J. Comp. Path. 1997 Vol. 116, 45-54

Identification of k Light Chain Amyloid in Eight Canine and Two Feline Extramedullary

Plasmacytomas

S.J. Platz, W. Breuer, O. Geisel , R. P. Linke* and W. H e r m a n n s

Institute of Veterinary Pathology, University of Munich, Veteriniirstrasse 13, D-80539 Munich and * Max-Planck-Institute of Biochemistry , Am Klopferspitz 18a, D-82152 Martinsried,

Germany

Summary Amyloid deposition in varying amounts and with variable patterns of dis- tribution (focal or diffuse) was demonstrated in eight canine and two feline extramedullary plasmacytomas expressing ~ light chains. Frequently, the neoplastic plasma cells had been displaced by the amyloid deposits. Foreign- body giant cells were regularly detected in the vicinity of the amyloid. In all 10 cases, Congo-red staining of the amyloid was resistant to potassium permanganate oxidation. Immunohistochemically, the amyloid reacted pos- itively with cross-reacting antibodies against human and equine A~ amyloids. Extramedullary plasmacytomas accompanied by localized AL amyloidosis have so far been described in human beings, dogs, cats and horses.

�9 1997 W.B. Saunders Company Limited

Introduction

Amyloidosis is characterized by proteinaceous extracellular deposits in various tissues and organs of the body with common morphological, structural and staining properties but variable protein composition. By light microscopy, amyloid has a homogeneous, eosinophilic appearance after staining with haematoxylin and eosin. It stains red with alkaline Congo-red dye, and under polarized light the deposits exhibit a red-green birefringence (Puchtler et al., 1962). Ultrastructurally, amyloid deposits are composed of non-branching fibrils of indefinite length and a diameter of 7"5 to 10 nm (Shirahama and Cohen, 1967; Eanes and Glenner, 1968). Within the fibrils, the protein subunits are organized as a p-pleated sheet. Depending on the type of precursor protein, and thus on the origin of the amyloid, a distinction is made between localized, organ-specific and systemic types of amyloid (Glenner, 1980; Husby, 1994). Classification of amyloidosis by protein type can be carried out by chemical, immunochemical or immunohistochemical methods, but the most reliable method is that of amino-acid sequence analysis.

Amyloid A (AA) or reactive amyloidosis and amyloid L (AL) amyloidosis have frequently been described in man, and have also been described in domestic animals (Linke et al., 1984; Van de Kaa et al., 1986; Lucke, 1987; Carothers et al., 1989; Rowland et al., 1991; Breuer et al., 1993). In the case

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46 S .J . P latz et al.

Table 1 Clinical data on eight dogs and two cats with EMPs and AL amyloidos is

Case no. Breed Age Sex Location of EMP Quantity of Form of amyloid (years) amyloid

1 West Highland terrier 9 M Digit* + + + Diffuse 2 Yorkshire terrier 11 F Lip* + Diffuse 3 Dachshund 11 M Rectum'~ + + Focal 4 Cocker spaniel 11 M Lip* + + + Diffuse 5 German shepherd 11 M Knee* + § Focal 6 Boxer 9 F Digit* § § Diffuse 7 Poodle 6 M Tongue]" + Focal 8 Cocker spaniel 11 Lip* + § Focal 9 Domestic shorthaired cat 13 M/s Throat* § + Diffuse

10 Domestic shorthaired cat 7 F/s Thorax* § + + Diffuse

M = male; F = female; /s = spayed; EMP = extramedullary plasmacytoma. Amount of amyloid; + + + = large; + + = moderate; + = small; - - = not known. * Skin; "~ mucosa.

o f AA amyloidosis, amino terminal port ions of the acute-phase prote in serum amyloid A are deposited (Benditt et al., 1971; Husebekk et al., 1985). AL amyloid, however , consists of amino terminal f ragments and complete im- munoglobul in light chains. These are usually derived f rom lymphoplasmacyt ic tumours , such as the multiple myeloma, lymphoplasmacyt ic l y m p h o m a (Wal- dens t r6m's macroglobul inaemia) and ext ramedul lary p l a smacy toma (EMP), which synthesize and secrete a monoc lona l prote in (Azzopardi and Lehner , 1966; Gertz et al., 1993). The latter forms amyloidogenic precursors, which then aggregate to form the amyloid deposits.

The aim of the present study was to classify the amyloid in canine and feline EMPs immunohis tochemica l ly with a panel o f cross-react ing antibodies against h u m a n and equine AA and AX amyloids.

M a t e r i a l s a n d M e t h o d s

The tumours that were studied comprised eight canine and two feline EMPs exhibiting amyloid deposition. The clinical data on the animals concerned are given in Table 1. The EMPs had previously been characterized immunohistochemically on the basis of their light-chain restriction; all tumours expressed ~, light chains, and the amyloid was stained by Congo red (Platz, 1995).

The tumours were fixed in 7% buffered formalin and embedded in paraffin wax. All the histochemical and immunohistochemical reactions were carried out on serial sections 4 gm thick. In addition to routine staining with haematoxylin and eosin, alkaline Congo-red staining was carried out (Puchtler et al., 1962) and resistance to oxidation with potassium permanganate was assessed (Wright et al., 1977).

Immunohistochemical demonstration of the expression of only one type of im- munoglobulin light chain (E or K chain) was performed by the avidin-biotin complex immunoperoxidase method (chromogen:diaminobenzidinetetrahydrochloride) (Hsu et al., 1981); the amyloid was classified by means of the peroxidase-anti-peroxidase method (chromogen: 3-amino 9-ethyl-carbazol) (Sternberger et al., 1970). All the sections were counterstained with haematoxylin. Reactions without a primary antibody or with non-immune sera were carried out as negative controls; human tissues with amyloid whose proteins had been classified by amino-acid sequence analysis served as positive controls.

AL Amyloidosis in Dogs and Cats 47

Primary antibodies consisted of the cross-reacting polyclonal antibodies rabbit anti- human ~. chain (Dako Diagnostika, Hamburg; diluted 1 in 750) and rabbit anti- human ~c light chain (Dako; diluted 1 in 500); cross-reacting polyclonal antibodies against the various amyloid types, namely, rabbit anti-human A)~ (HAR; diluted 1 in 8000), rabbit anti-human Ak (ULI; diluted 1 in 2000) and rabbit anti-equine A)v (HIP; diluted 1 in 2000) (for HAR and ULI see Linke [1985]; and for HIP see Linke et al. [1991]); and the monoclonal antibodies mouse anti-human AA (mcl; diluted 1 in 10) and mouse anti-human AA (mc129; diluted 1 in 10) (Linke, 1984).

R e s u l t s

The clinical histories provided no data concerning EMP recurrence or meta- stases. Macroscopically, all 10 tumours were nodular in shape and of firm consistency. Ulceration of the underlying epithelium was observed in eight of 10 tumours. Other details are given in Table 1.

On histological examination, the EMPs were found to be in the corium and subcutis of the skin or lamina propria of mucous membranes. They contained varying amounts of amyloid (Fig. 1). The tumour cells--atypical plasma cells as well as mono- and multi-nucleate giant cells--were arranged in clusters separated by collagen fibres.

The amyloid in four of the EMPs occurred focally in islet-like deposits in the tumour tissue. As these deposits increased in size, they displaced the surrounding connective tissue, forming a compression capsule around the amyloid. A second type of amyloid deposition, found in six of the EMPs, was characterized by diffuse distribution. In one case only small band-shaped deposits of amyloid were found between the collagen fibres, but in three cases extensive displacement of the tumour cells by the amyloid deposits was found, which meant that the tumour tissue could be demonstrated only in isolated foci. In the other two cases, amyloid and tumour tissue were present in equal amounts.

Macrophages and foreign-body giant cells were observed in all EMPs in the vicinity of the amyloid. The foreign-body giant cells differed from the mono- and multi-nucleate giant cells of the tumour insofar as their karyoplasm was more translucent and their cytoplasm more extensive.

Immunohistochemical demonstration of the monoclonal expression of )v light chains in the plasmacytic tumour cells confirmed the diagnosis of EMP in all 10 cases (Fig. 9). No reaction of the extracellular amyloid or the amyloid in the foreign-body giant cells with the anti-s antibodies was observed in any case.

The amyloid was stained with alkaline Congo red, the staining being resistant to oxidation by potassium permanganate (Fig. 3).

Immunohistochemically, the amyloid reacted positively with all antibodies against human and equine A)~ amyloid (Fig. 4). As amyloid was thus present in all 10 cases (Table 2). In addition to the extracellular amyloid, amyloid in the cytoplasm of the foreign-body giant cells was demonstrated with anti-A~. antibodies. Positive reactions of varying degree were also recorded in the tumour cells, non-tumorous plasma cells and plasma in the blood vessels. Of the three anti-A~ antibodies used, ULI elicited the strongest reaction from

48 S.J. Platz e t al .

AL Amylo idos i s in Dogs and Cats 49

the amyloid and tumour cells in all l0 EMPs; with HIP the reaction was of medium strength and with HAR it was only slight. No positive reaction with the antibodies against AA amyloid (me 1 and mc 129) was observed in any of the 10 EMPs.

D i s c u s s i o n

AL amyloidosis is characterized by the deposition of amyloidogenic proteins derived from )~ or ~ immunoglobulin light chains (Glenner, 1980). In the present study, amyloid deposits in eight canine and two feline EMPs were examined immunohistochemically with antibodies against various types of amyloid. All the EMPs exhibited tumour cells expressing ~, light chains, and the amyloid deposits invariably reacted with antibodies against A~,-type amyloid. It was thus possible to diagnose the lesions as localized A)~ amyloidosis induced by a plasmacytic neoplasm.

The localized type of AL amyloidosis must be distinguished from the systemic type, in which deposits are found in organs such as the kidneys, heart, liver and lungs (Gertz et al., 1993): Both forms of AL amyloidosis originate from lymphoplasmacytic neoplasms. Whereas a multiple myeloma or a malignant lymphoma of the lymphoplasmacytoid type can generally be demonstrated in cases of systemic amyloidosis (Glenner, 1980), plasmacytic tumours, especially EMPs, are frequently observed in cases of localized AL- type amyloidosis (Linke et al., 1991; Krishnan et al., 1993; Rowland and Linke, 1994). The fact that EMPs are rarely associated with systemic amyloid deposition is explained by their low paraproteinaemia (Patterson et al., 1988).

Cases in which localized AL amyloidosis has been demonstrated immuno- histochemically have been described in dogs (Geisel et al., 1990; Rowland and Linke, 1994), cats (Rowland and Linke, 1994) and horses (Van Andel et al., 1988; Linke and Trautwein, 1989; Linke et al., 1991). The presence of AL amyloid has already been demonstrated by amino-acid sequence analysis in dogs (Liepnieks et al., 1994), cats (Liepnieks et al., 1991) and horses (Linke et al., 1991; Kongsrud et al., 1994; Renning et al., 1994), as well as in man (Glenner et al., 1971).

In man, plasmacytic neoplasms associated with amyloid deposits represent about 10% of all cases of multiple myeloma (Azzopardi and Lehner, 1966)

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Canine extramedullary plasmacytoma with homogeneous eosinophilic amyloid deposits. Case 6. HE. x 150.

Immunohistochemical identification of )~ light chains in the tumour cells of the canine ex- tramedullary plasmacytoma. There is no reaction on the part of the amyloid deposits. Case 6. x 300.

Congo red-staining of a canine extramedullary plasmacytoma. All red areas within the tumour are identified as amyloid by green-birefringence in polarized light. Case 6. x 300.

Immunohistochemical identification of AX amyloid in a canine extramedullary plasmacytoma. Deposits show a strong reaction with anti-A~. (ULI). There is also an intracytoplasmic reaction with the native ~. light chains in plasmacytic tumour cells. Case 6. x 300.

50 S . J . P l a t z e t al .

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and 15% of all EMPs (Batsakis, 1983). In canine EMPs, a figure of 6% was arrived at in two separate studies (Rowland et al., 1991; Platz, 1995). In cats, amyloid deposits were found in two out of five EMPs (Platz, 1995).

In all cases in which AL amyloidosis in domestic animals has been examined immunohistochemically, the presence of )~ light chains as the amyloidogenic protein has been demonstrated. In dogs, cats and horses this may be due to the physiological light chain ratio. T h e )~:~: ratio of the plasma cells in lymphatic organs is quoted as 91:9 for dogs, 92:8 for cats and 96:4 for horses (Arun et al., 1996).

In our own immunohistochemical investigations with antibodies against human and equine A)~ amyloid (HAR, ULI and HIP), a clear reaction was obtained in all canine and feline tumours, but this reaction varied in intensity. The results of our study confirm the cross-reactivity of antibodies against human amyloid (HAR, ULI) and equine amyloid (HIP) with canine and feline AL amyloid. Similar findings were obtained by others (Linke and Trautwein, 1989; Geisel et al., 1990; Linke et al., 1991). In addition, our investigations confirm that antibodies against human and equine AL amyloid recognize epitopes of the native )~ light chains, whereas antibodies against light chains do not react with the A)~ amyloid (Linke et al., 1973).

Immunohistochemical demonstration of amyloid in the foreign-body giant cells, together with a lack of reaction on the part of the )~ light chains, confirms investigations indicating that AL amyloid is phagocytized by foreign-body giant cells (Vital and Vital, 1984).

Table 3 provides an overview of all cases of AL amyloidosis described in the available literature. A)~ amyloidosis has been diagnosed in nine horses, four cats and 15 dogs to date, including the animals in our study; Ate amyloidosis has not been demonstrated in any animal to date. In all the dogs and cats, the A)~ amyloidosis was associated with an EMP. The EMPs were located on the digits or in the oral cavity in 58% of the animals, most of which were no longer young (mean age 9-7 years).

The responsible neoplasm was described only in two out of the eight cases of horses with AL amyloidosis. One of the tumours was diagnosed as a lymphohistiocytic sarcoma (Van Andel et al., 1988), the other as an EMP (Linke et al., 1991). The amyloid deposits in all the horses were located in the mucosa of the nasal cavity. Three horses had deposits in the skin and in two of these cases also in the lymph nodes. The fact that the amyloidosis, but not the underlying lymphoplasmacytic tumour, was described in the overwhelming majority of cases probably indicates that the amyloid deposits overshadowed the tumour tissue. It must be assumed that what is referred to as "cutaneous amyloidosis" in horses is in reality an EMP with amyloid deposition.

Acknowledgments

The authors wish to thank Dr D. von Bomhard for providing some of the tumour material.

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Received, March 19th, 1996 1 Accepted, September 23rd, 19961