Protein dynamics in early disease Distinguishing myeloma from MGUS by m-protein and serum free light chain results alone Professor Mark Drayson, University of Birmingham 13th March 2019

Myeloma is the worst cancer for delayed diagnosis because it is rare in primary care and non-haem specialty consultations the symptoms are common, variable, and of low predictive value Of 32,236 newly diagnosed myeloma patients in England, 11,006 (34%) presented as emergencies; of these, 4,182 (38%) died within 6 months of diagnosis, accounting for 78% of all myeloma deaths within 6 months of diagnosis. Myeloma could be detected earlier because 99% of all myeloma patients have a serum m-protein and or an abnormal serum free light chain ratio that can be easily detected in blood

Inhibitions to laboratory testing: Cost of tests needs to be considered Testing also reveals 100% of the 100x more common MGUS MGUS must then be distinguished from myeloma at Great cost to the patient Major cost to the health service

MGUS 1% risk of progression to myeloma Prevalence of MGUS 100 fold that of the incidence of myeloma Are we testing for myeloma too little or too much New m-proteins in a hospital 15 years ago Mayo Malmo St Helier Diagnosis (1510) (930) (200) MGUS 51% 72% 74% Myeloma 24% 19% 16% WM 3% 2% 2% AL 11% 1% 1% Other 11% 6% 7%

Use of m-protein levels and serum FLC ratios to distinguish myeloma from MGUS m-proteins >30g/l are attributable to myeloma; this is the International guideline cut point for distinguishing myeloma from MGUS. 40% of myeloma patients have m-proteins <30g/l 10% have m-proteins <10 g/l. Over 90% of myeloma patients have an abnormal SFLCR so do some 40% of patients with MGUS. So do many patients who do not have a monoclonal gammopathy (renal impairment present in 25% of the myeloma demography; autoimmune disease SLE etc; infection; inflammatory disease) Accordingly neither m-protein nor SFLC ratio alone can provide good sensitivity and specificity for distinguishing myeloma from MGUS.

M-protein types and levels in UK Myeloma trials 1980-2013   Old trials (n = 2807) MIX (n = 1555) MXI (n = 1692) 1980-1997 2003-2008 To June 2013 Age, % ≥ 65 years 51 53.1 59.5 >65 years Intensive pathway – 12.8 18.9 M-protein type, % IgG 57.5 61.7 58.2 IgA 24.0 21.5 25.8 Light chain only 13.3 12.5 13.4 IgD 1.6 1.9 1.5 Non-secretory 3.6 1.0 0.5 IgM 0.3 Patients getting older Treatment intensifying Reduced % non-secretory

M-protein types and levels at diagnosis in UK Myeloma IX and XI M-protein levels in 1894 patients with IgG and 757 patients with IgA m-proteins 32.7% < 30 g/L 42.3% < 30 g/L

Combining high thresholds for serum FLC ratios and m-protein levels to facilitate appropriate and rapid referral for myeloma diagnosis Methods: retrospective cohort study Measured serum FLC ratio and m-protein concentrations in samples from 3177 newly diagnosed myeloma patients and 711 MGUS cases. Applied different thresholds for SFLC ratio range and m-protein levels at 5, 10 and 15 g/L to decide whether a patient should be considered at risk of myeloma and thus need further investigation.

Using an M-protein at any level and/or a SFLC ratio outside the normal range (0.26 – 1.65) then 3153/3177 (99%) of newly diagnosed myeloma patients were identified (0.8% were non-secretory). This strategy identified 100% of 771 MGUS cases for consideration of myeloma.

Inverse relationship between SFLC ratio and m-protein level Involved/uninvolved FLC ratio in 436 patients without an intact m-protein and 2717 patients with different m-protein concentrations

Light chain only (n= 418) oligosecretory The percentage of myeloma patients missed through stratification according to different serum FLC κ:λ ratios and M-protein levels All patients (N = 3177) Non-secretors (n = 24) Light chain only (n= 418) oligosecretory (n = 18) IgG/A/D/M (n = 2717) IgG/A/M/D   κ:λ ratio % (n) M-protein < 5g/L (n = 122) < 10g/L (n = 226) < 15g/L (n = 336) Normal ratio range (0·26–1·65) 5·2% (166) 0·8% (24) 4·7% (148) 0·4% (12) (23) 1·1% (29) Extended ratio range 1 (0·15–3·36) 10% (318) 0·5% (2) 10·8% (293) 0·60% (16) 1·3% (35) 1·8% (49) Extended ratio range 2 (0·08–7·41) 15% (477) (5) 16·5% (449) 0·6% 1·5% (40) 2·4% (65)

The percentage of 711 patients with MGUS that would be excluded from further assessment for myeloma using the stated cut-offs for m-protein level and reference ranges for serum free light chain κ:λ ratio M-protein cut-off % (n) Normal ratio range (0·26–1·65) Extended ratio range 1 (0·15–3·36) Extended ratio range 2 (0·08–7·41) M-protein ≥ 5g/L   66% (468) 0.4% 82% (581) 0.8% 91% (645) 1.1% M-protein ≥ 10g/L 81% (577) 0.6% 89% (630) 1.3% 94% (670) 1.8% M-protein ≥ 15g/L (646) 93% (663) 1.5% 96% (684) 2.4% Percentage of secretory myeloma patients missed

MGUS risk of progression to myeloma Mayo study (Blood 2005 Rajkumar) 1148 patients median 15 years follow up (8982 patient years follow up) M-protein >15 g/l HR 2.4 Abnormal FLC ratio HR 2.6 Non-IgG type m-protein HR 2.6 39% MGUS patients no factors abnormal absolute risk of progression at 20 years accounting for death as a competing risk, 2% 37% MGUS patients one factor abnormal absolute risk of progression at 20 years accounting for death as a competing risk, 10% Swedish study (Blood 2014 Turesson) 728 patients median 10 years follow up (7590 patient years follow up) M-protein >15 g/l HR 3.6 Abnormal FLC ratio HR 3.0 Immunoparesis HR 2.8

MGUS precedes myeloma (m-protein dynamics) American cancer screening study (Blood 2009 Landgren) 77,469 healthy adults; 71 developed myeloma. Stored serum from 2 – 9.8 years available pre myeloma diagnosis MGUS present in 100% 2 years prior; 82% 8 years prior M-protein concentration (g/dL) levels year by year prior to multiple myeloma diagnosis HALF RISING; HALF STABLE SAME FOR FLC

MGUS precedes myeloma (m-protein dynamics) American armed forces health study (Blood 2009 Weiss) 7 million army personel; 90 received transplants for myeloma; 30 had Stored serum from 2.2 – 15.3 years available pre myeloma diagnosis MGUS present in 27/30 M-protein concentration (g/dL) levels year by year prior to multiple myeloma diagnosis HALF RISING; HALF STABLE SAME FOR FLC

for a complete response to become evident Immunoglobulin half lives and response; can take several months for a complete response to become evident IgG 21 days IgA 5 days FLC 12 hours Short half lives of serum FLC allow real time evaluation of tumour kill

FLC response to 3 week cycles of myeloma therapy Facilitates real time evaluation of tumour kill A B Changes in FLC (A) and IgG paraprotein (B) levels during the first four cycles of induction chemotherapy if a 60% kill was achieved in each cycle. The FLC reduction at the end of each cycle is representative of the percentage kill of MM cells. Due to the long half-life of IgG, the reduction in the level of the IgG paraprotein represents a combination of IgG half-life and the MM kill.

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

Acknowledgements Chief Investigators: Professor Ian MacLennan Professor Tony Child Professor Gareth Morgan Professor Gordon Cook Professor Graham Jackson Clinical Trials Research Units: Leeds Professor Walter Gregory Professor David Cairns Dr Sue Bell Birmingham / Warwick Professor Janet Dunn Dr Gulnaz Begum Clinical Immunology Service Mr Tim Plant Mrs Nicki Newnham Mrs Karen Walker Mrs Alison Adkins Mr Zaheer Afzal Mrs Jean Giles Mr Mark Fellows Mr Ramesh Ramnatsing Mrs Tarana Ahm Miss Claire Backhouse R&D Dr Margaret Goodall Dr John Campbell Dr Jennifer Heaney Dr Ilaria Chicca Dr Hannah Giles MRD studies Dr Roger Owen Molecular/translational studies: Professor Faith Davies Dr Brian Walker Dr Martin Kaiser Cytogenetics Dr Fiona Ross All the patients and staff at over 100 centres throughout the UK whose participation made these studies possible. All principle investigators for their dedication and commitment to recruiting patients to the studies.