Monoclonal Immunoglobulins

EDWARD C. FRANKLIN, M.D.

Department of Medicine, New York University School of Medicine, New York, NY 10016

ARSTRACT

Precise immunologic analysis of monoclonal immunoglobulin compo nents is essential to allow proper diagnosis, monitoring of therapy and a clearcut understanding of the biosynthetic derangements in plasma cell neoplasms. Such analyses have resulted in the delineation of a number of well-defined clinical syndromes, such as the group of heavy chain diseases.

Introduction general increase in many of them in dis eases associated with a diffuse state of There are few if any cells in the body hypergammaglobulinemia, such as is that lend themselves as readily to precise often encountered in chronic infections, biochemical analyses as the plasma cells cirrhosis of the liver, systemic lupus ery and lymphocytes. This is largely due to thematosus (SLE), rheumatoid arthritis the fact that each clone of cells secretes a and a host of other diseases. single homogeneous protein that often Thus, electrophoresis and immuno- constitutes more than 20 percent of its electrophoresis, and also immuno total synthetic product, and that the type globulin quantitation, do not generally of protein produced generally remains detect specific changes in immuno constant for many generations. Because globulins and are consequently of little there exist (1) more than 40 know n classes value diagnostically in these instances. In and subclasses of heavy (H) chains, (2) at contrast, these techniques are very useful least eight subclasses of light (L) chains in those diseases (table I) where a single and (3) many thousands of H and L chains clone of plasma cells or lymphocytes pro differing in their primary structure, it liferates to produce large amounts of a will ultimately be possible to identify homogeneous immunoglobulin or and follow the fate of virtually every immunoglobulin fragment that is readily clone biochemically by examining the detectable as a narrow band on ordinary secreted immunoglobulins.1,2 The normal serum electrophoresis. Unfortunately, the immunoglobulin fraction consists of a concentration, mobility or appearance of large number of classes and subclasses of such an electrophoretic spike on paper or immunoglobulins and thousands of dif cellulose acetate electrophoresis rarely if ferent antibody molecules. There is a ever permits a classification of the type of 181 0091-7370/80/0500-0181 $00.60 © Institute for Clinical Science, Inc. 182 FRANKLIN

TABLE I A detailed biochemical and immuno Disorders of Lymphoid and Plasma Cells logic approach to the classification of Accompanied by the Production of Homogeneous Intact Immunoglobulins, Immunoglobulin plasma cell and lymphocyte neoplasms is Polypeptide Chains and Immunoglobulin Variants of value because first, it allows precise classification of the type of disorder, Intact Immunoglobulins With or Without Free Light which is not always possible on clinical or Chains Multiple myeloma (igG, igA, igD, IgE) pathologic grounds. In certain instances, Macroglobulinemia (IgM) such as macroglobulinemia, heavy-chain Benign monoclonal gammopathy diseases or L-chain production, this clas Immunoglobulin Variants Heavy chain diseases (y, a, u; ?6 & ?e) sification correlates with the clinical fea Myeloma with abnormal immunoglobulins or tures of the disease and, on occasion, also fragments* with the prognosis. In many instances, the type of protein produced and the amount *A variety of such molecules has been described, but to date the number is insufficient to define present may influence the type of therapy clinical syndromes related to them. to be used. Secondly, it permits insights into the biosynthetic processes occurring protein, and it is usually necessary to in normal and also in pathologic neoplas employ other techniques, especially tic cells and, hence, significantly in those relying on the antigenic features of creases our understanding of factors con these molecules, if one wishes more pre trolling immunoglobulin synthesis. cise characterization of the protein Thirdly, it may ultimately lead to clini produced.4 cal and biochemical correlations of which

TABLE II* Clinical and Laboratory Features of Diseases With Monoclonal Immunoglobulins and Typical Electrophoretic Patterns Encountered in Serum and Urine

Macroglobu Benign Monoclonal Heavy Chain Diseases Multiple Myeloma linemia Gammopathy

Bone lesions Lymphaden- Lymphaden- Intestinal CLL opathy opathy lymphoma Lymphadenopathy Anemia Hepatospleno- Hepatospleno- malabsorp Hepatospleno- megaly megaly tion megaly Infections Bone Marrow Plasma cellular Lymphocytos i s Moderate plasma- Plasma cells or Lymphocytosis or infiltrates- cytosis (<10%) lymphocytoid lymphocytoid sheets lymphocytoid plasma cells plasma cells plasma cells with vacuoles Eie ctrophoresis Serum

Immunoelectrophoresis 1 type of H chain y chain 1 type of H chain Y chain a chain (Y, a/ Ô or e) (Y, ct, 6 or e ) 1 type of L chain 1 type of L 1 type of L chain No L No L Free k or X (k or X) chain (k (< or X) in 2/3 or X) K or X K or X Rare k or X Y chain or X in 2/3

♦Original table by E. C. Franklin was published in Rheumatology and Immunology, Cohen, A.S., ed., New York, Grune & Stratton, Inc., 1979, p. 426. MONOCLONAL IMMUNOGLOBULINS 183 we are not yet aware on the basis of our of albumin or other lower molecular still-limited biochemical analyses. weight protein are present, the loss in the Fourthly, careful characterization and de urine of the myeloma protein. Quantita tailed studies of the products of these cells tion of immunoglobulins generally re have provided knowledge of the normal veals a marked increase (usually greater biochemistry and physiology of plasma than 2.5 or 3.0 g per dl) of one class of cells and lymphocytes and, in the last ten immunoglobulin often associated with a years, have been directly responsible for concomitant depression of the other virtually all the important advances in classes of immunoglobulins. This depres immunochemistry that have taken place. sion of background immunoglobulins is In table II are listed the major clinical and important in the diagnosis of myeloma, laboratory features of the diseases and helps in distinguishing it from benign are illustrated typical electrophoretic monoclonal gammopathy and probably patterns encountered in serum and urine. contributes to the susceptibility to infec tion commonly seen in the disease. In general, for routine follow-up, in M u l t i p l e M y e l o m a spection of the electrophoretic pattern al lows the ready detection of changes of Multiple myeloma is the most common both the myeloma protein and the remain of these disorders.1,2,4 Approximately 98 ing gamma globulin so that more accurate percent of patients with multiple myeloma have an abnormal homogeneous quantitation need not be done often. protein in the serum, in the urine or in Examination of the urine is carried out both. The electrophoretic pattern typi most easily by the heat test for Bence cally has a prominent homogeneous pro Jones proteins but more accurately by tein peak ranging in mobility from the electrophoretic and immunoelectro- slow gamma to the alpha globulins. The phoretic analyses. It should be remem homogeneity and narrowness of the peak bered that Bence Jones proteins are often are more important than the absolute con missed by the usual albustick test. Early centration. When such a monoclonal pro in the disease, only the Bence Jones pro tein is seen on routine electrophoretic tein is usually visible; however, as renal damage occurs, the Bence Jones protein analysis, immunoelectrophoresis or immunoglobulin class and subclass quan can be obscured by other serum proteins, including the myeloma protein, that spill titation are required for a precise classifi cation of heavy and light chain class. All in the urine. It should be noted that not types of multiple myeloma give a infrequently Bence Jones proteins can be homogeneous monoclonal spike with the produced in the absence of an intact myeloma protein. exception of certain IgA myelomas, which exist as a series of polymers and therefore give a broad peak in the beta or alpha 2 B e n i g n M o n o c l o n a l G a m m o p a t h y globulin region. Approximately 20 percent of patients Closely resembling multiple myeloma, w ith m yelom a produce a Benee Jones pro but much less severe, is the entity known tein in addition to a serum myeloma pro as benign monoclonal gammopathy, tein. The Bence Jones protein and the which can involve any of the immuno myeloma protein generally have different globulins with the exception of IgM .1,2,4 electrophoretic mobilities. If the The diagnosis of benign monoclonal mobilities of the serum and urine spike gammopathy is usually one of exclusion, are identical, it is purely coincidental or i.e. in a patient having a monoclonal spike may indicate, especially if large amounts in whom the diagnosis of multiple 1 8 4 FRANKLIN myeloma cannot be made definitively on present, the patients frequently have evi clinical grounds or bone marrow examina dence of hemolysis, often precipitated by tion. In general, the concentration of the exposure to the cold. Thirdly, rheumatoid abnormal protein is less than 2 to 2.5 g per factor tests should also be carried out in dl and, in most instances, there is no as patients with macroglobulinemia since sociated depression of the other classes of many macroglobulins are positive for immunoglobulins. Bence Jones proteins rheumatoid factor activity. As was the case are rarely seen, although a few instances in multiple myeloma, patients with have been reported. It is not possible to macroglobulinemia can, on occasion, also distinguish monoclonal gammopathy produce Bence Jones proteins which are from multiple myeloma simply on the then diagnosed in a manner similar to basis of biochemical analyses. The differ those in multiple myeloma. ential diagnosis is ultimately dependent In addition to identifying the type of upon the clinical features and the absence protein produced, sera in all three of these of a characteristic diffuse bone marrow in diseases should be carefully examined for filtrate by plasma cells. the presence of cold insoluble proteins, i.e. cryoglobulins. Cryoglobulins in pa M acroglobulinemia o f tients with plasma cellular or lymphoid W a l d e n s t r o m neoplasms are frequently asymptomatic On the basis of electrophoretic analysis and are discovered accidentally in the of serum, macroglobulinemia of Walden performance of a routine laboratory pro strom is indistinguishable from multiple cedure. Cryoglobulins are easy to detect m yelom a.1,4 Although in about 75 percent since they precipitate in the cold and go of the patients the monoclonal protein is back in solution when the serum is water insoluble, as indicated by a positive heated. Precipitation usually occurs be Sia water test, this property is neither uni tw een 20° and4°C; however, occasionally versally present nor specific since a a serum has to be cooled to 0°C so that the number of myeloma proteins are also protein will precipitate. In general, if a water soluble. The diagnosis of macro cryoglobulin is present, part or almost all globulinemia of Waldenstrom is therefore of the homogeneous protein is cold insol made on the basis of immunoelectro- uble and an analysis of the supernatant phoretic analysis and/or immunoglobulin serum will allow a good estimate of the quantitation by radial immunodiffusion. amount of cryoglobulin present. Because of the high likelihood of obtain

ing positive results and the importance in H e a v y C h a i n D is e a s e s planning a proper therapeutic regimen, several additional tests should be carried Heavy chain diseases provide rather out in any patient suspected of having characteristic electrophoretic or, at times, macroglobulinemia of Waldenstrom. The immunoelectrophoretic patterns so that first among these is serum viscosity, since they can be readily recognized in the the incidence of hyperviscosity syndrome laboratory.3 is very high in patients with macro Gamma (y) Heavy Chain Disease. globulinemia and the diagnosis should be Gamma heavy chain disease is invariably made early so that appropriate therapy can associated with the presence in the serum be initiated. Secondly, a search should be and usually also in the urine of a made for cold agglutinins, since a fair per heterogeneous spike most often having a centage of macroglobulins have antibody beta globulin mobility, but occasionally activity to the big I or occasionally the also migrating with the gamma globulins. little i antigen. If cold agglutinins are The amount of protein present in the MONOCLONAL IMMUNOGLOBULINS 1 8 5 serum and in the urine can vary from trace electrophoretic analysis of the serum amounts to up to 15 g per day in the urine which shows a protein reactive only with and 3 or 4 g per dl in the serum. Gamma antisera to mu chains in the alpha 2 to heavy chain disease can be suspected on alpha 1 globulin region. Unlike patients clinical grounds and the diagnosis has to with agamma and alpha chain disease, pa be confirmed by immunoelectrophoretic tients with mu chain disease frequently analysis which shows several characteris continue to produce light chains which tic findings: (1) electrophoretic mobility fail to assemble to the deleted mu chain. of the serum and urine protein are identi As a consequence, the serum often con cal and the protein bands are usually tains a Bence Jones protein which has a broad and heterogeneous; and (2 ) different mobility from the mu chain immunoelectrophoretic analysis demon fragment. Since the mu chain fragment strates a reaction with antisera to the Fc generally exists in the serum as a polymer, fragment of the heavy chain and no reac it is rarely, if ever, found in the urine. tion with antisera to light chains. Further However, large amounts of free light analysis of the proteins usually shows chains, usually of the R type, are often them to be deleted heavy chains with a encountered. Bone marrow examination molecular weight ranging from 30 to frequently shows vacuolated plasma cells. 40,000 for the monomer or 60 to 80,000 for Although these were considered patho- the dimer. The nature of these proteins genemonic for the disease, they have now has been clearly described in several re been encountered in one patient with cent reviews. myeloma and one individual with macro- Alpha (a) Heavy Chain Disease. Alpha globulinemia associated with monomers. heavy chain disease is marked by the presence of a relatively broad spike in the O t h e r I mmunoglobulin V a r ia n t s beta or alpha globulin region which can be present in amounts that make detection To date it is not possible to recognize easy or concentrations which make distinctly other immunoglobulin variants immunoelectrophoretic analysis mandat on the basis of characteristic electro ory for detection. Alpha chain protein may phoretic or immunoelectrophoretic fea not appear in the urine at all or may be tures. In most instances, these variants are excreted in trace amounts only. Because readily detected by additional analytical of the lymphomatous involvement largely procedures such as molecular weight de in the intestine, alpha chain disease pa terminations, examination of the protein tients frequently show the same abnormal under dissociating conditions, etc. These protein in the intestinal fluids. The diag are relatively sophisticated laboratory nosis, as was the case with gamma heavy techniques that are not yet in general or chain disease, is based on immuno clinical use. electrophoretic analysis, and the demon Patients suspected of having a mono stration of a spike in the serum and urine clonal protein should have the following reactive with antisera to IgA and unreac studies. Initially, electrophoretic analysis tive with antisera to light chains. using any one of the standard supporting M u (fj,) C hain Disease. Mu chain dis media such as cellulose acetate, paper or ease is generally difficult to diagnose be agarose, should be carried out. By this cause the amount of protein produced is procedure it is possible to detect the pres usually small and therefore not readily de ence of a monoclonal homogenous band tectable on routine cellulose acetate or in the serum and the urine. Secondly, paper electrophoresis. The diagnosis is when a monoclonal band is found, it must generally dependent upon immuno- be further classified by immunoelectro- 186 FRANKLIN

phoretic analysis using specific antisera to lerance, Raynauds phenomenon or gan the major classes of immunoglobulin grene of the extremities, it should be heavy chains and to kappa and lambda noted that in recent years cryoglobulins light chains. It should be remembered are more commonly encountered in a host that certain macroglobulins and IgA of other diseases where they exist in small myelomas have non-reactive light chains, amounts and often contain more than one so that it is difficult to classify precisely class of immunoglobulin. It is now gener the nature of the associated light chains. ally accepted that these cryoglobulins In addition, to confirm the immuno- represent immune complexes and that electrophoretic results and to help distin they contribute significantly to the sys guish the benign form from the more temic manifestation seen in these patients malignant ones, it is usually advisable to by causing vasculities, most often involv quantitate the levels of the three major ing the kidneys and skin but at times also classes of immunoglobulins and IgD and other vital organs. Studies of such cryo E when indicated. globulins, which by virtue of the solubility Treatment and progress of the disease properties are easy to isolate, have proven can be followed most easily by repeated of value in identifying and isolating cer electrophoresis since the major part of the tain infectious agents associated with un immunoglobulin band happens to be the explained systemic immune complex dis monoclonal protein. If one wishes to be eases. It should be mentioned that the more precise, quantitation of the diagnosis of each of these conditions is immunoglobulin levels is useful. Exami largely dependent on histological studies nation of the urine requires the heat test of the bone marrow or lymphoid tissues for Bence Jones proteins to be carried out, and that analysis of the serum and urine usually at pH 4 and pH 7, followed by proteins by themselves is not sufficient to electrophoresis and immunoelectro- permit a clearcut differential diagnosis. phoresis to define the nature of the pro tein. It should be remembered that many R eferences Bence Jones proteins do not register on 1. F r a n k l in , E. C.: Immunoglobulin diseases. the usual dip stick test. Seminars Hemat. i0:l-86, January 1973; Additional studies that should be car 87-176, April 1973. 2. F r a n k l in , E. C. and B u x b a u m , J.: Immuno ried out on all patients with monoclonal globulin structure, synthesis, secretion, and re proteins are the following: (1) serum vis lation to neoplasms of B cells. Clinics in cosity; (2) test for cold agglutinins, either Haematology, vol. 6. Philadelphia, W. B. Saun ders Co., 1977, pp. 503-532. if there is a clinical indication or in pa 3. F r a n k l in , E. C. and F r a n g io n e , B.: Structural tients with macroglobulinemia of Wal variants of human and murine immuno denstrom and (3) cryoglobulins.1 Though globulins. Contemporary Topics in Molecular Immunology, vol. 4. Inman, F . P. and Mandy, cryoglobulins were initially described in W. J., eds. New York, Plenum Publishing Com patients with monoclonal proteins and pany, 1975, pp. 89-126. generally felt not to contribute signifi 4. Rit z m a n n , S. E. and D a n ie l s , J. C.: Serum Protein Abnormalities: Diagnostic and Clinical cantly to the clinical picture with the rare Aspects. Boston, Little, Brown and Company, exception of patients showing cold into 1975.