of multiple myeloma patients treated with biologics · multiple myeloma 18% al amyloidosis 11% 4%...
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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)
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B lymphocyte
Bone marrow
Peripheral blood
Plasma Cell
Monoclonal gammopathy/plasma cell dyscrasia (e.g. multiple myeloma)
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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)
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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
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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
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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
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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
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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
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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 %
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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
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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
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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
…
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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...’
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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?
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‘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
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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.
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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
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Improved treatment regimes for MM patients
IMWG guideline: Ludwig et al. Leukemia 2013
Daratumumab
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Tumor specific antibodies
Carter et al. Nat Rev Cancer 2007
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*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.
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Monoclonal antibody therapy in multiple myeloma
Touzeau et al. Review. Leukemia 2017
Daratumumab
Elotuzumab
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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’
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Biologics for MM patients in clinical practice
Lokhorst et al. NEJM 2015 Mateos et al. NEJM 2018
Lonial et al. NEJM 2015
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Daratumumab Mechanisms of effect
Laubach et al. Clin Cancer Research 2015 Van de Donk et al. Blood 2018
Immunomodulatory effects
expansion
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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
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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
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Abrogate interference using mAb against biological
McCudden et al. Clin Chem Lab Med 2016
DIRA ‘DARA shift-assay’ Daratumumab-specific Immunofixation Reflex Assay
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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)
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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
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Daratumumab does not interfere with serum FLC testing
Rosenberg et al. Clin Biochem 2016
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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
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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
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Daratumumab interferes with blood group compatibility testing
Van de Donk et al. Blood 2018
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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…”
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Towards curative therapy for MM…
Barlogie et al. Blood 2014 Paiva et al. Blood 2015
…increases the need for detecting MRD
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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
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Bone marrow not preferred for monitoring MM
Sampling error caused by tumor heterogeneity
Cumbersome and time-consuming procedure for
repetitive monitoring
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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
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M-protein diagnostics = Personalized diagnostics
Questions?