Analysis of autoantibody reactivity and common idiotype PR4 expression of myeloma proteins

Sera from 75 patients with monoclonal gammopathies and with no clinical evidence of autoimmune disease have been screened for a wide range of autoreactivity including binding to DNA, cardiolipin, extractable nuclear antigen (ENA), rheumatoid factor activity and the presence of the common anti-DNA antibody idiotype PR4. The sera of 17/75 (23%) patients possessed autoreactivity: six were positive for anti-DNA activity, two had anticardiolipin activity and the PR4 ID was found in two sera (both of which possessed anti-DNA activity). Antibodies to ENA were found in one serum (anti-Ro) and anti-organ-specific antibodies in five. Using iso-electric focusing and immunoblotting we have shown that the PR4 ID and DNA binding activity are carried on the paraprotein and not on some other serum constituent. The IgG subclass distribution of 55 IgG paraproteins has also been investigated. The majority of IgG paraproteins belong to IgG1 subclass (55%), with the others, being IgG2 (4%), IgG3 (9%) and IgG4 (27%). In this study we have shown that sera from myeloma patients frequently possess autoreactivity, and that in many cases this can be attributed to the paraprotein.     

Cytology of myeloma cells.

A cytological, cytochemical, and cytometric study of plasma cells from 195 cases of multiple myeloma showed that, contrary to earlier reports, flaming cells, thesaurocytes, and intranuclear inclusions are not confined to IgA-secreting cases but are common also in IgG and Bence Jones varieties of myeloma. IgA-secreting cells are not larger, nor do they have a lower nuclear cytoplasmic ratio than other myeloma cells. On average, for a given mass of tumour, Bence-Jones, IgG, and IgA varieties of myeloma produce amounts of paraprotein in the ratio 1 to 1-6 to 2-7.     

Monoclonal gammopathy of undetermined significance: a morphologic and immunophenotypic study of the bone marrow.

A monoclonal paraprotein in the serum or urine raises the possibility of myeloma. However, in a significant proportion of individuals with serum paraproteins, particularly those with low levels of paraprotein, clinical and routine bone marrow evaluation is not diagnostic of an underlying neoplasm. The purpose of this study was to define the pathologic basis for monoclonal gammopathy in patients whose bone marrow biopsies showed no evidence of myeloma. We used immunofluorescence microscopy and flow cytometry of cell suspensions prepared from aspirated marrow, as well as immunohistochemistry of core biopsies, to perform immunopathologic evaluations of the bone marrow from 26 such patients. Eighteen patients with myeloma and seven without a serum paraprotein or evidence of myeloma were similarly studied. The data indicate that 17 of the 26 patients with monoclonal paraproteins whose routine bone marrow biopsies were normal or nondiagnostic had, in fact, a dispersed monotypic plasma cell population of concordant immunoglobulin heavy and light chain type in the bone marrow demonstrable by at least one of the three analytic methods. Among these, immunofluorescence microscopy of isolated bone marrow mononuclear cells was the most sensitive assay. Immunophenotypic evaluation of the bone marrow is useful for documenting and quantifying a monoclonal plasma cell population in patients with monoclonal gammopathy of undetermined significance.     

Multiple myeloma in association with sarcoidosis

The association of sarcoidosis with Hodgkin disease and non-Hodgkin lymphoma is well known. However, multiple myeloma also can occur rarely in association with sarcoidosis. We describe a patient with sarcoidosis who subsequently developed multiple myeloma. The patient was a 49-year-old woman with a 4-year history of severe, chronic, active sarcoidosis involving her lungs, lymph nodes, eyes, and bone marrow. During the initial clinical workup, a serum monoclonal paraprotein was detected and bone marrow examination revealed a slight increase in plasma cells (4%), in addition to noncaseating granulomas. Thus, the diagnoses of monoclonal gammopathy of undetermined significance and sarcoidosis were established simultaneously. She sought medical attention for her current illness when she developed low back pain and weakness of her lower extremities. Serum protein electrophoresis and immunofixation revealed a monoclonal paraprotein, immunoglobulin (Ig) G kappa type, and quantification revealed an IgG level of 46.67 g/L (normal, 5.88--15.73 g/L). Bone marrow aspiration and biopsy revealed multiple myeloma and sarcoidosis. Including this patient, 11 cases of sarcoidosis and multiple myeloma have been reported to date, including 3 patients with monoclonal gammopathy of undetermined significance preceding the onset of multiple myeloma. In this case, as in most of the cases reported previously, sarcoidosis preceded the development of multiple myeloma.     

Paraproteinaemia: use of idiotypic determinants as specific markers for tumour activity

Five patients with paraproteinaemia were investigated using anti-idiotype antibodies specific for their paraproteins. Sensitive assays were used to detect soluble paraprotein either in serum or in supernatants from cultures of peripheral blood mononuclear cells (PBMC) and bone marrow cells. It was found that (i) measurement of serum paraprotein by specific radioimmunoassay is a sensitive method of monitoring the course of multiple and solitary myeloma, (ii) morphologically normal bone marrow from a multiple myeloma patient contained paraprotein-secreting plasma cells, (iii) a patient with benign monoclonal gammopathy had at least one stage in her disease where pre-plasmacytic tumour cells were present in the peripheral blood, (iv) the PBMC of the patients with active myeloma contained suppressor monocytes, and (v) PBMC from treated myeloma patients (both those with still active disease and those in apparently complete remission) would not secrete paraprotein in culture under a variety of different culture conditions including partial monocyte depletion.     

Studies of human IgM anti-IgG cryoglobulins: II. Isoelectric focusing characteristics of the reactive antigen and residual serum IgG pools.

The co-isoelectric focusing characteristics of the antigen-IgG and residual IgG pools in the serum of patients with mixed IgM:IgG cryoglobulinemia have been determined. The antigen IgG purified from five IgM cryoglobulins and the residual IgG purified from serum depleted of cryoglobulin were radiolabelled and co-isoelectric focused in polyacrylamide gels in order to determine if the IgM anti-globulin reacted in order to determine if the IgM anti-globulin reacted with IgGs having unique physicochemical properties. Electrofocusing profiles of the IgG from four sera showed only minor differences when the antigen-IgG was compared to residual serum IgG. In one instance, the IEF profile of the antigen-IgG pool was markedly restricted and of a different charge dispersion than the residual serum IgG. This correlated with the limited antigenic specificity previously shown for the autologous IgM antiglobulin.     

Identification of the paraproteins and clinical significance of more than one paraprotein and clinical significance of more than one paraprotein in serum of 56 patients.

Sera from 56 patients with more than one paraprotein were investigated for immunoglobin class and light chain type of each paraprotein. The patients were divided into two groups according to the diagnosis, that is myeloma and macroglobulinaemia or others. The frequency of combinations of paraproteins was considered in the whole series and in the two groups. The laboratory and clinical findings were analysed to investigate the diagnostic and prognostic significance of more than one paraprotein in a serum. It is concluded that a lower percentage of patients with more than one paraprotein can definitely be shown to have myeloma than might be expected from studies on monoclonal paraproteinaemia, that patients having IgA paraproteins in the serum had the poorest prognosis, and that paraproteins with lambda light chains were more likely to be associated with myeloma or macroglobulinaemia. A discriminant analysis of ESR and total paraprotein levels in the two groups of patients showed that combinations of the two parameters were not more effective at distinguishing the groups than the ESR alone.     

Affinity for measles virus anti-haemolysin of a residual immunoglobulin M in sera of some patients with multiple sclerosis.

HEp2 cells persistently infected with measles virus were treated with trypsin to remove haemagglutinin (HA) and examined unfixed or fixed in acetone by fluorescent antibody methods, comparing those sera specific for structural antigens of the virus. Staining patterns combined with the blocking of specific immunofluorescence indicated that IgG specific for measles virus haemolysin could be recognized in multiple sclerosis (MS) sera and that in some sera from which rheumatoid factor had been removed, a residual IgM (MS-IgM) was absorbed to measles virus-infected cells and showed the same specificity in blocking tests as measles virus anti-haemolysin. MS-IgM could be removed from sera by absorption with latex particles coated with human IgG and would seem to be anti-globulin with preferential affinity for anti-haemolysin.     

Inhibition of the anti-globulin reaction by human immunoglobulin G and its fragments

The specificities of antibodies involved in the agglutination of anti-D sensitized human red cells was studied using IgG and its fragments Fc and Fab as inhibitors. It was shown that commercial anti-globulin sera and specific sheep anti-IgG serum react with the antigenic groups common to IgG and Fc. Anti-Fab serum on the other hand agglutinated anti-D sensitized red cells by reacting with the antigenic groups common to IgG and Fab. This result is interpreted to mean that antibodies reacting with Fab have different specificities depending on whether IgG or Fab was used as antigen.     

A highly conserved determinant on human rheumatoid factor idiotypes defined by a mouse monoclonal antibody

Different human IgM rheumatoid factor (IgM RF) idiotypes have been described defined by polyclonal rabbit anti-idiotypic antibodies. These antisera do not allow clear genetic analysis of the idiotypic determinants, be they cross-reactive or private. Therefore, we tried to obtain a set of monoclonal anti-idiotypic antibodies directed against RF idiotypes. Purified IgM RF serum from a patient with classical rheumatoid arthritis was used to immunize BALB/c mice. The spleen cells were fused with Sp 2/0 Ag 14, a nonsecreting mouse myeloma cell line, and a hybrid producing monoclonal anti-idiotypic antibody was selected. The mouse antibody, an IgG1 kappa, reacts with an identical or similar determinant located on (or close to) the binding site of all tested monoclonal or polyclonal IgM RF from totally unrelated patients with Waldenstr?ms's macroglobulinemias or rheumatoid arthritis. The monoclonal antibody also reacts with 2 rheumatoid arthritis patients' IgG RF and with a low proportion of normal polyclonal IgM without detectable RF activity. An hypothesis is proposed to explain the existence of a such highly conserved determinant on RF idiotypes.     

Increased sialylation of oligosaccharides on IgG paraproteins--a potential new tumour marker in multiple myeloma

AIMS: To investigate whether changes in carbohydrate structure of IgG are related to malignancy and stage of disease in myeloma and monoclonal gammopathy of uncertain significance (MGUS). METHODS: 61 patients were studied at diagnosis: 14 with MGUS, nine with stage I multiple myeloma, 11 with stage II, 21 with stage III, and five with solitary plasmacytoma. IgG was extracted from serum by protein G affinity chromatography. Oligosaccharides were cleaved from the protein backbone enzymatically by N-glycosidase F. Oligosaccharide analysis was performed by high pressure anion exchange chromatography with pulsed electrochemical detection (HPAE-PED). RESULTS: Up to 15 oligosaccharide peaks were identified in three major fractions: neutral, monosialylated, and disialylated. Patients with myeloma showed an increase in the proportion of sialylated oligosaccharides in comparison with patients with MGUS. The ratio of neutral to sialylated oligosaccharides (N:S) was reduced at all stages of myeloma compared with MGUS: MGUS, 11.35; myeloma stage I, 7.6 (p = 0.047); stage II, 5.20 (p = 0.035); stage III, 3.60 (p = 0.0002); plasmacytoma, 7.5 (p = 0.046). The N:S ratio was independent of paraprotein concentration (r = 0.05). CONCLUSIONS: The ratio of neutral to sialylated oligosaccharides may act as a new marker of malignancy in IgG paraproteinaemia and warrants further investigation.     

Waldenström''s macroglobulinaemia with anti-IgG activity: a series of five cases

Clinical, serological and cellular studies were carried out in five cases of Waldenström's macroglobulinaemia with cryoglobulinaemia of the mixed IgM–IgG type and extremely high serum levels of rheumatoid factor activity.

Clinically, no joint involvement or other distinctive features were found in these patients, as compared with other cases of primary macroglobulinaemia without anti-γ-globulin activity.

The isolation of the monoclonal IgM immunoglobulins, as well as absorption studies with IgG and immunocytological investigations demonstrated that the whole of the rheumatoid activity was associated with the Waldenström-type paraproteins.

Such cases seem to represent instances of a rheumatoid factor monoclonal gammapathy, in fact a subgroup of Waldenström's macroglobulinaemia, in view of the presence of an IgM paraprotein, possessing anti-IgG activity, synthesized by the lymphocytoid cells infiltrating the bone marrow.

     

Pathogenic role of a monoclonal IgA (κ) anti-IgG paraprotein associated with hemorrhagic diathesis,rheumatoid arthritis,vascular purpura,and acute membranoproliferative glomerulonephritis

Sixteen years earlier a 42-year-old woman with an IgA plasma cell neoplasm presented with bleeding disorder. Her prolonged course was complicated by subsequent development of rheumatoid arthritis, vascular purpura, and an acute membranoproliferative glomerulonephritis (MPGN). The paraprotein and its (Fab)₂ fragment showed affinity for a test myeloma IgG₂ () paraprotein. The patient's serum and the IgA-IgG complex separated by gel filtration did not exhibit cryoprecipitation. The complex also did not dissociate by ultracentrifugation. Electron microscopic and immunofluorescent studies of a renal biopsy sample taken during the episode of nephritis showed subendothelial deposits and a lacy fluorescent pattern strongly positive for IgA and IgG. The same immunoglobulins were eluted from the kidney at postmortem. A low concentration of monoclonal IgA (antibody) and excess unbound polyclonal IgG (antigen) were demonstrated in the patient's serum at the time of MPGN, apparently analogous to the conditions necessary for the induction of experimental immune complex nephritis.     

Serum paraproteins in chronic lymphocytic leukaemia.

The presence of paraproteins in the sera of 10 patients with chronic lymphocytic leukaemia (CLL) was investigated using immunoisoelectric focusing. Monoclonal immunoglobulins were found in nine of these 10 sera. Five sera contained a single monoclonal IgM paraprotein, one serum contained a single monoclonal IgG paraprotein, while three sera contained more than one monoclonal paraprotein--namely, IgM + IgD, IgM + IgG, and IgM + IgD + IgG. The results indicate that the malignant B cells of CLL may be at a later stage of differentiation than previously assumed and serum monoclonal immunoglobulin could be of value as a tumour marker.     

IgM–IgG cryoglobulinaemia with IgM paraprotein component

Four patients with mixed IgM–IgG cryoglobulinaemia are described. Clinically they all had some features of an autoimmune disease, while two of them had a lympho-epithelial tumour in the parotid gland. The mixed cryoglobulins of all patients contained an IgM paraprotein with the properties of a rheumatoid factor. They can be regarded as cryoprecipitates of a rheumatoid factor with autologous IgG. In one case the parotid tumour, and not the bone marrow, produced the IgM paraprotein. The clinical significance of the cryoglobulins is discussed. The IgM paraproteins with rheumatoid factor activity may be an expression of an underlying abnormality of the immunological system of these patients.     

IgM monoclonal gammopathy/Waldenstr?m's macroglobulinemia: a morphological and immunophenotypic study of the bone marrow

The presence of a monoclonal paraprotein in the serum or urine raises the possibility of myeloma or lymphoma/leukemia. Yet, in a significant proportion of individuals with serum paraproteins, particularly those with low levels of paraprotein, clinical and routine bone marrow evaluation is not diagnostic of an underlying neoplasm. The purpose of this study was to define the pathologic basis for macroglobulinemia in patients whose routine bone marrow biopsies were not diagnostic of a lymphoplasmacytic neoplasm. We used immunofluorescence microscopy and flow cytometry of cell suspensions prepared from aspirated marrow, as well as immunohistochemistry of core biopsies, to perform immunopathologic evaluations of the bone marrow from 16 such patients. Seven individuals without a monoclonal serum paraprotein, who were similarly studied, served as controls. Our data indicate that 13 of the 16 patients with monoclonal serum IgM paraproteins whose routine bone marrow biopsies were normal or showed nondiagnostic changes morphologically had a dispersed monotypic B lineage population of concordant immunoglobulin heavy and light chain type in the bone marrow. The immunophenotype of these cells spanned the range from mature B cell to plasmacytoid B cell to plasma cell. In four of these 13 patients a diagnosis of lymphoplasmacytic lymphoma could be made on the basis of greater than or equal to 20% monoclonal B lineage cells among bone marrow mononuclear cells.     

The Distinct Subgroup of Patients with Rheumatoid Arthritis Shown by IgG3-Reactive Rheumatoid Factor

The reactivity of rheumatoid factor (RF) with immunoglobulins of the IgG3 subclass was examined in 49 patients with rheumatoid arthritis (RA) using two types of IgG3 myeloma (routine and IgG3m-15 allotype). Among 49 patients, serum from eight cases showed positive reactivity with both types of IgG3 myeloma by radio-immunoassay (RIA). The isotype of IgG3-reactive RF was not specific; it belonged to the IgM class as well as the IgG subclasses IgG1, IgG2 and IgG4. The patients with IgG3-reactive RF belonged to the clinically-severe classification of RA, having a high erythrocyte sedimentation rate (ESR), high titre in the RA hemagglutination (RAHA) test, and above all they had low levels of complement. Generally, it is concluded that patients with IgG3-reactive RF have serious arthritis and that IgG3-reactive RF might play an important role in the inflammatory process. Furthermore, it was also shown that the RF-reactive site was not associated with the protein-A binding site of IgG3, since RF reacting with IgG3m-15 reacted similarly with routine IgG3, regardless of the difference of the protein-A binding activity. This was confirmed by adding protein-A to the reaction of RF and IgG3m-15 which binds with protein-A. ‘This suggests that the actual reactive site of RF is different to the site that binds protein-A.     

The distinct subgroup of patients with rheumatoid arthritis shown by Ig G3-reactive rheumatoid factor

The reactivity of rheumatoid factor (RF) with immunoglobulins of the IgG3 subclass was examined in 49 patients with rheumatoid arthritis (RA) using two types of IgG3 myeloma (routine and IgG3m-15 allotype). Among 49 patients, serum from eight cases showed positive reactivity with both types of IgG3 myeloma by radio-immunoassay (RIA). The isotype of IgG3-reactive RF was not specific; it belonged to the IgM class as well as the IgG subclasses IgG1, IgG2 and IgG4. The patients with IgG3-reactive RF belonged to the clinically-severe classification of RA, having a high erythrocyte sedimentation rate (ESR), high titre in the RA hemagglutination (RAHA) test, and above all they had low levels of complement. Generally, it is concluded that patients with IgG3-reactive RF have serious arthritis and that IgG3-reactive RF might play an important role in the inflammatory process. Furthermore, it was also shown that the RF-reactive site was not associated with the protein-A binding site of IgG3, since RF reacting with IgG3m-15 reacted similarly with routine IgG3, regardless of the difference of the protein-A binding activity. This was confirmed by adding protein-A to the reaction of RF and IgG3m-15 which binds with protein-A. This suggests that the actual reactive site of RF is different to the site that binds protein-A.     

Precision and reliability of paraprotein determinations by high-resolution agarose gel electrophoresis

Agarose gel electrophoresis has recently replaced cellulose acetate electrophoresis as the preferred technique for monitoring paraprotein levels in patients with plasma cell dyscrasias. The authors studied the accuracy and precision of this method for paraprotein determination. Twenty-seven serum samples with paraprotein concentrations ranging from 5 to 73 g/L were aliquotted and assayed on 20 separate occasions, and the mean and standard deviation for the paraprotein concentration in each serum was established. Linear regression analysis showed that the standard deviation of paraprotein concentration (SD) increased as a function of paraprotein concentration (PC). For IgG paraproteins, the regression equation was SD = 0.041 (PC) + 1.06; R = 0.942; standard error = 0.32. For non-IgG paraproteins the equation was SD = 0.101 (PC) - 0.04; R = 0.851; standard error = 0.5. The accuracy of paraprotein determinations by the agarose gel electrophoretic technique was assessed by comparison with values obtained with the use of a previously validated enzyme-linked immunosorbent assay (ELISA) method for quantitation of IgG subclasses. Results obtained by the two methods were similar and highly correlated: (concentration by electrophoresis) = 0.921 (concentration by ELISA) + 0.46; R = 0.988; standard error = 0.34. The laser densitometric scanning procedure showed a loss of linearity above 60 g/L, indicating the need to dilute sera with very high paraprotein concentrations in order to obtain accurate results. A table is presented that should help pathologists who interpret such scans to determine whether small changes in paraprotein measurements occurring over time represent true changes in paraprotein concentration or merely reflect the analytic variability inherent in the technique.     

Multiple myeloma with monoclonal IgG and IgD of lambda type exhibiting, under treatment, a shift from mainly IgG to mainly IgD.

A patient with multiple myeloma (MM), who initially presented with a predominant IgG lambda and a minor IgD lambda paraprotein pattern, is described. After chemotherapy, levels of the IgD lambda protein increased and the IgG lambda levels decreased. The following results were obtained when serum IgD was predominant. In the bone marrow, there were three plasma cell populations: a major one containing only delta chains, a minor one containing only gamma chains, and another minor one containing both delta and gamma chains. All these plasma cell populations contained lambda chains. Stimulation of circulating mononuclear cells with pokeweed mitogen (PWM) achieved differentiation of circulating B lymphocytes into plasma cells: 30% with only cytoplasmic delta lambda chains and 10% with only cytoplasmic gamma lambda chains. These IgG-containing plasma cells showed cytoplasmic reactivity with rabbit antiserum raised against monoclonal IgD which was shown to contain specificities recognizing both delta chains and idiotypic determinants present in both serum IgD lambda and IgG lambda. Circulating B lymphocytes were 'monoclonal': almost all expressed surface delta lambda chains, and a small proportion of them expressed both delta gamma and lambda chains. High levels of IgD were detected in the supernatants of all cultures, but high concentrations of IgG were only detected in those from PWM-stimulated cultures with very low levels of IgM and IgA. These findings suggest that plasma cells producing either IgD or IgG were derived from a common B-cell clone. Double paraproteinaemia exhibiting a shift in immunoglobulin production from IgG to IgD has not been previously described.