Multiple myeloma and related plasma cell proliferative disorders


Multiple myeloma and related plasma cell proliferative disorders


Multiple myeloma and related plasma cell proliferative disorders


Multiple myeloma and related plasma cell proliferative disorders

  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.

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

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).

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


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. 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 /910/.

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.


Serum: 1 mL


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


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.


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/.


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/.


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.


Serum: 1 mL


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 Urine immunofixation electrophoresis


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.


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.


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 /1213/.


Serum: 1 mL


Similar to IFE /1213/.

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 /1516/

Immunoglobulin free light chains are by-products of immunoglobulin 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 each 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.

Polyclonal FLCs

In normal individuals immunoglubulin 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 0.26–1.65. 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.

Monoclonal 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 re absorptive 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 /1718/

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. Serum free light chains


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.


Serum: 1 mL

Reference interval

Refer to References /161920/ 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/. 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.

22.4 Multiple myeloma (MM), precursor stages of MM and other plasma cell proliferative cell disorders

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/.

22.4.1 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/.

22.4.2 Smoldering multiple myeloma

Smoldering multiple myeloma (SMM) is an intermediate stage between MGUS and symptomatic multiple myeloma. The risk of progression to malignant disease is about 10% per year in the first 5 years after diagnosis but than decreases to 3% in the following 5 years and 1% per year thereafter. The SMM accounts for about 14% of all patients with multiple myeloma.

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.

22.4.3 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: 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 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. 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). 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/.


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. Prognosis and risk stratification

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.

22.5 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.

22.5.1 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.

22.5.2 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/.

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 /3940/.

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.

22.6 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.

22.7 Monoclonal gammopathy of renal significance

Monoclonal gammopathy of renal significance (MGRS) regroups all renal disorders caused by a monoclonal immunoglobulin secreted by a nonmalignant B-cell clone. By definition, patients with MGRS do not meet the criteria for overt multiple myeloma/B-cell proliferation, and the hematologic disorder is generally consistent with monoclonal gammopathy of undetermined significance (MGUS). However, MGRS is associated with high morbidity due to the severity of renal and sometimes systemic lesions induced by the monoclonal proteins /42/.

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.

22.7.1 Laboratory investigations in MGRS

MGRS patients can present with renal insufficiency or isolated proteinuria. Immunofixation electrophoresis (IFE) and the determination of free light chains are recommended. IFE in serum and/or urine allows detection of small amounts of monoclonal immunoprotein and truncated monoclonal heavy chain.

22.8 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.

22.8.1 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.

22.9 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.

22.9.1 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.


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.

22.9.2 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).

22.10 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.

22.11 Findings in clonal plasma cell proliferative disorders

Refer to Tab. 22-25 – Findings in clonal plasma cell proliferative disorders.

22.12 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.


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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.

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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.

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Table 22-1 Multiple myeloma and related plasma cell disorders

Malignant monoclonal gammopathies

  • Multiple myeloma
  • Plasmacytoma
  • Plasma cell leukemia
  • Waldenstroem’s macroglobulinemia

Premalignant monoclonal gammopathies

  • Monoclonal gammopathy of undetermined significance
  • Smoldering myeloma
  • Primary amyloidosis
  • Light chain deposition disease
  • POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes)

Table 22-2 Panels for screening of plasma cell proliferative disorders* according to Ref /8/














































* IFES is the primary investigation in many laboratories in Europe.

Abbreviations: SPE, serum protein electrophoresis; IFES, immunofixation electrophoresis in serum, FLCS, free light chains in serum; UPE, urine protein electrophoresis, IFEU, immunofixation electrophoresis in urine; MGUS, monoclonal gammopathy of undetermined significance; POEMS, polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes; LCDD, light-chain deposition disease.

Table 22-3 Tests for diagnosing and monitoring of monoclonal gammopathies


Diagnostic significance

Serum protein

  • Screening test for monoclonal gammopathy
  • Quantification and monitoring of M protein
  • Screening test for suspected antibody deficiency in light chain myeloma

Serum free light
chains (FL)

  • Screening test used in combination with SPE
  • Light chain myeloma, amyloidosis, non secretory myeloma
  • Treatment monitoring of monoclonal plasma cell proliferative disease
  • Test for recurrence following bone marrow transplantation


  • Classification and typing of M-protein (gold standard); also, screening test for monoclonal gammopathy
  • Confirmation of monoclonality of an M-gradient in SPE (classification and typing)
  • Required in suspected light chain, IgD, or IgE myeloma
  • Test for relapse following bone marrow transplantation
  • Differential diagnosis of monoclonal, biclonal, and oligoclonal gammopathies

and CZE*

  • Indications as for serum IFE

κ/λ ratio**

  • Clarification of questionable M-gradient in SPE
  • Monitoring of course and treatment in monoclonal plasma cell proliferative disorders

of Ig isotypes

  • Quantification of antibody deficiency

Urine protein

Quantification of monoclonal free light chains (Bence Jones protein) in concentrated urine

Urine IFE

  • Detection of Bence-Jones protein and M-protein in urine

* CZE, capillary zone electrophoresis; ** Scarcely relevant since assays for determining free light chains became available.

Table 22-4 Diagnostic accuracy (%) of test combinations and single tests for detection of monoclonal gammopathies /8/









All patients
















































































SPE, serum protein electrophoresis; IFES, immunofixation electrophoresis in serum, IFEU, immunofixation electrophoresis in urine; FLCS, free light chains in serum; UPE, urine protein electrophoresis, MGUS, monoclonal gammopathy of undetermined significance; POEMS, polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes; MM, multple myeloma; LCCD, light-chain deposition disease, WM, Waldenstroems macroglobulinemia

Table 22-5 Pseudo M-spikes that may be mistaken for monoclonal immunoglobulin spike in serum protein electrophoresis /10/




α2-hypermacroglobulinemia in nephrotic syndrome, pronounced acute phase reaction, hyperlipoproteinemia (e.g., Fredrickson types III and IV)

α2-β intermediate

Haptoglobin-hemoglobin complexes

β-globulin band

Elevated transferrin, increased C3, presence of free hemoglobin

β-γ intermediate

Fibrinogen (electrophoresis of plasma), bacterial contamination of sample


Rheumatoid factors, uremic serum, high lysozyme concentration

Table 22-6 Antisera used to precipitate M-proteins in immunofixation electrophoresis


Visualizable immunoglobulins

Anti human serum

IgG, IgA, and IgM as structurally intact Ig; very rarely IgD, IgE.

Light chains of type κ and λ.

Free γ-, α-, μ-heavy chains.

Trivalent anti human IgG, IgA, IgM

As in anti human serum, not IgD and IgE.

Monovalent antiserum specific for heavy chains (e.g. IgG/γ chain, IgA/α chain, IgM/μ chain)

Classification of M protein and free heavy chains; antiserum is directed against the Fc fragment of immunoglobulins; light chains are not detected.

Monovalent antiserum specific for light chains such as L chain type κ and L chain type λ

Typing of immunoglobulin bound light chains. Antiserum is directed against the outer antigenic determinants of light chains.

Monovalent antiserum specific for light chains; free L chains, type κ and free L chains, type λ

Free light chain typing in serum and urine; antiserum is directed against the inner antigenic determinants of light chains, which are not accessible in the intact immunoglobulin molecule.

* Special antisera are not listed. Antisera used in immunofixation electrophoresis must be highly purified.

Table 22-7 Indication for serum free light chain (FLC) determination /17/

Screening in combination with immunofixation electrophoresis

Baseline values for prognosis

  • Monoclonal gammopathy of undetermined significance
  • Smoldering myeloma
  • Symptomatic myeloma
  • Plasmacytoma
  • AL (primary) amyloidosis

Assessment of hematological response

  • AL amyloidosis
  • Non secretory myeloma
  • Stringent complete response in multiple myeloma
  • Light-chain deposition disease

Table 22-8 Reference intervals for serum free light chains (FLCs)



κ- FLC * /19/

3.3–19.4 mg/L

κ- FLC **

6.7–22.4 mg/L

λ FLC * /16/

5.7–26.3 mg/L

λ FLC **

8.3–27.0 mg/L

κ/λ ratio * /16/


κ/λ ratio **


κ/λ ratio in CKD * /20/


κ/λ ratio in CKD **


*Binding site, ** Siemens

Table 22-9 Diagnostic criteria for multiple myeloma and further plasma cell proliferative disorders /2223/

Clinical and laboratory findings


All three criteria must be met:

  • Serum monoclonal protein (non-IgM type) < 3 g/dL
  • Clonal bone marrow plasma cells < 10%
  • Absence of end-organ damage such as CRAB that can be attributed to the plasma cell proliferative disorder.

Smoldering multiple myeloma

Both criteria must be met:

  • Serum monoclonal protein (IgG or IgA ) ≥ 3 g/dL, or urinary monoclonal protein ≥ 500 mg/24 h
  • Absence of myeloma defining events or amyloidosis

Multiple myeloma

Both criteria must be met:

  • Bone marrow plasma cells ≥ 10% or biopsy proven bony or extramedullary plasmacytoma
  • Any one or more of the following myeloma defining events

a) Evidence of end organ damage that can be attributed to the underlying plasma cell proliferative disorder, specifically one of the CRAB features

b) Clonal bone marrow plasma cell percentage ≥ 60%

c) Involved/uninvolved serum free light chain (FLC) ratio ≥ 100 (involved FLC level must be ≥ 100 mg/L)

d) > 1 focal lesions on magnetic resonance imaging (MRI) studies (at least 5 mm in size)


All three criteria must be met:

  • Serum IgM monoclonal protein < 3 g/dL
  • Bone marrow lymphoplasmacytic infiltration < 10%
  • No evidence of anemia, constitutional symptoms, hyper viscosity, lymphadenopathy, or hepatosplenomegaly that can be attributed to the underlying lymphoproliferative disorder.

Light chain MGUS

All criteria must be met:

  • Abnormal FLC ratio (< 0.26 or > 1.65)
  • Increased level of the appropriate involved light chain (increased kappa FLC in patients with ratio > 1.65 and increased lambda FLC in patients with ratio < 0.26)
  • No immunoglobulin heavy chain expression on immunofixation
  • Absence of end organ damage that can be attributed to the plasma cell proliferative disorder
  • Clonal bone marrow plasma cells < 10%
  • Urinary monoclonal protein < 500 mg/24 h

Solitary plasmacytoma

All four criteria must be met:

  • Biopsy proven solitary lesion of bone soft tissue with evidence of clonal plasma cells
  • Normal bone marrow with no evidence of clonal plasma cells
  • Normal skeletal survey and MRI (or CT) of spine and pelvis (except for the primary solitary lesion)
  • Absence of end organ damage such as hypercalcemia, renal insufficiency, anemia or bone lesions (CRAB) that can be attributed to a lymph-plasma cell proliferative disorder.

Solitary plasmacytoma with minimal marrow involvement

All four criteria must be met:

  • Biopsy proven solitary lesion of bone soft tissue with evidence of clonal plasma cells
  • Clonal bone marrow plasma cells < 10%
  • Normal skeletal survey and MRI (or CT) of spine and pelvis (except for the primary solitary lesion)
  • Absence of end organ damage such as hypercalcemia, renal insufficiency, anemia or bone lesions (CRAB) that can be attributed to a lymph-plasma cell proliferative disorder.

Table 22-10 CRAB features /21/

  • Serum calcium > 0.25 mmol/L higher than upper reference interval value or > 2.75 mmol/L
  • Serum creatinine > 2.0 mg/dL (178 μmol/L)
  • Hemoglobin > 20 g/L below the lower reference interval value or < 100 g/L
  • Bone lesions, osteoporosis, or bone fractures

Table 22-11 Risk of progression from MGUS to symptomatic MM within 20 years /27/

Risk group (number
of risk factors)

risk (%)

risk (%)

risk (%)*

Low (0)




Low to intermediate(1)




Intermediate to high (2)




High (3)




Risk group 0: serum M-protein < 15 g/L, normal FLC ratio (0.26–1.65), monoclonal IgG

Risk group 1: one of the following criteria: M protein ≥ 15 g/L, FLC ratio < 0.26 or > 1.65 (The BInding Site), non IgG M protein

Risk group 2: two criteria from risk group 1

Risk group 3: all criteria from risk group 1

* Taking into account the average life expectancy

Table 22-12 Criteria for high risk smoldering myeloma progression to multiple myeloma /24/


High risk

Bone marrow
plasma cells

Bone marrow plasma cells ≥ 10% and any one more of the following finding

Serum M-Protein
and monoclonal
plasma cells

Serum M-protein ≥ 30 g/L /25/) MCP, mean count of plasma cells; MPT, mean progression time)

  • M protein ≥ 30 g/L and MCP ≥ 10%; MPT 2 years
  • M protein ≤ 30 g/L and MCP ≥ 10%; MPT 8 years
  • M protein ≥ 30 g/L and MCP ≤ 10%; MPT 19 years

Monoclonal IgA

IgA progression into IgA symptomatic multiple myeloma (MM)


Reduction of two uninvolved immunoglobulin isotypes

A study /28/presented the following criteria and results:

1) More than 95% of phenotypically abnormal plasma cells

2) Decline ≥ 25% of two non involved Ig isotypes.


  • Survival time 23 months if criteria 1 and 2 exist
  • Survival time 73 months in the presence of 1 criteria
  • Survival time > 73 months in the absence of both criteria

Free light chain
(FLC) ratio

Serum involved/uninvolved FLC ratio ≥ 8 but less than 100.

A study /29/ showed the following results: the refence range of κ/λ-Ratio was 0.26–165. The FLC ratio Involved/uninvolved was determined in cases with κ/λ-Ratio > 1,65 and κ/λ-Ratio < 0,26. A high risk SMM was defined in cases with κ/λ-Ratio ≥ 100. In these patients the time of progression into a MM was 15 months and 55 months with a κ/λ-Ratio < 100. The risk of progression of SMM into MM was 72% and into amyloidosis 79% within 2 years.

Bone marrow
plasma cells

Bone marrow plasma cells 50–60%.

A high risk of progression from SMM into MM was found in 3,2% of patients with bone marrow infiltration of plasma cells > 60% within 2 years /27/.

increase in M
protein level

Increase in serum mononoclonal protein by 25% on two successive evaluations within 6 months

Abnormal plasma
cell immuno-

Abnormal plasma cell immunophenotype (≥ 95% of bone marrow plasma cells are clonal) and reduction of one or more uninvolved immunoglobulin isotypes.


Gain of t(4;14) or del 17 p or 1q

In a study /31/ of 551 patients with SMM the following results were found: 43.9% trisomies, 36.2% IgH-translocations, 4% trisomies and IgH-translocations. 15.1% of patients showed no cytogenetic changes. The progression time of SMM into MM in t(4;14) cases was 28 months (high risk) in comparison to 34 months in patients with trisomy (average risk) and 55 months in cases with t(11;14), MAF-translocation or IgH-translocation (normal risik).

Circulating plasma

Increase to ≥ 5%


Diffuse abnormalities or 1 focal lesion


Focal lesion with increased uptake without underlying osteolytic bone destruction

Table 22-13 Clinical symptoms and findings in symptomatic multiple myeloma

Clinical symptoms

Prevalence (%)

Bone pain




Reduced performance


Weakness, fatigue


Susceptibility to infections




Gastrointestinal disorders


Weight loss


Skin abnormalities


Neurological disorders




Hemorrhagic diathesis


Pathological findings

Prevalence (%)

ESR > 30 mm/h


ESR > 90 mm/h


Free light chains in serum


Red blood cells < 4 × 1012/L


Hb level < 120 g/L


Total protein > 80 g/L


Creatinine > 1.5 mg/dL
(133 μmol/L)


Thrombocyte count
< 50 × 109/L


Spontaneous fractures


Leukocyte count < 4 × 109/L


Leukocyte count 10 × 109/L


Serum calcium

  • below 2.25 mmol/L


  • ≥ 2.75 mmol/L


Table 22-14 Revised international staging system for multiple myeloma /32/

Stage 1, all the following

  • Serum albumin ≥ 3.5 g/dL
  • Serum beta-2-microglobulin < 3.5 mg/L
  • No high risk cytogenetics
  • Normal serum lactate dehydrogenase

Stage 2, not fitting to stage 1 or 3

Stage 3, both of the following

  • Serum beta-2-microglobulin > 5.5 mg/L
  • High risk cytogenetics: t(4;14), t(14;16) or del (17p) or elevated serum lactate dehydrogenase

Table 22-15 Investigations for the diagnosis and differentiation of monoclonal plasma cell proliferative diseases /23/

Clinical and laboratory findings

Bone marrow

Multiple myeloma (MM): clonal bone marrow plasma cells (BPMC) are ≥ 60%, irrespective of the presence or absence of CRAB features. These patients require therapy. Only 3–5% of MM patients are associated with monoclonal BPMC of less than 10%. In these patients, the diagnosis of MM needs a repeat bone marrow biopsy showing 10% or more plasma cells, or an image guided (CT or MRI) biopsy of a bony or extramedullary lesion (plasmacytoma).

Smoldering multiple myeloma: clonal bone marrow plasma cells are in the range of 10–60%. Only 3% of patients had BMPC of 60% or greater, and 95% of these patients progressed to MM within 2 years /48/. Extreme plasmacytosis is unknown in smoldering multiple myeloma.

MGUS: clonal bone marrow plasma cells are below 60%

Other plasma cell proliferative disorders: patients with AL amyloidosis, POEMS syndrome, monoclonal and gammopathy associated proliferative glomerulonephritis might seem to have CRAB-like features despite clonal bone marrow plasma cells are below 10%.

Serum free light chains (FLC)

The presence of an abnormal FLC ratio, and the extent to which the FLC ratio is abnormal, predict risk of progression in MGUS, smoldering multiple myeloma, AL amyloidosis, and solitary plasmacytoma.

Multiple myeloma: approximately 90% of patients have an abnormal FLC ratio.

Smoldering multiple myeloma: a FLC ratio of at least 100 (involved/uninvolved FLC) is a predictor of imminent progression. To reduce possibility of error a minimum involved FLC level of at least 100 mg/l is recommended. The risk of progression to MM is 60–95% within 2 to 3 years.

MGUS: approximately one third of patients have an abnormal FLC ratio.

Magnetic resonance imaging

MGUS: the presence of more than one focal lesion is associated with a substantial increase in risk of progression.

CRAB features

CRAB features are associated with symptoms and the term symptomatic is used to refer to patients with clinical symptoms. The CRAB features used for diagnosis of MM must be attributable to the underlying plasma cell disorder.

Bone disease in MM: evidence of one or more sites of osteolytic bone destruction (≥ 5 mm in size) seen on CT or PET-CT.

Renal failure in MM: estimated GFR below 40 [mL × min–1 × (1.73 m2)–1] using MDRD or CKD-EPI formulae. Only renal failure caused by light chain cast nephropathy (based on typical biological changes or presumptive diagnosis based on the presence of high involved FLC levels, typically higher than 1500 mg/L) is regarded as a myeloma defining event. A renal biopsy is recommended in patients with suspected cast nephropathy if the serum involved FLC levels are less than 500 mg/L.

Hypercalcemia: in the absence of clear MM other causes such as hyperparathyroidism must be ruled out.

Anemia: in the presence of clear MM other causes such as anemia of infection, inflammation and chronic liver disease must be ruled out.

Future directions – Generally

Promising markers for further study are listed below.

– Monoclonal serum protein level

Data are insufficient to incorporate increasing monoclonal protein as a myeloma defining event.

Smoldering multiple myeloma: an increase in the serum monoclonal protein concentration by at least 10% on two successive investigations with a half year period has been associated with a 65% probability of progression /49/.

– Multiparametric flow cytometry /50/

Immunophenotyping is used to differentiate MM from MGUS. Antigen expression is determined using flow cytometry with monoclonal antibodies. Immunophenotyping of plasma cells is more useful for differential diagnosis than for prognosis.

Normal plasma cells are positive for CD138, CD19, and CD38 and negative for CD56.

Atypical plasma cells can be distinguished from normal plasma cells by detection of changes in two or more surface antigens (CD19, CD27–/lo, CD28+, CD38+/lo, CD45, CD56+, CD81–/lo, CD117+) and/or monotypic light chain expression. If there is doubt about the presence of a monoclonal cell population (e.g., in the diagnosis of a non secretory or low secretory myeloma) cytoplasmic light chain expression is investigated. Clonality of bone marrow plasma cells is established by demonstration of cells with kappa or lambda light chain restriction. The existence of monoclonal plasma cells is suggested by a kappa/lambda ratio over 4 or below 1.

Multiple myeloma: more than 95% of bone marrow plasma cells are clonal.

Smoldering multiple myeloma: patients who display an immunophenotypic pattern identical to MM have a higher risk of progression.

MGUS: a substantial proportion of polyclonal plasma cells exist.

– Specific genetic abnormalities

Smoldering multiple myeloma: specific cytogenetic abnormalities, especially translocation t(4;14), 1q gain, and deletion 17p, are associated with a high risk of progression.

– Cytogenetic abnormalities

Acquisition of del(13q14), Ig heavy chain (IgH) trans locations at chromosome 14q32-t(14q32), and numerical gains of different chromosomes (e.g. trisomies of chromosomes 3, 5, 7, and 9) are early cytogenetic events in monoclonal plasma cell proliferative diseases. Other molecular changes such as MYC dysregulation and RAS mutation are characteristic of malignant transformation and advanced stages of disease /51/. In a study /52/ of 351 patients with SMM, 43.9% had trisomies, 36.2% had Ig heavy chain (IgH) trans locations, 4% had both trisomies and IgH trans locations, and 15.1% had no abnormalities. Mean transition time period to symptomatic MM was 28 months in patients with the t(4;14) (high risk), compared with 34 months in patients with trisomy alone (intermediate risk), and 55 months in patients with t(11;14), MAF trans locations, or other IgH trans locations (standard risk).

Table 22-16 Prognostic facors in multiple myeloma /26/


Standard risk

High risk


Host factors

KPS > 70%

Renal function N

Organ function N


FCI 0, CCl 0

KPS > 70%

Renal failure
(eGFR < 30)

Other organ

GA reduced

Advanced age

HR pts typically require a decrease in treatment intensity


Durie and Salmon
stage I and II

Durie and Salmon
stage III

Limited; some stage I pts require no therapy (SMM) and some require radiation only (if solitary bone lesion)

Tumor biology

t(11;14), t(6;14),

  • t(4;14), t(14;16),
    t(14;20), 17p-,
  • High lactate dehydogenase
  • High PC
    proliferation rate
  • Presentation
    as PCL
  • Extramedullary disease
  • HR signature on

Treatment of high-risk patients remains unsatisfactory, but bortezomib* appears to overcome HR features

KPS, Karnovsky performance status; GA, geriatic assessment, FCI, Freiburg comorbidity index; CCl, Charlson comorbidity index; eGFR, estimated glomerular filtration rate; PC, plasma cell; PCL, plasma cell leukemia; GEP, gene expression profiling; ISS, International staging system; N, normal; * Some HR features such as t(4;14) or del 17p, or renal impairment are overcome by bortezomib

Table 22-17 International Myeloma Working Group response criteria for multiple myeloma /35/

Response category

Response criteria (2 criteria must be present)

Stringent complete response (sCR)

CR criteria as defined below plus:

  • Normal serum FLC ratio and
  • Absence of clonal cells in bone marrow by immunohistochemistry or immunofluorescence.

Complete response

  • Negative immunofixation electrophoresis on the serum and urine and
  • Disappearance of any soft tissue plasmacytomas and
  • Less than 5% plasma cells in bone marrow.

Very good partial response (VGPR)

  • Serum and urine M-protein detectable by immunofixation electrophoresis but not on serum protein electrophoresis or
  • 90% or greater reduction in serum M-protein plus urine M protein level < 100 mg/24 h.

Partial response (PR)

  • A ≥ 50% reduction of serum M-protein and reduction in 24-h urinary M protein by ≥ 90% or to less than 200 mg/24 h
  • If the serum and urine M-protein are unmeasurable, a ≥ 50% decrease in the difference between involved and uninvolved FLC levels is required in place of the M protein criteria
  • If the serum and urine M-protein are unmeasurable, and serum FLC assay is also unmeasurable, ≥ 50% reduction of plasma cells is required in place of M protein, provided baseline bone marrow plasma cell proportion was ≥ 30%
  • In addition to the above listed criteria, if present at baseline, a ≥ 50% reduction in the size of soft tissue plasmacytomas is also required.

Stable disease (SD)

Not meeting criteria for CR, VGPR, PR or progressive disease.

Table 22-18 Myeloma Working Group criteria for assessing response in monoclonal plasma cell proliferative diseases using FLC analysis /53/


Minimum measurable

Partial response

Complete response

Stable complete response (sCR)


MM without measurable M-protein in serum or urine

iFLC ≥ 100 mg/L and FLC ratio abnormal

50% reduction in dFLC

Not defined

Normal FLC ratio and CR by IFE and bone marrow

50% increase in dFLC

MM with measurable M-protein in serum or urine

FLC: not recommended

FLC: not recommended

FLC: not recommended

Normal FLC ratio and CR by IFE and bone marrow

FLC: not recommended

AL without measurable M-protein in serum or urine

iFLC ≥ 100 mg/L

50% reduction in iFLC

Normal FLC ratio and CR by IFE and bone marrow

Not defined

50% increase in iFLC to > 100 mg/L

AL with measurable serum or urine M-protein

Not defined

Not defined

Not defined

Not defined

Not defined

M-protein: > 10 g/L in serum and > 200 mg/L in urine in myeloma and > 100 mg/24 h in primary amyloidosis (AL). iFLC, involved FLC (κ for κ-type and λ for λ-type plasma cell proliferative disease); dFLC, difference between iFLC and polyclonal FLC.

Table 22-19 Variables negatively associated with 10 year survival /37/


Odds ratio

Age over 65 years


IgA isotype


Serum albumin < 35 g/L


Serum creatinine ≥ 2 mg/dL (177 μmol/L)


Hemoglobin < 100 g/L


Thrombocyte count < 150 × 109/L


Table 22-20 Monoclonal gammopathies of renal significance /54/

Clinical and laboratory findings

Amyloidosis – AL (AH and ALH) amyloidosis

Refer to Table 22-26 – Findings in systemic amyloidosis.

Monoclonal immunoglobulin deposition disease (MIDD)

The monoclonal gammopathy in MIDD is sometimes due to multiple myeloma (MM). Frequently, MM secretes more light chains of type κ. The most common form of MIDD is light-chain deposition disease (LCDD). The light chain precipitates are non amyloid by definition and are deposited on the glomerular basement membrane. 65% of cases of LCDD are associated with MM. In 96% of patients, the kidneys are involved. The diagnosis of LCDD is based on histological findings.

Laboratory findings: in 19 patients with LCDD, IFE was positive in only 12, while FLC determination was positive in 15 /19/.

Type I cryoglobulinemia

Type I cryoglobulinemia is a condition in which the blood contains large amounts of monoclonal immunoglobulin (mIg) that precipitates at low temperatures. It can occur in any disease that is associated with the production of intact mIg (MM, Waldenstroem’s macroglobulinemia, MGUS, B-cell lymphoproliferative disorders). Renal involvement is common if the cryoglobulin is mIgG but rare if the cryoglobulin is mIgM. Cryoglobulinemia presents as chronic glomerular disease with sudden onset nephritic syndrome, acute renal failure, or severe hypertension. Histological findings include membranoproliferative glomerulonephritis with thrombi and monoclonal cryoglobulin deposition. Refer also to Section 18.11 – Cryoglobulins and cryofibrinogen.

Type II cryoglobulinemia

Type II cryoglobulinemia is a mixed cryoglobulinemia in which the blood contains a mIG (usually IgM type κ) with rheumatoid factor activity in association with a polyclonal Ig. The renal manifestations are the same as in type I cryoglobulinemia but the glomerular deposits contain monoclonal Ig, polyclonal Ig, and complement components. Refer also to Section 18.11.

Immunotactoid glomerulopathy (ITG)

Glomerulonephritis characterized by organized tubular deposits of mIg and complement. Extrarenal manifestations are uncommon. Half of cases are due to chronic lymphocytic leukemia or a small B cell lymphocytic lymphoma. Clinical presentation: chronic kidney disease and hypertension.

Laboratory findings: proteinuria (often nephrotic), hematuria.

Proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID)

PGNMID is a chronic renal disease that is restricted to the glomeruli. It is characterized by non organized deposits of mIg, usually IgG3 type κ, in the glomeruli but not in the glomerular basement membrane. PGNMID broadly resembles immune complex glomerulonephritis.

Laboratory findings: FLCs detected in serum and urine in one third of cases; monoclonal plasma cell proliferation in bone marrow in less than 10%.

Acquired Fanconi syndrome (FS)

Acquired FS is characterized by an accumulation of κ-FLC crystalline inclusions within the endolysosomes of proximal tubular cells. Similar inclusions are also found in the cytoplasm of monoclonal plasma cells. FS complicates MGUS and small multiple myeloma of type κ. FS leads to proximal tubular dysfunction and, frequently, osteomalacia with bone pain.

Laboratory findings: moderate increase in creatinine, hypophosphatemia and hypouricemia.

Table 22-21 Findings in central nervous (CNS) system multiple myeloma /75/

Clinical and laboratory findings

The majority of CNS multiple myeloma (MM) are in patients who have received MM therapy prior to CNS involvement and whose survival is generally short and may depend on subsequent treatment. Data suggest that escape from the bone marrow is enabled by mutations to tumor suppressor genes such as Tp53, oncogenes such as RAS, and altered expression of adhesion molecules. MM with CNS involvement develops via hematogenous dissemination of malignant cells or contiguous spread of the tumor, often associated with plasma cell leukemia and cranial plasmacytoma, respectively.

Clinical findings: CNS-MM patients can present with impairment to sight, speech, motor and sensory functions, radicular pain, headache, confusion, dizziness and, less frequently seizures, vomiting hearing loss and incontinence.

Laboratory findings: Cytolological investigations using CSF cytology and flow cytometry detect monoclonal plasma cells (CD38/CD138) in about 90% of CNS-MM cases. Detection of plasma cells in CSF provides strong evidence of CNS-MM. Presence of monoclonal immunoglobulin including free light chains in CSF. Undetectable concentrations of monoclonal protein in the parallel analysis of serum is strong evidence that monoclonal immunoprotein detected in CSF originates from plasma cells in the CNS.

Table 22-22 Differential diagnoses of monoclonal IgM gammopathy /43/

Monoclonal gammopathy of undetermined significance

Waldenstroem’s macroglobulinemia

Other indolent lymphomas (CLL, follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma

IgM multiple myeloma

IgM amyloidosis

IgM related disorders: IgM neuropathies, cryoglobulinemia type I and type II, cold agglutinin disease, POEMS syndrome, Schnitzler syndrome, Pyoderma gangraenosum, Scleromyxedema, monoclonal gammopathy of renal significance

Table 22-23 Clinical symptoms and laboratory findings in Waldenstroem’s macroglobulinemia

Clinical symptoms

Pathological findings

Initial complaints: fatigue, weight loss, susceptibility to infections (30%)

Followed by:

  • Hepatomegaly (70%)
  • Hepatosplenomegaly (20–50%)
  • Lymphadenopathy (30–50%)
  • Hemorrhagic diathesis (40–60%)
  • Polyneuropathy (25%)
  • Edema (17%)
  • Peripheral circulatory disorders (40%)
  • Retinal disorders
  • Skin infiltrates (17%)
  • Mikulicz syndrome
  • Gastrointestinal symptoms such as diarrhea and malabsorption (20%)
  • SPE: M gradient often > 10 g/L
  • Serum IFE: IgM monoclonal gammopathy, type κ more common than type λ (4/1).
  • Serum FLCs detectable in 70% of cases
  • ESR: extremely accelerated
  • Total protein: often increased
  • Cold agglutinin test: often positive
  • Cryoglobulins: often detectable
  • Bleeding and clotting time: prolonged
  • Blood count: anemia, pseudo thrombocytopenia
  • Plasma viscosity: elevated
  • Skeletal X-ray: no osteolytic lesions
  • Bone marrow cytology and histology: diffuse and sometimes focal infiltration of lymphoplasmacytic cells (smaller cells, frequently with bare nuclei, and larger cells), the nuclei contain one or more nucleoli
  • Proliferation of tissue mast cells and sometimes plasma cells

Table 22-24 Variables of Waldenstroem’s macroglobulinemia (WM) and IgM monoclonal gammopathies /44/





Symptoms of











– (b)




– (b)

1) In MGUS, the serum IgM level rarely exceeds 30 g/L. 2) Patients with conclusive evidence of bone marrow (BM) infiltration have WM; those without BM infiltration have MGUS. Some patients show inconclusive morphological evidence of bone marrow (BM) infiltration. Further tests such as immunophenotyping or molecular biological investigations (e.g., PCR are required in these patients). 3) Symptoms include cytopenia(s) or organomegaly (lymph nodes, liver, spleen). 4) These disorders include symptomatic cryoglobulinemia, primary amyloidosis, and autoimmune phenomena such as cold-agglutinin disease and peripheral neuropathy.

Table 22-25 Findings in clonal plasma cell proliferative disorders (PCPD)

Clinical and laboratory findings

Monoclonal gammopathy of undetermined significance (MGUS)

MGUS is an asymptomatic clonal plasma cell proliferative disorder with a variable period of stable disease but which may progress to multiple myeloma with a risk of progression of approximately 1% per year.

When 241 patients with MGUS from the Mayo Clinic were monitored over 24–38 years, the following was observed /4/: the median M gradient concentration was 17 g/L (3–32 g/L); 73% were IgG, 14% IgM, 11% IgA, 2% biclonal, 62% κ chains, and 38% λ chains. Approximately 6% of the patients had Bence-Jones proteinuria but only one at a concentration of > 1 g/24 h. Only 2% showed a reduction in serum polyclonal Ig level compared to baseline and the mean plasma cell proportion in the bone marrow was 3%.

One third of the MGUS patients had an abnormal serum FLC ratio. This is an independent risk factor for progression in MGUS. The risk is even higher if the M gradient does not consist of IgG or is > 15 g/L. In one study, the risk factors for progression over 20 years were as follows /45/: (i) FLC less than 0.25 or greater than 4, (ii) M gradient ≥ 15 g/L, (iii) M gradient did not contain mIgG /53/.

The risk of progression was 58% if all three criteria were present (high-risk MGUS), 37% if two criteria were present (high-intermediate risk), and 21% if only one criterion was present (low-intermediate risk). If none of these criteria was present, the risk of progression was 2% (standard risk).

Monitoring /4/: MGUS should be monitored as follows, depending on the initial findings:

  • Intermediate-risk MGUS: annual SPE and serum FLC determination
  • In high-risk MGUS, a radiological skeletal survey should be performed to search for bone lesions. In IgM MGUS, computer tomography of the abdomen is recommended to look for evidence of Waldenstroem’s macroglobulinemia or lymphoproliferative disease. If investigation results are satisfactory, SPE and FLC determination should be repeated every three months or, if findings remain stable, every six months.

MGUS is often an isolated event but it can also occur together with other diseases that tend to cluster in older individuals. For example /58/:

  • Approximately 7% of patients with non Hodgkin lymphoma or chronic lymphocytic leukemia have a monoclonal gammopathy
  • Approximately 16% of patients with hairy cell leukemia have a monoclonal gammopathy
  • Approximately 56% of patients with monoclonal IgM have MGUS; of a cohort of MGUS patients followed up for 7 years, 17% developed malignant disease of the lymphatic system and 9% developed Waldenstroem’s macroglobulinemia
  • Approximately 10% of patients with sensorimotor peripheral neuropathy have MGUS; the M protein involved in half of these cases is mIgM, which binds to myelin-associated glycoprotein
  • Approximately 3% of patients with AIDS have a monoclonal gammopathy
  • Organ transplant recipients have a detectable monoclonal gammopathy; the rate is 13–30% following liver or kidney transplantation and 10–20% following bone marrow transplantation
  • Monoclonal gammopathy is not uncommon in patients with rheumatic diseases such as rheumatoid arthritis, polymyalgia rheumatica, or myasthenia gravis
  • Chronic liver disease is associated with monoclonal gammopathy. This was the case in 11% of 231 patients with hepatitis C virus (HCV) but in only one patient who was HCV-negative.

Smoldering multiple myeloma (SMM)

SMM is an asymptomatic plasma cell disorder defined by the International Myeloma Working group as the presence of a serum M protein of ≥ 30 g/L and/or ≥ 10% bone marrow plasma cells with no evidence of end organ damage (absence of CRAB features) /25/. The rate of progression is 10% per year. The estimated prevalence ranges from 15–44% per year. If a serum FLC ratio ≥ 100 was used to define high risk SMM the risk of progression to multiple myeloma within the first two years was 72% /23/. Refer also to Tab. 22-12 – Risk of progression smoldering multiple myeloma to symptomatic multiple myeloma.

Multiple myeloma (MM) – Generally

MM is a clonal B cell disorder in which a single neoplastic clone of plasma cells produces M protein and causes clinical symptoms (CRAB features).

– IgG myeloma, IgA myeloma /25/

In 80% of cases on average, an M gradient is detectable on SPE. Approximately 100% of cases of MM can be detected using a combination of serum IFE and serum FLC determination /53/. More than 95% of patients with MM have an abnormal serum FLC ratio. In 15–20% of cases of MM, only the FLCs are increased and the FLC ratio is abnormal; these patients have a light chain myeloma.

An M gradient ≥ 30 g/L and the presence of CRAB features usually indicates MM. With respect to the diagnostic criteria for MM, however, it must be noted that a large study /59/ found that at the time of diagnosis only 57% of patients had an M gradient ≥ 30 g/L, only 28% had hypercalcemia of > 10.2 mg/dL (2.55 mmol/L), and only 72% had a Hb level < 120 g/L. A reduction in polyclonal Ig classes, which can also can occur in MGUS, was seen in only 30% of the patients studied /59/.

The basal serum FLC ratio is a prognostic criterion in newly diagnosed MM. In a study /60/ of 94 patients with MM, patients with κ-type MM had a median κ/λ ratio of 3.57 and those with λ-type MM had a κ/λ ratio of 0.022. The 5-year survival rate was 82% if the FLC ratio was below the median and 30% if it was above the median. The FLC ratio also correlated with serum creatinine and LD activity and with the extent of plasma cell infiltration of the bone marrow.

The highest FLC concentrations are associated with light chain myeloma, creatinine values > 2 mg/dL (177 μmol/L), elevated LD, and β2-microglobulin levels of > 3.5 mg/dL.

– IgD myeloma /61/

IgD myelomas account for approximately 3% of all MM. Although the clinical symptoms are similar to those of IgG or IgA myeloma, they are further developed at the time of presentation. Some 10–20% of these patients have amyloidosis.

Laboratory findings: at the time of presentation, 96% of patients have FLC proteinuria of type λ /62/. The rarity of κ IgD molecules can be explained by a block in the assembly of the molecule in the endoplasmic reticulum as well as increased intracellular catabolism prior to secretion. The electrophoretic pattern seen in SPE and IFE is similar in many ways to that seen in light chain myeloma. The M gradient in SPE, if present, is modest and rarely exceeds 20 g/L. The M protein is more frequently of type λ and the κ/λ ratio is around 0.6. All patients with clinical symptoms excrete λ light chains, with 50% of them excreting > 1 g/24 h. In all cases that excrete λ light chains and in which precipitation does not occur in the presence of antiserum to a heavy chain, quantitative IgD determination should be performed before a diagnosis of light chain myeloma is made.

Some of the reasons why M-protein is not detected or why only a narrow band or hypogammaglobulinemia is present are /63/:

A low concentration of mIgD that escapes detection

Post synthetic degradation of mIgD. Due to the long hinge region between the Fc fragment and the Fab fragment, the molecule is susceptible to post synthetic degradation; as a result, the mIgD appears as diffuse bands on electrophoresis.

If antisera against FLCs are used in IFE, precipitation is absent or slight due to their low antibody titer. Whenever serum FLCs or FLC proteinuria are demonstrated without the presence of a monoclonal immunoglobulin, IFE should be performed using antisera against IgD and IgE before a diagnosis of light chain myeloma is made. IgD can cause problems in electrophoresis because it migrates to the anode faster than IgG and is hidden in the β-globulin fraction under the transferrin band or the C3 band. As a result of the severe kidney injury associated with IgD myeloma, mIgD is also excreted in the urine and the detection rate of IgD is usually much higher in the urine than in the serum. For this reason, both serum and urine should be examined if IgD myeloma is suspected. It is important to remain vigilant for the antigen excess phenomenon.

At the time of diagnosis, 50% of patients have a serum creatinine of > 2 mg/dL (177 μmol/L), more than one third have hypercalcemia, and > 40% have a Hb level < 100 g/L.

–IgE myeloma /64/

Clinical presentation: the IgE myeloma accounts for 0.1% of all MM. Patient age and clinical features in IgE myeloma are very similar to those in other types of multiple myeloma. The frequency of plasma cell leukemia is significantly higher in IgE myelomas (18%, compared to ≤ 2%). IgE myeloma has a worse prognosis compared to other myelomas because the monoclonal gammopathy is usually highly advanced at the time of diagnosis. However, cases of IgE MGUS have also been described.

Laboratory findings: in cases where an M gradient is present, 71% of these lie in the γ globulin fraction. An antibody deficiency syndrome is present in 83% of cases. Monoclonal IgE is detected using serum IFE with mono specific anti-IgE serum. There is a κ/λ type light chain ratio of 5 : 2 and serum FLCs and urine FLCs are positive in some 70% of cases.

– IgM myeloma

IgM myeloma is very rare. It can be distinguished from Waldenström’s macroglobulinemia (WM) by the pure MM plasma cell morphology, immunophenotypic findings, and the presence of lytic bone lesions. Lytic bone lesions are present in only 5% of WM patients; the incidence of renal failure is low in WM compared to IgM myeloma and if it does occur, is of glomerular origin /65/.

– Light chain myeloma

The presence of monoclonal free light chains (FLCs) of type κ or λ in the urine is referred to as Bence-Jones proteinuria (BJPU). Multiple myeloma in which only FLCs are produced are also known as Bence- Jones myelomas and have comparable clinical symptoms and findings. Approximately 10–20% of all patients with MM produce monoclonal FLCs only.

Laboratory findings: if a large plasma cell clone produces FLCs in quantities large enough to be detected as an M protein in serum SPE or IFE due to their reduced migration toward the anode, the likelihood of a light chain myeloma is high. This is also the case if BJP excretion is detectable in patients with hypogammaglobulinemia. In a study /60/ of 1027 myeloma patients, 9% produced only FLC type κ and 7% produced only FLC type λ as the monoclonal component. 42% of the patients had detectable monoclonal FLCs in the serum and 38% had BJP excretion of > 2 g/24 h. Decreased polyclonal Ig synthesis was observed in 87% and serum creatinine was > 2 mg/dL (177 μmol/L) in 35%. At the time of diagnosis, all patients had elevated serum FLC concentrations and an abnormal κ/λ ratio. The correlation between serum FLCs and BJP excretion is modest. 38% of patients with light chain myeloma have detectable serum FLCs in the form of an M gradient on serum protein electrophoresis, 50% have hypogammaglobulinemia (< 7 g/L), 87% have reduced polyclonal synthesis of one or more Ig classes, and 93% have BJP excretion. In a study /66/, 99% of patients showed a reduction in serum FLCs in response to chemotherapy and 95% showed a reduction in BJP excretion. When normalization of serum FLCs was used as the criterion for complete remission, only 11% of patients achieved complete remission in response to treatment. When BJP excretion was used as the criterion, this increased to 32%.

Solitary plasmacytoma of bone /67/

Approximately 3–5% of patients with myeloma present with a single bone lesion due to a monoclonal plasma cell infiltrate. The majority of patients are male and 10 years younger on average than patients with multiple myeloma.

Clinical presentation: the most common symptom is pain at the site of the bone lesion, which most commonly occurs in the vertebral bodies (in decreasing order of frequency: thoracic, lumbar, sacral, and cervical). The long bones can also be affected. Patients with a solitary vertebral myeloma may also present with symptoms of nerve root compression. Patients do not have end organ damage (CRAB features).

Laboratory findings: SPE and serum IFE show a small M-gradient or a monoclonal band in 70% of patients. Approximately half of patients have an abnormal serum FLC ratio. Clonal bone marrow monoclonal plasma cells are < 10%. An abnormal FLC ratio is associated with a higher rate of progression to MM. Thus the rate of progression within 5 years is 26% in the absence of an abnormal FLC ratio but 44% if an abnormal ratio is detected at the time of diagnosis /53/.

Extramedullary myeloma (EM) /67/

Approximately 3% of monoclonal plasma cell proliferation diseases present as an extramedullary myeloma (EM). The mean patient age is 60 years and up to 75% of patients are male.

Clinical presentation: EM can arise anywhere in the body but 90% occur in the head and neck region, where the main site of involvement is the upper respiratory tract (nasal cavities, para nasal sinuses, pharynx, and larynx). The tumors are localized in the submucosa and present with symptoms such as nasal discharge, epistaxis, nasal obstruction, oral thrush, and hoarseness. The diagnostic criteria are as follows:

  • Extramedullary plasma cell tumor
  • Normal plasma cell percentage in bone marrow and normal plasma cell morphology
  • No imaging evidence of osteolytic lesions.

Laboratory findings: a small M gradient in SPE, a monoclonal band in IFE, and serum FLCs are present in approximately 10% of patients. An M protein can be analyzed in approximately 20% of cases using a combination of laboratory diagnostic tests.

Non secretory myeloma

Non secretory myeloma is characterized by the absence of detectable M protein in serum and urine. This is thought to be due to an inability of the plasma cell to secrete immunoglobulins, limited synthetic capacity, or increased intracellular or extracellular degradation of immunoglobulins. Non secretory myeloma is thought to account for 1–5% of all myelomas. In 85% of cases, monoclonal Ig can be detected intracellularly using immunohistochemical techniques. The remaining cases are true non producers. Absence of organ damage (CRAB features).

Laboratory findings: in many cases, in serum IFE small quantities of monoclonal Ig can be detected (e.g., 6 out of 28 patients studied). The monoclonal bands were faint and diffuse and contained FLCs. Nine of the 28 patients did not show immunoprecipitation, although the serum FLC concentration was > 200 mg/L. In 70% of patients, immunonephelometry showed elevated serum FLCs and an abnormal κ/λ ratio. The reason for the low diagnostic sensitivity of IFE is thought to be the variable polymerization tendency of FLCs that is caused by smearing or a lack of a visible precipitate in electrophoresis /68/. Serum FLC determination is the most reliable assay for diagnosing non secretory myeloma. In 75% of patients, the M protein is of type λ. The reduction in polyclonal Ig is more marked than in MM.

Plasma cell leukemia (PCL)

Plasma cell leukemia is an aggressive malignancy that accounts for 1–2% of all multiple myelomas (MMs). Characteristic findings include ≥ 20% circulating plasma cells or a cell count of more than 2 × 109/L. This definition includes both primary PCL arising de novo in the absence of antecedent MM and secondary PCL which describes leukemia transformation of MM. Approximately 60% of PCLs are primary and 40% represent a leukemic transformation of a pre-existing MM /68/. The current definition of PCL is too restrictive. According to a study /69/ outcomes of patients with ≥ 5% circulating plasma cells were much poorer when compared with MM patients who did not have circulating plasma cells at diagnosis. Therefore the authors propose that the definition of PCL be revised to patients with ≥ 5% circulating plasma cells on peripheral blood smear, who otherwise meet diagnostic criteria for MM.

Clinical presentation: PCL has a more aggressive clinical presentation than MM. Extramedullary sites of plasma cell production in the liver, spleen, and lymph nodes also become involved. Unlike secondary PCL, primary PCL affects younger individuals. These patients have a higher tendency to hepatosplenomegaly and lymphadenopathy, a higher thrombocyte count, fewer osteolytic lesions, and a longer survival time.

Laboratory findings: M spike in SPE (smaller in primary PCL than in secondary), abnormal serum free light chain ratio. At the time of diagnosis, more than half of patients have a Hb level of < 100 g/L, thrombocytopenia of < 100 × 109/L, serum calcium of > 11 mg/dL (2.75 mmol/L), and a creatinine level of > 2 mg/dL (177 μmol/L) /68/.

POEMS syndrome /70/

POEMS syndrome is a monoclonal plasma cell proliferative disorder with polyneuropathy and:

  • Any one of the following three other major criteria: sclerotic bone lesions, Castleman’s syndrome, elevated levels of vascular endothelial growth factor (VEGF)
  • Any one of the six minor criteria: organomegaly (splenomegaly, hepatomegaly, or lymphadenopathy), extravascular volume overload (edema, pleura effusion, or ascites) endocrinopathy (adrenal, thyroid, pituitary, gonadal, parathyroid, pancreatic), skin changes (hyper pigmentation, hypertrichosis, glomeruloid hemangioma, plethora, acrocyanosis, flushing, white nails), papilloedema, thrombocytosis/polycythemia. The chronic inflammatory demyelinating polyneuropathy (CIDP) is associated with the same symptoms as Guillain-Barré syndrome but is progressive.

Laboratory findings: monoclonal Ig in low concentration, almost always type lambda. There is no evidence of plasma cell proliferation in the bone marrow. Laboratory diagnostic features of MM (CRAB) are absent. The concentration of VEGF is elevated (1500–2500 ng/L).

Heavy chain disease (HCD) – Generally

The heavy chain diseases (HCD) are a group of monoclonal plasma cell proliferative disorders characterized by the production of heavy chains without associated light chains by a malignant B cell clone. Three Ig classes may be involved; consequently, α HCD, γ HCD, and μ HCD exist. In most HCD, the protein produced consists of the Fc fragment of the heavy chain with a normal carboxyterminal end. Parts of the variable region, hinge region, and C1 region are absent (refer also to Fig. 18.9-1 – T form of Ig molecule in free solution and V form during antigen binding). The α and μ heavy chains are monomers with a molecular weight of 29–35 or 55 kDa; γ heavy chains exist as multiple polymers.

Laboratory findings: HCD is diagnosed on the basis of immunological evidence of a molecule with heavy chain antigenicity in the absence of associated light chains. IFE is the method used. SPE fails to detect one third of γ-HCD cases, half of α-HCD cases, and two-thirds of μ-HCD cases. In addition, SPE does not demonstrate an M-gradient, but instead a relatively broad band that, in α-HCD, for example, extends from the α2 to the β region, due to the strong tendency of α HCD protein to form polymers. Special features:

  • The γ HCD protein can occur as a monomer in the urine due to its low molecular weight
  • In two thirds of cases in which μ-HCD protein is detected, κ FLCs are also detected
  • In some cases of HCD, mIgG or mIgM are also detected
  • Polyclonal Ig may be decreased or, occasionally, increased.

– α-HCD

This disease occurs in individuals from the Mediterranean region, Central Africa and Southern Africa, and Central and South America. Most patients are 20–30 years old. The disease mainly affects the IgA secretory system of the whole length of the intestine or the proximal half only. Stage A includes a lymphoplasmacytic infiltration of the lamina propria with variable villous atrophy, stage C is characterized by ulcerated lymphoblastic tumors of the intestine, and stage B represents an intermediate stage. Clinical features include chronic diarrhea, abdominal pain, weight loss, malabsorption, and protein losing enteropathy. The isolated respiratory form of α-HCD is very rare.

Laboratory findings: mild anemia, hypokalemia, hypocalcemia, hypomagnesemia, hypoalbuminemia, increased alkaline phosphatase activity as a result of an increase in the intestinal isoenzyme fraction. Parasitic infections are common. Abnormalities are found on SPE in about 50% of cases only.

– γ-HCD

The γ-HCD generally occurs in the sixth decade of life but occasional cases have been reported in children. Fever is present in around 20% of patients. γ-HCD is divided into the following categories:

  • Disseminated lymphoproliferative disease is present in 57–66% of γ-HCD patients. The usual findings include lymphadenopathy (56–62%), splenomegaly (38–52%), and hepatomegaly (8–37%). Localized proliferative disease (25%). Patients present with extramedullary plasmacytoma of the thyroid, tonsil, or oropharynx.
  • No proliferative disease (9–17%). Autoimmune disorders such as rheumatoid arthritis, autoimmune hemolytic anemia, vasculitis, or lupus erythematosus are reported in these patients.

Laboratory findings: normochromic normocytic anemia. An M-gradient can be detected on SPE in 60–86% of patients, most commonly in the β1–β2 region. The value of the M-gradient in diagnosed patients ranges from 4–39 g/L. Biclonal gammopathy has been reported in 15% of patients. In 65% of patients, the γ-HCD protein is IgG1.

– μ-HCD

μ-HCD is a rare disease with approximately 50 documented cases. The median age at diagnosis is 58 years (range 15–80 years). Patients present with a lymphoplasmacytic lymphoproliferative disorder with clinical features of chronic lymphocytic leukemia, lymphoma, WM, or myeloma. Splenomegaly and hepatomegaly are common and lytic bone lesions and osteoporosis are detectable in about 10% of cases.

Laboratory findings: an M gradient is detected by SPE in one third of cases; 10% of patients have a biclonal gammopathy and 40% are hypogammaglobulinemic. Bence-Jones proteinuria is found in more than half of patients. However, μ chains are found in the urine of fewer than 10% of cases.

Waldenstroem’s macroglobulinemia (WM) /72/

WM is characterized by B cell proliferation and the production of mIgM. This broad definition includes IgM MGUS, lymphoma, primary amyloidosis, chronic lymphocytic leukemia, and WM /44/. The term “IgM related disorders” is used to describe patients with clinical symptoms of WM but no bone marrow infiltration (Tab. 22-22 – Differential diagnosis of IgM monoclonal gammopathies). WM is a rare disorder with an annual incidence of 2.5 per million white women and 6.1 per million white men. The incidence increases with age, from 0.1 per million < 45 years of age and 36.3 per million > 75 years.

Clinical presentation /7374/: the clinical manifestations are related to the specific properties of the monoclonal IgM and to bone marrow infiltration. Malignant B cells circulate throughout the lymphatic system, involving it in the disease process. The clinical findings result from the following (Tab. 22-23 – Clinical symptoms and laboratory findings in Waldenstroem’s macroglobulinemia):

  • The hyper viscosity syndrome. Due to its high molecular weight, mIgM is mainly intravascular. This leads to increased osmotic pressure and intravascular volume, with slowing of the blood flow. The hyper viscosity syndrome occurs when the mIgM level reaches > 30 g/L but is attributed to the interaction of IgM with thrombocytes and plasma proteins. Patients present with chronic epistaxis and, less commonly, gastrointestinal hemorrhage. Other features include headache, tinnitus, dizziness, ataxia, and visual and auditory disturbances.
  • Neurological disorders such as peripheral neuropathy, since monoclonal IgM reacts as an autoantibody with neural glycoproteins and glycolipids to produce demyelination.
  • An urticarial rash with maculopapular lesions caused by malignant plasma cell infiltrates in the skin (Schnitzler’s syndrome).
  • Less commonly, lytic bone lesions (5% of cases). Kidney involvement is also less common than in MM.

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

Laboratory findings: in SPE and IFE, mIgM always migrates to the γ-globulin fraction. Because mIgM is a cryoglobulin in some patients, blood samples should be stored at 37 °C and the serum separated. Any serum precipitate must be dissolved prior to electrophoresis. The mIGM is detected on SPE and serum IFE in all cases of WM. Monoclonal FLCs are also detectable in the serum in > 70% of cases. BJ proteinuria is common, although it is rarely > 1 g/24 h. Although polyclonal Ig synthesis may be reduced, it is not usually reduced as significantly as in MM. The majority of patients have a mild to moderate anemia.

The IgM concentration should be checked every 3–6 months in patients with asymptomatic WM, every 3 months in patients with IgM MGUS, and annually if concentrations remain stable /71/.

Treatment options for symptomatic patients include alkylating agents, purine analogs, and the anti-CD20 monoclonal antibody rituximab as well as autologous and allogeneic stem cell transplantation. A prognostic scoring system is recommended to optimize the treatment of WM patients. Five adverse characteristics have been identified /72/: (i) age > 65 years, (ii) Hb ≤ 115 g/L, (iii) thrombocyte count ≤ 100 × 109/L, (iv) β2-microglobulin > 3.0 mg/L, (v) M-protein > 70 g/L.

Clinical significance: the 5-year survival rate in patients with:

1. One adverse characteristic who are ≤ 65 years of age is 87% (low risk)

2. Two characteristics or who are > 65 years of age is 68% (intermediate risk)

3. More than 2 characteristics is only 36% (high risk).

IgM amyloidosis

In the case of monoclonal IgM associated amyloid, the protein is produced by lymphoplasmacytic cells /73/. The monoclonal IgM is biodegradable and recyclable. Misfolded IgM produced in excess by lymphoplasmacytic cells is no longer amenable to recycling, is carried in the circulation to different tissues, deposites there, and is now referred to as an amyloid deposit. Amyloid typical deposits in the tissues

  • of the heart causing the heart to function poorly and become inefficient
  • of the kidney causing the kidney to leak protein in the urine (nephrotic proteinuria) resulting in hypoalbuminemia and increase in serum cholesterol
  • of the lung causing shortness of breath
  • of the nerves resembling IgM associated neuropathy with numbness and tingling, burning and lower extremity weakness
  • of the liver and causes enlargement of the organ.

The prevalence of IgM amyloidosis affects 8 of one million people yearly, the majority of patients are over the age 60, two thirds of the patients are men. The symptoms of amyloid include swelling, weakness, weight loss, shortness of breath, diarrhea, easy bruising of the face or eyelids, tongue enlargement, and dizziness upon standing. The diagnosis of IgM amyloidosis is based on symptoms and specialized biopsies of the bone marrow, skin or the fat.

IgM- associated amyloidosis should be differentiated from monoclonal IgM-related light chain (AL) amyloidosis which accounts for 5–7% of all light chain amyloidosis cases /74/.

Table 22-26 Findings in amyloidosis /76/

Clinical and laboratory findings


Amyloidosis is a rare disease that is a consequence from the accumulation of a misfolded precursor protein (amyloid) that deposits as generalized disease in body tissues and organs. Amyloidosis is differentiated:

  • Localized forms are a result of amyloid in specific areas of the body e.g., deposits occur in the brain from Alzheimer’s patients or in various tissues, often with aging (senile amyloidosis)
  • Systemic forms that deposit amyloid throughout the body. Systemic amyloidosis has been classified into three major types: primary (now AL), secondary (AA) and hereditary (ATTR). The ATTR include amyloid apolipoprotein A1 or AApoA1, amyloid apolipoprotein A2 or AApoAII, AGel, Alys, AFib).

Amyloidosis can occur as:

  • Isolated disease e.g., immunoglobulin light chain or AL amyloidosis, formerly referred to as primary amyloidosis.
  • Familial (hereditary) amyloidosis e.g., ATTR, AApoA2, AFib; rare forms that are more common in patients of African descent.
  • Amyloid protein is unstable and auto aggregates to form amyloid fibrils. These fibrils are deposited in organs such as the liver, heart, and kidneys, in the gastrointestinal tract, and in the autonomic nervous system.
  • Systemic light chain amyloidosis (AL) is the most common type of amyloidosis. The second most common type is due to transthyretin (TTR) misfolding.

– AL (AH and ALH) amyloidosis

AL, AH, and ALH are usually associated with a small plasma cell clone that produces monoclonal λ free light chains (FLCs), monoclonal H chains, or intact immunoglobulins. In AL, λ-FLC production is associated with a mature plasma cell clone or an underlying B cell lymphoproliferative disorder. The incidence of AL is 10 cases per 1 million population per year.

Clinical presentation: Approximately 60% of patients are male and most are > 60 years of age. Only 1% are below the age of 40 years. Fatigue and weight loss are key features. Approximately 15% of patients have hepatomegaly without splenomegaly. Tests for amyloid include aspiration of subcutaneous abdominal fat and bone marrow biopsy. Abdominal fat aspirate detects 70–80% of patients with amyloidosis, while a combination of bone marrow biopsy and fat aspirate detects 90%.

Cardiac involvement occurs in 60–75% of AL patients and is a significant factor in the outcome of patients with amyloidosis and is classified using the following criteria /55/: Concentration difference between λ FLCs and κ FLCs of ≥ 180 mg/L, cTnT ≥ 0.025 μg/L, NT-proBNP ≥ 1,800 ng/L. Assessment is based on a score, where patients are assigned one point for each criterion. Patients with a score of:

  • 0 have stage I disease, with a mean survival time of 94.1 months
  • 1 have stage II disease, with a mean survival time of 40.3 months
  • 2 have stage III disease, with a mean survival time of 14 months
  • 3 have stage IV disease, with a mean survival time of 5.8 months.

Cardiac amyloidosis is mainly a disease of diastole, therefore ejection fraction is commonly preserved.

Renal involvement occurs in 50–70% of AL amyloidosis patients and may present as nephrotic syndrome or as nonselective proteinuria.

Gastrointestinal involvement occurs in about 10% of AL amyloidosis patients.

Purpura is usually above the nipple line and classical periorbital purpura is seen in below 20%.

Nerve involvement: Nerve involvement may include sensory (paresthesia, numbness and pain) and autonomic symptoms (gastroparesis, bladder or bowel dysfunction and orthostatic hypotension). The gold standard for diagnosing small fiber neuropathy is quantification of intraepidermal nerve fibers in a skin biopsy and has a 90% sensitivity and specificity.

Laboratory findings: Urine IFE is positive in 83% of patients with AL, an abnormal serum FLC ratio is present in 91%, and a combination of serum FLC assay and IFE is positive in 99% /53/. The distribution of monoclonal immune proteins detected in the serum is as follows: free λ chains 34%, IgG λ 16%, IgA λ 8%, IgG κ 4%, free κ 3%, IgM 2%, and 33% have no M-protein /56/. Only 12% of patients have an M gradient > 15 g/L. The percentage of plasma cells in the bone marrow is generally low. For the treatment of AL, see Ref. /57/.

Following stem cell transplantation, higher baseline FLC ratios are associated with a lower survival rate. In patients with a serum FLC concentration of > 100 mg/L, response is defined as a 50% reduction in FLC and progression is defined as a 50% increase in FLC /53/.There are three methods of amyloid typing: immunohistochemistry, immunoelectron microscopy and mass spectrometry. The most sensitive indicator of myocardial involvement in AL are NT-proBNP and BNP. Patients with renal involvement have nephrotic proteinuria and some have elevated creatinine. β2-microglobulin is a prognostic marker. Electromyography is a useful test for evaluating peripheral neuropathy that usually will demonstrate symmetrical axonal sensimotor polyneuropathy. The prevalence of light chain monoclonal gammopathy of undetermined significance (MGUS) above the age of 50 is 4.2%, so monoclonal proteins in the serum, urine or clonal bone marrow plasma cells are not diagnostic /76/.

Beta-2 microglobulin amyloidosis

Beta-2 microglobulin amyloidosis occurs when amyloid deposits develop in patients with chronic renal insufficiency on dialysis. The deposits are found around joints and are composed of beta-2 microglobulin.


Familial amyloidosies are rare forms and have an autosomal dominant inheritance. Familial amyloidosis predominantly affects the heart and the nerves.

– Transthyretin amyloidosis (ATTR)

Transthyretin (ATTR) amyloidosis is a life-threatening, gain-of-toxic-function disease characterized by extracellular deposition of amyloid fibrils composed of transthyretin (TTR). TTR protein destabilised by gene mutation of TTR is prone to dissociate from its native tetramer to monomer, and to then misfold and aggregate into amyloid fibrils, resulting in autosomal dominant hereditary amyloidosis, including familial amyloid polyneuropathy, familial amyloid cardiomyopathy and familial leptomingeal amyloidosis /77/. Transthyretin familial amyloid polyneuropathy (TTR-FAP) classically presents as a length dependent small fiber polyneuropathy in endemic countries like Portugal. In non endemic countries it may mimic a variety of chronic neuropathies. Cardiac involvement of various severities is common in FAP /78/. Liver transplantation remains the standard antiamyloid therapy.

ATTR can be caused by TTR mutations or by wild-type TTR deposition in the elderly. Analogous misfolding of wild-type TTR results in senile systemic amyloidosis, now termed wild-type ATTR amyloidosis, characterised by acquired amyloid disease in the elderly.

Laboratory findings: TTR gene sequencing appears the most pertinent first-line test for diagnosis.

– Apolipoproein A-I amyloidosis

Conformational plasticity and flexibility are key structural features of Apo A-I in lipid metabolism. Amyloidogenic single point mutation (Leu75Pro) is characterized by the deposition of ApoA-I in various organs and associated with incurable familial amyloidosis with fibril deposition in peripheral organs. Renal, hepatic, and testicular involvement has been demonstrated.

Laboratory findings: Proteomic analysis by mass spectrometry of the amyloid deposits combined with genetic analysis can provide accurate diagnosis of ApoA-I amyloidosis /79/.

– Fibrinogen Aα-chain amyloidosis

Hereditary fibrinogen Aα-chain amyloidosis is a type of autosomal dominant systemic amyloidosis caused by mutations in fibrinogen Aα-chain gene. The gene variants are in the C-terminal region (amino acid residues 517–555). Clinically the most prominent manifestation is excessive amyloid deposition in the renal glomeruli /80/.

AA amyloidosis (systemic)

Type AA amyloidosis, also known as inflammatory amyloidosis, is a complication of chronic inflammatory conditions and is characterized by the deposit of insoluble amyloid fibrils in the affected organs and tissues. The protein AA is mainly a degradation product of the acute phase SAA and the consequence of overproduction and aberrant processing of SAA (Tab. 19.6-3 – SAA in inflammatory diseases). Sustained abnormally high levels of SAA in the tissues, which is usually present at low concentrations in serum, are essential for the development of AA amyloidosis.

Clinical suspicion: Proteinuria in up to 95% of patients leading to nephrotic syndrome is the most frequent clinical manifestation of AA in patients with chronic inflammation. The clinical significance of hepatosplenomegaly is relatively minor in the early stages of disease. The gastrointestinal tract may be affected, causing malabsorption, intestinal pseudo-obstruction, diarrhea or bleeding. Peripheral neuropathy, restrictive myocardiopathy and skin soft tissue involvement are uncommon when compared with other types of systemic amyloidosis. Diagnosis of AA amyloidosis is confirmed based on clinical organ involvement and histological demonstration of amyloid deposits.

Figure 22-1 B cell maturation. The abnormal cell clone in multiple myeloma develops from B cells that have passed through the germinal centers of the lymph nodes and have undergone somatic hyper mutation and isotype class switching to become plasma cells. Waldenstroem’s macroglobulinemia (WM) is characterized by the presence of lymphoplasmacytic cells that have undergone somatic hyper mutation but have not undergone isotype class switching. Modified from Ref. /53/. MGUS, monoclonal gammopathy of undetermined significance.

Somatic hypermutation Germinal centerlymph nodes Bone marrow VDJ recombination Pre-B cell Plasma cell Isotypical class switch B cell Smoldering MM MM

Figure 22-2 An atypical plasma cell (e.g., a myeloma cell) binds to a stromal cell of the bone marrow via adhesion molecules. IL-6 produced by the stromal cell stimulates the growth of the myeloma. Myeloma cells produce IL-1β, which has osteoclast activating (OAF) and other effects. Modified with kind permission from Ref. /8/. Adhesion molecules: VLA, very late appearing antigen; LFA, lymphocyte function associated antigen; VCAM, vascular cell adhesion molecule; ICAM, intercellular adhesion molecule.

Bonelesions Stromal cell Cell growth Myeloma cell Adhesion molecules – Upregulation of adhesion molecules– Induction of lytic foci– Paracrine IL-6 stimulation VLA-4 LFA-1 IL-6 IL-1β OAF

Figure 22-3 Serum protein fractions following electrophoretic separation on cellulose acetate sheets. a) Serum protein electrophoresis on normal control serum; b) Broad based increase in the γ-globulin fraction in polyclonal gammopathy; c) Saw blade pattern of γ-globulin fraction in oligoclonal gammopathy; d) Narrow peak indicating increased monoclonal immunoglobulin (M gradient) /9/.

Normal serum proteinelectrophoresis a Polyclonalgammopathy b Oligoclonalgammopathy c Monoclonalgammopathy d IgG 11.4 g/LIgA 2.0 g/LIgM 0.9 g/L IgG 25.4 g/LIgA 4.5 g/LIgM 2.7 g/L IgG 13.4 g/LIgA 1.5 g/LIgM 2.1 g/L IgG 35.3 g/LIgA 0.6 g/LIgM 0.1 g/L

Figure 22-4 Immunofixation electrophoresis in polyclonal (a) and monoclonal (b) gammopathy. In polyclonal gammopathy diffuse immunoprecipitate zones are detectable. In monoclonal gammopathy (b) of IgG type κ, a narrow dense immunoprecipitate is visible in the IgG heavy chain region and in the κ-light chain region.

a b * IgG IgA IgM κ λ

Figure 22-5 Immunoglobulin (Ig) molecule (right) with two identical heavy chains and two identical light chains. A free light chain is shown on the left. An antibody directed against the Ig molecule may only bind to the outer antigenic determinants of the light chains (); when a free light chain is present, the antibody can also react with the inner antigenic determinants ().

Hidden antigens Exposedantigens Light chain Previously hidden antigens are now accessible by antibodies Heavy chain Free light chain Antibody molecule Antigen binding sites

Figure 22-6 Frequency distribution of monoclonal gammopathies at the Mayo Clinic in Rochester. With kind permission from Ref. /31/. MGUS, monoclonal gammopathy of undetermined significance; lymphoproliferative diseases (e.g., chronic lymphocytic leukemia); SMM,smoldering myeloma; Macro, Waldenstroem’s macroglobulinemia.

Others 3% Macro 2% Solitary myeloma 1% Lymphoproliferative disease 3% Amyloidosis 10% Multiplemyeloma 14% N = 1149 SMM 4% MGUS 63%

Figure 22-7 Monoclonal immunoglobulin types in 1,027 patients with multiple myeloma, from Ref. /31/.

Negative 7% Only free λ 7% Only free κ 9% Biclonal 2% IgD λ 1% IgD κ 1% IgM λ 0.2% IgM κ 0.3% IgA λ 8% IgA κ 13% IgG λ 18% IgG κ 34%

Figure 22-8 Courses of monoclonal gammopathies. MGUS, monoclonal gammopathy of undetermined significance

g/l Smoldering myeloma Multiple myeloma MGUS M-Protein 80 60 40 20 2 4 6 8 10 12 Years

Figure 22-9 Morphological pattern of myeloma cells. Classification is based on the dominant cell type in the biopsy and/or aspirate. The six types can be classified into the prognostic grades: low, intermediate, and high malignancy /31/.

Low Intermediate High