Hevylite assays de - convoluted. Dr Karthik Ramasamy Oxford University Hospitals

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1 Hevylite assays de - convoluted Dr Karthik Ramasamy Oxford University Hospitals

2 Overview Hevylite assay Introduction Clinical utility of Hevylite 2

3 Hevylite specifici.es

4 Hevylite specifici.es Hevylite epitopes

5 Hevylite specifici.es IgGκ IgGλ IgAκ IgAλ IgMκ IgMλ Isotype pairs

6 Immunoglobulins in mul.ple myeloma IgAκ tumour Polyclonal plasma cells Total IgA = 3 g/l ( )

7 Immunoglobulins in mul.ple myeloma IgAκ HLC IgAλ HLC 2.75 Involved g/l ( ) HLC Uninvolved 0.25 g/l ( ) HLC HLC ra9o (IgAκ/IgAλ) = 11.0 ( ) Monoclonal IgAκ with HLC pair suppression

8 Tradi.onal Intact Ig Analysis Serum Protein Electrophoresis: Immunofixa9on: IgGλ 24 g/l SPE IgG IgA IgM κ Specific an.sera λ Normal range (g/l) IgGκ IgGλ IgGκ/IgGλ Example 1 (g/l) Hevylite results: Monoclonal IgGλ of ~22g/l

9 Changes in plasma cell microenvironment Polyclonal plasma cells IgAκ tumour? Soluble mediators? Niche modifica.on IgAκ IgAλ HLC pair suppression

10 Hevylite definitions Term * Defini9on For an IgAκ pa9ent HLC ihlc ra9o Involved heavy e.g. IgAκ/IgAλ + light chain isotype IgAκ/IgAλ uhlc ihlc Uninvolved Involved heavy + + light chain isotype IgAλ IgAκ HLC uhlc ra9o Uninvolved heavy Ig κ/ig λ + light chain isotype IgAκ/IgAλ IgAλ dhlc ihlc - uhlc IgAκ-IgAλ HLC pair Suppression* * an abnormal HLC ra2o is required When the concentra.on of the uhlc is below the normal reference interval Reduced concentra.on of IgAλ

11 Clinical utility Comigrating bands Surrogate marker for improved clinical outcomes Early response to therapy Minimal residual disease Early relapse

12 When the M-protein cannot be measured Comigra9ng samples Co-migra9on:~40% of IgA bands co-migrate with the β region n= normal serum n * * * * n * * * * * *

13 Hevylite is able to reliably measure the size of the clonal protein Solid squares denote pa.ents with quan.fiable M-Igs by SPEP, whereas open squares denote pa.ents with anodal migra.on of their M-Igs Ludwig et al Leukemia Jan; 27(1):

14 Difficult to monitor paraproteins star.ng at <10g/l: 8.1% IgG & 12.1 % IgA 2,789 Myeloma 11 th trial pa.ents Paraprotein level g/l IgG % of 1,665 pts IgA % of 699 pts IgD % of 35 pts IgM % of 6 pts < < No difference between kappa and lambda light chain types > Pa.ents who had a whole paraprotein on serum immunofixa.on Slide courtesy Prof Mark Drayson 14

15 Clinical utility Comigrating bands Surrogate marker for improved clinical outcomes Early response to therapy Minimal residual disease Early relapse

16 PFS at Diagnosis Baseline HLC ra.os predicted PFS (p=0.0002) Blue: ra.os (n=199) Red: ra.os <0.01 or >200 (n=109) N=399, The more abnormal the HLCr the poorer the PFS for the pa.ent even in a diagnos.c sample M-protein levels were not significant as a measure of prognosis Leukemia Jan; 27(1):

17 Overall Survival at Diagnosis Baseline HLC ra.os predicted OS (p=0.016) Blue Line HLCr Red Line HLCr <0.022 or >45 The more abnormal the HLCr, the poorer the OS for the pa.ent even in a diagnos.c sample. M-protein levels were not significant as a measure of prognosis Ludwig et al Leukemia (2012)

18 Prognosis: IgG/ IgA Hevylite in MM HLC ra.os >200 or <0.01 β2m >3.5mg/L ISS Bradwell et al. Leukemia (2012) doi: /leu

19 PFS at Maximum Response VGPR or berer Drayson et al. MRC IX data Presented as poster at ASH 2012

20 Overall Survival at Maximum Response PR or berer Blue Line HLCr normal Red Line HLCr Abnormal Ludwig et al Leukemia Jan; 27(1):

21 Prognos.c value of Hevylite ra.os During monitoring n Time PFS OS Drayson Max response p< Tovar CR p<0.01 p=0.07 Ludwig PR - p=0.04 D Souza Pre-transplant p=0.02 p=0.01 Suehara VGPR - p=0.03 Drayson Blood 2012;120:3964a Tovar Biol Blood Marrow Transplant 2012;18: Ludwig Leukemia 2013;27: D Souza Br J Haematol 2016 doi: /bjh Suehara Haematologica 2015;100:E1251aD

22 Clinical utility Comigrating bands Surrogate marker for improved clinical outcomes Early response to therapy Minimal residual disease Early relapse

23 Electrophoresis Over-Es9mates Tumour Burden due to IgG Recycling CZE/IFE IgGk serum concentra.on Short IgG half-life ~3-5d Normal IgG half-life ~22d Poten.al to reduce treatment cycles? Prolonged IgG half-life upto 90d IgG 21 days IgA 5 days FLC 12 hours Time HLCr normalisa.on

24 Early response in Myeloma XI trial and the importance of Ig half life % Reduc9on in paraprotein/flc post -cycle 1 - three weeks INT/NON-INT COMBINED IgG paraprotein (FLC diff <100) IgG paraprotein (FLC diff >100) FLC IgG FLC (FLC diff >100) (N) Unpaire d T-test p=0.99 p= 5.1E-15 FLC IgA paraprotein (FLC diff <100) IgA paraprotein (FLC diff >100) IgA FLC (FLC diff >100) p=0.07 p= IgG IgA All IgG paraprotein All IgA paraprotein p= 1.4E-07 FLC FLC only 58 Slide courtesy Prof Mark Drayson 24

25 Clinical utility Comigrating bands Surrogate marker for improved clinical outcomes Early response to therapy Minimal residual disease Early relapse

26 Serum Electrophoresis is Not Sensi.ve Enough to Rule Out the Presence of Disease Detec.on of Residual Disease Ludwig et al Leukemia Jan; 27(1): IFE nega.ve HLC and FLC posi.ve

27 Is Hevylite ra.o normalisa.on a surrogate marker for MRD nega.vity Hypothesis: We hypothesise that a correla.on exists between MFC MRD nega.vity and the serological normalisa.on of the Hevylite and Freelite assays Prospec.ve pilot study N= 23 ( to include serological CR vs < CR) Pre transplant BM MRD by MFC Hevylite, Freelite Post Transplant ( 3 months) BM MRD by MFC Hevylite, Freelite Further Hevylite analysis at clinic.me points Blood :4633;

28 Diagnostic performance compared to flow MRD which is gold standard sensitivity 82.4% ( %), specificity 100% ( %), ROC Blood :4633;

29 Clinical utility Comigrating bands Surrogate marker for improved clinical outcomes Early response to therapy Minimal residual disease Early relapse

30 Electrophoresis is Not Sensi.ve Enough to Rule Out the Presence of Disease Detec.on of Relapse IFE and FLC nega.ve HLC posi.ve Ludwig et al Leukemia (2012)

31 HLC provides early indica.on of relapse IgAκ MM SPE IFE Hevylite IgAκ IgAκ nega9ve nega9ve nega9ve IgAκ IgAκ IgAλ IgAκ/IgAλ IgAκ Ludwig Leukemia 2013;27:213-9 Reprinted by permission from Macmillan Publishers Ltd: Leukemia, months

32 Clinical utility Comigrating bands Surrogate marker for improved clinical outcomes ( when HLC ratio is applied at Diagnosis and maximal response) Early response to therapy ( Particularly in IgG Patients) Minimal residual disease ( Blood assay) Early relapse ( high risk disease)

33 Kumar Lancet Oncol 2016;17:e The Hevylite assay is useful in pa9ents with oligo-secretory disease and can overcome limita.ons associated with monitoring β- migra.ng monoclonal IgA by electrophoresis.

34 the use of heavy/ light chain ra9os might have an important role in the defini.on of a minimal residual disease-nega9ve state. Kumar Lancet Oncol 2016;17:e "Reprinted from Lancet Oncology, 17, Kumar et al, International Myeloma Working Group consensus criteria for response and minimal residual disease assessment in multiple myeloma, Copyright (2017), with permission from E

35 NICE Guideline NG35

36 Acknowledgements Dale Powner, Binding site Prof Mark Drayson Lauren Campbell ( MRD) 36