Introduction

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Chapter

1

SECTION 1 - Immunoglobulin free light chains and their analysis

Introduction
Figure 1.0 Three dimensional structure of a κ light chain molecule.

Multiple myeloma (MM) is a disease with many faces. It usually presents in old age but may occur in youth. Bone pain and fractures are characteristic yet soft tissue involvement by plasmacytomas may also occur. Patients may die within weeks of presentation while others "smoulder" for years. Patients may develop renal failure, acute and chronic infections or AL amyloidosis, and many will require stem cell transplantation or intensive chemotherapy. Consequently, many specialists, including haematologists, nephrologists, immunologists, orthopaedic surgeons and chemical pathologists become involved in disease management. Furthermore, the prevalence of MM is increasing due to a slowly rising incidence and a longer life expectancy [1][2][3]

Despite the complexity of this disease, one feature has been a great lighthouse in the fog, alerting the unwary to the diagnosis and guiding the hand of management: the presence of monoclonal immunoglobulins. Produced in excess, and in a variety of shapes and sizes, these molecules have been linked to MM since they were first identified by Henry Bence Jones over 150 years ago[4][5]. Notwithstanding their substantial history and great utility, measurement of these tumour markers, particularly free light chains (FLC), remained imperfect for many years. Techniques used for their measurement had failed to keep pace with analytical developments in other fields until the 21st century when serum FLC analysis was introduced.

Tumour Type Cancer Deaths (USA)[3] Tumour Markers Specificity Sensitivity Tumour Detection Clinical Utility
Lung + bronchus 28% Neuron specific enolase Poor Poor Late Poor
Colon + rectum 9% Carcinoembryonic antigen Poor Modest Late Modest
Breast 7% CA 15-3; CEA Poor Modest Late Modest
Pancreas 6% CA 19-9; CEA Poor Poor Late Poor
Prostate 5% Prostate-specific antigen Modest Good Good Good
Stomach 2% CEA; CA 19-9 Modest Modest Late Poor
Ovary 2.5% CA 125; PLAP Modest Modest Intermediate Good
Liver 3% Alpha feto-protein (αFP) Good Good Intermediate Good
Myeloma 1.9% Monoclonal protein/FLC Good Good Early Very good
AL amyloidosis 0.3% Monoclonal protein/FLC Good Good Early Very Good
Germ cell ~0.1% αFP; human chorionic gonadotrophin Good Good Early Very Good
Choriocarcinoma <0.1% Human chorionic gonadotrophin Good Good Early Very Good
Neuroendocrine <0.1% Chromogranin A, gastrin Modest Good Early Very Good

Table 1. Some common serum tumour markers and their clinical utility. All of these analytes were measured using highly sensitive immunoassays apart from monoclonal proteins. FLC = free light chains

Most serum cancer tests are now based on state-of-the-art immunoassays and are highly automated. (A selection of the more common serum markers is shown in Table 1). In contrast, tests for MM and AL amyloidosis are primarily based on serum and urine electrophoretic techniques. These are relatively insensitive, require considerable experience for interpretation and are often labour-intensive. How ironic that the first tumour marker to be identified should be the last to benefit from modern technology.

It is, perhaps, not surprising that these techniques produce errors in FLC measurements[6]. And, why use urine? It is hard to imagine a less attractive fluid in which to evaluate these molecules. An important function of the kidneys is to prevent the loss of FLCs and other small protein molecules into the urine. Furthermore, urine samples are voluminous, difficult to obtain, awkward to transport and need to be concentrated prior to analysis.

An alternative strategy is to measure FLCs in serum. In 1981, it was shown that serum concentrations of FLCs were elevated when Bence Jones proteinuria occurred (Chapter 2), and that measuring serum rather than urine was diagnostically more accurate in patients with renal failure [7][8]. Why, therefore, have serum immunoassays not been used before? It is now apparent that the overriding barrier was the difficulty in developing satisfactory antibodies for use in the assays. To function correctly, these antibodies must not only be of high affinity to allow measurement of low concentrations of serum FLCs, but must also be highly specific. Serum FLC concentrations are several orders of magnitude lower than serum light chains bound to intact immunoglobulins, so even minor antibody crossreactivity produces unacceptable results. Only recently have suitable antibodies been developed that bind exclusively to the hidden epitopes of FLC molecules (Chapter 4) . These antibodies have facilitated the development of serum FLC assays that are specific, sensitive and quantitative [9][10].


Serum FLC immunoassays include the following benefits:
  • Better sensitivity and precision than current electrophoretic assays for FLC molecules.
  • Numerical results for disease monitoring.
  • Convenience of using serum as a test medium.
  • Sensitive measurement of FLCs in AL amyloidosis and nonsecretory multiple myeloma patients who have no detectable monoclonal proteins by conventional tests.
  • Accurate marker of disease remission.
  • Short half-life marker for rapid assessment of treatment responses.
  • Marker of increased risk of progression in individuals with MGUS.
  • More sensitive than urine tests when screening symptomatic patients.
  • Diagnosis and monitoring of patients with myeloma kidney.

General statements have been formulated to describe the clinical applications of cancer markers [11]. Most of these have now been applied to FLC immunoassays, as follows:

1. Differential diagnosis in symptomatic patients. Serum FLC analysis is most helpful in the differential diagnosis of patients with bone pain, fractures, unexplained renal impairment and other features of MM and AL amyloidosis (Section 2 and 4).
2. Clinical staging of disease. Serum FLC concentrations show a relationship with the staging of monoclonal diseases and are helpful in assessing residual disease after treatment (Chapter 10 and 12).
3. Estimating tumour burden. Serum FLC concentrations correlate poorly with other tumour markers at the time of diagnosis but changing concentrations correlate well with changing tumour burden during treatment (Section 2).
4. Prognostic indicator for disease progression. Serum FLC concentrations are of prognostic value in MM at the time of clinical presentation. Also, patients who are in remission but have elevated FLC levels, in the absence of other abnormalities, are at risk of early relapse. Of particular interest is the use of the assays for predicting risk of progression of monoclonal gammopathy of undetermined significance (MGUS). Studies from the Mayo Clinic indicate that elevated monoclonal FLCs are a sensitive risk factor for progression to myeloma and other plasma cell dyscrasias (Section 2) .
5. Evaluating the success of treatment. FLC analysis is particularly helpful in assessing treatment responses in patients with AL amyloidosis, light chain multiple myeloma (LCMM) and nonsecretory multiple myeloma (NSMM). There are also indications that the short half-life of FLCs can be used to assess early treatment responses in nearly all patients with monoclonal proteins (Section 2).
6. Detecting the recurrence of cancer. Serum FLCs are useful for detecting disease recurrence and are often more sensitive than other tests in patients with AL amyloidosis, LCMM and NSMM. They are useful in a proportion of patients with intact immunoglobulin myeloma and some other monoclonal gammopathies (Section 2).
7. Screening symptomatic patients. Historically, patients with symptoms of MM or related disorders were screened for monoclonal proteins using serum and urine electrophoretic tests. Several recent studies have shown that serum FLC analysis in combination with serum electrophoresis and/or serum immunofixation identifies more patients and can replace urine tests in most instances (Chapter 23 and 24) .
8. Screening the general population. There is evidence that serum FLC measurements identify a new set of MGUS patients in otherwise healthy people (Chapter 19). Indeed, progression to light chain and nonsecretory myeloma has been observed in patients whose only preceding abnormalities were abnormal sFLC ratios. Because the criteria for a successful screening test include a beneficial outcome for the population screened, it will be many years before it is clear whether serum FLCs are useful in this context.

In addition, serum FLC assays have allowed new guidelines to be written for the diagnosis and monitoring of patients with MM, AL amyloidosis and other diseases producing excess clonal FLCs (Chapter 25)

In the following chapters, the discovery of Bence Jones protein, the structure and synthesis of light chain molecules, and assays for serum FLC quantification are described. A detailed account of the current use of the assays in clinical and laboratory practice is presented, together with potential applications in other settings. Appendices for guidance in their clinical and laboratory use are also provided. In addition, Chapter 32 provides an introduction to the analysis of heavy chain/light chain pairs (Hevylite™). These are new nephelometric and turbidimetric immunoassays that measure the concentrations of different light chain types of each immunoglobulin class, and provide a ratio that may assist in the diagnosis, staging and monitoring treatment responses of MM and other plasma cell dyscrasias.

Overview Back to Contents Page Chapter 2

References

  1. ↑ Katzel JA, Hari P, Vesole DH. Multiple myeloma: Charging toward a bright future. CA Cancer J Clin 2007; 57: 301 – 318 PMID: 17855486
  2. ↑ Kumar SK, Rajkumar SV, Dispenzieri A, Lacy MQ, Hayman SR, Buadi FK, Zeldenrust SR, Dingli D, Russell SJ, Lust JA, Greipp PR, Kyle RA, Gertz MA. Improved survival in multiple myeloma and the impact of novel therapies. Blood 2008;111:2516-20. PMID: 17975015
  3. ↑ 3.0 3.1 Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin. 2009; 59: 225 – 49. PMID: 19474385 PMID: 19474385
  4. ↑ Jones HB. Papers on Chemical Pathology, Lecture III. Lancet 1847; 2: 88 - 92
  5. ↑ Jones HB. On the new substance occurring in the urine of a patient with mollities ossium. Phil Trans R Soc B 1848; 138: 55 – 62
  6. ↑ Ward AM, White PAE, Beetham R. Monoclonal Protein Identification Distribution 986. UK NEQAS Sheffield, 1998.
  7. ↑ Solling K. Free light chains of immunoglobulins. Scand J Clin Lab Invest Suppl 1981; 157: 1 – 83 PMID: 6797039
  8. ↑ Sinclair D, Dagg JH, Smith JG, Stott DI. The incidence and possible relevance of Bence-Jones protein in the sera of patients with multiple myeloma. Br J Haematol 1986; 62: 689 – 94 PMID: 3964561
  9. ↑ Bradwell AR, Carr-Smith HD, Mead GP, Tang LX, Showell PJ, Drayson MT, Drew R. Highly sensitive, automated immunoassay for immunoglobulin free light chains in serum and urine. Clin Chem 2001; 47: 673 – 80 PMID: 11274017
  10. ↑ Katzmann JA, Clark RJ, Abraham RS, Bryant S, Lymp JF, Bradwell AR, Kyle RA. Serum reference intervals and diagnostic ranges for free kappa and free lambda immunoglobulin light chains: relative sensitivity for detection of monoclonal light chains. Clin Chem 2002; 48: 1437 – 44 PMID: 12194920
  11. ↑ Chan DW, Schwartz MK. Tumor markers: Introduction and general principles. In: Diamandis EP, Fritsche HA, Lilja H, Chan DW, Schwartz MK, eds. Tumor markers: Physiology, pathology, technology and clinical applications, AACC Press, 2002: Chapter 2: 9 – 17
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