Monoclonal gammopathies—their identification and biological significance

Clinica Chimica Acta, 180 (1989) 1-22 Elsevier

CCA 04370

Critical Review

Monoclonal gammopathiestheir identification and biological significance C. Kirk Osterland ofMedicine, Montreal,

McGill University School

Quebec (Canadn)

(Received 28 March 1988; revision received 3 October 1988; accepted 10 October 1988) Key words: Monoclonal gammopathy; B lymphocyte; M-protein

Introduction A clinically prominent group of malignant disorders of B lymphocytes are termed plasma cell dyscrasias (PCD). They are characterized by the presence of increased numbers and abnormal function or regulation of the marrow B lymphocytes and plasma cells, frequent bone lesions, and the presence of increased amounts of gammaglobulin in the serum and urine. In spite of the excess amount of serum g~aglobul~s, the patients are in fact deficient in protective antibody synthesis and they have enhanced susceptibility to the development of recurring infections. The most characteristic abnormality in the serum proteins of PCD patients is the presence of an abnormal homogeneous band in the globulin region (previously referred to as a paraprotein, now, more correctly, as a monoclonal gammopathyM-protein). The M-protein is the product of an abnormally expanded single clone of B-cells and plasma cells. The isolated M-protein shows homogeneity that stands out in particular contrast to the characteristically heterogeneous immune globulin proteins. It was for this reason that the abnormal globulin was initially referred to as a ‘paraprotein’ in the belief that it was a gammaglobulin whose structure was altered as part of the malignant process. There was concern that the use of M-proteins as models for the study of the structure of antibody proteins would provide spurious info~ation. The demonstration of the presence of antibody combining specificities in the paraproteins [1,2] was an important addition to the extensive immunochemical studies done on paraproteins that had already led to an understanding of immunoglobulin structure, isotypic subclass variation, and the chemistry of the antibody-antigen contact region. The careful clinical laboratory study of serum proteins provides one of the best ways of screening for and diagnosing the plasma cell dyscrasias. In this review, the

Correspondence to: Dr. C. Kirk Osterland, Director, Division of Clinical Immunology/Rheumatology, Royal Victoria Hospital, Montreal, Quebec, Canada H3A 1Al. ~9-8981/89/$03.50

Q 1989 Etsevier Science Publishers B.V. (Biomedical Division)

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focus will be on the diagnostic methods used for serum protein studies with discussion of some aspects of particular interest relating to the monoclonal proteins themselves. The spectrum of diseases with M-proteins

Monoclonal gammaglobulins become detectable in the serum when a single clone of mature and differentiated B lymphocytes (plasma cells) has proliferated to the degree that gammaglobulin production is sufficient for the sensitivity of the immunochemical procedures. This probably implies the presence of a tumor burden surpassing 10” cells. (The total number of lymphocytes is about 1012.) Each of the M-protein producing cells exhibits the functional feature of allelic exclusion in the expression of chromosomes that contain the genes coding for immunoglobulins. This is a characteristic of the plasma cells-for all allelic allotypic specificities that can be tested for and detected on an individual’s total gammaglobulins, there is clear evidence that only one gene haplotype (one of the two potentially functional chromosomes) is functioning in the cells synthesizing the M-protein. The heterogeneity of the immunoglobulins is thus pre-empted by the selective increase of a single uniform population of molecules-the M-protein. Generally speaking, the presence of a serum monoclonal gammopathy indicates that the abnormal clone consists of lymphocytes that have reached a maturation stage at, or close to the terminally differentiated plasma cell stage at which point the most active immunoglobulin synthesis takes place. A basic classification of monoclonal gammopathies is indicated in Table I. The presence of the visible M-protein indicates the expansion of an immunoglobulin secreting clone of B lymphocytes to a size sufficient to be detectable. This single clone may be part of a primary malignant process involving B lymphocytes and plasma cells or it may be the result of a secondary monoclonal B-cell expansion in association with cancer, auto immune diseases and many other clinical conditions [3-71. The term ‘benign’ monoclonal gammopathy is also referred to as ‘monoclonal gammopathy of unknown significance’. This latter designation gives emphasis to the

TABLE

I

Classification

of diseases

with monoclonal

I. Primary malignant disorders 1. Plasma dyscrasias. 2. Lymphomas. II. Disorders with secondary 1. Carcinomas. 2. Autoimmune diseases. 3. Drug reactions. 4. h4iscellaneous. III. ‘Benign’ monoclonal

affecting

gammopathies the B lymphocyte

clonal expansion

gammopathy.

lineage.

of B lymphocytes.

3 TABLE II Some clinical and laboratory gammopathy

features that help determine

the ‘benign’ nature of a monoclonal

I. Clinical features. 1. Lack of anemia, normal serum albumin. 2. Normal serum calcium. 3. Absence of bone lesions. 4. < 5% plasma cells in marrow, few mitoses and showing only weak acid phosphatase staining. 5. Stable-clinically. II. Immunology laboratory findings. 1. M-protein less than 1.5 gm/lOO ml serum. 2. Normal immunoglobulins (isotypes other than the M-protein)not quantitatively reduced. 3. Urine B,M < 3 mg/l. 4. Absent or low Bence-Jones proteinuria. 5. Stable value for M-protein quantitation over time. 6. Biclonal peaks. 7. Presence of Ig fragments more common in malignant disease. III. Miscellaneous laboratory findings. 1. Plasma cell DNA labelling index normal. 2. Low values for idiotype positive B lymphocytes in peripheral blood. 3. Normal value for Leu 1 B subset of lymphocytes in peripheral blood.

fact that some of those initially benign primary B lymphocyte hyperplasias may in fact undergo malignant change to become true plasma cell dyscrasias. From a clinical standpoint one of the most important distinctions to make regarding a monoclonal gammopathy is whether or not the basic underlying cause is benign or malignant. Some of the clinical and laboratory guidelines that can be useful in making this distinction are listed in Table II. No one laboratory test or clinical feature is always present or decisive. Benign monoclonal gammopathy is the most common type of monoclonal protein detected by routine serum protein electrophoresis [4] though the condition may not always be or remain benign, as the name suggests. A number of centres have reported on patients diagnosed as BMG, who, when subsequently followed for a .number of years, showed evolution of their disorder into a true B lymphocyte malignant process [14]. The diagnosis BMG must therefore always be looked upon as a potentially pre-malignant condition, that has to be assessed periodically with repeat immunochemical testing to detect changes in the parameters that help distinguish the benign and the malignant state. Older criteria suggested that a quantitative value of 3 gm % for an M-protein was the level which best distinguished between benign and malignant monoclonal gammopathies. However, many workers consider that when the M-proteins are in a concentration of greater than 1.5 gm 5%they will eventually be associated with malignant disease. When the B-cell clonal expansion is ‘benign’ there appears to be greater control of the normal regulatory factors that influence B-cell growth, maturation and immunoglobulin secretion. The DNA labelling index (Table II) of the lymphocytes

4

and plasma cells in the marrow of the BMG patients is also much lower than in the cells of the malignant plasma cell dyscrasias, presumably also reflecting regulatory differences. While it is possible to detect some light chain excretion even in normal urine, significant amounts of light chain (or Bence Jones proteinuria) is not regularly present even in BMG. Heavy B.J. proteinuria usually is indicative of a malignant PCD. A clinical syndrome referred to as idiopathic Bence Jones proteinuria can behave for a prolonged period as though it is benign, but most often it ultimately develops the clinical picture of true multiple myeloma [12,14]. Light chain and &-microglobulin excretion are low in BMG, (T&M < 3 mg/l) higher levels also being associated only with the high tumor burden and increased metabolic activity of the malignant B-cell dyscrasias [8,9]. Abnormal molecular forms of immunoglobulins such as half molecules and free heavy or light chains also occur much less commonly in the BMG [lO,ll]. Approximately 90% of BMGs are of the IgG-I subclass and while only about 50% of myeloma proteins are the same subclass this is not a reliable distinguishing feature, As will be discussed in a subsequent section, most, if not all monoclonal gammaglobulins probably have antibody activity which can only be defined if the correct antigen could be selected and screened against. Some antibody specificities of M-proteins have been defined and they can, at times, contribute to the clinical picture of the patient. IgM benign gammopathies with cold agglutinin activity can relate to cold sensitivity syndromes and anemia; IgG 4 M-proteins in BMG with anti-factor VIII activity, IgG 3 antiplatelet antibodies, and IgG 2 antidextran antibodies have all been described to contribute to clinical symptoms by promoting thromboses or hemorrhagic phenomena [11,13,15]. Though there is not extensive study on this point it seems likely that similar antibody activities occur in both benign and malignant gammopathies. Sometimes it remains difficult to make the distinction between a benign monoclonal gammopathy and one secondary to malignant disease. Approximately 1% of the population over the age of 25 has a detectable serum monoclonal protein and this number rises to 10% in subjects over age 75. The ‘BMG’ is, therefore, a common form of immunoglobulinopathy. When the diagnosis is made, patients should be re-studied at 6-mth intervals with repeat characterization and quantitation of the serum immunoglobulins. When such monitoring is supplemented by bone marrow, radiological and other clinical studies, a conversion of a benign to a malignant state should be picked up at the earliest point possible and appropriate measures taken. Much remains unknown about this transition. In fact, the nature of benign monoclonal B-cell expansion and the persisting influence of effective growth controls on the benign clone is an incompletely understood phenomenon. When a monoclonal gammopathy is secondary to the presence of a primary malignant tumor, there may be lymphocyte infiltration of the tumor itself and it may be attractive to suggest that the monoclonal protein might be antibody directed against a discrete tumor antigen as part of the host defense. However, there has been very little solid evidence to support this thesis and the cause and role of the plasma clonal expansion and M-protein with a primary tumor remains unknown. The transient monoclonal garmnopathies seen occasionally as part of an infec-

5

tious process, in a post immunization state, during particular drug therapy or in the post bone marrow transplant recovery phase, all would seem to represent clonal expansion of relevant, specific antibody-producing cells. Clearly, the clones remain under normal regulatory controls of the immune system and uncontrolled growth (or PCD) does not follow. The occurrence of monoclonal gammopathies in chronic inflammatory or immunological disorders such as rheumatic diseases remains somewhat enigmatic. One explanation given for this is the emergence of a dominant clone in the presence of chronic antigen stimulation representing the selective enhanced growth of a single clone making high affinity antibody for some discrete antigen important to the primary disease. The emergence of monoclonal gammopathies on a background of polyclonal gammaglobulin increase has also been noted in experimental animals receiving repeated immunization with a single antigen [16] and genetic factors have been shown to be important determinants for this type of response. In humans, there are reports of the familial occurrence of gammopathies. A surprisingly large number of human IgM M-proteins have been shown to exhibit

Fig. 1. Zone electrophoresis on cellulose acetate demonstrating a number of forms of M-proteins as they may appear on this type of examination. (Anode to right.) 1, Normal; 2, Slow IgG 3 M-protein, reduced normal Ig; 3, mid-zone IgG, small; 4, B-M-peak, IgA; 5, IgG-reduced normal Ig; 6; q-L.-chain M-protein with small IgG M-protein; 7, biclonal M-protein; 8, blurred M-protein, immune complexes.

6

rheumatoid factor activity. Some recent studies on the rheumatoid factors produced generally, indicates that these auto antibodies may regularly be immunochemically less heterogeneous than the total antibody population. The evidence suggests some limitations on the number V region gene segments that are used in the synthesis of both the heavy and light chains of IgM globulins with anti-immunoglobulin activity [17,18]. Whether or not there is similar restriction of V region segments utilized for the synthesis of other auto or natural antibodies awaits further study, but it does appear that some specific antibody immunoglobulins may not be as immunochemitally heterogeneous as has previously been supposed. The immunochemistry

of M-proteins

synthesized

in disease

Normal immunoglobulin production in humans includes 5 major gammaglobulin classes, which may be further divided immunochemically into subclasses based on either genetic characteristics on the chains or some other form of chemical microheterogeneity. Additional Ig heterogeneity comes from the presence of other immunoglobulin associated polypeptides such as J chain, secretory piece and perhaps even B,-microglobulin though this is not strictly speaking an Ig structure, but rather a protein that has evolved from the same primordial genes and is therefore a member of the same family of proteins. A monoclonal protein is usually identified when it becomes visible as a narrow or discrete band in the routine zone electrophoretic separation of serum proteins (Fig. 1). The band most frequently occupies a particular portion of the broad gamma region but alpha- and beta-located monoclonal proteins may occur, particularly when the M-protein is free light or free heavy chains. Very slow or post gamma peaks are most often seen in the case of the IgG subclass 1 or 3 M-protein or with IgM subunits. The M-proteins with special properties such as cold precipitability may also be initially detected as a result of this property causing a failure to migrate properly on electrophoresis. Variation features

in immunochemical

types

of M-proteins

and their associated

clinical

Table III lists the pathological conditions that are most commonly associated with the presence of a serum monoclonal gammopathy. In all cases the ‘abnormal protein can contribute to the clinical manifestations of the basic disease, either through its physical properties (e.g. viscosity, cryoproteins) or though their actions as antibodies. Cold agglutinins usually exist as IgM kappa proteins, and are listed separately in Table III under secondary monoclonal B-cell expansion. In fact, cold agglutinin diseases may occur as a primary disorder and could also be included as one of the benign monoclonal gammopathies (a particular one in which the specific antibody activity leads to clinical manifestations such as vasospasm, thrombosis or even vasculitis). Papular mucinosis is a skin condition in which small papules or larger placques of slcerederma like skin appears. There is, pathologically, hyperfunction of fibroblasts with increased mucin formation in the dermis. A monoclonal gammopathy, typically IgGh may form. The location of the clonal expansion of

TABLE III Pathological basis for monwlonal gammopathies 1. Benign monoclonal gammopathy (most common). 2. Primary plasma cell dyscrasias. Myeloma. Macroglobulinemia. Heavy chain diseases. Lymphomas. Primary amyloid. 3. Monoclonal plasma cell expansion with miscellaneous diseases. i. Malignant. - Cancer. ii. Benign. - Cold agglutinin. - Papular mucinosis. - Autoimmune disorders. .. . 111.Transient. - Drug induced. - Infection related.

plasma cells is not clear - only some moderate marrow plasmacytosis has been described. A serum factor that stimulates fibroblast activity could conceivably have some effect on lymphoid cells as well. Primary amyloid can have some clinical features that are similar to papular mucinosis. However, primary amyloidosis is generally considered to be a form of malignant plasma cell dyscrasia and a myeloma picture may be seen in this disease. Monoclonal gammopathies are most often IgGX, like the papular mucinosis and unlike myeloma where IgGK is the most common paraprotein. Table IV lists the frequency of occurrence of individual immunoglobulin classes as the protein involved in the monoclonal gammopathy. The frequency of occurrence of each immunoglobulin class as an M-protein correlates fairly well with the amount of that particular immunoglobulin present in the serum, and this, in turn, correlates with the number of B lymphocytes committed to making the particular class of immunoglobulin. However, this is not a strictly accurate rule and other TABLE IV Frequency of occurrence of various protein changes in monoclonal gammopathies IgG IgA IgM IgD IgE L chain H chain Miscellaneous subunits, half molecules

60% 20% 9% cl% il% 9% Cl% cl%

8

factors must also be important. IgM is synthesized for example only at an early stage of lymphocyte clonal development, so that the clonal expansion producing this form of M-protein would be expected to be of cells arrested in some stage of their developmental growth pathway. As another example, when the serum M-protein consists of free light chains only the bone marrow plasma cell morphology is frequently seen to be more active and differentiated than usual. Some of the least frequently occurring immunoglobulin changes may have considerable theoretical interest. It is worthwhile where at all possible to fully characterize the monoclonal protein in the clinical immunology laboratory. The different isotype classes of immunoglobulin M-proteins and other molecular forms such as free chains or fragments are associated with distinctive clinical features. While it is not the intention to give a complete clinical description of disorders associated with M-protein production, some brief remark will follow to illustrate some particular clinical features often associated with immunoglobulinopathy. One of the important aspects of the gammaglobulin assessment during therapy of a PCD patient, is the use of M-protein levels as a guide to the quantitative assessment of residual tumor. This is a reasonably valid approach in serial assessments of the patient during treatment. However, it must also be noted that absolute values for M-protein levels do not give the same quantitative information for all patients. There can be striking differences in the amount of monoclonal protein produced by given plasma cell tumors depending in part on the immunoglobulin isotype M-protein being synthesized. Characteristically, for example, the IgD and IgE M-protein levels are much lower than for the major immunoglobulin classes-G, A, M. This reflects features of the normal distribution in body fluid compartments of these immunoglobulins as well as protein degradation rates. Plasma cell neoplasms have been staged by correlating estimates of total tumor cell mass with clinical features [43]. This has involved measuring catabolic rate and tissue distribution to calculate total synthesis rate of the M-protein. This value is used along with the in vitro measurements of M-protein synthesis by a known number of cells to calculate a value for the plasma cell tumor mass. Patients at stage 1 who have minimal symptoms and systemic changes may have, nonetheless, 6 x 10” myeloma cells. By the time a patient has lOi myeloma cells, he is in an advanced clinical state. The staging method by calculation of tumor mass has provided considerable useful information. The situation is complex as illustrated by the fact that cellular synthesis in vitro may vary widely from case to case (2.5-38 pg/cell per day) and the cell survival duration of the involved plasma cells may also vary-both factors that will markedly influence the appearance of the serum M-protein. The fraction of cells undergoing active DNA synthesis can be judged by exposing marrow cells to tritiated thymidine, followed by auto radiography (Labelling Index -Table II, [43]). Normal values of < 5% is seen in cases of BMG; the more aggressive forms of plasma cell dyscrasias rise only to levels of about l-58. The labelling index numbers rise to high values only following rapid tumor cell destruction with chemotherapy and has suggested modified regimens with cycle specific and cycle non specific chemotherapeutic agents [44].

9

The symptoms of myeloma patients with IgG gammopathies can be similar regardless of which particular IgG subclass is being produced. Myeloma patients are deficient in their normal humoral antibody responses and frequently other components of the host defense mechanisms; this does not differ significantly amongst the isotype groups. The IgG 3 immunoglobulin has a marked tendency to aggregate and to be affected by temperature. It has been the most frequently seen M-protein in myeloma patients with cold related symptoms or hyperviscosity-a complication which is more usually associated with abnormal IgM proteins. Myeloma patients producing IgA immunoglobulin are usually clinically similar to those with IgG though IgA is more likely to aggregate or complex with other proteins and cause hyperviscosity. Some reports have claimed that IgA myeloma patients have higher tumor burdens at diagnosis, extra medullary plasma cell tissue and show more rapid disease progression [lO,ll]. The light chain type of the M-protein and the corresponding light chain Bence Jones proteinuria may result in clinically different effects. Lambda light chains, especially those with lower isoelectric points, have been claimed to be associated with more severe disease and in its metabolism in renal tubules seems more often to be associated with a nephrotoxic effect than does kappa type but this is not necessarily the case. With IgM immunoglobulin M-protein, problems are with bleeding, hyperviscosity syndrome and frequently cryoglobulin related symptoms. Significant Bence Jones proteinuria is relatively unusual in Waldenstrom’s macroglobulinemia. Low molecular weight fragments of the IgM are commonly seen in the serum of macroglobulinemic patients, and IgM subunits may be the main protein abnormality in some of the less well differentiated lymphomas. Occasionally, a well-differentiated plasma cell myeloma continues to secrete IgM as its M-protein. Clinically, it has been found that such patients seem to behave more like a macroglobulinemia patient and exhibit a somewhat slower and more benign disease course. However, the opposite may also occur and such patients may exhibit the same bone changes and other clinical features characteristic of IgG secreting myeloma. A myeloma tumor secreting IgD most often will also produce lambda light chains. The IgD monoclonal protein is often labile and is readily degraded by proteolytic enzymes in the serum, contributing to the low amounts of serum M-protein that are difficult to detect. It is easy to miss IgD monoclonal proteins unless one is alert to their possibility through some knowledge of the clinical aspects of the case. IgD myelomas are often associated with large extra medullary plasma cell lesions and tumors and they are a relatively aggressive form of multiple myeloma, presenting sometimes as a plasma cell leukemia. Though the tumor burden is quite large, IgD myelomas are rarely associated with large M-protein peaks unless the tumor is multifocal and diffusely present in bone marrow. The various B lymphocyte diseases in which the B lymphocytes synthesize Ig chains or fragments have some distinctive clinical features and provide important insight into phenomena relating to immunoglobulin chain synthesis. Light chain disease (kappa or lambda) is usually a fairly aggressive form of multiple myeloma. Even the ‘benign’ form of light chain disease more often than not ultimately evolves into a true plasma cell tumor [12,14]. The large amount of filtered light chains may

10

have noxious effects on the proximal tubular epithelium and produce a renal tubular Fanconi-like syndrome. Gamma heavy chain disease is associated often with a relatively broad based M-protein on the zone electrophoretic examination [19,20]. The cellular pathology of this condition is a rather mixed type of lymphoma. It frequently evolves on a background of chronic rheumatic disease and/or Sjogren’s syndrome. Mu heavy chain disease is a form of chronic lymphocytic leukemia with production of free Mu chains and often free light chains resulting in the presence of Bence Jones proteinuria. The excess plasma cells in the marrow of these patients may exhibit crystal-like presumably protein structures within their cytoplasm. Alpha heavy chain disease affects a younger age population with what is really a gastrointestinal lymphoma accompanied by a malabsorption syndrome [26]. The serum M-protein may be very small in amount and broad based, requiring special immunochemical procedures to demonstrate with any accuracy. It must be remembered that IgA immunoglobulin often may not react well with anti light chain antisera, a fact which may confound the correct diagnosis of alpha chain disease [21]. IgD immunoglobulin may also not type well with antisera against kappa or lambda antigens, further making the distinction among these gammopathies more difficult. Many different forms of B cell lymphoma may be associated with other quantitatively small monoclonal proteins. These M-proteins may be free chains, low molecular weight subunits of IgM or whole IgM protein molecules. The proteins of the heavy chain diseases show immunochemical evidence of delation in the V-region of the genome. The abnormal H-chains produced, therefore, lack normal hypervariable region, have no specific antibody activity and are of no use in normal host defense mechanisms. The production of normal immunoglobulins is often further suppressed in these patients. The reason that light chains are not synthesized in most of the heavy chain disease states remains a mystery; however it does appear that completion of the rearrangement of heavy chain region genes represents a critical signal for light chain V region activity and synthesis. The less common conditions associated with M-proteins such as lichen myxedematosus usually have relatively small concentrations of M-protein, most often the IgG type. The papular lesions of lichen myxedematosus are infiltrated with plasma cells that are the source of the M-protein itself. The transient M-protein appearing during a regular immune response or along with some drug therapy and drug reactions has considerable similarity to the M-proteins that have been induced in experimental animals undergoing chronic hyperimmunization with antigens such as streptococcal vaccines [7,16]. These immunoglobulinopathies can disappear very quickly following withdrawal of the stimulus, presumably reflecting the short life span (48 h) of the plasma cells themselves. Identification of the M-proteins

Table V lists the procedures that many clinical immunology laboratories use in order to help identify and characterize monoclonal proteins. Zone electrophoresis remains the easiest and a very sensitive means of ruling out the presence of a monoclonal protein [27]. The electrophoresis is usually carried out on cellulose

11 TABLE

V

Identification

of M-proteins

1. Zone electrophoresis - Serum - Urine, CSF 2. Immunochemical test on proteins - Ig class quantitation - Light chain class typing and quantitation - Immune electrophoresis and immunofixation - Separation steps followed by immunochemistry - Cryoglobulin analysis 3. Other - Immunofluorescence of cells and tissues - Molecular probe analyses

acetate or on thin layer plates of agar or agarose. The pH of the electrophoresis buffer is usually around 8.5 and the support material for the electrophoresis usually generates enough electroendosmosis that the gammaglobulins move towards the cathode. The cathodal migration has some advantage for the analysis of immunoglobulins since they migrate away from the rest of the serum proteins giving the clearest possible visual picture of the gamma zone. Agar or agarose electrophoresis has some greater resolving power of protein bands, and oligoclonal banding in CSF may not be seen on the cellulose acetate electrophoresis but show up on agar. The cellulose acetate is however very simple to run and economical; strips are simple to stain and their is a uniformity in the staining that is sometimes difficult to achieve with the agarose plate. Both methods can show up M-protein bands of concentration 0.5 mg/ml or even less. It is arguable how much resolution is optimal for routine electrophoretic examination of serum proteins. In spite of the hundred or so proteins in the serum only about 5-7 electrophoretic bands are ordinarily identified with each of them containing several proteins. It is possible that some of the high resolution methods for separation could provide a pattern in which many individual proteins could be directly identified and quantitated from the strip without resorting to individual immunochemical measurements, but this has yet to become a reality. When the zone electrophoretic patterns are stained and scanned by densitometer, the quantitative results for the alpha, beta, gamma regions can be recorded and distributed to the clinics. However, the most sensitive method for examining zone electrophoresis for the presence of an M-protein is a simple visual examination. The presence of small bands are obscured on densitometric scanning. Visual examination will also give information regarding the quantitative status of the non-M-protein or normal immunoglobulins. Many laboratories no longer do routine scanning nor give scanning values for serum electrophoresis. While zone electrophoresis remains the best method for detecting the presence of M-proteins, the pitfalls must be kept in mind. M-proteins of unusual mobility may occur and be hidden in alpha- and beta-protein bands. All of the heavy chain

12

diseases, light chain disease and IgD and IgE myelomas may produce M-proteins that are either broad based or of such low concentration that they do not show up as M-bands on the zone electrophoresis. Also, electrophoresis may not show any characteristic change if Ig fragments are present. It is important to have clinical information if one is to have a sufficient index of clinical suspicion to do the necessary detailed analyses to diagnose the less common or obscured monoclonal protein changes. It is not economically feasible to analyse all sera in such complete detail. Immunoglobulin quantitation is a useful adjunct to the serum protein electrophoresis pattern. The presence of markedly elevated individual immunoglobulin classes in the presence of reduced levels of the other major classes is practically diagnostic of some forms of plasma cell dyscrasia. If IgD and IgE globulins are measured quantitatively and found to be markedly elevated this also is suggestive of myeloma. Serial measurements of the specific M-protein class can be a useful method for following treatment results in a given patient. The rarity with which myeloma M-proteins completely disappear with therapy underscores the fact that treatment of these disorders remains unsatisfactory. Quantitative methods may also be applied to determine ratios of kappa to lambda proteins; striking alterations from the normal 2: 1 ratio provide diagnostic evidence for the presence of a monoclonal protein. If screening procedures identify a M-protein, subsequent steps for characterizing it are immunochemical procedures. Up until recently, the most useful of these was immunoelectrophoresis (IEP). This now, however, is being largely superceded by the immunofixation (IF) procedure which is quicker, probably more sensitive and even more accurate in identifying and characterizing immunoglobulin bands [22-241. These procedures are not used for the purpose of screening sera for the presence of monoclonal proteins, but rather as a means of identifying the immunochemical nature of the protein abnormality. As clinical laboratory procedures go they are expensive and labor intensive. It must also be borne in mind that both IEP and IF procedures are only as good as the antisera available are specific. Commercially available serum must be regularly evaluated for their specificities as these may not remain constant. Immunoelectrophoresis is best done in noble agar. While agarose can be used at a lower percent gel concentration and therefore allow freer migration of the serum proteins, agar has the salutory presence of greater electroendosmosis which moves the immunoglobulins towards the cathodal direction away from the other serum proteins. By setting voltage and time conditions correctly, this can produce long extended immunoglobulin arcs, a feature which facilitates recognizing abnormalities in the immunoelectrophoretic patterns. The use of agar or agarose preparations that leave the immunoglobulins close to the origin after electrophoresis or give only slight anodal migration, do not provide satisfactory arcs for immunoelectrophoretic identification of pattern abnormalities. The immunoelectrophoresis method is standard and will not be reviewed. The antisera chosen to develop the immunoplates will depend on information derived from the immunoglobulin quantitation studies and immunoelectrophoretic screening analyses. The abnormalities that can be

13

detected consist of arc distortion due to bowing or local thickening, apparent absence of a portion of an arc due to immune precipitate solubilization in marked antigen in excess or double line formation and Ig fragmentation. A number of variations of technique with the addition of extra antigen troughs, interrupted antibody troughs, and others have been used and may improve the diagnostic power of the method, but certainly at the expense of both cost and time required. Immunoelectrophoresis can be an excellent procedure for demonstrating the presence of free light chains in serum using antiserum specific for free chain determinants only. It also can give diagnostic patterns in the various heavy chain diseases and for IgD or IgE monoclonal gammopathies. IgM immunoglobulins, however, may fail to migrate into the gel as a result of its molecular size and relative insolubility in the low ionic strength buffer of the gel. Mild reduction of a serum with either cysteine or dithiothreitol and IEP analysis before and after reduction may reveal a major IgM band. Kappa and lambda typing of M-proteins is usually straightforward on immunoelectrophoresis and particularly so if the patient has Bence Jones proteinuria. The exception is the problem of light chain class typing of IgA and IgD M-proteins previously referred to in which some sort of steric hindrance effect interferes with the reactivity of the light chains of these molecules [21]. Light chain reactivity may be brought out by mild reduction of the M-protein or pre-treatment with a mild acid. Immunofixation is now usually done routinely on commercially available immunofixation plates. This provides a sensitive method of characterizing M-proteins. It avoids the umbrella effect seen in IEP in which the high concentration of IgG obscures the antibody reaction with smaller IgM or A M-proteins. Biclonal gammopathies, free light chains and immunoglobulin fragments are also very successfully demonstrated if the right antisera are available for the analysis [25] (though Ig fragments may also be visualized on IEP). One can save on the expense of the procedure due to the need for large amounts of specific antisera by applying the antiserum only to selected areas of the electrophoresis track rather than flooding the entire strip. The interpretation of the IF results is usually straightforward if antiserum is specific (examples are seen in Fig. 2). The main variation that one frequently sees is the solubilization of the middle part of the M-protein bands due to an excess concentration of antigen in a large band (Fig. 2). It can however serve as a low sensitivity method for identifying soluble immune complexes. IgM M-protein with rheumatoid factor activity can cause typing problems by reacting with the Fc of typing sera, but this is not common. The IF procedure may not give adequate information with regard to the possibility of higher molecular weight polymers of an M-protein or of subunits or fragments of an IgM monoclonal protein. A simple procedure for helping to clarify this is to separate the serum specimen on a standardized G-200 Sephadex or Sepharose column. This will allow reproducible separation of serum into three distinctive protein peaks according to their molecular size. One can usually, without concentrating the samples, apply representative samples from the three G-200 separation peaks to agar double diffusion plates reacting them against various specific antisera. The presence of large amounts of IgM globulin well on into the second G-200 peak indicates the presence of low

14

a

1

C

1

2

2

3

3

4

bl

2

3

di

2

3

Fig. 2. Examples of immunofixation analysis of some serum proteins containing M-proteins. Anode up-cathode down. (a) M-band in track 1 is shown to be IgG (track 2) Alight chains (track 4). Both K reactivity (track 3) and normal Ig is reduced. (b) Large M-band (track 1) causes characteristic antigen excess solubilization in the IgG (track 2) K (track 3) bands. This can occasionally cause a band to be missed. (c) A broad IgG band (track 1) is resolved to 2 monoclonal bands K (track 2) and h (track 3). (d) An IgG (track 2) X (track 3) band with no reduction of normal immunoglobulins.

molecular weight IgM subunits. The presence of IgG antigens well on into the third (albumin) peak of the G-200 curve implies the possible presence of a heavy chain protein abnormality or Ig fragments. The presence of cryoprecipitability of the M-protein facilitates its purification. The cryoprotein can be precipitated and centrifuged out in the cold then redissolved in warm dilute saline or electrophoresis buffer for further analyses. It must be remembered that the electrophoresis itself may have to be run at elevated temperatures, particularly if the cryoprotein has a high thermal amplitude of precipitability. Other procedures can be brought in to assist in the diagnosis of monoclonal gammopathies and associated plasma cell dyscrasias. Immunofluorescence analysis of bone marrow or tissue cells, is useful in cases of myeloma where the protein is synthesized but not secreted by the malignant cell. The tumor cells from PCD patients frequently exhibit chromosomal alterations, particularly in the chromosomes with the genes related to heavy or light chain synthesis [14,2,22] and/or

15

the regulation of total immunoglobulin synthesis (6, XY). Molecular probe analyses of DNA extracted from the tumor tissue have been used to distinguish between B and T cell lymphomas by demonstrating evidence of molecular rearrangement of gene segments coding for the variable regions of immunoglobulins (B cell) or of the gene segments coding for the T-cell receptor (T cell). These are powerful new techniques which may find much more use in clinical diagnostic immunology as time goes on, but they do require a sample of the tumor tissue for DNA extraction and processing. Pseudo-M-proteins

Sometimes an M-protein is suspected from the zone electrophoresis but cannot be identified by the immunochemical testing procedures. In the unlikelihood that it represents a new, undescribed Ig subclass (although J chain M-protein has yet to be described) these M-proteins are referred to as pseudo M-proteins. The causes of the pseudo M-proteins often have special medical interest of their own, and they include: (1) presence of immune complexes. Immune complexes may appear as a darker band of restricted heterogeneity often on a background of normal or increased Ig. The band is usually small and with less distinct borders than a true myeloma related protein. Ig values by scanning may be higher than the nephelometric quantitation value and IF analysis may indicate IgG, M, K and in the peak; (2) marked elevation of transferrin or alpha-2 macroglobulin; (3) marked hemolysis with hemoglobin providing the pseudo M-band; (4) the presence of fibrin monomers due to abnormal clotting or the presence of active fibrinolysis; (5) large amounts of C-reactive protein; (6) infected serum with subsequent protein alteration due to denaturation and proteolysis. One must always be aware of the possibility of a denatured protein that stays at the application point and does not migrate. Clinical symptoms of plasma cell dyscrasias that are related to the M-proteins

There have now been a large number of human monoclonal immunoglobulins described, with specific antibody activities (Table VI). Prior to the demonstration of the antibody activity, monoclonal proteins were referred to as paraproteins, believed to be structurally different from normal immunoglobulins, i.e. the abnormal product of malignant plasma cells. Normal antibodies were characterized by their electrophoretic heterogeneity. Immunochemical work in this field, using purified. M-proteins and antisera specific for them led to the delineation of the component classes and subclasses of immunoglobulins that go to make up the heterogeneous normal gammaglobulins. The identification of antibody activity in M-proteins provided important vindication for their use as true models of antibody structure and studies of the combining site region [1,2]. Studies on M proteins have provided many important new insights into the immune response, clonal growth, expression of autoimmunity as well as the fine chemistry of the antibody combining site itself. It is likely that all structurally intact M-proteins could be shown to possess some antibody combining activity. However this can only be detected by laboriously

16 TABLE Antibody

VI activities

in human

M-proteins

1. Against protein antigens. Rheumatoid factors. Clotting factors. Others. 2. Against cellular membrane, and intracellular Nucleoprotein, DNA. RBC. Lymphocytes. Cytoskeletal proteins. Cold agglutinins. Cardiolipin. Myelin associated glycoprotein. Platelet glycoprotein III.

antigens

3. Microbial antigens. Streptococcal. Staphylococcal. Transient - many infections 4. Miscellaneous. Dinitrophenol. Phosphoryl choline. Riboflavin. Dextran. Ca, Cu. DNA (sugar-phosphate

or base specificity).

screening against a variety of antigens [28], unless the M-protein antibody activity leads to some related clinical manifestations. Interestingly, the M-protein produced by the malignant lymphocyte-plasma cells is often the cause of major symptoms of the PCD either partly because of their concentration and chemical properties, or as a result of their specific antibody activity. It was initially of interest to study human M-protein antibodies to see whether or not the pattern of combining activities would shed light on the basic etiology of the various plasma cell dyscrasias, or if information of the specificity of the abnormal clone and therefore B cell antigen receptor, would provide any new avenues for regulating the growth and activity of the malignant clone. This has not been the case, but several interesting facts about the antibody specificities have emerged as well as some surprising data showing the ‘degeneracy’ of the antigen combining site-region [29-331. The first detailed studies on the kinetics of combining activity of a human myeloma protein reactive with DNP determinants showed complete homogeneity of the molecular interactions [l]. This was unique; antibody preparations had always shown the expected heterogeneity of kinetic binding effects. However, in spite of this homogeneity, the expected antigen monospecificity was not observed [l]. The isolated M-protein could be shown to bind to multiple chemical determinants with

17 TABLE VII Clinical systems in patients with PCD related to the M-proteins 1. Physiochemical properties of the M-proteins related. i. Hyperviscosity syndrome. ii. Cryoglobulinemia - cutaneous, hemorrhage. iii. Nephropathy - amyloid. - nephrosis. - tubular casts. - Fanconi syndrome. - Radio contrast media induced anemia. 2. ‘Hypogammaglobulinemia’ - Infections.

related.

3. Related to antibody properties. - Clotting factors-bleeding. - Anemia-cold agglutinins. - Hypercholesterolemia, xanthoma. - Peripheral neuropathy. 4. Metabolic properties. - Amyloidosis.

varying affinity. Some of the determinants bound would not be predicted to have similar surface characteristics to explain why they should be recognized by the homogeneous antibody. This has been termed a ‘degeneracy’ of the combining site. The phenomenon of multispecific antibodies is now well recognized and may have important clinical implications in autoimmunity [29]. It is due either to the combining site region having different adjacent or overlapping subregions capable of recognizing different epitopes [29] or to various unrelated proteins (or other biochemical substances) exhibiting a similar surface epitope determinant. For antibodies that do bind to multiple different substances it must be proven that this is always true antigen antibody interaction and with an affinity high enough to have biological significance, and not merely non-specific hydrophobic interactions [34]. There is considerable interest in monoclonal gammaglobulins with antibody activities against autoantigens [30-33,35,41,42]. Many of these are showing similar patterns of cross reactivity with multiple autoantigens and exhibit idiotype cross reactivity [17,18]. A highly conserved utilization of selected V-region genes for generating these autoantibody activities is suggested from the results. This is opening up new paths of study into the nature of the V-region rearrangements, as well as the origin of the generation of autoantibody diversity. Symptoms of patients with PCD are often directly related to the abnormal globulins. Some of the ways that M-proteins contribute to clinical symptoms or complications are listed in Table VII. In many instances the symptoms improve by therapy reducing M-protein synthesis and serum concentration, whether or not the disease itself is basically altered, though there is little doubt that the M-protein serum levels reflect the size of the tumor in the patient.

18

Perhaps the most dramatic example of a M-protein causing clinical symptoms is in the hyperviscosity syndrome that can be a medical emergency which is very successfully managed with plasmapheresis and chemotherapy. Some M-proteins cause life threatening complications as a result of their (auto)antibody activities and are also successfully managed with aggressive therapy to lower the M-protein level (examples are coagulation defects and protein-protein interactions as in mixed cryoglobulinemic disorders). There is, of course, a tendency towards certain patterns of clinical complications seen with different isotypic forms of the M-proteins. IgM macroglobulin is characteristically the offending protein in hyperviscosity syndromes, although both IgG and IgA M-proteins may also cause this when they are present in particularly high serum concentrations [40]. IgG 3 subclass abnormalities have the greatest tendency to show cryo properties and some molecular self association, producing a protein of larger molecular mass. Similarly IgA typically can exist in dimeric (secretory) form or as complexes with a number of other serum proteins. Any of the M-proteins can have cryoglobulinemic properties although the most typical are IgM (the M-protein itself or as part of a mixed cryoglobulinemia) or IgG 3. Cold agglutinin and cold reactive rheumatoid factor activities may also be exhibited by these same isotype classes [2,11,13]. The effects of M-proteins on the kidney are complex. In the event of large glomerular losses of light chains there may be precipitation and protein cast formation in the tubules. Some light chains filtered by the glomerulus have the action of producing some direct noxious damage on the proximal tubules and a Fanconi syndrome (glycosoria, aminoaciduria) may ensue. The amino terminal half

TABLE

VIII

Factors

responsible

for restricted

heterogeneity

of gammaglobulin

on electrophoresis

Factors

Examples

1. Nature of the antigen. (Limited antigenic determinants.)

Streptococcal antibodies. Acute viral infections. Some drug reactions.

2. Host factors. (Immunological immaturity.) (Primary immune deficiencies.)

Antibody response to infections. IgM predominance in fetal and newborn infections. During immune restitution.

3. Specific host-antigen interactions. i. Exposure of antigen to mucosal sites only. ii. Exposure of Ag to sites poor in immune responding cells. iii. Chronic low grade persistent antigen exposure. (Clonal emergence.) (Immune exhaustion.)

IgA elevations. Ig banding

in CSF.

Restricted heterogeneity with background. Ig that is normal or increased.

19

of the light chain is also the major constituent of the secondary amyloid that may complicate plasma cell dyscrasias. Antibody with specificity for a myelin associated glycoprotein (MAG) and glycolipid antigen has been found in patients with an IgM-M-protein and the clinical changes of a sensorimotor neuropathy [36,37]. Another syndrome with the acronym POEMS because of the involvement of pleura, otosclerosis, endocrinopathies, and skin, is characterized by an IgM-monoclonal gammopathy. Other types of autoantibody activities are frequently found in M-proteins such as rheumaoid factor and antibodies specific to nuclear or cytoplasmic constituents but these are not associated with outward signs of arthritis or lupus erythematosus. Nonetheless, the frequently finding of activity in M-proteins with specificity for the host’s own cellular antigens is intriguing [30-33,35,38,39]. Are these activities with marker cross reactivities, the M-protein from a clone that is really specific for some extrinsic antigen or are M-protein activities implying that a major part of the B-cell repertoire is really reactive against epitopes expressed on autologous tissue structures, and that these accordingly show up with high frequency in the malignant transformed clones? Monoclonal gammopathies are an important clinical laboratory finding that can be the result of a wide variety of host conditions (Tables VII, VIII). A careful analysis of these M-proteins has in the past yielded considerable information about clinical aspects of the patients and a great deal of insight into the biology of the immune system itself. Acknowledgement

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