(CANCER RESEARCH 51. 349-353. January I. 1991]

Fibrinogen Deposition without Thrombin Generation in Primary Human BreastCancer Tissue1

Vincenzo Costantiâ„¢,2Leo R. Zacharski,3 Vincent A. Memoli, Walter Kisiel, Bohdan J. Kudryk,

and Sandra M. RousseauDepartments of Mediane /C. C, L. R. Z.] and Pathology ¡V.A. M.], Dartmouth Medical School, Hanover, \ew Hampshire; t'eterans Administration Medical Center,White River Junction, Vermont 05001 ¡V.C., L. R. Z., S. M. R.]; Department of Pathology, L'nirersity of New Mexico School of Medicine, Albuquerque, New Mexico87131 ¡W.K.]; and Plasma Protein-Coagulation Laboratory, New York Blood Center. New York, New York 10021 ¡B.J. K.J

ABSTRACT

The occurrence and distribution of components of coagulation pathways in situ were determined using immunohistochemical techniquesapplied to 10 cases of primary carcinoma of the breast, normal breasttissue obtained from two patients undergoing reductive mammoplasty,and three patients with benign breast tumors. Tumor cells stained forfactor X and thrombomodulin but not for tissue factor, factor V, factorVII, or factor XIII. Rare nonneoplastic duct epithelial cells stained forthrombomodulin, but these tissues did not otherwise stain for any ofthese antigens. Macrophages within the tumor stroma stained for tissuefactor, factor VII, and factor XIII but not for factor V or factor X. Thesefeatures of macrophages were the same in malignant and nonmalignantbreast tissue. Fibrinogen was present in abundance throughout the connective tissue in breast cancer but not in nonmalignant tissues. Bycontrast, no staining was observed using fibrin-specific antibodies. Theseresults suggest that an intact coagulation pathway does not exist in breastcancer tissue and that thrombin capable of transforming fibrinogen tofibrin is not generated in significant amounts in this tumor type. Whilefibrin is not a feature of the connective tissue stroma in breast cancer, itis conceivable that the abundant fibrinogen present in the tumor connective tissue (and factor XIII present in connective tissue macrophages)might contribute to the structural integrity of breast tumor tissues.

INTRODUCTION

Carcinoma that arises from the epithelial cells lining theducts of the mammary gland will develop in approximately 1of 14 American women (1). Endocrine, dietary, and hereditaryfactors as well as ionizing radiation have been incriminated inthe development of breast cancer, but the details of the patho-genesis of this common malignancy are largely unknown (1).While a measure of success in controlling breast cancer hasbeen realized with surgical excision, chemotherapy, hormonaltherapy, and radiation therapy, this tumor tends to disseminaterelatively early in its course in many women and such treatments are of limited efficacy ( 1). There is ample motivation toobtain new information concerning the pathogenesis of thisdisorder.

Attention has been directed to mechanisms of coagulationactivation in cancer because of interest in understanding thenature of the coagulopathy commonly associated with malignancy and also because such activation may play an integral

Received 8/13/90; accepted 9/24/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by the Veterans Affairs Medical Research Service; research

grants from the National Institutes of Health HL35246 (W. K.). HL21465(B. J. K.). and BRSG S07RR05392 (L. R. Z.): and Blood Systems. Inc. (W. K.).

2V. C. is a Visiting Scientist. Dartmouth Medical School and recipient of afellowship from the Italian National Research Council-NATO. Permanent address: Istituto di Semeiotica Medica. Università di Perugia. Via E. dal Pozzo.06100 Perugia. Italy.

3To whom requests for reprints should be addressed, at the VA Medical and

Regional Ottice Center. White River Junction, VT 05001.

role in tumor cell proliferation, invasion, and metastasis (2).The latter concept has been tested in controlled clinical trialsof treatment designed to interrupt such pathways (3-6). Thesetrials have met with success in certain tumor types but not inothers (7, 8).

In an attempt to sort out mechanisms of coagulation activation in cancer, immunohistochemical techniques have beenapplied to several human tumor types in an attempt to determine the occurrence and cellular distribution of components ofpathways of thrombin formation and fibrinolysis in situ (9-15).The goal of these studies has been to determine whether thecellular elements of tumors (e.g., the tumor cells themselvesversus stremai cells such as macrophages) are associated withreactants that might contribute to such pathways.

Data available so far suggest that coagulation activation incancer may arise by at least two different mechanisms. "Direct"

activation may result from assembly of coagulation pathwaycomponents on tumor cells themselves that leads to thrombingeneration and conversion of fibrinogen to fibrin adjacent toviable tumor nodules. Direct activation may occur in SCCL4

because both coagulation pathway intermediates and fibrin havebeen discovered in situ in this tumor type by means of immunohistochemical techniques (9-11). By contrast, "indirect" ac

tivation may result when a tumor produces a soluble substancethat triggers the production of coagulation initiators on hostcells (e.g., macrophages or endothelial cells) at sites distantfrom the tumor. Evidence suggests that an indirect mechanismof coagulation activation may exist in acute myelogenous leukemia (16). Other mechanisms of coagulation activation incancer may also exist.

It is well known that systemic activation of coagulation,manifested by changes in peripheral blood tests of coagulationor by thromboembolism, occurs commonly in breast cancer(17-20). Evidence has also been presented suggesting that fibrinmay be formed within breast cancer tissue (21). However, doubtremained because reagents available earlier were not capable ofdistinguishing with certainty between fibrinogen and fibrin.Furthermore, an intact coagulation pathway has not been demonstrated in human breast cancer. The present study was undertaken in an attempt to resolve these uncertainties.

MATERIALS AND METHODS

Studies were performed on AMeX-fixed tissue (22) prepared fromfresh surgically excised primary breast cancers from 10 cases (9 ofwhich were infiltrating ductal carcinomas and one of which was aninfiltrating lobular carcinoma). No patient had received treatment forher disease. Comparisons were made with normal breast tissue obtainedduring reductive mammoplasty surgery from two cases, from benignfibroadenomas in two cases, and from benign fibrocystic disease in one

4The abbreviations used are; SCCL. small cell carcinoma of the lung; TF,

tissue factor; TM. thrombomodulin.

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case. Staining procedures and controls for the avidin-biotin complextechnique (9-15) using reagents (Vectastain Kits; Vector Laboratories.Burlingame, CA) and for the indirect immunofluorescence technique(23) have been described previously. Antigen staining was detected bythe dark brown (peroxidase reaction with diaminobenzidine) or red(alkaline phosphatase reaction with Red Vector) reaction productsobtained with the avidin-biotin complex procedure. This contrastedwith the dark blue nuclei of cells and the pale pink appearance ofunstained cells and stroma.

The double-labeling technique was used to visualize simultaneouslytwo different antigens in the same section. For demonstration of twoantigens on the same cell, an immunoenzymatic step that gave a coloredreaction product was applied to unstained sections, and this step wasfollowed by the indirect immunofluorescence procedure (24). Whenboth first and second primary antibodies were raised in the same species,the specificity for binding of the fluorescein-conjugated secondary antibody applied to the second primary antibody is preserved because theperoxidase-staining procedure applied to the first primary' antibody

prevents its binding to the second secondary antibody (25).Procedures used monospecific, purified rabbit antibodies to the fol

lowing: recombinant human TF, factor V, factor VII, factor X, the "a"

subunit of factor XIII, protein C, and protein S. Rabbit polyclonalantibodies were prepared from protein antigens that were >99% pure.Antibodies were purified from crude antiserum by affinity chromatog-raphy either on an antigen-Sepharose column (for antibodies to factorVII and factor X) or on a protein A-Sepharose column (for antibody to

TF). Antibody specificity was demonstrated on the basis of activityneutralization. Western immunoblot analysis, and immunoprecipita-tion studies using '"I-labeled antigen or proteins labeled metabolicallyin vivo using |"S]methionine. The antibody to TF apoprotein was

capable of immunoprecipitating TF apoprotein from cell extracts andstained a protein of M, about 44,000 on Western blots. Monoclonalantibodies specific for epitopes on fibrin(ogen) or their degradationproducts included the following: antibody 1-8C6 that requires an intact14 arginine-17 glycine bond in the B/i chain of fibrinogen and thereforereacts with fibrinogen or fibrin I (des-fibrinopeptide A-type fibrin) butnot fibrin II (des-fibrinopeptide B-type fibrin) (26): antibody T2GI thatreacts with the amino-terminal part of the Bßchain only followingremoval by thrombin of fibrinopeptide B (B/j 1-14) and thus with fibrinbut not fibrinogen (27); and antibody GC4 that reacts with fragment Dof fibrinogen as well as n-dimer from cross-linked fibrin but not witheither fibrinogen or fibrin (28). The reactivity of these antibodies inimmunohistochemical procedures has been verified independently (29).Mouse monoclonal antibodies to the thrombin cleavage sites on the A«and ßßchains of fibrinogen and a goat antibody to rabbit TM thatcross-reacted with human TM were obtained from American Diagnostica (New York, NY). In our hands, the anti-TM antibody recognizedTM in the syncytiotrophoblast of human placenta and in the endothe-lium of small blood vessels. The macrophage-specific monoclonal antibody IBM] 1 and rabbit polyclonal antibody to antithrombin III wereobtained from Dako Corp. (Santa Barbara. CA). We have shownpreviously that reactivity of these antibodies is similar in fresh frozenand AMeX-fixed tissue (9-15).

Antibodies were tested on control and tumor tissues in concentrations that provided maximal staining intensity with minimal background staining. Controls consisted of omission from the procedure ofthe primary' antibody and use of antibodies developed in the same

species but with different or irrelevant specificities. Results of studieson breast cancer tissues were interpreted in association with stainingprocedures performed on normal control (i.e., normal breast, placenta,and liver), benign breast disease, and other neoplastic tissues (e.g.,SCCL, renal cell carcinoma, and colon cancer) processed simultaneously. Particularly, the fibrin-specific antibody T2G1 stained material

adjacent to viable tumor cells in SCCL (11) and renal cell carcinoma(13) and adjacent to tissue macrophages in lymphoproliferative disorders. These tissues served as positive controls for this antibody and theresults in these tumor types will be reported separately.

RESULTS

Tumor cell bodies stained for factor X to a variable extent in5 of the 10 cases of breast cancer, but no tumor cell stainingwas detected for TF, factor V, factor VII, or factor XIII "a"

subunit. Results for factor X are illustrated in Fig. 1. Focalstaining of breast cancer cells (and occasional nonneoplasticduct epithelial cells) for TM was also observed. Otherwise, nostaining was observed for any of these antigens on epithelialcells from normal tissues or benign breast disease. Double-labeling procedures revealed that macrophages scatteredthroughout the connective tissue in malignant, benign, andnormal breast tissues stained for TF, factor VII, and factor XIII"a" subunit. Scattered fibroblasts present in the connectivetissue also stained for factor XIII "a" subunit (Fig. 2). There

was no macrophage staining for factor V or factor X. Thesefeatures of macrophages were the same for malignant andnonmalignant tissues. Macrophage staining for factor VII in acase of benign fibroadenoma is illustrated in Fig. 3.

Patchy but diffuse staining of the tumor connective tissuewas observed for factor V, and more uniform, diffuse strongstaining of the connective tissue was observed for fibrinogenusing the fibrinogen-specific monoclonal antibody I-8C6 (26).The appearance of the fibrinogen is illustrated in Fig. 4. No

Fig. 1. Specific tumor cell staining (arrows) by the peroxidase technique usingdiaminoben/.idine as substrate (that gave a brown reaction product) for factor Xin primary breast cancer (a). Staining was absent (cell bodies appeared pale blue)in preparations handled identically but from which the primary antibody wasomitted (b). Hematoxylin counterstain; original magnification, x 250.

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staining for factor V or fibrinogen was observed in the connective tissue of nonmalignant disease. In addition, no connectivetissue staining was observed using fibrin-specific antibodies ineither malignant or nonmalignant tissues. Staining with ami

Fig. 2. Staining by the peroxidase reaction for factor XIII "a" subunit in

fibroblasts (straight arrows) and macrophages (curved arrow) in the connectivetissue stroma of primary breast cancer. Hematoxylin counterstain; original magnification, x 250.

Fig. 3. Coincident staining of macrophages within the same microscopic fieldin a case of benign fibroadenoma of the breast using the macrophage-specificantibody EBM-II with the peroxidase reaction (a) and using antibody to factorVII with fluorescence (b). Specimens are not counterstained: original magnification, X 400.

Fig. 4. Specific staining (with antibody 1-8C6) by the peroxidase techniqueusing diaminobenzidine as substrate for Pibrinogen distributed throughout theconnective tissue stroma (arrows) in primary breast cancer. Staining was absentfrom specimens handled identically but from which the primary antibody wasomitted. The appearance of unstained tumor connective tissue is illustrated inFig. \h. Hematoxylin countcrstain; original magnification, x 250.

body GC4 that reacts with fragment D of fibrinogen as well asD-dimer of cross-linked fibrin was observed in a patchy distribution throughout the connective tissue. This was interpretedas consistent with the existence of plasmin-degraded fibrinogenrather than cross-linked fibrin because fibrin itself could not bedetected. Of course, other interpretations for these findingsexist because fibrinogen may be degraded by enzymes unrelatedto the coagulation mechanism. (Interactions of breast cancertissues with pathways of fibrinolysis will be reported separately.)Little or no staining of these tissues was observed for proteinC, protein S, or antithrombin III. Apart from the sporadicfindings for factor X, results of these immunohistochemicalprocedures were consistent among cases.

DISCUSSION

The pathogenesis of the coagulopathy of malignancy is complex and incompletely understood. However, emerging datasuggest that such activation may result in certain tumor typesfrom a direct interaction between tumor cells themselves andcoagulation factor intermediates such that thrombin is generated in situ adjacent to tumor deposits (9-13). In other tumortypes, the tumor appears to activate coagulation indirectlythrough production of a soluble mediator (cytokine) that induces procoagulant activity on host cells such as macrophagesor endothelial cells (16).

While systemic activation of coagulation in breast cancer hasbeen abundantly documented (17-20), the relative contributionof tumor cells versus tumor products, treatment (19, 20), andhost organ dysfunction (30) have not been clearly distinguished.In fact, fibrinopeptide A and fibrin degradation product levels(indicative of increased systemic formation of thrombin andplasmin, respectively) have been found in carefully conductedstudies to be elevated to a similar extent in both benign andmalignant breast disease (17, 31). Thus, coagulation activationin breast cancer may not be a feature that is specific for themalignant cell. It is conceivable that coagulation activation inboth benign and malignant breast disease may be cytokinemediated. Results presented in the present study suggest thatconditions that exist in situ in primary human breast cancermay not be conducive to local thrombin production. While

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factor X was detected on tumor cell bodies in some (but notall) cases, the tumor cells did not manifest TF, factor VII, orfactor V. Stromal macrophages manifested TF and factor VIIbut not factor V or factor X, and these features of macrophageswere similar in benign and malignant tissues. The tumor cellsalso stained for thrombomodulin. Furthermore, monoclonalantibodies capable of distinguishing fibrinogen from thrombin-cleaved fibrinogen (fibrin) have revealed exposure of thrombin-specific cleavage sites on fibrinogen in various tissues (29),including SCCL (10, 11) and renal cell carcinoma (13), butsuch cleavage sites were not demonstrable in breast cancer. Inthis regard breast cancer resembles colon cancer ( 14) and meso-thelioma (15). The limited information available suggests acorrespondence between the existence of an intact coagulationpathway and evidence for local thrombin formation in situ inthe tumor types examined thus far (9-15). The lack of an intactcoagulation pathway associated with either tumor cells orstromal macrophages, the existence of tumor cell-associatedTM, and the absence of exposure of thrombin cleavage sites onthe fibrinogen that is otherwise present in abundance in theconnective tissue adjacent to deposits of viable breast cancer isconsistent with the concept that thrombin is not formed insignificant quantities in primary human breast cancer tissue.These observations do not support the concept that local fibrinformation either enhances the vascularization of breast cancertissue (32) or serves as a barrier that prevents invasion of thetumor by host inflammatory cells (33, 34). The antigen detectedby others in breast cancer tissue by means of polyclonal antibodyto fibrinogen may, in fact, have been fibrinogen rather thanfibrin (34, 35) as confirmed in the present study. Reports ofvariable procoagulant activity of cultured breast cancer cells(36, 37) should be interpreted with caution based on the presentdata.

Although fibrin was not detected in the breast cancer stroma,it is possible that the fibrinogen present may have a role inextracellular matrix organization in this disease. It has beenshown that factor XIII (that we have detected here associatedwith stromal macrophages and fibroblasts in breast cancertissue) is capable of inducing gelation of fibrinogen and offibrinogen combined with fibronectin in the absence of thrombin and without release of either fibrinopeptide A or B (38, 39).The potential ability of such stable gels to support cell adhesionhas been discussed (40).

The significance of breast carcinoma tumor cell staining forfactor X is speculative. Extracts of breast cancer tissue havebeen shown to possess an activator of factor X termed cancerprocoagulant (41). It is conceivable that factor X derived fromthe plasma may have become bound to such a substance presentwithin the tumor cells or that the tumor cell might have synthesized factor X. However, it does not appear that this factorX participates in a thrombin-generating pathway.

An additional and unexpected finding in the present studieswas the observation of TM associated with breast cancer cells.TM is known to occur in the syncytiotrophoblast of placentaand vascular endothelium (42) and has been described in cho-riocarcinoma (43) and in tumors of vascular origin (44). Wedetected TM in tissue macrophages in a case of large celllymphocytic lymphoma5 but we are unaware of reports of this

substance in other tumor types. It is conceivable that this TM

' V. Costantini, L. R. Zacharski. V. A. Memoli. W. Kisiel. B. J. Kudryk, S.M. Rousseau, and D. C. Stump. Fibrinogen deposition and macrophage-associ-ated fibrin formation in malignant and nonmalignant lymphoid tissue, submittedfor publication. 1990.

might be capable of regulating the functional state of anythrombin formed to further limit in situ procoagulant activityof breast cancer tissue, but the significance of TM in this diseaseremains to be determined.

The present findings may be of importance for planningtherapeutic trials. Initiation of thrombin formation in situ bySCCL tumor cells has been thought to contribute to growthregulation of this tumor because interruption of thrombin generation by warfarin therapy ameliorates the course of thisdisease (3, 4). Warfarin has been administered to patients withbreast cancer in pilot studies (45, 46), but this drug has notbeen proven to be effective in this tumor type. Based upon thepresent findings, warfarin therapy might not be expected toexert an effect by limiting local thrombin formation in situ, butit is conceivable that this drug could influence cell behavior bymodifying the activity of other vitamin K-dependent proteinsthat might exist in these cells. Alternatively, warfarin mayinfluence the properties of embolie tumor cells within thecirculation.

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