cysteine and serine proteases in gastric cancer

367

Cysteine and Serine Proteases in Gastric Cancer Mario Plebani, M.D.,* Lliszlo' Hersze'nyi, M.D.,t Rornilda Cardin, Ph.D.,S Giovanni Roveroni, M.D.,* Paolo Carraro, M.D.,* Massimo D. Paoli, M.D.,§ Massimo Rugge, M.D., 1) Walter F. Grigioni, M.D.,T Donato Nitti, M.D.,§ Remo Naccarato, M.D.,$ and Fabio Farinati, M.D.S

Background. Cysteine lproteases (cathepsin B [CATB] and cathepsin L [CATL]), tlhe serine protease urokinase- type plasminogen activator (UPA), and plasminogen activator inhibitor type-1 (PAI-1) are thought to play an important part in cancer invasion and metastasis. The aims of this study were to measure CATB, CATL, UPA, and PAI-1 in gastric cancer (GC) and normal mucosa distant from the tumor (NORM); to evaluate whether tissue levels are related to tumor stiige, grade, or histotype; to as- sess their prognostic relevance; and to examine UPA and PAI-1 expression immunohistochemically.

Methods. Gastric cancer and NORM samples were obtained from 25 patients with gastric cancer patients un- dergoing surgery (17 males, 8 females; mean age, 62 years; range, 31-84 years). Antigen concentrations were measured using the enzyme-linked immunosorbent assay method. Immunohistochennistry was performed using monoclonal UPA and PAI-'I antibodies.

Results. Significantly higher antigen levels were found: (1) in GC vs. NORM (CATB, CATL, UPA, PAI-1) tissues; (2) in GC with versus without metastasis (CATB, CATL, UPA); (3) in poorly or moderately versus well differentiated GC; and (4) in diffuse versus intestinal-type GC (CATB, CATL). Urokinase-type plasminogen activa-

From the *Dipartimento di Medicina di Laboratorio, Laboratorio Centrale, Padova, Italy; tCattedra Malattie Apparato Digerente, Isti- tuto di Medicina Interna, Padova, Italy, and Department of Medicine, Semmelweis University, Budapest, Hungary; SCattedra Malattie Ap- parato Digerente, Istituto di Medicina Interna, Padova, Italy; IICatte- dra di Istochimica e Immuno-Istochimica Patologica, Istituto di Ana- tomia Patologica, Padova, Italy; 7Istituto di Anatomia Patologica, Universiti di Bologna, Bologna, Italy; 5Istituto di Clinica Chirurgica 11, Universiti di Padova, Padova, Italy.

Supported in part by the Veneto Region grant No. 394/01/93, Diagnosi precoce del cancro di stomaco e pancreas

This work was performed under the auspices of the R. Farini Foundation for Gastroenterological Research.

Address for reprints: Fabio Farinati, M.D., Cattedra Malattie Ap- parato Digerente, Istituto di Medicina Intema, Policlinico Universita- rio, Via Giustiniani 2,35128 Padova, Italy.

Received November 8, 1995; revisions received February 22, 1995, and April 13, 1995; accepted April 13, 1995.

tor, PAI-1 and CATB levels had a significant prognostic impact. Cancer and stromal cells, showed immunoreactivity to anti-UPA and anti-PAI-1 antibodies.

Conclusions. These results confirm the important role of CATB, CATL, UPA and PAI-1 in gastric cancer progression. Higher levels are detected in GC with metastases, poorer differentiation, and diffuse histotype, thus identifying patients with a worse prognosis Cancer 1995; 76:367-75.

Key words: gastric cancer, protease, cathepsins, urokinase-type plasminogen activator, plasminogen activator inhibitor type-1, enzyme-linked immunosorbent assay, prognosis.

Proteolytic enzymes, such as cysteine and serine proteases, play an important part in physiologic processes (e.g., protein turnover) and in pathologic conditions.' For instance, the proteolysis of tissue barriers is an es- sential step in tumor spread and has been linked to the production by stromal and/or cancer cells of degrading enzymes2 It has been suggested, therefore, that cysteine proteases-cathepsin €3 (CATB) and cathepsin L (CATL)-have an important role in the process of cancer invasion and metastasis through the destruction of surrounding extracellular matrix components, including collagen, fibronectin, proteoglycans, and elastin,3J4 as well as basement membrane specific components, including type IV collagen and laminin.5

Plasminogen activators (PA) form part of the group of serine proteases and are also involved in many protein-degrading processes, converting plasminogen into active plasmin. The tissue-type plasminogen activator (TPA) is a key enzyme in the fibrinolytic cascade, whereas the urokinase-type plasminogen activator (UPA) plays a major part in extracellular matrix degradation, tumor invasion, and the metastatic spread of malignant Plasminogen activator activity is controlled by plasminogen activator inhibitors, which are members

368 CANCER August 2,2995, Volume 76, No. 3

of the serine protease inhibitors (serpin) family." The PA inhibitor type-1 (PAI-1) is produced by vascular endo- thelial cells and inhibits both TPA and UPA by forming a covalent inhibitor-enzyme complex."*'2

Several published reports indicate that protease synthesis is a process involved in cancer development and progression. Ohta et al.13 demonstrated that pancreatic ductal adenocarcinomas express CATB immuno- reactive peptides, thus suggesting that CATB may be active in the process of pancreatic cancer invasion and metastasis. Shuja et al.I4 reported that CATB and CATL activity is significantly higher in colorectal cancer than in normal tissue distant from the tumor. Hirano et al.15 suggested that serum and urinary CATB levels may be indicators of malignancy. Higher concentrations of UPA have been found in a number of solid tumors, such as cancer of the breast,16-'l lung," pr~state, '~ brain,24 and C O ~ O ~ . ~ ~ - ~ ~

An increased PAI-1 has been found in patients with cancer of the b r e a ~ t , ' ~ , ' ~ , ~ ' br ain,24 and colo- reCtum,27.29-32 strongly suggesting that PAI-1 also may be very important in tumor invasion and metastasis. The best evidence to support a role for PA in metastasis comes from experiments with malignant cell lines in vi- tro and with tumor cells transplanted into laboratory animals. These studies demonstrated that increased UPA levels correlate with the invasive and metastatic potential of malignant tumor, and anti-UPA antibodies strongly inhibit metastatic In the specific case of gastric cancer, however, relatively few studies have been published on tissue cathepsins, UPA, and PAI- 1 .9*35-41 These studies have suggested that measuring proteases in gastric cancer tissue could be one of the most effective means for revealing the relation between the tumor and its process of invasion and metastasis.

Some of the above studies also have pointed to the possible prognostic role of tissue proteases. For instance, the importance of UPA and PAI-1 determina- tion has been demonstrated in patients with breast can-

and also in patients with completely resected gastric ~arcinoma.~' However, to our knowledge, the prognostic relevance of cathepsins in gastric cancer has not been evaluated or compared with that of UPA and

The aim of the present study was to measure the levels of cysteine proteases (CATB, CATL), the serine protease UPA, and its inhibitor PAI-1 gastric cancer (GC) in normal tissue obtained from a tumor free area, and to evaluate any relation these proteases may have with the presence or absence of metastasis, the differentiation, histotype, and prognosis of the cancer.

Patients and Methods The study involved 25 patients with gastric cancer un- dergoing gastroresective surgery (1 7 males, 8 females;

cer16-19,21

PAI- 1.

mean age, 62 years; range, 31-84 years). Informed con- sent was obtained from all patients involved in the study. Immediately after removal of the stomach, fresh samples of tumor and of normal tissue (taken more than 10 cm from the macroscopic border of the tumor) were obtained. After removing fat and muscle layers, the samples were frozen at -7OOC for later use (always within 15 days of sampling).

All patients had histologically confirmed advanced adenocarcinoma. From the macroscopic point of view, the gastric cancer could be classified according to Borr- mann4' as follows: 5 type I (exophytic), 11 type I1 (ulcer- expansive), 5 type I11 (ulcer-infiltrative), 4 type IV (scir- rhus-infiltrative). Pathologic staging was obtained for the presence (n = 13) or absence (n = 12) of metastasis; for differentiation (well differentiated G1 [n = 41, moderately differentiated G2 [n = 141, or poorly differentiated G3 [n = 7]), and for histotype (intestinal-type [n = 131 or diffuse-type [n = 12]), according to Lauren.43

Extracts of resected tissues were prepared from 50- 100 mg wet weight tissue samples. Essentially, the specimens were homogenized for 2 minutes in melting ice using a Teflon homogenizer in 1 ml (vol/vol) Tris Tween buffer (0.1 M, 0.1% Tween 80, pH 7.5) per 60 mg wet tissue. After centrifugation for 10 minutes at 10.000 X g at 4OC, the supernatants were stored at -7OOC in 200 pl aliquots before assay.

Protein concentrations of the supernatants were determined by the Bradford method44 (Bio-Rad, Munchen, Germany). Antigen levels were measured using the enzyme linked immunosorbent assay (ELISA) method as described below.

Assay for Cathepsin B and Cathepsin L

Briefly, the Cathepsin quantitative enzyme immunoas- say is a solid-phase ELISA based on the sandwich principle (BioAss, Diesen, Germany). One hundred pl tissue extract is added to a polyclonal, immunoselected antihuman-cathepsin antibody, immobilized in polysty- rene microtiter wells, and incubated. A second antica- thepsin antibody labeled with horseradish peroxidase (conjugate) is added. The conjugate binds immunologi- cally to the cathepsin in the well, resulting in the cathepsin molecules being sandwiched between the solid phase and the enzyme-linked antibody. The lowest de- tectable concentration is estimated to be 10 ng/ml for CATB and 2 ng/ml for CATL.

Assay for Urokinase-Type Plasminogen Activator

Antigen quantification was performed using the TintEl- ize UPA ELISA (Biopool, Umea, Sweden). A mouse monoclonal anti-urokinase-type plasminogen activator

Tissue Proteases in Gastric Cancer/Plebani et al. 369

was used as a catching anti.body. After incubation with the tissue homogenates, a second goat antihuman UPA, conjugated with horseradish peroxidase, was used to form a ”sandwich” for ELISA and ortho-phenylene di- amine was added as a substrate. The amount of UPA antigen in the samples was calculated from a 6-point standard curve of UPA (0-4 ng/ml). The detection limit is about 0.1 ng/ml for UPA.

Assay for Plasminogen-Activator Inhibitor Type-1

Plasminogen activator inhibitor type-1 antigen was determined using Asserachrom PAI-1-ELISA (Diagnos- tics Stago, AsniGres-sur-Seines, France) with mouse monoclonal anti-human PAI-1 as a catching antibody. A second mouse monocl!onal anti-PAI-1 is coupled with peroxidase and binds to another antigenic deter- minant at a distance frorn the first, thus forming the “sandwich.“ The bound enzyme peroxidase is then revealed, in the same way as for UPA, in the presence of hydrogen peroxidase. Absolute quantities of PAI-1 antigen on the samples were calculated from a 5-point standard curve of PAI-1 (0 or 6.25-100 ng/ml). The detection limit is about 2.5 ng/ml for PAI-I. Antigen concentrations were expressed as ng of antigen per mg of protein. Results are given as mean values -t standard deviation.,

Immunohistochemistry

This part of the study was performed on samples from 10 randomly selected patients (six males and four females; mean age, 65 years; age range, 53-74 years; five cases of intestinal-type and five of diffuse-type GC, according to Lauren).43 The central part of the tumor from each resection specimen was embedded in OCT-com- pound (Miles, Naperville, IL) and snap-frozen in liquid nitrogen at -7OOC.

The cryopreserved tumor samples were cut into 5 pm serial sections, which were placed on slides, treated with polylysine, dried in air overnight, rehydrated in Tris-buffered saline (TBS), incubated with 5 mmol/l pe- riodic acid (Sigma, St. Louis MO) for 10 minutes to in- activate endogenous peroxidase and then washed five times in TBS. To eliminate nonspecific background staining, the sections were incubated with 10% nonim- mune rabbit serum (Dako Corporation, Santa Barbara, CA) for 30 minutes. Two specific monoclonal mouse antibodies (MoAb) were used (i.e., an antiurokinase plasminogen activator AAoAb [American Diagnostica, New York, NY], 1:100, and an antihuman plasminogen activator inhibitor type- 1 MoAb [Biopool, Umea, Fin- land] 1:50). The sections were incubated at 4OC overnight and then treated with rabbit antimouse immuno-

globulin (2259, Dako) and with mouse peroxidase antiperoxidase immunoglobulin (P850, Dako) according to Sternberger.45 The sections were developed in a 0.25% diaminobenzidine (DAB, Sigma) solution, containing 0.003% hydrogen peroxide for 5 minutes, counter- stained in hematoxylin, dehydrated in alcohol, and mounted in Eukitt (0. Kindler, GMbH & Co., Freiburg, Germany).

Two independent observers screened the sections to evaluate the immunostaining semiquantitatively for UPA and PAI- 1. A five-grade score was employed according to the percentage of labeled tumor cells in each section: ++++ = more than 75% of positive tumor cells; +++ = 50%-75% of labeled cells; ++ = 25%- 50% of labeled cells; + = less than 25%; and 0 = negative.

Survival and Statistics

Differences between groups were statistically tested using Student’s t test on paired and unpaired data, or the Mann-Whitney U test, where applicable. Differences were considered as significant with 2P < 0.05. The re- ceiver operating characteristic curves were used to determine the optimal cutoff values (with the Youden J- test for overall accuracy). To determine the prognostic value of CATB, CATL, UPA, and PAI-1 by comparison with histomorphologic prognostic factors, survival was analyzed accordmg to Cox’s proportional hazard model. Patients were enrolled in a follow-up study between August 1992 and December 1994. Inclusion cri- teria were macroscopically and histologically complete surgical removal of primary gastric cancer and a survival time longer than 3 months. Patients were followed either directly or through their attending physicians.

Twelve patients (48%) died of tumor recurrence. Their median survival time was 8 months (range, 4-1 7 months). At the end of the follow-up period, 13 patients (52%) were still alive; their median follow-up is 18 months, (range, 8-27 months). The median survival time, calculated over all patients, is 12.3 months.

Group-oriented curves for survival were calculated according to the Mantel-Cox model for CATB levels; CATL; UPA; PAI-1; T stage; N stage; grade; Borrmann classification; Lauren classification; age; sex; and tumor site.

Finally, stepwise logistic regression analysis was used to evaluate the most significant independent prognostic variables.

Results

1. Antigen concentrations for cysteine and serine proteases and for PAI-1 in GC tissue and normal mu-


Table 1. Cathepsins, Serine Protease UPA, PAI-1 in Gastric Cancer, and Normal Tissue

% Norm (n = 25) GC (n = 25) Increase* P

CATB 155.0 f 180.5 325.9 f 427.10 110 40.05 CATL 27.5 f 19.30 43.6 2 24.50 58 40.005 UPA 0.43 k 0.37 1 . 8 5 f 1.05 311 co.001 PAIL1 0.50 & 0.30 2.35 * 2.69 3 70 co.001 Values are ng/mg protein, mean + standard deviation, for all parameters. UPA: urokinase-type plasminogen activator; PAI- 1: plasminogen activator inhibitor type-1; CATB: Cathepsin B; CATL Cathepsin L; Norm: normal tissue; GC: gastric cancer.

GC vs. Norm.

cosa, expressed as ng/mg protein in the 25 patients, are shown in Table 1. Cathepsin 8, CATL, UPA and PAI-1 were significantly higher in GC tissue than in normal mucosa. The highest percentage of increase was observed for PAI-1 (370%).

2. Cathepsin B, CATL and UPA were significantly higher in specimens from patients with lymph node (n = 11) or liver metastases (n = 2) than from patients with no metastases (CATB = P < 0.05; CATL = P < 0.05; UPA = P < 0.05) (Fig. 1). Plasminogen activator inhibitor type-1 was also higher, though not significantly so, in GC with metastasis than in GC without metastasis.

3. Significantly higher CATB and CATL antigen levels were seen in poorly differentiated than in well differentiated tumors. Urokinase-type plasminogen activator and PAI-1 were significantly higher, however, in patients with moderately versus well differentiated tumors (Table 2).

4. Cathepsins were significantly higher in diffuse-

CATB,CATL: ngfmg protein _-___ -1

UPA,PAI-l: 10 x ng/mg protein

-1 ____

p 0.05 vs NO METAST

CATL UPA MI-1

m WITH METASTASIS RSY NO METASTASIS (n-13) (n.12)

Figure 1. Cathepsins, urokinase-type plasminogen activator, and plasminogen activator inhibitor type-I in gastric cancer: k

Table 2. Cathepsins, Serine Protease UPA, PAI-1, and Differentiation of Gastric Tumors

Well Moderately Poorly G1 (n = 4) G2 (n = 14) G3 (n = 7)

CATB 96.00 & 90.95 297.85 f 262.32 513.28 f 705.92* CATL 23.85 k 14.60 44.87 f 22.37 52.44k 29.13: UPA 0.86 f 0.34 2.29 f 0.917 1.94 -C 2.03 PAI-1 0.79 k 0.30 3.00 f 3.21t 1.53 f 0.99 Values are ng/mg protein, mean ? standard deviation, for all parameters. UPA: urokinase-type plasminogen activator; PAI- 1: plasminogen activator inhibitor type-1; CATB Cathepsin B; CATL: Cathepsin L.

* Poorly vs. Well: P < 0.05. t Moderatelv vs. Well: P < 0.005 (serine orotease UPAl P i 0.05 (PAl-1).

versus intestinal-type GC (CATB = P < 0.05; CATL = P < 0.05). No significant differences were observed with respect to UPA and PAI-1 (Fig. 2). We then subgrouped patients with the diffuse or intestinal histotype according to any presence or absence of metastases and according to grade. Ca- thepsin B and CATL levels were significantly higher in the diffuse than in the intestinal histotype when patients without metastases were considered (Table 3). No significant differences were observed with respect to the remaining subgroups even though a trend toward higher levels in diffuse type was also present with respect to CATB (with metastases) and UPA (with metastases); on the other hand, a trend toward higher levels in intestinal type was observed for UPA (without metastases) and PAI-1 (both with and without metastases). As for grade, only patients with moderately differentiated tumors were compared, given the small number of patients with well or poorly differentiated cancers: CATB and

CAT6,CATL: nglmg protein UPA,MI-I: io-'x ng/mg pr0t.l

6oo I p < 0.05 vs INTESTINAL

400

300

200

100

n " CAT6 CATL UPA

= INTESTINAL DIFFUSE (n-13) (n.12)

Figure 2. Cathepsins, urokinase-type plasminogen activator, plasminogen activator inhibitor type-I, and histotype of gastric

metastasis. tumors.


Table 3. Cathepsins, Serine Protease UPA, and PAI-1 According to Histotype and Metastatic Status (+/-) of Tumors Status CATB CATL UPA PAI-1

DIFFUSE+ (n = 8) 558.2 2 630.1 50.1 k 22.4 2.3 k 0.6 2.0 F 1.7 DIFFUSE- (n = 4) 211.7f 71.5* 53.7 i 33.2* l . l + 0 . 9 1.6 i 0.8

1NTEST.- (n = 8) 152.8 f 239.6* 27.6 F 15.6* 1.8 * 1.0 2.4 F 2.8

Values are ng/mg protein; mean 2 standard deviation. U P A urokinase-type plasminogen activator; PAI-I: plasminogen activator type-1; CATB: Cathepsin B; CATL: Cathep~ sin L; INTEST: intestinal.

INTEST.+ (n := 5) 370.8 f 311.8 50.8 i 26.2 1.8 * 1.1 3.4 f 4.5

* CATB. CATL: DIFFUSE- VS. INTEST.-: P < 0.05.

CATL levels were, here again, significantly higher in diffuse than in intestinal-type cancer (CATB =

diffuse- vs. intestinal-type: 338.6 k 154 vs. 165 k 133.5, P < 0.05; CATL = diffuse- vs. intestinal- type: 59.8 * 27.7 vs. 37.1 * 15.4, P < 0.05). No difference was detected with respect to UPA and PAI-1 (data not shown).

5. A significant correlation was found in GC levels of CATB and CATL (r = 0.52; P = 0.006) and of UPA and PAI-1 (r = 0.50, P = 0.01). No correlation was found between the twlo subgroups.

6. Cancer cells showed both apical and cytoplasmic staining with MoAb anti-UPA (Fig. 3). Some in- flammatory cells, mainly macrophages and scattered stromal elements like fibroblasts, also showed cytoplasmic immunoreactivity. The monoclonal antibody anti-PAI- 1 revealed a similar distribution of immunoreactivity (Fig. 4). The percentage of elements positive for PAI-1 was lower than for UPA.

7.

Urokinase-type plasminogen activator antigen expression was slightly more evident in the intestinal- type adenocarcinomas (+++ to ++++) than in the diffuse type (+ to +++). No differences were found between intestinal- and diffuse-type adenocarcinoma in terms of PAI-1 expression (++ to +++). The optimal cutoff value for CATB was 265 ng/ mg protein (J = 0.63). This discriminated 15 patients (60%) below and 10 (40%) above the cutoff. As for CATL, a cutoff of 45 ng/mg protein (J = 0.39) was found, discriminating 17 patients (68%) with CATL I 45 ng/mg protein and 8 patients (32%) having CATL > 45 ng/mg protein. The cutoff for UPA was placed at 1.25 ng/mg protein (J = 0.70), with 8 patients (32%) below and 17 (68%) above said level. For PAI-1, the selected value of 0.9 ng/mg protein (J = 0.66) dis- tinguished between 6 patients (24%) below and 19 (76%) above the cutoff. The Mantel-Cox survival analysis curves showed

Figure 3. lmmunohistochemical staining with monoclonal antibody anti-urokinase-type plasminogen activator of intestinal-type gastric cancer. A large number of malignant cells show cytoplasmic staining. Some scattered stromal elements also are immunostained

Figure 4. Immunohistochemical staining with monoclonal antibody antiplasminogen activator inhibitor type-1 of diffuse-type gastric cancer. Cancer cells and scattered stromal cells are immunostained

(peroxidase-antiperoxidase immunostaining, original magnification X 60). X 60).

(peroxidase-antiperoxidase immunostaining, original magnification


100 \ 4 \ 100

80

60

40

-

\ * = C U T - O F F

(n.15)

\

< CUT - OFF (n=8)

2:L PA CUT-OFF = 1.25 ng/mg protein

0 3 8 9 12 15 18 21 24 27

MONTHS

p 0.0001, MANTEL-COX Figure 5. Survival curves stratified by urokinase-type plasminogen activator in patients with gastric cancer (n = 25).

that the following parameters were significantly correlated with survival time (in order of significance): UPA, (P = 0.0001) (Fig. 5); PAI-1, (P = 0.0004) (Fig. 6); CATB, (P = 0.002) (Fig. 7); Staging N, (P = 0.0022); Borrmann classification, (P = 0.0024); Staging T (P = 0.005); Grade

No significant correlation with survival was observed with respect to CATL (P = 0.17), age, sex, tumor site, or histotype, according to Lauren.

Urokinase-type plasminogen activator was selected as the single independent variable by the stepwise logistic regression analysis (F value 12.624; P = 0.0001).

(P = 0.02).

Discussion

Proteolytic mechanisms, such as those depending on cysteine and serine proteases, have been suggested to

% SURVIVAL 100 ' CUT - OFF 80 (n=6)

60

40

20 1 CUT - OFF

- 0 3 6 9 12 15 18 21 24 27

MONTHS

p 0.0004, MANTEL-COX Figure 6 . Survival curves stratified by plasminogen activator inhibitor type-1 in patients with gastric cancer (n = 25).

% SURVIVAL

\

[CATB CUT-OFF 265 ng/ mg protein 0

0 3 6 9 12 15 18 21 24 27

MONTHS

p 0.002, MANTEL-COX Figure 7. Survival curves stratified by Cathepsin B in patients with gastric cancer (n = 25).

represent crucial factors in the process of tumor invasion and A number of studies also have been published on gastric cancer but none evaluated both cysteine and serine proteases in the same stom- achs. In the present study, therefore, we surveyed the relation between CATB, CATL (cysteine proteases), UPA (serine protease), and its inhibitor PAI-1 in human gastric cancer tissues.

Tissue Levels of Cathepsin B, Cathepsin L, Urokinase- Type Plasminogen Activator, and Plasminogen Activator Inhibitor Type-1

Our demonstration that CATB, CATL, and UPA are significantly higher in homogenates of GC tissue than in those of cancer free samples obtained from the same stomach confirm earlier studies reporting increased levels of CATB, CATL,35-37 and UPA9,38-4' in GC tissue, also clearly indicating that these two enzyme families are involved in gastric cancer development and growth. In our experience, PAI-1 antigen was also significantly higher in GC tissue, confirming findings reported by Ta- naka et al.,31 Nekarda et al.,40 and Nakamura et aL41

Sier et al.,39 Nekarda et al.,40 and Nakamura et al.41 used ELISA to measure UPA and PAI-1, whereas activity tests were used in the other references cited here. Enzyme-linked immunosorbent assay for UPA has been used in breast cancer ~ t u d i e s ' ~ * ' ~ and the results obtained were of greater interest than those deriving from activity assays.I6 Because of these differences in the methods adopted by different authors, it is difficult to compare our data on cathepsins with other reports in the literature.

Conversely, concerning UPA and PAI-1, our findings are in the same range as those reported by Tanaka


et aL3' and Nekarda et aL4' In our experience, the increase observed in PAI-1 was the most dramatic. This may be because various factors are involved in PAI-1 synthesis and release in gastric cancer, as well as in other tumors. Under normal physiologic conditions, UPA forms inactive enzyme complexes with the inhibitor PAI-1, if the latter is present.",'* The exact role of PAI-1 in tumor biology is not well established, however. It has been suggested that PAI-1 may represent a specific protein of transformed malignant It has also been claimed that PAI-1 may serve to protect cancer tissue against the proteolytic degradation trig- gered by the tumor on surrounding normal Another possible explanation is that the inhibitor might have a role in angiogenesis, which has an important part to play in tumor ~ p r e a d . ~ , ~ ~ Whatever the principal mechanism, we believe that PAI-1 levels are high in gastric cancer tissue as a result of a multifactorial stim- ulation.

Our immunohistochemical study on UPA and PAI- 1 expression in a subgroup of patients, which indicated that both cancer and stromal cells are positive for the two antigens, apparently Iconfirms data previously reported by Buo et al.32 concerning colon cancer. There are also conflicting reports, however. Nakamura et al.,41 for instance, could detect no staining in stromal cells in gastric cancer. Whether UPA or PAI-1 are synthesized and released by stromal, parenchymal, or both types of cell is still open to debate.3e The topic will probably have to be addressed using molecular biology techniques such as in situ hybridization, as we used in a previous investigation on metalloproteinases in gastric can~er ,~ ' because a nonspecific immunohistochemical result due to antigen uptake by macrophages (or even by cancer cells) cannot be ruled out. 'This part of our study was not intended to solve the problem, but simply to provide morphologic support for our data. Another finding of our study was that stromal and cancer cells were both positive for UPA and PAI-1 at the same time. This prompts several hypotheses: (1) that the invasive properties of tumor tissue are the net result of the balance between an aggressive activity and a protective factor; (2) that an excessively high level of PAI-1 may have an aggressive role; or (3) that cancer cells may synthesize PAI-1 as a self-defense mechanism in relation to the risk of a u t o d i g e s t i ~ n . ~ ~ ~ ~ ~

Factors Influencing Cathepsin B, CATL, Urokinase- Type Plasminogen Activator, and Plasminogen Activator Inhibitor Type-2 Tissue Levels

In this study, variations in CATB, CATL, UPA, and PAI-1 levels in the neoplastic tissue were also considered in relation to clinical and pathologic parameters of

prognostic relevance, such as the presence of metastasis, tumor grade, and histotype.

The finding of significantly higher CATB, CATL, and UPA levels in specimens from patients with lymph node or liver metastases than in samples from patients without metastases confirms the correlation between cysteine and serine protease synthesis and tumor invasion potential. Significantly higher antigen levels were also found in poorly (CATB, CATL) and moderately differentiated (UPA, PAI-1) tumors by comparison with well differentiated cancers. Finally, cathepsins were significantly higher in diffuse- than in intestinal-type gastric cancer, whereas no significant differences were found with respect to UPA and PAI-1, though the intestinal type tended to have slightly higher UPA and PAI- 1 levels. When patients were subgrouped according to the presence or absence of metastases, CATB and CATL levels were higher in diffuse type, even though significantly so only in patients without metastases. Urokinase-type plasminogen activator levels did not present clearcut changes, whereas PAI-1 levels tended to be higher in the intestinal histotype. This data confirm that cathepsins levels are higher in patients with diffuse gastric cancer, by large independently of metastases status, thus indicating that the more aggressive tumors feature a more enhanced cathepsins synthesis. Similarly, when patients with the same degree of differentiation are considered, the diffuse type again correlates with higher levels of CATB and CATL.

Conversely, the UPA/PAI-1 system appears to be activated to the same extent in intestinal and in diffuse gastric cancer. The demonstration of similar or slightly increased levels of CPA and PAI-1 levels in the intestinal-type cancer is also supported by our preliminary immunohistochemical data. Intestinal-type cancers in- deed also have similar or slightly higher UPA and PAI-1 immunohistochemical expression. It is well known (and has also been confirmed by our own research that Lauren's diffuse or Ming's infiltrating type gastric cancer is associated with a worse prognosis than the intestinal/expanding type.43 Our data, therefore, suggest that the more negative prognosis-and possibly also the morphologic behavior of these two tumor types- could be related to the synthesis and release of cathepsins.

Prognostic Xole of Urokinase-Type Plasrninogen Activator, Plasminogen Activator Inhibitor Type-1, and Cathepsin B

In our experience, UPA, PAI-1 and CATB levels were significant in the prediction of survival and, to be more specific, correlated better with survival than TNM stage, tumor grade, or the Borrmann classification. Concern-


ing UPA and PAI-1, the survival curves of patients with levels below or above the cutoff differed dramatically, revealing a 100% 2-year survival in patients with low tissue levels of plasminogen activator pathway versus 10%-20% among patients with concentrations above the cutoff levels. Urokinase-type plasminogen activator proved the only and most significant independent predictor variable in stepwise logistic regression analysis. Given the relatively small number of patients involved, the data reported in this section must be considered as preliminary, though they are quite promising.

These findings are relevant from the biologic and clinical point of view. Gastric cancer is one of the most lethal cancers of the gastrointestinal tract in terms of cure rate and survival. It would therefore be very useful for clinicians to have more accurate prognostic indica- tions available, for instance, to identify patients needing adjuvant treatment.

Our findings concerning gastric cancer appear to be supported by experience with other tumors: recent studies on breast carcinoma have shown that high UPA and PAI-1 levels in tissue extracts are associated with an aggressive tumor and a poor prognosis, and appear to be independent prognostic fa~tors . ’ ’ -~~,~~ Our report now adds the finding that not only UPA and PAI-1, but also CATB, may serve as a prognostic factor for survival in patients with GC.

These data also appear to be reproducible in nu- meric terms, as our selected cutoff for UPA (1.25 ng/mg protein) and PAI-1 (0.9 ng/mg protein) are comparable with those previously reported by Nekarda et al.40

Measuring proteases is not the only approach to this problem. The histotype is a relevant prognostic variable43; intracellular mucous production has been suggested as an important predictor of outcome53 and, in one of our previous studies?l the presence of vascular invasion and ploidy proved to be the most significant prognostic parameters after pathologic staging. Pre- sumably, measuring tissue proteolytic enzyme levels will also facilitate the identification of high risk patients with gastric cancer who are candidates for extensive resection and/or adjuvant or neoadjuvant therapy, and a comparison between the former and latter parameters could be attempted.

In conclusion, our results confirm that cysteine, serine proteases, and PAI- 1 may have a crucial role in gastric cancer progression. Considering the valid correlation between higher antigen levels, the presence of metastases, the diffuse histotype (for cathepsins), and poor differentiation in gastric carcinomas, and given the ca- pacity of these biologc markers to predict patients’ survival, measuring these proteases and PAI-1 in gastric cancer tissue might prove to be of great value in identifying patients with a poor prognosis.

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