1. Director of Scientific Affairs
Understanding Serum Free Light Chain (Freelite®) and Heavy Chain-Light Chain (Hevylite®) Tests
Judith A. Finlay Ph.D.
Director of Scientific Affairs
The Binding Site, Inc.
San Diego, CA
Some of the subject matter and data contained or addressed in the following presentation may involve off-label or forward-looking use of Freelite® kits, Hevylite® kits and other Binding Site products. As such, no off-label use of Freelite, Hevylite or other Binding Site products can be supported by Binding Site Inc.

2. Outline Background Tests used to Assess Multiple Myeloma
Advantages of serum Free Light Chain Tests (Freelite®)
Practical Considerations
New Hevylite® tests
Summary

3. Multiple Myeloma: a cancer of the Plasma Cells
Cancerous plasma cells accumulate in the bone marrow and cause symptoms:
Increased Infections
Kidney Damage
Anemia
Bone Damage
Use Testing to monitor disease and guide therapy

4. Plasma Cells are in the Bone Marrow
Red Blood
Cells
White Blood
Cells (B
lymphocytes/
Plasma cells)
Soft blood-forming tissue that fills the cavities of bone
Platelets
The soft blood-forming tissue that fills the cavities of bones and contains fat and immature and mature blood cells, including white blood cells, red blood cells, and platelets. Diseases or drugs that affect the bone marrow can affect the total counts of these cells.

5. Plasma Cells Produce Antibodies and Light Chains
Kappa
Lambda
Plasma cells produce twice as many kappa as lambda light chains, but the median K/L ratio is 0.6 (meaning more lambda than kappa in the serum). This seeming paradox is explained by the slower clearance of lambda from the serum, such that its half life is somewhat longer and hence it has higher concentrations.

6. Antibodies are Made up of Heavy Chains and Light Chains

7. Normal vs Myeloma Lots of different types of whole antibodies Normal
IgG κ
IgG L
IgA κ
IgA L
IgD κ
IgD L
IgE κ
IgE L
IgM κ
IgM L
IgG κ
IgG L
IgA κ
IgA L
IgD κ
IgD L
IgE κ
IgE L
IgM κ
IgM L
Lots of different types of whole antibodies
Normal
IgG κ
Intact Ig Myeloma
IgG κ
Whole Ab and excess light chains – and too many of both!

8. FEWER MYELOMA CELLS = FEWER ANTIBODIES AND LIGHT CHAINS
Myeloma Therapy
THERAPY
KILLS MYELOMA CELLS
FEWER MYELOMA CELLS = FEWER ANTIBODIES AND LIGHT CHAINS 8

9. There Are Two Types Of Free Light Chains – Kappa And Lambda
FL measure both types
Kappa Free
Light Chains
Lambda Free
Light Chains
The FreeLite Test Measures Free Light Chains

10. Advantages of Freelite Tests
Serum test specific for free light chains
Very sensitive for detection of free light chains
Early detection of response to treatment
Earlier detection of relapse

11. Tests Measure Antibodies and Light Chains
Measured by SPEP and IFE
Measured by Serum free light chain test = Freelite
Plasma Cell
Free Light Chains

12. Advantages of Freelite Tests
Serum test specific for free light chains
Very sensitive for detection of free light chains
Early detection of response to treatment
Earlier detection of relapse

13. Normal Ranges for the Freelite tests
Units (mg/L)
Kappa:
3.3–19.4 mg/L
Lambda:
5.7–26.3 mg/L
k/l ratio:
0.26–1.65
Units (mg/dL)
Kappa:
0.33–1.94 mg/dL
Lambda:
0.57–2.63 mg/dL
k/l ratio:
0.26–1.65

14. Types of monoclonal proteins

15. What type of MM do you have?
Type of MM depends on type of abnormal protein found in blood or urine
IgG kappa, IgG lambda, IgA kappa, IgA lambda
Light chain only (kappa or lambda)
Non-secretory (low to no protein)
Involved light chain: one that is elevated:
E.g. IgG kappa, it is kappa
Determined by a combination of laboratory tests:
SPE
sIFE
Freelite

16. Why is your MM protein type important?
If you have high levels of light chains you may be more prone to kidney damage
If you are an IgA MM you will benefit most from the Hevylite test
You will be able to tell if your MM protein type changes
E. g. Light chain escape can be detected

17. Advantages of the Freelite Tests
Serum test specific for free light chains
Very sensitive for detection of free light chains
Early detection of response to treatment
Earlier detection of relapse

18. Freelite Detects Response Earlier than SPEP
Patient with IgG Kappa Multiple Myeloma
Adapted from: Mead et al. BJH :348

19. Advantages of the Freelite Tests
Serum test specific for free light chains
Very sensitive for detection of free light chains
Early detection of response to treatment
Earlier detection of relapse

20. Freelite detects relapse earlier than IFE+ + + + + + _ - - -
IFE is either positive (+) or negative (-). 15 17 19 21 23 25 27 29
Relapse: FLC levels start increasing 4 months before IFE becomes positive.
Adapted from: Mosbaurer et al. Haematologica 2007:92:275

21. Monitoring Therapy with Serum Free Light Chains

22. Advantages of the Freelite Tests
Serum test specific for free light chains
Very sensitive for detection of free light chains
Early detection of response to treatment
Earlier detection of relapse

23. Lab Reports

24. Units are important!
May
= 385 mg/L
July

25. Practical Considerations for the Measurement of Serum Free Light Chains
Laboratory variability
Day to day variation in a lab
Normal lab to lab variation
Different chemistry instruments
(Different kits for different instruments)
Biologic variability
Clinical changes are much greater

26. Changing Laboratories
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
May note of units lab reports (mg/L or mg/dL)
The change in your absolute sFLC level should not effect the clinical interpretation

27. Total Light Chain Assay
“The total light chain assays are no longer used for diagnosis. Only FLC measurements have relevance” David Keren ASCP Press 2012

28. Binding Site’s New Hevylite™ Tests
Cleared by FDA
In studies in the USA
Early in tests’ development
We expect these tests to add value to Freelite
We expect these tests to be especially helpful in IgA MM patients
Durie and IMF has mentioned Hevylite
2013 Publications: Mayo Clinic, Austria, TBS-UK
Myeloma Canada summary

29. When you measure IgA
You are measuring
IgAk +IgAl
(IgAk +IgAl)

30. IgG, A and M assays measure IgGk and IgGl, etc., together
IgAk
IgAL
IgMk
IgML
Now IgAk and IgAl can be measured independently with Hevylite®
Why is this important?

31. What is HevyLite? IgAk v. IgAl IgGk v. IgGl IgMk v. IgMl
Hevylite at test that measures the heavy and light chains together
So what?
Can distinguish:
IgAk v. IgAl
IgGk v. IgGl
IgMk v. IgMl
Hevylite antibodies were designed to recognize conformational epitopes that occur when a specific heavy chain (e.g. gamma) pairs with a specific light chain (e.g. kappa). There are probably multiple unique epitopes formed by any individual heavy/light chain pairing. Most of the epitopes would be formed between the CH1 region of the heavy chain and the CL region of the light chain. However, conserved sequences in the VH and VL regions of immunoglobulin could also form epitopes recognized by Hevylite reagents. Since there has been no epitope mapping done (nor is there likely to be) we should stay away from any descriptions of how many residues on each chain are recognized.

32. Additional Resources www.wikilite.com (web version of our “red book”)
Google “Binding Site” us
Call The Binding site
We have CESs & DSAs across the US and Europe
Understanding Serum Free Light Chain Assays (IMF booklet)
IMF website (myeloma.org)

33. Summary Multiple Myeloma Plasma cells produce Measure with lab tests
cancer of the plasma cells
found in the bone marrow
Plasma cells produce
Antibodies
free light chains
Measure with lab tests
Including free light chain test (Freelite)
To monitor therapy
Fewer light chains means fewer plasma cells
New test for MM: Hevylite