Paraproteins and response assessments – what have we learnt from MRC trials? Professor Mark Drayson, University of Birmingham 19th March 2015

Paraprotein type at presentation: 2,789 Myeloma 11th trial patients. Number patients Percent total Kappa : lambda ratio   IgG 1,665 60 2.25 IgA 699 25 1.78 Light Chain Only 361 13 1.34 IgD 35 1.3 0.46 Non Secretor 23 0.8 n/a IgM 6 0.2 2.00 Light Chain Only (6% urine fix negative) serum FLC dif >100mg/l (>500mg/l in 85%) Non Secretor Immunofixation negative and serum FLC dif <100mg/l (15/23 pts <10mg/l; 5/23pts >50mg/l)) Same findings in Myeloma 9 and in Myeloma 4 - 8

Paraprotein level at presentation: 32.7% < 30 g/L 42.3% < 30 g/L

Difficult to monitor paraproteins starting at <10g/l Paraprotein level g/l IgG % of 1,665 pts IgA % of 699 pts IgD % of 35 pts IgM % of 6 pts <5 3.7 6.4 54.2 16.7 5 - <10 4.4 5.7 17.1 10 - 20 9.8 14.3 20 - 40 49.0 42.5 11.4 >40 33.1 31.0 66.7 No difference between kappa and lambda light chain types Patients who had a whole paraprotein on serum immunofixation

FLC enable monitoring of 75% patients with PP <10g/l FLC Level k/l difference mg/l IgG <10g/l % 134pts IgG >10g/l 1,531pts IgA <10g/l 85pts IgA >10g/l 614pts IgD <10g/l 25pts IgD >10g/l 10pts   <100 28 32 26 37 4 100-500 20 34 25 24 30 >500 52 49 35 72 70 FLC are higher when paraprotein <10g/l (no variation above 10g/l) IgD patients have low paraprotein and high FLC levels – usually lambda

Suppression of polyclonal immunoglobulin Normal range B Normal range C Normal range n (%) IgG (n =1302) IgA (n = 2471) IgM (n = 3228) Below NR 1049 (80.6) 1981(80.2) 2871 (88.9) Within NR 253 (19.4) 479 (19.4) 349 (10.8) Above NR 0 (0.0) 11 (0.4) 8 (0.2) Median (range) g/L 3.90 (0–15.65) 0.34 (0–5.60) 0.16 (0–3.56)

FLC levels are higher when the light chain type is lambda FLC Level k/ldifference mg/l IgG kappa % 1,153pt IgG lambda 512pts IgA kappa 448pts IgA lambda 251pts LCO kappa 207pts lambda 154pts   <100 32 30 40 25 100-500 34 29 27 15 >500 41 33 46 85 Lambda higher than kappa analysing as continuous variables. Equally nephrotoxic

eGFR by difference between involved and uninvolved FLC (dFLC) clone at presentation in mg/L Dr Punit Yadav analysis of 1,595 Myeloma 9 patients

Immunoglobulin half lives and response IgG 21 days IgA 5 days FLC 12 hours Short half lives of serum FLC allow real time evaluation of tumour kill

Paraprotein response to 3 week cycles of myeloma therapy 100 40 g/l 80 60 IgG paraprotein g/l 20 g/l 8 g/l 40 10 g/l 8g/l 5.0 g/l 20 4 g/l 2.0 g/l 1.6 g/l 0.8 g/l 0.3 g/l 1st cycle 80% kill 2nd cycle 80% kill 3rd cycle 80% kill 4th cycle 80% kill (0.16% residue)

FLC response to 3 week cycles of myeloma therapy 5000 4000 3000 2000 1000 1st cycle 80% kill 2nd cycle 3rd cycle 4th cycle 80% kill (0.16% residue) Dif FLC mg/l 4000 mg/l 800 mg/l 160 mg/l normalise

Does a reducing kill rate indicate clonal heterogeneity and predict poor survival? 5000 4000 3000 2000 1000 1st cycle 80% kill 2nd cycle 20% kill 3rd cycle 4th cycle 0% kill (3.2% residue) Dif FLC mg/l 4000 mg/l 200 mg/l 160 mg/l normalise

FLC response at end of cycle 1 predicts later response Correlation coefficient=0.588 Correlation coefficient=0.155 227 Myeloma 11 patients with IgG and IgA paraproteins >10g/l and dFLC >100mg/l

Early response in myeloma 11 and the importance of Ig half life Unpaired T-test (N) 531 117 211 756 328 58 225 % Reduction in paraprotein/FLC post -cycle 1 - three weeks p=0.99 p=0.07 p= 5.1E-15 p= 0.005 p= 1.4E-07 FLC IgG IgA

IgG level does not appear to affect IgG half-life in myeloma 11 IgG level does not appear to affect IgG half-life in myeloma 11. (FcR neonatal receptor saturated at about 30g/l) IgG FLC IgG FLC IgG FLC IgA FLC FLC

No difference in early paraprotein and FLC responses between Intensive and Non-intensive patients 11th trial Figure 1: Percentage reduction in paraprotein and sFLC light chain levels for patients treated in the MRC Myeloma XI trial at the end of the first cycle of induction chemotherapy. Whisker boxplots showing the median, 25th and 75th centiles. Tails represent the 5th and 95th centiles. The diamonds represent the means. Mann Whitney U tests were performed to assess the statistical significance of the differences between the groups. RCD=lenalidomide, cyclophosphamide and dexamethasone (320mg/cycle); CTD=cyclophosphamide, thalidomide and dexamethasone (320mg/cycle); RCDa=lenalidomide, cyclophosphamide and dexamethasone (160mg/cycle) and CTDa=cyclophosphamide, thalidomide and dexamethasone (160mg/cycle). p=0.11 p=0.30 p=0.38 p=0.40 p=0.24

No difference in maximum responses to induction therapy between Intensive and Non-intensive patients 9th & 11th trial p=0.07 p=0.77 p=0.70 p=0.13 Figure 2: Percentage reduction in paraprotein and sFLC light chain levels for patients treated with CTD or CTDa in the MRC Myeloma IX trial and RCD/CTD or RCDa/CTDA in the MRC Myeloma XI trial at the end of induction chemotherapy. Whisker boxplots showing the median, 25th and 75th centiles. Tails represent the 10th and 95th centiles. The diamonds represent the means. Mann Whitney U tests were performed to assess the statistical significance of the differences between the groups. RCD=lenalidomide, cyclophosphamide and dexamethasone (320mg/cycle); CTD=cyclophosphamide, thalidomide and dexamethasone (320mg/cycle); RCDa=lenalidomide, cyclophosphamide and dexamethasone (160mg/cycle) and CTDa=cyclophosphamide, thalidomide and dexamethasone (160mg/cycle).

Response at end of induction versus 100d post HDM HDM deepens response but that is not completely evident by 100 d for IgG paras FLC Response Paraprotein Response IgA 45 IgG 117 LCO 44 Percentage reduction in sFLC and paraprotein levels at the end of induction chemotherapy compared to at 100 days post high dose melphalan for patients treated in the MRC Myeloma IX trial. Whisker boxplots showing the median, 25th and 75th centiles. Tails represent the 10th and 95th centiles. The diamond represents the mean. Mann Whitney U tests p<0.01 for all pairs

Response at end of induction versus 100d post HDM HDM deepens response but that is not completely evident by 100 d for IgG paras FLC Response Paraprotein Response LCO 105 IgA 97 IgG 249 IgA 97 IgG 249 Percentage reduction in sFLC and paraprotein levels at the end of induction chemotherapy compared to at 100 days post high dose melphalan for patients treated in the MRC Myeloma XI trial. Whisker boxplots showing the median, 25th and 75th centiles. Tails represent the 10th and 95th centiles. The diamond represents the mean. Mann Whitney U tests p<0.01 for all pairs

At relapse there were three types of relapse:- Serum free immunoglobulin light chain evaluation as a marker of impact from intraclonal heterogeneity on myeloma outcome Blood 123(22):3414-3419 The first 520 patients in myeloma 9 who had an IgA or IgG paraprotein at presentation At relapse there were three types of relapse:- 183/520 (35.2%) patients had a significant increase in both paraprotein and FLC levels 258/520 (49.6%) patients only had a significant increase in their paraprotein levels Paraprotein only relapse 3. 54/520 (10.4%) patients relapsed with only an increase in their FLC levels: Free light chain escape 24/369 (6.5%) IgG and 30/151 (19.9%) IgA patients relapsed with FLC escape

Effect of Relapse Type on Survival

Model of Darwinian Evolution in Multiple Myeloma

Is this type of clonal heterogeneity present in response? IgG and FLC>100mg/l Post cycle one results IgA and FLC>100mg/l Post cycle one results

Key findings IgG 60%; IgA 25%; LCO 13%; IgD 1.3%; NS 0.8% (LCO missed in older pts?) In 10% of patients whole paraprotein <10g/l but can be monitored by FLC Polyclonal Igs are below normal level in 80% and above normal level in <0.4% (3,228pts) FLC levels are higher when type is lambda but kappa and lambda FLC are equally likely to be nephrotoxic at the same level. Many patients have non-toxic FLC. FLC levels need to be >500mg/l to have nephrotoxicity. Ig half life is important when assessing response IgG 21days (7-10d on dex); IgA 5 days FLC <12 hrs Early FLC response indicates kill rate and can predict max response. Changing kill rate with subsequent cycles of therapy may indicate clonal heterogenity and poor prognosis No difference in speed of response or final depth of response between patients on 20 and on 40 mg dex Light chain escape in 6% IgG and 20% IgA patients. Different relapse patterns indicate clonal heterogeneity. Patients relapsing with FLC survive worse from relapse than paraprotein only relapses Some patients have asynchronous FLC and paraprotein response which might also indicate clonal heterogeneity and poor survival