M-protein diagnostics of Multiple Myeloma patients treated with biologics
JFM (Hans) Jacobs
Radboud university medical center (The Netherlands)
Laboratory Specialist Medical Immunology ([email protected])
EFLM webinar March 27th 2018
Moderated by Christopher McCudden (Ottawa, Canada)
B lymphocyte
Bone marrow
Peripheral blood
Plasma Cell
Monoclonal gammopathy/plasma cell dyscrasia (e.g. multiple myeloma)
Serum protein electrophoresis (SPE)
the separation of charged proteins in an electrical field
g:
b:
a2:
Albumin
a1: - Orosomucoid
- Haptoglobin
- Transferrin
- Immunoglobulins (IgG)
• SPE characterized by albumin, α1-, α2-, β- and γ-fraction • Band-intensity corresponds to its concentration
- a1 Antitrypsin
- a2 Macroglobulin - a Lipoprotein - Ceruloplasmin
- C3 Complement - Hemopexin
- b Lipoprotein
Anode + Pole
Cathode - Pole
- Immunoglobulins (IgA)
M-protein diagnostics, gel electrophoresis
Alb
α-1
α-2
β
Normal γ
Serum protein
electrophoresis (SPE)
#1 #2
Detection
#1 normal
#2 M-protein
Densitometry
Quantification
ELP G A M κ λ
Serum #2
Immunofixation
electrophoresis (IFE)
IgG-κ
Typing/Characterisation
M-protein = Myeloma protein = Paraprotein = Monoclonal component = M spike
Diagnosed at Mayo Clinic 2002
Multiple Myeloma 18% (273)
Amyloidosis (AL) 11% (167)
Lymphoma 4% (55)
Smouldering myeloma 6% (87)
Solitary or extramedullary plasmacytoma
1% (23)
Waldenström’s Macroglobulinemia
3% (43)
Other 6% (93)
MGUS 51% (769)
IgA (21%) Biclonal
(1%)
IgE (0.01%)
IgG (59%) IgD (1%)
FLC 15%
Nonsecretory myeloma (3%)
Monoclonal gammopathy / Plasma cell dyscrasia
M-protein quantification is required for
Classification: f.e. MGUS, smouldering myeloma (sMM) or multiple myeloma (MM)
Staging of symptomatic myeloma (stage I, II and III)
To monitor disease / therapy-response / or disease evolution (f.e. from MGUS > sMM > MM)
M-protein quantification: Why?
Durie et al. Leukemia 2006 Rajkumar et al. Blood 2015
M-protein quantification: How?
Alb. α-1 α-2 β γ
M-protein ‘M-spike’ 20% of total serum protein
T G A M κ λ
SPE Immunofixation
IgG-kappa
For example: • Total serum protein = 80 g/L • M-spike = 20% of total serum protein
• IgG-kappa M-protein = 16 g/L
SPE scanning densitometry
ELP G A M κ λ
2011.1A IgG-kappa
100% IgG-kappa Mean: 42.4 g/L VC: 13 %
Dutch External Quality Assessment (EQA) M-protein diagnostics
National program organized by Radboudumc (75 participating labs)
2011.2C IgG-kappa
2011.4B IgG-kappa
>95% IgG-kappa Mean: 2.7 g/L VC: 29 %
92% IgG-kappa Mean: 5.5 g/L VC: 40 %
ELP G A M κ λ
Monitoring patients, requires good test reproducibility…
(n=75 labs) 2009.3A Mean: 6.9 g/L
CV: 23 %
ELP G A M K L
2009.4A Mean: 6.7 g/L CV: 22 %
2010.4A Mean: 6.4 g/L CV: 22 %
2012.1A Mean: 6.4 g/L CV: 22 %
2014.3B Mean: 6.4 g/L CV: 23 %
ELP G A M K L
Over 5 measurements average within-lab VC : 14 %
Bone marrow and lymphoid organs Polyclonal FLCs produced approx. 500 mg/day
Kidney Capacity to absorb and metabolize 10-30 gram/day
T1/2 varies from 2-6 hrs to (2-3 days with renal failure)
Katzmann et al. Clin Chem 2002
Free Light Chain biology
FLC normal ranges (Freelite, TBS)
Kappa: 3.3 – 19.4 mg/L Lambda: 5.7 – 26.3 mg/L Ratio: 0.26 – 1.65
Kappa Lambda
Diagnosed at Mayo Clinic 2002
Multiple Myeloma 18% (273)
Amyloidosis (AL) 11% (167)
Lymphoma 4% (55)
Smouldering myeloma 6% (87)
Solitary or extramedullary plasmacytoma
1% (23)
Waldenström’s Macroglobulinemia
3% (43)
Other 6% (93)
MGUS 51% (769)
IgA (21%) Biclonal
(1%)
IgE (0.01%)
IgG (59%) IgD (1%)
FLC 15%
Nonsecretory myeloma (3%)
Monoclonal gammopathy
Free Light Chain
λ or κ
Multiple myeloma and renal impairment
Multiple myeloma at initial presentation
• 18-50% renal impairment (serum creat ↑) • 12-15% acute renal failure • 8% become dialysis dependent
Dimopoulos et al. J Clin Oncol 2010 Basnayake et al. Kidney Int 2011
hyperCalcemia, Renal impairment, Anemia, Bone disease
(CRAB diagnostic criteria MM)
Block urine flow Interstit. inflam.
Pathology
• Cast nephropathy (myeloma kidney)
• Light chain (AL) amyloidosis
• Light chain deposition disease
• Hypercalcemia
• Nephrotoxic drugs
• Hyperviscosity syndrome
…
Monoclonal Free Light Chains: not always a monoclonal band
‘hidden epitope’
Free Light Chain λ or κ
ELP G A M κ λ FLC FLC κ λ
FLC’s = short T1/2 = low serum concentration = often no ‘M-spike’…
Henry Bence Jones
Bence Jones proteins The very first cancer biomarker: The Lancet; 1847
‘When urine is heated, a white cloud appears and a precipitate forms. The precipitate disappears on boiling and reappears on cooling...’
REF values (freelite) Free kappa: 3,3 – 19,4 mg/l Free lambda: 5,7 – 26,3 mg/l Ratio: 0,26 – 1,65
sFLC nephelometry
Bead
Bead
Patient 192 mg/l 6.6 mg/l 29
Diagnosis: FLC kappa plasmacytoma-Th12
If clinical suspicion of: FLC multiple myeloma AL amyloidosis
ELP G A M κ λ
Dispenzieri et al. Leukemia 2009
No M-protein… no monoclonal gammopathy?
‘Diagnostic requirement: additional band’
Sensitivity 500-2.000 mg/L 150-500 mg/L
M-protein diagnostics: summary
‘hidden epitope’
Free Light Chain λ or κ
Immunoassay (neph/turb/elisa)
Bead
Bead
‘Diagnostic requirement: abnormal FLC κ/λ ratio’
Bradwell et al. 2001 Clin Chem ‘immunoassay for quantification of FLC’ Drayson et al. 2001 Blood ‘identifying and monitoirng ‘non-secretory MM’ Dispenzieri et al. 2009 Leukemia ‘FLC in international guidelines’
Sensitivity 1-3 mg/L
15% LCMM
• Early detection LCMM • Improved monitoring • Prognostic value
85% intact M-protein
Multiple Myeloma 18%
AL Amyloidosis 11%
Lymphoma 4%
Smouldering myeloma 6%
Other 6% (93)
MGUS 51%
plasmacytoma 1%
Waldenström’s
Macroglobulinemia 3%
M-protein diagnostics: screening, diagnosis and staging
Diagnosed at Mayo Clinic 2002
Diagnostic criteria Disease staging
*
* Or myeloma defining event.
Rajkumar et al. Lancet Oncology 2014.
M-protein diagnostics: follow-up and response evaluation
Durie et al. Leukemia 2006 Rajkumar et al. Blood 2015
MGUS MM
Diagnosis Diagnosis
Progression to MM
1st relapse
2nd relapse
Improved treatment regimes for MM patients
IMWG guideline: Ludwig et al. Leukemia 2013
Daratumumab
Tumor specific antibodies
Carter et al. Nat Rev Cancer 2007
*List is not extensive…
Anti-cancer antibodies used in the clinic*
Scott et al. Nat Rev Cancer 2012
1. Direct tumor cell killing
3. Vascular and stromal cell ablation
2. Immune-mediated tumor cell killing
Daratumumab 2015 CD38 Multiple myeloma
Elotuzumab 2015 SLAMF7 Multiple myeloma
4. Immune modulation tumor micro-environm.
Monoclonal antibody therapy in multiple myeloma
Touzeau et al. Review. Leukemia 2017
Daratumumab
Elotuzumab
Production and humanization of monoclonal antibodies
Techniques:
1) Merge binding portion of monoclonal mouse antibody with human antibody producing DNA.
Use cell cultures to express this DNA product
2) Genetically engineered mice that produce ‘human’ antibodies / Human hybridomas
Risk of immunological rejection
Risk of loss of specificity
‘…momab’ ‘…ximab’ ‘…zumab’ ‘…umab’
Biologics for MM patients in clinical practice
Lokhorst et al. NEJM 2015 Mateos et al. NEJM 2018
Lonial et al. NEJM 2015
Daratumumab Mechanisms of effect
Laubach et al. Clin Cancer Research 2015 Van de Donk et al. Blood 2018
Immunomodulatory effects
expansion
Xu et al. Clin Phar Ther 2017 Clemens et al. Clin Pharmacokinet 2017
daratumumab
Human IgG1-kappa mAb biologic
Daratumumab pharmacokinetics
16 mg/kg Weekly
16 mg/kg Every 2 wks
16 mg/kg Every 4 wks
Reaching serum [dara] up to 1 g/L
PE G A M κ λ
Patient 1 Before Daratumumab
Enlarged γ-region
Patient 1 Before daratumumab
M-spike 20.4 g/L
1A
Daratumumab and M-protein interference
G κ λ
Patient 1 After daratumumab
Patient 1 After Daratumumab
Enlarged γ-region
Dara-spike 0.4 g/L
M-spike 10.6 g/L
B
G κ λ
Daratumumab spiked in saline
Van de Donk et al. Clin Chem Lab Med 2016
a.o. IFE negative…
IMWG response criteria (Durie et al. 2006)
SP G κ SP G κ
Before daratumumab
After daratumumab
Abrogate interference using mAb against biological
McCudden et al. Clin Chem Lab Med 2016
DIRA ‘DARA shift-assay’ Daratumumab-specific Immunofixation Reflex Assay
Perform DIRA (or similar shift assay)
Indication to use DIRA or similar shift-assay
Adapted from Van de Donk et al. Blood 2018
SP G κ SP G κ
Before daratumumab
After daratumumab
Daratumumab Hydrashift assay (=pos example)
(+ M-protein comigrates with dara)
Outlook: synergistic effect of combined mAb in MM patients ??
Adpated from Touzeau et al. Review. Leukemia 2017
Alternative techniques to multiplex M-protein / mAb monitoring
Zajec et al. J Proteome Res 2018 Willrich et al. CCLM 2016
Daratumumab does not interfere with serum FLC testing
Rosenberg et al. Clin Biochem 2016
Dira spiked sample in Dutch EQA
100% IgG-kappa M-protein Mean M-spike (n=66): 4.9 g/L Inter-lab CV: 22 %
Dara spiked at 5 g/L
M-spike 5.2 g/L
98% IgG-kappa M-protein Mean M-spike (n=44): 1.7 g/L Inter-lab CV: 46% Many labs don’t spike such small M-proteins and reported <2 g/L
Dara spiked at 1 g/L
All participants report a normal [FLC-kappa]: which is in line with observation of Rosenberg et al.: no monoclonal FLC kappa in Daratumumab
Theoretically yes, but they often go unnoticed….
Can mAb’s used for other indications also interfere with serum protein electrophoresis?
Daratumumab in MM patients
• SPE performed to monitor disease • Dara dosed at high concentrations (16 mg/kg i.v. weekly in first 8 weeks)
• Hypogamma globulinemia (caused by disease process and therapy)
Adalimumab (α-TNF) in Rheumatoid Arthritis
• SPE not commonly performed in RA • Adalimumab dosed at lower concentrations (40 mg s.c. weekly or every 2 weeks)
• Hypergamma globulinemia (caused by disease process)
Low background: easy to detect small bands
High background: difficult to detect small bands
McCudden et al. Clin Chem 2010
Daratumumab interferes with blood group compatibility testing
Van de Donk et al. Blood 2018
Minimal Residual Disease in multiple myeloma
THERAPY
Daratumumab
MRD: few remaining cancer cells after therapy, often below detection limit, eventually cause cancer relapse.
Kumar et al. Lancet Oncology 2016
“…>50% of patients achieve sCR…”
Towards curative therapy for MM…
Barlogie et al. Blood 2014 Paiva et al. Blood 2015
…increases the need for detecting MRD
MRD R&D focus on molecular assays on bone marrow
Landren et al. Am J Hematol 2014; Paiva et al. Blood 2016 (flow cytometry) Puig et al. Leukemia 2014 (ASO q-PCR) Martinez-Lopez et al. Blood 2014 (next generation sequencing) Mailankody et al. Nat Reviews 2015
Paiva et al. Blood 2015
Multicolor flow cytometry ASO qPCR Next Generation Sequencing
Focus: Rearranged B-cell receptor on MM cells
Bone marrow not preferred for monitoring MM
Sampling error caused by tumor heterogeneity
Cumbersome and time-consuming procedure for
repetitive monitoring
Mass spectrometry as alternative sensitive assay to detect M-proteins in serum*
Based on unique M-protein mass
*List of publications is not extensive…
Based on unique M-protein peptides
M-protein diagnostics = Personalized diagnostics
Questions?