Diagnosis and Follow-up of Multiple Myeloma and Related Disorders: The role of the laboratory

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1 Diagnosis and Follow-up of Multiple Myeloma and Related Disorders: The role of the laboratory Anne L Sherwood, PhD Director of Scientific Affairs The Binding Site, Inc.

2 Learning Objectives Compare traditional myeloma testing methods with current recommended protocols and describe how kappa & lambda free light chain analysis plays a key role. Discuss the updated IMWG criteria for diagnosis of Multiple Myeloma and new recommendations for inclusion in routine practice. Explain why it is necessary to monitor both intact immunoglobulin paraprotein and serum free light chains in multiple myeloma.

3 Disclaimer Some of the subject matter and data contained or addressed in this session may involve off-label or forward-looking use of Freelite

4 In This Talk Introduction Depth of Response Multiple Myeloma Biology of FLCs Monitoring with Freelite Updated MM Diagnostic Criteria

5 Multiple Myeloma A Cancer of the Plasma Cells in the Bone Marrow Multiple Myeloma: Abnormal bone marrow with sheets of plasma cells 1% vs up to 90%

6 Definition of MGUS, SMM and Symptomatic Multiple Myeloma Ghobrial and Landgren. Blood, Oct 2014, prepub.online; doi: /blood

7 Plasma cells secrete intact antibody and free light chains Light Chain Heavy Chain Kappa, k Lambda, l Kappa releasing Lambda releasing

8 Monoclonal gammopathies Disorders associated with monoclonal proteins Asymptomatic e.g. MGUS Malignant e.g. MM, AL amyloidosis

9 Any type of Multiple Myeloma can express free light chains Intact Immunoglobulin 80% Light chain only 15-20% Nonsecretory 1-2% 89% 100% 68% Abnormal k/l sflc ratio

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

11 Sensitivity of SPE at diagnosis Multiple myeloma AL amyloidosis 88% 66% Intact immunoglobulin Light chain Nonsecretory 97% 61% 0% Katzmann Clin Chem 2009;55: Jeong CCLM 2013;51:e187-9

12 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

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

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

15 Interpretation of Free Light Chain Results k l k/l Ratio mg/l mg/l Normal Normal or Decreased Monoclonal k Normal or Decreased Monoclonal l Normal or Slightly Increased Normal Renal Impairment Polyclonal Gammopathy Jagannath Clin Lymphoma Myeloma, 7(8):518-23; 2007

16 Higher risk of malignant plasma cell disorder is associated with more extreme k/l ratio Lambda secretor Normal Kappa Secretor k/l ratio k/l ratio : Risk : Likely Possible Very Low Inconclusive Possible Likely Katzmann J, Clin Chem : 1437 Vermeersch Clin Chem Acta : 54-58

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

18 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:

19 Use of the FLC assay has significantly changed the way that patients with AL amyloidosis are diagnosed and monitored..the serum FLC assay is critical for evaluating patients with AL, as many patients lack a measurable circulating intact immunoglobulin. Comenzo Leukemia 2012;26:

20 Updated criteria for diagnosis of myeloma Clonal bone marrow plasma cells 10% or biopsy-proven bony or extramedullary plasmacytoma and one/more myeloma defining events: Any one or more biomarkers of malignancy (SLiM) Related end organ damage (CRAB) Clonal bone marrow plasma cell percentage 60% (Sixty %) Involved/uninvolved serum Free Light Chain ratio 100* >1 focal lesion on MRI studies HyperCalcemia Renal insufficiency Anemia Bone lesions Rajkumar Lancet Oncol 2014;15:e538-e548 Reprinted from Lancet Oncology, 2014, with permission from Elsevier. *By Freelite iflc must be 100 mg/l

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

22 Term iflc (involved) uflc (uninvolved) k/λ sflc ratio Involved/uninvolved sflc ratio dflc (difference) FLC definitions 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 k sflc k sflc / λ sflc l sflc / k sflc l sflc - k sflc

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

24 dflc less affected by renal impairment k sflc l sflc k/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 k/l sflc ratio Not recommended for serial monitoring

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

26 Summary of IMWG recommendations Diagnosis k/l sflc ratio involved/ uninvolved sflc ratio iflc or dflc Monitoring k/l sflc ratio Define clonality Define biomarker of malignancy Dispenzieri Leukemia 2009;23: Rajkumar Lancet Oncology 2014;15:e538-e548 Assess response Define scr

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

28 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

29 k 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 k sflc Monoclonal IgG Time from Presentation (days) Courtesy of G. Mead

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

31 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

32 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

33 Urine κ BJP (g/24hr) k Urine BJP (g/24h) Serum k sflc κ FLC (mg/l) (mg/l) Urine κ BJP (g/24hr) k Urine BJP (g/24h) Serum k 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) k Urine BJP (g/24h) Serum k 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.

34 dflc (mg/l) UPE densitometry (mg/24h) dflc (mg/l) UPE densitometery (mg/24h) Freelite provides equivalent or superior information to urine BJP A uife Presentation 1 2Cycle 2 3Cycle 4 4 ASCT 5 B dflc ubjp uife - - dflc ubjp sife Cycle 2 G A M κ λ G A M κ λ 1 0Presentation 1 Cycle 2 2 Cycle 3 4 4ASCT 5 Dejoie Haematologica 2016;101: T. Dejoie, et al., Comparison of serum free light chain and urine electrophoresis for the detection of the light chain component of monoclonal immunoglobulins in light chain and intact immunoglobulin multiple myeloma. Haematologica 2016;101:361 0

35 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

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

37 sflc detects relapse earlier than IFE 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

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

39 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

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

41 Why do we need multiple tools to monitor?

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

43 At diagnosis, myeloma patients exhibit clonal heterogeneity IgGk multiple myeloma IgGk k 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

44 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.

45 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:

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

47 Light chain escape 6.5% 19.9% IgG IgA Only ½ of LCE patients were ubjp positive n=520 pts Brioli Blood 2014;123:3414-9

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

49 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

50 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)

51 Overall survival (%) Median survival from relapse (months) Relapse with FLC indicates poorer survival FLC & Intact Ig FLC escape Intact Ig only p= Time (months) Brioli Blood 2014;123: Republished with permission of the American Society of Hematology, permission conveyed through Copyright Clearance Center, Inc.

52 Progression free survival Overall Survival Normalization of sflc ratio improves survival across all response levels Progression Free Survival Normal k/l sflc ratio Abnormal k/l sflc ratio Overall Survival Follow up from diagnosis (months) 29 vs. 16 months (P <.001) Follow up from diagnosis (months) 91 vs. 58 months (P <.001) Moustafa Leukemia 2015;29: Reprinted by permission from Macmillan Publishers Ltd: Leukemia, 2009.

53 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 Depth of sflc response correlates with survival

54 Any Questions? (206)

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