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Thrombomodulin inhibits intrahepatic spread in human hepatocellular carcinoma
Thrombomodulin Inhibits Intrahepatic Spread in Human Hepatocellular Carcinoma TAKETOSHI SUEHIRO, MITSUO SHIMADA, TAKASHIMATSUMATA,AKINOBU TAKETOMI, KAZUHARUYAMAMOTO, AND KEIZO SUGIMACHI
T h r o m b o m o d u l i n (TM) c o n v e r t s t h r o m b i n from procoa g u l a n t into a n t i c o a g u l a n t p r o t e i n to activate p r o t e i n C. T h r o m b i n also plays an i m p o r t a n t role in the m e t a s t a t i c p r o c e s s o f c a n c e r cells. We p e r f o r m e d an i m m u n o h i s t o c h e m i c a l a n d c l i n i c o p a t h o l o g i c a l study of TM in 141 cases w i t h r e s e c t e d h e p a t o c e i l u l a r c a r c i n o m a (HCC) m e a s u r i n g less t h a n 6 c m in diameter. Twenty-five specim e n s (17.73%) s t a i n e d positive for TM. TM w a s f o u n d in the c y t o p l a s m a n d surface o f c a n c e r cells. The clinicop a t h o l o g i c a l findings a c c o r d i n g to the positive of TM are e x a m i n e d in HCC. The p r e o p e r a t i v e p l a s m a TM level o f t h e p a t i e n t s w i t h tissue that s t a i n e d positive for TM w a s significantly h i g h e r t h a n that o f the patients w i t h negative results; for the p o s t o p e r a t i v e TM level, there w e r e n o differences b e t w e e n them. In addition, t h e frequencies of i n t r a h e p a t i c metastasis, t u m o r t h r o m b u s in the portal vein, a n d c a p s u l a r infiltration w e r e significantly l o w e r in p a t i e n t s w h o s e tissue s t a i n e d positive for TM t h a n in p a t i e n t s w h o s e tissue s t a i n e d n e g a t i v e for TM. The r e c u r r e n c e f r e e d o m rate w a s significantly h i g h e r in p a t i e n t s w h o s e tissue s t a i n e d positive for TM t h a n patients w h o s e tissue s t a i n e d n e g a t i v e for TM. Thus, TMp r o d u c i n g HCC s h o w s a s l o w ~ t r a h e p a t i c spread. Therefore, t h e s e findings s u g g e s t ~hat TM m a y inhibit the a d h e s i o n of t u m o r cells to the portal v e i n b e c a u s e o f a n t i c o a g u l a n t activity a n d t h u s p r e v e n t the spread o f i n t r a h e p a t i c metastasis. (HEPATOLOGY 1995;21:12851290.)
Tumor metastasis is a complex series of ~,ulti-tep processes. ~'2 In these steps, two processes are important. One process is the detachment of cancer ~'ells into the blood circulation, and another is the a d b ~sion of such cancer cells to endoth ~lial cells. Blood coa~:;ulation and fibrinolysis have been shown to play an important role in these two metastatic processes. It has been reported t h a t administration of warfarin, which
Abbreviations: TM, thrombomodulin; HCC, hepatocellular carcinoma; PBS, phosphate-buffered saline. From the Department of Surgery II, Faculty of Medicine, Kyu~ ~ University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan. Received February 22, 1994; accepted December 3, 1994. Address reprint requests to: Taketoshi Suehiro, MD, Departm .~J ,: Surgery II, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higa: :.~ ,~1, Fukuoka 812, Japan. Copyright © 1995 by the American Association for 11,~ Sty:. of Liver Diseases. 0270-9139/95/2105-001053.00/0
prevents the synthesis of the vitamin K - d e p e n d e n t procoagulants (factors II, VII, IX, and X), significantly lowered the numbers of tumor metastasis. 3't Fibrinolytic agents, such as plasminogen activator or urokinase, are thought to play an important role in tumor metastasis, because these agents are able to lyse tumor-associated fibrin clots and cause the destruction of the extracellular matrix and basement membrane. ~'6 Thrombin plays an important role in the adhesion of cancer cells to endothelial cells through fibrin formation and platelet activation, 7,s and thrombin stimulates tumor cell bindings to endothelial cells2 Thrombomodulin (TM) is the endothelial cell surface protein t h a t converts thrombin from a procoagnlant protein into the activator for protein C. ~o From histological evidence of experimental metastasis, several investigator,-~ have previously reported on the relationship between coagulation activity and tumor metastasis. ~~5 They have suggested t h a t fibrin acts a~ a glue t h a t facilitates tumor cell adhesion to the endothelial ell and therefore is critical to the pathogenesis oftum(~r growth and metastasis formation. TM is known to i~hibit this fibrin formation and thus may protect agai,~st the adhesion of tumor cells to endothelial cells. The aim of this study is to compare the positivity of TM with clinicopathologic findings and discuss the relationship between TM and the intrahepatic spread of he mtocellular carcinoma (HCC). PATIENTS AND METHOD,~ P a t i e n t s . One hundred and forty-one pat~e~ts with HCC who underwent hepatic resections durin~ th~ ~riod between January 1991 and August 1992 at the D~part~~ n t of Surgery II, Kyushu University Hospital, were used in ~:},~sstudy. They included 111 men and 30 women. Their age at op. ,'ation ranged from 37 to 79 years (mean, 63 years). The types of hepatic resection performed were a partial hepatectomy (64 pa*ients), subsegmentectomy (17 patients), segmentectomy (1 ~ patients), lobectomy (41 patients), and an extended lobecto~,y (5 patients). The patients were strictly followed up using monthly measurements of a-fetoprotein and des-T-carboxy prothrombin, monthly bedside ultrasonography and ultrasonography, dynamic computed tomogr~ phy by radiologists every 3 months, and angiographic examinations performed after admission whenever recurrence was strongly suspected. A n t i b o d i e s . The antibody used is an anti-TM monoclonal antibody derived from mice (MFTM-5; Fuji Pharmaceutical
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Co, Ltd, Tokyo, Japan). This antibody recognizes the epidermal growth factor-like domain of TM, which is the binding site for thrombin and protein C. 16 Immunohistochemical Study. Formaldehyde-fixed paraffin-embedded six-micron sections were used in this study. The paraffin sections were dewaxed according to ordinary procedures and then immersed in 3% hydrogen peroxide in methanol for 30 minutes to block the endogenous peroxidase activity. The sections were subsequently washed in phosphate-buffered saline (PBS). For the immunohistochemical study, the avidin-biotin immunoperoxidase method was used 17with the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). After treatment with normal horse serum, each section was incubated with mouse monoclonal antibodies at room temperature for 1 hour and then rinsed with PBS. Mouse anti-TM monoclonal antibody was used at a concentration of 20 #g/mL. The section was then incubated with biotinylated horse anti-mouse immunoglobulin for 30 minutes and washed with PBS. Avidinbiotin-horseradish peroxidase complex was applied for 30 minutes, and then the section was washed with PBS. The sections were incubated with a peroxidase substrate solution (mixture of 0.02% [wt/vol] hydrogen peroxide and 0.1% [wry col] diaminobenzidine tetrahydrochloride in PBS; pH 7.4), and then were counterstained with methyl green. For negative control, 1% bovine serum albumin-PBS and normal rat serum were used as primary antibody. The percentage of cells that stained positive for TM was classified in four categories: - , (<25%); +, (25% to 50%); + +, (50% to 75%); + + +, (> 75%), and we defined + to + + + grades as having stained positive for TM. For the estimation of interobserver variation, the percentage of cells that stained positive for TM of all cases was examined by another experienced surgical pathologist without any knowledge of the clinical outcome or the first observer's determination of the percentage of cells that stained positive for TM. The mean of the two counts of each observer was considered to be the percentage of cells that stained positive for TM. Histopathological Findings. For the histopathological examination, the sections were stained with hematoxylin-eosin. The degree of cell differentiation was classified according to the criteria of Edmondson and Steiner. is Other pathological findings were classified according to the Japanese general rules for primary liver cancer. 19 Determination of TM and Protein C, Blood sampling was done 1 day before surgery and on the 7th day after surgery. The blood was drawn into sterile tubes (one containing 3.8% trisodium citrate and the other containing nothing). Citrate plasma was used to determine the protein C activity, and the serum was used for determining the soluble TM. The protein C activity was determined using a STACHROM PROTEIN C test kit (DIAGNOSTICA STAGO, France) (normal range, 70% to 140%). The soluble TM was then determined using the one-step sandwich enzyme immunoassay described by Kodama S e t al 2° (Fuji Pharmaceutical Co, Ltd., Tokyo, Japan). Renal function (serum creatinine level and creatinine clearance) and urine volume of each patient was also recorded, because plasma TM level is easily influenced by renal clearance. Statistical Analysis. A statistical evaluation was made using the Mann-Whitney test, Yates' continuity-corrected X2 test, and McNemar's X2 test. The recurrence freedom survival rate was estimated using Kaplan and Meier's actuarial method. The probability values for comparing two sets of lifetable data were determined using the generalized Wilcoxon
HEPATOLOGYMay 1995
FIG. 1. (A) The immunohistochemical distribution of TM in HCC. A granular reaction was noted in the cytoplasm of cancer cells. (Original magnification ×200.) (B) Control.
test. All statistical analyses were considered significant at a P value of <.05. RESULTS
D i s t r i b u t i o n o f T M in HCC. TM w a s r e c o g n i z e d in 17.7% of H C C s m e a s u r i n g less t h a n 6 cm in d i a m e t e r (25/141). Cells t h a t s t a i n e d positive for TM w e r e obs e r v e d diffusely, w h e r e a s a g r a n u l a r r e a c t i o n w a s obs e r v e d in t h e c y t o p l a s m of t h e c a n c e r cells (Fig. 1). B o t h t h e i n t e n s i t y of positive s t a i n i n g a n d t h e n u m b e r of cells t h a t s t a i n e d positive v a r i e d f r o m a r e a to a r e a in i n d i v i d u a l cases. I n s u r r o u n d i n g n o n c a n c e r ous tissue, TM w a s n o t e d on t h e c a p i l l a r y e n d o t h e l i a l cells in t h e t u m o r a n d / o r a r o u n d t h e t u m o r (Figs. 2 a n d 3). The R e l a t i o n s h i p B e t w e e n the C l i n i c a l F i n d i n g s a n d Tissue TM Positivity. Table 1 shows the clinical feat u r e s of 141 p a t i e n t s with H C C m e a s u r i n g less t h a n 6 cm in d i a m e t e r according to positivity of TM staining. The s e r u m total bilirubin level was significantly lower in the p a t i e n t s with tissue t h a t s t a i n e d positive for TM t h a n with tissue t h a t s t a i n e d negative for TM. In the o t h e r clinical findings, t h e r e were no differences be-
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HEPATOLOGY Vo|. 21, No. 5, 1995
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TABLE 1. T h e R e l a t i o n s h i p B e t w e e n t h e C l i n i c a l F i n d i n g s a n d T i s s u e S t a i n i n g P o s i t i v i t y for TM Positive (n - 25) Mean age (yr) ICG (%) Albumin (mg/dL) Total bilirubin (mg/dL) GOT (IU/L) GPT (IU/L)
18.9 3.82 0.76 71.8 66.8
63 ± 10.1" ± 0.41 _+ 0.32 _-2 35.5 _+ 40.2
Negative (n = 116)
19.4 3.75 0.88 71.7 69.7
62 ± 9.5 _+ 0.36 _+ 0.35 ± 35.2 ± 44.0
P
.7947 .4459 .0326 .9339 .8233
Abbreviations: ICG, indocyanine green retention test; GOT, glutamate oxaloacetic transaminase; GPT, glutamate pyruvic transaminase. * Data represent mean ± SD.
FIG. 2. The immunohistochemical distribution of TM in HCC. Although almost all cancer cells stained positive for TM, surrounding noncancerous hepatocytes stained negative for TM.
tween the patients with staining results that were positive and negative. The R e l a t i o n s h i p Between the P l a s m a TM Level a n d Tissue TM Positivity. All patients showed good renal function (creatinine <2.0 mg/dL, creatinine clearance >70 mL/min). There was no difference between the patients with positive and negative staining for TM in renal function (Table 2). Figure 4 shows the changes in the plasma TM levels of the 141 patients with HCC measuring less than 6 cm in diameter according to the staining positivity for TM. The preoperative plasma TM level of the patients with tissue that stained positive for TM was significantly higher than those with tissue that stained negative for TM (4.922 _+ 1.221 ng/mL vs. 3.290 _+ 0.664 ng/ mL; P = .0208). In the patients with tissue that stained positive for TM, the plasma TM level at the 7th day
after surgery was significantly lower than the day before surgery (4.922 _+ 1.221 ng/mL vs. 3.400 _+ 0.932 ng/mL; P = .0293). The postoperative plasma TM levels showed no differences between the patients with tissue stained positive and negative for TM. Table 3 shows the relationship between the coagulofibrinolytic characteristics and positive staining for TM in tissue. There were no differences between the patients staining positive and negative for TM regarding coagulofibrinolytic characteristics. The Relationship Between Pathological Prognostic Factors and Tissue Staining Positive for TM. The relationship between the pathological prognostic factors and tissue's TM reactivity in 141 HCC measuring less than 6 cm in diameter is summarized in Table 4. Tumor thrombus in the portal vein, intrahepatic metastasis, and capsular infiltration were all more frequently observed in patients whose tissue stained negative for TM than in patients whose tissue stained positive for TM (tumor thrombus in the portal vein: P = .0378, intrahepatic metastasis: P = .0320, capsular infiltration: P = .0156). Macroscopic classification, histological classification, Edmondson's classification, and tumor diameter showed no significant differences between the patients in which staining for TM was positive and negative. The R e l a t i o n s h i p Between the Recurrence-Freedom Rate and Tissue Staining Positive for TM. The recurrence-freedom rate was calculated by the Kaplan-Meier method for 141 patients with resected HCC measuring less than 6 cm in diameter. The number of recurrencefreedom days ranged from 29 to 927 days. Figure 5 represents the recurrence-freedom curves of patients with respect to tissue staining positive for TM. The recurrence-freedom rate was significantly higher in the patients whose tissue stained positive for TM than in those whose tissue stained negative for TM (P = .0085). DISCUSSION
FIG. 3. The immunohistochemica] distribution of TM in noncancerous tissue. TM was noted on the capillary endothelial cells and sinusoidal endothelial cells in a cirrhotic liver. (Original magnification × 100.)
In this study, we investigated the relationship between HCC and TM both clinicopathologically and immunohistochemically. TM was seen both in cancer cells and on capillary endothelial cells in and/or around
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HEPATOLOGY May 1995
TABLE 2. The Relationship B e t w e e n Renal F u n c t i o n and Tissue Staining Positivity for TM 1 Day Before Surgery
Tissue stained positive Tissue stained negative P
25 116 --
7 Days
After Surgery
Plasma TM (ng/mL)
Serum Cr (mg/dL)
CCr
Plasma TM
Serum Cr
CCr
(mL/min)
(ng/mL)
(mg/dL)
(mL/min)
4.922 ± 1.221 3.290 _+ 0.664 .0208
0.95 _+ 0.13 1.01 _+ 0.16 .8766
86.6 _+ 15.1 88.2 _ 17.2 .8012
3.400 ± 0.932 3.392 + 0.895 .8923
0.93 ± 0.11 0.96 ± 0.15 .9743
87.2 ± 18.6 87.8 ± 18.3 .9893
Abbreviations: Cr, creatinine; CCr, endogenous creatinine clearance.
HCC. The immunohistochemical positivity of TM in the 141 cases of HCC measuring less than 6 cm in diameter was 17.7% (25/141). In addition, the tissue that stained positive for TM in HCC cases showed a higher plasma TM level than did the tissue that stained negative for TM (4.922 +_ 1.221 ng/mL vs. 3.290 _+ 0.664 ng/mL: P -- .0208); however, after resection, the plasma TM level of the tissue that stained positive for TM in HCC cases immediately decreased to the same level of the tissue that stained negative in HCC cases (3.400 +_ 0.932 _+ 3.392 _+ 0.895: P = .9378). From these findings, some HCC are considered to produce TM that is released into the vessels. In general, intrahepatic metastasis and tumor thrombus in the portal vein are the most determinant prognostic factors for HCC. 21'22By analyzing the pathological prognostic factors and the reactivity of TM, we showed that the frequency of intrahepatic metastasis and tumor thrombus of the portal vein were both inversely correlated to the reactivity of TM. It has been established that TM is widely distributed on the vascular endothelium. 2327 TM has two distinct anticoagulant functions: (1) it inhibits the ability of thrombin to clot fibrinogen and to activate factor V, 2s'29
=
6.0"
T
= tissue TM positive (n=25) - - - 0 - - tissue TM negative (n=116)
_/ a,b ~
a p=0.0208 b p=0.0293
and (2) it accelerates the formation of the anticoagulant, activated protein C. 1° Once converted to activated protein C, this protein serves as a major anticoagulant in the blood because of its ability to inactivate the activated cofactors of the coagulation cascade, factors Va and VIIIa. 2s-3°M a r u y a m a et a123investigated the distribution of TM in h u m a n tissue using immunohistochemical methods and reported that TM was found on the endothelial cells of the arteries, veins, capillaries, and lymphatics, but not on the sinusoidal lining cells. DeBault et a124 also investigated the immunohistochemical distribution of TM in rabbits and reported that the vascular endothelium of the liver stained positive for TM, but they did not discuss in detail the TM positivity of the sinusoidal and capillary endothelial cells. In addition, Drake et a127 reported that TM was seen in the sinusoidal endothelial cells of a baboon, but only showed weak staining. They used polyclonal antibodies for immunohistochemistry but failed to discuss the functional activity of the antibodies. On the other hand, TM is present in various kinds of tumor cells that originate from vascular endothelial cells, such as angiosarcoma cells 31 and choriocarcinoma cells. ~2 In addition, Tamura et a133 reported that TM was also found in h u m a n lung cancer cells, but there were no differences found in the clinicopathologic findings. Recently, soluble TM has been measured, and its significance has been reported. Ishii et a134 reported that the level of soluble TM was elevated under conditions of endothelial injury. Several investigators have also TABLE 3. The Relationship B e t w e e n Coagulofibrinolytic Characteristics and Tissue S t a i n i n g Positivity for TM
e-
I-
,
E W m n
2.0 PRE OPE
POST OPE
FIG. 4. Changes in the plasma TM levels according to the staining positivity of TM. The preoperative plasma TM level in patients with tissue t h a t was stained positive for TM was significantly higher t h a n in those with tissue t h a t stained negative for TM (P = .0208), although in tissue that stained positive for TM, the postoperative plasma TM level was significantly lower t h a n the preoperative plasma TM level (P = ,0293). However, regarding the postoperative plasma TM levels, there were no differences between the patients with tissue that stained positive or negative for TM.
Thrombomodulin (ng/mL) Protein C activity (%) AT III activity (%) TAT (ng/mL) Fibrinogen (mg/dL) t-PA (ng/mL) PAI-1 (ng/mL) Platelets (×104/mL) PT (%)
Positive
Negative
( n = 25)
( n = 116)
4.9 58.8 75.5 5.2 253.3 5.5 12.9 13.4 86.3
+_ 1.2 _+ 15.7 _+ 17.3 _+ 3.8 ± 63.6 ± 3.6 ÷ 8.8 ± 6.7 _+ 11.4
3.3 58.4 77.3 3.8 256.7 5.8 14.8 14,9 78,4
_+ 0.7 _+ 29.4 _+ 23.3 _+ 2.3 _+ 95.8 _+ 4,4 _+ 9.0 _+ 6,3 _+ 17.7
P
.0208 .9274 .8899 .4831 .7692 .8693 .6538 .5092 .1643
Abbreviations: AT III, antithrombin III; TAT, plasma thrombinantithrombin III complex; t-PA, tissue plasminogen activator antigen; PAI-1, plasminogen activator inhibitor-l; PT, prothrombin activity.
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SUEHIRO ET AL
reported that elevated serum-soluble TM also exists in various conditions, such as systemic lupus erythematosus, cirrhosis, disseminated intravascular coagulation, pulmonary thromboembolism, adult respiratory distress syndrome, chronic renal failure, and acute hepatic failure. 2°'3~ The patients with tissue that stained positive for TM showed a significantly higher preoperative soluble TM level than the patients with tissue that stained negative. However, the postoperative soluble TM level of the patients with tissue that stained positive for TM was the same as that of the patients with tissue that stained negative for TM. These findings suggest that the HCC produced TM and released it into the vessels. Sarode et a136 reported that metastatic tumors had a significantly higher procoagnlant activity compared with nonmetastatic tumors. Nierodzik et al 7,s showed that thrombin stimulated tumor-platelet adhesion in vitro, as well as metastasis in vivo, and these phenomenon were inhibited with specific inhibitors ofthrombin, such as hirudin and dansyl-arginine N-(3-etyl-l,5 pentanediyl) amide. TM is also a specific thrombin inhibitor, and thus is thought to inhibit thrombin-related tumor-platelet adhesion and metastasis. The protein recognized by MFTM-F5 has a functional activity as a TM and inhibits thrombin binding and protein C activation. 18 Although the mechanism of production for TM in HCC is still unclear, TM-producing HCC shows a lower incidence of tumor thrombus and intrahepatic metasta-
TABLE 4. R e l a t i o n s h i p B e t w e e n P a t h o l o g i c a l P r o g n o s t i c F a c t o r s a n d T i s s u e S t a i n i n g P o s i t i v i t y for TM
Macroscopic classification Nodular Massive Histological classification Trabecular Others Edmondson's classification Grades I and II Grades III and IV vp vv im fc fc-inf sf Cirrhosis Tumor diameter (mm)
~'-.,..
i
80"
|
60 ~
Eo
1289
I
p=0.0085
"'--',-~., L'-I.._.,
b
c
40-
I,,,.
n,,'
20 0
I
Tissue TM positive (n=25) I ....... Tissue TM negative (n=116) I
0 100 2()0 300 4()0 5()0 600 7()0 8()0 9()0 Days after operation
FIG. 5. The recurrence-freedom curves as calculated by the Kaplan-Meier method for the 25 patients whose tissue stained positive for TM and the 116 patients whose tissue stained negative for TM. The difference was statistically significant (P = .0085).
sis and a higher recurrence freedom rate than HCC that does not produce TM. These findings suggest that TM may inhibit the adhesion of tumor cells to the portal vein because of the existence of some anticoagulant activity. Thus, TM modulates the invasiveness and metastatic activity of HCC.
Acknowledgment: We thank B. Quinn, Kyushu University, for critical comments on the manuscript and useful suggestions; and M. Sonoda for help in preparing experiments. REFERENCES
No. of Patients
Positive (n = 25)
Negative (n = 116)
112 29
17 (68.0) 8 (32.0)
95 (81.9) 21 (18.1)
.1189
112 29
18 (72.0) 7 (28.0)
94 (81.0) 22 (19.0)
.3107
68 73 34 25 42 87 68 40 94
14 (56.0) 11 (44.0) 2 (8.0) 2 (8.0) 3 (12.0) 19 (76.0) 11 (57.8) 8 (32.0) 17 (68.0) 34.4 _+ 14.8
54 (46.5) 62 (53.5) 32 (27.6) 23 (19.8) 39 (33.6) 68 (58.6) 57 (83.8) 32 (27.6) 77 (66.4) 34.2 _+ 14.5
.3912
P
.0378 .1602 .0320 .1049 .0156 .6570 .8761 .9672
NOTE. The X2 test was used with a continuity correction along with the Mann-Whitney test. Abbreviations: vp, tumor thrombus in the portal vein; vv, tumor thrombus in the hepatic vein; ira, intrahepatic metastasis; fc, capsule formation; fc-inf, capsular infiltration; sf, septal formation.
1. Nicolson GL. Cancer metastasis: tumor cell and host organ properties important in metastasis to specific secondary sites. Biochim Biophys Acta 1988;948:175-208. 2. Fidler IJ. Tumor heterogeneity and the biology of cancer invasion and metastasis. Cancer Res 1978;38:2651-2660. 3. Brown JM. A study of the mechanism by which anticoagulation with warfarin inhibits blood-bone metastasis. Cancer Res 1973;33:1217-1224. 4. Zacharski LR, Henderson WG, Rickles FR, Forman WB, Cornell J r CJ, Forcier RJ, et al. Effect of warfarin anticoagulation on survival in carcinoma of the lung, colon, head and neck, and prostate; final report of VA cooperative study #75. Cancer 1984; 53:2046-2052. 5. Carlsen SA, Ramshaw IA, Warrington RC. Involvement of plasminogen activator production with tumor metastasis in a rat model. Cancer Res 1984;44:3012-3016. 6. Marutsuka K, Suzumiya J, Kataoka H, Komada N, Koono M, Sumiyoshi A. Correlation between urokinase-type plasminogen activator production and the metastatic ability of human rectal cancer cells. Invasion Metastasis 1991; 11:181-191. 7. Nierodzik MLR, Kajumo F, Karpatkin S. Effect of thrombin treatment of tumor cells on adhesion of tumor cells to platelets in vitro and tumor metastasis in vivo. Cancer Res 1992;52:32673272. 8. Nierodzik MLR, Plotkin A, Kajimo F, Karpatkin S. Thrombin stimulates tumor-platelet adhesion in vitro and metastasis in vivo. J Clin Invest 1991;87:229-236. 9. Klepfish A, Greco MA, Karpatkin S. Thrombin stimulates melanoma tumor-cell binding to endothelial cells and subendothelial matrix. Int J Cancer 1993;53:978-982.
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10. Esmon CT. The role of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem 1989;264:47434749. 11. Rickles FR, Edwards RL. Activation of blood coagulation in cancer: Trousseau's syndrome revisited. Blood 1983;62:14-26. 12. Rickles FR, Edwards RL, Barb C, Cronlund M. Abnormalities of blood coagulation in patients with cancer: fibrinopeptide A generation and tumor growth. Cancer 1983;51:301-310. 13. Karpatkin S, Pearlstein E. Role of platelets in tumor cell metastasis. Ann Intern Med 1981;95:636-643. 14. Jones JD, Wallace AC, Fraser EF. Sequence of events in experimental metastasis of Walker 256 tumors: light, immunofluorescent and electron microscopic observations. J Natl Cancer Inst 1971;46:493-502. 15. Gasic GJ, Gasic TB, Galanti N, Johnson T, Murphy S. Platelettumor cell interactions in mice. The role ofplatelets in the spread of malignant disease. Int J Cancer 1973; 11:704-710. 16. Hayashi T, Suzuki K. Monoclonal antibodies for human thrombomodulin which recognize binding sites for thrombin and protein C. J Biochem 1990; 108:874-878. 17. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques; a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem 1981;29:577-582. 18. Edmondson HA, Steiner PE. Primary carcinoma of the liver; a study of 100 cases among 48,900 necropsies. Cancer 1954; 7:462482. 19. Liver Cancer Study Group of Japan. The general rules for the clinical and pathological study of primary liver cancer. Jpn J Surg 1989; 19:98-129. 20. Kodama S, Uchijima E, Nagai M, Mikawatani K, Hayashi T, Suzuki K. One-step sandwich enzyme immunoassay for soluble human thrombomodulin using monoclonal antibodies. Clin Chim Acta 1990; 192:191-199. 21. The Liver Cancer Study Group of Japan. Primary liver cancer in Japan: clinicopathologic features and results of surgical treatment. Ann Surg 1990;211:277-292. 22. Scudamore CH, Ragaz J, Kluftinger AM, Owen DA. Hepatocellular carcinoma: a comparison of Oriental and Caucasian patients. Am J Surg 1988; 155:659-666. 23. Marnyama I, Bell E, Majeus PW. Thrombomodulin is found in endothelium of arteries, veins, capillaries, lymphatics, and on syncytio-trophoblast of human placenta. J Cell Biol 1985; 101: 363-372.
HEPATOLOGYMay 1995 24. DeBault LE, Esmon NL, Olson JR, Esmon CT. Distribution of the thrombomodulin antigen in the rabbit vasculature. Lab Invest 1986;54:172-178. 25. Suzuki K, Kusumoto H, Deyashiki Y. Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C. EMBO J 1987;6:1891-1897. 26. Kumada T, Dittman WA, Majeus PW. A role for thrombomodulin in the pathogenesis of thrombin-induced thrombomodulin in mice. Blood 1987;71:728-736. 27. Drake TA, Cheng J, Chang A, Taylor FB. Expression of tissue factor, thrombomodulin, and E-selectin in baboons with lethal Escherichia coli sepsis. Am J Pathol 1993; 142:1458-1470. 28. Suzuki K, Stenflo J, Dahlback B, Tedosson B. Inactivation of human coagulation factor V by activated protein C. J Biol Chem 1983;258:1914-1919. 29. Fulcher CA, Gardiner JE, Griffin JH, Zimmerman TS. Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. Blood 1984;63:486-493. 30. Esmon CT, Esmon NL, Harris KW. Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation. J Biol Chem 1982;357:7944-7949. 31. Yonezawa S, Maruyama I, Sakae K, Igata A, Majeus PW, Sato E. Thrombomodulin as a marker for vascular tumors: comparative study with Factor VIII and Ulex europaeus I lectin. Am J Clin Pathol 1987;88:405-411. 32. Yonezawa S, Maruyama I, Tanaka S, Nakamura T, Sato E. Immunohistochemical localization of thrombomodulin in chronic diseases of the uterus and choriocarcinoma of the stomach: a comparative study with the distribution of human chorionic gonadotropin. Cancer 1988;62:569-576. 33. Tamura A, Matsubara O, Hirokawa K, Aoki N. Detection of thrombomodulin in human lung cancer cells. Am J Pathol 1993; 142:79-85. 34. Ishii H, Majerus PW. Thrombomodulin is present in human plasma and urine. J Clin Invest 1985;76:2178-2183. 35. Takano S, Kimura S, Ohdama S, Aoki N. Plasma thrombomodulin in health and diseases. Blood 1990; 76:2024-2030. 36. Sarode R, Marwaha N, Gupta NM. Procoagulant activity of human tumors: existence of Xa and thrombin-like activities. Br J Cancer 1990; 61:29-31.
T h r o m b o m o d u l i n (TM) c o n v e r t s t h r o m b i n from procoa g u l a n t into a n t i c o a g u l a n t p r o t e i n to activate p r o t e i n C. T h r o m b i n also plays an i m p o r t a n t role in the m e t a s t a t i c p r o c e s s o f c a n c e r cells. We p e r f o r m e d an i m m u n o h i s t o c h e m i c a l a n d c l i n i c o p a t h o l o g i c a l study of TM in 141 cases w i t h r e s e c t e d h e p a t o c e i l u l a r c a r c i n o m a (HCC) m e a s u r i n g less t h a n 6 c m in diameter. Twenty-five specim e n s (17.73%) s t a i n e d positive for TM. TM w a s f o u n d in the c y t o p l a s m a n d surface o f c a n c e r cells. The clinicop a t h o l o g i c a l findings a c c o r d i n g to the positive of TM are e x a m i n e d in HCC. The p r e o p e r a t i v e p l a s m a TM level o f t h e p a t i e n t s w i t h tissue that s t a i n e d positive for TM w a s significantly h i g h e r t h a n that o f the patients w i t h negative results; for the p o s t o p e r a t i v e TM level, there w e r e n o differences b e t w e e n them. In addition, t h e frequencies of i n t r a h e p a t i c metastasis, t u m o r t h r o m b u s in the portal vein, a n d c a p s u l a r infiltration w e r e significantly l o w e r in p a t i e n t s w h o s e tissue s t a i n e d positive for TM t h a n in p a t i e n t s w h o s e tissue s t a i n e d n e g a t i v e for TM. The r e c u r r e n c e f r e e d o m rate w a s significantly h i g h e r in p a t i e n t s w h o s e tissue s t a i n e d positive for TM t h a n patients w h o s e tissue s t a i n e d n e g a t i v e for TM. Thus, TMp r o d u c i n g HCC s h o w s a s l o w ~ t r a h e p a t i c spread. Therefore, t h e s e findings s u g g e s t ~hat TM m a y inhibit the a d h e s i o n of t u m o r cells to the portal v e i n b e c a u s e o f a n t i c o a g u l a n t activity a n d t h u s p r e v e n t the spread o f i n t r a h e p a t i c metastasis. (HEPATOLOGY 1995;21:12851290.)
Tumor metastasis is a complex series of ~,ulti-tep processes. ~'2 In these steps, two processes are important. One process is the detachment of cancer ~'ells into the blood circulation, and another is the a d b ~sion of such cancer cells to endoth ~lial cells. Blood coa~:;ulation and fibrinolysis have been shown to play an important role in these two metastatic processes. It has been reported t h a t administration of warfarin, which
Abbreviations: TM, thrombomodulin; HCC, hepatocellular carcinoma; PBS, phosphate-buffered saline. From the Department of Surgery II, Faculty of Medicine, Kyu~ ~ University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan. Received February 22, 1994; accepted December 3, 1994. Address reprint requests to: Taketoshi Suehiro, MD, Departm .~J ,: Surgery II, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higa: :.~ ,~1, Fukuoka 812, Japan. Copyright © 1995 by the American Association for 11,~ Sty:. of Liver Diseases. 0270-9139/95/2105-001053.00/0
prevents the synthesis of the vitamin K - d e p e n d e n t procoagulants (factors II, VII, IX, and X), significantly lowered the numbers of tumor metastasis. 3't Fibrinolytic agents, such as plasminogen activator or urokinase, are thought to play an important role in tumor metastasis, because these agents are able to lyse tumor-associated fibrin clots and cause the destruction of the extracellular matrix and basement membrane. ~'6 Thrombin plays an important role in the adhesion of cancer cells to endothelial cells through fibrin formation and platelet activation, 7,s and thrombin stimulates tumor cell bindings to endothelial cells2 Thrombomodulin (TM) is the endothelial cell surface protein t h a t converts thrombin from a procoagnlant protein into the activator for protein C. ~o From histological evidence of experimental metastasis, several investigator,-~ have previously reported on the relationship between coagulation activity and tumor metastasis. ~~5 They have suggested t h a t fibrin acts a~ a glue t h a t facilitates tumor cell adhesion to the endothelial ell and therefore is critical to the pathogenesis oftum(~r growth and metastasis formation. TM is known to i~hibit this fibrin formation and thus may protect agai,~st the adhesion of tumor cells to endothelial cells. The aim of this study is to compare the positivity of TM with clinicopathologic findings and discuss the relationship between TM and the intrahepatic spread of he mtocellular carcinoma (HCC). PATIENTS AND METHOD,~ P a t i e n t s . One hundred and forty-one pat~e~ts with HCC who underwent hepatic resections durin~ th~ ~riod between January 1991 and August 1992 at the D~part~~ n t of Surgery II, Kyushu University Hospital, were used in ~:},~sstudy. They included 111 men and 30 women. Their age at op. ,'ation ranged from 37 to 79 years (mean, 63 years). The types of hepatic resection performed were a partial hepatectomy (64 pa*ients), subsegmentectomy (17 patients), segmentectomy (1 ~ patients), lobectomy (41 patients), and an extended lobecto~,y (5 patients). The patients were strictly followed up using monthly measurements of a-fetoprotein and des-T-carboxy prothrombin, monthly bedside ultrasonography and ultrasonography, dynamic computed tomogr~ phy by radiologists every 3 months, and angiographic examinations performed after admission whenever recurrence was strongly suspected. A n t i b o d i e s . The antibody used is an anti-TM monoclonal antibody derived from mice (MFTM-5; Fuji Pharmaceutical
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Co, Ltd, Tokyo, Japan). This antibody recognizes the epidermal growth factor-like domain of TM, which is the binding site for thrombin and protein C. 16 Immunohistochemical Study. Formaldehyde-fixed paraffin-embedded six-micron sections were used in this study. The paraffin sections were dewaxed according to ordinary procedures and then immersed in 3% hydrogen peroxide in methanol for 30 minutes to block the endogenous peroxidase activity. The sections were subsequently washed in phosphate-buffered saline (PBS). For the immunohistochemical study, the avidin-biotin immunoperoxidase method was used 17with the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). After treatment with normal horse serum, each section was incubated with mouse monoclonal antibodies at room temperature for 1 hour and then rinsed with PBS. Mouse anti-TM monoclonal antibody was used at a concentration of 20 #g/mL. The section was then incubated with biotinylated horse anti-mouse immunoglobulin for 30 minutes and washed with PBS. Avidinbiotin-horseradish peroxidase complex was applied for 30 minutes, and then the section was washed with PBS. The sections were incubated with a peroxidase substrate solution (mixture of 0.02% [wt/vol] hydrogen peroxide and 0.1% [wry col] diaminobenzidine tetrahydrochloride in PBS; pH 7.4), and then were counterstained with methyl green. For negative control, 1% bovine serum albumin-PBS and normal rat serum were used as primary antibody. The percentage of cells that stained positive for TM was classified in four categories: - , (<25%); +, (25% to 50%); + +, (50% to 75%); + + +, (> 75%), and we defined + to + + + grades as having stained positive for TM. For the estimation of interobserver variation, the percentage of cells that stained positive for TM of all cases was examined by another experienced surgical pathologist without any knowledge of the clinical outcome or the first observer's determination of the percentage of cells that stained positive for TM. The mean of the two counts of each observer was considered to be the percentage of cells that stained positive for TM. Histopathological Findings. For the histopathological examination, the sections were stained with hematoxylin-eosin. The degree of cell differentiation was classified according to the criteria of Edmondson and Steiner. is Other pathological findings were classified according to the Japanese general rules for primary liver cancer. 19 Determination of TM and Protein C, Blood sampling was done 1 day before surgery and on the 7th day after surgery. The blood was drawn into sterile tubes (one containing 3.8% trisodium citrate and the other containing nothing). Citrate plasma was used to determine the protein C activity, and the serum was used for determining the soluble TM. The protein C activity was determined using a STACHROM PROTEIN C test kit (DIAGNOSTICA STAGO, France) (normal range, 70% to 140%). The soluble TM was then determined using the one-step sandwich enzyme immunoassay described by Kodama S e t al 2° (Fuji Pharmaceutical Co, Ltd., Tokyo, Japan). Renal function (serum creatinine level and creatinine clearance) and urine volume of each patient was also recorded, because plasma TM level is easily influenced by renal clearance. Statistical Analysis. A statistical evaluation was made using the Mann-Whitney test, Yates' continuity-corrected X2 test, and McNemar's X2 test. The recurrence freedom survival rate was estimated using Kaplan and Meier's actuarial method. The probability values for comparing two sets of lifetable data were determined using the generalized Wilcoxon
HEPATOLOGYMay 1995
FIG. 1. (A) The immunohistochemical distribution of TM in HCC. A granular reaction was noted in the cytoplasm of cancer cells. (Original magnification ×200.) (B) Control.
test. All statistical analyses were considered significant at a P value of <.05. RESULTS
D i s t r i b u t i o n o f T M in HCC. TM w a s r e c o g n i z e d in 17.7% of H C C s m e a s u r i n g less t h a n 6 cm in d i a m e t e r (25/141). Cells t h a t s t a i n e d positive for TM w e r e obs e r v e d diffusely, w h e r e a s a g r a n u l a r r e a c t i o n w a s obs e r v e d in t h e c y t o p l a s m of t h e c a n c e r cells (Fig. 1). B o t h t h e i n t e n s i t y of positive s t a i n i n g a n d t h e n u m b e r of cells t h a t s t a i n e d positive v a r i e d f r o m a r e a to a r e a in i n d i v i d u a l cases. I n s u r r o u n d i n g n o n c a n c e r ous tissue, TM w a s n o t e d on t h e c a p i l l a r y e n d o t h e l i a l cells in t h e t u m o r a n d / o r a r o u n d t h e t u m o r (Figs. 2 a n d 3). The R e l a t i o n s h i p B e t w e e n the C l i n i c a l F i n d i n g s a n d Tissue TM Positivity. Table 1 shows the clinical feat u r e s of 141 p a t i e n t s with H C C m e a s u r i n g less t h a n 6 cm in d i a m e t e r according to positivity of TM staining. The s e r u m total bilirubin level was significantly lower in the p a t i e n t s with tissue t h a t s t a i n e d positive for TM t h a n with tissue t h a t s t a i n e d negative for TM. In the o t h e r clinical findings, t h e r e were no differences be-
SUEHIRO ET AL
HEPATOLOGY Vo|. 21, No. 5, 1995
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TABLE 1. T h e R e l a t i o n s h i p B e t w e e n t h e C l i n i c a l F i n d i n g s a n d T i s s u e S t a i n i n g P o s i t i v i t y for TM Positive (n - 25) Mean age (yr) ICG (%) Albumin (mg/dL) Total bilirubin (mg/dL) GOT (IU/L) GPT (IU/L)
18.9 3.82 0.76 71.8 66.8
63 ± 10.1" ± 0.41 _+ 0.32 _-2 35.5 _+ 40.2
Negative (n = 116)
19.4 3.75 0.88 71.7 69.7
62 ± 9.5 _+ 0.36 _+ 0.35 ± 35.2 ± 44.0
P
.7947 .4459 .0326 .9339 .8233
Abbreviations: ICG, indocyanine green retention test; GOT, glutamate oxaloacetic transaminase; GPT, glutamate pyruvic transaminase. * Data represent mean ± SD.
FIG. 2. The immunohistochemical distribution of TM in HCC. Although almost all cancer cells stained positive for TM, surrounding noncancerous hepatocytes stained negative for TM.
tween the patients with staining results that were positive and negative. The R e l a t i o n s h i p Between the P l a s m a TM Level a n d Tissue TM Positivity. All patients showed good renal function (creatinine <2.0 mg/dL, creatinine clearance >70 mL/min). There was no difference between the patients with positive and negative staining for TM in renal function (Table 2). Figure 4 shows the changes in the plasma TM levels of the 141 patients with HCC measuring less than 6 cm in diameter according to the staining positivity for TM. The preoperative plasma TM level of the patients with tissue that stained positive for TM was significantly higher than those with tissue that stained negative for TM (4.922 _+ 1.221 ng/mL vs. 3.290 _+ 0.664 ng/ mL; P = .0208). In the patients with tissue that stained positive for TM, the plasma TM level at the 7th day
after surgery was significantly lower than the day before surgery (4.922 _+ 1.221 ng/mL vs. 3.400 _+ 0.932 ng/mL; P = .0293). The postoperative plasma TM levels showed no differences between the patients with tissue stained positive and negative for TM. Table 3 shows the relationship between the coagulofibrinolytic characteristics and positive staining for TM in tissue. There were no differences between the patients staining positive and negative for TM regarding coagulofibrinolytic characteristics. The Relationship Between Pathological Prognostic Factors and Tissue Staining Positive for TM. The relationship between the pathological prognostic factors and tissue's TM reactivity in 141 HCC measuring less than 6 cm in diameter is summarized in Table 4. Tumor thrombus in the portal vein, intrahepatic metastasis, and capsular infiltration were all more frequently observed in patients whose tissue stained negative for TM than in patients whose tissue stained positive for TM (tumor thrombus in the portal vein: P = .0378, intrahepatic metastasis: P = .0320, capsular infiltration: P = .0156). Macroscopic classification, histological classification, Edmondson's classification, and tumor diameter showed no significant differences between the patients in which staining for TM was positive and negative. The R e l a t i o n s h i p Between the Recurrence-Freedom Rate and Tissue Staining Positive for TM. The recurrence-freedom rate was calculated by the Kaplan-Meier method for 141 patients with resected HCC measuring less than 6 cm in diameter. The number of recurrencefreedom days ranged from 29 to 927 days. Figure 5 represents the recurrence-freedom curves of patients with respect to tissue staining positive for TM. The recurrence-freedom rate was significantly higher in the patients whose tissue stained positive for TM than in those whose tissue stained negative for TM (P = .0085). DISCUSSION
FIG. 3. The immunohistochemica] distribution of TM in noncancerous tissue. TM was noted on the capillary endothelial cells and sinusoidal endothelial cells in a cirrhotic liver. (Original magnification × 100.)
In this study, we investigated the relationship between HCC and TM both clinicopathologically and immunohistochemically. TM was seen both in cancer cells and on capillary endothelial cells in and/or around
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SUEHIRO ET AL
HEPATOLOGY May 1995
TABLE 2. The Relationship B e t w e e n Renal F u n c t i o n and Tissue Staining Positivity for TM 1 Day Before Surgery
Tissue stained positive Tissue stained negative P
25 116 --
7 Days
After Surgery
Plasma TM (ng/mL)
Serum Cr (mg/dL)
CCr
Plasma TM
Serum Cr
CCr
(mL/min)
(ng/mL)
(mg/dL)
(mL/min)
4.922 ± 1.221 3.290 _+ 0.664 .0208
0.95 _+ 0.13 1.01 _+ 0.16 .8766
86.6 _+ 15.1 88.2 _ 17.2 .8012
3.400 ± 0.932 3.392 + 0.895 .8923
0.93 ± 0.11 0.96 ± 0.15 .9743
87.2 ± 18.6 87.8 ± 18.3 .9893
Abbreviations: Cr, creatinine; CCr, endogenous creatinine clearance.
HCC. The immunohistochemical positivity of TM in the 141 cases of HCC measuring less than 6 cm in diameter was 17.7% (25/141). In addition, the tissue that stained positive for TM in HCC cases showed a higher plasma TM level than did the tissue that stained negative for TM (4.922 +_ 1.221 ng/mL vs. 3.290 _+ 0.664 ng/mL: P -- .0208); however, after resection, the plasma TM level of the tissue that stained positive for TM in HCC cases immediately decreased to the same level of the tissue that stained negative in HCC cases (3.400 +_ 0.932 _+ 3.392 _+ 0.895: P = .9378). From these findings, some HCC are considered to produce TM that is released into the vessels. In general, intrahepatic metastasis and tumor thrombus in the portal vein are the most determinant prognostic factors for HCC. 21'22By analyzing the pathological prognostic factors and the reactivity of TM, we showed that the frequency of intrahepatic metastasis and tumor thrombus of the portal vein were both inversely correlated to the reactivity of TM. It has been established that TM is widely distributed on the vascular endothelium. 2327 TM has two distinct anticoagulant functions: (1) it inhibits the ability of thrombin to clot fibrinogen and to activate factor V, 2s'29
=
6.0"
T
= tissue TM positive (n=25) - - - 0 - - tissue TM negative (n=116)
_/ a,b ~
a p=0.0208 b p=0.0293
and (2) it accelerates the formation of the anticoagulant, activated protein C. 1° Once converted to activated protein C, this protein serves as a major anticoagulant in the blood because of its ability to inactivate the activated cofactors of the coagulation cascade, factors Va and VIIIa. 2s-3°M a r u y a m a et a123investigated the distribution of TM in h u m a n tissue using immunohistochemical methods and reported that TM was found on the endothelial cells of the arteries, veins, capillaries, and lymphatics, but not on the sinusoidal lining cells. DeBault et a124 also investigated the immunohistochemical distribution of TM in rabbits and reported that the vascular endothelium of the liver stained positive for TM, but they did not discuss in detail the TM positivity of the sinusoidal and capillary endothelial cells. In addition, Drake et a127 reported that TM was seen in the sinusoidal endothelial cells of a baboon, but only showed weak staining. They used polyclonal antibodies for immunohistochemistry but failed to discuss the functional activity of the antibodies. On the other hand, TM is present in various kinds of tumor cells that originate from vascular endothelial cells, such as angiosarcoma cells 31 and choriocarcinoma cells. ~2 In addition, Tamura et a133 reported that TM was also found in h u m a n lung cancer cells, but there were no differences found in the clinicopathologic findings. Recently, soluble TM has been measured, and its significance has been reported. Ishii et a134 reported that the level of soluble TM was elevated under conditions of endothelial injury. Several investigators have also TABLE 3. The Relationship B e t w e e n Coagulofibrinolytic Characteristics and Tissue S t a i n i n g Positivity for TM
e-
I-
,
E W m n
2.0 PRE OPE
POST OPE
FIG. 4. Changes in the plasma TM levels according to the staining positivity of TM. The preoperative plasma TM level in patients with tissue t h a t was stained positive for TM was significantly higher t h a n in those with tissue t h a t stained negative for TM (P = .0208), although in tissue that stained positive for TM, the postoperative plasma TM level was significantly lower t h a n the preoperative plasma TM level (P = ,0293). However, regarding the postoperative plasma TM levels, there were no differences between the patients with tissue that stained positive or negative for TM.
Thrombomodulin (ng/mL) Protein C activity (%) AT III activity (%) TAT (ng/mL) Fibrinogen (mg/dL) t-PA (ng/mL) PAI-1 (ng/mL) Platelets (×104/mL) PT (%)
Positive
Negative
( n = 25)
( n = 116)
4.9 58.8 75.5 5.2 253.3 5.5 12.9 13.4 86.3
+_ 1.2 _+ 15.7 _+ 17.3 _+ 3.8 ± 63.6 ± 3.6 ÷ 8.8 ± 6.7 _+ 11.4
3.3 58.4 77.3 3.8 256.7 5.8 14.8 14,9 78,4
_+ 0.7 _+ 29.4 _+ 23.3 _+ 2.3 _+ 95.8 _+ 4,4 _+ 9.0 _+ 6,3 _+ 17.7
P
.0208 .9274 .8899 .4831 .7692 .8693 .6538 .5092 .1643
Abbreviations: AT III, antithrombin III; TAT, plasma thrombinantithrombin III complex; t-PA, tissue plasminogen activator antigen; PAI-1, plasminogen activator inhibitor-l; PT, prothrombin activity.
HEPATOLOGYVol. 21, No. 5, 1995
SUEHIRO ET AL
reported that elevated serum-soluble TM also exists in various conditions, such as systemic lupus erythematosus, cirrhosis, disseminated intravascular coagulation, pulmonary thromboembolism, adult respiratory distress syndrome, chronic renal failure, and acute hepatic failure. 2°'3~ The patients with tissue that stained positive for TM showed a significantly higher preoperative soluble TM level than the patients with tissue that stained negative. However, the postoperative soluble TM level of the patients with tissue that stained positive for TM was the same as that of the patients with tissue that stained negative for TM. These findings suggest that the HCC produced TM and released it into the vessels. Sarode et a136 reported that metastatic tumors had a significantly higher procoagnlant activity compared with nonmetastatic tumors. Nierodzik et al 7,s showed that thrombin stimulated tumor-platelet adhesion in vitro, as well as metastasis in vivo, and these phenomenon were inhibited with specific inhibitors ofthrombin, such as hirudin and dansyl-arginine N-(3-etyl-l,5 pentanediyl) amide. TM is also a specific thrombin inhibitor, and thus is thought to inhibit thrombin-related tumor-platelet adhesion and metastasis. The protein recognized by MFTM-F5 has a functional activity as a TM and inhibits thrombin binding and protein C activation. 18 Although the mechanism of production for TM in HCC is still unclear, TM-producing HCC shows a lower incidence of tumor thrombus and intrahepatic metasta-
TABLE 4. R e l a t i o n s h i p B e t w e e n P a t h o l o g i c a l P r o g n o s t i c F a c t o r s a n d T i s s u e S t a i n i n g P o s i t i v i t y for TM
Macroscopic classification Nodular Massive Histological classification Trabecular Others Edmondson's classification Grades I and II Grades III and IV vp vv im fc fc-inf sf Cirrhosis Tumor diameter (mm)
~'-.,..
i
80"
|
60 ~
Eo
1289
I
p=0.0085
"'--',-~., L'-I.._.,
b
c
40-
I,,,.
n,,'
20 0
I
Tissue TM positive (n=25) I ....... Tissue TM negative (n=116) I
0 100 2()0 300 4()0 5()0 600 7()0 8()0 9()0 Days after operation
FIG. 5. The recurrence-freedom curves as calculated by the Kaplan-Meier method for the 25 patients whose tissue stained positive for TM and the 116 patients whose tissue stained negative for TM. The difference was statistically significant (P = .0085).
sis and a higher recurrence freedom rate than HCC that does not produce TM. These findings suggest that TM may inhibit the adhesion of tumor cells to the portal vein because of the existence of some anticoagulant activity. Thus, TM modulates the invasiveness and metastatic activity of HCC.
Acknowledgment: We thank B. Quinn, Kyushu University, for critical comments on the manuscript and useful suggestions; and M. Sonoda for help in preparing experiments. REFERENCES
No. of Patients
Positive (n = 25)
Negative (n = 116)
112 29
17 (68.0) 8 (32.0)
95 (81.9) 21 (18.1)
.1189
112 29
18 (72.0) 7 (28.0)
94 (81.0) 22 (19.0)
.3107
68 73 34 25 42 87 68 40 94
14 (56.0) 11 (44.0) 2 (8.0) 2 (8.0) 3 (12.0) 19 (76.0) 11 (57.8) 8 (32.0) 17 (68.0) 34.4 _+ 14.8
54 (46.5) 62 (53.5) 32 (27.6) 23 (19.8) 39 (33.6) 68 (58.6) 57 (83.8) 32 (27.6) 77 (66.4) 34.2 _+ 14.5
.3912
P
.0378 .1602 .0320 .1049 .0156 .6570 .8761 .9672
NOTE. The X2 test was used with a continuity correction along with the Mann-Whitney test. Abbreviations: vp, tumor thrombus in the portal vein; vv, tumor thrombus in the hepatic vein; ira, intrahepatic metastasis; fc, capsule formation; fc-inf, capsular infiltration; sf, septal formation.
1. Nicolson GL. Cancer metastasis: tumor cell and host organ properties important in metastasis to specific secondary sites. Biochim Biophys Acta 1988;948:175-208. 2. Fidler IJ. Tumor heterogeneity and the biology of cancer invasion and metastasis. Cancer Res 1978;38:2651-2660. 3. Brown JM. A study of the mechanism by which anticoagulation with warfarin inhibits blood-bone metastasis. Cancer Res 1973;33:1217-1224. 4. Zacharski LR, Henderson WG, Rickles FR, Forman WB, Cornell J r CJ, Forcier RJ, et al. Effect of warfarin anticoagulation on survival in carcinoma of the lung, colon, head and neck, and prostate; final report of VA cooperative study #75. Cancer 1984; 53:2046-2052. 5. Carlsen SA, Ramshaw IA, Warrington RC. Involvement of plasminogen activator production with tumor metastasis in a rat model. Cancer Res 1984;44:3012-3016. 6. Marutsuka K, Suzumiya J, Kataoka H, Komada N, Koono M, Sumiyoshi A. Correlation between urokinase-type plasminogen activator production and the metastatic ability of human rectal cancer cells. Invasion Metastasis 1991; 11:181-191. 7. Nierodzik MLR, Kajumo F, Karpatkin S. Effect of thrombin treatment of tumor cells on adhesion of tumor cells to platelets in vitro and tumor metastasis in vivo. Cancer Res 1992;52:32673272. 8. Nierodzik MLR, Plotkin A, Kajimo F, Karpatkin S. Thrombin stimulates tumor-platelet adhesion in vitro and metastasis in vivo. J Clin Invest 1991;87:229-236. 9. Klepfish A, Greco MA, Karpatkin S. Thrombin stimulates melanoma tumor-cell binding to endothelial cells and subendothelial matrix. Int J Cancer 1993;53:978-982.
1290
SUEHIRO ET AL
10. Esmon CT. The role of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem 1989;264:47434749. 11. Rickles FR, Edwards RL. Activation of blood coagulation in cancer: Trousseau's syndrome revisited. Blood 1983;62:14-26. 12. Rickles FR, Edwards RL, Barb C, Cronlund M. Abnormalities of blood coagulation in patients with cancer: fibrinopeptide A generation and tumor growth. Cancer 1983;51:301-310. 13. Karpatkin S, Pearlstein E. Role of platelets in tumor cell metastasis. Ann Intern Med 1981;95:636-643. 14. Jones JD, Wallace AC, Fraser EF. Sequence of events in experimental metastasis of Walker 256 tumors: light, immunofluorescent and electron microscopic observations. J Natl Cancer Inst 1971;46:493-502. 15. Gasic GJ, Gasic TB, Galanti N, Johnson T, Murphy S. Platelettumor cell interactions in mice. The role ofplatelets in the spread of malignant disease. Int J Cancer 1973; 11:704-710. 16. Hayashi T, Suzuki K. Monoclonal antibodies for human thrombomodulin which recognize binding sites for thrombin and protein C. J Biochem 1990; 108:874-878. 17. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques; a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem 1981;29:577-582. 18. Edmondson HA, Steiner PE. Primary carcinoma of the liver; a study of 100 cases among 48,900 necropsies. Cancer 1954; 7:462482. 19. Liver Cancer Study Group of Japan. The general rules for the clinical and pathological study of primary liver cancer. Jpn J Surg 1989; 19:98-129. 20. Kodama S, Uchijima E, Nagai M, Mikawatani K, Hayashi T, Suzuki K. One-step sandwich enzyme immunoassay for soluble human thrombomodulin using monoclonal antibodies. Clin Chim Acta 1990; 192:191-199. 21. The Liver Cancer Study Group of Japan. Primary liver cancer in Japan: clinicopathologic features and results of surgical treatment. Ann Surg 1990;211:277-292. 22. Scudamore CH, Ragaz J, Kluftinger AM, Owen DA. Hepatocellular carcinoma: a comparison of Oriental and Caucasian patients. Am J Surg 1988; 155:659-666. 23. Marnyama I, Bell E, Majeus PW. Thrombomodulin is found in endothelium of arteries, veins, capillaries, lymphatics, and on syncytio-trophoblast of human placenta. J Cell Biol 1985; 101: 363-372.
HEPATOLOGYMay 1995 24. DeBault LE, Esmon NL, Olson JR, Esmon CT. Distribution of the thrombomodulin antigen in the rabbit vasculature. Lab Invest 1986;54:172-178. 25. Suzuki K, Kusumoto H, Deyashiki Y. Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C. EMBO J 1987;6:1891-1897. 26. Kumada T, Dittman WA, Majeus PW. A role for thrombomodulin in the pathogenesis of thrombin-induced thrombomodulin in mice. Blood 1987;71:728-736. 27. Drake TA, Cheng J, Chang A, Taylor FB. Expression of tissue factor, thrombomodulin, and E-selectin in baboons with lethal Escherichia coli sepsis. Am J Pathol 1993; 142:1458-1470. 28. Suzuki K, Stenflo J, Dahlback B, Tedosson B. Inactivation of human coagulation factor V by activated protein C. J Biol Chem 1983;258:1914-1919. 29. Fulcher CA, Gardiner JE, Griffin JH, Zimmerman TS. Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V. Blood 1984;63:486-493. 30. Esmon CT, Esmon NL, Harris KW. Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation. J Biol Chem 1982;357:7944-7949. 31. Yonezawa S, Maruyama I, Sakae K, Igata A, Majeus PW, Sato E. Thrombomodulin as a marker for vascular tumors: comparative study with Factor VIII and Ulex europaeus I lectin. Am J Clin Pathol 1987;88:405-411. 32. Yonezawa S, Maruyama I, Tanaka S, Nakamura T, Sato E. Immunohistochemical localization of thrombomodulin in chronic diseases of the uterus and choriocarcinoma of the stomach: a comparative study with the distribution of human chorionic gonadotropin. Cancer 1988;62:569-576. 33. Tamura A, Matsubara O, Hirokawa K, Aoki N. Detection of thrombomodulin in human lung cancer cells. Am J Pathol 1993; 142:79-85. 34. Ishii H, Majerus PW. Thrombomodulin is present in human plasma and urine. J Clin Invest 1985;76:2178-2183. 35. Takano S, Kimura S, Ohdama S, Aoki N. Plasma thrombomodulin in health and diseases. Blood 1990; 76:2024-2030. 36. Sarode R, Marwaha N, Gupta NM. Procoagulant activity of human tumors: existence of Xa and thrombin-like activities. Br J Cancer 1990; 61:29-31.