International consensus on serum free light chain analysis Alex Legg PhD Scientific Affairs Manager The Binding Site Distributor in Poland BIOKOM beata.olsz@biokom.com.pl

Serum free light chain immunoassay Heavy chain Kappa Light chain Exposed surface Hidden surface Lambda The serum FLC assay is a latex-enhanced immunoassay that uses sheep polyclonal antisera specific for the epitopes shown here in yellow. These are conformational epitopes found on the constant domain of the light chain. These epitopes are hidden in the intact immunoglobulin molecule but exposed in unbound light chains. Therefore, the serum FLC assay is specific for kappa or lambda free light chains. 2

Serum free light chain immunoassay Heavy chain Kappa Light chain Exposed surface Hidden surface Lambda The serum FLC assay is a latex-enhanced immunoassay that uses sheep polyclonal antisera specific for the epitopes shown here in yellow. These are conformational epitopes found on the constant domain of the light chain. These epitopes are hidden in the intact immunoglobulin molecule but exposed in unbound light chains. Therefore, the serum FLC assay is specific for kappa or lambda free light chains. 3

Sensitivity of light chain analysis techniques 1000 SPE CZE 100 sIFE Light chain concentration (mg/L) 10 UPE Normal range in serum uIFE This slide shows the relative analytical sensitivities of techniques used to detect light chains. In green, the serum based techniques of serum protein electrophoresis, capillary zone electrophoresis and immunofixation are at best sensitive down to 100 mg/ml. In contrast. the serum free light chain assay is the only assay able to measure through the normal range . It is also a quantitative assay that can be used for monitoring. In yellow, urine protein electrophoresis and urine immunofixation are more sensitive than their equivalent serum electrophoresis counterparts,. However, serum and urine immunofixation are only quantitative and the concentration of light chains in urine does not necessarily reflect their serum concentration, due to renal metabolism. 1 sFLC

Renal Metabolism of FLC reabsorption & breakdown FLC - 25 kDa FLC - 50 kDa Glomerulus 40-60 kDa pores 10-30g/day reabsorption & breakdown This slide show a cartoon of a nephron. Free light chains are relatively small proteins,. The molecular weight of monomeric kappa is 25 kDa and the dimeric lambda is 50 kDa. Light chains can pass relatively easily through the glomerulus and into the proximal tubule where they are absorbed and metabolised. In a healthy individual, ~ half a gram of light chain is produced per day. As the kidney has a capacity to reabsorb 10 – 30 gram protein per day, very little light chain enters the urine. In a patient producing low quantities of monoclonal FLC, the renal reabsorption capacity may not be exceeded and urinary FLC may not be detected. Urine

Renal Metabolism of FLC reabsorption & breakdown FLC - 25 kDa FLC - 50 kDa Glomerulus 40-60 kDa pores Renal metabolism results in sFLC half-life of 2-6 hours This is in contrast to intact immunoglobulins which have half-lives of 5-21 days 10-30g/day reabsorption & breakdown As a result of this renal metabolism, the half life of free light chains in serum is quite short – around 2-6 hours. This contrasts with the 5-21 day half-lives of intact immunoglobulins. Urine

Light Chain Multiple Myeloma Normal sera Kappa LCMM Lambda LCMM Renal impairment (CKD2) Hutchison, unpublished data; Bradwell, Lancet 2003; 361: 489-491

LCMM – Renal Threshold Patient 2 Patient 1 Patient 3 Patient 4 sFLC Thalidomide HDC ASCT Patient 1 Patient 2 Severe osteolytic lesions HDC ASCT Patient 3 Patient 4 sFLC 24 hr urine BJP excretion Alyanakian Am J. Hematol. 2004; 75: 246 – 248

Nonsecretory Multiple Myeloma 10000 ABCM VAD HDM/ PBSCT 1000 Kappa sFLC (mg/L) 100 NR 10 Here is an example of a NSMM patient that has been monitored using sFLC results. This young patient initially presented with a fractured rib following a mild trauma. However, due to the non-specific symptoms and normal biochemistry tests, the initial diagnosis of NSMM was delayed. Seven months later bone scans and x-rays showed extensive osteolytic lesions, she had hypercalcaemia but electrophoresis showed immuneparesis but no monoclonal Ig. A biopsy was performed, leading finally to the diagnosis of NSMM. The patient received ABCM (adriamycin, busulphan, cyclophosphamide, melphalan) chemotherapy which resulted in clinical remission. Seven months later the patient reattended hospital because of increasing symptoms but routine biochemistry/ haematology tests showed no abnormality or M protein spike. sFLC test was now requested and showed a ratio of 51, with a greatly raised kappa of 330mg/L. The sFLC result was questioned and was requested again 2 and 3 weeks later, showing a steady increase in kappa to 470 and 525mg/L, confirming reoccurence of the disease. It was now that previous serum samples were analyses retrospectively, and results correlated strongly with disease diagnosis and remission. From then on the patient was monitored prospectively and used to follow response to new treatments. This patient highlights the difficulties of diagnosing and monitoring these patients and how sFLC tests can provide clinically important information. 10 20 30 40 50 60 70 80 90 100 Number of weeks since presentation

Intact Immunoglobulin MM No correlation between Ig’ and sFLC n = 120 IgGl MM patients Serum l FLC (mg/L) Total IgG (g/L) Mead et al. Br J Haematol 2004;126 : 348 – 54

Rapid evaluation of response to chemotherapy Dispenzieri et al. Retrospective analysis of ECOG trial E9486 VBMCP  IFN or  cyclophosphamide 399 patients Assessed sFLC and M-protein responses to therapy “FLC response after 2 months of therapy was superior to early M-protein measurement to predict overall response.” ECOG = Eastern Cooperative Oncology Group Trial = VBMCP  IFN or  high dose cyclophosphamide VBMCP = vincristine, carmustine [BCNU], melphalan, cyclophosphamide, and prednisone Dispenzieri et al. Blood 2008; 111: 4908 - 4915

Light chain escape “Rising monoclonal free light chain production at relapse without increased monoclonal intact immunoglobulin” Drayson et al. Myeloma IX trial Estimate incidence: 5% for IgG MM 15% for IgA MM First observed in the 1960s First coined by Prof JR Hobbs who studied MRC I trial data. Back then, urine electrophoresis was used to monitor patients for LCE. Now we can use the sFLC assay…. Drayson, M.T., et al., Clin Lymphoma Myeloma, 2009. February: 346a.

IgA (g/L), creatinine (mg/dL) 6xMP PD 7xMP No therapy HD-Dex 800 20 HD-Mel Tx 18 700 16 600 14 500 12 sFLC (mg/L) IgAλ paraprotein Serum lambda FLC Creatinine IgA (g/L), creatinine (mg/dL) 400 10 8 300 6 200 4 Patient diagnosed with an IgA lambda IIMM 100 2 BM infiltration rate 100% 9/01 10/02 4/03 10/03 4/04 12/04 1/05 2/05 Date Kϋhnemund, A., et al. J Cancer Res Clin Oncol, 2009. 135(3): p. 477-84

Dual plasma cell subsets Stain with: anti- IgG Anti-Kappa anti-BrDU Plasma cell Populations: IgG  FLC Looked at approx. 100 patients 18% demonstrated dual subsets For example we have a patient with an IgGk myeloma. Where the cytoplasm stained green – indicating IgG Heavy chain production Where the cytoplasm stained red – indicated kappa FLC production Cells that are producing IgG and K stain with both antibodies and appear yellow Cells that produce only kappa stain red. Green nuclear staining shows cells that are DNA replication. (S-phase) Ayliffe Haematologica 2007; 92: 1135 - 1138

Model of light chain escape IgA (g/L) Serum kappa FLC (mg/L) This data can be used to explain light chain escape: At the beginning there may be two populations of plasma cells present producing intact immunoglobulin and light chain only. Both populations decrease in response to chemotherapy. On relapse there is a dramatic increase in the number of LC only plasma cells but not intact immunoglobulin plasma cells.. So the concentration of LC increases whilst intact Ig remains constant. Time

International guidelines for sFLC analysis in MM and related disorders Assessment of response Serial sFLC should be routinely performed in: Oligosecretory MM/ NSMM AL amyloidosis LCDD (personal experience of authors) * * Periodic urine or sFLC assessment for LCE Dispenzieri et al Leukemia (2009) 23, 215–224

sFLC identify residual disease Normal sera Serum λ FLC (mg/L) IFE negative MM Normal / Abnormal / Serum κ FLC (mg/L)

International guidelines for sFLC analysis in MM and related disorders Assessment of response All MM patients to define a stringent CR CR Stringent CR Negative S/U IFE BM plasma cells ≤ 5% Normal sFLC ratio Absence of clonal cells in BM Dispenzieri et al. Leukemia 2009: 23, 215–224 Durie et al., Leukemia, 2006. 20, 1467-73

Screening for Monoclonal Gammopathies Serum electrophoresis → Monoclonal intact immunoglobulins Urine electrophoresis → Monoclonal free light chains Screening for Monoclonal Gammopathies In terms of screening, if a monoclonal gammopathy is suspected, it is traditional to perform both serum and urine electrophoresis for the detection of any abnormal paraproteins. The serum electrophoresis is required for the detection of monoclonal intact immunoglobulins and urine for the detection of monoclonal free light chains. As I have already shown, in the published literature, the serum FLC has a higher sensitivity for monoclonal free light chains than urine electrophoresis. Therefore, do you still need a 24 hour urine sample when screening for monoclonal gammopathies or should you use a serum FLC test instead? Can the serum FLC assay replace urine electrophoresis? 19 19

Screening for monoclonal gammopathy sFLC + CZE 9 additional B-cell disorders identified from 1003 consecutive unknown samples Bakshi et al. Am J Clin Path 2005; 124: 214-218 Adding the serum FLC assay to screening protocol increased tumour detection rate by 56% sFLC + SPE 8 additional monoclonal gammopathies identified from 923 unknown samples Hill et al. Clin Chem 2006; 52: 1743-1748 Slide 10 - Screening Adding sFLC to screening protocol increased tumour detection rate by 56% The addition of the serum free light chain assay to electrophoretic techniques (either capillary zone electrophoresis (CZE) or serum electrophoresis (SPE)) in screening protocols for monoclonal gammopathies has been the subject of several trials – electrophoresis must be retained in order to identify monoclonal protein in the 4% of intact immunoglobulin myeloma patients with no monoclonal FLC production. Data from the 3 studies listed on this slide demonstrated increased numbers of B-cell disorders detected following addition of the serum free light chain assay to either CZE or SPE in primary screens. These additional disorders would have been missed if the electrophoresis technique had been used alone. The third of these trials also examined the possibility of replacing urine Bence Jones protein analysis with the serum free light chain assay in primary screening for monoclonal gammopathies. The authors concluded that “As no substantial pathology would have been missed by replacing urine Bence Jones proteins with serum free light chains we no longer require a urine sample as part of the initial screen.” The hospital that performed the study, the Derbyshire Royal Infirmary in the UK, has not tested urine as part of their primary screen since May 2005.

Replacement of urine tests with sFLC 428 urine samples positive by uIFE On paired serum samples: Serum electrophoresis + sFLC missed only 2 urine positive light chain MGUS patients No significant pathology was missed Numerous studies have looked at this question and the largest study to date was by Katzmann and colleagues at the Mayo clinic. Out of 428 samples all positive by urine immunofixation, a combination of serum electrophoresis and a serum FLC test on the paired serum sample only missed two urine positive light chain MGUS patients. MGUS is a benign disease and so does not required any treatment. Therefore, the Mayo clinic concluded that a serum FLC assay can be used as a direct replacement to urine when screening for diseases. Katzmann et al. Mayo Clin Proc 2006; 81: 1575 - 1578 21

International guidelines for sFLC analysis in MM and related disorders Screening Recommended in combination with serum electrophoresis sFLC can replace 24 h urine IFE* International guidelines for sFLC analysis in MM and related disorders *For AL screening, 24 h urine IFE still recommended Dispenzieri et al. Leukemia 2009: 23, 215–224

Serum FLCs are prognostic in: MGUS progression Myeloma outcome Smouldering MM progression Plasmacytoma progression AL amyloidosis outcome B-CLL outcome Waldenstrom’s outcome Serum FLCs are prognostic in:

? MM MGUS WM AL amyloid IgM lymphoma Risk of progression 1% per year LCDD WM CLL ? Solitary plasmacytoma Follow up MGUS patients: How frequently?

Monoclonal Gammopathy of Undetermined Significance (MGUS) Risk Factors for progression: Serum M protein >15g/L Serum M protein NOT IgG Monoclonal gammopathy of undetermined significance – or MGUS – is found in ~2% of the population over the age of 50 and carries with it a 1% per year risk of progression to myeloma or a related disorder, MGUS accounts for the majority of patients diagnosed with a monoclonal protein and patients followed up annually. A long term prospective study carried out by the Mayo clinic following up over 1,300 inviduals for up to 35 years with MGUS identified……….M protein size and type as two independent risk factors for progression of MGUS. Kyle R. NEJM 2002; 346: 564-569

Monoclonal Gammopathy of Undetermined Significance (MGUS) Risk Factors for progression: Serum M protein >15g/L Serum M protein NOT IgG sFLC ratio In 2005 the study was extended with retrospective analysis of sFLC in the presentation sample. The sFLC ratio was found to be an additional independent risk factor for progression of MGUS. Kyle R. NEJM 2002; 346: 564-569 Rajkumar Blood 2005; 106: 812-817

MGUS progression Abnormal FLC ratio (/ = <0.26 or >1.65) Years Cumulative probability of progression (%) 0 10 20 30 40 50 60 0 5 10 15 20 25 30 Normal FLC ratio (/ = 0.26 – 1.65) Abnormal FLC ratio (/ = <0.26 or >1.65) Rajkumar Blood 2005; 106: 812-817

No. of abnormal risk factors Absolute risk of progression at 20 years* MGUS risk stratification model incorporating M-protein size, type and FLC ratio Risk of progression No. of abnormal risk factors No. patients Absolute risk of progression at 20 years* Low 449 2% Low-Intermediate 1 420 10% High-Intermediate 2 226 18% High 3 53 27% The Mayo have now proposed this risk stratification model that incorporates M-protein size, type and sFLC ratio…. Walk people through the slide! This useful table demonstrates that patients with all 3 risk factors have a much higher risk of progression to MM than those patients with no or only 1 risk factor. The patients that can be categorised as low risk was around 40%. This study was of significant importance for clinicians to aid in the management of patients with MGUS. These findings suggest that the well documented 1% annual average risk of MM progression among MGUS patients is highly heterogeneous and emphasizes the fact that the risk of developing MM varies greatly among individuals diagnosed with MGUS * Accounting for death as a competing risk Rajkumar Blood 2005; 106: 812-817

FLC-MGUS may evolve into LCMM -16 - 8 Years prior to diagnosis of MM No M-protein by sIFE, SPEP or sFLC sFLC positive sFLC and sIFE positive Weiss et al. , Blood ePub Feb 20, 2009

IgA (g/L), creatinine (mg/dL) IgA paraprotein Serum kappa FLC Creatinine PD DTC No therapy 5 VAD 20 HD-Mel 4.5 18 Tx 4 HD-Mel 16 Tx 3.5 14 3 12 IgA (g/L), creatinine (mg/dL) sFLC (g/L) 2.5 10 2 8 1.5 6 1 4 20% 0.5 2 IgA kappa patient Actually diagnosed 2001 as MGUS AND had stable IgA levels In 12 2004, patient went into unexpected acute renal failure – this was correlated with high K FLC levels and 60% abnormal BM plasma cells Patient started on thalidomide induction therapy, 60% BM infiltration rate 4/04 7/04 10/04 12/04 1/05 2/05 3/05 7/05 Date Kϋhnemund, A., et al. J Cancer Res Clin Oncol, 2009. 135(3): p. 477-84

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 2009: 23, 215–224

Summary of IMWG sFLC Guidelines Intact Ig MM Light chain Non secretory Screening + serum electrophoresis Prognosis Monitoring (Oligosecretory) (LCE) (sCR) In conclusion, the serum free light chain test provides valuable information in numerous monoclonal gammopathies. For patients with myeloma, the guidelines recommend screening with a serum only panel of serum protein electrophoresis and a serum FLC test. The only exception to this rule is AL amyloidosis, where urine immunofixation should be performed alongside the serum panel. There are 2 significant advantages for this screening panel. Firstly, if a patient is subsequently diagnosed as MGUS, you already know the M protein size, type and the serum FLC ratio for risk stratification and subsequent follow up. Secondly, the serum FLC test provides valuable baseline prognostic information in all these diseases and helps identify patients with more aggressive disease. Finally, the assay is important for monitoring numerous diseases. For patients with intact immunoglobulin and light chain multiple myeloma, the assay must be used to monitor the patients without a quantifiable M protein – the so called oligosecretors. It must also be used to define a stringent complete response once the patients are immunofixation negative. Finally, the intact myeloma patients should be monitored for the optimal detection of light chain escape, which can occur at any time point of the disease. Finally, the serum FLC test is essential for monitoring all non secretory multiple myeloma and AL amyloidosis patients. Dispenzieri, A., et al. Leukemia, 2009. 23(2): p. 215-24 Kyle, R.A. and S.V. Rajkumar. Leukemia, 2009. 23(1): p. 3-9

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Serum free light chain immunoassay platforms BECKMAN COULTER IMMAGE, IMMAGE 800 BINDING SITE SPAPLUS OLYMPUS AU400 / 640 / 2700 / 5400 ROCHE MODULAR P Cobas Integra 400 / 800 Cobas c501 ROCHE (Hitachi) 911, 912 , 917 SIEMENS (Bayer) ADVIA 1650 / 1800 / 2400 SIEMENS (Dade Behring) BNII, Prospec

MGUS progression       Relative Risk of Progression Normal    0.5 1.0 5.0 10.0 50.0 500.0 Relative Risk of Progression …..the risk of progression was increased with more abnormal ratios. In this graph the central line represents the relative risk of progression. And the outer lines represent 95% confidence intervals 0.01 0.10 0.26 1.0 1.65 10.00 100.00 Free Light Chain Ratio Rajkumar Blood 2005: 106: 812-817

Multiple myeloma survival Years Overall survival % 20 40 60 80 100 2 4 6 8 10 12 14 risk factors* med. survival 51 m 39 m 30 m 22 m 1 3 In multiple myeloma, there is the international staging system that uses albumin and Beta 2 microglobulin as 2 risk factors for reduced survival. In this snozek study, patients with a highly abnormal serum free light chain ratio also demonstrated reduced survival and this was independent of the international staging system. As shown in this graph, Snozek and colleagues have incorporated these three independent risk factors of albumin, B2M and the free light chain ratio. Patients with no risk factors follow this top line and have a median survival of over 4 years. Patients with all 3 risk factors follow the bottom line and their survival is under 2 years. * sFLC / <0.03 or >32, albumin < 35 g/L, 2MG ≥ 3.5 mg/L Snozek, C.L., et al., Leukemia, 2008. 22:1933-7

Risk stratification summary: Risk Factors No. Serum FLC ratio Other risk factors MGUS 3 <0.26 or >1.65 M-protein size M-protein type SPB 2 SMM <0.125 or >8 % BM plasma cells MM <0.03 or >32 Albumin 2-microglobulin

Renal Metabolism of FLC reabsorption & breakdown FLC - 25 kDa FLC - 50 kDa Glomerulus 40-60 kDa pores 10-30g/day reabsorption & breakdown This slide show a cartoon of a nephron. Free light chains are relatively small proteins,. The molecular weight of monomeric kappa is 25 kDa and the dimeric lambda is 50 kDa. Light chains can pass relatively easily through the glomerulus and into the proximal tubule where they are absorbed and metabolised. In a healthy individual, ~ half a gram of light chain is produced per day. As the kidney has a capacity to reabsorb 10 – 30 gram protein per day, very little light chain enters the urine. In a patient producing low quantities of monoclonal FLC, the renal reabsorption capacity may not be exceeded and urinary FLC may not be detected. Urine

Light chain escape “Rising monoclonal free light chain production at relapse without increased monoclonal intact immunoglobulin” Kühnemund et al: Largest case series of LCE Estimate incidence is 2.5% MM at relapse 5/10 cases of LCE had renal impairment First observed in the 1960s First coined by Prof JR Hobbs who studied MRC I trial data. Back then, urine electrophoresis was used to monitor patients for LCE. Now we can use the sFLC assay…. Kuhnemund, A., et al., J Cancer Res Clin Oncol, 2009. 135, 477-84

Light chain escape Courtesy of Effie Liakopoulou, Christies Hospital IgA paraprotein Serum lambda FLC Velcade Patient with IgAL paraprotein in response to velcade, IgA concentrations fall – shown by green line. Lambda FLC concentrations also fall. Several months after velcade treatment the IgA paraprotein concentrations remain stable but the concentration of lambda FLC is dramatically rising, indicating LC escape. Courtesy of Effie Liakopoulou, Christies Hospital

Prevalence of MGUS 2 4 6 8 10 50 60 70 80 90 Prevalence of MGUS (%) 4 6 8 10 50 60 70 80 90 Prevalence of MGUS (%) Age (years) Kyle NEJM 2006, 354: 1362 – 1369