1. Clinical interpretation of Serum Free Light Chain assays 22 Feb 2013 Dr. Eric Chan Consultant Immunologist Queen Mary Hospital Hong Kong

2. Figure 3.6. Diagrammatic representation of plasma cells producing intact immunoglobulins with monomeric κ and dimeric λ FLC molecules. (Serum Free Light Chain Analysis. AR Bradwell. 5th edition, 2008)

3. Clinical uses of serum free light chain (SFLC) assays Screening of diseases with monoclonal gammopathy  SPE (serum protein electrophoresis) + SFLC provide a simple and effective screen  Can replace SPE+UPE (urine protein electrophoresis)  UPE and serum IFE (immunofixation) can be ordered more selectively e.g. for amyloidosis and for typing of myeloma respectively Monitoring  Light chain multiple myeloma – potentially can replace 24-hour urine light chain quantitation  Intact immunoglobulin multiple myeloma – for light chain escape  Non-secretory or oligo-secretory multiple myeloma Prognosis  MGUS progression (monoclonal gammopathy with unknown significance)  IMWG guidelines: Smouldering MM progression plasmacytoma progression Myeloma outcome AL amyloidosis outcome B-CLL outcome Wald. Macro. outcome

4. 1. LYC (F/59) Breast carcinoma Skull lesion ? Metastasis, biopsy: plasmacytoma Bone marrow: plasma cell myeloma, Kappa restriction SPE, UPE: no monoclonal detected (NMD) IgG, A, M: immunosuppression SFK ↑, SFL ↓

5. Discussion Questions:  Mis-match between UPE and SFLC?  Light-chain Myeloma or Non-secretory Myeloma? High SFK levels may be due to polymerisation. This results in an over-estimation of the SFK level. Polymerisation would also cause a false negative result in UPE because the polymerised proteins are of different charges. By definition this is non-secretory myeloma. There has been no modification of the definition which is based on SPE/UPE/IFX. But this patient is more likely to have LCMM. 1

6. 2. CKY (M/58) June 2005 – diagnosed Lambda light chain myeloma  BM - markedly hypercellular marrow for age. Sheets of abnormal plasma cells are seen. Jan 2006 – post BMT BM: residual myeloma Collect Date : 29/06/05 02/07/05 03/07/06 30/07/06 14/08/06 ------------------------------------------------------------------------------------------------------------------------------ IgG 633 L -- 767 L 776 L 1030 819 – 1725 mg/dl IgA 61 L -- 133 111 136 70 - 386 mg/dl IgM 29 L -- 68 86 156 55 - 307 mg/dl SPE Weak MD -- NMD weak MD weak MD Serum IFX free L -- -- weak GK Total protein 66 -- -- 51.0 61.0 g/l % Paraprotein 3 -- -- 4.2 5.2 % Paraprotein 2 -- -- 2.1 3.2 g/l S. Free Kappa -- -- -- -- 21.90 H 3.30-19.40 mg/l S. Free Lambda -- -- -- -- 61.90 H 5.71-26.30 mg/l S. K/L Ratio -- -- -- -- 0.35 0.26-1.65 UPE -- MD -- -- -- Urine IFX -- FL -- -- -- Urine protein -- 0.47 -- -- -- <0.15 g/D Urine % paraprotein -- 21% -- -- -- Jul 2006 – weak IgG/K band -> oligoclonal reconstitution Discussion  Weak monoclonal or oligoclonal responses are not uncommon when the bone marrow regenerates after treatment by chemotherapy  Raised SFK and SFL, SFLC ratio normal  Renal function normal

7. Nov 2006 – relapse with  sheets of plasma cells in BM  But SFLC mildly elevated and UPE weak Trephine biopsy shows markedly hypercellular marrow diffusely infiltrated by abnormal plasma cells. Many show nuclear immaturity and prominent nucleoli. Little erythroid and myeloid activity are recognised. Megakaryocytes are not seen. Bony trabeculae are unremarkable. Reticulin fibres are moderately coarsened. 2

8. Discussion Discrepancy between bone marrow and serological findings (weak SPE or UPE bands, low SFLC levels, but marked immunosuppression) In general paraprotein levels reflect tumour load. The tumour cells of this patient is oligo-secretory 2

9. 21/06/0716/08/0707/01/0826/03/0925/06/0930/09/0912/04/1023/11/10 S Free Kappa13,60022.727.721.522.812.011.217.0 S Free Lambda3.211.232.024.824.318.318.017.6 S K/L Ratio421.532.030.87 0.940.660.620.97 urea6.7 3.57.56.75.67.66 creatinine219118113125145104110115 Treatment VADPBSCT BM exam pleomorphic plasma cells predominate no p'cytosis regenerating marrow mild p'cytosis Questions & Discussion: Reasons of ↑ SFK and SFL There is a mild degree of renal impairment. Hence both SFK and SFL are elevated. Other causes: polyclonal activation Normal ratio but still ↑SFK Although the ratio is normal the elevated SFK levels indicate there is still residual disease Continuously falling indicates complete remission 3. HWM (M/53)

10. "Serum free light chain measurement aids the diagnosis of myeloma in patients with severe renal failure" BMC Nephrology 2008;9:11 doi: 10.1186/1471-2369-9-112

11. 07/03/201105/09/201124/10/201126/04/201228/05/201212/10/201212/11/2012 S Free Kappa26.134.353.5182.082.0190.0435.0 S Free Lambda52.531.426.839.923.517.421.6 S K/L Ratio0.51.124.63.510.920.1 urea5.66.36.15.110.275.7 creatinine109111110111106117102 Treatment thal Len/dx BM exam no plasma- cytosis PET ++ no plasma- cytosis Discussion: ↑ S Free Kappa and S Free Lambda with normal ratio initially Similar explanations as before Continuously rising S Free Kappa indicates relapse Discrepancy between serum levels and bone marrow Bone marrow negative PET – extensive lytic lesions throughout the skeleton 3

12. 4. Light chain escape (TWF) Collect Date : 26/10/07 26/10/07 06/12/07 26/12/07 15/01/08 ------------------------------------------------------------------------------------------------------------------------------ IgG -- 4870 H 1580 853 880 819 - 1725 mg/dl IgA -- 40 L 22 L 27 L 27 L 70 - 386 mg/dl IgM -- 23 L 19 L 14 L 14 L 55 - 307 mg/dl B2M -- 2.56 H -- -- -- < 1.42 ug/ml SPE -- MD MD MD MD Serum IFX -- GL -- -- -- Total protein -- 100.0 68.0 61.0 68.0 g/l % Paraprotein -- 27.5 18.8 9.9 8.3 % Paraprotein -- 27.5 12.8 6.0 5.6 g/l UPE MD -- -- -- -- Urine IFX FL -- -- -- -- %Parapro.(Ur) 14.5 -- -- -- -- % 2007 – present as IgG myeloma 2008-2009 – partial remission

13. 4 Collect Date : 05/02/09 26/03/09 26/03/09 26/05/09 26/05/09 ---------------------------------------------------------------------------------------------------------------------------------------- IgG -- -- 1190 -- 944 819 - 1725 mg/dl IgA -- -- 89 -- 54 L 70 - 386 mg/dl IgM -- -- 97 -- 59 55 - 307 mg/dl SPE -- -- WMD -- NMD % Paraprotein -- -- WQ -- -- % UPE NMD NMD -- MD -- %Parapro.(Ur) -- -- -- 44.1 -- % S. Free Kappa -- -- 0.64 -- 7.44 3.30-19.40 mg/L S. Free Lambda -- -- 127H -- 523H 5.71-26.30 mg/L S. Free K/L Ratio -- -- 0.005 -- 0.014 retrospective assay 5/2009: Clinical deterioration

14. 4 Collect Date : 12/08/09 20/08/09 28/08/09 28/09/09 27/10/09 ------------------------------------------------------------------------------------------------------------------------- IgG -- -- 646 L 521 L 394 L 819 - 1725 mg/dl IgA -- -- 24 L 18 L 10 L 70 - 386 mg/dl IgM -- -- 28 L 21 L 15 L 55 - 307 mg/dl SPE -- -- MD MD MD Total protein -- -- 79.0 68.0 64.0 g/l % Paraprotein -- -- 3.0 3.0 2.1 % Paraprotein -- -- 2.4 2.0 1.3 g/l UPE MD -- -- -- -- %Parapro.(Ur) 88.2 -- -- -- -- % S. Free Kappa -- -- -- 6.86 -- 3.30-19.40 mg/L S. Free Lambda -- -- -- 5790 -- 5.71-26.30 mg/L S. K/L Ratio -- -- -- 0.0012 -- IgG paraprotein levels stable UPE & SFL increasing

15. 4 Collect Date : 05/07/10 05/07/10 05/07/10 19/07/10 23/07/10 ------------------------------------------------------------------------------------------------------------------------------ IgG -- 267 L -- 445 L 448 L 819 - 1725 mg/dl IgA -- 14 L -- 35 L 37 L 70 - 386 mg/dl IgM -- 8 L -- 23 L 21 L 55 - 307 mg/dl S. Free Kappa -- -- <1.07 -- -- 3.30-19.40 mg/L S. Free Lambda -- -- >3000.00 H -- -- 5.71-26.30 mg/L S. K/L Ratio -- -- <0.0004 -- -- SPE -- WMD -- WMD WMD Total protein -- 54.0 -- 67.0 61.0 g/l % Paraprotein -- 1.2 -- 2.4 2.7 % Paraprotein -- 0.6 -- 1.6 1.6 g/l UPE MD -- -- -- -- %Parapro.(Ur) 69.0 -- -- -- -- % SFL continuously ↑ → death (2010)

16. http://www.bindingsite.com/lightchainescape