A n n a l s o f C l i n i c a l a n d L a b o r a t o r y S c i e n c e , Vol. 4, No. 3 Copyright © 1974, Institute for Clinical Science

Experimental Plasmacytomas in Relation to Human Multiple Myeloma

HENRY A. AZAR, M.D.

Laboratory Service, VA Hospital, Tampa, FL 33612and

Department of Pathology, University of South Florida College of Medicine, Tampa, FL 33620

ABSTRACT

Among animal models of plasma cell tumors, that induced in B A L B /c mice by means of intraperitoneal injections of oils remains the most reproducible and the most intensively studied.

The BA LB /c oil-induced and transplantable plasmacytomas resemble hu­man myeloma in their ability to produce a monoclonal immunoglobulin or Bence Jones protein. Bence Jones type nephrosis in BA LB /c mice closely mimics its human counterpart. There are also similarities in background of immunodeficiency and in antigen-binding affinity of monoclonal immuno­globulins, as well as in interesting interrelationships with malignant lympho­mas. Unlike the BA LB /c tumor model, human myeloma is, however, prin­cipally a skeletal disease, not a gut-oriented or peritoneal plasmacytoma. The intriguing presence of intracistemal type A virus-like particles in BA LB/c plasmacytoma cells and their absence in human myeloma is another major difference between the two forms of growth.

The pathogenesis of human myeloma remains obscure but the availability of experimental plasmacytoma models offers a means of systematically analyz­ing events leading to the neoplastic transformation of antibody-forming cells.

In tro d u ctio n

In addition to man, several mammals were observed to develop plasma cell tu­mors spontaneously. For example, the mul­tiple myeloma of the dog resembles human myeloma and features osteolytic lesions, monoclonal gammapathy and Bence Jones type nephropathy.16 Multiple myeloma has been also observed in a cat.10 Two plasma­cytomas have been described in the Syrian hamster: one arose in the neck and the

other in a mesenteric lymph node.4 These spontaneous tumors constitute uncommon though fascinating events. Plasma cell tu­mors can be induced in a predictable fashion in mice, particularly the BA LB /c strain. The reader is referred to Potters comprehensive review of experimental plasma cell tumors of mice.18

The aim of this presentation is to outline the characteristics of induced plasma cell tumors of mice, particularly the BA LB /c

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TA BLE I

P l a s m a c y t o m a s o f M i c e

"P lasm a C e ll Leukem ia" i n S t r e e t S t r a i n Mice 1951

R a s k - N ie l s e n , R . and G orm sen, H.

I l e o c e c a l P lasm acytom as o f C3H M ice 1954

Dunn, T .B .

In d u ced P e r i t o n e a l P lasm acytom as (BALB/c M ice) 1960

P o t t e r , M. and R o b e r t so n , C .L .

P lasm a C e ll Tumors i n NZB and NZBX BALB/c Mice 1966

W erner, N. e t a l

P leo m o rp h ic Lymphomas in S JL /J Mice 1969

M urphy, E .D .

strain, and relate these to the problem of human myeloma.

P la sm acy to m as o f M ice

The more important experimental plasma cell tumor models of mice are listed in table I. Although the reader’s attention will be drawn to the oil-induced peritoneal plasmacytomas of BA LB /c mice, a brief outline of other models of plasmacytic and related lymphoreticular growths is in order.

The “plasma cell leukemia” in Street strain mice described by Rask-Nielsen and and Gormsen20 in 1951 has the morphologic features of reticulum cell sarcoma with plasmacytic differentiation. In later studies, (C BA X D BA /2) F I hybrids, including mice injected with a variety of cell-free material, developed a high incidence of “plasma cell leukemia.”21 Several of the transplanted reticular and plasmacytic neo­plasms transplanted by Ebbesen and Rask- Nielsen9 were associated with “parapro­teinemia” and amyloidosis.

Thelma Dunn6 reported in 1954 that sev­eral mice had an inflammatory lesion of the cecum. In some of these lesions, plasma­cytic neoplasms arise in the ileocecal region and in relation to the cecitis. A later sur­vey7 indicated that ileocecal plasmacyto­mas were commoner in C3H mice than in other strains and that they occurred more frequently in old mice.

Murphy15 described in 1963 a new in- bred strain of mice (S JL / J ) with a high incidence of reticulum cell neoplasms. Fifty percent of these neoplasms were asso­ciated with “paraproteinemia.” Unfortu­nately, the production of monoclonal im­munoglobulin has been unstable following transplantation of these tumors. Fujinaga et al11 transmitted “S JL /J disease” to BA LB/c mice with cell-free extracts. They succeeded in producing reticulum cell neo­plasms but not plasmacytomas.

Plasmacytomas are successfully induced by intraperitoneal injection of oily sub­stances in BA LB/c and NZB mice as well as in (B A LB /c X NZB) F I hybrids.18 The NZB strain is also genetically susceptible to the development of nephritis and auto­immune phenomena. The oil-induced plas­macytomas are by far the most widely studied model of immunoglobulin-produc­ing experimental tumors. Primary and transplanted BA LB /c plasmacytomas are under active investigation in our labora­tory. They are considered to be a depend­able and readily available source of tumor that strikingly resembles human myeloma.

O il-Induced P la sm acy to m as o f B A L B /c M ice

In an experiment designed to test the survival of allogeneic grafts as well as the behavior of M.T.A. virus, Merwin and Al- gire13 implanted estrogen-treated BA LB/c mice with Millipore diffusion chambers containing C3H mammary tumor. Six months later, the BA LB/c recipients de­veloped hemorrhagic ascites owing to per­

E X P E R IM E N T A L PLA SM A CYTO M A S 1 5 9

itoneal plasma cell tumor or fibrosarcoma.8 Empty chambers, leucite discs or borings also caused the development of plasma cell tumors.14 Subsequently, Potter and Boyce19 reported that plasma cell tumors could be induced by means of intraperitoneal injec­tions of mineral oil alone or adjuvant mix­tures. A variety of oils have been tested since. More recently, pristane (2,6,10,14- tetramethylpentadecane) was found to be an extremely potent inducer of plasmacy­tomas in BA LB /c mice.1

The experimental model of oil-induced plasmacytomas has been described in de­tail elsewhere.18 This model has been ex­tensively exploited to demonstrate the chain of events leading to the synthesis of specific classes and types of immunoglob­ulin. The same model has been used to study the effect of hereditary and hormonal influences on the induction of plasma cell tumors. Since the majority of oil-induced plasmacytomas are immunoglobulin-form­ing tumors, the role of intestinal bacteria and bacterial antigens on the differentia­tion of plasmacytomas has also been exten­sively investigated.

M o r p h o l o g ic F e a t u r e s

The sequence of events following the intraperitoneal injections of mineral oil or pristane starts with an acute inflammatory reaction which first covers the peritoneal surfaces and surrounds oil droplets. No plasma cells or lymphocytes are noted at this stage. At a later period, from one to three months after the initial injection of the oil, individual or small clusters of plasma cells begin to form within the oil granulomas. From five months to eight months following the injection of the oil, larger clusters of plasma cell precursors are observed. Such aggregates are particularly noted under the diaphragm and over the surface of the liver. Ultimately, these gran­ules grow into small tumors which become autonomous plasmacytomas that can be

T A B L E II

N e o p l a s m s F o u n d i n B A L B / c M i c e F o l l o w i n g I n t r a p e r i t o n e a l I n j e c t i o n o f A d j u v a n t

Germ-f r e e

Ex—Germ-f r e e

Con-v en -t i o n -a l

P lasm a c e l l 2 24 28tum or

P leo m o rp h ic 16 3 4r e t ic u lu mc e l l sarcom a

Monomorphic 7 1 1re t ic u lu mc e l l sarcom a

Mixed r e t ic u lu m 2c e l l sarcom a

L ym phocytic 1■ neoplasm

Pulm onary 2 2adenoma

M y o ep ith e lio m a 1 1 1U te r in e adenoma 1R e t i c u l a r 3 4

h y p e r p la s ia(no tum or) — — —

T o ta l 33 31 40

A f te r M c ln t i r e and P r i n c l e r : Immunology 1 7 :4 8 1 , 1969.

readily transplanted. Primary or induced plasmacytomas may vary in size from one mm to two or three cm in greater diameter. Transplantable plasmacytomas grow into BA LB/c hosts or BA LB/c hybrids to attain a size of two to three cm at which point they frequently ulcerate or kill their host as a result of bowel strangulation or of Bence Jones type nephrosis.

Bence Jones type nephrosis with typical hard, fragmented or laminated casts are seen in animals bearing k or A polypeptide- producing tumors. Metastases are not seen as a rule in primary or induced plasma-

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cytomas but do develop in animals bearing ulum cells except for their basophilic andlarge transplanted plasmacytomas. pyroninophilic cytoplasm. Seldom do they

Plasmacytoma cells often resemble retie- appear to be frankly plasmacytic. Trans-

F i c u i i e 1. Electron micrograph of portion of Pristane-induced plasmacytoma cell illustrating a large number of intracisternal type A particles. Some of these appear to be budding from the endoplasmic reticulum lining the cisternae. Uranyl acetate and lead citrate, X 51,000.

E X PER IM EN T A L PLASM ACYTOM AS 1 6 1

planted plasmacytomas are even more ana­plastic looking than primary plasma cell tumors. On electron microscopy, plasma­cytoma cells generally display an abun­dance of rough endoplasmic reticulin as well as free polyribosomes.

T y p e A — I n t r a c i s t e r n a l P a r t i c l e s

Whereas the normal lymphoid tissue of the BA LB/c mouse may contain type B or C RNA viruses with well-formed nucleoids, one does not readily observe type A par­ticles in either lymphocytes or plasma cells. In contrast to the lack of type A intra­cisternal particles within reactive plasma cells, these particles are found in abun­dance within the cisternae of plasmacy­toma cells.2’5 In figure 1 is illustrated the intracisternal clustering of type A particles within a BA LB/c pristane-induced plasma­cytoma cell. It appears that type A par­ticles are markers for plasmacytoma cells although their significance is far from known. These particles are non-infective; they lack a nucleoid and are poor in RNA. They are known to be rich in DNA-poly- merase which is thought to represent a de­fective tumor virus enzyme.22 It should be emphasized that human myeloma cells are free of type A particles. Furthermore, there have been no consistent or well-docu­mented observations of any type of virus within human myeloma cells.

M o n o c l o n a l I m m u n o g l o b u l i n s

As in human myeloma, the majority of oil-induced primary plasma cell tumors are associated with production of an immuno­globulin and/or of a light-chain polypep­tide. The majority of immunoglobulins pro­duced have been monoclonal IgA. There is also a marked predominance of k over A polypeptides.18 Some of the monoclonal immunoglobulins produced by B A LB/c plasmacytomas as well as in human my­eloma cases were found to have antigen- binding properties.17’18

H0PC1 S

RPC5 S

TEPC183 S M0PC321 S RPC20 S

NP2 S M0PC315 S I

F i g u r e 2. Electrophoretic cellulose acetate pat­terns of sera from BALB/c mice bearing trans­plantable plasmacytomas. Note the M component in each of the three upper patterns.

The electrophoretic serum patterns of seven transplantable plasma cell tumors are shown in figure 2. A distinct M compo­nent is revealed in the upper three pat­terns: HOPC-1 and RPC-5 produce, re­spectively, an IgG A or k monoclonal protein; TEPC-138, an IgM k . MOPC-321 produces k polypeptide alone and its serum electrophoretic pattern shows no M com­ponent; its urine contains Bence Jones pro­tein. RPC-20 produces A polypeptide alone. Its serum as well as its urine electropho­retic patterns revealed an M component. NP-2 is a “non-producer” and shows a nor­mal pattern. MOPC-315 is an IgA pro­ducer; no M component is apparent on this electrophoretic pattern.

The monoclonicity of the class of im­munoglobulin produced by these BA LB/c transplantable plasmacytomas is revealed in the immunoelectrophoretic studies illus-

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Control S

H0PC1 S

F ig u r e 3 . Immunoelectrophoretic thin-gel agar­ose tracings of sera of normal mouse and of two BALB/c mice (RPC-5 and HOPC-1) bearing an IgG-producing monoclonal protein.

trated in figure 3. Compared to control serum, both RPC-5 and HOPC-1 demon­strate the characteristic “scooping-out” of the IgG arc which set them apart from the diffusely spread-out and even arc of the polyclonal IgG of the control serum.

E f f e c t o f B a c t e r i a l E n v i r o n m e n t

Mclntire and Princler12 reported that 60 to 80 percent of conventional and ex-germ- free BA LB/c mice develop plasma cell tumor following the injection of mineraloil into the peritoneal cavity, whereas germ-free mice develop a high incidence of lymphoreticular neoplasms of a type more primitive than plasma cell tumors. These findings of Mclntire and Princler are summarized in table I. The results suggest the importance of microbacterial flora in the development and differentiation of plasma cells in a genetically susceptible host.

G e n e t i c M a k e - U p

The BA LB/c mouse appears to be uniquely susceptible to the development of oil-induced plasma cell tumors. This ge­netic susceptibility was further studied by observing the incidence of induced plasma cell tumors in a variety of hybrid mice.

Among the various combinations tested, only the (NZB X BA LB/c) F I hybrid ap­pears to be highly susceptible to induction of plasma cell tumors. The NZB mouse it­self has a relatively high incidence rate of 20 to 30 percent. This could probably be raised if it were not for the susceptibility of this strain to early death from nephritis and autoimmune disorders.

C o m p a r i so n w ith H u m a n M u lt ip le M y e lo m a

The morphologic characteristics of hu­man multiple myeloma have been de­scribed in detail elsewhere.3 There are certain obvious similarities between the experimental plasmacytomas and human myelomas: Both forms are usually associ­ated with production of monoclonal immu­noglobulins and of Bence Jones polypep­tides. When the latter are produced in mice, the kidneys develop a Bence Jones type nephrosis which is strikingly similar to its human counterpart. Amyloidosis is not a common development in oil-induced plasma cell tumors of BA LB/c mice. It was, however, frequently observed by Eb- besen and Rask-Nielsen9 in association with their sublines of murine “plasma cell leuke­mia.” As in human myeloma, there is an interesting interrelationship with reticulum cell sarcoma which is particularly demon­strated in the spectrum of lymphoreticular neoplasms induced in germ-free mice12 and in the pleomorphic lymphomas of S JL /J mice.15

Although the pathogenesis of human my­eloma is obscure, there appears to be a background of hypogammaglobulinemia and immunodeficiency which is particu­larly evident in the “non-secretory” forms of myeloma.3 The BA LB/c mouse model may offer a similar example of immuno­deficiency. There is an initial modest rise in serum immunoglobulins following the three customaiy intraperitoneal oil injec­tions. Several months later, the oil-treated

E X P E R IM E N T A L PLA SM ACYTO M AS 1 6 3

mice develop a relative hypogammaglob­ulinemia that ushers the production of plasma cell tumors.

There are also some marked differences between the experimental mouse plasma­cytomas and human myelomas. The type A intracistemal particles have been consist­ently observed in oil-induced as well as in transplanted plasmacytoma cells but not in myeloma cells.2’5 Granulomas and other states of chronic inflammation are generally absent in the clinical period preceding hu­man myeloma. Hormonal and genetic influ­ences appear to be unimportant or of ob­scure significance in man. Finally, human myeloma is principally a skeletal disease and, as such, it is more akin to the canine or feline myeloma than to the BA LB /c oil- induced peritoneal plasmacytomas.

R eferences1. A n d e r s o n , P . N. a n d P o t t e r , M.: Induction

of plasma cell tumours in BA LB/c mice with 2,6,10,14-tetramethylpentadecane ( Pristane). Nature 222:994-995, 1969.

2. A z a r , H. A.: Significance of adjuvant-induced plasmacytomas of mice in relation to the pathogenesis of human multiple myeloma. Ann. Allerg. 26:293-298, 1968.

3. A z a r , H. A . a n d P o t t e r , M., eds.: Multiple Myeloma and Related Disorders, Vol. 1. Harper & Row, Hagerstown, MD, 428 pp.,1973.

4. C o t r a n , R. S. a n d F o r t n e r , J. G.: Serum protein abnormality in a transplantable plas­macytoma of the Syrian golden hamster. J. Nat. Cancer Inst. 28:1193-1205, 1962.

5. D a l t o n , A. J., P o t t e r , M . , a n d M e r w i n ,

R. M . : Some ultrastructural characteristics of a series of primary and transplanted plasma cell tumors of the mouse. J. Nat. Cancer Inst. 26:1221-1268, 1961.

6. D u n n , T. B.: Normal and pathologic anatomy of the reticular tissue in laboratory mice with a classification and discussion of neoplasms. J. Nat. Cancer Inst. 14:1281—1433, 1954.

7. D u n n , T. B. a n d D e r i n g e r , M. K.: Reticulum cell neoplasms type B or the “Hodgkin’s like lesion” of the mouse. J. Nat. Cancer Inst. 40: 771-821, 1968.

8. D u n n , T. B., P o t t e r , M . , F a h e y , J. L., a n d

M e r w i n , R. M.: Morphology and serum pro­

tein changes in plasma cell neoplasms in mice. Arch. De Vecchi Anat. Patol. 31:67-77, 1960.

9. E b b e s e n , P. a n d R a s k - N i e l s e n , R . : On amy­loidosis and paraproteinemia in seven trans­plantation sublines of a murine plasma cell leukemia. J. Nat. Cancer Inst. 38:723-739,1967.

10. F a r r o w , B. R. H. a n d P e n n y , R.: Multiple myeloma in a cat. J . Amer. Vet. Med. Assoc. 158:606-609, 1971.

11. F u j i n a g a , S., P o e l , W . E., W i l l i a m s , W . C., a n d D m o c h o w s k i , L .: Biological and morpho­logical studies of S JL /J strain reticulum cell neoplasms induced and transmitted serially in low leukemia strain mice. Cancer Res. 30:729- 742, 1970.

12. M c In t i r e , K. R. a n d P r i n c l e r , G . L .: Pro­longed adjuvant stimulation in germ-free BA LB /c mice: development of plasma cell neoplasia. Immunology 17:481-487, 1969.

13. M e r w i n , R. M. a n d A l g i r e , G . H.: Induction of plasma-cell neoplasms and fibrosarcomas in BA LB /c mice carrying diffusion chambers. Proc. Soc. Exp. Biol. Med. 101:437-439, 1959.

14. M e r w i n , R . M . a n d R e d m o n , L. W.: Induc­tion of plasma cell tumors and sarcomas in mice by diffusion chambers placed in the per­itoneal cavity. J . Nat. Cancer Inst. 31:998- 1017, 1963.

15. M u r p h y , E. D.: S JL /J, a new inbred strain of mouse with a high early incidence of retic­ulum-cell neoplasms. Proc. Amer. Assoc. Can­cer Res. 4:46, 1963.

16. O s b o r n e , C. A., P e r m a n , V., S a u t t e r , J. H . ,

S t e v e n s , J. B., a n d H a n l o n , G . F . : Multiple myeloma in the dog. J. Amer. Vet. Med. Assoc, i 53:10, 1300-1319, 1968.

17. P o t t e r , M.: Antigen-binding myeloma pro­teins in mice. Ann. NY Acad. Sci. 190:306- 321, 1971.

18. P o t t e r , M.: Experimental plasma cell tumors and other immunoglobulin-producing lympho- reticular neoplasms in mice. Multiple My­eloma and Related Disorders, Vol. I, Azar,H. A. and Potter, M., eds. Harper & Row, Hagerstown, MD, pp. 153-194, 1973.

19. P o tte r , M. a n d Boyce, C. R.: Induction of plasma-cell neoplasms in strain BA LB /c mice with mineral oil and mineral oil adjuvants. Nature ¿93:1086-1087, 1962.

20. R a s k - N i e l s e n , R . a n d G o r m s e n , H.: Sponta­neous and induced plasma cell neoplasia in a strain of mice. Cancer 4 :387-397, 1951.

21. R a s k - N i e l s e n , R . a n d G o r m s e n , H.: On the occurence of plasma-cell leukemia in various strains of mice. J. Nat. Cancer Inst. i6 :1137- 1147, 1956.

22. W i l s o n , S. H. a n d K u f f , E. L .: A novel DNA polymerase activity found in association with intracistemal A-type particles. Proc. Nat. Acad. Sci. 69:1531-1536, 1972.