Chain Test. Linda Traylor, Ph.D. Technical Director The Binding Site, Inc. San Diego, CA

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1 Understanding the Serum Free Light Chain Test Linda Traylor, Ph.D. Technical Director The Binding Site, Inc. San Diego, CA

2 Objectives Plasma Cell Involvement and Immunoglobulin Proteins Importance of Serum Free Light Chains in Plasma Cell Dyscrasias Serum Free Light Chain Test - Freelite Provide Patient Examples of Monitoring Disease Practical Considerations Lab Results

3 Multiple Myeloma And Amyloidosis Are Both Cancers Of The Plasma Cell Cancerous plasma cells can accumulate in the bone marrow and cause: Low blood counts (e.g., anemia) Bone damage Abnormally high protein levels in the blood and/or urine Increased risk of infection In amyloidosis, proteins can deposit on critical organs (e.g., heart, kidneys) and cause damage to those organs Testing is important to monitor the disease so that it can be treated before any of these things occur

4 Bone Marrow Aspirate

5 Plasma Cells Produce Immunoglobins (also called antibodies) Plasma Cell Antibodies (Immunoglobulins)

6 There Are Three Immunoglobulins Commonly Associated with Myeloma/Amyloidosis IgA IgG IgM

7

8 Plasma Cells Produce Fully Formed Immunogloblulins li and Free Light Chains Immunoglobulins SPE and IFE Free light chain test = Freelite Plasma Cell Free Light Chains

9 Methods to Detect and/or Quantify Immunoglobulins and Free Light Chains Protein electrophoresis Serum (SPEP) Urine (UPEP) Immunofixation electrophoresis (IFE) Capillary zone electrophoresis (CZE) Nephelometry and turbidimetry Quantitative immunoglobulins: measure levels of IgG, IgA, IgM, IgE, IgD Serum free light chain assays: measure levels of kappa, lambda, and their ratio

10 Serum Protein Electrophoresis Alb α1 α2 β γ Normal Monoclonal Disease

11 Serum Immunofixation Electrophoresis (IFE) Normal Monoclonal Disease

12 Immunoglobulins Are Made Up Of Heavy Chains And Light Chains Immunoglobulin Light Chains Heavy Chains

13 There Are Two Types Of Free Light Chains Kappa And Lambda Kappa Free Light Chains Lambda Free Light Chains

14 Normal Levels of Polyclonal Immunoglobulins li and Free Light Chains IgG: 7,000 16,000 mg/l IgA: 700 4,000 mg/l IgM: 400 2,300 mg/l IgD: 0 80 mg/l IgE: 0.01 mg/l Free κ: mg/l Free λ: mg/l In multiple myeloma, the concentration of monoclonal proteins may exceed 100,000 mg/l, or the level of monoclonal proteins may be so low as to be hidden within the polyclonal proteins, or the tumor may be truly nonsecretory

15 Monoclonal Plasma Cells in Bone Marrow by Immunoflourescence ou with Specific c Anti-lambda or Anti-kappa Antibodies

16 Free Light Chain Assays: Polyclonal Ab to Sequestered Light Chain Epitopes Kappa Sequestered surface Heavy chain Antibody target target Light chain Lambda

17 The FreeLite Test Measures Serum Free Light Chains Latex Reagent: consisting of monospecific polyclonal antibody coated onto polystyrene latex Mixed with serum sample and analyzed using a nephelometer (light scatter) or turbidimeter (light absorption) Quantified: kappa concentration, ti lambda concentration and kappa/lambda ratio

18 Manufacturing of Sheep Antibodies Expose American Sheep to Human Proteins (Antigens) using specific immunization and tolerization protocol Queens Award for Innovation Patented and Trade Secret Specific Polyclonal Sheep Antibodies to Human Proteins Collect sheep plasma in USA and ship to UK for processing Purification and Rolling Pool Process

19 Why is it important to measure free light chains?

20 The Free Light Chain Test Serum test specific for free light chains More sensitive for detection of free light chains than SPE or IFE May indicate if therapy is working, sometimes earlier than SPE or IFE May detect relapse of disease earlier than other diagnostic tests.

21 Sensitivity of Assays for Light Chains 1000 tein conc centratio on, mg/l SPEP CZE Normal range in serum sife UPEP uife M-prot 1 Normal range in urine sflc Adapted from Bradwell et al. Serum Free Light Chain Analysis. 3 rd ed The Binding Site, Ltd.

22 The Free Light Chain Test Serum test specific for free light chains More sensitive for detection of free light chains than SPE or IFE May indicate if therapy is working, sometimes earlier than SPE or IFE May detect relapse of disease earlier than other diagnostic tests.

23 Patient: sflc and IgG Levels During Treatment IgG κ Multiple Myeloma Kappa FLC Monoclonal IgG n, ncentratio /L sflc κ Co mg CVAMP HDM/PBSCT 0.1% Plasma cells Dex IgG, g/l Mo onoclonal Time from Presentation ti (days) Mead et al BJH :348

24 The Free Light Chain Test Serum test specific for free light chains More sensitive for detection of free light chains than SPE or IFE May indicate if therapy is working, sometimes earlier than SPE or IFE May detect relapse of disease earlier than other diagnostic tests.

25 Serum Free Light Chains: An Earlier Indication of Relapse Than IFE, mg/l 4-6-mo Earlier Indication of Relapse Mosbauer et al. Haematologica 2007:92:

26 Practical Considerations

27 Normal Ranges for Serum Free Light Chains Units (mg/l) Units (mg/dl) Kappa: mg/l Kappa: mg/dl Lambda: mg/l k/l ratio: Lambda: mg/dl k/l ratio:

28 Examples May - Lab A 385 mg/l 385 mg/l July Lab B

29 Laboratory Variability Absolute values in the serum free light chain assay can vary from lab to lab Different chemistry instruments Different chemistry instruments Different kits for different instruments Normal lab to lab variation

30 Laboratory Variability If you change the laboratory where your serum free light chains are measured: If possible, obtain some of the last sample and run it along with the new sample or Establish a new baseline for your serum free light chain levels. The change in your absolute sflc level should not effect the clinical interpretation.

31 International guidelines for sflc analysis in MM and related disorders Assessment of response Serial sflc should be routinely performed in AL amyloidosis Oligosecretory MM All MM patients to define a stringent CR Negative serum and urine IFE Absence of clonal cells in BM Normal sflc ratio Dispenzieri et al. Leukemia epub 20 Nov 2008

32 International guidelines for sflc analysis in MM and related disorders Screening Recommended in combination with serum electrophoresis sflc can replace 24 h urine IFE* *For screening for AL amyloidosis, 24 h uife still recommended Dispenzieri et al. Leukemia epub 20 Nov 2008

33 Interpretation of sflc Results: Updates to Guidelines sflc testing is considered abnormal when the clonal free light chain is increased above normal and the ratio is abnormal (Durie BG, Harousseau JL, Miguel JS, et al. International uniform response) Measurable disease with free light chain testing has been defined as a clonal free light chain concentration of at least 100 mg/l with an abnormal ratio (Durie BG, Harousseau JL, Miguel JS, et al. International uniform response)

34 International guidelines for sflc analysis in MM and related disorders Prognosis sflc should be measured at diagnosis for all patients with MGUS, SMM or MM, Solitary plasmacytoma and AL amyloidosis Dispenzieri et al. Leukemia epub 20 Nov 2008

35 Thank you! Questions?

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