ABC of laboratory techniques for diagnosis and follow-up of monoclonal gammopathies. An Hendrickx MSc. Scientific Advisor

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1 ABC of laboratory techniques for diagnosis and follow-up of monoclonal gammopathies An Hendrickx MSc. Scientific Advisor

2 In this talk introduction biology of free light chains laboratorium investigation of monoclonal proteins SPE (serum, urine) IFE (serum, urine) serum free light chains (Freelite ) guidelines for monitoring

3 Monoclonal gammopathy

4 Monoclonal gammopathies λ κ MGUS SMM Multiple myeloma AL amyloidosis Intact immunoglobulin Light chain Non-secretory 80% 15 20% 1 2%

5 Clonal plasma cells in the bone marrow (in aspirate/biopsy) > 10% and at least one of the following: Myeloma defining events Related end organ damage Any one or more biomarkers of malignancy iflc must be 100mg/L; values based on Freelite assay Details HyperCalcaemia Renal insufficiency Anaemia Bone lesions Clonal bone marrow plasma cell percentage 60% Involved/uninvolved serum FLC ratio 100 >1 focal lesion on MRI studies Rajkumar et al. Lancet Oncology 2014;15:e538-e548

6 Laboratory investigation for monoclonal proteins associated with multiple myeloma g/l Intact Ig MM Serum SPE + electrophoresis Serum Freelite mg/l Light Chain MM Urine Serum Freelite electrophoresis

7 Traditional tools for detecting M-proteins Serum protein electrophoresis (SPE) Monoclonal protein in γ region Immunofixation electrophoresis (IFE) IgGl monoclonal protein

8 Traditional tools for detecting M-proteins: SPE -1 anti-trypsine -2 glycoprotéine -2 macroglobuline haptoglobuline hémopexine, transferrine, -lipoprotéine, complément C3 IgA IgM IgG + Alb Total proteins: g/l Albumine % g/l -1 glob. 1,5 4 % 1 3 g/l -2 glob % 5 8 g/l -glob % 4 10 g/l -glob % 5 12 g/l

9 A few examples

10 Disease patterns Control Nephrotic pattern Alb 2 macroglobulin Liver disease Alb + broad peak due to bilirubin binding fusion/bridging ( polyclonal IgA) globulins ( polyclonal IgG)

11 Hypergammaglobulinemia : Ex : Polyclonal increase in serum Ig T G A M l T G A M l Ex : monoclonal Ig, IgG lambda

12 Others: Hyper 2- -glob. Hypo -glob. a) monoclonal IgA moved to the 2- -glob. fraction T G A M l T G A M l b) Normal SPE, but monoclonal IgA moved to -glob. fraction

13 double peak 1 2 a) Monoclonal IgM and IgM l T G A M l Hyper -glob. T G A M l b) Oligoclonal aspect of IgG et IgM

14 Special cases: Peak between and - glob. 1 2 T G A M l Normal Immunofixation think of fibrinogen Treat the serum with thrombase and redo the SPE Redo IFE using an anti-fibrinogen

15 Presence of a cryoglobulin or a polymerized Ig? Anomaly T G A M l T G A M l Redo the immunofixation after warming the serum at 37 C Redo the immunofixation after depolymerization by a reducing agent (mercapto-ethanol)

16 Conclusion

17 Quantification of a monoclonal Ig Integration of the peak at the SPE: yes, but... Nephelometric assay (IgG, IgA, IgM) or IgE and IgD (radial immunodiffusion = RID) Nephelometric measurement of free light chains (FLC)

18 Freelite immunoassay: polyclonal antisera to monoclonal proteins Heavy chain FLC Light chain Hidden surface l FLC Exposed surface

19 Freelite immunoassay: polyclonal antisera to monoclonal proteins FLC FLC l FLC mg/l mg/l Exposed surface /l sflc ratio Range = Median = 0.6 l FLC Katzmann Clin Chem 2002;48:

20 Why a polyclonal assay to detect FLC?

21 Light chains are polymorphic Polymorphism due to light chain genetics: Recombination of gene segments Isotypic variation Allotypic variation

22 Light chain structure Variable domain Constant domain Light chain proteins are encoded by 3 types of gene segment: V J and C

23 Light chain structure Variable domain Constant domain Amino acids that contribute to Freelite epitopes directly indirectly via 3D protein structure

24 Light chain structure V J 1-5 C 1 Vl 1-30 Jl 1-4; Cl 1-4 V, J and C arrangements confer high sequence diversity V J C FLC ~ 1500 l FLC ~ 500 V L C L

25 Light chain variability Idiotype Isotype Allotype V L C L V L C L V L C L V L C L V L C L V L C L Multiple V and Vl genes + subtypes (V I-IV; Vl I-VI) Cl - Mcg, Kern Mcp & Oz serotypes C - Km1, Km2 or Km3 serotypes

26 Post translational modification Modification of protein following translation Attachment of functional biochemical groups Phosphate Acetate Lipids Carbohydrates Immunoglobulin constant regions are glycosylated

27 A free light chain assay must recognise this variation

28 Polyclonal or monoclonal antibodies for a FLC immunoassay?

29 Binding Site polyclonal technology Tolerise Immunise Polyclonal sheep antisera thousands of unique clones Enormous variety of epitopes recognised

30 Binding Site polyclonal technology Tolerise Immunise Specific polyclonal antisera Affinity and adsorption chromatography

31 Freelite Polyclonal antisera Multiple epitopes recognised Variable domain Constant domain

32 Monoclonal antibody Immunise Monoclonal mouse antibody Single epitope recognised

33 Monoclonal antibody Single epitope recognised Variable domain Constant domain Only monoclonal FLC containing this specific epitope will be detected Pathological patient monoclonal FLC are highly variable The specific epitope recognised by the monoclonal antisera may be absent

34 Monoclonal or Polyclonal Abs Binding Site Monoclonal Polyclonal Epitope(s) recognised Single epitope Multiple epitopes Specificity +++ or Recognition of polymorphic antigen? No Yes

35 Polyclonal antibodies: immune complexes ++ cross-linking of antigen ++

36 Polyclonal antibodies on a bead: immune complexes ++++ cross-linking of antigen ++++

37 Monoclonal antibody: small soluble immune complexes no cross-linking of antigen

38 Monoclonal antibody on a bead: No cross-linking of antigen

39 Monoclonal or Polyclonal Abs Binding Site Monoclonal Polyclonal Epitope(s) recognised Single epitope Multiple epitopes Specificity +++ or Recognition of polymorphic antigen? Immune complex formation Light scattering in nephelometric assay No Small soluble complexes Yes Large insoluble complexes +/- ++++

40 Light chain concentration (mg/l) Analytical sensitivity Freelite is more sensitive than electrophoresis SPE CZE sife 10 Normal range in serum 1 Freelite

41 SPE alone is insensitive at diagnosis Screening algorithm Diagnostic sensitivity (%) SPE sife + uife SPE + sflc MM AL MM AL MM AL Testing with Freelite in addition to SPE improves detection rates and eliminates the reliance on urine testing Katzmann Clin Chem 2009;55:

42 Freelite challenges for interpretation 1. Accuracy and standardisation 2. Batch-to-batch concistency 3. Rare cases of antigen excess 4. Polymerisation 5. Non-Linearity

43 1. Accuracy and standardisation

44 4. Lot to Lot Reproducibility Production Target: Effective - recognise all monoclonal FLC variants. Consistent - antibody-antigen binding affinity between lots. Dealing with biological systems Reproducibility was a historical problem following release of Freelite Fact: Binding Site s continuous improvement of quality control and manufacturing procedures in recent years significantly improved ensuring consistency in breadth of response between reagent lots minimising variation in the reactivity of the reagents

45 Volume (L) Improved Manufacture Previously used individual pools of antibody now use larger rolling pools of affinity purified polyclonal antisera 2L from each pool A B C D E F G H I J New Batch Pool Number

46 Improved Quality Testing <5% variability between two consecutive batches Every batch calibrated with the same Internal reference preparation Large panel of samples tested on each batch and compared to previous results

47 Reduced Lot to Lot Variation 18% 13% Percentage of all Freelite complaints logged due to batch reproducibility issues (includes non-valid complaints) 8% 3% %

48 Investigating Batch Variation Check Parameters and lot specific details have been correctly updated Deterioration of the materials being tested? Instrument maintenance? Consider the sample - Katzmann J.A., et al. Identified upto 27% variation due to biological variation (Clin Chem 2011; 57(12): )

49 Ensuring Lot Reproducibility Manage customer stock levels- 2 batches of Freelite produced every year minimize batch-batch changes Make a comment highlighting batch change when reporting the results If possible, retest previous samples on the new batch for comparison Assess in conjunction with other clinical tests if no indication of change test sample sooner reset baseline

50 Response 3. Antigen Excess < 1% : Antibody excess Equivalence Antigen excess Antigen Concentration

51 How to detect antigen excess? Prozone parameters on some instruments The 4-fold rule Use the defold (initial) dilution and the the dilution to check for antigen excess (see product insert) If result is less than 4x (initial result) report initial result If result is greater than 4x (initial result) report 1/2000 result

52 4. Polymerisation

53 4. Polymerisation Not/poorly detectable by SPE or IFE due to the presence of variably sized FLC polymers; despite high serum FLC values, BenceJones may be negative various FLC polymers also possess different electrophoretic mobilities Prevents the formation of a single clear band of restricted mobility Not/poorly detectable by the electrophoretic methods. Mead et al; Clinical Chem 2010; doi

54 4. Advising Customers: polymerisation Since polymerisation is due to abnormal amino acid sequences it consequently will always be present in future samples from that patient Polymerisation does not detract from the clinical utility of the assay - able to use Freelite to monitor FLC concentrations have been shown to change in patients who present with a polymerised form of pathological, monoclonal FLC Polymer half life much longer due to reduced renal clearance so levels do not decrease as quickly

55 5. Sample non-linearity

56 Large variation cause non-linearity in some samples

57 Handling sample non-linearity Follow sample dilution protocol Report the first plausible result

58 Conclusion Freelite results should not be interpreted in isolation and other laboratory findings and clinical symptoms should be considered when evaluating the status of the patient.

59 Monitoring The same characteristics that make Freelite an effective addition to multiple myeloma diagnosis make it an important element in monitoring

60 FLC definitions Term iflc (involved) uflc (uninvolved) /λ sflc ratio Involved/uninvolved sflc ratio dflc (difference) Use Tumor load / response to therapy Polyclonal light chain, may be influenced by renal function Clonality and confirmation of response Define biomarker of malignancy Tumor load / response to therapy For a patient with monoclonal l sflc l sflc sflc sflc / λ sflc l sflc / sflc l sflc - sflc

61 dflc provides better assessment of response to therapy sflc (mg/l) l sflc (mg/l) /l sflc ratio Normal range dflc (mg/l) Baseline st time point /l sflc ratio The same pre- and post-therapy Therapy failure? iflc or dflc 90% reduction Therapy successful

62 dflc less affected by renal impairment FLCs produced 0.5-1g/day reabsorptive capacity 10-30g/day Urine FLC levels are a function of production minus reabsorption

63 Renal failure in MM is linked to FLC toxicity AL Amyloidosis LCDD Cast Nephropathy Fanconi Syndrome Presence of RF in > 50% MM at presentation 1 Hutchison Nat Rev Neph, Wirk BMT, 2011

64 PRODUCTION REMOVAL Normal/ healthy Κ monomers (25kDa) Serum κ < λ Bone marrow (normal) 2 κ plasma cells for each λ λ dimers (50kDa) Healthy Kidney

65 PRODUCTION REMOVAL Normal/ healthy Κ monomers (25kDa) Serum κ > λ Bone marrow (normal) 2 κ plasma cells for each λ λ dimers (50kDa) Damaged Kidney

66 sflc and Renal Impairment Kappa FLC Lambda FLC Polyclonal FLCs when GFR Hutchison et al. Clin J Am Soc Nephrol 2008

67 dflc less affected by renal impairment sflc l sflc /l sflc (mg/l) (mg/l) ratio Normal range dflc (mg/l) Baseline st time point Renal impairment develops 2 nd time point rd time point /l sflc ratio Not recommended for serial monitoring

68 for serial measurements, either the involved FLC or the difference between the involved and uninvolved (dflc) should be used. Dispenzieri Leukemia 2009;23:

69 When do I use FLC ratio vs dflc? κ/λ sflc ratio dflc (iflc-uflc) Diagnosis Establish Clonality Monitoring Quantify response to therapy Remission Absence of MRD Relapse Detect returning clonal disease

70 Short half-life Depth of response Why monitor with Freelite Enhanced sensitivity Clonal change Residual disease / Early relapse

71 The short half-life of sflcs allows rapid response assessment 0 24 hour 1 week 1 month 3 months κ FLC λ FLC 2-6 hours IgA IgM 5-7 days IgG ~21 days

72 sflc (mg/l) IgG (SPE, g/l) Change in iflc demonstrates response to therapy more rapidly than SPE Rx 1 Rx 2 Rx 3 0.1% Plasma cells sflc Monoclonal IgG Time from Presentation (days) Courtesy of G. Mead

73 Short half-life Depth of response Why monitor with Freelite Enhanced sensitivity Clonal change Residual disease / Early relapse

74 Light chain concentration (mg/l) Analytical sensitivity Freelite is ~ 10-fold more sensitive than uife SPE CZE sife 10 Normal range in serum UPE uife 1 Freelite

75 FLCs are filtered, reabsorbed and catabolised in the kidney FLCs produced 0.5-1g/day reabsorptive capacity 10-30g/day Urine FLC levels are a function of production minus reabsorption

76 Urine κ BJP (g/24hr) Urine BJP (g/24h) Serum sflc κ FLC (mg/l) (mg/l) Urine κ BJP (g/24hr) Urine BJP (g/24h) Serum sflc κ FLC (mg/l) (mg/l) Urine λ BJP (g/24hr) l Urine BJP (g/24h) Serum l sflc λ FLC (mg/l) (mg/l) Urine κ BJP (g/24hr) Urine BJP (g/24h) Serum sflc κ FLC (mg/l) (mg/l) Freelite provides equivalent or superior information to urine BJP HDC, ASCT Time Time (months) Thalidomide Time (months) sflc concentration urine BJP (24hr) Severe osteolytic lesions HDC, ASCT Time Time (months) Time (months) 0 Alyanakian Am J Hematol 2004;75: Wiley Periodicals, Inc.

77 Three advantages of serum Freelite vs. urine electrophoresis Freelite 10 X more sensitive than UPE/uIFE FLCs are not always found in urine Renal metabolism Inadequate sample Analytical sensitivity Urine compliance Freelite vs. urine analysis

78 Short half-life Clonal change Why monitor with Freelite Enhanced sensitivit y Residual disease / Early relapse

79 sflc detects relapse earlier than IFE IFE negative IFE positive Mosbauer Haematologica 2007;92: U. Mosbauer, et al., Monitoring serum free light chains in patients with multiple myeloma who achieved negative immunofixation after allogeneic stem cell transplantation. Haematologica 2007;92:275

80 Abnormal FLC ratios indicate residual disease in Intact Ig Multiple Myeloma 31 IFE negative IIMM patients Reid Bone Marrow Transplant 2004;33:623a

81 Abnormal FLC ratios indicate residual disease in Light Chain Multiple Myeloma 35 IFE negative LCMM patients + Normal sera CR Normal ratio CR Abnormal ratio Reid Bone Marrow Transplant 2004;33:623a Reid Clin Chem 2004;50:C34a

82 IMWG Defining Response For patients with measurable disease by electrophoresis Complete response (CR) Stringent complete response (scr) Negative serum/urine IFE BM plasma cells 5% Negative serum/urine IFE Absence of clonal cells in BM Normal sflc ratio Rajkumar Blood 2011;117:4691-5

83 Overall Survival (Probability) Attaining scr improves survival scr improved overall survival Kapoor J Clin Oncol 2013;31: Originally published by the American Society of Clinical Oncology. Time since transplantation (years)

84 Short half-life Clonal change Why monitor with Freelite Enhanced sensitivit y Residual disease / Early relapse

85 Why do we need multiple tools to monitor?

86 sflc (mg/l) sflcs and intact Igs are independent tumor markers n=164 IgG MM R 2 = IgG (g/l) IFM data courtesy of H. Avet-Loiseau κflc measured using Freelite IgGκ measured using Hevylite

87 At diagnosis, myeloma patients exhibit clonal heterogeneity IgG multiple myeloma IgG FLC dual populations are an example of intra-clonal heterogeneity in monoclonal gammopathies Ayliffe J Clin Pathol 2012;65: Ayliffe Haematologica 2007;92: M. Ayliffe, et al., Demonstration of changes in plasma cell subsets in patients with multiple myeloma. Haematologica 2007;92:1136

88 Sensitive techniques can identify clonal change Clonal populations change during therapy 5 unique clones at diagnosis Adapted from Keats Blood 2012;120: Republished with permission of the American Society of Hematology, permission conveyed through Copyright Clearance Center, Inc.

89 At relapse Clonal diversity at relapse Monoclonal protein at diagnosis FLC & intact Ig FLC only Intact Ig only 22% 68% 10% 100% 22% 40% 17% 38% 83% (n =126) 44% patients changed their monoclonal protein type Adapted from Zamarin Bone Marrow Transplant 2012;48:

90 Light chain escape rising monoclonal free light chain production at relapse without increased monoclonal intact immunoglobulin Courtesy of Christie Hospital, UK

91 Summary Freelite enhances MM monitoring Rapid evaluation of response Enhanced sensitivity compared to SPE and UPE Detects residual disease and early relapse Detects clonal change when combined with monoclonal intact Ig measurements

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