Path. Res. Pract. 187,73-77 (1991)

Histochemical and Immunohistochemical Morphology of Carcinoid Heart Disease L. Lundin 1, K. Funa2 , H. E. Hansson 3 , E. Wilander4 and K. Oberg 2 Departments of Internal Medicine 1, Thoracic surgery3, Pathology4 and Ludwig Institute for Cancer Research2, University Hospital, Uppsala, Sweden

SUMMARY Histochemical and immunohistochemical investigations were performed on tissue obtained from the right heart side in three patients subjected to valve, replacement operations because of severe carcinoid heart disease. Extensive fibrotic changes were present on the endocardium ofthe right atrium, the papillary muscles ofthe tricuspid valve and the leaflets ofthe tricuspid and pulmonic valves ofall patients. The main constituent of the lesions was a stroma with abundant acid mucopolysaccharides and collagen but devoid ofstainable elastic components. The lesions were in some areas sharply delineated from the normal endocardium, but often also extended into the endocardium and myocardium. Small to medium sized vessels were demonstrated histochemically in the lesions and confirmed by positive immunoreaction against endothelial and smooth muscle cells. The moderate number ofmesenchymal cells within the lesions had immunoreactivity consistent with muscle cells which seemed to have a very low proliferating activity. The histochemical and immunohistochemical techniques used confirmed some earlier observations in carcinoid heart disease but also rendered new information contradicting previous findings. The infiltrative nature ofthe carcinoid plaque gives a new dimension to the carcinoid heart disease. The etiology still remains obscure and well known growth factors for connective tissue such as platelet derived growth factor (PDGF) do not seem to be directly involved in the process. Introduction Classical carcinoid syndrome is frequently associated with characteristic endocardial fibrotic heart lesions, which in almost all cases are found on the right side of the heart7,10,19,21, but in about 25% of patients described in the literature also on the left side. Since the first reported case in 19303 the morphology of the lesions has been described in several reports and predominantly in autopsy materiaI4,11,13,19,21,22. Ultrastructural studies have been performed in a limited number of cases5, 14. Although the etiology behind the lesions still remains unclear, there seems to be agreement about the typical morphological changes of the lesions in carcinoid heart disease. Thus, the plaque-like thickenings are reported to be superimposed ©

1991

by Gustav Fischer Verlag, Stuttgart

on unaffected endocardium of the valvular leaflets and the cardiac chambers and sometimes on the intima of the great veins, the coronary veins and the aorta in close proximity to the heart I9 ,21. The major part of the lesions is composed of a stromal tissue rich in acid mucopolysaccharides, reticulum fibers and collagen, but typically devoid of elastic components. The most frequent cell type in the carcinoid plaques expresses ultrastructural characteristics of smooth muscle cells as well as fibroblasts or fibrocytes, resembling the myofibroblasts observed in e.g. granulation tissue 6 • The present study was performed in order to further characterize the carcinoid heart lesions by means of immunohistochemical techniques on fresh cardiac tissue obtained at valvular surgery in three patients with 0344-0338/91/0187-0073$3.50/0

74 . L. Lundin et al. advanced carcinoid heart disease. Immunohistochemical investigation of carcinoid heart lesions has not been reported previously.

Immunohistochemistry

Material and Methods

Tissue Processing Heart tissue was obtained from three female patients aged 47, 56 and 68 years with classical carcinoid syndromes due to midgut carcinoid tumors with liver metastases. They all had developed symptoms and signs of advanced right heart failure and card~ac ultrasound and hemodynamic findings were consistent With severe carcinoid heart disease with pressure and volume load of the right heart side. During tricuspid and pulmonic valve replacement surgery tissue material was obtained from the deformed tricuspid and pulmonic valves, the enlarged right atria, and parts of the tricuspid papillary muscles. One part of each fre~h tissue specimen was frozen in liquid nitrogen (-150°C), while another part was directly fixed in 4% paraformaldehyde and later embedded in paraffin. The frozen tissue was stored in -70 °C un~il cryosectioned (2800 Frigocut, Reichert-Jung, West Germany) In 6 Ilm sections. The cryosections were subsequently fIxed In acetone at 4°C for 10 minutes. The pataffin embedded tissues were also cut in 6 Ilm sections which were placed on glass slides and deparaffinized in graded alcohols. In addition, tumor tissue was obtained at operation of the primary intestinal tumor in one of the above patients and in two other patients with carcinoid syndrome.

Histologic Investigations Sections from the paraffin embedded blocks were stained with haematoxylin-eosin, alkaline Congo red l ? and toluidine blue l6 . In order to assess the presence and interrelation of acid mucopolysaccharides, collagen and elastin, a combination staining (AVE)

Table 1. Antibodies used and immunostaining results in cardiac carcinoid plaques Antigen

Antibody

Dilution

Immunostaining in carcinoid plaque

Muscle actin filament

HHF 35

1/25 000

++

Desmin intermediate filament in muscle cells

Anti-Desmin

1/100

+

Vimentin intermediate fila-Anti-Viment in cytoskeleton of mentin mesenchymal cells

11100

++

Nuclear antigen expressed KI67 on all proliferating cells

1110

Platelet derived growth factor B-type receptor

including alcian blue, van Gieson's staining and Miller's elastic stain was used on all tissue sections. By this staining method acid mucopolysaccharides are stained blue, elastin dark blue/black, collagen red and muscle yellow l8 .

PDGFR-B 21120

Blood group H antigen on UEAI lectin 11200 endothelial cell surface + anti-Ulex antibody Surface antigen on mono- LEU-M 5 1/160 cytes/macrophages

+

Avidin-biotin-coupled immunoperoxidase staining was performed on formalin-fixed, paraffin-embedded or cryosectioned acetone-fixed material by using a Vectastain ABC kit (Vector Laboratories, Burlingame, CAl according to the vendor's description. 3,3-diaminobenzidine tetrahydrochloride (DAB) or 3amino-9-ethyl-carbazole (AEC) was used as chromogen in all the immunoperoxidase stainings. A negative control staining was performed in each assay by substituting a corresponding normal serum of the same species of animal for the primary antibody and previously known positive tissues served as positive control. A mouse monoclonal antibody, HHF 35, which detects an epitope common to the actin isotypes of all muscle cell types, but not to the actin isotypes of non-muscle cells, was used on paraffin-embedded tissues 23 • The antibody was kindly provided by Dr. Gown from the UniversityofWashingron, Seattle, WA. An antidesmin mouse monoclonal antibody reacting with the intermediate filament protein desmin of both striated and smooth muscle was used on tissue cryosections for further characterization of the muscle components of the heart lesions. Another intermediate filament protein, vimentin, present in cells of mesenchymal origin was stained by a mouse monoclonal antivimen tin antibody. For staining of endothelial cells Ulex europaeus agglutinin I lectin (UEAI) and biotinylated rabbit anti-Ulex antibody (Vector Laboratories) were used on paraffin-embedded tissues. A mouse monoclonal antibody (KI 67) reacting with human nuclear antigen, expressed only on proliferating cells, was used to estimate cell proliferating activity. The antidesmin, antivimentin and KI 67 antibodies were purchased from Dakopatts, Glostrup, Denmark. A mouse monoclonal antibody, anti-Leu-M5 (Becton Dickinson, Mountain View, CAl reactive to an epitope on the alpha chain of CD lIe on the cell surface of monocytes and macrophages was used for t~e detect~on of t~ese cells 9 . In addition a mouse monoclonal antibody raised against porcine PDGF B-;ype receptors, PDGFR-B2, which specificially bind to the extracellular domain of PDGF-B type receptors, was used to screen a presence of PDGF receptors 20 • A summary of all antibodies and the dilutions used is found in Table 1.

Results Carcinoid heart lesions were present both macroscopically and microscopically in tissue samples from the three patients with some difference in the extent of the.lesi.ons. The lesions were sufficiently similar to allow theJf hIstochemical and immunohistochemical properties to be analysed collectively. . The AVE stainings in Fig. 1 demonstrate the endocardIal localization of the carcinoid plaques in the right atrial wall, on the tricuspid papillary muscle, and on a pulmonary cusp with a typically embedded curly edge. The main constituent of the lesions was a stromal tissue of acid mucopolysaccharides and collagen intermingled with a moderate number of cells. These cells were elongated, slender and orientated parallel to the endothelial su~face in the ~ajor part of the lesions. Closer to the underlymg endocardIUm, however, the cells often had a more irregular stellate shape

Histological Morphology of Carcinoid Heart Disease· 75

Fig. 1. Photomicrographs of a tricuspid papillary muscle, a right atrial wall and a pulmonic cusp from patients with severe carcinoid heart disease. - A) A cross-sectioned tricuspid papillary muscle with a carcinoid lesion mainly consisting of a stroma of acid mucopolysaccharides (blue) and collagen (red) which completely encircles the central muscle (yellow). AVE-staining (x 20). - B) Extensive infiltration of the carcinoid lesion (blue) through the endocardium into right atrial wall myocardium (yellow) forcing the muscle fibers apart. AVE-staining (x 130). - C) Pulmonic cusp with a typically curly edge embedded in a thick carcinoid lesion (blue and red) mainly located on the arterial aspect of the cusp. The fairly well delineated native leaflet seems to contain more than normal acid mucopolysaccharides (blue). AVE-staining (x 20). - D) Positive immunoreaction with HHF 35 antibodies demonstrating presence of muscle actin in the cells of the carcinoid lesion. Lower left part of A (x 260).

and seemed to be more randomly orientated. The lesions which lacked typical elastic components were in some areas sharply delineated from the normal underlying tissue by an elastic membrane, but frequently the acid mucopolysaccharides stained by the alcian blue were extended

from the carcinoid plaque through the endocardium and the elastic membrane into the myocardium of the papillary muscle and the atrial wall (Fig. lB). The fibrotic lesions on the valve cusps were mainly located on the ventricular aspect of the tricuspid valve leaflets and on the arterial side

76 . L. Lundin et al.

of those of the pulmonic valve. The edges of the leaflets of both valves were often curled and embedded in the fibrous plaque, which seemed to immobilize the cusps (Fig. 1C). Although the native leaflets of both the pulmonary and tricuspid valves seemed to a great extent sharply separated from the attached fibrous lesion by an elastic membrane, they contained an increased amount of acid mucopolysaccharides as judged by staining. This staining, furthermore, visualized that the carcinoid lesion also extended through the elastic membrane of the native cusps. Some scattered inflammatory cells were observed in the endocardium with the hematoxylin-eosin staining and immunostaining with anti-Leu-M5-revealed presence of a few monocytes/macrophages in the cardiac tissue. The carcinoid lesions, however, lacked these kinds of cells. Staining with toluidine blue detected single rare mast cells located on the epicardial side of the atrial wall. No mast cells were observed within the fibrous lesions which, however, stained intensely metachromatic. The positive staining by alcian-blue and metachromatic reation with toluidine blue indicates a possible presence of amyloid in the carcinoid lesions. Alkaline Congo-red staining could, however, not confirm this conjecture when examined both by ordinary and polarised light. In all carcinoid heart lesions the cytoplasm of the fibroblast-like cells was immunoreactive with the muscle actin-specific monoclonal antibody (Fig. ID). Some variation in staining intensity was present within the lesions but more intensely stained areas were scattered throughout the specimens. The smooth muscles of the intracardiac vessel walls and the myocardium, which served as positive control for the muscle actin immunoreaction stained strongly in all tissue specimens. The anti-desmin antibody regularly stained the myocardium and vessel wall smooth muscles, and some areas of the fibrous lesions. A single endothelial cell layer was demonstrated on the cavity side of endocardium with and without carcinoid lesions by staining with DEAl, which binds to a blood group H antigen on the surface of endothelial cells. Endothelium of blood vessels within the cardiac tissue was also detectable as were vessels located within the carcinoid lesions close to the endocardium. The vessels within the carcinoid lesions had a varying wall thickness some of them having a muscle wall layer which stained positively with the muscle-actin antibody. The mesenchymal origin of the cardiac carcinoid plaque was further demonstrated by immunohistochemical detection of cytoplasmatic vimentin filaments throughout the lesions. No cell stained positively with the KI 67 antibody, which indicates a very low proliferating activity of the cells throughout the carcinoid lesions. Neither did the cardiac fibrous lesions stain with the PDGFR-B2 antibody. AVE-staining of the intestinal tumor revealed the presence of abundant connective tissue with staining characteristics quite different from the carcinoid cardiac plaques. The stromal tissue was rich in dense collagen, elastin was observed frequently but acid mucopolysaccharides were missing. Muscle actin was immunohistochemically shown to be present in the string-like tissue within and around the tumors, probably reflecting separation of normal intestinal smooth muscle by the tumor.

Discussion The present study demonstrates a common morphology and histochemical composition of carcinoid heart lesions in tissue specimens obtained from different parts of the right side of the heart of three patients with severe carcinoid heart disease. In accordance with earlier reports 4,S, 11, 14,22, the major component of the lesions was found to be an elastin-deficient stroma rich in acid mucopolysaccharides and collagen, embedding a moderate to low number of cells. Although valvulitis was described in some of the early case reports2,12 more recent studies have found low numbers of inflammatory cells, and in this study no inflammatory cells were found in the carcinoid plaques. In contrast to previous studies, the present investigation demonstrates continuous extension of the carcinoid fibrous lesion through a discontinuous elastic membrane into the myocardium separating the individual myocardial fibers of the atrial and papillary muscles. Similar extensions of the carcinoid lesion were observed through the native delineation of the valvular leaflets. Furthermore, the native cusps often contained abnormal fibrous material which had staining characteristics similar to the more superficial carcinoid plaques. The presence of these extensions of the carcinoid lesions into the underlying tissue differs from observations in several previous studies which state that the lesions are superimposed on normal endocardium and intima and delineated from them by an intact elastic membrane 4,s, 11, 19,21. The positive immunostaining of the cells within the carcinoid plaque with the anti-actin and anti-desmin antibodies suggests the presence of muscular characteristics. This accords with previous studies which describe the presence of both usual smooth muscle cells and myofibroblasts with characteristics of smooth muscles as well as fibroblasts s. The specific lectin binding to endothelial cells confirmed previous findings that the carcinoid lesions are covered by an endothelial cell layer. Presence of some vessels within plaques was questionable on some locations due to the fact that the lesions extend into underlying tissues making it hard to clearly delineate the plaque. However, some vessels were also found in carcinoid lesions sharply separated from the endocardium by an intact lamina elastica interna. The location of vessels in the fibrous tissue adjoining the endocardium accords with observations in previous investigations 1,22. The pathogenesis of the carcinoid heart lesions still remains obscure. Connective tissue within or close to carcinoid tumors and their liver metastases has recently been shown to exhibit an increased number of PDGF-B receptors indicating a role of PDGF in the production and growth of the often substantial fibrous tissue in association with these tumors (Funa et al. Cancer Res.: in press). The inability to demonstrate PDGF B-type receptors in the cardiac lesions, however, argues against PDGF as a common pathogenetic mechanism. Furthermore, the difference in morphology and staining characteristics of the connective tissue of the carcinoid tumor and of carcinoid heart lesions supports a difference in pathogenetic mechanisms. Tachykinins have been reported to stimulate fibroblast growth in vitro 15 • These vasoactive substances

Histological Morphology of Carcinoid Heart Disease . 77

are produced and released from carcinoid tumors and are present in high concentrations in the right heart. A possible stimulatory activity by these substances on cardiac endocardial cells justifies further studies in vitro and animal models as well as receptor studies in the cells of carcinoid plaques. The disability to stain carcinoid lesions with the KI 67 antibody, however, indicates a very low proliferation rate of these cells. Several growth factors have recently been found to stimulate fibroblasts to synthesize extracellular matrix 8 . The cardiac lesions are in fact characterized by their great amount of mucopolysaccharides intermingled with collagen fibers. We therefore tentatively suggest that some of these growth factors are involved in the development of the heart lesions and act by stimulating not proliferation but matrix synthesis. In summary, the present study using new techniques confirms some of the earlier observations concerning the carcinoid heart lesion, such as content of mesenchymal cells, acid mucopolysaccharides and collagen. A new observation is the infiltrative nature of the carcinoid plaque projecting into the myocardium. The presence of vessels within the carcinoid plaques has been immunohistochemically confirmed. The proliferation rate of the cells within the lesions is very low and the pathogenetic mechanisms remain obscure.

Acknowledgements This work was supported by the Faculty of Medicine, University of Uppsala, the Ludwig Institute for Cancer Research, University Hospital, Uppsala, Torsten and Ragnar Soderberg Research Foundation and the Swedish National Association against Heart and Chest Diseases.

References 1 Amoury RA (1970) Heart disease and the malignant carcinoid syndrome. A case report and review of pathophysiologic and pathogenetic mechanisms. Am J Surg 119: 585-594 2 Biorck G, Axen D, Thorsson A (1952) Unusual cyanosis in a boy with congenital pulmonary stenosis and tricuspid insufficiency. Fatal outcome after angiocardiography. Am Heart J 44: 143-148 3 Cassidy MA (1930) Post-mortem findings in case shown on October 10, 1930, as one of abdominal carcinomatosis with probable adrenal involvement. Proc Roy Soc Med 24: 920-921 4 Cosh J, Cates JE, Pugh DW (1959) Carcinoid heart disease. Br Heart J 21: 369-380 5 Ferrans VJ, Roberts WC (1976) The carcinoid endocardial plaque. An ultrastructural study. Hum Pathol 7: 387-408

6 Gabbiani G, Hirschel BJ, Ryan GB, Statkov PR, Maino G (1972) Granulation tissue as a contractile organ. A study of structure and function. J Exp Med 135: 719-734 7 Howard RJ, Drobac M, Rider WD, Keane TJ, Finlayson J, Silver MD, Wigle ED, Rakovski H (1982) Carcinoid heart disease: Diagnosis by two-dimensional echocardiography. Circulation 66: 1059-1065 8 Ignotz RA, MassagueJ (1986) Transforming growth factor-~ stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J BioI Chern 261: 4337-4345 9 Lanier LL, Arnaout MA, Archwarting R, Warner NL, Ross GD (1985) p 150/95, third member of the LFA-1/CR3 polypeptide family identified by anti-Leu M5 monoclonal antibody. Eur J Immunol15: 713-718 10 Lundin L, Norheim I, Landelius J, Oberg K, TheodorssonNorheim E (1988) Carcinoid heart disease: relationship of circulating vasoactive substances to ultrasound-detectable cardiac abnormalities. Circulation 77: 264-269 11 MacDonald RA, Robbins SL (1957) Pathology of the heart in the carcinoid syndrome. A comparative study. A. M. A. Arch Pathol 63: 103-112 12 McKusick VA (1955) Carcinoid cardiovascular disease. Bull John Hopkins Hosp 98: 13-36 13 Millman S (1943) Tricuspid stenosis and pulmonary stenosis complicating carcinoid of the intestine with metastasis to the liver. Am Heart J 25: 391-398 14 Miiller HG, Siebenmann RE (1981) Ultrastructur der Endocardveranderungen beim Carcinoidsyndrom. Virch Arch A 391: 33-44 15 Nilsson J, von Euler AM, Dalsgaard CJ (1985) Stimulation of connective tissue cell growth by substance P and substance K. Nature 315: 61-63 16 Pearse AGE (1985) Histochemistry. Theoretical and Applied. Volume 2: Analytical Technology, p 742. Churchill Livingstone, London 17 Puchtler H, Sweat F, Levine M (1962) On the binding of Congo red by amyloid. J Histochem Cytochem 10: 355-364 18 Reynolds GJ, Pomerance A, Eames K (1978) A combined stain for elastin and acid mocopolysaccharides in large blood vessels. Histopathology 2: 225-226 19 Ross EM, Roberts WC (1985) The carcinoid syndrome: Comparison of 21 necropsy subjects with carcinoid heart disease to 15 necropsy subjects without carcinoid heart disease. Am J Med 79: 339-354 20 Ronnstrand L, Terracio L, Claesson-Welsh L, Heldin CH, Rubin K (1988) Characterization of two monoclonal antibodies reactive with the external domain of the platelet-derived growth factor receptor. J Bioi Chern 262: 2929-2932 21 Strickman NE, Rossi PA, Massumkhani GA, Hall RJ (1982) Carcinoid heart disease: a clinical, pathologic, and therapeutic update. Curr Probl Cardiol 6: 1-42 22 Trell E, Rausing A, Ripa J, Torp A, Waldensttom J (1973) Carcinoid heart disease. Clinicopathologic findings and followup in 11 cases. Am J Med 54: 433-444 23 Tsukada T, Tippens D, Gordon D, Ross R, Gown AM (1987) HHF 35, a muscle-actin-specific monoclonal antibody. I. Immunocyto-chemical and biochemical characterization. Am J Pathol126: 51-60

Received January 8, 1990 . Accepted March 14, 1990

Key words: Carcinoid heart disease - Endocardial fibrosis - Acid mucopolysaccharides - Elastic fibres - PDCF Lennart Lundin, M.D., Department of Cardiology, Medical Clinic, University Hospital, S-75185 Uppsala, Sweden

Histochemical and immunohistochemical morphology of carcinoid heart disease.

Histochemical and immunohistochemical investigations were performed on tissue obtained from the right heart side in three patients subjected to valve,...
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