Alimentary tract and pancreas

Alimentarni trakt i pankreas

Fenyvesi Attila,

Department of Pathology, General Hospital,Health

Center " Dr Gero István"

Senta, Serbia

ARCH GASTROENTEROHEPATOL 2003; 22 (No 1 - 2): 12 – 17

Assessment of the matrix

metalloproteinase-9 (MMP-9)

expression in colorectal

cancer: absence of correlation

with prognosis and clinicopathological

factors

Procena ekspresije metaloproteinaze-9 ( MMP-9)

u kolorektalnom karcinomu: otsustvo korelacije

sa prognozom i klinikopatološkim faktorima

 (accepted May 19st, 2001)

Key Words:

colorectal carcinoma,

matrix metalloproteinase-9 (MMP-9),

prognosis.

Abstract

Matrix metalloproteinases family of endopeptideses, degradative enzymes are considered to play an

important role in tumor invasion and metastasis. Type IV collagen is a major structural protein in the

basement membrane and extrecellular matrix, thus, much attention has been focused on the type IV

collagenases, (gelatinases, matrix metalloproteinase-2 and matrix metalloproteinase-9), which

specifically degrade the basement membrane. To assess the validity of matrix metalloproteinase-9 as

a prognostic marker of colorectal carcinoma, we performed immunohistochemical analysis on tissues

from 40 invasive colorectal adenocarcinomas. We investigated the relationship between clinicopathological

features of colorectal carcinoma and matrix metalloproteinase-9 immunoreactivity of

the tumor cells and their potential relationship with patients prognosis. Matrix metalloproteinase-9

immunoreactivity was assesed semiquantitatively at the invasive front of colorectal cancer. There

were no significant associations between intratumoral matrix metalloproteinase-9 expression with

major clinicopathological factors and with overall survival of patients with colorectal carcinoma. By

univariate Cox hazard-model stage of disease and histological grade of colorectal carcinoma were a

prognostic factor, while expression of matrix metalloproteinase-9 did not show prognostic significance.

Acknowledgement: Author wishes to thank Toth Tibor MD, PhD (Department of Pathology and Clinical Cytology, General Hospital Falun,

Sweden) for his kind assistance in immunohistohemical staining.

Sazetak

Matriks metaloproteinaze su degradiraju}i fermenti is familije endopeptidaza, za koje se pretpostavlja

da igraju zna~ajnu ulogu u procesu tumorskog napredovanju i nastanku metastaza. Kako je

kolagen tip IV glavni strukturni protein bazalne membrane i ekstra}elijskog matriksa glavna pa`nja

je usmerena na tip IV kolagenaza (gelatinaze, matriks metaloproteinazu-2 i matriks metaloproteinazu-

9) koje specifi~no razgradjuju bazalnu membranu. U cilju procene validnosti aktivnosti enzima

metaloproteinaze-9 kao prognosti~kog pokazatelja kolorektalnog karcinoma, mi smo sproveli

imunohistohemijsko ispitivanje tkiva 40 invazivnih kolrektalnih karcinoma. Ispitivali smo odnos

izmedju klinikopatolo{kih pokazatelja kolorektalnih karcinoma i imunoreaktivnosti matriks metaloproteinaze-

9 u tumorskim }elijama kao i mogu}u povezanost sa prognozom obolelih

osoba.Imunoreaktivnost matriks metaloproteinaze-9 je procenjivana semikvantitativno u

invazivnom segmentu kolorektalnog karcinoma. Nije utvrdjena zna~ajna korelacija izmedju intratumorske

ekspresije matriks metaloproteinaze-9 i klju~nih klinopatolo{kih obele`ja odnosno pre`vljavanja

pacijenata sa kolorektalnim karcinomom. "Cox-hazad" model procene stepena uznapredovalnosti

bolesti i histolo{ki stadijum kolorektalnog karcinoma su operiraju}i prognosti{ki faktori, dok

ekspresija matriks metaloproteinaze-9 nije pokazala prognosti~ki zna~aj.

Kljucne reci:

kolorektalni karcinom,

matriks metaloproteinaza-9 (MMP-9),

prognoza.

INTRODUCTION

The colorectal carcinoma (CRC) is the most common

malignant tumor of the alimentary tract and second major

cause of cancer-associated morbidity and mortality in

Europe (1). At the time of diagnosis, CRC usually shows

extensive local invasion and metastasis. Although many

factors regulate malignant tumor growth and spread, interactions

between a tumor and its surrounding microenvironment

result in the production of important protein products

that are crucial to each step of tumor progression (2).

Degradation of extracellular matrix (ECM) is essential in

physiological processes and several pathological conditions.

Tumor invasion, metastatsis and angiogenesis

require controlled degradation of ECM and increased

expression of matrix metalloproteinases (MMPs) (3).

MMPs are a family of zinc-dependent endopeptideses,

degradative enzymes with clear links to malignancy. These

enzymes are associated with tumor cell invasion of the

basement membrane (BM) and stroma, tumor-induced

angiogenesis, blood vessel penetration and metastasis (4).

Currently, this family of MMPs has at least 17 different

members in humans, collectively capable of degrading

almost all ECM components. According to their structures

and substrate specificities, MMPs can be classified into

subgroups of collagenases, gelatinases, stromelysins,

membrane-type MMPs and other MMPs (5). The degradative

ability of tumor can be through both enzymatic activity

of the tumor cell and through cellular components of

the tumor stroma (3). The expression of MMPs in tumors

is regulated in paracrine manner by growth factors and

cytokines secreted in continuous cross talk processes

between tumor, stromal and inflammatory cells during the

invasion (5). Type IV collagen is a major structural protein

in the BM and ECM (6), thus, much attention has been

focused on the type IV collagenases, (gelatinases A and B,

MMP-2 and MMP-9), which specifically degrade the BM

(3,5). MMP-9 is considered to be one of the key enzymes

involved in tumor invasion and metastasis (7). Several

recent studies revealed higher expression of MMP-9 in:

agressive tumor cell lines of human lung carcinoma (8),

advanced stage of melanoma (9), human giant cell tumors

of bone (10). There are many literature data about extensive

proteolytic activity in CRC (11) and its association

with stage, grade of tumor and patients prognosis (12-14).

The aims of this study were investigated by immunohistochemistry

the expression of MMP-9 and their potential

relationship with patients prognosis, metastatic behavior

of tumor and classical clinicopathological prognostic

factors of CRC.

PATIENTS AND METHODS

The study population was composed of 40 patients

underwent curative resection of CRC at the Department of

Surgery of General Hospital Senta from 1990 to 1995.

None of them had received chemotherapy or radiation therapy

before surgery. The operation were standard colon or

rectum resection with regional lymph node dissection.

The tumors were typed and graded according to the criteria

of the World Health Organization classification (15).

The extent of the tumor invasion and presence of metastasis

were based on the Astler-Coller modification of the

Dukes classification system (16). Patients died within 30

days after surgery were excluded. Only patients classified

as stage C received postoperative chemotherapy using 5-

fluorouracil and leucovorin.

Slides were obtained from the invasive front of tumor

(17). Three-micrometar-thick sections from the selected

original paraffin blocks were cut and rehydrated as usual.

Immunohistochemical staining was carried out in the

Ventana ES automated Immunohistochemistry System

(Ventana Medical System Inc., Tucson, AZ, USA) using

original Ventana reagents, with the exception of the primary

antibodies, MMP-9 (Novocastra, lyophilised monoclonal

NCL-MMP-9; Novocastra Laboratories Ltd., Newcastle

upon Tyne, UK). Antigen retrieval was performed in 0.1 M

citric acid puffer, pH 7,3, with heating from room temepature

to 97oC. Primary antibody MMP-9 was used at the

dilution of 1:25 with reaction time of 32 minutes. The

slides were weakly counterstained with hematoxylin and

were mounted routinely. The intratumoral expression of

MMP-9 was analyzed at the tumor-stromal border and

assessed semiquantitatively. The absence of staining reaction

in the tumor cells was assessed as a negative result

(score 0). Staining in less than 10% of the tumor cells was

considered as focal positivy (score 1), and staining in more

than 10% of the tumor cells was considered as diffuse positivy

(score 2), as ilustrated in figure 1.

All patients were followed up until death or every third

month for 3 years after surgery and subsequently every

sixth month for at least 5 years.

Statistical analysis were performed using computer system

for biomedical investigation MedCalc (MedCalc

Software, Mariakerke, Belgium). A value of p<0,05 was

considered statisticaly significant. Student's t-test was conducted

to evaulate differences the means of age. Using the

non-parametric Kruskal-Wallis test, expression of MMP-9

was compared to various clinical and histopathological

parameters. Survival curves were calculated using Kaplan-

Meier method and analysed by log rank test. Univariate

Cox regression analysis were performed to determine if the

prognostic variables were predictiv of overall survival.

Cox analysis were made using BMDP (BMDP Statistical

Software Inc., Los Angeles, CA, USA) program package.

Table 1. Description of clinociopathological data and associated

distribution of matrix metalloproteinase-9 expression

counts in colorectal cancer.

Table 1. Prognostic influence of clinicopathological variables by

univariate Cox's proportional hazard regression model for

overall survival in patients with colorectal cancers, with

standard error (SE), 95% confidence interval (CI) and

statistical significance (p).

RESULTS

The study was based on 40 patients with CRC out of

which 24 were male and 16 were female. The age of the

patients ranged from 33 to 82 years, with mean age of

62,7±8,94 years. Seventeen tumors were located in the rectum,

12 in left colon and 11 in right colon. Histologically

among CRC, there were 9 well differentiated, 19 were

moderately, and 6 were poorly differentiated adenocarcinomas.

MMP-9 activity were identified by redish brown

intracytoplasmatic coloration with MMP-9 antibody. The

colon epithelial cells themselves showed negative reaction.

Expression of MMP-9 were not identified in stromal cells

(i.e., fibroblasts, inflammatory cells, endothelial cells).

There were no differences in staining intensity between the

invasive front of carcinoma compared with central parts. In

the whole study population negative MMP-9 expression in

the colorectal tumor was found in 16 (32%), focal positivity

in 8 (16%), and diffuse positivity in 16 (32%) cases.

Table 1. shows the major clinicopathologic variables and

associated distribution of MMP-9 expression counts in

CRC. There were no statistically significant associations

between MMP-9 expression and sex, age, localization, stadium

of disease, histological type and histological grade of

CRC. There were increased expressions of MMP-9, but not

statistically significant, in tumor of distant localization

(diffuse positivity in 2 cases of tumor in right colon, 5

cases in left colon and 9 cases of rectum) and in advanced

stage of disease (diffuse positivity in 5 cases of B stadium

and 11 cases of C stadium of CRC). Median survival from

the time of surgery in whole population was 44.875±19.04

months. The 5 year survival of study population was 55%.

The average overall survival in patients with no expression

of MMP-9 in CRC was 45.75±17.61 months, in patients

with focal positivity of MMP-9 expression 46.62±13.49

months, and in patients with diffuse expression of MMP-9

in tumor tissue 43.12±23.27 months. The 9 (56.25%)

patients with negative MMP-9 expression, 3 (37.5%)

patients with focal positive MMP-9 expression and 10

(63%) with diffuse MMP-9 expression were alive five

years after operation. The Kaplain-Meier survival curves

showed no statistically significant differences in the overall

survival rates between patients with different level of

MMP-9 expression in CRC tumor cells (Graph 1.). The

univariate Cox-models revealed that stage of CRC disease

and histologic grade of tumor were prognostic factors for

overall survival, while MMP-9 expression in tumor tissue

of CRC failed to attain statistical significance (Table 2.).

Figure 1. The expression of MMP-9 in tumor cells of colorectal cancer: a.) absence of staining reaction in the tumor cells (score 0), b.)

staining in less than 10% of the tumor cells (score 1), c.) staining in more than 10% of the tumor cells (score 2). (MMP-9, x400)

(arrow-head: intracytoplasmatic expression of MMP-9 in tumor cells).

DISCUSSION

Metastatic spread of tumors continues to be a major

obstacle to successful treatment of malignant tumors.

Metastasis is a complex multistep process that requires

sequential interactions between the invasive cell and the

ECM (4). Several studies have been done to try and characterize

the phenotypic and enzymatic profiles of more

agressive tumor cell lines (3). Deteched malignantly transformed

cells migrate and cross structural barriers including

BM and surrounding collagenous ECM. Cellular motility

is associated with controlled proteolysis which involves

interaction between tumor cells and ECM (4). In vivo

expression of MMPs is localized in both tumor and stromal

cells at the invading margin of the tumor, providing a

mechanism for highly concerted degradation of ECM. It

may be that both cellular components contribute to a different

part of the metastatic cascade. The tumor cell MMPs

may contribute to the invasive growth of the tumor while

the stromal element contribute to the remodeling process

and desmoplastic reaction that occur in the tissue adjacent

to the tumor (3). Both gelatinases MMP-2 and MMP-9 are

abundantly expressed in various malignant tumors. MMP-

9 is mainly expressed by malignant cells, but also by

inflammatory cells, including tissue macrophages and

eosinophils (18). Regulation of the MMPs occurs on three

levels: alteration of gene expression, activation of latent

zymogens, and inhibition by tissue inhibitors of matrix

metalloproteinases (TIMPs). Alteration of all three levels

of control have been associated with tumor cell progression

(5). Most MMPs are secreted as latent precursors

(zymogen) that are proteolytically activated in the extracellulare

space. The activity of MMPs in the extracellular

space is specifically inhibited by TIMPs, which bind to the

zinc binding site of active MMPs at molar equivalence

(19). In malignant disease MMPs are considered to be

overexpressed or TIMPs to be underexpressed, leading to

increased proteolytic activity (3-5). A great deal of evidence

has accumulated in recent years for an important but

complex role of proteases in tumor development.

Inhibition of MMPs provides one attractive target for a

novel class of therapeutic agents to control tumor progression

and metastatic spreading (20). Experimental and preclinical

data are seen to strongly support the ability of

MMPs inhibitors to reduce invasion and spontaneous

metastases and significantly prolonged survival.(21,22)

Colorectal adenocarcinoma is the most frequent cancer

in occidental countries. The mean 5 year relative survival in

Europe remains poor, 40%, in spite of progress in surgery

and chemotherapy (23). Prognosis in patients with CRC

conventionally has been determined by a staging system

based on the extent of primary tumor and the presence or

absence of metastasis, as in Dukes' classification (24). Yet,

in each Dukes' class, the survival of individual patients may

vary considerably. This may occur because the classification

reflects a stage in course of CRC rather than the biologic

behavior of the neoplasma (6). The differences in the

survival rate of patients with the same stage of the CRC disease

induced a search for new diagnostic methods with

prognostic relevance. One of the major field of interest in

the tumor biology is molecular and enzymatic processes in

the invasive margin of tumor. The aim of our study was to

assess the validity of expression of MMP-9 which specifically

degrade the BM as a prognostic marker of CRC.

Breakdown of BM is believed to be an essential step for

tumor invasion and metastases. Despite of many, partly

controversial literature data about intensive proteolytic

activity in CRC in our study we did not find correlation of

MMP-9 expression and clinicopathological factor and overall

survival of patients with CRC. Zeng et al. described correlation

of MMP-9 expression with metastatic potential of

human CRC, and he first demonstrated by duoble

immunostaining morphological evidence that type IV collagen

degradation correlates with local elevations in MMP-9

expression (25,13). In the study of Ambriu et al, patients

with high type IV collagenase activity in CRC tissue had a

significantly shorter disease-free and overall survival time.

With respect to overall survival, only type IV collagenase

activity status provided significant predictive value in multivariate

analysis (26). Garbet et al. reported greater both

proteinase and inhibitor expression in the tumour tissue

when compared with the corresponding normal colorectal

tissue and suggested that increased extracellular proteinase

concentrations and activity may encourage tumour invasion

and metastasis (11). Liabakk et al. found significantly higher

expression of MMP-9 in Dukes' stage A than in stage B

carcinoma, but no correlation with tumor location, differentiation

and disease-free 5-year survival, in contrast to

Karakiulakis et al. who described correlation of MMP-9

activity to tumor grade, and he also noted enhanced proteolytic

activity in metastases more than in primary tumor

(12,14). Several another members of MMPs family have

previously been investigated in CRC. MMP-2 was frequently

studied in pair with MMP-9 (11-14). Leeman et al.

documented that high MMP-13 staining score showed a

trend towards poorer survival of CRC patients and pointed

on central position of MMP-13 in the MMPs activation cascade,

both activating and being activated by several MMPs

(27). Adachi et al. noted significant correlation of

matrilysin (MMP-7) expression in CRC with the deapth of

invasion, lymph node metastasis, lymphatic invasion,

advanced Dukes' stage and poorer outcome. Matrilysin

expression was a significant prognostic variable for predicting

overall survival in multivariate analysis (28).

Shiozawa et al. investigated MMP-1 immunoreactivity in

CRC found significant correlation with the presence of

lymph node and hepatic metastasis and with increasing

stages of Dukes' classification. The main structural components

of the stroma in the gastrointestinal tract are collagens

of types I and III. Carcinoma cells must break down these

structural components for further invasion through the

bowel wall; such degradation is effected mainly by MMP-

1. Thus, overexpression of MMP-1 is considered to play a

key role in the process of local tumor invasion (29).

In conclusion, our study did not show significant associations

between intratumoral MMP-9 expression and

major clinicopathological factors and overall survival of

patients with CRC. By univariate Cox hazard-model

expression of MMP-9 did not show prognostic significance.

We think that immunohistochemically staining, that

allowes registration only intracytoplasmatyc, pro forme

(92 kDA) of MMP-9 has not shown benefit in pathology

analysis of CRC speciments. Maybe measurement of both

pro and active forme of MMPs, and identification of more

than one MMPs or simultaneously measurement of MMPs

and TIMPs will give better results.

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