American Association for Clinical Chemistry
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August 2010 Clinical Laboratory News: Multiple Myeloma

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August 2010: Volume 36, Number 8


Multiple Myeloma
Laboratory Testing for Plasma Cell Proliferative Processes

By David F. Keren, MD


Photo courtesy of Steven H. Kroft, MD,
Professor of Pathology, Director of Hematopathology,
Medical College of Wisconsin

Although it’s not widely known today, multiple myeloma was the first disease for which the clinical laboratory could measure a quantifiable tumor marker, the Bence Jones protein. First described in 1847 by the English physician Henry Bence Jones and later characterized as a monoclonal free immunoglobulin light chain (mFLC), today the Bence Jones protein and related monoclonal (M) protein markers are invaluable for detecting, classifying, staging, and monitoring multiple myeloma.

Multiple myeloma, a highly lethal neoplasm of plasma cells, accounts for 10% of hematologic malignancies. Last year, 20,000 new cases of the disease were diagnosed in the U.S, with a median age at diagnosis of 62 years. Not surprisingly, as the U.S. population ages, the incidence of multiple myeloma has been increasing. However, about 2% of cases are diagnosed in individuals under the age of 40.

A continuous progression from monoclonal gammopathy of undetermined significance (MGUS), to smoldering (asymptomatic) multiple myeloma (SMM) and eventually to symptomatic (active) multiple myeloma parallels the evolution of colonic polyps to colon cancer, suggesting a random two-hit etiology. Genomic alterations, including hyperdiploidy and translocations at the immunoglobulin chain locus (14q32) in most cases of MGUS, support this hypothesis.

Pathology of Multiple Myeloma

Multiple myeloma originates from a single clone of plasma cells in the bone marrow. As the neoplastic plasma cells proliferate, bone tissue is destroyed and normal marrow elements are displaced. The neoplastic clone produces a unique immunoglobulin molecule composed of two identical heavy chains and two identical light chains that are covalently linked by disulfide bonds. Because the light chain type produced early in B-cell maturation does not change, it helps to define the clonal nature of the monoclonal process.

Normal plasma cells synthesize more light chains than are needed to combine with the heavy chains. These excess unbound light chains pass into the serum as free light chains (FLCs). Due to their relatively small size, 22 kDa for the κ-chain and 44 kDa for the λ-chain, the proteins also clear the kidneys, passing into the glomerular filtrate. The majority of the chains are reabsorbed by the proximal tubules of the kidney, and the rest are excreted into the urine. The small amount of circulating normal polyclonal FLCs causes no damage to the kidneys; however, excessive production of mFLC can lead to formation of β-pleated sheets of AL amyloid, deposition of non-amyloid light chains (light chain deposition disease; LCDD), injury of proximal tubular cells (acquired Fanconi syndrome), or precipitation and obstruction of the distal nephron (cast nephropathy).

In the vast majority of multiple myeloma cases, the clonal plasma cells produce a large M protein—often with excess mFLC—but in about 15% of cases, the M protein is only an FLC.

Prior to development of the serum FLC assay, laboratories could not detect the M protein by immunofixation (IFE) of serum or urine in about 1–2% of cases. These cases were therefore classified as “nonsecretory myeloma.” Now, however, the improved sensitivity and precision of the serum FLC assay allows laboratories to detect an altered ratio of free κ/λ (rFLC) light chains in slightly more than half of such patients. Cases in which the M protein is detectable only by the serum FLC assay are classified as “oligosecretory.”

Although detection of the M protein is a key indicator of multiple myeloma, it is important to realize that the disease is a member of a large family of plasma cell proliferative disorders. Multiple myeloma stands out as the most malignant member of the family, but it is far from the most prevalent member of this family of diseases that produce an M protein (Table 1). The range of presenting clinical features varies widely from none in MGUS and SMM to anemia, lytic bone lesions, and renal failure in multiple myeloma.

Table 1
Conditions with Monoclonal Gammopathies
Condition
Clinical Effect
Multiple Myeloma Severe
Waldenström’s Macroglobulinemia Moderate
AL Amyloid Severe
Smoldering Multiple Myeloma (SMM) Asymptomatic with risk of progression
Monoclonal Gammopathy of Undetermined Significance (MGUS) Asymptomatic with risk of progression
Transient monoclonal Usually reactive (infection, occasionally autoimmune)
M Protein-Associated Neuropathy Moderate to Severe
Plasmacytoma None to moderate

Who Should be Tested?

The acronym CRAB emphasizes the most useful clinical and laboratory features of symptomatic multiple myeloma: elevated Calcium, Renal insufficiency, Anemia, and Bone lesions. The clinical features of multiple myeloma are inconsistent, however, and mild, early symptoms can mimic many other diseases (Table 2). Bone pain, which is reported by about 60% of patients at the time of diagnosis, is the symptom that most immediately focuses clinicians’ attention on multiple myeloma. On the other hand, in a population that is typically in the late 50’s or older at disease onset, bone pain is also a common and nonspecific finding.

Table 2
Common Clinical Features Associated with Multiple Myeloma
Clinical Feature
Common Cause(s)
Nausea, confusion, polyuria Elevated calcium, renal insufficiency
Fatigue Anemia, renal insufficiency
Bone pain Osteolysis, pathologic fractures
Paraplegia Spinal cord compression
Confusion and blurred vision Hyperviscosity
Bleeding Thrombocytopenia
Skin nodules Plasma cell tumors
Clinical features of amyloidosis AL amyloid deposits
Bacterial Infections Immune deficiency

Given the general nature of clinical features in patients with multiple myeloma and the vast difference between the occurrence of the disease (incidence of 4:100,000) and that of MGUS (>1 out of 20 in individuals older than age 70), screening tests ordered to evaluate malaise, weakness, bone pain, or even potentially relevant organ-related symptoms dredge up considerably more cases of MGUS than multiple myeloma. Indeed, such serendipitous findings, which show up on serum protein electrophoresis (SPE), are the only way to detect SMM or MGUS.

Progression to Multiple Myeloma: Subcategorizing Risks in MGUS or SMM

MGUS and SMM are asymptomatic, and each carries a considerably different potential for progression to multiple myeloma. By definition, patients with MGUS have <3 g/dL M protein in serum and <10% of plasma cells in the bone marrow, but they do not have hypercalcemia, anemia, lytic bone lesions, or organ failure. Overall, the risk of MGUS progressing to multiple myeloma or other treatable plasma cell proliferative processes is about 1% per year.

In SMM, the clinical and laboratory findings are the same as MGUS except that the serum M protein is ≥3 g/dL and/or the bone marrow plasma cells are ≥10%. The risk of SMM progressing to symptomatic multiple myeloma or AL amyloidosis is about 10% per year for the first 5 years after diagnosis, about 3% per year for the next 5 years, and 1% per year for the succeeding 10 years. The cumulative probability for progression is 73% at 15 years.

By looking at three key factors, clinicians can more precisely estimate the risk of MGUS progression to multiple myeloma. Cases with IgG M proteins are less likely to progress than those with IgA or IgM M proteins. Those with M proteins <1.5 g/dL are also less likely to progress than those with higher levels, and individuals in which the M proteins have a normal rFLC are less likely to progress than those with an abnormal ratio (Figure 1A). The actual risk, however, is less when competing factors for death are included.

Figure 1A
20 Year Progression of MGUS to MM

Data from Rajkumar et al. 2005 (used with permission)

Clinicians also evaluate progression to multiple myeloma for individuals diagnosed with SMM based on assignment to a risk group. Patients are classified into risk groups based on the proportion of bone marrow plasma cells and concentration of M protein (Figure 1B). By using a slightly broader range of normal for rFLC, clinicians also can refine the prognosis for progression of SMM.

Figure 1B
15 Year Progression of SMM to MM

Data from Kyle et al. 2007 (used with permission)

Initial Detection and Identification of Treatable Monoclonal Processes

When clinical features suggest the presence of multiple myeloma, laboratory evaluation should include several routine clinical tests: a complete blood count with differential and peripheral blood smear review; a chemistry panel that includes calcium and creatinine; albumin; total protein; and IgG, IgA, and IgM.

With the exception of the 1–2% of cases that are nonsecretory, all individuals with multiple myeloma have the telltale signatures of M protein immunoglobulin product and/or its mFLC counterpart. Therefore, laboratory testing procedures should be directed toward detecting these molecules. The five main tests currently used for detecting these tumor markers are: SPE; IFE of serum; urine protein electrophoresis (UPE), optimally a 24-hour collection, but an early morning void also can provide a useful qualitative specimen; IFE of urine; and the serum FLC assay.

Which of the five methods should clinicians order to find the M protein, and should they order all five for every patient? In the vast majority of multiple myeloma cases, laboratories can readily detect large quantities of intact M proteins by SPE and/or excess mFLCs from concentrated UPE. In the 15% of cases that demonstrate only mFLC, laboratory evaluation should include the serum FLC assay, as well as SPE and serum IFE. This combination circumvents the need for the often difficult-to-obtain initial urine specimen.

Some multiple myeloma cases are more difficult to detect. For example, if the intact M protein is produced in relatively small amounts and co-migrates with prominent β- or α-region proteins, it may elude detection by SPE. The presence of subtle β-region bands, “fuzzy bands”, or increased β-1 or β-2 bands by SPE, without an obvious explanation such as a liver disease pattern, should trigger a reflex IFE test.

Laboratories should also consider the testing recommendations from the International Myeloma Working Group. For example, the group recommends that patients with clinical suspicion for symptomatic multiple myeloma be tested by IFE of serum and urine when a protein electrophoresis study is negative. Because clinical suspicion and the symptoms for multiple myeloma are rather broad considerations, some of the recommendations on optimal laboratory testing to detect treatable M protein cases have generated considerable debate in the clinical community.

An analysis by Katzmann and colleagues of 1,877 patients provides a better perspective on the capabilities of the five main laboratory tests for M proteins. In this study, the researchers performed all five M protein assays on all patients. By performing only SPE and FLC, they were able to detect all cases of multiple myeloma and Waldenström’s disease. However, to optimize detection of other treatable conditions such as AL amyloid, LCDD, plasmacytoma, and M protein-associated neuropathy, IFE of serum and/or urine was necessary (Table 3).

Table 3
Clinical Study: Detection of M Proteins+
Condition (n)
Detected by using All 5* tests % (n)
Detected by using SPE and FLC % (n)
Detected by Serum IFE % (n)
MM (467)
100 (467)
100 (467)
94.4 (441)
Waldenström’s (26)
100 (26)
100 (26)
100 (26)
SMM (191)
100 (191)
99.5 (190)
98.4 (188)
MGUS (524)
100 (524)
88.7 (465)
92.8 (486)
Plasmacytoma (29)
89.7 (26)
86.2 (25)
72.4 (21)
POEMS (31)
96.8 (30)
74.2 (23)
96.8 (30)
Extramedullary
plasmacytoma (10)
20.0 (2)
10.0 (1)
10.0 (1)
Primary AL (581)
98.1 (570)
96.2 (559)
73.8 (429)
LCDD (18)
83.3 (15)
77.8 (14)
55.6 (10)

*SPE, UPE, serum IFE, urine IFE, FLC

Abbreviations: multiple myeloma (MM), smoldering multiple myeloma (SMM), monoclonal gammopathy of undetermined significance (MGUS), peripheral neuropathy, organomegaly, endocrine disorders, M protein, skin changes (POEMS)

+Data from 1877 patients (Katzmann, 2009)

If no M protein is detected by IFE in serum or urine or by the FLC method, yet active MM is still suspected, a bone marrow or relevant tissue biopsy needs to be performed to detect nonsecretory MM, some cases of AL amyloidosis, and most cases of plasmacytoma.

When the laboratory detects an M protein in a patient, it should also identify the M protein isotype to enable the clinician to follow the patient’s response to therapy. Most often this is accomplished by serum and urine IFE with gel electrophoresis, or immunotyping (IT) (immunosubtraction) with capillary electrophoresis (CE). While IT can identify most M proteins, it is inherently less sensitive than IFE and cannot be used to document complete remission or to rule out neuropathy associated M proteins such as POEMS (defined in Table 3). For cases with oligosecretory mFLC, the serum FLC assay is the best way to identify the light chain type. Individuals with absolute nonsecretory myeloma require immunohistochemistry or immunofluorescence on bone marrow plasma cells.

Classifying and Measuring Monoclonal Processes

With gel electrophoresis, laboratories should measure the M protein by densitometry, using total protein determined by various total protein methods. However with CE, the M protein percent is measured on the electropherogram by peptide bond absorbance. Alternatively, laboratories may choose to measure M proteins indirectly by nephelometric techniques.

Unfortunately, results from these methods show systematic differences. Gel electrophoresis relies on staining by a protein dye. At high concentrations of the M protein, staining saturation of the M protein yields lower M protein values and higher albumin values. CE is not affected by this issue; therefore, laboratories may record higher M protein values on CE versus gel SPE from the same patient. However, with CE, occasional IgM M proteins migrate erratically, resulting in markedly decreased values from those observed by gel electrophoresis or nephelometric measurements. Nephelometric techniques, though not plagued with problems of dye saturation or migration issues, can be affected by self-aggregation of M proteins, are prone to overestimate IgM M proteins, and can suffer from antigen excess, the so-called high-dose hook effect.

Small quantities of M proteins obscured by normal proteins in β- or α-globulin fractions cannot be estimated reliably by densitometry or CE. For those situations, measuring the total isotype of the involved M protein may be the best way to follow them. Recently, a promising method, Hevylite, from the Binding Site Group has been become available. This method measures the combined heavy and light chain combinations, meaning it can quantify IgGκ molecules separately from IgGλ molecules. Further independent studies are needed, however, to document the utility of this technique in clinical practice.

With light chain multiple myeloma, even if the serum FLC test shows an abnormal ratio, 24-hour urine measurements are recommended to quantify the M protein. For cases of oligosecretory multiple myeloma, FLC measurements may decrease the need for serial bone marrow studies to follow the response to therapy.

Prognosis Stratification of Multiple Myeloma

Two staging systems are currently in use to stratify prognosis of multiple myeloma: Durie-Salmon staging system (DSS) and the international staging system (ISS) (Table 4). Both are predictive for progression-free survival and overall survival. Other factors that indicate higher risk include increased LDH, IgA isotype, extramedullary disease, renal failure, abnormal rFLC, plasma cell leukemia, and plasmablastic disease.

Table 4
Staging Systems for Multiple Myeloma
System
Stage I
Stage II
Stage III
Durie-
Salmon

All of the following:

Hemoglobin >10 g/dL

Serum Calcium ≤12 mg/dL

Normal skeletal survey or solitary plasmacytoma

Low quantity of M protein
IgG <5 g/dL
IgA <3 g/dL
Bence Jones <4 g/24h

Not
Stage I
nor
III

One or more of the following:

Hemoglobin <8.5g/dL

Serum Calcium >12 mg/dL

≥3 lytic bone lesions

High quantity of M protein
IgG >7 g/dL
IgA >5 g/dL
Bence Jones >12 g/24h

Inter-
national

ß-2 microglobulin
<3.5 mg/L and

Serum albumin ≥3.5 g/dL

Not
Stage I
nor
III
ß-2 microglobulin
>5.5 mg/L

Fluorescence in situ hybridization (FISH) and cytogenetic studies can stratify individuals into standard- and high-risk groups. Those with standard-risk have a survival of 3–6 years, tend to have an IgGκ M protein, and usually present with lytic bone lesions. High-risk individuals have a mean survival less than 3 years, often have an IgAλ M protein, and are less likely to present with skeletal manifestations.

Following Treatment

Most likely, patients in whom M proteins are detected will be cases of MGUS. A follow-up measurement of the M protein a few months after initial detection provides an estimate of the rate of growth of the neoplasm and helps to rule out a case of active multiple myeloma. Subsequent intervals of follow-up depend on how stable the M protein and clinical picture are. With active multiple myeloma, in addition to following the M protein, measurements of creatinine and BUN, serum calcium levels, and hematologic parameters by the CBC are needed. The intervals vary widely depending on the clinical course.

Standard chemotherapeutic regimens typically produce a measurable reduction of the M protein, occasionally to undetectable levels, by serum and urine IFE determination and a decrease in normal γ-globulins. In addition, 2–3 months post autologous stem cell transplantation (ASCT), patients may exhibit γ-region oligoclonal bands that are occasionally prominent enough to suggest the possibility of a new M protein. These are transient, however, and usually disappear within 2 years. Their presence reflects recovery of normal immunoglobulin producing clones and is a favorable feature for event-free and overall survival.

Finally, laboratories are increasingly using serum FLC assays to follow oligosecretory multiple myeloma and AL amyloidosis. According to the International Myeloma Working Group, the assay results are one of the criteria for complete and stringent and complete patient response.

A Brighter Outlook

Although still not considered a curable disease, ongoing trials with unique combinations of therapies are extending the lives of individuals affected by multiple myeloma. Treatment with thalidomide, development of new drugs such as bortezomib and lenalidomide, and use of ASCT have lengthened periods of complete and stringent complete remission.

Clinical laboratory diagnosis of multiple myeloma continues to improve and evolve. The concept of measuring FLC, totally unproven merely a decade ago, is now a routine test that can replace urine IFE for the initial diagnosis, detect most cases of formerly “nonsecretory” multiple myeloma, and help confirm stringent complete remission. Better protein resolution and more efficient SPE, CE, IFE, and IT now allows laboratories to effectively triage cases and to detect subtle M proteins in the α- and β-regions. In sum, today labs possess a greater arsenal of methods to measure M proteins and are more keenly aware of the disparities of measuring M proteins by different methods.

As noted at the beginning of this article, M proteins are the granddaddy of all tumor markers. But their detection and measurement by the newest technologies play a vital role as medicine strives towards better outcomes for patients with multiple myeloma.

ADDITIONAL READING

Bradwell AR, Harding SJ, Fourrier NJ, Wallis GLF, et al. Assessment of monoclonal gammopathies by nephelometric measurement of individual immunoglobulin κ/λ ratios. Clin Chem 2009;55:1646–55.

Dispenzieri A, Zhang L, Katzmann JA, Snyder M, et al. Appraisal of immunoglobulin free light chain as a marker of response. Blood 2008;111:4908–15.

Katzmann JA, Dispenzieri A, Kyle RA, Snyder MR, et al. Elimination of the need for urine studies in the screening algorithm for monoclonal gammopathies by using serum immunofixation and free light chain assays. Mayo Clin Proc 2006;81:1575–78.

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

Kumar SK, Mikhael JR, Buadi FK, Dingli D, et al. Management of newly diagnosed symptomatic multiple myeloma: updated Mayo stratification of myeloma and risk-adapted therapy (mSMART) consensus guidelines. Mayo Clin Proc 2009;84:1095–110.

Kyle RA, Rernstein ED, Therneau TM, Dispenzieri A, et al. Clinical course and prognosis of smoldering (asymptomatic) multiple myeloma. NEJM, 2007;356:2582–90.

Kyle RA, Rajkumar SV. Treatment of multiple myeloma: a comprehensive review. Clin Lymphoma Myeloma 2009;9:278–88.

Landgren, O, Kyle RA, Pfeiffer RM, Katzmann JA, et al. Monoclonal gammopathy of undetermined significance (MGUS) consistently precedes multiple myeloma: a prospective study. Blood 2009;113:5412–17.

Palumbo A, Sezer O, Kle R, Miguel JS, et al. International Myeloma Working Group guidelines for the management of multiple myeloma patients ineligible for standard high-dose chemotherapy with autologous stem cell transplantation. Leukemia 2009;23:1716–30.

Rajkumar SV, Kyle RA, Therneau, TM, Melton III, LJ, et al. Serum free light chain ratio is an independent risk factor for progression in monoclonal gammopathy of undetermined significance. Blood 2005;106:812–17.

WEB SOURCES

National Cancer Institue website. Multiple myeloma and other plasma cell neoplasms treatment (PDQ®). Online. 12-11-2009.

Rajkumar SV. Treatment of Myeloma: cure vs control. Online. 10-2008.


David F. Keren, MD, is medical director of Warde Medical Laboratory, a member of the Department of Pathology at St. Joseph Mercy Hospital, Ann Arbor, Mich., and adjunct professor of pathology at the University of Michigan. Email: kerend@wardelab.com.


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