-year survival was 64.8% in MMP-9 positive patients compared to 63.5% in those MMP-9 negative, (p = 0.418). In subgroup analysis of Dukes’ stages there were no significant differences in survival according to stage.

                Neither MMP-2 immunoexpression (p = 0.477) nor MMP-8 immunoexpression (p = 0.719) associated in univariate analysis with prognosis.

                Discussion

                It is clinically relevant to identify a prognostic marker in Dukes’ B (stage II) disease. This stage is classified as local disease, but 20% will still die from this, with surgery curing 80%. We urgently need biomarkers to identify those 20% at risk in order to offer them adjuvant treatment. Here we show that MMP-9 tumour expression is an independent prognostic factor in Dukes’ B colorectal cancer. In the other stage groups, no significant difference in survival emerged. In Dukes’ B, the difference was so great, that it affected analysis of the whole cohort. In Dukes’ B and C of the validation series, we noticed poorer prognosis among patients with MMP-9 negative tumours, but the differences were not significant.

                The overall survival was better in the validation series. The difference in survival may have several reasons. First, pathologic routines have changed and more lymph nodes are nowadays evaluated, which may lead to stage migration. Second, the surgical technique has changed, especially in rectal cancer treatment. Third, the more frequent use of adjuvant treatment might have improved survival.

                Our results differ from those in a recent study of stage II CRC in which high MMP-9 expression associated with higher recurrence rate, shorter disease-free survival, and also shorter disease-specific survival, but association with disease-specific survival was not significant in multivariate analysis [21]. In CRC patients, high MMP-9 expression has been associated with liver metastasis [22]. We also analyzed high versus low immunoexpression, but found no association with survival (data not shown). Elevated MMP-9 mRNA levels have been associated with poor disease-free and overall survival [14]. On the other hand, some studies using immunohistochemistry have failed to show any correlation between MMP-9 and survival or clinicopathological parameters [16, 23]. In other cancer forms, such as lung cancer, head and neck squamous cell carcinoma, and gastric cancer, MMP-9 associates with poor prognosis [2426]. Interestingly, in early breast cancer, elevated MMP-9 associates with better prognosis [27].

                In cancer, the host response, like the intra- or peritumoral inflammation reaction or desmoplasia, can predict better prognosis [68]. Theoretically, some matrix-degrading proteinases may play a defensive role by supporting the immune/inflammatory response. Stromal expression of MMP-9 inversely associates with liver metastasis and tumour infiltration in CRC [28]. Stromal MMP-9 positivity also inhibits metachronous haematogenic metastasis in Dukes’ B and C colorectal cancer patients [29]. Here, we did not evaluate stromal MMP-9 expression, because we used tissue arrays with punches taken from cancerous regions, and our samples were unsuitable for reliable stroma evaluation. The TMA is not as reliable as analysis of whole tissue sections because of intra-tumoural heterogenity of immunohistochemical expression. However, the results of evaluation of TMAs and those of whole tissue sections have been shown to be in concordance [30].

                Tumour expression of MMP-2 has been associated with poor prognosis in colorectal [1113, 31], gastric [25], ovarian [32], and breast cancer [33]. Here, we found no correlation between MMP-2 immunoexpression and clinicopathological variables or prognosis of CRC. Results similar to ours have emerged in CRC [34]. Thereby the role of MMP-2 in CRC is still controversial.

                In breast cancer, MMP-8 changes metastatic potential in vitro[15], and in melanoma and lung cancer it inhibits metastasis formation by modulating cancer cell invasion and adhesion [14]. It was expected that MMP-8 might be a marker for improved prognosis in colorectal cancer, but we noticed no association between MMP-8 immunoexpression and survival.

                Conclusion

                The role of MMP-9 immunoexpression in colorectal cancer is dual; it plays a role in matrix degradation enabling tumour invasion, but it also seems to act as a supportive factor for hosts’ defensive mechanisms against cancer spread. Here we show that immunoexpression of MMP-9 is a promising prognostic marker in Dukes’ B (Stage II) CRC, the group of patients for whom we need new markers to identify those at risk who need adjuvant treatment.

                Ethics approval

                The study has been approved by the Surgical Ethics Committee of Helsinki University Central Hospital (Dnro HUS 226/E6/06) and National Supervisory Authority for Welfare and Health.

                Abbreviations

                BM: 

                Basement membrane

                CRC: 

                Colorectal cancer

                EMC: 

                Extracellular matrix

                H&E: 

                Haematoxylin-eosin

                MMP: 

                Matrix metalloproteinase

                mRNA: 

                Messenger ribonuclein acid

                TMA: 

                Tissue microarray.

                Declarations

                Acknowledgements

                We thank Päivi Peltokangas, Tuire Koski, and Elina Aspiala for excellent technical assistance.

                Funding

                This study was supported by Finska Läkaresällskapet, Medicinska Understödsföreningen Liv och Hälsa, Stiftelsen Dorothea Olivia, Karl Walter och Jarl Walter Perkléns minne, the Sigrid Jusélius Foundation, and a special governmental subsidy for research and training.

                Authors’ Affiliations

                (1)
                Department of Surgery, Helsinki University Central Hospital
                (2)
                Department of Pathology, Haartman Institute, University of Helsinki
                (3)
                Institute for Molecular Medicine Finland (FIMM), University of Helsinki
                (4)
                Institute of Dentistry, University of Helsinki and Department of Oral and Maxillofacial Diseases, Helsinki University Central Hospital

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                35. Pre-publication history

                  1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1472-6890/​12/​24/​prepub

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