- Email: [email protected]
Tumor interaction with the fibrinolytic system
JOURNAL
OF SURGICAL
RESEARCH
26,
581-589 (1979)
Tumor Interaction J. M. MALONE, The Department
with the Fibrinolytic
System’
M.D.,2 A. S. GERVIN, M.D., W. S. MOORE, M.D., F.A.C.S., AND K. KEOWN, M.A.
of Surgery
of the University
of Arizona
College of Medicine,
Tucson, Arizona
85724
Presented at the Annual Meeting of the Association for Academic Surgery, Cleveland, Ohio, November 12- 15, 1978 Tumor activation and inhibition of fibrinolysis appear to be related to the pathophysiology of metastatic tumor spread. Our data demonstrate a correlation between the incidence of tumor metastases and both increasing tumor fibrinolytic activator activity and decreasing tumor fibrinolytic inhibitory activity. Nonmalignant lesions and normal tissue, other than liver, had activation/ inhibition ratios of zero. Most malignant neoplasms without metastatic spread had activation/ inhibition ratios less than 7, while most tumors with metastatic spread had activation/inhibition ratios greater than 7. Calculation of mean activation/inhibition ratios for tumors with and without metastatic spread demonstrated a statistically significant difference between their activation/ inhibition ratios (P = 0.001). Therefore, tumor activation/inhibition ratios would appear to have statistical reliability as biologic markers for the presence or absence of metastatic tumor spread. These data have significant clinical and therapeutic implications with respect to the utilization of tumor activation/inhibition ratios as biologic markers of tumor spread, and they also warrant further investigation into the mechanisms of tumor interaction with the fibrinolytic system.
INTRODUCTION
Neither histologic nor clinical staging always correlates with either patient survival or the time course of tumor metastatic spread. Anticancer treatment is based upon norms for specific tumor cell types, and treatment often cannot be individualized. In order to accurately assess tumor growth and/or tumor recurrence a biological tumor marker is needed. To a limited extent, and for specific tumors, the carcinoma embryonic antigen and the a-fetoprotein have provided assessment of the adequacy of tumor resection and tumor recurrence. However, there is no general biological tumor marker which is able to distinguish those patients with microscopic residual cancer who will develop tumor recurrence, and therefore require adjuvant anticancer treatment, from those patients cured as a result of their 1 Supported in part by V.A. Career Development Grant 678-5447-01 and V.A. Grant 622-5798-03. * To whom requests for reprints should be addressed: Department of Surgery, Arizona Health Sciences Center, Tucson, Arizona 85724.
primary treatment and who, therefore, do not require additional anticancer therapy. Factors important to tumor growth include host immunological response, host hormonal environment, the intrinsic biologic potential of the tumor, dormancy, spontaneous regression, and selective organ enhancement [31, 321. The potential of tumors to metastasize has also been correlated with both activation and inhibition ofthefibrinolyticsystem [1,4,10,13,14,1720, 24, 26, 27, 29, 311. The purpose of this report is to evaluate tumor interaction with the fibrinolytic system and to investigate the potential of tumor activation and inhibition of fibrinolysis as biologic markers for the presence or absence of metastatic disease. MATERIALS
AND METHODS
Biopsies of both normal tissue and tumor were obtained from fresh surgical specimens of patients undergoing curative or palliative tumor resection at the Tucson V.A. Hospital from January 1,1978, through April 15, 1978. Biopsies were often limited
581
0022-4804/79/05058l-09$1.00/0 Copyright 0 1979 by Academic Press. Inc. All rights of reproductmn in any form reserved.
582
JOURNAL OF SURGICAL RESEARCH: VOL. 26, NO. 5, MAY 1979
in size so as not to interfere with routine histological and clinical staging. When possible, biopsy specimens were taken from gross tumor, grossly normal tissue around tumor, and normal tissue at the surgical margins of resection. All tissue samples were transported to our research laboratory in normal saline, and warm ischemia time was limited to 1 hr. Initially all tissue samples were analyzed immediately upon arrival in the laboratory. However, since the analysis requires 6-10 hr, and since many specimens were not available until late afternoon, we divided our early samples into two groups to compare fresh and cryopreserved tissue with respect to activation and inhibition of fibrinolysis. The cryopreserved tissue was thawed and analyzed in bimonthly batches. Storage time varied, but averaged 2 weeks. The cryopreservation techniques utilized in this project have been previously published and will be reviewed only briefly [2, 61. All tissue samples were cut into 2-mm slices. A solution of 15% dimethylsulfoxide and 0.5% methylprednisolone was used as a cryopreservative. Tissue samples were soaked in the cryopreservative solution for 10 min, then transferred to plastic storage bags (UCAR Blood Freezing Bags, Style 7450-2) with 8- 10ml of the cryopreservative solution. The specimen bags were then heatsealed (Vertrod Thermal Impulse Heat Sealer, Model 14A-CAB), rapidly frozen (5°C per second) in liquid nitrogen (- 196”(Z), and stored in liquid nitrogen vapor (- 170°C). Prior to analysis the specimen bags were rapidly thawed (5°C per second) in a 37°C water bath. Prior to tissue analysis the cryopreservative solution was discarded, and the tissue samples were rinsed twice in saline. Tumor activation of fibrinolysis was measured by a standard fibrin-plate technique [8, 11, 151.Human fibrinogen (Merck Sharp & Dohme) was dissolved in a sodium barbital buffer and precipitated with bovine thrombin in a loo-mm petri dish. Each fibrin plate contained 30 mg of fibrinogen, and the
plates were stable for 3 weeks at 4°C and for longer than 48 hr at 37°C. Tissue activation of fibrinolysis was measured by placing a 7-mm punch biopsy of tissue in the center of a plate, which was then incubated at 37°C for 24 hr. Tissue fibrinolytic activity was terminated by adding 8- 10 ml of 1% trichloroacetic acid to the fibrin plate after completion of incubation. The plate was allowed to stand with trichloroacetic acid for 5 min, and then the acid was decanted. The zones of fibrinolysis were measured with an electronic planimeter (Lasico, Model 1250E) and expressed in square millimeters. Examples of tissue activation of fibrinolysis are shown in Fig. 1. A new technique, not previously described in the literature, was developed in our laboratory for the measurement of tissue inhibition of fibrinolysis. Fibrin plates made from an unstable fibrinogen (Calbiothem) underwent spontaneous lysis over a plate fibrinogen concentration-dependent time course. Plate fibrinogen concentration was varied from 15 to 150 mg, and all fibrin plates demonstrated spontaneous lysis at both 4 and 37°C. Fifteen-milligram plates were chosen for the inhibitor assay because of their relatively short time of spontaneous lysis (6 hr), and because tumor inhibition of fibrinolysis appeared to be inversely related to the concentration of plate fibrinogen. Because these fibrin plates undergo spontaneous lysis over 5-7 days at 4”C, fresh fibrin plates were used for all inhibitor analyses. It would appear that this unstable fibrinogen may be contaminated with plasminogen activators, because the addition of .+aminocaproic acid to the fibrinogen solution prior to thrombin precipitation blocks spontaneous plate lysis in a dose-dependent fashion. The fact that drop placement of this fibrinogen solution on a plasminogen-free fibrin plate caused fibrinolysis in a dosedependent manner suggests that this fibrinogen is also contaminated with plasmin Tissue inhibition of fibrinolysis was measured by placing a 7-mm punch biopsy of tissue in the center of a plate and incubating the plate at 37°C until complete lysis
MALONE ET AL.: TUMOR INTERACTION
WITH THE FIBRINOLYTIC
SYSTEM
FIG. 1. Zones of fibrinolysis around both tissue specimens on a fibrin plate are easily noted.
demonstrate either activation or inhibition of fibrinolysis (P = 0.001). There was no statistically significant difference between fresh and cryopreserved tissue with respect to either activation or inhibition of fibrinolysis (P = 0.001). Normal tissue from most thoracic and abdominal organs was analyzed for both activation and inhibition of fibrinolysis. All normal tissue demonstrated activation of fibrinolysis (Table 1). The amount of activity varied from 25 (liver) to 979 mm2 (lung). Most normal tissue demonstrated moderate amounts of fibrinolytic activity (200-600 mm*), while lung, colon, skin, and stomach routinely demonstrated very high levels of fibrinolytic activator activity (greater than 600 mm*). No normal tissue other than liver demonstrated inhibition of fibrinolysis. Several types of benign lesions were analyzed for both activation and inhibition of fibrinolysis. All benign lesions demonRESULTS strated significant activation of fibrinolysis The cryopreservative solution, both be- but no inhibition of fibrinolysis (Table 2). fore and after cryopreservation, did not Compared to normal liver, cirrhotic liver
of control plates, without tissue specimens, was noted. The incubation time averaged 610 hr. The analysis was terminated on the test plates by the addition of 10 ml of 1% trichloroacetic acid. In the absence of tissue inhibition of fibrinolysis, the tissue specimens were found floating freely in the dissolved fibrin. The presence of tissue inhibitors of fibrinolysis resulted in a zone of unlysed fibrin around the tissue specimen. The areas of unlysed fibrin (inhibition of fibrinolysis) were measured with an electronic planimeter (Lasico, Model 1250E) and expressed in square millimeters. An example of tumor inhibition of fibrinolysis is shown in Fig. 2. Statistical analysis utilized the t test for nonpaired means, and all data were analyzed with a programmable electronic calculator (HP-97).
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JOURNAL
OF SURGICAL
FIG. 2. A zone of unlysed fibrin (inhibition
RESEARCH:
of fibrinolysis)
demonstrated increased activation and decreased inhibition of fibrinolytic activity (Table 3). Twenty-five malignant neoplasms were analyzed. All malignant neoplasms, except two superficial spreading melanomas, demonstrated inhibition of fibrinolysis (range 33-312 mm*) (Table 4). Except embryonal cell carcinoma, all malignant neoplasms demonstrated activation of fibrinolysis (range 12-1508 mm*) (Table 4). The grossly normal tissue around tumors did not consistently demonstrate inhibition of fibrinolysis; however, there usually was decreased fibrinolytic activator activity (range 20-40%) compared to normal tissue at the surgical margin of the same specimen (P
VOL. 26, NO. 5, MAY
1979
is readily apparent around the tumor biopsy.
= 0.05). Analysis of histologically normal liver in two patients with extrahepatic primary abdominal tumors demonstrated both increased hepatic inhibition and decreased hepatic activation of fibrinolysis. compared to normal liver tissue in patients without known malignancies (Table 5). There appeared to be a reasonably linear correlation between measured activation of fibrinolysis and tumor spread. With the exception of several tumor cell types (superficial spreading melanoma, pancreatic adenocarcinoma, histiocytic lymphoma, embryonal cell carcinoma, and nodular melanoma), most malignant lesions without metastatic spread had fibrinolytic activity levels less than 600 mm2, while most tumors
MALONE
ET AL.: TUMOR
INTERACTION
WITH THE FIBRINOLYTIC
TABLE FIBRINOLYTIC
Tissue type
ACTIVATOR
ACTIVITY
Number of samples
Adrenal Colon Inferior vena cava Liver Lung Parathyroid Spleen Skeletal muscle Skin Stomach
2
2 1 2 3 1 3 2 2 2
AND
1
Mean activation (mm’)
Mean inhibition (mm*)
481 736 160 25 979 470 220 370 820 737
0 0 0 64 0 0 0 0 0 0
ACTIVITY
Tissue type
Number of samples
Keloid Scar Splenic hemangioma Villous adenoma
2 2 1 3
Activation/inhibition (A/Z) ratio 0 0 0 0.39 0 0 0 0 0 0
earlier, there was a direct linear correlation between tumor activation/inhibition ratios and tumor metastatic spread. All nonmalignant lesions and normal tissue had activation/inhibition ratios of 0.3 Most nonmetastatic malignant neoplasms had A/Z ratios less than 7, while most tumors with metastatic spread had A/Z ratios greater than 7 (Table 4). Excluding adenocarcinoma of the pancreas, histiocytic lymphoma, embryonal cell carcinoma and both superficial spreading and nodular melanomas, when all other malignant neoplasms were subdivided into two groups based upon the presence or absence of metastatic spread, tumors without metastatic spread had an average A/Z ratio of 4.09 * 0.8 (mean 5 1 SD), and tumors with metastases had an 3 Tissues which did not exhibit inhibition of fibrinolysis were arbitrarily assigned an activation/inhibition ratio of 0, rather than the mathematical value of infinity.
TABLE ACTIVATOR
585
INHIBITION OF FIBRINOLYSIS:NORMAL TISSUE
with metastatic spread had fibrinolytic activity levels greater than 600 mm2 (Table 4). The correlation between the measured level of inhibition of fibrinolysis and the presence of tumor metastases was inverse and somewhat linear. In general, excluding the specific tumor cell types noted above, tumors with inhibition of fibrinolysis greater than 100 mm2 did not show histologic evidence of either nodal, regional, or distant organ metastatic spread, while most malignant neoplasms with inhibition of fibrinolysis less than 100 mm2 demonstrated metastatic spread (Table 4). Since tumor spread appeared to correlate directly with tumor activation of fibrinolysis and inversely with inhibition of fibrinolysis, activation/inhibition ratios (A/Z ratios) were calculated for each tumor analyzed. Excluding the specific tumor cell types noted
FIBRINOLYTIC
SYSTEM
2
AND INHIBITION
OF FIBRINOLYSIS:
Mean activation (mm’)
Mean inhibition (mm2)
245 369 1105 542
0 0 0 0
BENIGN
LESIONS
Activation/inhibition (A/I) ratio 0 0 0 0
586
JOURNAL OF SURGICAL RESEARCH: VOL. 26, NO. 5, MAY 1979 TABLE 3
FIBRINOLYTIC
ACTIVATOR
ACTIVITY
AND INHIBITION
OF FIBRINOLYSIS:
NORMAL
Tissue type
Number of samples
Mean activation (mm*)
Mean inhibition (mm7
Normal liver Cirrhotic liver
2 2
25 450
64 0
average A/I ratio of 18.36 +- 3.7 (mean k 1 SD). The differences in mean A/Z ratios of tumors with and without metastases are statistically significant (P = 0.001). DISCUSSION
Normal tissue demonstrated activation of fibrinolysis (range 25-979 mm2), but no
AND CIRRHOTIC
LIVER
Activation/inhibition (A/Z) ratio 0.39 0
evidence of inhibition of fibrinolysis was found. In addition, benign lesions demonstrated activation but not inhibition of fibrinolysis. Excluding normal liver, which demonstrated low levels of inhibition of fibrinolysis, the presence of tissue inhibition of fibrinolysis was diagnostic of malignancy. Metastatic tumor spread appeared to cor-
TABLE 4 FIBRINOLYTIC
ACTIVATOR
ACTIVITY
AND INHIBITION
OF FIBRINOLYSIS:
Mean activation (mm*)
Tumor type
Metastases
Superficial melanoma Superficial melanoma Embryonal cell carcinoma* Melanoma* Adenocarcinoma of pancreas* Adenocarcinoma of pancreas* Adenocarcinoma of pancreas* Adenocarcinoma of colon Adenocarcinoma of rectum Squamous cell carcinoma of trachea Histiocytic lymphoma* Adenocarcinoma of colon Adenocarcinoma of rectum Adenocarcinoma of cecum Adenocarcinoma of colon Adenocarcinoma of cecum Adenocarcinoma of breast Melanoma Gastric adenocarcinoma Adenocarcinoma of colon Osteosarcoma Adenocarcinoma of breast Adenocarcinoma of breast Adenosquamous carcinoma Adenocarcinoma of breast
No No Yes Yes Yes Yes Yes No No
350 112 0 23 12 40 121 346 394
No Yes No No No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes
147 120 345 658 364 214 342 755 812 829 640 691 858 767 1105
MALIGNANT
Mean inhibition (mm? 0 0
174 305 57 123 235 312 277 74 57 73 121 64 35 55 94 75 60 38 40 47 37 52 33
LESIONS
Activation/inhibition (A/Z) ratio 0 0 0
0.08 0.21 0.33 0.51 1.11 1.42 1.99 2.10 4.74 5.44 5.69 6.11 6.22 8.03 10.83 13.82 16.86 17.28 18.26 20.72 21.25 41.26
a Except those followed by an asterisk, lesions with All less than 7 do not have metastasis, and all lesions with A/Z greater than 7 do have metastasis.
MALONE
ET AL.: TUMOR
INTERACTION
WITH THE FIBRINOLYTIC
TABLE FIBRINOLYTK
Tissue Normal liver Normal liver extrahepatic
type
with tumor
OF FIBRINOLYSIS: TUMOR
Mean activation
Mean inhibition
(mm21
(mm2)
2
25
64
2
0
167
relate directly with activation of fibrinolysis and inversely with inhibition of fibrinolysis. Nonmalignant lesions and normal tissue, except liver, had activation/inhibition ratios of 0,3 while most tumors without metastases had activation/inhibition ratios less than 7, and most tumors with metastases had activation/inhibition ratios greater than 7. Excluding several specific cell types (adenocarcinoma of the pancreas, histiocytic lymphoma, embryonal cell carcinoma, and both superficial spreading and nodular melanomas), tumors without metastases had an an average A/Z ratio of 4.09 + 0.8 (mean t 1 SD), and tumors with metastases had an average A/Z ratio of 18.36 ? 3.7 (mean t 1 SD). The difference in the mean A/Z ratios for tumors with and without metastases is statistically highly significant (P = 0.001). Therefore, tumor activation/inhibition ratios would appear to have validity as biologic tumor markers with respect to the presence or absence of metastatic disease. Histologically normal liver in patients with extrahepatic primary abdominal tumors demonstrated increased liver inhibition of fibrinolysis and decreased liver activation of fibrinolysis compared to normal liver in patients without known malignancy (Table 5). The significance of these hepatic changes is unknown. As previously noted, the metastatic spread of adenocarcinoma of the pancreas, histiocytic lymphoma, embryonal cell carcinoma, and both superficial spreading and nodular melanomas did not correlate well with their respective activation/inhibition ratios. How-
587
5
ACTIVATOR ACTIVITY AND INHIBITION WITH AND WITHOUT EXTRAHEPATIC Number of samples
SYSTEM
LIVER,
Activation/inhibition (A/Z) ratio
0.39 0
ever, all tumors, excluding the superficial spreading melanomas, demonstrated inhibition of fibrinolysis, a finding not observed with any normal tissue or benign lesion except normal liver. The lack of tumor inhibition of fibrinolysis by the superficial spreading melanomas may actually correspond with the A/Z ratio format since, although histologically malignant, these lesions appeared clinically benign (no evidence of metastatic spread during 10 years of follow-up), and therefore probably should not demonstrate inhibition of fibrinolysis. Finally, most malignant neoplasms studied in this report were adenocarcinomas of either the breast or the gastrointestinal tract, and due to small sample size we cannot comment on the differences, if any, between adenocarcinomas, sarcomas, melanomas, lymphomas, and primitive cell carcinomas with respect to their intrinsic fibrinolytic activator activity or fibrinolytic inhibitor activity. Our data are consistent with the literature. Franklin et al. noted both tumor-related activation and inhibition of fibrinolysis [8]. However, two important distinctions between our data and those of Franklin et al. are that all tumors demonstrated inhibition of fibrinolysis with our assay, and that individual tumor inhibition of fibrinolysis could be directly quantitated with our assay. Several researchers have suggested that tumor fibrinolytic activator activity is related to tumor metastases [4, 5, 8, 9, 12, 21, 221. The proposed mechanism involves increased primary tumor cell shedding with
588
JOURNAL
OF SURGICAL
RESEARCH:
increasing levels of tumor fibrinolytic activator activity. Peterson et al. found that intravascular tumor invasion in patients with bronchogenic carcinoma correlated directly with tumor fibrinolytic activity [25]. Similar findings were noted by Newstead et al., who reported that the hematogenous spread of tumor cells in patients with colorectal carcinoma increased with increasing levels of tumor fibrinolytic activator activity [20]. Most clinical and experimental data suggest, however, that tumor inhibition of fibrinolysis, and not tumor activation of fibrinolysis, is best correlated with the occurrence of tumor metastases. In fact, both fibrinolytic agents and anticoagulation drugs have been demonstrated to decrease the incidence of tumor metastases in multiple experimental and clinical studies [3, 7, 12, 13, 16, 18, 23-25, 28, 291. Most recently, Thornes reported a beneficial effect on patient survival in a clinically controlled setting which utilized warfarin as an adjunctive chemotherapeutic agent in patients with lymphosarcoma, adenocarcinema of the breast, and adenocarcinoma of the ovaries [29]. In addition, Thornes reported that streptokinase treatment, as an adjunct to surgical resection for advanced carcinoma of the colon, appeared to have a beneficial effect compared to surgery alone [29]. Similarly, Hoover and Ketcham have found decreased metastases in patients with osteogenic sarcoma when anticoagulant drugs were begun preoperatively [13]. Tumor cell inhibition of fibrinolysis, at least at the cell surface, appears to be directly related to capillary endothelial adherence, tumor cell invasion of perivascular spaces, tumor cell defense against host immune mechanisms, and the successful establishment of both nodal and organ metastases [13, 14, 18, 28, 29, 31, 321. Theoretically, drugs or agents which either increase fibrinolytic activity or alter the coagulation system might be expected to increase primary tumor cell shedding; however, almost all the available data suggest that fibrinolytic, anticoagulant, or antiplate-
VOL. 26, NO. 5, MAY
1979
let agents, in fact, decrease tumor cell survival and decrease the incidence of successful tumor cell implantation [l , 3, 7, 9, 10, 12, 13, 18-20, 24-291. Although there are some inconsistencies in the literature, an overview of available clinical and experimental data suggests the following mechanism of tumor interaction with the fibrinolytic system: (1) Tumor cell activation of fibrinolysis is probably important in respect to a decrease or loss of intracellular adhesiveness and subsequent primary tumor cell shedding [4, 5, 8, 9, 17, 20-22, 251; and (2) individual tumor cell inhibition of fibrinolysis and the subsequent formation of a fibrin-platelet matrix around individual tumor cells would appear to be of overwhelming importance in the successful establishment of tumor metastatic foci after tumor cell shedding [3, 7, 12, 13, 16, 18, 23-26, 28, 291. The fibrin-plate assay utilized for tumor fibrinolytic activator activity in this report has been used extensively for measurement of tissue fibrinolytic activator activity by several groups of investigators, and therefore the assay has undergone reasonable scientific scrutiny with respect to both technique and reliability of results [4, 8, 11, 151. However, the same cannot be said of our assay for inhibition of fibrinolysis. The inhibitor assay was developed in our laboratory specifically for this tumor project, and the technique and results have not been substantiated by other investigators. In addition, since the inhibitor assay utilizes an unstable fibrinogen which has a more rapid, spontaneous fibrinolytic activity than that of the tissue being analyzed, the assay measures relative inhibition of fibrinolysis, not absolute inhibitory activity. It is therefore impossible to know if we are measuring antiplasms, antiplasminogens, or a combination of fibrinolytic inhibitors. Nevertheless, the assay can be quantitated; inhibitory activity was noted only with malignant lesions, and the data are statistically significant based upon even a small sample size. Obviously, the assay needs
MALONE ET AL.: TUMOR INTERACTION
WITH THE FIBRINOLYTIC
SYSTEM
589
refinement, but the preliminary data suggest IS. Malone, J. M., Gervin, A. S., Keown, K., et al. Venous distention and fibrinolytic activity. C/in. that further detailed investigation of tumor Res. 26: 250A. 1978. interaction with the fibrinolytic system 16 Moroz, L. A. Increased blood fibrinolytic activity is warranted. after aspirin ingestion. N. Engl. J. Med. 2%: 525, 1977. 17. Merskey, C. Pathogenesis and treatment of altered blood coagulability in patients with malignant I. Agostino, D., and ClitBon, E. E. Anticoagulants and tumors. Ann. NY Acud. Sci. 230: 289, 1974. the development of pulmonary metastases. Anticoagulant effect on Walker 256 carcinosarcoma in 18. Mootse, G., Agostino, D., and Cliffton, E. E. Alterations in fibrinogen, plasminogen, and inhibitors rats. Arch. Surg. 84: 449, 1962. of plasmin in the growth of V2 carcinoma in the 2. Boren, C. B., Roon, A. .I., and Moore, W. S. Mainrabbit. .I. Nutl. Cancer Inst. 35: 567, 1965. tenance of viable arterial allografts by cryopreser19. Nagy, B., Ban J., and Brollar, B. Fibrinolysis vation. SurgeT 83: 382, 1978. associated with human neoplasia. Production 3. Cliffton, E. E., and Grossi, C. E. Effect of human ofplasminogen activation by human tumors. Int. J. plasmin on the toxic effects and growth of bloodCancer 19: 614, 1977. borne metastases of the Brown-Pearce carcinoma and the V2 carcinoma of the rabbit. Cancer 9: 20. Newstead, G. L., Griffiths, J. D., and Salsbury A. J. Fibrinolytic activity of carcinoma of the 1147, 1956. colorectum. Surg. Gynecol. Obstet. 143: 61, 1976. 4. Cliffton, E. E., and Grossi, C. E. Fibrinolytic activity of human tumors as measured by the fibrin 21. O’Meara, R. A. Q. Coagulation properties of cancer. Ir. J. Med. Sci. 396: 474, 1958. plate method. Cancer 8: 1146, 1955. 5. Coman, D. R. Adhesiveness and stickiness: Two 22. O’Meara, R. A. Q., and Jackson, R. D. Cytological observations on carcinoma. Ir. J. Med. Sci. 391: independent properties of the cell surface. Cancer 327, 1958. Res. 21: 1436, 1961. 6. Dent, T. L., Weber, T. R., Lindenauer, S. M., et 23. Pechet, L. Fibrinolysis. N. Engl. J. Med. 273: 966, 1965. al. Cryopreservation of vein grafts. Surg. Forum 25: 241, 1974. 24. Peterson, H. I., Kjantansson, I., Korsan-Bengtsen, 7. Elias, E. G., Sepulvada, F., and Mink, B. InK., et al. Fibrinolysis in human malignant tumors. creasing the efficiency of cancer chemotherapy Acta Chir. Stand. 139: 219, 1973. with heparin. “Clinical Study.” J. Surg. Oncol. 5: 25. Peterson, H. I., Larsson, S., and Zettergren, L. 189, 1973. Fibrinolysis in human bronchogenic carcinoma. 8. Franklin, J. D., Gervin, A. S., Bowers, D. B., et al. Bib/. Anat. 13: 315, 1975. Fibrinolytic activator activity in human neo- 26. Ryan, J. J., Ketcham, A. S., and Wexler, H. plasms. Plust. Reconstr. Surg. 61: 241, 1978. Reduced incidence of spontaneous metastases 9. Gasic, G. J., Gasic, T. B., Talanti, N., er al. with long term coumadin therapy. Ann. Surg. 168: Platelet tumor cell interactions in mice. The role of 163, 1968. platelets in the spread of malignant disease. Inr. J. 27. Soong, B. C. F., and Miller, S. P. Coagulation Cancer 11: 704, 1973. disorders in cancer. III. Fibrinolysis and inhibitors. IO. Gasic, G. J., Gasic, T. B., and Stewart, C. C. Cancer 25: 864, 1970. Antimetastatic effects associated with platelet 28. Sugarbaker, E. V., and Ketcham, A. S. Mechareduction. Proc. Nutl. Acad. Sci. USA 61: 46, nisms and prevention of cancer dissemination. An 1968. overview. Semin. Oncol. 4(l): 19, 1977. 11. Gervin, A. S., and Butler, B. M. Fibrinolytic activity in autologous jugular vein. Loss and 29. Thomes, R. D. Adjunct therapy of cancer via the cellular immune mechanisms or by induced tibrinolrestoration. Amer. Swg. 41: 726. 1975. ysis and oral anticoagulants. Cancer 35: 91, 1975. 12. Grossi, C. E., Agostino, D., and Cliffton, E. E. 30. Walter, J. B., and Israel, M. S. Genera/Pathology. The effect of human fibrinolysin on pulmonary Baltimore: Williams&Wilkins, 1970.Pp. 607-609. metastases of Walker 256 carcinosarcoma. Cancer 31. Wood, S. Jr. Experimental studies on the intraRes. 20: 605, 1960. vascular dissemination of ascites V2 cancer cells 13. Hoover, H. C., Jr., and Ketcham, A. S. Techniques in the rabbit with special reference to fibrinogen for inhibiting tumor metastases. Cancer 35: 5, 1975. and fibrinolytic agents. Bull. Swiss Acud. Med. Sci. 14. Lai, K., Tyler, H. M., and Yancey, S. T. Clot 20: 92, 1964. forming and stabilizing enzymes from mouse tumor YPC-1. Biochem. Biophys. Res. Commun. 24: 776, 32. Young, S. W. Determinants of malignant tumor 1966. growth. Resident StuffPhys. 24(4): 89, 1978.
REFERENCES
OF SURGICAL
RESEARCH
26,
581-589 (1979)
Tumor Interaction J. M. MALONE, The Department
with the Fibrinolytic
System’
M.D.,2 A. S. GERVIN, M.D., W. S. MOORE, M.D., F.A.C.S., AND K. KEOWN, M.A.
of Surgery
of the University
of Arizona
College of Medicine,
Tucson, Arizona
85724
Presented at the Annual Meeting of the Association for Academic Surgery, Cleveland, Ohio, November 12- 15, 1978 Tumor activation and inhibition of fibrinolysis appear to be related to the pathophysiology of metastatic tumor spread. Our data demonstrate a correlation between the incidence of tumor metastases and both increasing tumor fibrinolytic activator activity and decreasing tumor fibrinolytic inhibitory activity. Nonmalignant lesions and normal tissue, other than liver, had activation/ inhibition ratios of zero. Most malignant neoplasms without metastatic spread had activation/ inhibition ratios less than 7, while most tumors with metastatic spread had activation/inhibition ratios greater than 7. Calculation of mean activation/inhibition ratios for tumors with and without metastatic spread demonstrated a statistically significant difference between their activation/ inhibition ratios (P = 0.001). Therefore, tumor activation/inhibition ratios would appear to have statistical reliability as biologic markers for the presence or absence of metastatic tumor spread. These data have significant clinical and therapeutic implications with respect to the utilization of tumor activation/inhibition ratios as biologic markers of tumor spread, and they also warrant further investigation into the mechanisms of tumor interaction with the fibrinolytic system.
INTRODUCTION
Neither histologic nor clinical staging always correlates with either patient survival or the time course of tumor metastatic spread. Anticancer treatment is based upon norms for specific tumor cell types, and treatment often cannot be individualized. In order to accurately assess tumor growth and/or tumor recurrence a biological tumor marker is needed. To a limited extent, and for specific tumors, the carcinoma embryonic antigen and the a-fetoprotein have provided assessment of the adequacy of tumor resection and tumor recurrence. However, there is no general biological tumor marker which is able to distinguish those patients with microscopic residual cancer who will develop tumor recurrence, and therefore require adjuvant anticancer treatment, from those patients cured as a result of their 1 Supported in part by V.A. Career Development Grant 678-5447-01 and V.A. Grant 622-5798-03. * To whom requests for reprints should be addressed: Department of Surgery, Arizona Health Sciences Center, Tucson, Arizona 85724.
primary treatment and who, therefore, do not require additional anticancer therapy. Factors important to tumor growth include host immunological response, host hormonal environment, the intrinsic biologic potential of the tumor, dormancy, spontaneous regression, and selective organ enhancement [31, 321. The potential of tumors to metastasize has also been correlated with both activation and inhibition ofthefibrinolyticsystem [1,4,10,13,14,1720, 24, 26, 27, 29, 311. The purpose of this report is to evaluate tumor interaction with the fibrinolytic system and to investigate the potential of tumor activation and inhibition of fibrinolysis as biologic markers for the presence or absence of metastatic disease. MATERIALS
AND METHODS
Biopsies of both normal tissue and tumor were obtained from fresh surgical specimens of patients undergoing curative or palliative tumor resection at the Tucson V.A. Hospital from January 1,1978, through April 15, 1978. Biopsies were often limited
581
0022-4804/79/05058l-09$1.00/0 Copyright 0 1979 by Academic Press. Inc. All rights of reproductmn in any form reserved.
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in size so as not to interfere with routine histological and clinical staging. When possible, biopsy specimens were taken from gross tumor, grossly normal tissue around tumor, and normal tissue at the surgical margins of resection. All tissue samples were transported to our research laboratory in normal saline, and warm ischemia time was limited to 1 hr. Initially all tissue samples were analyzed immediately upon arrival in the laboratory. However, since the analysis requires 6-10 hr, and since many specimens were not available until late afternoon, we divided our early samples into two groups to compare fresh and cryopreserved tissue with respect to activation and inhibition of fibrinolysis. The cryopreserved tissue was thawed and analyzed in bimonthly batches. Storage time varied, but averaged 2 weeks. The cryopreservation techniques utilized in this project have been previously published and will be reviewed only briefly [2, 61. All tissue samples were cut into 2-mm slices. A solution of 15% dimethylsulfoxide and 0.5% methylprednisolone was used as a cryopreservative. Tissue samples were soaked in the cryopreservative solution for 10 min, then transferred to plastic storage bags (UCAR Blood Freezing Bags, Style 7450-2) with 8- 10ml of the cryopreservative solution. The specimen bags were then heatsealed (Vertrod Thermal Impulse Heat Sealer, Model 14A-CAB), rapidly frozen (5°C per second) in liquid nitrogen (- 196”(Z), and stored in liquid nitrogen vapor (- 170°C). Prior to analysis the specimen bags were rapidly thawed (5°C per second) in a 37°C water bath. Prior to tissue analysis the cryopreservative solution was discarded, and the tissue samples were rinsed twice in saline. Tumor activation of fibrinolysis was measured by a standard fibrin-plate technique [8, 11, 151.Human fibrinogen (Merck Sharp & Dohme) was dissolved in a sodium barbital buffer and precipitated with bovine thrombin in a loo-mm petri dish. Each fibrin plate contained 30 mg of fibrinogen, and the
plates were stable for 3 weeks at 4°C and for longer than 48 hr at 37°C. Tissue activation of fibrinolysis was measured by placing a 7-mm punch biopsy of tissue in the center of a plate, which was then incubated at 37°C for 24 hr. Tissue fibrinolytic activity was terminated by adding 8- 10 ml of 1% trichloroacetic acid to the fibrin plate after completion of incubation. The plate was allowed to stand with trichloroacetic acid for 5 min, and then the acid was decanted. The zones of fibrinolysis were measured with an electronic planimeter (Lasico, Model 1250E) and expressed in square millimeters. Examples of tissue activation of fibrinolysis are shown in Fig. 1. A new technique, not previously described in the literature, was developed in our laboratory for the measurement of tissue inhibition of fibrinolysis. Fibrin plates made from an unstable fibrinogen (Calbiothem) underwent spontaneous lysis over a plate fibrinogen concentration-dependent time course. Plate fibrinogen concentration was varied from 15 to 150 mg, and all fibrin plates demonstrated spontaneous lysis at both 4 and 37°C. Fifteen-milligram plates were chosen for the inhibitor assay because of their relatively short time of spontaneous lysis (6 hr), and because tumor inhibition of fibrinolysis appeared to be inversely related to the concentration of plate fibrinogen. Because these fibrin plates undergo spontaneous lysis over 5-7 days at 4”C, fresh fibrin plates were used for all inhibitor analyses. It would appear that this unstable fibrinogen may be contaminated with plasminogen activators, because the addition of .+aminocaproic acid to the fibrinogen solution prior to thrombin precipitation blocks spontaneous plate lysis in a dose-dependent fashion. The fact that drop placement of this fibrinogen solution on a plasminogen-free fibrin plate caused fibrinolysis in a dosedependent manner suggests that this fibrinogen is also contaminated with plasmin Tissue inhibition of fibrinolysis was measured by placing a 7-mm punch biopsy of tissue in the center of a plate and incubating the plate at 37°C until complete lysis
MALONE ET AL.: TUMOR INTERACTION
WITH THE FIBRINOLYTIC
SYSTEM
FIG. 1. Zones of fibrinolysis around both tissue specimens on a fibrin plate are easily noted.
demonstrate either activation or inhibition of fibrinolysis (P = 0.001). There was no statistically significant difference between fresh and cryopreserved tissue with respect to either activation or inhibition of fibrinolysis (P = 0.001). Normal tissue from most thoracic and abdominal organs was analyzed for both activation and inhibition of fibrinolysis. All normal tissue demonstrated activation of fibrinolysis (Table 1). The amount of activity varied from 25 (liver) to 979 mm2 (lung). Most normal tissue demonstrated moderate amounts of fibrinolytic activity (200-600 mm*), while lung, colon, skin, and stomach routinely demonstrated very high levels of fibrinolytic activator activity (greater than 600 mm*). No normal tissue other than liver demonstrated inhibition of fibrinolysis. Several types of benign lesions were analyzed for both activation and inhibition of fibrinolysis. All benign lesions demonRESULTS strated significant activation of fibrinolysis The cryopreservative solution, both be- but no inhibition of fibrinolysis (Table 2). fore and after cryopreservation, did not Compared to normal liver, cirrhotic liver
of control plates, without tissue specimens, was noted. The incubation time averaged 610 hr. The analysis was terminated on the test plates by the addition of 10 ml of 1% trichloroacetic acid. In the absence of tissue inhibition of fibrinolysis, the tissue specimens were found floating freely in the dissolved fibrin. The presence of tissue inhibitors of fibrinolysis resulted in a zone of unlysed fibrin around the tissue specimen. The areas of unlysed fibrin (inhibition of fibrinolysis) were measured with an electronic planimeter (Lasico, Model 1250E) and expressed in square millimeters. An example of tumor inhibition of fibrinolysis is shown in Fig. 2. Statistical analysis utilized the t test for nonpaired means, and all data were analyzed with a programmable electronic calculator (HP-97).
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FIG. 2. A zone of unlysed fibrin (inhibition
RESEARCH:
of fibrinolysis)
demonstrated increased activation and decreased inhibition of fibrinolytic activity (Table 3). Twenty-five malignant neoplasms were analyzed. All malignant neoplasms, except two superficial spreading melanomas, demonstrated inhibition of fibrinolysis (range 33-312 mm*) (Table 4). Except embryonal cell carcinoma, all malignant neoplasms demonstrated activation of fibrinolysis (range 12-1508 mm*) (Table 4). The grossly normal tissue around tumors did not consistently demonstrate inhibition of fibrinolysis; however, there usually was decreased fibrinolytic activator activity (range 20-40%) compared to normal tissue at the surgical margin of the same specimen (P
VOL. 26, NO. 5, MAY
1979
is readily apparent around the tumor biopsy.
= 0.05). Analysis of histologically normal liver in two patients with extrahepatic primary abdominal tumors demonstrated both increased hepatic inhibition and decreased hepatic activation of fibrinolysis. compared to normal liver tissue in patients without known malignancies (Table 5). There appeared to be a reasonably linear correlation between measured activation of fibrinolysis and tumor spread. With the exception of several tumor cell types (superficial spreading melanoma, pancreatic adenocarcinoma, histiocytic lymphoma, embryonal cell carcinoma, and nodular melanoma), most malignant lesions without metastatic spread had fibrinolytic activity levels less than 600 mm2, while most tumors
MALONE
ET AL.: TUMOR
INTERACTION
WITH THE FIBRINOLYTIC
TABLE FIBRINOLYTIC
Tissue type
ACTIVATOR
ACTIVITY
Number of samples
Adrenal Colon Inferior vena cava Liver Lung Parathyroid Spleen Skeletal muscle Skin Stomach
2
2 1 2 3 1 3 2 2 2
AND
1
Mean activation (mm’)
Mean inhibition (mm*)
481 736 160 25 979 470 220 370 820 737
0 0 0 64 0 0 0 0 0 0
ACTIVITY
Tissue type
Number of samples
Keloid Scar Splenic hemangioma Villous adenoma
2 2 1 3
Activation/inhibition (A/Z) ratio 0 0 0 0.39 0 0 0 0 0 0
earlier, there was a direct linear correlation between tumor activation/inhibition ratios and tumor metastatic spread. All nonmalignant lesions and normal tissue had activation/inhibition ratios of 0.3 Most nonmetastatic malignant neoplasms had A/Z ratios less than 7, while most tumors with metastatic spread had A/Z ratios greater than 7 (Table 4). Excluding adenocarcinoma of the pancreas, histiocytic lymphoma, embryonal cell carcinoma and both superficial spreading and nodular melanomas, when all other malignant neoplasms were subdivided into two groups based upon the presence or absence of metastatic spread, tumors without metastatic spread had an average A/Z ratio of 4.09 * 0.8 (mean 5 1 SD), and tumors with metastases had an 3 Tissues which did not exhibit inhibition of fibrinolysis were arbitrarily assigned an activation/inhibition ratio of 0, rather than the mathematical value of infinity.
TABLE ACTIVATOR
585
INHIBITION OF FIBRINOLYSIS:NORMAL TISSUE
with metastatic spread had fibrinolytic activity levels greater than 600 mm2 (Table 4). The correlation between the measured level of inhibition of fibrinolysis and the presence of tumor metastases was inverse and somewhat linear. In general, excluding the specific tumor cell types noted above, tumors with inhibition of fibrinolysis greater than 100 mm2 did not show histologic evidence of either nodal, regional, or distant organ metastatic spread, while most malignant neoplasms with inhibition of fibrinolysis less than 100 mm2 demonstrated metastatic spread (Table 4). Since tumor spread appeared to correlate directly with tumor activation of fibrinolysis and inversely with inhibition of fibrinolysis, activation/inhibition ratios (A/Z ratios) were calculated for each tumor analyzed. Excluding the specific tumor cell types noted
FIBRINOLYTIC
SYSTEM
2
AND INHIBITION
OF FIBRINOLYSIS:
Mean activation (mm’)
Mean inhibition (mm2)
245 369 1105 542
0 0 0 0
BENIGN
LESIONS
Activation/inhibition (A/I) ratio 0 0 0 0
586
JOURNAL OF SURGICAL RESEARCH: VOL. 26, NO. 5, MAY 1979 TABLE 3
FIBRINOLYTIC
ACTIVATOR
ACTIVITY
AND INHIBITION
OF FIBRINOLYSIS:
NORMAL
Tissue type
Number of samples
Mean activation (mm*)
Mean inhibition (mm7
Normal liver Cirrhotic liver
2 2
25 450
64 0
average A/I ratio of 18.36 +- 3.7 (mean k 1 SD). The differences in mean A/Z ratios of tumors with and without metastases are statistically significant (P = 0.001). DISCUSSION
Normal tissue demonstrated activation of fibrinolysis (range 25-979 mm2), but no
AND CIRRHOTIC
LIVER
Activation/inhibition (A/Z) ratio 0.39 0
evidence of inhibition of fibrinolysis was found. In addition, benign lesions demonstrated activation but not inhibition of fibrinolysis. Excluding normal liver, which demonstrated low levels of inhibition of fibrinolysis, the presence of tissue inhibition of fibrinolysis was diagnostic of malignancy. Metastatic tumor spread appeared to cor-
TABLE 4 FIBRINOLYTIC
ACTIVATOR
ACTIVITY
AND INHIBITION
OF FIBRINOLYSIS:
Mean activation (mm*)
Tumor type
Metastases
Superficial melanoma Superficial melanoma Embryonal cell carcinoma* Melanoma* Adenocarcinoma of pancreas* Adenocarcinoma of pancreas* Adenocarcinoma of pancreas* Adenocarcinoma of colon Adenocarcinoma of rectum Squamous cell carcinoma of trachea Histiocytic lymphoma* Adenocarcinoma of colon Adenocarcinoma of rectum Adenocarcinoma of cecum Adenocarcinoma of colon Adenocarcinoma of cecum Adenocarcinoma of breast Melanoma Gastric adenocarcinoma Adenocarcinoma of colon Osteosarcoma Adenocarcinoma of breast Adenocarcinoma of breast Adenosquamous carcinoma Adenocarcinoma of breast
No No Yes Yes Yes Yes Yes No No
350 112 0 23 12 40 121 346 394
No Yes No No No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes
147 120 345 658 364 214 342 755 812 829 640 691 858 767 1105
MALIGNANT
Mean inhibition (mm? 0 0
174 305 57 123 235 312 277 74 57 73 121 64 35 55 94 75 60 38 40 47 37 52 33
LESIONS
Activation/inhibition (A/Z) ratio 0 0 0
0.08 0.21 0.33 0.51 1.11 1.42 1.99 2.10 4.74 5.44 5.69 6.11 6.22 8.03 10.83 13.82 16.86 17.28 18.26 20.72 21.25 41.26
a Except those followed by an asterisk, lesions with All less than 7 do not have metastasis, and all lesions with A/Z greater than 7 do have metastasis.
MALONE
ET AL.: TUMOR
INTERACTION
WITH THE FIBRINOLYTIC
TABLE FIBRINOLYTK
Tissue Normal liver Normal liver extrahepatic
type
with tumor
OF FIBRINOLYSIS: TUMOR
Mean activation
Mean inhibition
(mm21
(mm2)
2
25
64
2
0
167
relate directly with activation of fibrinolysis and inversely with inhibition of fibrinolysis. Nonmalignant lesions and normal tissue, except liver, had activation/inhibition ratios of 0,3 while most tumors without metastases had activation/inhibition ratios less than 7, and most tumors with metastases had activation/inhibition ratios greater than 7. Excluding several specific cell types (adenocarcinoma of the pancreas, histiocytic lymphoma, embryonal cell carcinoma, and both superficial spreading and nodular melanomas), tumors without metastases had an an average A/Z ratio of 4.09 + 0.8 (mean t 1 SD), and tumors with metastases had an average A/Z ratio of 18.36 ? 3.7 (mean t 1 SD). The difference in the mean A/Z ratios for tumors with and without metastases is statistically highly significant (P = 0.001). Therefore, tumor activation/inhibition ratios would appear to have validity as biologic tumor markers with respect to the presence or absence of metastatic disease. Histologically normal liver in patients with extrahepatic primary abdominal tumors demonstrated increased liver inhibition of fibrinolysis and decreased liver activation of fibrinolysis compared to normal liver in patients without known malignancy (Table 5). The significance of these hepatic changes is unknown. As previously noted, the metastatic spread of adenocarcinoma of the pancreas, histiocytic lymphoma, embryonal cell carcinoma, and both superficial spreading and nodular melanomas did not correlate well with their respective activation/inhibition ratios. How-
587
5
ACTIVATOR ACTIVITY AND INHIBITION WITH AND WITHOUT EXTRAHEPATIC Number of samples
SYSTEM
LIVER,
Activation/inhibition (A/Z) ratio
0.39 0
ever, all tumors, excluding the superficial spreading melanomas, demonstrated inhibition of fibrinolysis, a finding not observed with any normal tissue or benign lesion except normal liver. The lack of tumor inhibition of fibrinolysis by the superficial spreading melanomas may actually correspond with the A/Z ratio format since, although histologically malignant, these lesions appeared clinically benign (no evidence of metastatic spread during 10 years of follow-up), and therefore probably should not demonstrate inhibition of fibrinolysis. Finally, most malignant neoplasms studied in this report were adenocarcinomas of either the breast or the gastrointestinal tract, and due to small sample size we cannot comment on the differences, if any, between adenocarcinomas, sarcomas, melanomas, lymphomas, and primitive cell carcinomas with respect to their intrinsic fibrinolytic activator activity or fibrinolytic inhibitor activity. Our data are consistent with the literature. Franklin et al. noted both tumor-related activation and inhibition of fibrinolysis [8]. However, two important distinctions between our data and those of Franklin et al. are that all tumors demonstrated inhibition of fibrinolysis with our assay, and that individual tumor inhibition of fibrinolysis could be directly quantitated with our assay. Several researchers have suggested that tumor fibrinolytic activator activity is related to tumor metastases [4, 5, 8, 9, 12, 21, 221. The proposed mechanism involves increased primary tumor cell shedding with
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JOURNAL
OF SURGICAL
RESEARCH:
increasing levels of tumor fibrinolytic activator activity. Peterson et al. found that intravascular tumor invasion in patients with bronchogenic carcinoma correlated directly with tumor fibrinolytic activity [25]. Similar findings were noted by Newstead et al., who reported that the hematogenous spread of tumor cells in patients with colorectal carcinoma increased with increasing levels of tumor fibrinolytic activator activity [20]. Most clinical and experimental data suggest, however, that tumor inhibition of fibrinolysis, and not tumor activation of fibrinolysis, is best correlated with the occurrence of tumor metastases. In fact, both fibrinolytic agents and anticoagulation drugs have been demonstrated to decrease the incidence of tumor metastases in multiple experimental and clinical studies [3, 7, 12, 13, 16, 18, 23-25, 28, 291. Most recently, Thornes reported a beneficial effect on patient survival in a clinically controlled setting which utilized warfarin as an adjunctive chemotherapeutic agent in patients with lymphosarcoma, adenocarcinema of the breast, and adenocarcinoma of the ovaries [29]. In addition, Thornes reported that streptokinase treatment, as an adjunct to surgical resection for advanced carcinoma of the colon, appeared to have a beneficial effect compared to surgery alone [29]. Similarly, Hoover and Ketcham have found decreased metastases in patients with osteogenic sarcoma when anticoagulant drugs were begun preoperatively [13]. Tumor cell inhibition of fibrinolysis, at least at the cell surface, appears to be directly related to capillary endothelial adherence, tumor cell invasion of perivascular spaces, tumor cell defense against host immune mechanisms, and the successful establishment of both nodal and organ metastases [13, 14, 18, 28, 29, 31, 321. Theoretically, drugs or agents which either increase fibrinolytic activity or alter the coagulation system might be expected to increase primary tumor cell shedding; however, almost all the available data suggest that fibrinolytic, anticoagulant, or antiplate-
VOL. 26, NO. 5, MAY
1979
let agents, in fact, decrease tumor cell survival and decrease the incidence of successful tumor cell implantation [l , 3, 7, 9, 10, 12, 13, 18-20, 24-291. Although there are some inconsistencies in the literature, an overview of available clinical and experimental data suggests the following mechanism of tumor interaction with the fibrinolytic system: (1) Tumor cell activation of fibrinolysis is probably important in respect to a decrease or loss of intracellular adhesiveness and subsequent primary tumor cell shedding [4, 5, 8, 9, 17, 20-22, 251; and (2) individual tumor cell inhibition of fibrinolysis and the subsequent formation of a fibrin-platelet matrix around individual tumor cells would appear to be of overwhelming importance in the successful establishment of tumor metastatic foci after tumor cell shedding [3, 7, 12, 13, 16, 18, 23-26, 28, 291. The fibrin-plate assay utilized for tumor fibrinolytic activator activity in this report has been used extensively for measurement of tissue fibrinolytic activator activity by several groups of investigators, and therefore the assay has undergone reasonable scientific scrutiny with respect to both technique and reliability of results [4, 8, 11, 151. However, the same cannot be said of our assay for inhibition of fibrinolysis. The inhibitor assay was developed in our laboratory specifically for this tumor project, and the technique and results have not been substantiated by other investigators. In addition, since the inhibitor assay utilizes an unstable fibrinogen which has a more rapid, spontaneous fibrinolytic activity than that of the tissue being analyzed, the assay measures relative inhibition of fibrinolysis, not absolute inhibitory activity. It is therefore impossible to know if we are measuring antiplasms, antiplasminogens, or a combination of fibrinolytic inhibitors. Nevertheless, the assay can be quantitated; inhibitory activity was noted only with malignant lesions, and the data are statistically significant based upon even a small sample size. Obviously, the assay needs
MALONE ET AL.: TUMOR INTERACTION
WITH THE FIBRINOLYTIC
SYSTEM
589
refinement, but the preliminary data suggest IS. Malone, J. M., Gervin, A. S., Keown, K., et al. Venous distention and fibrinolytic activity. C/in. that further detailed investigation of tumor Res. 26: 250A. 1978. interaction with the fibrinolytic system 16 Moroz, L. A. Increased blood fibrinolytic activity is warranted. after aspirin ingestion. N. Engl. J. Med. 2%: 525, 1977. 17. Merskey, C. Pathogenesis and treatment of altered blood coagulability in patients with malignant I. Agostino, D., and ClitBon, E. E. Anticoagulants and tumors. Ann. NY Acud. Sci. 230: 289, 1974. the development of pulmonary metastases. Anticoagulant effect on Walker 256 carcinosarcoma in 18. Mootse, G., Agostino, D., and Cliffton, E. E. Alterations in fibrinogen, plasminogen, and inhibitors rats. Arch. Surg. 84: 449, 1962. of plasmin in the growth of V2 carcinoma in the 2. Boren, C. B., Roon, A. .I., and Moore, W. S. Mainrabbit. .I. Nutl. Cancer Inst. 35: 567, 1965. tenance of viable arterial allografts by cryopreser19. Nagy, B., Ban J., and Brollar, B. Fibrinolysis vation. SurgeT 83: 382, 1978. associated with human neoplasia. Production 3. Cliffton, E. E., and Grossi, C. E. Effect of human ofplasminogen activation by human tumors. Int. J. plasmin on the toxic effects and growth of bloodCancer 19: 614, 1977. borne metastases of the Brown-Pearce carcinoma and the V2 carcinoma of the rabbit. Cancer 9: 20. Newstead, G. L., Griffiths, J. D., and Salsbury A. J. Fibrinolytic activity of carcinoma of the 1147, 1956. colorectum. Surg. Gynecol. Obstet. 143: 61, 1976. 4. Cliffton, E. E., and Grossi, C. E. Fibrinolytic activity of human tumors as measured by the fibrin 21. O’Meara, R. A. Q. Coagulation properties of cancer. Ir. J. Med. Sci. 396: 474, 1958. plate method. Cancer 8: 1146, 1955. 5. Coman, D. R. Adhesiveness and stickiness: Two 22. O’Meara, R. A. Q., and Jackson, R. D. Cytological observations on carcinoma. Ir. J. Med. Sci. 391: independent properties of the cell surface. Cancer 327, 1958. Res. 21: 1436, 1961. 6. Dent, T. L., Weber, T. R., Lindenauer, S. M., et 23. Pechet, L. Fibrinolysis. N. Engl. J. Med. 273: 966, 1965. al. Cryopreservation of vein grafts. Surg. Forum 25: 241, 1974. 24. Peterson, H. I., Kjantansson, I., Korsan-Bengtsen, 7. Elias, E. G., Sepulvada, F., and Mink, B. InK., et al. Fibrinolysis in human malignant tumors. creasing the efficiency of cancer chemotherapy Acta Chir. Stand. 139: 219, 1973. with heparin. “Clinical Study.” J. Surg. Oncol. 5: 25. Peterson, H. I., Larsson, S., and Zettergren, L. 189, 1973. Fibrinolysis in human bronchogenic carcinoma. 8. Franklin, J. D., Gervin, A. S., Bowers, D. B., et al. Bib/. Anat. 13: 315, 1975. Fibrinolytic activator activity in human neo- 26. Ryan, J. J., Ketcham, A. S., and Wexler, H. plasms. Plust. Reconstr. Surg. 61: 241, 1978. Reduced incidence of spontaneous metastases 9. Gasic, G. J., Gasic, T. B., Talanti, N., er al. with long term coumadin therapy. Ann. Surg. 168: Platelet tumor cell interactions in mice. The role of 163, 1968. platelets in the spread of malignant disease. Inr. J. 27. Soong, B. C. F., and Miller, S. P. Coagulation Cancer 11: 704, 1973. disorders in cancer. III. Fibrinolysis and inhibitors. IO. Gasic, G. J., Gasic, T. B., and Stewart, C. C. Cancer 25: 864, 1970. Antimetastatic effects associated with platelet 28. Sugarbaker, E. V., and Ketcham, A. S. Mechareduction. Proc. Nutl. Acad. Sci. USA 61: 46, nisms and prevention of cancer dissemination. An 1968. overview. Semin. Oncol. 4(l): 19, 1977. 11. Gervin, A. S., and Butler, B. M. Fibrinolytic activity in autologous jugular vein. Loss and 29. Thomes, R. D. Adjunct therapy of cancer via the cellular immune mechanisms or by induced tibrinolrestoration. Amer. Swg. 41: 726. 1975. ysis and oral anticoagulants. Cancer 35: 91, 1975. 12. Grossi, C. E., Agostino, D., and Cliffton, E. E. 30. Walter, J. B., and Israel, M. S. Genera/Pathology. The effect of human fibrinolysin on pulmonary Baltimore: Williams&Wilkins, 1970.Pp. 607-609. metastases of Walker 256 carcinosarcoma. Cancer 31. Wood, S. Jr. Experimental studies on the intraRes. 20: 605, 1960. vascular dissemination of ascites V2 cancer cells 13. Hoover, H. C., Jr., and Ketcham, A. S. Techniques in the rabbit with special reference to fibrinogen for inhibiting tumor metastases. Cancer 35: 5, 1975. and fibrinolytic agents. Bull. Swiss Acud. Med. Sci. 14. Lai, K., Tyler, H. M., and Yancey, S. T. Clot 20: 92, 1964. forming and stabilizing enzymes from mouse tumor YPC-1. Biochem. Biophys. Res. Commun. 24: 776, 32. Young, S. W. Determinants of malignant tumor 1966. growth. Resident StuffPhys. 24(4): 89, 1978.
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