22 Multiple myeloma and related plasma cell proliferative disorders

Lothar Thomas

Multiple myeloma and related plasma cell disorders (plasma cell dyscrasias) are a heterogeneous group of disorders caused by the monoclonal proliferation of plasma cells or lymphoplasmacytic cells (Tab. 22-1 – Multiple myeloma and related plasma cell disorders).

22.1 B cell development and proliferation

Plasma cells are a type of immune cells that makes specific antibodies and result from the terminal differentiation of B cells /1/. Through the rearrangement of the V, D, and J segments of the genes that encode heavy chains and light chains, the B cell develops the ability to produce antibodies. All cells of a naive B cell clone produced in the bone marrow have the same VDJ segment sequence. Cells of this clone then migrate to the lymph nodes, where they develop into IgM secreting B cells following primary contact with an antigen. Following secondary antigen contact in the lymph nodes, mutations in the VDJ sequence occur, resulting in improved antigen specificity /2/. This process is called somatic hyper mutation.

The somatically mutated B cells develop in one of two directions:

• Most undergo isotype class switching to become IgA and IgG secreting plasma cells. They leave the germinal centers of the lymph nodes and return to the bone marrow to become antibody secreting plasma cells.
• A minority of plasmablasts develop into memory cells in niches of the bone marrow and may persist as memory cells for months or even years /3/.

Multiple myeloma and related plasma cell disorders result from the neoplastic transformation and proliferation of a memory plasma cell in the bone marrow. The plasma cell clone produces an intact immunoglobulin, free light chains or Ig fragments.

B-cell maturation antigen /95/

The B cell maturation antigen (BCMA) is a glycoprotein on the surface of normal and malignant plasma cells and some mature B-cells. The membrane protein has an extracellular domain, intracellular ligand binding domain and a transmembrane domain.

BCMA is a pivotal therapeutic target and has been used to treat multiple myeloma using the following substances:

• Balantamab mafodotin as a BCMA-targeted antibody-drug conjugate
• idecabtagene-vicleucel and ciltacabtagenea as anti-BCMA chimeric antigen receptor therapy
• teclistamab-cqyv as BCMA-targeted bispecific T cell engager.

Polyclonal cell proliferation

The immune stimulation by microbes and macromolecular antigens results in activation, multiplication and differentiation of a heterogenous population of B lymphocyte clones which leads to plasma cells producing a broad pattern (polyclonal pattern) of antibodies. The electrophoretic separation of serum proteins is characterized by a broad γ-globulin fraction.

Oligoclonal cell proliferation

In some circumstances immune stimulation results only in a restricted number of clones producing a limited pattern of immunoglobulins. The electrophoretic separation is characterized by a saw blade pattern of the γ-globulin fraction.

Monoclonal cell proliferation

The unknown stimulation is restricted to a single clone with increased proliferation. The electrophoretic pattern is characterized by an M-spike in the γ-globulin fraction or the β-globulin fraction.

In multiple myeloma and related plasma cell disorders, the malign cell clone develops from B cells that have passed through the germinal centers of the lymph nodes, have already undergone somatic hyper mutation and isotype class switching, and rest as memory plasma cells in niches of the bone marrow. Homing to the bone marrow is thought to be promoted by the expression of adhesion molecules such as NCAM (CD56), syndecan (CD138), and PECAM1 (CD31) on the transformed B cell.

Refer to Fig. 22-1 – B cell maturation.

Waldenstroem’s macroglobulinemia is characterized by the presence of lymphoplasmacytic cells that have undergone somatic hyper mutation but have not undergone isotype class switching. These cells secrete monoclonal IgM.

IL-6 and IL-1β are important factors in the pathogenesis of monoclonal plasma cell proliferative diseases. IL-1β is produced by the abnormal plasma cells and has strong osteoclast activating factor (OAF) activity /5/. In addition, IL-1β stimulates the expression of adhesion molecules on the abnormal plasma cells and activates bone marrow stromal cells to secrete IL-6 /6/. IL-6 promotes the proliferation of the abnormal plasma cell clone; there is a high degree of correlation between the IL-6 concentration and the plasma cell labeling index.

Refer to Fig. 22-2 – Binding of an atypical plasma cell to a stromal cell of the bone marrow.

Multiple myeloma is the most important plasma cell proliferative disease of terminally differentiated plasma cells. Evidence of monoclonal gammopathy is a key criterion in the diagnosis of multiple myeloma /7/.

22.2 Gammopathies

The increase in γ-globulin fraction (gammopathy) may result in a polyclonal, oligoclonal or monoclonal pattern in serum protein electrophoresis.

Polyclonal gammopathy

Immunoglobulin (Ig)-producing plasma cells are highly specialized. Each B cell clone produces proteins of one immunoglobulin class, subclass, allotype class and light chain type. This means that each clone synthesizes identical Ig molecules with the same antibody specificity.

In disease states (infection, liver disease, autoimmune disorder), a wide range of antigens are released that induce antibody production by many different cell clones. This results in a large number of specific antibody molecules with differing primary structures, functions, and physicochemical properties that have both the same and different Ig classes and light chain types. An abnormality in plasma protein composition of the serum (dysproteinemia) results characterized by a broad-based peak in the γ-globulin region on serum protein electrophoresis (immunoreactive constellation). This is known as polyclonal gammopathy and is typically seen in infections, acute and chronic inflammation, liver disease, autoimmune disorders, and systemic malignancy

Monoclonal gammopathy

Monoclonal gammopathy is defined by the presence of monoclonal immunglobulin in plasma, urine or both that is produced most often by clonal plasma cells and less common by lymphocytes. In contrast to the reactive, heterogeneous increase in the γ-globulin fraction observed in polyclonal gammopathy, abnormal proliferation of plasma cells or lymphoplasmacytic cells leads to excessive synthesis of monoclonal proteins (M-proteins) i.e., structural intact immunoglobulins, free light chains, and free heavy chains with identical antigens and structures. Because they are produced monoclonally, M-proteins are:

• Intact Ig of one class and one light chain type
• Light chains of one type: kappa (κ), lambda (λ)
• Heavy chains of one class (α, γ, μ, δ, ε).

Monoclonal Igs have the same structure as polyclonal Igs and are assigned to physiological Ig classes and types according to their structure. They are classified into the classes IgG, IgA, IgM, IgD, and IgE and the light chain types κ and λ. The homogeneity of M-proteins manifests as a narrow gradient on serum protein electrophoresis (known as an M-gradient or M-peak). The M-gradient remains within the area where polyclonal Ig is normally seen, that is, within the γ and β area. M-proteins are also known as paraproteins.

M-proteins appear on serum immunofixation electrophoresis as:

• Ig of one class and one type (e.g., IgG-κ, IgA-λ)
• Free light chains of one type, κ or λ
• A combination of monoclonal Ig and free light chains
• A free heavy (H) chain (e.g., α-, γ-, μ-chain or an Fc fragment)
• Various M-proteins (e.g., in biclonal, triclonal, or multi clonal gammopathy).

The hematologic criteria for classifying monoclonal gammopathies are based on the presence of a clonal mass and end-organ damage.

Diagnosis, prediction of progression and indication of treatment of monoclonal gammopathies are dependent on a clonal mass and end-organ damage /7/:

• Symptomatic multiple myeloma: Criteria are > 60% bone marrow plasma cells, elevated serum free light chain ratio with the level of the involved free light chain > 100 mg/L, and more than one bone lesion on magnetic resonance imaging. Refer to Table 22-9 – Diagnostic criteria for multiple myeloma and further plasma cell proliferative disorders.
• Smoldering multiple myeloma: Patients who do meet the tumor burden criterion but do not have evidence of endorgan damage. Refer to Table 22-9 – Diagnostic criteria for multiple myeloma and further plasma cell proliferative disorders.
• Monoclonal gammopathy of undetermined significance (MGUS): Patients who neither meet the clonal burden criterion nor have end-organ damage. Refer to Table 22-9 – Diagnostic criteria for multiple myeloma and further plasma cell proliferative disorders.
• Monoclonal gammopathy of renal significance (MRGS): The MRGS represents any B-cell or plasmacell clonal disorder but produces a nephrotoxic monoclonal immunoglobulin that directly or indirectly results in kidney disease or injury.
• Lymphoplasmacytic lymphoma (formerly Waldenstroems macroglobulinemia). Treatment is indicated when bone marrow involvement is 10% or more and at least one of the following is present: anemia or thrombocytopenia, cryoglobulinemia, and hyperviscosity syndrome.
• IgM-MGUS: Lymphoplasmacytic lymphoma clones or monoclonal B-cell lymphocytosis are present.
• Chronic lymphocytic leukemia (CLL): More than 5 × 10⁹ leukemic cells per liter and the presence anemia or thrombocytopenia or the presence of symptomatic lymphadenopathy, splenomegaly or both.

22.3 Laboratory diagnostics of monoclonal gammopathy

The goals of tests for the diagnosis of monoclonal immune protein (M-protein) production are:

• Detection of monoclonal gammopathy
• Immunochemical characterization and confirmation of monoclonality
• Quantitative determination of the M-protein.

Screening tests used to diagnose monoclonal plasma cell proliferative diseases /8/:

Tab. 22-2 – Screening panels for diagnosis of monoclonal plasma cell proliferative diseases.

Investigations to confirm monoclonal gammopathy:

Tab. 22-3 – Tests for diagnosing and monitoring of monoclonal gammopathies.

Methods of determination:

Tab. 22-3 – Tests for diagnosing and monitoring of monoclonal gammopathies /8/.

Suspicion for plasma cell dsycrasia

Suspicion for abnormal plasma cell proliferation is based on clinical and laboratory findings.

22.3.1 Serum protein electrophoresis

Indication

Serum protein electrophoresis (SPE) is a suitable screening test for monoclonal gammopathies because an M-gradient is usually present in IgG and IgA multiple myeloma and in Waldenstroem’s macroglobulinemia. A prominent peak in the β-region often demonstrates a myeloma protein. Immunofixation electrophoresis (IFE) is used to confirm monoclonality. If SPE is used as a screening test for monoclonal gammopathy, serum free light chains should also be determined. This is because small M-gradients in SPE are not be registered by haptoglobin increase in the α₂ region or by transferrin or C3 increase in the β region. In addition, some M-proteins (e.g., IgM, may not be separated in SPE as a result of self-aggregation on the sample application side). Thus, in one study /9/, these and similar abnormalities were found in SPE in 13.2% of cases (Tab. 18.3-4 – Reflex testing upon abnormalities in serum protein electrophoresis). Additional serum IFE identified the abnormality as an M-protein in 43% of these cases. Serum free light chain determination or urine IFE should be performed even if SPE is negative because SPE is usually unable to detect an M-gradient in light chain, IgD, or IgE myeloma or in non secretory myeloma or amyloidosis. In light chain myeloma and the other entities mentioned, secondary antibody deficiency often appears as hypogammaglobulinemia in SPE.

The M-protein concentration can be calculated based on the area of M-gradient in the SPE printout and the total protein concentration. For quantitative determinations of M-protein, SPE is more reliable than immunonephelometric and immunoturbidimetric measurements since M-proteins are quantified independently from antigen-antibody binding by means of a reaction with dyes /9, 10/. To correctly differentiate a patient with very good partial response (≥ 90% reduction in measurable intact M-protein) from partial response (≥ 50%), minimal response (≥ 25%) and progressive disease (≥ 25% increase), it is crucial that the variability in M-protein measurements is small /35/. The Dutch External Quality Assessment program for M-protein diagnostics revealed a large variation in reported M-protein concentration between different laboratories. The data indicated that M-protein quantification to monitor patients is appropriate, when subsequent testing is performed within the same laboratory. The average between laboratory CV was 25%. M protein concentration < 2 g/L were not assessable /81/.

Method of determination

Principle: serum proteins are separated on a cellulose acetate or agarose gel medium according to their isoelectric points (Section 18.3 – Serum protein electrophoresis) /9/.

Detection limit: a serum M-protein level of greater than 2 g/L produces a detectable M-gradient whose detection depends on the position of the M-gradient in the electrophoretogram and the concentration of polyclonal Ig. Low concentrations of M-protein (especially IgD, IgE, or free light chains) may be missed. In terms of percentages, the electrophoretic fraction may lie within the reference interval, despite the presence of a visible M-gradient. For a comparison between the diagnostic sensitivity of SPE in comparison to other electrophoreses, refer to Section 18.3.

Specimen

Serum: 1 mL

Assessment

On the printouts of cellulose acetate serum protein electrophoresis, M-protein presents as a dense, discrete band and in the densitometric evaluation of the print-out as a narrow peak (Fig. 22-3 – Serum protein fractions on cellulose acetate sheets). The M-gradient is normally located in the γ fraction, less commonly in the β fraction, and rarely in the α₂ fraction. It is not possible to determine the M-protein class based on the electrophoretic mobility of the M-gradient.

A broad-based increase in the γ-globulin fraction, sometimes with the development of a shoulder between β- and γ fraction, is present in polyclonal gammopathy (Fig. 22-3 – Serum protein fractions on cellulose acetate sheets).

Oligoclonal gammopathy is characterized by the presence of multiple discrete bands in SPE while the densitometric evaluation printout reveals an irregular pattern of the γ-globulin fraction (Fig. 22-3 – Serum protein fractions on cellulose acetate sheets).

An abnormal broad band found in the α₂- or β-globulin region in serum protein electrophoresis or immunofixation electrophoresis is usually associated with a heavy chain disease.

The frequency of detection of monoclonal gammopathies depending on the test used is shown in Tab. 22-4 – Diagnostic accuracy of tests and test panels for detection of monoclonal gammopathies.

Comments and problems

In order to correctly identify an M-protein, it is advantageous to directly, visually assess the electrophoresis sheet /9/. The printout of the densitometry results is less sensitive and less specific than direct assessment of the separation on the cellulose acetate or agarose sheet. Tab. 22-5 – Pseudo M-gradients that may be mistaken for M-gradients in serum protein electrophoresis lists technical artifacts as well as so-called “pseudo” M-gradients.

When calculating the M-gradient, it is important to consider potential abnormalities in the total protein concentration (e.g., falsely high results following the use of plasma expanders).

If no attention is paid to possible presence of cryoglobulinemia, false negative results,may occur; this also applies to immunofixation electrophoresis and capillary zone electrophoresis.

22.3.2 Urine protein electrophoresis

Indication

Quantitative determination of the excretion of monoclonal free light chains (Bence Jones protein, BJP).

Method of determination

Principle: as for SPE. Prior to electrophoresis, the urine is concentrated some 200 times using a concentrator to produce a final urine protein concentration of 20–80 g/L /10/. Following electrophoresis and protein staining, densitometric evaluation of the electropherogram is performed. BJP does not produce a clear M-gradient like it does in SPE; two BJP gradients are also possible.

Specimen

Morning spontaneous urine or 24-hour collection of urine*: 100 mL

* Stabilized by the addition of 1 g sodium azide to prevent bacterial degradation of BJP.

Reference interval

Densitometric evaluation of concentrated urine electrophoresis: less than 50 mg/L or less than 75 mg /24 h /10/.

Assessment

An M-gradient suggests the presence of BJP as well as monoclonal Ig; classification and typing are accomplished by immunofixation electrophoresis.

22.3.3 Immunofixation electrophoresis

Serum IFE

Immunofixation electrophoresis (IFE) is regarded as the gold standard for detecting M-protein in serum and urine /11/.

Indication

IFE is a qualitative method that is approximately 5–10 times more sensitive than SPE for identifying monoclonal gammopathies. It is also used to confirm the presence of an M-gradient in SPE and to classify and type M-proteins. In addition, it can differentiate between monoclonal, biclonal, and oligoclonal gammopathies.

Method of determination

Principle: proteins are separated according to their charge and identified through immunoprecipitation with monovalent antisera. The sheets used are agarose gels. Following electrophoresis, a paper strip soaked with antiserum is applied onto the gel along the migration axis. The resulting antigen-antibody complexes adhere to the pore structure of the gel and after a step of washing to rinse off non precipitated proteins, are stained with Coomassie Blue /11/.

For precipitation highly purified monovalent antisera are used directed against the constant region of the heavy chains γ, α, and μ as well as against the constant region of the κ and λ light chains (Tab. 22-6 – Antisera used to precipitate M- proteins in immunofixation electrophoresis). Detection limit: 0.2–0.6 g/L.

Specimen

Serum: 1 mL

Assessment

The following diagnostic interpretations are made based on the band patterns /11/:

• A diffuse increase in precipitate with one or more heavy chain antisera and both light chain antisera indicates a polyclonal gammopathy (Fig. 22-4 – Immunofixation electrophoresis in polyclonal and monoclonal gammopathies (a))
• A densely stained band in a diffuse IgG, IgA, or IgM precipitate along with a corresponding densely stained κ or λ precipitate suggests the presence of an M-protein (Fig. 22-4 – Immunofixation electrophoresis in polyclonal and monoclonal gammopathies (b))
• Monoclonal free light chains are indicated by the formation of a narrow precipitate with one of the two antisera to light chains
• Heavy chain gammopathies are characterized by the presence of a dense, homogeneously stained precipitate with one of the heavy chain antisera whereas no corresponding precipitation occurs with antisera to light chains
• Oligoclonal gammopathies are characterized by at least three narrow bands: either a discrete band is present within the precipitate of a heavy chain and of both light chains or multiple bands are present within the precipitates involving heavy chain and light chain antisera.

Comments and problems

To avoid misinterpretation, it is important to note the following:

• If free light chains are detected, it is important to check for a narrow zone of precipitate with IgD and IgE antisera to ensure that an IgD and IgE myeloma is not missed
• Consideration must be given to the fact that biclonal gammopathy may be mimicked by a prozone phenomenon since immunoprecipitation fails to occur in the presence of high antigen excess within the central area of the band (the area with the highest antigen concentration). To avoid the prozone phenomenon, the IFE should be repeated at various dilutions.
• Immune complexes in the patient’s serum (e.g., IgM) aggregates, remain at the application site and form narrow precipitation bands, however, they are identifiable as artifacts by their reaction with both light chain antisera
• Antisera to heavy chains, in particular the ε chain, but also the λ chain can demonstrate cross reactivity with fibrinogen /11/.
• Some reagents, especially anti-IgM and anti-IgA, have cross reactivity with other proteins

Multiple M-spikes can result /46/:

• Due to the presence of two or more plasma cell clones. Commonly however, when the light chains are identical for the two IgG bands the finding is considered likely to present a single gene product with differential post translational processing formation of dimers.
• Due to different degrees of Ig polymerization (e.g., monomeric IgA, dimeric IgA, dimeric IgA in folded form)
• Complex formation with serum proteins, and self-aggregates. In such cases, mercaptoethanol should be added to the serum (0.5–1% final concentration) before applying the sample. Polymeric forms of Ig are then transformed into single Ig molecules.
• Under- or over dilution of serum prevents recognition of light chains. Antigen excess or a prozone effect may lead to false negative effects For patients with a total immunoglobulin 20 g/L a 3-fold dilution of serum is recommended

Monitoring the M-protein of multiple myeloma patients treated with therapeutic monoclonal antibody /83/

Therapeutic monoclonal antibodies Daratumumab and Nivolumab become visible on IFE as two IgG kappa bands that migrate close to each other at the cathodal end of the Gamma-region. In case the M-protein co-migrates with these therapeutic monoclonal antibodies. The M-protein can be distinguished from both therapeutic monoclonal antibodies by use of a double hydrashift assay.

22.3.3.1 Urine immunofixation electrophoresis

Indication

Detection of monoclonal free light chains in urine (Bence-Jones protein, BJP).

Method of determination

Principle: as with serum IFE. The urine is first concentrated some 100 times using a concentrator to a final protein concentration of 1–10 g/L.

Specimen

Morning spontaneous urine or 24 hour collection of urine*: 100 mL

* Stabilized by the addition of 1 g sodium azide to prevent bacterial degradation of BJP.

Assessment

The detection limit in urine is around 100 mg/L, which corresponds to 200 mg/24 hour urine. In theory, it should be possible to detect 1 mg of BJP in urine that has been concentrated 100 fold. BJ proteinuria is seen in around 75% of cases of multiple myeloma.

22.3.4 Immunosubtraction (IS) and capillary zone electrophoresis (CZE)

If an M-gradient has been detected with SPE or CZE, a combination of immunosubtraction and CZE can be used in a similar way to IFE to classify and type the serum M-protein. Refer also to Section 18.3 – Serum protein electrophoresis.

Method of determination

Principle: the sample is incubated with antibody-coated Sepharose beads that bind IgG, IgA, IgM, κ, or λ molecules. Following precipitation, the respective supernatant is separated in comparison with the untreated sample using CZE (refer also to Section 18.3). The M-protein class and type are identified based on their disappearance following incubation with the corresponding antibody. Detection limit: 0.5 g/L /12, 13/.

Specimen

Serum: 1 mL

Assessment

Similar to IFE /12, 13/.

Comments and problems

Quantitative measurement of serum M-protein is an important tool for monitoring disease activity in multiple myeloma. IF M-protein quantification by CZE was compared to densitometric scanning of high resolution agarose gel electrophoresis there is a clear bias /14/:

• Densitometry gave a higher value at low M-gradient (< 20 g/L)
• CZE gave a higher value at large M-gradient (> 20 g/L).

22.3.5 Free light chains (FLCs)

Biology of FLC production /15, 16/

Immunoglobulin free light chains are by-products of immunoglobulin (Ig) synthesis and in healthy individuals are released in the circulation in small quantities. To achieve the correct assembly of intact immunoglobulin, the production rate of light chains must be 40% higher than that of heavy chains. Excess FLCs are released into the circulation and excreted by the kidneys. Nearly twice as many plasma cells produce intact immunoglobulins of isotype κ as of isotype λ and the ratio of immunoglobulin κ to λ in serum is 1 : 1.81. The κ FLCs have a molecular mass of 22.5 kDa and a plasma half-life of 2 to 4 hours. The λ FLCs exist in dimeric forms and have a molecular mass of 45 kDa and half-life of 3 to 6 hours. The 500 mg of FLCs normally produced by the lymphatic system every day are catabolized in the proximal tubule following glomerular filtration. FLC reabsorption takes place through non-specific binding to megalin/cubulin scavenger receptors. The system is so efficient that up to 30 g of FLCs can be reabsorbed daily. Usually, light chains exist as monomeric or dimeric forms, but rarely, larger molecules, such as trimers or tetramers have been reported in the serum.

The determination of FLCs and their ratio may be applied on patients with:

• benign monoclonal gammopathy
• in monoclonal gammopathy of undetermined significance (MGUS) to symptomatic patients, as in multiple myeloma (MM) or amyloid light chain (AL) amyloidosis. FLCs measured on serum samples obtained fom such patients can be used for monitoring disease progression and patient management.

The NICE guidelines for the management of patients with myeloma, the International Myeloma Working Group guidelines, and NCCN Clinical Practice Guidelines in Oncology recommend the use of FLCs for initial workup of multiple myeloma. Today measurement of FLCs is part of the standard care of patients with suspected gammopathies. Method comparison has shown good agreements for kFLC, λFLC and FLC ratio /90/.

The plasma cell clones of about 20% of multiple myeloma produce only immunoglobulin light chains (light chain multiple myeloma, LCMM) Similar to MGUS there exists a light chain MGUS as precursor condition.

FLCs in MGUS

The criteria of MGUS are shown in Tab. 22-9 – Diagnostic criteria for multiple myeloma and further plasma cell proliferative disorders. The prevalence of MGUS in Germany is 3.5% (95% confidence interval 3.0–4.1) in the median age group of 63 years (range 47–75 years) /91/. Studies have shown that black individuals have a 3-fold higher prevalence of MGUS than white individuals /92/.

Similar to MGUS there exists a light chain MGUS as precursor condition leading to light chain multiple myeloma, defined by use of the FLC ratio. The light chain MGUS was found to be 0,8% in the general population of 50 years of age or older /93/.

Polyclonal synthesized FLCs

In normal individuals immunoglobulin (Ig) and light chain synthesis is polyclonal and only 1–10 mg of FLCs are excreted in the urine per day. Inflammation/infection or chronic kidney disease (CKD) can result in serum concentrations of polyclonal FLCs that are many times greater than normal. Inflammation/infection does not affect the serum κ/λ ratio, which remains within the reference interval of about 1.5. In chronic kidney disease (CKD), the serum FLC concentration increases as the glomerular filtration rate (GFR) declines and by stage 4 of CKD is more than 5 times higher than normal. As the GFR continues to decline, the reticuloendothelial system also becomes involved, eliminating FLCs by pinocytosis. Because pinocytosis is independent of molecular mass, the serum κ/λ ratio shifts in the direction of the normal production rate from a mean value of 0.58 in healthy individuals to a mean value of 1.19 in patients with stage 5 CKD. The reference interval for the κ/λ ratio in CKD changes to 0.37–3.1. An increase in the concentration of normal polyclonal λ light chains is associated with an increase in the conversion of monomers to dimers and tetramers /89/.

Monoclonal synthesized FLCs /17/

Monoclonal FLC production by a plasma cell clone causes a shift in the κ/λ ratio. A clone that produces κ FLCs increases the ratio while a clone that produces λ FLCs decreases it. In plasma cell dyscrasia, the production of monoclonal FLCs can increase by up to 100 fold, exceeding the tubular reabsorptive capacity. Monoclonal FLCs have toxic effects on proximal tubular cells and block the transport of glucose, amino acids, and phosphate. Excessive endocytosis of monoclonal FLCs in the proximal tubule triggers a range of inflammatory responses that can lead to isolated tubular cell toxicity, tubulo interstitial nephritis, and myeloma kidney (myeloma cast nephropathy). There is a risk of acute renal failure if the urinary excretion of FLCs increases to over 2 g per day. The pattern of kidney injury depends on the structural peculiarities of monoclonal FLCs, particularly of the variable (V) domain, as well as factors such as the pH of the primary urine, urea concentration, and local tissue proteolysis.

Antisera for FLC determination /17, 18/

Ideally, FLCs should be determined using antisera that are specific for FLCs and do not recognize light chains bound to heavy immunoglobulin chains. Antisera to FLCs only recognize the hidden inner antigenic determinants in the constant region of immunoglobulin light chains These determinants are located between the light and heavy chain of an immunoglobulin and are only available to react with antibodies when the light chain is separated from the heavy chain. Antisera that do not differentiate between FLCs and immunoglobulin bound light chains recognize both the accessible external and internal hidden antigenic determinants and therefore measure immunoglobulins in addition to FLCs.

Refer to Fig. 22-5 – Immunoglobulin molecule with two identical heavy chains and two identical light chains.

22.3.5.1 Serum free light chains

Indication

The indications for serum FLC determination according to the recommendations of the International Myeloma Working Group guidelines /17/ are listed in Tab. 22-7 – Indication for serum free light chain determination.

Method of determination

Principle: latex-enhanced immunoassay. The patient sample is incubated with antiserum (polyclonal or monoclonal antibodies to free κ chains or free λ chains fixed to latex particles) and immune complex formation is detected using kinetic nephelometry or kinetic turbidimetry. Reactivity between the antisera used and the bound light chains of immunoglobulins is negligible /18/. The concentration of κ and λ FLCs is determined and the κ/λ ratio is calculated. The κ/λ ratio has a higher diagnostic significance than the FLC level which is not specific for monoclonal FLCs because polyclonal FLCs are also detected.

Specimen

Serum: 1 mL

Reference interval

Refer to References /16, 19, 20/ and Tab. 22-8 – Reference ranges of free light chains in serum.

Clinical significance

Patients with a κ/λ ratio > 1.65 have excess κ FLCs and are presumed to be producing monoclonal κ FLCs. Patients with ratios < 0.26 have excess λ FLCs and are presumed to be producing monoclonal λ FLCs.

The diagnostic accuracy of serum FLCs for detection of monoclonal gammopathies is shown in Tab. 22-4 – Diagnostic accuracy of tests and test panels for detection of monoclonal gammopathies.

Serum FLC determination has a diagnostic specificity of 96–98.5%.

About a third of patients with MGUS, 70% of patients with smoldering multiple myeloma, and more than 90% of patients with multiple myeloma have altered FLC serum ratios that indicate production of clonal FLC by the proliferating plasma cell population

Comments and problems

The detection limit of serum FLCs is:

• 0.5–2 g/L for serum protein electrophoresis
• 0.15–0.5 g/L for immunofixation electrophoresis
• Approximately 1 mg/L for immunoassay.

Assays using polyclonal FLC antiserum have poor dilution linearity compared to assays using monoclonal antiserum. For example, patient samples with excess of κ FLCs were underestimated in the initial dilution and were 15–43% higher in the next highest dilution /21/. When both assay were compared, correspondence of 81% was shown for κ FLCs and 74% for λ FLCs. The results from 5% of patient samples were discordant. It is thought that monoclonal antibodies are unable to detect FLCs in some patients /22/.

Serum free light chain (sFLC) measurements have inherent analytical limitations impacting sFLC interpretation. In a study /88/ analytical and diagnostic performance of three polyclonal sFLC assays on four analytical platforms were investigated. Method comparison showed excellent correlation with Freelite/Optilite method for all assays. Significant differences in reference values and diagnostic performance hamper interchangeability of sFLC assays.

22.3.5.2 Urine free light chains

Monoclonal FLCs in the urine are also known as Bence-Jones protein. Urine FLC immunoassays are not recommended for monitoring patients with monoclonal plasma cell proliferative disease or for assessing their response to treatment. This is because FLC concentrations are overestimated in some 75% of cases and there is no correlation between the concentrations in serum and urine /17/.

Polyclonal FLC concentrations in the urine correlate with polyclonal FLC concentrations in the serum. However, the same is not true for monoclonal FLCs. This reflects divergent handling of monoclonal and polyclonal FLCs in the kidney. Proximal tubular reabsorption of FLCs is mediated by megalin-cubulin receptor endocytosis. Depending on the renal disease, the receptor has a differential affinity for monoclonal and polyclonal FLCs /20/.

22.3.6 Quantitative determination of immunoglobulin isotypes

Commercially available antisera are suitable for detecting normal immunoglobulins using immunonephelometric and immunoturbidimetric assays. However, discrepancies are likely if they are used to quantify M-proteins. Monoclonal IgM concentrations as measured using these antisera are 10–20 g/L higher than those calculated using the M-gradient in SPE and the total protein. Monoclonal IgG and IgA concentrations are also overestimated slightly /10/. The reasons for this are unknown. The absolute M-protein concentration should therefore be determined using SPE. Immunonephelometric and immunoturbidimetric assays can be used to monitor changes in the M-protein concentration over time, provided the same method and same antiserum are used for all determinations.

22.3.7 Plasma cell proliferative cell disorders

Features of plasma cell proliferative cell disorders are:

• a clonal proliferation of plasma cells
• the clonal proliferative plasma cells produce monoclonal immunoglobulins and light chains that are determined in serum/plasma or urine
• plasma cell proliferative cell disorders range clinically from asymptomatic conditions such as MGUS to smoldering multiple myeloma and to symptomatic conditions such as multiple myeloma and plasmacytoma.

Plasma cell proliferative disorders and monoclonal gammopathies are reported in the following diseases and conditions:

• Symptomatic multiple myeloma
• Non IgM monoclonal gammopathy of undetermined significance (MGUS)
• Smoldering multiple myeloma
• Non secretory multiple myeloma
• Solitary plasmacytoma of bone
• Extramedullary plasmacytoma
• Solitary plasmacytomas with minimal marrow involvement
• Plasma cell leukemia.

The 2013 International Myeloma Working Group consensus /23/ updated criteria for multiple myeloma and related plasma cell disorders. The Working group criteria /24/ are shown in Tab. 22-9 – Diagnostic criteria for multiple myeloma and related plasma cell disorders.

According to the 2003 International Myeloma Working Group consensus /25/, the CRAB criteria (hypercalcemia, renal insufficiency, anemia, bone lesions) distinguish between symptomatic multiple myeloma and asymptomatic forms (Tab. 22-10 – CRAB features). Symptomatic multiple myeloma arises from an asymptomatic precursor condition, either MGUS or smoldering multiple myeloma. Patients with MGUS or smoldering myeloma should be followed carefully for the development of myeloma related organ or tissue impairment (ROTI), the most common being CRAB features /26/.

22.3.8 Monoclonal gammopathy of undetermined significance (MGUS)

MGUS is present in 3–4% in the population over the age of 50 years. The incidence increases with age and is higher in African-Americans than in Caucasians. The risk of progression to symptomatic myeloma is approximately 1% per year /23/. A study of 1384 individuals with MGUS conducted between 1960 and 1994 determined the relative risk of progression to the following diseases (X times higher compared to individuals without MGUS) /27/:

• Multiple myeloma (25)
• IgM myeloma (2.4)
• Primary amyloidosis (8.4)
• Waldenstroem’s macroglobulinemia (46)
• Plasmacytoma of bone (8.5)
• Chronic lymphocytic leukemia (0.9).

Diagnostic criteria: refer to Tab. 22-9 – Diagnostic criteria for multiple myeloma and related plasma cell disorders.

The risk of MGUS depends on the class of the M-protein and the height of the M-gradient. Par example in patients:

• The risk of progression is twice as high for individuals with monoclonal IgA or IgM than for those with IgG
• The risk of progression for individuals with an M-gradient > 15 g/L is twice as high, and for those with an M-gradient of > 25 g/L is 4.6 times as high as for individuals with an M-gradient of 5 g/L.

In oncologic clinics, over half of patients with monoclonal gammopathy have MGUS and 15–20% have symptomatic multiple myeloma or smoldering multiple myeloma. In one study /28/, M-protein levels of < 10 g/L were measured in 63.5% of individuals studied and concentrations of ≥ 20 g/L were measured in only 4.5%; light chain proteinuria was detected in 21.5%. The isotype of the monoclonal protein was IgG in 68.9%, IgM in 17.2%, IgA in 10.8%, and biclonal in 3%. The urinary light chains were 62% κ chains and 39.9% λ chains. The progression of MGUS to smoldering multiple myeloma was independent of age and did not increase with the duration of MGUS. A risk stratification model for MGUS is shown in Tab. 22-11 – Risk of progression from MGUS to symptomatic MM within 20 years.

The presence of an abnormal free light chain (FLC) ratio, and the extent to which the FLC ratio is abnormal, predict risk of progression in MGUS /28/.

22.3.9 Smoldering multiple myeloma

According to the updated International Myeloma Working Group criteria smoldering multiple myeloma (SMM) is an asymptomatic plasma cell disorder characterized by an M-component > 3 g/dL, bone marrow plasma cell infiltration > 10% and < 60%, and absence of any myeloma defining event. Active multiple myeloma is preceded by SMM, with a median time to progression of approximately 5 years. Cases of SMM range from the extremes of “monoclonal gammopathy of undetermined significance (MGUS) like”, in which patients never progress during their lifetimes to “early multiple multiple myeloma”, in which transformation into symptomatic disease, based on genomic evolution, may be rapid and devastating /29/.

SMM is a clinically defined but biologically heterogenous entity /23/ and includes:

• Patients with a very low rate of progression such as similar to MGUS
• Patients who develop organ damage within the first 2 years of diagnosis.

Overall, the cumulative probability of progression to symptomatic myeloma at 15 years is over 70%. It is important to identify patients who are at risk of progression 2–3 years after diagnosis. Continuous monitoring is essential in this patients.

Diagnostic criteria of SMM: refer to Tab. 22-9 – Diagnostic criteria for multiple myeloma and related plasma cell disorders.

The risk of progression from SMM to symptomatic multiple myeloma is represented in Tab. 22-11 – Risk of progression from SMM to symptomatic MM.

The progression of SMM is shown in Fig. 22-8 – Courses of monoclonal gammopathies.

High risk disease in SMM was defined in a study /34/ as:

• Plasma-cell bone marrow infiltration of at least 10%
• Monoclonal component defined as an IgG level ≥ 3 g/dL, an IgA level ≥ 2 g/dL, or a urinary Bence Jones protein level > 1 g/24 hours
• One of the criteria described above, plus at least 95% phenotypically aberrant plasma cells in the bone marrow plasma cell compartment with reductions in one or two uninvolved immunoglobulins of more than 25%, as compared with normal values.

22.3.10 Symptomatic multiple myeloma

Symptomatic multiple myeloma (MM) is a malignant disease characterized by the criteria Tab. 22-9 – Diagnostic criteria for multiple myeloma and related plasma cell disorders.

A neoplastic plasma cell clone proliferates in the bone marrow and spreads through the bone, resulting in bone destruction, pain, and fractures. Based on the Swedish myeloma registry /30/ the age adjusted incidence was 6.8 myeloma cases per 100,000 inhabitants per year. Among initially symptomatic patients 77% had osteolytic lesions or compression fractures, 49% had anemia, 18% impaired kidney function, and 13% hypercalcemia. In active myeloma, the median relative survival of patients aged 65 years or under was 7.7 years, and 3.4 years in patients aged 66 years and over.

The median age at clinical presentation is 70 years in most cases. Approximately 15% of cases occur below the age of 60 years and fewer than 2% occur below the age of 40 years. The incidence of MM is higher among Afro-Caribbean individuals than among Caucasians. Most cases occur primarily but a small proportion of cases develop from MGUS.

Clinical symptoms

The symptoms of MM are listed in Tab. 22-12 – Clinical symptoms and findings in symptomatic multiple myeloma.

The CRAB criteria are essential criteria for myeloma-related organ dysfunction (Tab. 22-10 – CRAB features).

Refer to:

• Fig. 22-6 – Frequency distribution of monoclonal gammopathies
• Fig. 22-7– Monoclonal immunoglobulin types.

Symptomatic multiple myeloma involves bone marrow, lymph nodes, spleen, liver, and kidney. The incidence of symptomatic myeloma increases gradually with age of patients. More than 90% of cases occurring in patients aged more than 50 years and median age of 70 years in patients at diagnosis. In addition to identification and determination their percentage of plasma cells, knowledge of abnormal morphology of plasma cells is helpful for the diagnosis of plasma cell myeloma. Par example the flaming plasma cells. Flaming plasma cells are very large with fine chromatin and sometimes with nucleoli. Large areas of the cytoplasm show homogeneous deposits which present with either flaming red or blue color or both, with the red color seen in the periphery of the abnormal plasma cell /94/.

22.3.10.1 Diagnostic investigations

If MM is suspected following the detection of an M- protein, an investigative workup is initiated to include:

• Clinical investigations and basic laboratory tests for the assessment of whole body status
• Tests to confirm diagnosis and assessment of tumor burden (International staging system)
• Prognosis and risk stratification
• Assessment of myeloma related tissue damage

22.3.10.1.1 Basic tests used to assess whole body status

Complete blood count, creatinine, Na⁺, K⁺, Ca²⁺, albumin, uric acid, erythrocyte sedimentation rate, serum protein electrophoresis, quantitative determination of IgG, IgA, and IgM.

Imaging of symptomatic regions of the skeleton.

The clinical symptoms and laboratory findings are presented in Tab. 22-12 – Clinical symptoms and findings in symptomatic multiple myeloma.

22.3.10.1.2 Tests to confirm diagnosis and assessment of tumor burden

Laboratory tests

Investigation for the presence of monoclonal (M) proteins: Serum protein electrophoresis, serum immunofixation electrophoresis, and quantitative FLC determination in serum.

Bone marrow studies

Bone marrow studies include:

• Counting of the plasma cell proportion in the bone marrow /31/. A proportion of ≥ 10% plasma cells of the nucleated bone marrow cells and plasma cell atypia suggest multiple myeloma (Fig. 22-9 – Morphological pattern of myeloma cells). In approximately 25% of myeloma patients, however, the plasma cell proportion at diagnosis is below 10%. Because plasma cells are not distributed homogeneously throughout the bone marrow, the plasma cell proportion is highly dependent on the particular puncture site chosen. This is why it is also important to assess plasma cell morphology. Various plasma cell subtypes exist.
• Fluorescent in situ hybridization (FISH) probes to detect t(11;14). t(4;14), t(14; 16), t(6;14), t(14;20), trisomies, and del(17p). About 40% MM is characterized by the presence of trisomies in the neoplastic plasma cells, while most of the rest have a translocation involving the immunoglobulin heavy chain (IgH) locus on chromosome 14q32. A small proportion of patients have both trisomies and IgH translocations /24/

Imaging studies

Imaging studies of the axial skeleton. Characteristic osteolytic lesions are found in 45% of cases, isolated osteoporosis in 10%, and osteoporosis in combination with other skeletal abnormalities in 60%.

Tumor burden in multiple myeloma is assessed using the International staging system /32/ (Tab. 22-14 – Staging system for multiple myeloma).

22.3.10.1.3 Assessment of myeloma related tissue damage

Laboratory markers in routine practice for the assessment of tissue damage in symptomatic myeloma are presented in Tab. 22-15 – Investigations for the diagnosis and differentiation of monoclonal plasma cell proliferative diseases.

Complete blood count

Half of all patients with MM have mild to moderate anemia (Hb about 100 g/L) and some 20% have Hb levels below 80 g/L /27/. The WBC is usually normal and 10–15% have thrombocytopenia, with a thrombocyte count of below 100 × 10⁹/L.

Erythrocyte sedimentation rate (ESR)

Two-thirds of patients with MM have an ESR of over 50 mm/h, as a result of hypergammaglobulinemia and the rouleaux phenomenon. Some 10% have an ESR of below 20 mm/h; this is particularly common in patients with light-chain myeloma /27/.

Serum creatinine

Levels > 2 mg/dL (178 μmol/l) are indicators of free light chain caused renal damage

Serum calcium

Concentrations > 2.75 mmol/l are indicators of bone involvement

Patients under treatment

Investigation of its prognostic significance has produced varying meaning /27/.

LD

Increased LD activity is measured in 5–11% of patients with MM. Elevated values are associated with a poor prognosis /27/.

Quantitative determination of non isotypic immunoglobulins

A decrease in the concentration of non-monoclonal immunoglobulin is a sign of humoral immune paresis.

22.3.10.1.4 Prognosis and risk stratification

The International Myeloma Working Group defined three criteria that predict the progression of symptomatic myeloma:

• More than 60% bone marrow plasma cells
• A serum free light chain ratio > 100 with the level of the involved free light chains > 100 mg/L
• More than one bone lesion on magnetic resonance imaging.

There is major variation in survival depending on host factors, tumor burden (stage), biology (cytogenetic abnormalities) and response to therapy. The international staging system combines elements of tumor burden and disease biology (presence of high risk cytogenetic abnormalities or elevated lactate dehydrogenase level) to create the unified prognostic index /26/ shown in Tab. 22-16 – Prognostic factors in multiple myeloma.

22.4 Treatment of newly diagnosed myeloma

According to the International Myeloma Working Group /23/ treatment should be initiated in patients with symptomatic multiple myeloma and with smoldering multiple myeloma. A trial found that early therapy with lenalidomide and dexamethasone in patients with symptomatic multiple myeloma was associated with a benefit in progression free survival but not overall survival /33/. However, early therapy with lenalidomide and dexamethasone in patients with high risk smoldering multiple myeloma can prolong overall survival /34/.

Patients on treatment should be monitored for response to therapy. After initiation of therapy, patients are monitored monthly, whereas during follow-up or maintenance the frequency of monitoring can be reduced to every 2–3 months.

22.4.1 Laboratory tests for myeloma management

Besides clinical evaluation the following biochemical markers can be important:

• Complete blood count
• Estimated GFR and serum calcium, albumin, and LD
• M-protein quantification (electrophoresis and nephelometry for IgA monoclonal proteins), keeping in mind that in IgG and in the rare case of IgM myeloma the amount of monoclonal protein may be overestimated by nephelometry
• Serum free light chains (FLCs). An increase in the involved FLCs accompanied by changes in the serum FLC ratio indicate progressive disease. This refers also in patients with light-chain escape, where progressive disease is not reflected in changes in the heavy-chain level.
• Twenty-four-hour urine monoclonal protein excretion may be included in case of light-chain myeloma and measurable urine M-gradient, as well as in patients with suspected renal amyloidosis or light-chain escape.

22.4.2 Response criteria

The International Myeloma Working Group response criteria /35/ are listed in Tab. 22-17 – International Myeloma Working Group response criteria for multiple myeloma.

The working group has also developed new criteria for assessing response using serum FLCs (Tab. 22-18 – Myeloma Working Group criteria for assessing response in monoclonal plasma cell proliferative diseases using FLC analysis).

Response assessment of multiple myeloma is based upon the reduction of monoclonal protein in serum and urine protein electrophoresis, with different levels and/or speed of response showing an association with clinical outcome in the majority of patients /36/.

Survival of myeloma patients has greatly improved with the use of high-dose melphalan-autologous stem cell transplantation (HDM-ASCT) /37/ and novel agents such as proteasome inhibitors, immunomodulatory drugs and monoclonal antibodies /38/. This is especially true for younger and HDM-ASCT eligible patients where pre-transplant induction therapy is able to provide > 60% rates of very good practical response or better.

In a multi-institutional, international retrospective analysis of HDM-ASCT eligible MM patients /37/ the clinical predictors of complete response (long term survival in MM > 10 years) in terms of their presenting features were investigated (Tab. 22-19 – Variables negatively associated with 10-year survival). The variables presented in the table appeared to be negatively associated with 10-year survival. The data identified complete response as an important predictor of long-term survival for HDM-ASCT eligible MM patients.

Definition of measurable disease

Response criteria for all categories and subcategories of response, except complete remission are applicable only to patients who have multiple myeloma or smoldering multiple myeloma defined by the International Myeloma Working Group criteria /23/.

Response criteria for complete remission (CR)

The response criteria are applicable for patients who have abnormalities on one of the three measurements Tab. 22-17 – International Myeloma Working Group response criteria for multiple myeloma. Note that patients who do not meet any of the criteria listed above can only be assessed for stringent CR, and cannot be assessed for any of the other response categories /35/.

Minimal residual disease (MRD)

Current methods to assess MRD in multiple myeloma are flow cytometric techniques or next generation sequencing (NGS-MRD) performed on bone marrow aspirates. Mass spectrometry (MS-MRD) in blood samples is a sensitive, minimally invasive alternative to measure multiple myeloma disease activity. In a study for determination MRD status /39/ per M-protein 2 optimal clonotypic peptides were selected (1 for the heavy chain and 1 for the light chain). The authors concluded MRD status obtained in bone marrow provides information that cannot be achieved by MS, such as clone evolution and bone marrow reconstitution. As such they anticipate that in the future MS will not replace existing MRD tests on bone marrow, but will have clinical value as a companion method especially for minimally invasive, longitudinal monitoring of MRD in blood.

Light-chain escape from plateau phase (LEPP)

Extensive treatment of myeloma can result in changes in the biological behavior of plasma cell clones. During treatment, the production of immunoglobulin switches from intact immunoglobulin to FLC production due to increasing malignant transformation of plasma cells, leading to a sharp increase in FLCs from the plateau phase within 1–2 months. It is therefore advisable to monitor serum FLCs throughout the course of the disease /39, 40/.

An alternative strategy for monitoring monoclonal proteins is the determination of heavy and light chains (HLCs) using immunoassays which separately measure the intact immunoglobulin of each light chain type and from kappa/lambda ratios to provide an indication of clonality. A study /36/ found a good agreement between electrophoretic and HLC assays for response assignment, with the significant exception of patients at conventional very good partial response, where HLC ratios had normalized and bone marrow plasma cells were below 5% in approximately 50% of cases, suggesting a deeper level of response (≥ complete response) in these patients.

22.5 Myeloma kidney

Around 50% of patients with MM have kidney disease at the time of diagnosis. Up to 10% of patients have severe acute kidney disease that requires dialysis; 90% of have myeloma kidney. The majority of patients who require dialysis remain dialysis dependent and have a poor prognosis. Most die within a year /16/.

From a laboratory diagnostic standpoint, up to 70% of patients with MM have proteinuria at the time of diagnosis, 50% have elevated serum creatinine, and 20% have serum creatinine levels of ≥ 2 mg/dL (177 μmol/L) /38/. Monoclonal serum FLCs are present in 95% of patients with MM and intact monoclonal immunoglobulin. Patients with FLC concentrations of > 1,000 mg/L (10–15% of patients with IgG or IgA myeloma) have an increased risk of kidney injury.

Kidney disease in MM is the result of increased FLC concentration. FLCs have a toxic effect on the proximal tubules and cause proximal tubular injury, cast nephropathy, or a combination of both.

The underlying pathology in myeloma kidney (also known as cast nephropathy) is intratubular obstruction due to the precipitation of FLCs in the lumen of the distal nephron, which results in interstitial inflammation and fibrosis. The characteristic feature of myeloma kidney is the presence of dense, waxy, lamellated casts in the distal tubules. The casts are surrounded by multinucleated syncytial giant cells that can be demonstrated using monoclonal antibody HAM56. The formation of casts correlates with the extent of urinary FLC excretion. Nephrotoxic monoclonal FLCs cause kidney injury at a very early stage, before the other manifestations of multiple myeloma appear. FLCs can also occur as droplets in the tubular cells, leading to atrophy of the cells.

Less is known about two other forms of kidney injury associated with MM:

• Proximal tubulopathy (acute tubular necrosis)
• An inflammatory tubular interstitial process without casts that has morphological features identical to those seen in classical acute tubulointerstitial nephritis.

22.6 Monoclonal gammopathy of renal significance

The monoclonal gammopathy of renal significance (MGRS) represents any B-cell or plasma cell clonal disorders that does not meet current criteria of a symptomatic multiple myeloma. MRGS is associated with a nephrotoxic monoclonal immunoglobulin that directly or indirectly results in kidney disease or injury /7/. This diagnostic category includes MGUS and smoldering hematologic diseases (low-grade CLL and lymphoplasmacytic lymphoma). MRGS-associated kidney diseases are characterized by three features /7/:

• The diseases do not respond well to immunosuppressive regimens used in the treatment of autoimmune nephropathies
• MRGS affected patients have a high rate of recurrence after kidney transplantation if the monoclonal gammopathy is not eliminated before or immediately after transplantation
• Affected patients are at risk for progression to the corresponding hematologic cancer.

Refer to Tab. 22-20– Monoclonal gammopathies of renal significance.

The renal pathology includes /41/:

• MGRS lesions with fibrillar deposits
• MGRS lesions with microtubular structure
• MGRS lesions with deposition of crystals
• MGRS lesions with granular (non-organized) deposits.

MGRS lesions with fibrillar deposits

IgG related amyloidosis: this type accounts for more than 80% of cases of renal amyloidosis in the US. The amyloidosis in most cases is derived from fragments of monoclonal immunoglobulin light chains and rarely from fragments of immunoglobulin heavy chains. Ultrastructurally, amyloid deposits appear as randomly arranged, non branching fibrils which can be seen within the mesangium, glomerular basement membranes, vessels, interstitium, and/or tubular basement membranes /42/.

Non amyloid fibrillar glomerulonephritis (FGN)

Monotypic glomerular deposits staining for IgG (predominantly IgG₄ and IgG₁) are the manifestation. In light microscopy mesangial hyper cellularity, duplication of glomerular basement membrane, and glomerular non congophilic deposits are typical.

MGRS lesions with microtubular structure

Immunotactoid glomerulopathy: this disease is defined by glomerular deposition of microtubules that have distinct hollow centers. They are arranged focally in parallel arrays and stain for immunoglobulins, in the absence of clinicopathologic diagnosis of cryoglobulinemic glomerulonephritis or lupus nephritis. The two glomerular patterns of glomerular injury are membranous glomerulonephritis (deposits are present in the subepithelial space) and membranoproliferative glomerulonephritis (deposits are preferentially located in the subendothelial zone) /42/.

Type I cryglobulinemic glomerulonephritis: this type of glomerulonephritis shows membranoproliferative or endocapillary proliferative glomerulonephritis and large intraluminal immunoprotein deposits (hyaline thrombi). In cristal cryoglobulinemia, monoclonal Ig deposits are organized into intracellular or extracellular crystals in the cytoplasm of glomerular endothelial, mesangial cells and/or in glomerular subendothelial spaces and within vascular lumens (refer to Section 18.11 – Cryoglobulins and cryofibrinogen).

MGRS lesions with deposition of crystals

Light chain (LC) proximal tubulopathy: this disorder was previously referred to as LC Fanconi syndrome. The disease is characterized by the presence of rod shaped or rhomboid shaped hyper eosinophilic and PAS-negative crystals with localization in the proximal tubular cells. Patients with LC proximal tubulopathy with crystals may or may not have a complete or partial Fanconi syndrome /42/.

MGRS lesions with granular (non organized) deposits 

Mononoclonal immunoglobulin deposition of the Randall type (MIDD): depending on the composition of the deposits kappa light chain, lambda light chain or heavy chain deposits are differentiated. Thickening of tubular basement membranes and nodular mesangial sclerosis accompanied by variable degrees of tubular atrophy, interstitial fibrosis, and inflammation are characteristic features /42/.

Proliferative glomerulonephritis with monoclonal IgG deposits: this disorder shows membranoproliferative or endocapillary proliferative glomerulonephritis, with or without membranous features, and less commonly pure mesangial proliferative glomerulonephritis. Contrary to the Randall type, the deposits are restricted to glomeruli and contain an intact monoclonal immunoglobulin.

C3 glomerulopathy with monoclonal gammopathy: two pathologic entities are encompassed, the C3 glomerulonephritis and dense deposit disease. Both entities are due to a deregulation of the complement alternative pathway resulting from functional inhibition or mutations of complement regulating proteins. The glomerulopathy shows mesangial proliferative, membranoproliferative or endocapillary proliferative glomerulonephritis.

22.6.1 Laboratory investigations in MGRS

MGRS-related diseases are found in 40 to 45% of patients with monoclonal gammopathy who undergo a kidney biopsy.

Laboratory findings are /7/:

• Small amounts of monoclonal immunoprotein and truncated monoclonal heavy chain
• Proteinuria > 1.5 g/L
• Hematuria
• An abnormal serum free light chain ratio
• A rapid loss of glomerulum filtration rate.

22.6.2 Multiple myeloma with central nervous system involvement

Extra medullary disease develops in up to 5% of multiple myeloma (MM) arising via hematogenous spread or through the bone cortex into contiguous tissues. MM with CNS involvement is a rare form of extramedullary disease characterized by plasma cell infiltration of the central nervous system (CNS), meninges or cerebrospinal fluid /75/. Refer to Tab. 22-21 – Clinical and laboratory findings in multiple myeloma with central nervous system involvement.

22.6.3 Multiple myeloma related polyneuropathy

Approximately 8–37% of patients with multiple myeloma (MM) develop a symptomatic neuropathy. While neuropathy is well recognized as a specific clinical presentation in MM, its relationship to MGUS is still the subject of some debate. The most common neurological complications seen in MM are compressive radiculopathies and peripheral neuropathies /5/. The incidence of compressive radiculopathy is 5%. Intracranial myelomas are diagnosed by the presence of an identical pattern of monoclonal bands in the cerebrospinal fluid and serum.

22.7 Monoclonal IgM gammopathy

Monoclonal IgM gammopathies are the result of the clonal proliferation of B cells at a particular stage of maturation into plasma cells that produce monoclonal IgM (mIgM) (Fig. 21.1-9 – Class switch recombination following contact between B2 cell and antigen). The B cells express the pan B cell markers CD19, CD20, CD22, CD79, and FMC7 but do not express CD10 and CD23; CD5 is expressed in only 15–20% of cases. The bone marrow smear shows diffuse proliferation of lymphoplasmacytic cells with deep blue cytoplasm and lymphocyte like nuclei.

Characteristic molecular features of IgM are:

• IgM immunoglobulins are large, asymmetric molecules that are 80% intravascular and can form aggregates. An increased plasma IgM leads to hyper viscosity, which is characterized by chronic bleeding from the nose, gums, and occasionally from the gastrointestinal tract. Most patients with symptoms of hyper viscosity have an M-gradient of > 40 g/L.
• IgM molecules can form aggregates and have a large carbohydrate component, which enables them to bind water, resulting in an increased osmotic pressure, increased resistance to blood flow, and impaired microcirculation
• IgM molecules have a high binding capacity. They bind to platelets and coagulation factors, resulting in prolonged bleeding time and clotting time
• IgM antibodies may behave as cryoglobulins, cold agglutinins, or autoantibodies (refer also to Section 18.11 – Cryoglobulins and cryofibrinogen). In around 10% of patients with monoclonal IgM, the molecules behaves as cold agglutinins and react with red blood cell antigens to produce a mild extravascular hemolysis. The cold agglutinin titer is usually > 1 : 1000 and the protein involved is usually IgM κ.

The most frequent differential diagnosis in IgM monoclonal gammopathy /43/ is presented in Tab. 22-22 – Differential diagnosis of monoclonal IgM gammopathy.

Approximately 20% of chronic lymphatic leukemia and 7% of marginal zone lymphoma patients exhibit IgM monoclonal gammopathy.

22.7.1 Waldenstroem’s macro­globulinemia

Waldenstroem’s macroglobulinemia (WM) is a distinct clinicopathological entity that should only include patients who present with a lymphoplasmacytic lymphoma, bone marrow infiltration, and monoclonal IgM synthesis. WM accounts for 1–2% of hematologic neoplasms. WM is a heterogeneous disease not only clinically but also genetically. The most common acquired mutation affects the gene MYD88, which is mutated in more than 90% of patients. A second common mutation affects the gene CXCR4, which carries a genetic change in about 30% of patients /43/.

The following genotypes have been described:

• Genotype 1: MYD88 mutated/CXCR4 wild type
• Genotype 2: MYD88 mutated/CXCR4 mutated
• Genotype 3: MYD88 wild-type/CXCR4 wild type

Classifying mutations into genotypes could have clinical implications.

The clinical and laboratory findings in WM are variable /44/ and depend on which IgM property predominates in individual patients (Tab. 22-23 – Clinical symptoms and laboratory findings in Waldenstroem’s macroglobulinemia).

Symptomatic WM

Most patients with WM have elevated IgM and exhibit clinical manifestations of monoclonal IgM or tumor infiltration and are therefore classified as having symptomatic WM.

IgM MGUS

Patients with IgM MGUS are asymptomatic, their laboratory findings are consistent with WM, and bone marrow infiltration is absent. This is the most commonly seen constellation in patients with monoclonal IgM. Patients in whom B cells are detectable in the bone marrow on flow cytometry but in whom morphologically discernible infiltration is absent. The relative risk of developing WM in a patient with MGUS of IgM type is twice as high as the relative risk of developing multiple myeloma in a patient with MGUS of IgG or IgA type /45/.

IgM-associated disorders

These disorders have the same clinical and laboratory diagnostic findings as MW but infiltration of the bone marrow with lymphoplasmacytic cells is absent. The patients frequently have cold-agglutinin disease, cryoglobulins, amyloidosis, or peripheral neuropathy.

The variables of WM are shown in Tab. 22-24 – Variables of Waldenstroem’s macroglobulinemia and IgM monoclonal gammopathies.

Transformed Waldenstroem macroglobulinemia (WM)

Histological transformation of WM to diffuse large B-cell lymphoma has been reported to occur in 2–10% of WM patients. Most transformed patients present with high-risk features such as extranodal disease, elevated serum lactate dehydrogenase (LD) level and high international prognostic index scores. A prognostic index predicting 2-year survival was created using the following adverse covariants: elevated serum LD (2 points), thrombocyte count < 100 × 10⁹/L (1 point) and any previous treatment for WM (1 point). Three risk groups were defined: low risk (0–1 point), intermediate risk (2–3 points), and high risk (4 points). Two year survival rates were 81%, 47% and 21%, respectively /84/.

22.7.2 IgM-related polyneuropathy

Two-thirds of patients with distal demyelinating symmetric neuropathy have IgM monoclonal gammopathy. The monoclonal IgM antibodies often show reactivity to MAG (myelin-associated glycoprotein). High titers of anti-MAG antibodies are always associated with a chronic, predominantly sensory demyelinating neuropathy with a slowly progressive course. In terms of life expectancy, patients with anti-MAG antibodies often have a favorable prognosis.

In 6–8% of patients with monoclonal IgM gammopathy, the monoclonal IgM shows reactivity to anti-sulfatide antibodies. Anti-sulfatide antibodies are predominantly associated with sensory axonal neuropathies but can also be associated with sensorimotor demyelinating neuropathies.

The POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin abnormalities) can occur in association with osteosclerotic myeloma. The dominant clinical feature is a chronic progressive neuropathy (Tab. 22-25 – Findings in clonal plasma cell proliferative disorders).

22.8 Heavy chain diseases

Heavy chain diseases are rare monoclonal plasma cell proliferative diseases characterized by the infiltration of certain organs by mature plasma cells or lymphoplasmacytic cells. Heavy chain diseases involving the three immunoglobulin classes have been described. Alpha (α)-heavy chain disease is the most common and has the most uniform presentation; γ and μ-heavy chain diseases have variable clinical presentations and histopathological features.

In heavy chain diseases, the constant-1 domain of the immunoglobulin heavy chain molecule, which is responsible for light chain binding, is truncated. This results in the production of abnormal heavy chains without corresponding light chains /46/.

An abnormal broad band found in the α₂- or β-globulin region in serum protein electrophoresis or immunofixation electrophoresis is usually associated with a heavy chain disease.

Heavy chain diseases can be thought of as variants of non Hodgkin lymphoma /47/:

• α-heavy chain disease is an extra nodal marginal zone lymphoma of mucosa associated lymph node tissue
• γ-heavy chain disease is a lymphoplasmacytic non Hodgkin lymphoma
• μ-heavy chain disease is a small lymphocytic non Hodgkin lymphoma or chronic lymphocytic leukemia.

α-chain protein

The molecular weight of the basic unit is 29–34 kDa. The polypeptide component is approximately half to two thirds as long as in the normal α chain. The α heavy chain protein belongs to the α₁ subclass; besides the light chain, the V region is also absent (Fig. 18.9-1 – T form of Ig molecule in free solution and V form during antigen binding) and the amino acid sequence at the amino terminal end is changed. The α-chain disease typically affects the gastrointestinal system.

γ-chain protein

The molecular weight of the basic unit is 27–49 kDa. The polypeptide component varies but is approximately half to two thirds as long as in the normal γ chain. The γ heavy chain protein has a V region that begins normally but is cut short or interrupted. The CH1 region is absent and the polypeptide starts with the hinge or CH2 region (Fig. 18.9-1). The γ-chain disease has a heterogenous clinical presentation, ranging from an asymptomatic state to aggressive lymphoproliferative process.

μ-chain protein

The molecular weight of the basic unit is 26–158 kDa. It results from polymerization of the μ-chain fragments. In these fragments, a large part of the V region is absent while the constant regions are often normal (Fig. 18.9-1). The u-chain disease has features resembling chronic lymphocytic leukemia or small lymphocytic lymphoma.

22.9 Findings in clonal plasma cell proliferative disorders

• Refer to Tab. 22-25 – Findings in clonal plasma cell proliferative disorders
• Refer to Ref. /87/.

22.10 Amyloidosis

Amyloidosis is a heterogenous group of disorders in which proteinaceous fibrils accumulate in certain organs and compromise their structure and function. In hematoxylin and eosin staining, an amorphous eosinophilic deposit is detected, and when viewed under polarized light after Congo red stain, green birefringence is seen. The most common types of systemic amyloidosis are immunoglobulin light chain (AL), reactive (AA), mutant or wid type transthyretin (ATTR), Fibrinogen (AFib) and apolipoprotein A-1 (AApoAI) /76/. Refer to Tab. 22-26 – Findings in amyloidosis.

References

1. Hasbold J, Corcoran LM, Tarlinton DM, Tangye SG, Hodgkin PD. Evidence from the generation of immunoglobuln G-secreting cells that stochastic mechanisms regulate lymphocyte differentiation. Nature Immunol 2004; 5: 55–63.

2. Fairfax KA, Kalies A, Nutt SL, Tarlinton DM. Plasma cell development: from B-cell subsets to long-term survival niches. Semin Immunol 2008; 20: 49–58

3. Nutt SL, Hodgkin PD, Tarlinton DM Corcoran LM. The generation of antibody secreting plasma cells. Nature Rev Immunol 2015; 15: 160–71.

4. Lust JA, Donovan KA. The role of interleukin 1β in the progression of multiple myeloma. Hematol/Oncol Clin North Am 1999; 13: 1117–25.

5. Dispensieri A, Kyle RA. Neurological aspects of multiple myeloma and related disorders. Best Practice & Research Clinical Haematology 2005; 18: 673–88.

6. Kyle RA, Rajkumar SV. Monoclonal gammopathies of undetermined significance. Hematol Oncol North Am 1999; 13: 1181–1202.

7. Leung N, Bridoux F, Nasr SH. Monoclonal gammopathy of renal significance. N Engl J Med 2021; 384 (20): 1931–41.

8. Katzmann JA, Kyle RA, Benson J, Larson DR, Snyder MR, Lust JA, et al. Screening panels for detection of monoclonal gammopathies. Clin Chem 2009; 55: 1517–22.

9. Thomas L, ed. Eiweiss-Elektrophorese, Grundlagen, Technik, Fehlermöglichkeiten und Bewertung der Eiweisstrennung auf Celluloseacetatfolie. München: Urban & Schwarzenberg, 1981.

10. Katzmann JA, Stankowski-Drengler TJ, Kyle RA, Lockington KS, Snyder MR, Lust JA, et al. Specificity of serum and urine protein electrophoresis for the diagnosis of monoclonal gammopathies. Clin Chem 2010; 56: 1899–1900.

11. Kehren DF, ed. High resolution electrophoresis and immunofixation. Second Edition. Boston: Butterworth-Heinemann 1994.

12. Katzmann JA, Clark R, Sanders E, Landers JP. Kyle RA. Prospective study of serum protein capillary zone electrophoresis and immunotyping of monoclonal proteins by immunosubtraction. Am J Clin Pathol 1998; 110: 503–9.

13. Bienvenu J, Graziani MS, Arpin F, Bernon H, Blessum C, Marchetti C, et al. Multicenter evaluation of the Paragon CZE 2000 capillary zone electrophoresis system for serum protein electrophoresis and monoclonal component typing. Clin Chem 1998; 44: 599–605.

14. Mussap M, Pietrogrande F, Ponchia S, Stefani PM, Sartori R, Plebani M. Measurement of serum monoclonal components: comparison between densitometry and capillary zone electrophoresis. Clin Chem Lab Med 2006; 44: 609–11.

15. Hutchinson CA, Basnayake K, Cockwell P. Serum free light chain assessment in monoclonal gammopathy and kidney disease. Nature Rev 2009; 5: 621–7.

16. Hutchinson CA, Batuman V, Behrens J, Bridoux F, Sirac C, Dispenzieri A, et al. The pathogenesis and diagnosis of acute kidney injury in multiple myeloma. Nature Rev 2012; 8: 43–51.

17. Dispenzieri A, Kyle R, Merlini G, Miguel JS, Ludwig H, Hajek R, Palumbo A, et al. International myeloma working group guidelines for serum-free light chain analysis in multiple myeloma and related disorders. Leukemia 2009; 23: 215–24.

18. Bhole MV, Sadler R, Ramasamy K. Serum-free light-chain assay: clinical utility and limitations. Ann Clin Biochem 2014; 51: 528–42.

19. Katzmann JA, Clark RJ, Abraham RS, Bryant S, Lymp JF, Bradwell AR, Kyle RA. Serum reference intervals and diagnostic ranges for free κ and free λ immunoglobulin light chains: relative sensitivity for detection of monoclonal light chains. Clin Chem 2002; 48: 1437–44.

20. Hutchinson CA, Harding S, Hewins P, Mead GP, Townsend J, Bradwell AR, et al. Quantitative assessment of serum and urinary polyclonal free light chains in patients with chronic kidney disease. Clin J Am Soc Nephrol 2008; 3: 1684–90.

21. Jacobs JFM. Effect of sample dilution on two free light chain nephelometric assays. Clin Chim Acta 2012; 413: 1708–9.

22. Lock BJ, Saleem R, Roberts EG, Wallage MJ, Pesce TJ, Rowbottom A, et al. A multicentre study comparing two methods for serum free light chain analysis. Ann Clin Biochem 2013, 50: 255–61.

23. Rajkumar SV, Dimopoulos MA, Palumbo A, Blde J, Merlini G, et al. International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma. Lancet Oncology 2014; 15 (12): e538–e48.

24. Rajkumar SV. Multiple myeloma: 2016 update on diagnosis, risk stratification and management. Am J Hematol 2016; 91: 719–34.

25. Rajkumar SV, et al. International Myeloma Working Group. Criteria for the classification of monoclonal gammopathies, multiple myeloma and related disorders: a report of the International Myeloma Working Group. Br J Haematol 2003; 121: 749–57.

26. Engelhardt M, et al. Europen myeloma network recommendations on the evaluation and treatment of newly diagnosed patients with multiple myeloma. Haematologica 2014; 99: 232–42.

27. Kyle RA, Durie BGM, Rajkumar SV, Landgren O, Blade J, Merlini G, et al. Monoclonal gammopathy of undetermined significance (MGUS) and smoldering (asymptomatic) multiple myeloma: IMWG consensus perspectives risk factors for progression and guidelines for monitoring and management. Leukemia 2010; 24: 1121–7.

28. Larsen JT, Kumar SK, Dispenzieri A, Kyle RA, Katzmann JA, Rajkumar SV. Serum free light chain ratio as a biomarker for high-risk smoldering multiple myeloma. Leukemia 2013; 27: 941–6.

29. Musto P, Engelhardt M, Caers J, Bolli N, Kaiser M, van de Donk N, et al. 2021 European Myeloma Network review and consensus statement on smoldering multiple myeloma: how to distinguish (and manage) Dr. Jekyll and Mr Hyde. Hematologica 2021; 106 (11): 2799–2812.

30. Bilmark CH, Turesson I, Genell A, Ahlberg L, Björkstrand B, Carlson K, et al. Outcome and survival of myeloma patients diagnosed 2008–2015. Real-world data on 4904 patients from the Swedish myeloma registry. Haematologica 2018; 103: 506–13.

31. Bartl R, Frisch B, Fateh-Moghadam A. Histologic classification and staging of multiple myeloma. Am J Clin Pathol 1987; 87: 342–55.

32. Palumbo A, Avet-Loiseau H, Oliva S, Lokhorst HM, Goldschmidt H, Rosinol C, et al. Revised international staging system for multiple myeloma: a report from international myeloma working group. J Clin Oncol 2015; 33: 2863–9.

33. Mateos MV, Hernandez MT Giraldo P, de la Rubia J, de Arriba 34. Witzig TE, Laumann KM, Lacy MQ, Hayman SR, Dispenzieri A, Kumar S, et al. A phase III randozide trial of thalidomide plus zoledronic acid versus zoledronic acd alone in patients with asymptomatic multiple myeloma.Leukemia 2013; 27: 220–5.

34. Mateos MV, Hernandez MT, Giraldo P, de la Rubia J , Arriba F, Corral LL, et al. Lenalidomide plus dexamethasone for high-risk smoldering multiple myeloma. N Engl J Med 2013; 369: 438–47.

35. Durie BGM, Harousseau JL, Miguel JS, Blade J, Barlogie B, Anderson K, et al. International uniform response criteria for multiple myeloma. Leukemia 2006; 20: 1467–73.

36. Michallet M, Chapuis-Cellier C, Dejoie T, Lombard C, Caillon H, Sobh M, et al. Heavy and light chain monitoring correlates with clinical outcome in multiple myeloma patients. Leukemia 2017; doi: 10.1038/leu.2017.209.

37. Usmani SZ, Hoering A, Cavo M, San Miguel J, Goldschmidt H, Hajek R, et al. Clinical predictors of long term survival in newly diagnosed transplant eligible multiple myeloma: an IMWG research project. Blood cancer J 2018; 8. doi: 10.1038/s41408-018-0155-7.

38. Gay F, Engelhardt M, Terpos E, Wäsch R, Glaccone L, Auner HW, et al. From transplant to novel cellular therapies in multiple myeloma: European Myeloma Network guidelines and future perspectives. Haematologica 2018; 103: 197–211.

39. Langerhorst P, Noori S, Zajec M, De Rijke YB, Gloerich J, van Gool Aj, Caillon H, et al. Multiple myeloma minimal residual disease detection: targeted mass spectrometry in blood vs next-generation sequencing in bone marrow. Clin Chem 2021; 67 (12): 1689–98.

40. Kühnemund A, Liebisch P, Bauchmüller K, zur Hausen A, Veelken H, Wäsch R, et al. Light chain escape multiple myeloma – an escape phenomenon from plateau phase: report of the largest patient series using LC-monitoring. J Cancer Res Clin Oncol 2008. doi: 10.1007/s00432-008-0470-7.

41. Kyle RA. Monoclonal proteins and renal disease. Annu Rev Med 1994; 45: 71–7.

42. Fermand JP, Bridoux F, Kyle RA, Kastritis E, Weiss BM, Cook MA, et al. Diagnosis of monoclonal gammopathy of renal significance. Kidney Int 2015; 87: 698–711.

43. Grunenberg A, Buske C. Monoclonal IgM gammopathy and Waldenstroem’s macroglobulinemia. Dtsch Arztebl Int 2017; 114: 745–51.

44. Owen RG, Treon SP, Al-Katib A, Fonseca R, Greipp PR, McMaster ML, et al. Clinicopathological definition of Waldenström’s Macroglobulinemia: Consensus Panel Recommendations from the Second International Workshop on Waldenström’s Macroglobulinemia. Semin Oncol 2003; 30: 110–5.

45. Rajkumar SV, Kyle RA, Therneau TM, et al. Serum free light chain ratio is an independent risk factor for progression in monoclonal gammopathy of undetermined significance. Blood 2005; 106: 812–7.

46. Yu M, Bruns DE, Katzmann JA, Silverman LM, Murray DL. Restricted IgG kappa and free alpha-heavy- chain bands in an asymptomatic 62-year-old man. Clin Chem 2018; 64: 265–9.

47. Wahner-Roedler DL, Kyle RA. Heavy chain disease. Best Practice & Research Clinical Haematology 2005; 18: 729–46.

48. Rajkumar SV, Larson D, Kyle RA. Diagnosis of smoldering multiple myeloma. N Engl J Med 2011; 365: 474–75.

49. Rosinol L, Blade J, Esteve J, et al. Smoldering multiple myeloma: natural history and recognition of an evolving type. Br J Haematol 2003; 123: 631–6.

50. Chakraborty R, Muchtar E, Kumar S, Jevremovic D, Buadi FK, Dingli D, et al. Serial measurement of circulating plasma cells before and after induction of therapy have an independent prognostic impact in patients with multiple myeloma undergoing upfront autologous transplantation. Haematologica 2017; 102: 1439–45.

51. Schmidt-Hieber M, Gutierrez ML, Perez-Andres M, Paiva B, Rassilo A, Tabernero MD, et al. Cytogenetic profiles in multiple myeloma and monoclonal gammopathy of undetermined significance: a study in highly purified aberrant plasma cells. Haematologica 2013; 98: 279–87.

52. Rajkumar SV, Gupta V, Fonseca R, Dispenzieri A, Gonsalves WI, Larson D, et al. Impact on primary molecular cytogenetic abnormalities and risk of progression in smoldering multiple myeloma. Leukemia 2013; 27: 1738–44.

53. Dispenzieri A, Kyle R, Merlini G, Miguel JS, Ludwig H, Hajek R, Palumbo A, et al. International myeloma working group guidelines for serum-free light chain analysis in multiple myeloma and related disorders. Leukemia 2009; 23: 215–24.

54. Fermand JP, Bridoux F, Kyle RA, Kastritis E, Weiss BM, Cook MA, et al. How I treat monoclonal gammopathy of renal significance. Blood 2013; 122: 3583–90.

55. Kumar S, Dispenzieri A, Lacy MQ, Hayman SR, Buadi FK, Colby C, et al. Revised prognostic staging system for light chain amyloidosis incorporating cardiac biomarkers and serum free light chains. J Clin Oncol 2012; 30: 989–95.

56. Gertz MA, Lacy MQ, Dispensieri A. Amyloidosis. Hematol/Dittrich T, Benner A, Kimmich C, aus dem Siepen C, Veelken K, von Kirsten A, et al. Performance analysis of AL amyloidosis cardiac biomarker staging systems with special focus on renal failure and atrial arrhythmia.Heamatologica 2019; www.haematologica. org/node/81273. full. print

57. Dittrich T, Benner A, Kimmich C, aus dem Siepen C, Veelken K, von Kirsten A, et al. Performance analysis of AL amyloidosis cardiac biomarker staging systems with special focus on renal failure and atrial arrhythmia.Heamatologica 2019; www.haematologica. org/node/81273. full. print

58. Van de Donk NECJ, Palumbo A, Johnsen HE, Engel hardt M, Gay F, Gregersen H, et al. The clinical relevance and management of monoclonal gammopathy of undetermined significance and related disorders: recommendations from the European Myeloma Network. Hematologica 2014; 99: 984–96.

59. Kyle RA, Gertz MA, Witzig TE, Lust JA, Lacy MQ, Dispensieri A, Fonseca R, et al. Review of 1027 patients with newly diagnosed multiple myeloma. Mayo Clin Proc 2003; 78: 21–33.

60. Kyrtsonis MC, Vassilakopoulos TP, Kafasi C, Sachanas S, Tzenou T, Papadogiannis A, et al. Prognostic value of serum free light chain ratio at diagnosis in multiple myeloma. Br. J Haematol 2007; 137: 240–3.

61. Shinamoto Y. IgD myeloma: clinical characteristics and a new staging system based on analysis of Japanese patients. Cancer Detect Prev 1995; 19: 426–35.

62. Sinclair D. IgD myeloma: clinical, biological and laboratory features. Clin Lab 2002; 48: 617–22.

63. Blade J, Lust JA, Kyle RA. Immunoglobulin D multiple myeloma: presenting features, response to therapy, and survival in a series of 53 cases. J Clin Oncol 1994; 12: 2398–404.

64. Alexander Jr RL, Roodman ST, Petruska PJ, Tsai CC, Janney CG. A new case of IgE myeloma. Clin Chem 1992; 38: 2328–32.

65. Kondo H, Yokoyama K. IgM myeloma: different features from multiple myeloma and macroglobulinemia. Eur J Haematol 1999; 63: 366–8.

66. Bradwell AR, Carr-Smith HD, Mead GP, Harvey TC, Drayson MT. Serum test for assessment of patients with Bence Jones myeloma. Lancet 2003; 361: 489–91.

67. United Kingdom Myeloma Forum. Guidelines on the diagnosis and management of solitary plasmacytoma of the bone and solitary extramedullary plasmacytoma. Br J Haematol 2004; 124: 717–26.

68. Blade J, Kyle RA. Nonsecretory myeloma, immunoglobulin D myeloma, and plasma cell leukemia. Hematol/Oncol North Am 1999; 13: 1259–72.

69. Ravi P, Kumar SK, Roeker L, Gonsalves W, Buadi F, Lacy MQ, et al. Revised diagnostic criteria for plasma cell leukemia: results of a Mayo Clinic study with comparison of outcomes to multiple myeloma. Blood cancer J 2018; 8: 116. doi: 10.1038/s41408-018-0140-1.

70. Levinson SS. POEMS syndrome: importance of the clinical laboratory practitioner’s role. Clin Chim Acta 2012; 413: 1800–7.

71. Morel P, Duhamel A, Gobbi P, Dimopoulos MA, Dhodapkar MV, McCoy J, et al. International prognostic scoring system for Waldenström macroglobulinemia. Blood 2009; 113: 4163–70.

72. Ghobrial IM, Gertz MA, Fonseca R. Waldenströms macroglobulinemia. Lancet Oncol 2003; 4: 679–85.

73. Gertz MA, Buadi FK, Hayman SR. IgM amyloidosis: clinical features in therapeutic outcomes. Clin Lymphoma Myeloma Leuk 2011. doi: 10.3816/CLML.2011.n.034.

74. Milani P, Merlini G. Monoclonal IgM-related AL amyloidosis. Best Practice &Research Clinical Hematology 2016; 29: 241–8.

75. Egan PA, Elder PT, Deighan WI, O’Connor SJM, Alexander HD. Multiple myeloma with central nervous system relapse. Haematologica 2020; 105 (7): 1780–90.

76. Vaxman I, Dispenzieri A, Muchtar E, Gertz M. New developments in diagnosis, risk assessment and management in systemic amyloidosis. Blood Reviews 2019. doi: 10.1016/j.blre.2019.100636.

77. Sekujima Y. Transthyretin (ATTR) amyloidosis: clinical spectrum, molecular pathogenesis and disease-modifying treatments. J Neurol Neurosurg Psychiatry 2015; 86 (9): 1036–43.

78. Adams D, Theaudin M, Cauquil C, Algalarrondo V, Slama M. FAP neuropathy and emerging treatments. Clin Neurol Neurosci Rep 2014; 2014; 14 (3): 435. doi: 10.1007/s11910-013-0435-3.

79. Lu C, Zuo K, Lu Y, Liang S, Husang X, Zeng C, et al. Apolipoprotein A-I related amyloidosis 2 case. Reports and review of the literature. Medicine (Baltimore) 2017; 96 (39): e8148.

80. Yazaki M, Yoshinaga T, Sekijama Y, Kametani F, Okumura N. Hereditary fibrinigen Aα-chain amyloidosis in Asia: clinical and molecular characteristics. Int J Mol Sci 2018: 19 (1): 320. doi: 10.3390/ijms19010320.

81. De Kat Angelino CM, Jacobs JFM. External quality assessment of M-protein diagnostics: a realistic impression of the accuracy and precision of M-protein quantification. Clin Chem Lab Med 2021; 59 (6): 1063–8.

82. Kastritis E, Palladinini G, Minnema MC, Wechalekar AD, Jaccard A, Lee HC, et al. Daratumumab-based treatment for immunoglobulin light-chain amyloidosis. N Engl J Med 2021; 385 (1): 46–58.

83. Noori S, Verkleij CPM, Zajec M, Langerhorst P, Bosman PWC, de Rijke YB, et al. Monitoring M-protein of multiple myeloma patients treated with a combination of monoclonal antibodies: the laboratory solution to eliminate interference. Clin Chem Lab Med 2021; 59 (12): 1963–71.

84. Durot E, Kanagaratnam L, Zanwar S, Kastritis E, D’Sa S, Garcia-Sanz R, et al. A prognostic index predicting survival in transformed Waldenström makroglobulinemia. Hematologica 2021; 106 (11) 2940–6.

85. Jean J, Liuri MV, Chambliss AB.No monoclonal protein in a patient with CRAB features. Clin Chem 2021; 67 (12) 1584–9.

86. Langerhorst P, Noori S, Zajec M, de Rijke YB, Gloerich J, van Gool AJ, et al. Multiple myeloma minimal residual disease detection: Targeted mass spectrometry in blood vs next-generation sequencing in bone marrow. Clin Chem 2021; 67 (12): 1689–98.

87. Piechotta V, Skoetz N, Engelhardt M, Einsele H, Goldschmidt H, Scheid C. Patients with multiple myeloma or monoclonal gammopathy of undetermined significance – diagnosis, treatment, and follow-up. Dtsch Arztebl Int 2022; 119: 253–60. doi: 10.3238/arztebl.m2022.0149.

88. Van Hoovels L, Vercammen M, Nevejan L, Cornette M, Briers P-J, Deeren D, et al. Serum free light chain analysis: persisting limitations with new kids on the block. Clin Chem Lab Med 2022; 60 (9): 1440–8.

89. Abraham RS, Charlesworth MC, Owen BAL, Benson LM, Katzmann JA, Reeder CB, Kyle RA. Trimolecular complexes of lambda light chain dimers in serum of a patient with multiple myeloma. Clin Chem 2002; 48 (10): 1805–11.

90. Rollborn N, Kultima K, Larsson A. Reference intervals for plasma free Kappa and Lambda chains and Kappa/Lambda ratio using PENIA. Clin Lab 2023; 69: 1687–91.

91. Eisele L, Düring J, Hüttmann A, Dührsen U, Assert R, Bohof B, et al. Prevalence and progression of monoclonal gammopathy of undetermined significance and light-chain mGUS in Germany Ann Hematol 2012; 91: 243–8.

92. Landgren O, Gridley G, Turesson I, Caporaso NE, Goldin LR, Baris D, et al. Risk of monoclonal gammopathy of undetermined significance (MGUS) ans subsequent multiple myeloma among African American and white veterans in the United States. Blood 2006; 107: 904–6.

93. Dispenzieri A, Katzmann JA, Kyle RA, Larson DR, Melton LJ 3rd, Colby CL, et al. Prevalence and risk of progression of light chain monoclonal gammopathy of undetermined significance: a retrospective population-based cohort study Lancet 2010; 375: 1721–8.

94. Bao J, Li G, Zhang J, Xu J, Zuo H. Flaming plasma cells in a 28-year old man with non-secretory multiple myeloma. Clin Lab 2022; 68: 1277–81.

95. Shen X, Dong X, Shi J,Chen H, Lan Y, Lim AC, et al. Deciphering the exact sequence of endogenous soluble B cell maturation antigen and unbiased quantitation in multiple myeloma patient samples by LC-MS. Clin Chem 2024; 70 (1): 339–349.