Paraproteinemias

Paraproteinemias J. VIVIAN WELLS H. HUGH FUDENBERG

T A B L E OF C O N T E N T S GLOSSARY OF TERMS USED IN TIlE TEXT IMMUNOGLOBULIN

STRUCTURE

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INVESTIGATION OF PARAPROTEINEMIA

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CLASSIFICATION OF b,|ONOCLONAL GAMMOPATIIIES

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ETIOLOGY

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TIIERAPY

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CONCLUSION

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was graduated in Medicine (M.B., B.S.) in 1960 from Sydney University, Australia and was trained in Internal Medicine to attain membership in the Royal Australasian College of Physicians (M.R.A.C.P.). He then performed 3 years in immunology research for his Doctorate Thesis before moving to the United States in 1969 for I year with Dr. A. Nisonoff, Chairman of Biochemistry, University of Illinois at the Medical Center. Since then, he has worked with Doctor Fudenberg in San Francisco, and in 1972 was elected F.R.A.C.P. and elected to the faculty of the University of California, San Francisco. In 1973 he was admitted as a Fellow of the Royal College of Pathologists of Australia (F.R.C.P.A.). Ills major interests are monoclonal gammopathies, immunodeficiency, tumor immunology and immunogenetics.

is Professor of Medicine at the University of California School of Medicine in San Francisco and Professor of Bacteriology and Immunology at the University of California in Berkeley. Itle received his M.D. from the University of Chicago and served his internship at the University of Utah tlospital. His residency training was taken at Mr. Sinai Hospital in New York and Peter Bent Brigham Hospital in Boston. Doctor Fudenberg has added extensively to the literature and is coeditor of two recently published books, Phagocytic Mechanisms in Health and Disease and Basic immunogenetlcs.

Glossary of Terms Used in the Text BJP BMG C-region CLL EPG H chain HCD HGG HVS IEP L chain MM PV RIA V-region WM

Bence Jones protein benign monoclonal gammopathy constant region of immunoglobulin chronic lymphocytic leukemia electrophoretogram heavy chain heavy chain disease human gamma globulin hyperviscosity syndrome immunoelectrophoresis light chain multiple myeloma plasma volume radioimmunoassay variable region of immunoglobulin Waldenstr~Sm's macroglobulinemia

THE TERM "paraprotein" was introduced by Apitz in 1940 to denote an "abnormal" serum protein of restricted electrophoretic mobility, which is seen in the electrophoretogr~im as a narrow band or spike. By common usage, it has been applied to a monoclonal serum immunoglobulin that represents the protein synthesized by a single clone of lymphoid cells. The associated clinical disorders are known collectively as the "paraproteinemias" or "plasma cell dyscrasias." In 1848, the first published association between a plasma cell malignancy and a paraprotein led to the urinary protein being known as "Bence Jones protein" (BJP), unfortunately not acknowledging the contributions of the attending clinician and pathologist. However, in the past 20 years there have been numerous examples of cooperation between clinicians and scientists studying these patients and their proteins, culminating in the joint Nobel Prize in Medicine in 1972 to Drs. G. M. Edelman and R. R. Porter for the elucidation of the structure of immunoglobulins. The malignant diseases associated with paraproteins are not common, comprising only 1.5% of all malignancies. However, there are several reasons to justify the great attention they have received and to indicate that they represent an excellent model for further analysis in neoplastic diseases. For example, the tumor arises from a single cell (monoclonal gammopathy); it secretes a well-characterized and well-analyzed synthetic product (paraprotein) that can be measured accurately in tissues, serum and urine; the paraprotein contains tumor-specific antigenic determinants that are unique for that protein (idiotype); the neoplastic cells can be readily sampled and studied in in-vitro cultures; there are animal models of these'diseases in mice, dogs and rats; and there are chemotherapeutic agents available that are effective in these diseases and can be studied both in vivo and in vitro ( 1, 4, 16, 18, 23, 26, 28, 31 ). In this monograph we will discuss the investigation of the subject with a paraprotein, the clinical features of the various diseases, the 'newer concepts of the etiology and nature of plasma cell dyscrasia and the therapeutic management of the individual patient. Where possible, attempts will be made to explain the clinical features of the diseases in terms of the known pathophysiologic effects of the immunoglobulins. Our

discussions will be prefaced with a summary of relevant basic knowledge of the structure and function of the immunoglobulins while noting that much of this knowledge has come from studies in those types of patients that we will be analyzing later. The various WHO terms for the immunoglobulins and their components will be used wherever possible.

Immunoglobulin Structure Tile basic immunoglobulin molecule represents a unit comprising four polypeptide chains joined by disulfide bonds and ' noncovalent interactions (16, 23). Two are identical heavy (H ) chains of approximately 446 amino acid residues and molecular weight of 50,000-55,000 and two are identical light (L) chains of approximately 214 amino acid residues and molecular weight of 23,000 (Fig. 1). The immunoglobulins are divided into five main classes (lgG, IgA, lgM, IgD and IgE) according to the antigenic determinants in the H chains, which are designated 7, a, tt, ~ and ~, respectively. There are two classes of L chains designated ,~ and ,~, and any of the H chains can combine with either type of L chain. The classic experiments conducted by Porter with the enzyme papain on lgG led to the identification of three f r a g m c n t s i t w o Fab and one Fc. Each Fab contained an L chain and half an H chain and was able to bind antigen whereas the Fc fragment did not bind antigen. Several biologic functions reside in the Fc fragment, including the fixation of complement, binding to membranes, passage across the placenta and membranes, the rate of catabolism, etc. Studies of the amino acid sequences show that the amino-tetminal (Nterminal) residue in both the H and L chains denotes the start of the variable (V-) region, which comprises the first half of the L chain or the first quarter of the H chain. The V-region is approximately 110-115 anaino acid residues in length and display~ a high incidence of variations or substitutions in the residues, especially in the hypervariable segments, which, when folded, contribute to the combining site for antigen. The remainder of the H or L chain is the constant (C-) region, which in the H chain is composed of three "domains" of approximately equal length.

vH

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F[o. l.--Diagrammatic representation of the basic 4-polypeptide chain immunoglobulin unit. Va denotes the variable region and C,1, C.2 and C.3 the three domains in the constant region of the heavy chain. VL and CL are the corresponding parts of the light chain. The combining site for antigen is composed of segments of the folded Va and VL regions.

Recent studies have confirmed that at least two structural ~. genes are involved in the synthesis of an lmmunoglobulin polypeptide chain, one for the V-region and one for the C-region. A V-region gene that appears to represent a particular antibody specificity for a particular antigen can combine with C-region genes for tL and ), to produce IgM and IgG class antibodies with the same antibody specificity. The actual genetic mechanisms are not known. The H and L chains in an Fab region are folded so that the hypervariable segments in the V-region are in close proximity to comprise the antigen-binding site. The antigenic determinants of the combining site in a myeloma protein are,

with rare exception, u n i q u e to that p r o t e i n and a r e called "ind i v i d u a l l y specific" or " i d i o t y p i c . " T h e i m m u n o g l o b u l i n s of the different classes h a v e distinctive p h y s i c a l - c h e m i c a l , genetic, biologic and functional p r o p e r t i e s ; these are s u m m a r i z e d in T a b l e 1. I n c r e a s i n g attention is b e i n g given to the subclasses of l g G ( I g G 1, 2, 3 a n d 4 ) , each of which is under s e p a r a t e genetic control a n d is associated with a specific set of inherited antigenic d e t e r m i n a n t s ( G m f a c t o r s ) as well as subclass specific d e t e r m i n a n t s and s h a r e d i n t e r s u b class d e t e r m i n a n t s . T h e levels of the I g G subclasses in n o r m a l s e r u m a r e a p p r o x i m a t e l y 7 5 % , 1 1 % , 1 0 % and 4 % of total I g G . T h e different subclasses h a v e different biologic p r o p e r t i e s ; e.g., IgG 3 is c a t a b o l i z e d at a rate m o r e t h a n twice that of the

TABLE |.----SUMblARYOF THE PROPERTIES OF IMMUNOGLOBULINS lgG

Heavy chain class and subclasses Light chain type Molecularformula (for ~ type) Designation Sedimentation coeff. (S20. ~) Molecular weight J chain Carbohydrate ( % ) Complement fixation Genetic markers Biologic survival (plasma T 8 9 days) Serum concentration (mean; mg/100 mi) % Total Ig located in plasma Turnover in adult (gm/day) Placental transfer Reaginic activity Antibacterial lysis Viral inhibition

IgA

IgM

7 a tt ~'1'~.27g~4 al a,., /~1 p2 •andh ~candX ~r (7,c),, (a,c).o ( ( ~ ) 2 ) 5 IgG-~ 7 150,000 0 3 + Gm 21

IgA-x 7

IgM-g 19

IgD

lgE

/~ ~r (,~K)z

randX (oc)2

IgD-~c IgE-g 7 8

170,000 900,000 180,000 200,000 0 + 0 0 8 12 12 I1 0 + 0 0 Am Mm 7 7 6 5 3' 2

1000

200

120

3

0.5

50

60

70

60

60

2.3

1.7

0.5

0.03

0.0014

+

0

0

0

?

0

0

0

+ +

+ +++

+++ +

7 7

0 ++++ 7 7 9

other subclasses and binds complement the most efficiently, whereas IgG 4 does not bind complement. IgG, IgD, IgE and generally IgA and occasionally IgM occur in the serurri as the monomer. The great majority of IgM occurs as the pentamer. In secretions, the lgA is the major immunoglobulin and occurs as an I IS dimer that includes a polypcptide secretory T-piece (mol. wt. 60,000). It is synthesized in situ in the mucosa and attached to the newly synthesized immunoglobulins to facilitate their passage through and stability in the exocrine secretions. The two subclasses of IgA have striking structural differences. IgA1 comprises approximately 90% of serum lgA, and IgA2 (which lacks the disulfide bonds that link the H and L chains in all other immunoglobulins) comprises up to 50% of salivary IgA. Genetic factors have been defined on IgA2 molecules and designated A2m(1) and A,,m(2). The synthesis and assembly of the imnmnoglobulins occur within the plasma cells. It is known that H and L chains are synthesized on separate polyribosomes. Although some attachment of the L chain to the H chain may occur while the latter is still on the polyribosome, the major assembly of the whole protein occurs after the component chains have been released into the cisternae of the endoplasmie reticulum. The various assembly intermediates include HL pairs, which may bind another free H chain or may dimerize directly to H,,L,,. Whichever assembly pathway is the one operating for a particular myeloma protein, excess L chains are produced in approximately 75% of cases. In clinical multiple myeloma, the markedly increased excess of L chains is detected in the urine as BJP (16).

Investigation of Paraproteinem|a There are two broad situations in which investigations are considered: First, when a patient presents with features that suggest paraproteinemia and, second, when a paraprotein is detected during routine investigation of a patient for an apparently unrelated complaint or reason. The time-honored sequence entails taking a history and then a full physical examination before the various clinical laboratory tests and specialized investigations are performed. The relevant clinical and 10

general findings will be discussed in the classification of paraproteinemias. The basic investigations should include the following procedures: routine complete blood cottltt and hematologic tests, including crythrocyte sedimentation rate; rotttine clinical chemistry, including serum levels of total protein, albumin, globulin, calcium, phosphorus, alkaline phosphatase, urea, cholesterol, electrolytes; hemostatic prol~le (20, 25), including bleeding and clotting tests, platelet numbers and function studies and, if an abnormality is detected, specific factor assays; serum viscosity compared to distilled water in an Ostwald viscometer; bone marrow aspiration or biopsy for routine microscopy; radiology, including routine chest film and skeletal bone survey; renal Junction study, including examination of urine for protein, red cells, casts and sugar, measurement of serum creatinine and, if indicated, a creatinine clearance test. Obviously, detection of an abnormality in one or more of the above procedures will prompt further investigation. We will discuss briefly here the immunologic tests necessary for assessing a patient with paraproteinemia. The initial screening test is serttm electrophoresis after separation of tile serum at 37~ to avoid the loss of a paraprotein as a cryoprecipitate. The resulting pattern is viewed after staining of the electrophoresed proteins and is the serum electrophoretogram (EPG). The procedure may be performed on a variety of supporting materials, but the best material currently available for routine use is cellulose acetate. The paraprotein is seen as a narrow, dense band or, if an attachment is used to give a densitometric tracing, as a narrow-based spike, indicating a protein or population of molecules of restricted electrophoretic properties. Examples of paraproteins in EPGs are shown in Figure 2. Scanning of the EPG after staining permits calculation of the serum level of the paraprotein if the total serum protein concentration is known. lmmtmoelectrophoresis (IEP) is the technic that is employed in routine laboratories to type the paraprotein (Fig. 3). This is routinely performed with anfisera specific for -/, ,1, v, ~, ~. chains, and, if these are negative and the other evidence remains suggestive of a malignant paraproteinemia, antisera to 8 and c chains should be tested. The features that suggest a paraprotein 11

Notmo| Serum

II WM

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FIG. 2.--Patterns of serum electrophoretograms (EPGs) from 9 patients with paraproteinemia and I normal control, In each case the anode is on the right. The heavy chain class determined by immunoelectrophoresis (IEP) is marked below each EPG. Note the variation in electrophoretie mobility of the various paraproteins, the inability to predict the immunologic type from the EPG alone and the fact that the "scalloping" appearance is not specitie for IgM paraproteins (4 JE).

12

Flo. 3.--IEP or normal human serum (upper and center wells) and serum from a patient with an IgG paraprotein (lower well). The upper trough contains antiserum to whole human serum and the lower trough antiserum to 7 chains. Note the difference between the gradually curving precipitin arc with normal lgG and the "seagull" or "batwing" slurred arc with the lgG paraprotein. in the I E P pattern are a change in the angle of the precipitin line, i.e., the traditional "arc," " b o w " or "seagull" deformity; a marked reduction in the length or a m o u n t of " n o r m a l " precipitate for the same class ,as a possible arc deformity; a marked change in the density of the precipitate, which m a y show a typical arc deformity when the serum is rerun in dilutions; and a localized r e d u p l i c a t i o n of precipitin lines. It is crucial that, with routine I E P patterns for the great majority of all paraproteins tested, there should be a corresponding arc deformity with either anti-K or anii-X but not with both. Detection o] Bence Jones protein (BJP). This is an important and necessary part of the assessment of a patient with paraproteinemia but it is only very infrequently tested for properly 13

and adequately. The traditional heating test is quite unsatisfactory, as it yields a high percentage of false negatives, even with significant amounts of BJP in the urine. Bradshaw's test is satisfactory as a general screening test and is performed by layering the urine over concentrated hydrochloric acid (16). The best definitive procedure for the detection of BJP is electrophoresis of a concentrated urine sample and staining for protein. Preferably, it is run at the same time as the serum EPG to permit direct comparison of the relative electrophoretic mobilities of any proteins detected. If any BJP is found, it can be typed and confirmed by IEP of the same urine concentrate, again comparing it directly to the serum sample from the same patient (16). BJP should be of one type, cither ,c or ,X, but not both. Abnormal narrow bands in the serum EPG that must be distinguished from paraproteins include denatured proteins in old or uremic serum, fibrinogen, free hemoglobin, excess transferrin and excess lipoproteins. Bands in the urine EPG that may be misinterpreted initially include free hemoglobin, transferrin, lysozyme, excess al-glycoproteins or, rarely, other mucoproteins in malignant disorders (16). Consideration of the age and color of the test sample, attention to the electrophoretic mobility of the band on the EPG and especially accurate interpretation of tile IEPs should clarify any doubts on a narrow band in a particular EPG. Measurement of the serum levels of the individual immunoglobulins usually is performed by radial immunodiffusion (RID) in specific antibody-agar plates (2, 6, 16, 19). The method of radioimmunoassay (RIA) is achieving wider use, especially for IgD and IgE with their much lower normal serum levels. The application of immunofluorescent technics is rapidly growing in the study of bone marrow specimens, lymph node antisera are labeled with fluorescein or rhodamine and the disbiopsies and lymphocytes in peripheral blood (27). Specific tribution of cells synthesizing the respective types of immunoglobulins is sought, studying both intracytoplasmic and surface membrane immunoglobulin in conjunction with routine microscopy. Ancillary investigations should include the following in appropriate cases: Euglobulhz or SILl water-test. Euglobulins 14

are proteins that precipitate in solutions of low ionic strength and are detected routinely by adding 1 or 2 drops of serum to 5 ml of distilled water in a test tube. Any resulting precipitate can be isolated by centrifugation and its amount determined. It is most frequently positive with IgM paraproteins (approximately 55% are positive) but is quite nonspecific and is positive in many sera with polyclonal hyperimmunoglobulinemia. Cryoprecipitation is tested by placing 1 ml of serum (after separation at 37 ~ C) in a test tube in a 4" refrigerator for 72 hours. Any resulting precipitate is dissolved in warm saline and its concentration determined. The nature of the precipitate with both euglobulins and cryoglobulins should be verified by IEP with both polyvalent and specific antisera. Rheumatoid ]actor and cold agglutinin tests are performed on the serum by routine methods and, if positive, the paraprotein should be isolated in pure form and retested to determine whether the antibody activity resides in the paraprotein fraction. Analytic ultracentri[ugation of serum is an ancillary investigation the use of which depends primarily on its availability. It is not required as a routine but can be quite helpful in equivocal cases in which information concerning the molecular size of a paraprotein may be contributory. It permits one to determine the presence of an excess of 7S protein, 19S protein, the occurrence of polymers in the range 9-13S (such as occurs with some IgA paraproteins), 13S polymers, which occur in Waldenstr~Sm's hypergammaglobulinemic purpura due to monoclonal lgG binding autologous IgG, or complexes of rheumatoid factors greater than 19S. Radioisotope studies may be indicated for me~urement of whole blood, plasma and red cell volumes (21), especially if there is evidence of an increased serum viscosity. Assessment of the immune status of the patient may be required if infection is the main clinical presentation and recurrent infections continue as an important clinical feature. Cellular immunity may be assessed by skin testing for delayed hypersensitivity to specific antigens, lymphocyte transformation and rosette tests; humoral immunity may be assessed by measuring the antibody response to defined antigens; and granulocyte function may be assessed by neutrophil counts and granulocyte 15

function tests for mobilization, adherence, phagocytosis and killing (25). Specialized immunologic studies, such as measurement of immunoglobulin metabolism in vivo with radio-labeled immunoglobulins (33) and immunoglobulin synthesis by biopsy material (28), will be described later, as will the application of selected examples of these procedures to the management of individual patients with specific chemotherapy.

Classification of Monoclonal Gammopathies There are several detailed published reports of large studies and investigations of subjects with confirmed paraproteinemia and there are, accordingly, several conflicting published classifications (16-19, 34). The one we currently use in our laboratory is summarized in Table 2. A classification, such as this should not be "nailed to the door" and defended as a tenet of faith; we use it only as a convenient means of expressing our current state of knowledge, our ideas on the nature of the diseases, our appreciation of the various clinicopathologic correlations and the most reasonable approaches to the various forms of management of the individual patient with paraproteinemia. The two diseases that classically are considered first when a serum paraprotein is detected are multiple myeloma (MM) and Waldenstr~Sm's macroglobulinemia (WM). The key features from our studies are listed in Table 3 and are in close agreement with published data from several studies. MM classically displays recurrent serious infections, lytic'bone lesions, anemia, renal abnormalities and BJP in the urine in a high percentage of patients. WM classically presents with a variety of hemostatic disturbances, especially recurrent epistaxes and gastrointestinal bleeding, lymphoid organomegaly and the stigmata of the hyperviscosity syndrome (HVS, Table 4). HVS is seen in most patients with WM requiring in,patient care but is also seen in a small but significant percentage of MM patients. Severe HVS warrants immediate attention as a medical emergency. A serum viscosity greater than 2.0 is significantly increased and a level greater than 3.0 usually is accompanied by symptoms. Symp16

TABLE 2.------CLASSIFICATIONOF

DISORDERS WITI! I'ARAPROTEINS (MoNoCLONAL IMMUNOGLOBULINS)

I.

Malignant Monoclonal Gammopathy

1. 2. 3. 4. 5. 6. 7.

Multiple rnyeloma (MM) Waldenstr/Sm'smacroglobulinemia (WM) Solitary immunocytoma Amyloidosis Heavy chain diseases Malignant lymphoma Chronic lymphocytic leukemia (CLL)

II. Associated l~lonoclonaI Ganznlopathy I. Neoplasia of nonimmunocytes 2. Monocytic leukemia 3. Liver cirrhosis 4. Chronic inflammatory disease 5. Autoimmunity 6. Cold agglutinin syndrome 7. Papular mucinosis 8. Immune deficiency III. Benign Monoclonal Gammopathy (BMG) 1. Iatrogenic 2. Transient 3. Persistent IV. Pseudomyeloma toms other than fatigue usually do not occur until the serum viscosity is greater than 4.0, and occasionally the viscosity may be well tolerated up to a level of 6.0. In general, however, a serum viscosity greater than 4.0 produces some of the symptoms listed in Table 4 and the level of IgM paraprotcin df 2 g m / 1 0 0 ml approximately corresponds to this degree of viscosity (10, 21). HVS in MM is associated most frequently with polymer IgA paraproteins or with IgG paraproteins (especially IgG 3). The factors contributing to HVS in MM include a high serum concentration of paraprotein, abnormal shape and polymer formation, and aggregate formation, whereas the factors in WM include a high serum concentration of paraprotein, its large size (19S and greater) and its largely intravascular localization ( 7 0 80% ). If the paraprotein has cryoglobulin properties, this may contribute further to the HVS ( 1 5 ) . 17

T A B L E 3.----COMPARISON OF TIIE ~IAIN FEATURES OF ~,tULTIPLE I~iYELOMA (1~,11~1) AND ~VALDENSTR6M'S ~IACROGLOBULINEMIA ( W ~ l ) PARAMETER

Recurrent infections Bone pain Lytic bone lesions Mucosal bleeding llepatomegaly Lymphadenopathy Neuropathy Mental changes Alterations in visual acuity Fundal abnormalities Leukopenia Anemia Thrombocytopenia tlypercalcemia Serum hyperviscosity Renal insufficiency

M3,i

Vq~f

+++ +q-q+++ + -q+ -t+ -+ +++ + +++ + +++

---+++ +++ +-I-q-+++ +++ +++ -+++ --+++ --

TABLE 4.--FEATURESOF TIlE IIYPERVISCOSITYSYNDROME (HVS) General:

Weakness, fatigue, anorexia

Cardiovascular:

Congestive cardiac failure, hypervolemia Headache. dizziness, vertigo, nystagmus, deafness, postural hypotension, somnolence, stupor, coma, generalized seizures, EEG abnormalities

Central nervotts system: Hematologic:

Ocular:

Epistaxis, oral mucosal bleeding, hematuria, hematemesis, melena, postoperative prolonged bleeding, anemia Loss of visual acuity (may be complete), distended tortuous retinal veins, retinal hemorrhages, papilledema

A n e m i a is a very c o m m o n p r o b l e m in both M M and W M and has a multifactorial etiology. I n M M , one must consider the effects of the extensive infiltration of the b o n e m a r r o w with plasma cells a n d the effects of serious repeated infections. Blood loss contributes to the a n e m i a in both M M a n d W M and in m a n y cases is due to a b n o r m a l i t i e s in hemostasis. We have also noted a previously unrecognized finding in that a history of a rela18

tively recently confirmed peptic ulcer is" not uncommon in patients presenting with WM. The hemostatic abnormalities include thrombocytopenia, qualitatively abnormal platelet function, suppression of one or more of the specific coagulation factors or nonspecific effects. In some cases ( 2 5 ) , these abnormalities appear to be due to the physical presence of the paraprotein. In rare cases, they may occur because the paraprotein has antibody activity to one of the factors (e.g., factor A H G - V I I I ) , although in many cases the mechanisms are not known. The latter often is the case when it is clearly abnormal bleeding associated with a marked HVS. An increased incidence of thromboembolic phenomena in paraproteinemia has been emphasized recently (20). A variable feature that contributes to the degree of anemia based simply on measurement of the hemoglobin level is the degree of hemodilution. An increase in whole blood volume due to an increase in plasma volume ( P V ) is not uncommon while at the same time there is only a small reduction in red cell mass. The changes are mainly related to the increased PV, which is more marked and more frequent in WM. Whereas the normal PV in adults has a mean around 41 ml/kg, values in MM are not uncommon in the range 45-55 ml/kg and values over 100 ml/kg are not uncommon in patients presenting with established WM (10, 16, 21, 32, 33). The importance of these observations will become evident when we discuss the management of these patients. Of course, WM represents excessive proliferation of a clone producing an IgM paraprotein whereas MM exists with IgG, IgA, IgD, lgE, ,, or A paraproteins or combinations thereof. When these immunochemical designations are examined in conjunction with routine morphologic analysis of bone marrow or other tissues, several problems arise. It still remains basically true in WM that the cell populations that are increased resemble "activated" o r relatively immature or "plasmacytic" lymphoid cells or lymphocytes, whereas in MM the increased cell population comprises plasma tzells. However, the plethora of terms used in studying the cells in WM emphasizes the pleomorphism of cells variously involved in IgM synthesis. Such bone marrows also may show increased numbers of fairly normal mature plasma cells and mature lymphocytes. An IgM paraprotein can be associated with a variety of clinical and histopathologic find19

ings. Thus, one may see increased lymphocytes and an IgM paraprotein with a classic presentation of WM, in a patient who is diagnosed as having malignant lymphoma or in a lpatient diagnosed as having CLL. In a recent study of 50 cases of CLL, not a single patient was found who had completely normal serum immunoglobulin levels. Hobbs (16) has suggested that the paraproteinemia in patients with these varying clinical syndromes associated with monoclonal lgM proteins warrants the designation "malignant" when there is a significant amount of the protein existing in the monomer 8S form. The concept is that the immunocytoma in such patients is not capable of secreting a normally synthesized IgM 19S pentamer and this suggests functional dedifferentiation. It should be noted that increased amounts of 7S polyclonal IgM occur in the serum of patients with connective tissue or such autoimmune disorders as systemic lupus erythematosus. The application of immunofluorescent technics with specific antisera to the analysis of bone marrow and other tissues of WM has produced very interesting data (27). In lymph nodes and bone marrow, the plasma cells usually stained for IgM in the cytoplasm but a surprisingly high number of the lymphocytes in bone marrow showed no staining at all, so that the over-all incidence of IgM-positive cells in lymphoid cells was low. The hypothesis was presented that those cells that contained no intracytoplasmic IgM bore surface-bound membrane monoclonal IgM; this has been demonstrated now in several examples of patients with CLL. In the future, consideration should be given to the routine analysis of bone marrow specimens and peripheral blood smears with immunofluorescent technics. When MM is the presumptive diagnosis, it is not sufficient to note merely an increased number of plasma cells (a plasma cell count over 5% nucleated bone marrow cells represents a significant increase). Inflammation frequently is accompanied by this degree of plasmacytosis; in MM, the plasma cell count generally is over 10%, with an average of 32% (34). The main point, then, is to look for the predominance of abnormal, multinucleated, immature plasma cells especially distributed in sheets or packets. These are rare in simple inflammatory plasmacytosis. Bone marrow biopsy may be required to confirm the disposition 20

of immature plasma cells in some patients with MM. Whereas the above observations on cytologic abnormalities have been found to be consistently reliable, this certainly is not true when simple morphologic study is used to "type" a paraprotein. Occasionally it is correct; e.g., with some cases of "flaming cell" plasma cells and IgA paraproteins, but the sensible way to type a paraprotein is by IEP. Determination of the type or class of a paraprotein is an integral part of diagnosis. Rarely, the clinical features of MM, including renal and bone abnormalities, may be associated with an lgM paraprotein or, more frequently, the clinical features of WM and HVS may be associated with an IgG paraprotein with no obvious renal or bone abnormalities at the time of presentation. Typing is more important, however, when one considers the various parameters in MM with different types of paraproteins. In this respect, BJP-MM refers to those patients with clinical and histologic evidence of MM in whom the only detectable paraprotein is ~ or ~. BJP in serum and/or urine. We have summarized in Table 5 some published data of Hobbs (16) in 212 cases of proved MM that confirm the general clinical impression that BJP-MM and, to a lesser extent, IgA-MM represent more extensive or more quickly growing tumors with more serious complications with renal damage, bone lesions and hypercalcemia. Rarely, MM occurs without paraproteins (3). Renal damage is a frequent finding in paraproteinemia. This damage is mediated through several mechanisms. In approximately half of the cases, the lesion is directly related to the presence of the paraprotein itself (16). Thus, myel~ma kidney refers to blockage of the distal tubules with the casts containing paraprotein (especially BJP) and it is this syndrome that has beenassociated with acute renal failure following fluid restriction for intravenous pyelography. Other ways in which renal damage can occur include amyloidosis, hypercalcemia, actual invasion of the kidney by tumor, cryoprccipitates, possibly hyperuricemia and other as yet unexplained methods. In HVS, it is possible that this, per se, produces a loss in concentratingdiluting ability but the superimposition of HVS on the above established renal abnormalities certainly exacerbates the renal 2]

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abnormalities (5, 16). Typing the paraproteins thus aids in prognosis. A crucial consideration is the relationship between malignant paraproteinemias and the group of subjects with benign monoclonal gammopathy ( B M G ) . It should be emphasized here that the diagnosis of MM is the prerogative of the attending clinician. It is based on evidence of clinical features, the presence of a paraprotein, histologic evidence of increased numbers of atypical plasma cells and radiologic evidence of discrete bone lesions. In several patients, the four groups of criteria may not be satisfied on initial presentation (or, rarely, during his entire course), but the diagnosis is not warranted on the basis of only one piece of evidence. BMG refers to the finding of a paraprotein in the serum E P G without the accompanying clinical stigmata and pathologic features that would permit a diagnosis. The second crucial consideration in all these discussions is time. This is a "temporal" diagnosis, since, not infrequently, one finds that with observation and continued study other manifestations arise that indicate the correct diagnosis. There are several studies of the incidence of BMG and although there is a range of values, an average of approximately 3 % of Caucasians over the age of 50 have BMG (16-19, 24, 34). The incidence is close to 1% at the age of 50 and increases with increasing age. With observation, some of these undergo "transition" to a disease that warrants diagnosis as MM but this "latent period" may be 24 years ( 2 4 ) . The majority, however, remain essentially as BMG and it therefore becomes of major importance to try to classify the patient initially as "malignant" paraproteinemia or BMG, since the latter need not be treated with cytotoxic therapy. Basically, ' no single observation or measurement is adequate, and one relies on an assessment of five points to distinguish malignant and benign paraproteinemia: l. Serum paraprotein level > 1 gm/100 ml (ratio of malignant to benign 6.1 to I). 2. Reduced serum levels of normal immunoglobulins (ratio of malignant to benign 9.8 to 1). 3. Progressive increase in serum paraprotein in untreated subjects (ratio of malignant to benign 99 to 1). 23

4. Presence of immunoglobulin fragments (ratio of malignant to benign 84 to 1). 5. Increased serum levels of free L chains > 20/~g/ml (ratio of malignant to benign 2.4 to 1). Clearly, the patient with a higher and increasing serum paraprotein level, low serum levels of normal immunoglobulins and marked amounts of BJP in serum and urine is at highest risk to present the stigmata of MM within a relatively short time. Solitary immunocytoma is another diagnosis that requires consideration of the temporal factor. We have seen examples of IgG paraproteinemia with a solitary bone lesion (or, more rarely, an extramedullary plasmacytoma) containing sheets of atypical plasma cells, which remained the only stigma of MM for periods of observation up to 12 months, when obvious signs of dissemination appeared. In one patient with solitary plasmacytoma of the manubrium sterni, irradiation of the lesion was accompanied by disappearance of small IgG-A paraprotein from the serum EPG but the patient relapsed within 6 months with paraproteinemia, multiple bone lesions and classic MM. The control mechanisms for the prolonged existence of paraproteins in some patients with no clinical sequelae are not known. Amyloidosis has long been an intriguing and unsolved puzzle to pathologists. For years, research into this disease was limited to defining its clinical associations, geographic distribution, anatomic localization and tinctorial properties. When methods eventually were developed that permitted solubilization of the amyloid fibrils that represent file basic unit of amyloid tissue, several interesting findings appeared that have contributed greatly to our understanding of this disease. In many but not all cases of amyldidosis, the major protein of the characteristic amyloid fibril was found to be derived from immunoglobulin. Conflicting claims had been published earlier by different laboratories on the detection of irnmunoglobulins in amyloid tissue by means of immunofluorescent anti-immunoglobulin antisera. The problem was that the great majority of those antisera had antibodies mainly directed to C-region determinants whereas an antiserum directed to idiotypic determinants in the V-region clearly reacted with amyloid tissue and with a serum component. Amino acid sequence analysis of material purified from amyloid 2,1

fibrils" conlirmed that in many cases the" main component comprised parts of L chains, especially the V-regions of ,~ chains. This may occur in both primary and secondary amyloidosis by one or more of four mechanisms. First, antigen-antibody complexes that are likely to be seen especially in patients with chronic infections and reticuloendothelial hyperplasia may be degraded in macrophages in liver and spleen in such a way as to be deposited as amyloid fibrils in secondary amyloidosis. Second, excess circulating intact L chains or whole immunoglobulin synthesized by patients with MM may precipitate as amyloid fibrils in the cardiovascular system as primary amyloidosis. Third, deletions in the L chain gene(s) may produce fragments of L chains analogous to the fragments in heavy chain disease (see below). Fourth, there might be synthesis of Vregions and C-regions by discrete V- and C-region L chain genes in special circumstances (14). It is possible to take a series of BJP from patients with MM or WM and subject each BJP to various enzymes and temperature and pH conditions. A small number of the BJPs (especially ,x. type) will produce a product that is indistinguishable in microscopic appearance, birefringence, antigenicity and tinctorial properties from native amyloid fibrils in vivo (14). Osserman has suggested that the BJPs from patients who develop amyloid tissue show a significantly higher affinity for binding to certain tissues in vitro comp,qrcd to nonamyloidogenic BJP. The clinical counterparts to these observations are obvious. Amyioid is seen in increased incidence in patients with paraproteinemia, especially those who are clearly designated as having malignant paraproteinemia. It may appear late in tile course as a complication, and otherwise unexplained neuropathy, cardiopathy, malabsorption or nephropathy in a patient with proved MM should be investigated for amyloidosis. The prognosis in such cases generally is poor. as there is as yet no effective treatment for established amyloidosis. Heavy chain diseases"(HCD) comprise a group of rare but interesting diseases characterized by the presence in the serum of a paraprotein composed of incomplete H chains without L chains (9, 29). The three types correspond to the H chains of the three major immunoglobulins and are designated yHCD, 25

first described in 1963 (30 known cases), aHCD, first described in 1968 (approximately 40 known eases), and ~HCD, first described in 1970 (only 3 known cases). Although the diagnosis is suspected by certain clinical and laboratory features, it can be established only by analysis of the HCD protein. This can easily be missed in a routine EPG, as it tends to give a broad rather than a narrow band, but the protein often is found in appreciable amounts in the urine. In many cases, no abnormality is seen on routine inspection of the EPG (29). IEP is essential and must be run with specific antisera. The component must be shown to react with antisera to 7, '~ or # but not ,c or ~.. Clinically, none of the reported cases of ~,HCD or ,~HCD resemble MM. There is marked variation in the clinical features of 7HCD, but the most common presentation is that of a lymphoproliferative disorder with hepatosplenomegaly, lymphadenopathy and uvular and palatal edema (9, 29). Some patients, however, do not present with lymphoproliferation but with recurrent febrile episodes. The onset and course of the disease may be sudden or insidious, lasting a few weeks or up to 5 years to death. Infection is tile most common cause of death. Anemia is almost invariable and leukopenia is common, with atypical peripheral blood lymphocytosis or plasmacytosis. The clinical picture in aHCD is quite uniform, with a severe malabsorption syndrome with chronic diarrhea, weight loss, steatorrhea, hypocalcemia, abdominal lymphadenopathy and histologic evidence of involvement of the larger part of the small bowel with mature plasma cells, lymphocytes and, to a lesser extent, reticulum cells. With progression of the disease, the plasma cells appear more atypical, and extension outside the lamina propria occurs. Two cases of ,~HCD have occurred that involved the respiratory tract in a similar fashion (9). Only 3 cases of tLHCD have been reported and all occurred in long standing C L L with progressive hepatosplenomegaly. In each case, tile bone marrow contained unusual plasma cells with vacuoles. Studies of the abnormal proteins in the HCD have yielded important information concerning the genetics of the synthesis of the immunoglobulins. The HCD may be found with a partial deletion in the Fd fragment of the H chain (with normal se26

quence from residue 216 onward), fi deletion in the hinge region or a combination of the two abnormalities. Cryoglobulins were noted earlier as proteins that precipitate when stored at 4 ~ C for 72 hours, and our discussion will be directed to cryoimmunoglobulins, while noting that other plasma proteins may show cryoactivity, including cryofibrinogen, a C-reactive protein-albumin complex, heparin-precipitable protein and a nonclotting component of Cohn Fraction I-1 (15). It should be emphasized that during routine separation of blood at room temperature some cryoimmunoglobulins may be missed, as they already may have precipitated by that temperature. The term cryocrit refers to the ratio of cryoprecipitate to whole serum after centrifugation of serum stored in 2 X 15 mm capillary tubes. With some proteins, the precipitate is readily visible within a few hours whereas others may require the full 72 hours to become obvious. Frequently it is not appreciated that a small amount of cryoimmunoglobulin is very common in normal subjects, up to 80 t~g/ml. The term usually is applied to subjects with pathologic amounts of such cryoprecipitate. The cryoimmunoglobulins are classified by the nature of the precipitate into three main types, designated monoclonal, mixed and polyclonal, according to the results of IEP and EPG studies of the purified cryoprecipitate. Monoclonal proteins constitute approximately 25% o f a large series of cryoimmtinoglobulins and the majority are either IgM or IgG (by definition, of only one L chain type). Occasionally a monoclonal immunoglobulin may be composed of IgA, ~ chains or A chains. The second type, mixed cryoimmunoglobulins, refers to the fact that cryoprecipitation occurs only when a monoclonal protein (almost always IgM, of one L chain type) is complexed with autologous IgG of mixed L chain types. Ultracentrifugal studies have indicated that although the monoclonal IgM often has rheumatoid factorlike activity, the strength of the binding between the IgM and the IgG in the cryoprecipitate apparently is weaker than that of classic rheumatoid factors with the formation of large complexes (22S and higher). The third type generally is the most frequent, the cryoprecipitate being composed of polyclonal immunoglobulin. The majority of cases contain IgM and IgG; occasionally it is composed of IgM only or a combination of 27

IgM, IgA and IgG. The latter are seen most frequently in chronic infections, autoimmune diseases, such as systemic,lupus erythematosus, or other diseases associated with chronic reticuloendothelial hyperplasia. Cryoimmunoglobulin activity has been found in 7% of a series of patients with WM whereas it is found less frequently in MM. The clinical features directly attributable to the cryo nature of the protein vary in individual patients. On occasions, it produces features that are clearly distinct from those usually found in the parent disease. For example, a patient with a monoclonal cryo-IgM presented with gastrointestinal symptoms that subsequently were confirmed as marked malabsorption due to the precipitation of the IgM in the bowel. The more usual clinical features include cold intolerance with typical Raynaud's phenomenon, dependent purpura, cutaneous vasculitis with pain t h a t may progress to frank ulceration, retinal hemorrhages and thrombotic phenomena. Cold urticaria has n o t been directly proved to be a manifestation of cryoimmunoglobulin. As more studies are performed, it is becoming obvious that cases of type 1 occurring in WM or MM frequently are in fact type 2, with small amounts of autologous lgG in a weakly bound complex. Originally, the interest in mixed cryoimmunoglobulins arose with studies of a syndrome of essential mixed cryoimmunoglobulinemia with dependent purpura, arthralgia, weakness and frequent hepatosplenomegaly, lymphadenopathy and mild anemia. Sj/Sgren's syndrome, progressive glomerulonephritis and myositis occurred occasionally and these findings were all interpreted as indicating the existence of circulating immune complexes in vivo (without etiologic significance) (15): As a result, it has become more sensible to consider cryoimmunoglobulins from two points of view in addition to characterizing the nature of the cryoprecipitate. First, at what temperature does significant precipitation occur? Second, is the cryoimmunoglobulin associated with anti-7-globulin or rheumatoid-like activity? Those that possess no anti-:/-globulin activity must rely on special properties of their structure to account for their abnormal insolubility at lower temperatures. Those that possess anti-v-globulin activity appear to have the immune complexes as a key consideration in pathophysiology, and currently studies are directed toward de28

fining the nature of the antibody activity of the monoclonal protein and the determinants toward which it is directed. Biclonal gammopathy refers to the existence in one patient of two distinct monoclonal proteins. It does not include the situation in which the patient has, for example, IgG-K protein and x BJP. It occurs in approximately only 1% of a large series of paraproteinemias, but the incidence increases in any study in which they are sought more frequently with more sophisticated technics. Other causes that might give the appearance of two distinct paraproteins must be excluded, including polymerization of IgA, so that a notched, slurred or even double appearance is seen in the EPG; complexing of the paraprotein with other proteins such as albumin; deamidization of the paraprotein after secretion; and degradation of the protein, for example, with bacterial contamination (16). Two main groups then remain-one in which the proteins are products of two distinct clones and one in which the paraproteins are the products of cells with some sharing of genetic material. Studies of the latter proteins demonstrate that they have the same V-region but this is combined with different C-regions, for example, the same binding specificity for antigen for monoclonal IgG and IgM proteins in the same patient. In general, the clinical features are those of the original or predominant monoclonal protein. Thus, clinical WM is seen with IgM and IgG paraproteins, with lgM representing the main paraprotein. It appears that CLL is a potentially frequent precursor of the existence of biclonal gammopathy. The most frequent combinations appear to be IgM and lgG and IgG and lgA. Very rarely, triclonal gammopathy may be found. Associated monoclonal gammopathy refers to various diseases or syndromes in which the existence of paraproteinemia is observed more frequently than is expected in a particular disease. The primary disease possibly is of pathogcnetic significance or the paraprotein appears to have antibody activity. Analysis of these various syndromes has aided our understanding of tile possible etiology of paraproteinemia (see Etiology, below). All published reports of large series of paraproteins have noted the presence o f paraproteinemia in patients with various malignancies of nonimmunocytes, especially carcinoma of the 29

rectosigmoid region, prostate, breast, stomach and lung (16-19, 34). In the great majority of cases, the clinical features are those of the primary malignancy and not of the paraproteinemia. A very interesting, special, but as yet not fully understood, relationship has been recognized between acute leukemia and MM. There now are at least 25 fully documented cases of acute leukemia occurring in malignant paraproteinemia (23 MM, 1 WM, 1 amyloidosis). The acute leukemia generally is diagnosed 1-10 years after the paraproteinemia, and the main problem is deciding whether it is part of the basic immunocyte abnormality, which is obvious only when the survival of the patient has been significantly prolonged by therapy, or whether it has been induced by the therapy. This issue is not yet settled but it is possible that it is due to chromosomal aberrations induced by the cytotoxic therapy. The leukemia has been defined as acute monocytic or acute myelomonocytic leukemia in 24 cases and as erythroleukemia in 1 case. No case of lymphocytic leukemia has been reported. It is a serious complication, with none of the patients surviving 6 months thus far. Chronic inflammatory diseases, especially chronic biliary tract disease and chronic allergic disease, have long been implicated by some investigators in the development of paraproteinemia (17, 34), but not all series have emphasized this association. Another interesting association is that of paraproteinemia and autoimmunity diseases, such as systemic lupus erythematosus, rheumatoid arthritis, etc., although here, too, it may be induced by therapy. The cold agglutinin syndrome denotes clinicopathologic features associated with the existence of a lYaraprotein (classically IgM-~) with antibody specificity for an antigen in the red cell membrane I system. The paraproteins in these patients show many similarities but also, in some cases, differences in clinical severity, the temperature at which their maximal effects are seen on red cells and their specificity. Papular mucinosis is a rare dermatologic syndrome (formerly called lichen myxedematosus) in which the patient has outbreaks of small, nontender papules on the face, limbs and trunk that gradually are replaced over the years by a "sclerodermatous" appearance with fibrosis. These patients almost invariably 30

have a very basic IgG 1-A monoclonal protein that is postulated rebut not yet proved--to be a monoclonal antibody to an acidic component in the papules. The final interesting example of associated monoclonal gammopathy we will mention is immune deficiency. The majority of examples of paraproteinemia observed in infants or children occur in patients with proved immune deficiency. It is possible that these paraproteins represent monoclonal antibodies to determinants of an infecting organism and are indicative of the restricted capacity of the subject's immune system so that a normal polycional response is not possible. BMG has been discussed above as a persistent paraproteinemia that warrants distinction from malignant paraproteinemia, the latter probably requiring active therapy. Two additional points will be mentioned. One is the rare report of paraproteinemia following therapy with a sulfa drug, which disappears with cessation of drug therapy (iatrogenic BMG). The other is the occasional instance in which a paraprotein is detected that gradually disappears spontaneously under supervision (transient BMG).

Etiology Despite extensive" studies, especially in the past decade, the basic mechanisms involved in the etiology of paraproteinemia still remain not known. In particular, we do not know the events that initiate the emergence of the clone, the factors that indicate abnormal control with continued replication and protein synthesis, the mechanisms involved in defining whether a particular clone will be benign, malignant or progress from benign to malignant or the exact nature of the changes that occur in the abnormal cells when they become resistant to chemotherapy. Many aspects of our understanding of paraproteinemia have come from extensive studies in animal models (26). Mice and rats are the animals most studied as models for human paraproteinemias, but it should be pointed out that although these species have many similarities in terms of the monoclonal proteins, mice and rats do not develop the clinical features we have described in human patients with MM or WM. In dogs, how31

ever, one may see a disease with paraproteinemia that closely resembles the human form of MM. The studies in mice extended our knowledge of growth rates and growth characteristics of these tumors and the correlations between tumor mass and the amount of paraprotein. Observations on the doubling times for serum levels of paraproteins in such animals led to the view that the initial "insult(s)" to the cell that have been postulated to initiate the abnormal clone must have occurred 10-20 years before the product of the clone--the paraprotein---could be detected in sufficient quantity in the serum. Measurement of the amount o[ paraprotein synthesized in vivo in individual human patients has been performed by metabolic turnover studies with isotope-labeled protein (33). Although increased synthesis clearly is a characteristic feature, there was no direct correlation between the IgG synthesis rate (mg of monoclonal protein synthesized/kg body weight/day) and the clinical features. Patients with MM may demonstrate IgG synthesis rates more than 10 times that of normal IgG in normal subjects, which is 20-60 (mean 36) mg/kg/day. Salmon (28) has employed turnover studies in the whole body in conjunction with in-vitro measurement of the secretion of monoclonal protein by short-term cultures of cells obtained by bone marrow aspiration. By the latter method it is hoped to measure the secretion of protein per plasma cell and thereby eventually estimate the whole body tumor mass. Such estimates might then prove helpful in planning the most effective forms of therapy. The use of such studies has not yet been realized in routine clinics that are responsible for the management of patients with MM. One view concerning the implications of monoclonal proteins is that they are "antibodies in search of an antigen." Other workers have been more intrigued by the possible relationship to autoantigens and have raised the possibility that in many cases a monoclonal protein is an antibody that is being produced by an autoantibody clone of cells. Although these suggestions have yet to be proved, it certainly is true that it has been possible to demonstrate binding of known antigens by many monoclonal proteins. These proteins remain the minority, but much of our knowledge of the structure and genetics of 32

immunoglobulin molecules has come from analysis of these proteins. We have already mentioned the existence of mixed cryoimmunoglobulins in which a monoclonal lgM or IgG binds autologous IgG. In addition, there are many other examples of monoclonal "rheumatoid factors" that are not cryoimmunoglobulins. Binding activity for red blood cell antigens has been known for some time in the classic monoclonal IgM cold agglutinins. Currently we are studying in our laboratories monoclonal proteins that appear to bind blood groups A and B antigens and carcinoembryonic antigen. Other monoclonal proteins have been shown to bind bacterial antigens from streptococci (streptolysin O), Klebsiella, Brucella, etc. Finally, there is an increasingly long list of scattered reports of monoclonal proteins binding nonimmunoglobulin proteins, such ,as serum albumin, horse a2-macroglobulin, human a2-macroglobulin, lipoproteins, fibrin monomer, clotting factor VIII, clotting factor XIII and transferrin, and other compounds including dinitrophenyl groups, nucleic acid derivatives, phosphoryl choline, cardiolipids and heparin. We have intentionally used the term "binding" rather than directly referring to them as "antibodies." We believe that first it should be established that the binding is localized to the Fab fragment, binding does not occur with the Fc fragment, the reaction is inhibitable by an excess of the supposed antigen in free form and some estimate can be made of the strength of the binding. These reports of antibody activity in monoclonal proteins remain of great interest but still fail to provide an explanation for the etiology of these proteins.

Therapy While acknowledging the gaps in our basic knowledge concerning paraproteinemias, it is important to emphasize that the management of these patients has improved significantly in the past 15 years. Space will not permit a detailed discussion of each disorder outlined in Table 2, but we will reiterate some earlier comments that apply to many of the disorders. The therapy in many cases will consist of detailed investigation and then regular supervision. The diagnosis of MM remains the pre33

rogative of the clinician, as MM is not an immunochemical diagnosis per se but requires evidence of immunoglobulin abnormality, abnormal plasma cells and certain clinical features. In the absence of malignant gammopathy and in the absence of any of the primary diseases that would warrant it being considered "associated" monoclonal gammopathy, the approach then is for regular review to detect any possible progression with appearance of features of MM. In many of the examples given for associated gammopathy, treatment obviously will be indicated for the primary disorder, such as a solid-tissue tumor, liver cirrhosis, chronic inflammatory disease or immunodeficiency. Patients with paraproteinemia associated with malignant lymphoma or CLL should be treated along conventional lines for the primary disease based on the usual clinical and staging criteria. Unfortunately, in HCD and in amyloidosis, the over-all results of treatment have remained disappointing. Although occasionally there are reports of patients with amyloidosis responding to cytotoxic therapy, extensive systemic involvement in this disease generally indicates a poor prognosis (14). Solitary immunocytoma is best treated with local radiotherapy, although partial or even full surgical excision often is performed as the first stage when the lesion has been diagnosed by frozen section analysis during open surgical biopsy. It should be emphasized that it is the rule for patients with "solitary" immunocytoma to present later (generally within months) with features suggesting the onset of generalized MM. The majority of symptoms in WM, especially those associated with HVS, are directly attributable to the effects of the excess plasma IgM. As we indicated earlier, plasmapheresis is the correct procedure to remove this excess IgM, and it is the treatment of choice. In severe cases, initial management may necessitate the performance of daily plasmaphereses of 2 units for several days. These usually are performed by conventional methods with "sterile, enclosed blood-collecting plastic packs that permit readministration of the patient's red cells after centrifugation. Reference centers occasionally use the Automatic Cell Separator for plasmapheresis over a single period of several hours to handle a much greater volume of plasma, but close 3,t

medical supervision is required in older "patients with abnormal circulatory systems. Once the patients have been initially controlled, the aim is to keep them as symptom-free as possible, with their relative serum viscosity preferably under 3.0 and certainly under 4.0. The patients generally know when they need repeat plasmapheresis, as they quickly note the return of fatigue, weakness and hearing and visual impairment. Regular plasmapheresis of 2 units every 3 or 4 weeks is sufficient in many patients whereas others require it every 2 weeks. A period of less than 2 weeks between each plasmapheresis generally is too time-consuming and indicates that the patient should either be plasmapheresed for a greater volume on the Automatic Cell Separator at 2-4-week intervals or be treated with chemotherapy. We prefer to use small doses of chlorambucil, and 2 m g / d a y frequently is sufficient. This cautious approach to chemotherapy is warranted in older patients, who are especially prone to develop bone marrow toxicity on otherwise "conventional" doses. Rarely, one sees an unusual response to plasmapheresis, as in the following illustrative case. P. B. was a 27-year-old Negro female who presented at another hospital with a 2-month history of low back pain and anterior chest pain worsened by coughing or movement. Investigation revealed an lgA myeloma protein in the serum with no osteolytic lesions on x-ray. Laminectomy was performed when lumbar puncture indicated a block, and a soft, extradural plasmacytoma was fonnd and removed. Six months later, she was referred to the University of California, San Francisco, with malaise, weakness and frequent severe epistaxes and vaginal bleeding. She had had recurrent urinary infections. Investigation revealed multiple osteolytie lesions and a total serum protein level of 11.4 gm/100 ml. Her creatinine clearance was measured as 7-9 ml/min on different occasions. Clinically she had uremic frost. Intensive plasmapheresis was instituted and her general condition improved. Her serum creatinine fell to 2.6 rag/100 ml and then 2.0 mg/100 ml while her creatinine clearance rose to 30 ml/min. She was discharged to a peripheral hospital on a regimen of weekly plasmapheresis. On review 5 months later she was found to have continued her improvement (serum creatinine 1.2 nag/100 ml, creatinine clearance 75 ml/min). Two months later her values were 1.3 mg/100 ml and 123 ml/min, respectively. Un35

fortunately, a change in staff in the peripheral hospital at that stage resulted in suspension of her plasmapheresis program without our knowledge. Within 2 weeks she had relapsed, with poor renal function, urinary infection and pulmonary infiltrates and died from bronchopneumonia and renal failure within another week. Although there are several factors contributing to reduced renal function in a patient with IgA-MM, we believe that in this particular patient the time relationship between renal function studies and regular plasmapheresis strongly suggested a beneficial therapeutic effect by the latter in facilitating increased effective renal plasma flow. The treatment of MM has improved considerably in the past 15 years. Median survival was found to be 17 months from the onset of symptoms and 7 months from the onset of therapy for 600 patients treated prior to 1960 with radiotherapy and supportive measures but without alkylating agents. The use of cytotoxic therapy, combined with over-all better general management, has produced a 3-7-fold increase in median survival times, with improved quality of life. It is clear that cytotoxic therapy is warranted in proved MM, but unfortunately the various treatment regimens tested have not yet produced an accepted single protocol. One problem has been the marked variation in the various aspects of organ involvement. A second is that the various investigators have failed to agree on a definition of what can be considered a remission (8). Whereas Osserman notes mean improvement in serum or urinary monoclonal protein levels, hemoglobin level, pain and performance status, other groups require quantitative assessments of the decreases in monoclonal proteins, rise in hemoglobin level, improvement in renal function and radiologic evidence of healing of lytic bone lesions (1, 4, 7, 8, 12, 13). The main approaches to the treatment of MM are listed in Table 6. Farhangi and Osserman (8) urge early ambulation, even in those with extensive bone involvement, but have not seen spinal t:ord complications in their patients as a result of early ambulation. Hypercalcemia may presen t as a medical emergency in patients with MM, with vomiting, dehydration, azotemia, coma and cardiac dysrhythmias. A recent study of various methods of treating hypercalcemia demonstrated that 36

T A B L E 6.--I~,IANAGEMENT OF PATIENTS WITH ,3,|ULTIPLE ~IYELOMA

Ambulation Analgesia Infections---Antibiotics Therapeutic human gamma globulin Fluid-electrolyte intake Renal failure management--Conservative Hemodialysis tlyperuricemia--Allopu rinol Hypercalcemia--Fluids Phosphate infusion Prednisone Plasmapheresis Radiotherapy Chemotherapy Melphalan Cyclophosphamide Procarbazine Prednisone General

the best results were obtained with 6-8 hour infusions of phosphate (11). The latter study revealed that the degree of reduction of hypercalcemia was related to the amount of phosphate infused. All are in general agreement that rehydration is necessary, but it must be noted that phosl~hate or sulfate infusions may pose the problem of salt overloading in some patients. For these reasons, corticosteroid therapy generally is employed as a short-term measure in patients who do not respond quickly to rehydration. Allopurinol is also generally given to prevent damage to the kidneys by urate deposition and to avoid acute arthritis, but hyperuricemia generally does not preseht as serious a problem in MM as it does in other forms of malignant disease treated with cytotoxic therapy. An early part of the management of MM is adequate analgesia to permit ambulation, and with large lytie bone lesions this means local palliative radiotherapy (1200-1500 rads). Since recurrent infections are noted frequently in MM, full antibiotic therapy is indicated for any infection. With severe infections, it may also be necessary to administer therapeutic human gamma globulin ( H G G ) , but there is no support for the routine administration of HGG as a prophylactic measure over 37

a prolonged period. One reason is that sufficient HGG cannot be given by intramuscular injection and the rapid IgG catabolism in MM patients applies to the injected HGG. In selected cases, infusion of normal plasma (with IgA and IgM) may be indicated. Conventional measures are required for the management of the renal abnormalities, including those associated with Bence Jones proteins, hypercalcemia, hyperurieemia and amyloidosis. It has been the usual view that the patient presenting initially with MM and acute renal failure should be treated with conservative measures, as the prognosis appears very poor. There are, however, published reports of such patients who have been placed on a program of hemodkilysis and full chemotherapy and subsequently have had a full remission, with return of.renal function sufficient to suspend further hemodialysis. If facilities are available, it is therefore worthwhile considering this approach in selected patients, together with vigorous plasmapheresis. Hemostatic abnormalities are not infrequently seen in MM, with some of the clinical manifestations listed in the HVS (Table 4) and a serum viscosity in the range 2.5-4.0. We believe that plasmapheresis is warranted initially in such patients. If indicated, specific chemotherapy can be started. We believe that it may be possible in the future to manage several of these patients by weekly plasmapheresis in the absence of chemotherapy and its possible side-effects. Clearly, plasmapheresis is essential in patients with severe acute HVS in whom it represents a medical emergency. The management of HVS in MM is complicated by the fact that mental changes also occur with dehydration, azotemia and hypercalcemia, and all these aberrations may be present. One of the problems in specific chemotherapy of MM is that the patient requires some degree of individualization at the outset of treatment. We had noted for some time that the routine use of prednisone in the initial attempt to obtain a remission in MM often was associated with a poor outcome, especially in those with obvious renal failure on their initial presentation. A recently published study of 189 patients demonstrated the statistical basis for this impression (7). The patients in the 38

latter study initially were assessed as "good risk" or "poor risk." The criteria for good risk were as follows: 1. 2. 3. 4. 5 6.

Blood urea nitrogen <30 mg/100 ml. Serum calcium <12 mg/100 ml. Absence of serious infection on presentation. WBC count >4000/#1. Platelet count > 100,000/M. Estimated survival > 2 months.

If the patient failed to meet all these criteria, he was classified as a poor risk for the purposes of the study. In good-risk patients, the use of melphalan and prednisone was compared with the use of melphalan alone, with prednisone being given over the initial 10 weeks. The combination gave a significantly higher percentage of good responses ( 5 5 % vs 2 3 % ) and longer survival (53 months vs 30 months). In poor-risk patients, however, the combination gave no improvement in response rate but rather was associated with a much shorter survival (9 months vs 21 months). Clearly, therefore, prednisone is not indicated as a primary remission-inducing drug in patients with M M who present with evidence of significant renal or bone marrow impairment. Several other points have arisen from combined trials of the treatment of MM, including one conducted by the Medical Research Council in the United Kingdom (12). Not unexpectedly, a poor outcome was noted in patients presenting with poor renal function, high BJP excretion and low hemoglobin. Thus, more than 50% of patients with an initial blood urea level over 80 m g / 1 0 0 ml were dead within 2 months. A low serum albumin level was also an' indication of a poor prognosis and was not simply an index of poor renal function. It appeared to indicate a poor prognosis in that it signified a very "active" or rapidly growing tumor and this led to the suggestion that the serum albumin was acting as a "food" for the tumor. It is interesting in this regard that we found that unusually rapid catabolism of albumin may be found in some but not all cases of MM (32). Serial studies of serum levels of the myeloma protein showed that they could decline rapidly, decline slowly or show no significant change over several weeks (6, 16). Although not all groups have agreed with this 39

exact classification, all appear to agree that the "fast responders," who have the most rapid falls in serum levels of myeloma proteins, have the poorest prognosis. These "fast responders" tend to have low serum albumin levels and it now is considered that they signify an active tumor, which may show an initial response with lowering of the immunoglobulin level, although the over-all survival of the patient is much shorter. It is interesting to note that corticosteroids increase IgG catabolism and probably would contribute to the more rapid rate of fall of the serum level of an IgG myeloma protein. To indicate the complexity of the problem in the selection of drugs in the treatment of MM, we have summarized some of the specific treatment regimens published by different groups (Table 7). The two major decisions are the choice of drugs and whether the administration should be continuous or intermittent. F a r h a n g i and Osserman (8) recently reviewed the published data on median survival times and, in our opinion, neither issue is definitely settled. This includes the contention that continuous therapy is preferable to reduce the risk of drug resistance, since a similar benefit is claimed by some for intermittent therapy. We have already mentioned one problem in comparing, viz., the lack of a widely accepted set of criteria to define partial and full remission. Our preference is for therapy based on the initial assessment of the patient. Good-risk patients are treated with melphalan and prednisone for an initial loading period of 7-10 days and then are treated with a course of both drugs for 4 days every 4-6 weeks (see Table 7). Whichever regimen the ~/ttending physician selects, it is essential that the 'patient receive close clinical, laboratory, immunologic and especially hematologic monitoring. With close supervision, it soon is clear whether the patient can tolerate the drug courses every 4 weeks or whether they will have to be given at the more usual period of 6 weeks. We believe that any irregularity or unwillingness of'the patient to adhere to his regimen is a major contraindication to the use of continuous therapy with higher risks of toxicity. We prefer that poor-risk patients not be treated with prednisone as a primary drug and we use melphalan, by either the continuous or the intermittent system. A final point of dispute is the actual 40

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drug dosage, and the alternatives are summarized in Table 7. Bergsagel (4) has reviewed several of the factors involved in deciding the dose of melphalan. With intermittent doses of 0.25 mg/kg/day for 4 days, this means a total dose of 1 mg/kg, which some have suggested is too myelosuppressive. However, the data of the SWCCSG (see Table 7) contradict this, as they found that the WBC count fell below 1000//d in only 2% of their patients, the majority of whom were receiving more than 1 mg/kg (over the 4 days). Farhangi and Osserman (8) are the main proponents of continuous therapy, and after a loading dose of 6-10 mg daily for 7-10 days they continue with 2 mg/day. Usually this is accompanied by WBC counts in the range 20003000/td and platelet counts in the range 50,000-100,000//d. We have already mentioned that myelosuppression is the major sign of toxicity and that regular supervision is the best approach. Allied with the myelosuppression is infection, which is the main cause of death from toxicity (7). Other manifestations of toxicity include gastrointestinal abnormalities, such as nausea and vomiting and skin rashes. Cyclophosphamide may be associated with hemorrhagie cystitis or alopecia, which warrants withdrawal of this drug. Hence, we prefer to use melphalan. Procarbazine has not yet been studied in sufficient numbers of cases for full assessment of its role in the treatment of MM. There are two further points that warrant discussion. The first is the problem of resistance to the initial drug therapy and the second is the occurrence of "tumor escape" or relapse after an initial response. These are closely interrelated, as they reflect the factors of the patient, his plasma, cell tumor and the drug(s). It is clear that resistance to one drug does not automatically indicate resistance to another drug (e.g., melphalan any cyclophosphamide), and initial drug failure (or tumor escape) warrants a change in drugs. Of course, a "slow response" is to be desired in view of the recent findings in fast and slow responders, and the decision to change drugs should not be madetoo early. "Tumor escape" was noted in 60% of patients in a Medical Research Council study (12, 16). It may be indicated by a change in growth rate of the primary tumor, by the appearance 42

of an increase in BJP production, by th~ appearance of amyloid or by other factors that are not yet understood. Our understanding is complicated by our lack of knowledge of the normal control of plasma cell differentiation and immunoglobulin synthesis. Studies by Salmon (28) and others indicate that it soon should be possible to attempt analysis of each patient by studying the effects of drugs on in-vitro bone marrow cultures. Differential effects have been observed with melphalan on the myeloma cells and hematopoietic stem cells. Moreover, the effects of different drugs can be assessed. Such studies would provide a rational basis for the selection of the appropriate drugs, but they have not yet been accepted and probably will become available only at selected centers. Conclusion

It is clear that the study of paraproteinemias has been especially fruitful in many areas. Much of our knowledge of basic immunology and immunoglobulin structure has come from analysis of these plasma cell neoplasms and their synthesized products. Although our understanding of this miscellaneous group of disorders has improved, the basic mechanisms for the development, progress and elimination of plasma cell dyscrasias remain unproved. Future progress is likely to be rapid, however, as it is being realized that the paraproteinemias represent a suitable model for the analysis of neoplasia. The widespread availability of reliable and reproducible immunologic laboratory procedures has improved the diagnosis of various paraproteinemias, but the diagnosis of'the disease (e.g., MM or WM) should remain one based finally on clinical assessment. In the most common of the malignant gammopathies, MM and WM, we are fortunate that the past 15 years have seen considerable advancements in both general management and specific chemotherapy. I n particular we would stress the efficacy of plasmapheresis to reduce serum viscosity in both WM and MM and measures to combat hypercalcemia and renal damage in MM. Although the various regimens in MM have not yet been consolidated into a single accepted protocol, it is clear that closely supervised patients receiving chemotherapy 43

a n d a d e q u a t e s u p p o r t i v e t h e r a p y live l o n g e r a n d have a b e t t e r quality of life in that p e r i o d t h a n u n t r e a t e d patients without specific c h e m o t h e r a p e u t i c m a n a g e m e n t . REFERENCES 1. Alexanian, R., Bonnet, I., Gehan, E., Haut, A., Hewlett, J., Lane, M., hionto, R., and Wilson, H.: Combination chemotherapy for multiple myeloma, Cancer 30:382, 1972. 2. Alexanian, R., and hiigliore, P. J.: Normal immunoglobulins in multiple myeloma: Effect of melphalan chemotherapy, I. Lab. & Clin. Med. 75:225, 1970. 3. Azar, H. A., Zaino, E. C., Pham, T. D., and Yannopoulos, K.: "Nonsecretory" plasma cell myeloma: Observations on seven cases with electron microscopic studies, Am. J. Clin. Path. 58:618, 1972. 4. Bergsagel, D. E.: Plasma cell myeloma: An interpretative review, Cancer 30:1588, 1972. 5. Bloch, K. I., and Maki, D. G.: Hyperviscosity syndromes associated with immunoglobulin abnormalities, Seminars Hematol. 10:113, 1973. 6. Caggiano, V., Cuttner, J., and Solomon, A.: Myeloma proteins, Bence Jones proteins and normal immunoglobulins in multiple myeloma, Blood 30:265, 1967. 7. Costa, G., Engle, R. L., Jr., Schilling, A., Carbone, P., Kochwa, S., Nachman, R. L., and Glidewell, O.: Melphalan and prednisone: An effective combination for the treatment of multiple myeloma, Am. J. Med. 54:589, 1973. 8. Farhangi, M., and Osserman, E. F.: The treatment of multiple myeloma, Seminars Hematol. 10:149, 1973. 9. Frangione, B., and Franklin, E. C.: Heavy chain diseases: Clinical features and molecular significance of the disordered immunoglobulin structure, Seminars Hematol. 10:53, 1973. 10. Fudenberg, H. H.: Waldenstr/Sm's Maeroglobulinemia, in Cancer Chemotherapy 11, The Twenty-second Hahne4nann Symposium (New York: Grune & Stratton, Inc., 1973). 11. Fulmer, D. H., Dimich, A. B., Rothschild, E. O., and Myers, W. P. L : Treatment of hypercalcemia, Arch. Int. l~led. 129:923, 1972. 12. Galton, D. A. G.: Treatment of myelomatosis---M. R. C. Trial, Brit. h !. J. 2:323, 1971. 13. George, R. P., Doth, J. L., Gordon, D., and Schrier, S. L.: Multiple myeloma--intermittent combination chemotherapy compared to continuous therapy, Cancer 29:1665, 1972. 14. Glenner, G. G., Terry, W. D., and Isersky, C.: Amyloidosis: Its nature and pathogenesis, Seminars Hematol. 10:65, 1973. 15. Grey, H. hi., and Kohler, P. F.: Cryoimmunoglobulins, Seminars Hematol. 10:87, 1973. 44

16. Hobbs, J. R.: Immunoglobulinsin clinical chemistry, Adv. Clin. Chem. 14:219, 1971. 17. Isobe, T., and Osserman, E. F.: Pathologic conditions associated with plasma cell dyscrasias: A study of 806 cases, Ann. New York Acad. Sc. 190:507, 1971. 18. Kanoh, T.: The behaviour of immunoglobulin in monoclonal gam~ mopathies and their classification and pathogenesis, Tohoku J. Exper. Med. 102:369, 1970. 19. Kanoh, T.: Significance of M-components in plasma cell myeloma and related disorders, Tohoku J. Exper. Med. 102:341, 1970. 20. Lackner, H.: Hemostfitic abnormalities associated with dysproteinemias, Seminars Hematol. 10:125, 1973. 21. MacKenzie, M. R., and Fudenberg, H. H,: Macroglobulinemia: An analysis of forty patients, Blood 39:874, 1972. 22. Meyers, B. R., Hirschman, S. Z., and Axelrod, J. A.: Current patterns of infection in multiple myeloma, Am. J. Med. 52:87, 1972. 23. Natvig, J. B., and Kunkel, H. G.: Human immunoglobulins: Classes, subclasses, genetic variants, and idiotypes, Adv. Immunol. 13:1, 1973. 24. Norgaard, O.: Three cases of multiple myeloma in which the preclinical asymptomatic phases persisted throughout 15 to 24 years, Brit. J. Cancer 25:417, 1971. 25. Penny, R., Castaldi, P. A., and Whitsed, H. M.: Inflammation and haemostasis in paraproteinemias, Brit. J. Haematol. 20:35, 1971. 26. Potter, M.: The developmental history of the neoplastic plasma cell in mice: A brief review of recent developments, Seminars Hematol. 10:19, 1973. 27. Preud'homme, J. L., Hurez, D., and Seligmann, M.: Immunofluorescence studies in Waldenstr/Jm's macroglobulinemia, Rev. Europ. Etud. Ciin. et Biol. 15:1127, 1970. 28. Salmon, S. E.: Immunoglobulin synthesis and tumor kinetics of multiple myeloma, Seminars Hematol. 10:135, 1973. 29. Seligmann, M.: Heavy chain diseases, Rev. Europ. Etud. Clin. et Biol. 17:349, 1972. 30. Seligmann, M., and Brouet, J. C.: Antibody activity' of human myeloma globulins, Seminars Hematol. 10:163, 1973. 31. Solomon, A., and McLaughlin, C. L.: Immunoglobulin structure determined from products of plasma cell neoplasma, Seminars Hematol. 10:3, 1973. 32. Wells, J. V., and Fudenberg, H. H.: Metabolism of radioiodinated human serum albumin in patients with monoclonal gammopathy, Blood 38:66, 1971. 33. Wells, J. V., and Fudenberg, H. H.: Metabolism of radioiodinated IgG in patients with abnormal serum IgG levels. I. Hypergammaglobulinemia, Clin. Exper. Immunol. 9:761, 1971. 34. Zawadzki, Z. A., and Edwards, G. A.: Non-myelomatous monoclonal immunoglobulinemia, Prog. Clin. Immunol. 1:105, 1972. 45