Jerry Katzmann
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Transcript of Jerry Katzmann
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
Jerry Katzmann, Ph.D. Dept. Laboratory Medicine & Pathology
Mayo Clinic
Laboratory Perspectives in the Diagnosis and Monitoring in Plasma Cell/B Lymphoid
Dyscrasias
June 8, 2012, Royal Free London
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Department of Laboratory Medicine and Pathology
DISCLOSURES:
Travel expenses, The Binding Site, Ltd.TBS FLC & HLC immunoassay reagents at no cost for clinical studies
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
Serum Protein Electrophoresis & Immunofixation Electrophoresis
Normal Serum MM Serum
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Department of Laboratory Medicine and Pathology
Monoclonal Gammopathies: Mayo Clinic 2010 (n=1,609)
MGUS 52% (836)
Multiple myeloma 19% (307)
Amyloidosis (AL) 9% (152)
Lymphoproliferative 2% (35)
SMM 4% (66)Solitary or extra- medullary 2% (33)
Macro 4% (56)Other 8% (124)
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Department of Laboratory Medicine and Pathology
Laboratory problems for electrophoretic diagnosis and monitoring of plasma cell
dyscrasias
Free light chains: identification & monitoring•
FLC quantitation
Monitoring very large M-spikes on agarose•
Ig and/or HLC quantitation
Monitoring small M-proteins in γ or β fractions•
HLC quantitation
Broadly migrating M-proteins : identification & monitoring
•
HLC quantitation
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Department of Laboratory Medicine and Pathology
Serum & Urine Protein Electrophoresis and Immunofixation Electrophoresis
LCMM Serum LCMM Urine
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Department of Laboratory Medicine and Pathology
1027 Newly Diagnosed Multiple Myeloma: Monoclonal Serum Proteins
IgG 52%
IgA 21%
IgM 0.5%
IgD 2%
Biclonal 2%
Free Light Chain only 20%
Nonsecretory Myeloma 2.8%
Kyle et. al., Mayo Clin Proc 2003
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Department of Laboratory Medicine and Pathology
Serum & Urine Protein Electrophoresis and Immunofixation Electrophoresis:
Primary Amyloid
AL, Serum AL, Urine
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Department of Laboratory Medicine and Pathology
Principle of FLC assay
antigen
binding
sites
exposed surface
hidden surface
hinge region
carbohydrate
light chain
heavy chain
Previously
hidden surface
and antibody
target
exposed surface
Lambda
Kappa
The Binding Site
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Department of Laboratory Medicine and Pathology
Free Light Chain Quantitation: Reference Intervals
Reference Range(95% interval)
Diagnostic Range(100% interval)
Kappa FLC 0.33-1.94 mg/dL
Lambda FLC 0.57-2.63 mg/dL
FLC k/l ratio 0.3-1.2 0.26-1.65
Katzmann, J. Clin Chem, 2002
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Department of Laboratory Medicine and Pathology
1
10
100
1000
10000
100000
1 10 100 1000 10000 100000
Free light chain serum Kappa (mg/L)
Free
ligh
t cha
in s
erum
Lam
bda
(mg/
L)
Normals <70yrs
Normals >70yrs
KappaLCMM
LambdaLCMM
NSMM
Polyclonal
The Binding Site
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Department of Laboratory Medicine and Pathology
Abnormal rFLC in Plasma Cell DisordersDiagnosis n Abnormal
rFLC, %Multiple myeloma (MM)
Intact Ig MM 1706 96
“Non secretory” MM 33 73
Light chain MM 252 100
MGUS 1262 33
AL amyloidosis 467 95
LCDD 28 93
Smoldering MM 345 90
Plasmacytoma 116 47
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
Katzmann, J. Clin Chem, 2009
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
Diagnostic Screening Panels: Sensitivity
Diagnosis (n) Serum PEL/IFE/FLC1
Urine PEL/IFE
(all 5 assays)
Serum PEL/IFE/FLC
(all 3 serum assays)
Serum PEL/FLC
(2 serum assays)
MM2
(467) 100% 100% 100%
WM (26) 100% 100% 100%
AL (581) 98.1% 97.1% 96.2%
SMM (191) 100% 100% 99.5%
MGUS (524) 100% 97.1% 88.7%
1PEL=protein electrophoresis; IFE=immunofixation electrophoresis;
FLC=quantitative free light chain2MM=multiple myeloma; WM=Waldenström's macroglobulinemia; AL=primary amyloidosis: SMM=smoldering multiple myeloma; MGUS=monoclonal gammopathy of undetermined significance
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
IMWG Guidelines for Quantitative FLC: Impact on the Laboratory
Diagnosis:•
Screening panel: serum PEL/IFE & FLC unless suspect AL. ›
Serum PEL & FLC is sufficient for initial
screen
Prognosis:
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Department of Laboratory Medicine and Pathology
1384
patients in SE MN were identified with MGUS between 1960 and 1994. Patients were followed for an average of 8 years (11,009 total patient years).
115 progressed
to MM, lymphoma, AL, Macroglobulinemia, CLL, or plasmacytoma
~1% of MGUS patients progress/year.
Initial size of M-spike and heavy chain isotype are prognostic for progression
A Long Term Study of Prognosis in MGUS
Kyle, et al, NEJM, 2002
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Department of Laboratory Medicine and Pathology
FLC and Progression in MGUS
years
perc
ent
0 5 10 15 20 25 30
010
2030
4050
60p y g p g
Normal FLC ratio (K/L 0.26-1.65)Abnormal FLC ratio (K/L <0.26 or > 1.65)
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Department of Laboratory Medicine and Pathology
Multivariate Analysis of Prognostic Factors for Progression of MGUS
Prognostic factor Hazard ratio(95% C.I.)
p value
Abnormal FLC ratio 2.6 (1.7,4.2) <0.001
Serum M protein size 2.4 (1.7,3.5) <0.001
IgA, IgM, or biclonal IgA plus IgM
2.6 (1.7,4.0) <0.001
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and PathologyYears
Perc
ent
0 5 10 15 20 25 30
020
4060 Serum M-spike <1.5 gm/dL, IgG Subtype and normal FLC ratio
Any 1 factor abnormal
Any 2 factors abnormal
All 3 factors abnormal
Prognosis of MGUS: Risk Stratification using M spike size, type and FLC ratio
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Department of Laboratory Medicine and Pathology
Risk Stratification Model Incorporating All 3 Predictive Factors
27%58%20.853High
All 3
factors abnormal
18%37%10.1226High/Intermediate
Any 2
factors abnormal
10%21%5.4420Low/Intermediate
Any 1
factor abnormal
2% (0.1%/yr)5%1449
Low Risk1. M protein <1.5 gm/dL, 2. IgG subtype,3. normal FLC ratio
20-year ROP after other causes of
death
Absolute
ROP at
20 yearsRelative risk
Number of patients
Risk Group
ROP: risk of progressionRajkumar, SV, Blood,2005
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Department of Laboratory Medicine and Pathology
Diagnostic Screening Panels: SensitivityDiagnosis (n) Serum
PEL/IFE/FLC1
Urine PEL/IFE
(all 5 assays)
Serum PEL/IFE/FLC
(all 3 serum assays)
Serum PEL/FLC
(2 serum assays)
MM2
(467) 100% 100% 100%
WM (26) 100% 100% 100%
AL (581) 98.1% 97.1% 96.2%
SMM (191) 100% 100% 99.5%
MGUS (524) 100% 97.1% 88.7%
1PEL=protein electrophoresis; IFE=immunofixation electrophoresis;
FLC=quantitative free light chain2MM=multiple myeloma; WM=Waldenström's macroglobulinemia; AL=primary amyloidosis: SMM=smoldering multiple myeloma; MGUS=monoclonal gammopathy of undetermined significance
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
IMWG Guidelines for quantitative FLC: Impact on the Laboratory
Diagnosis:
•
Screening panel: serum PEL/IFE & FLC unless AL suspected.›
Do we need IFE?
Serum PEL & FLC are sufficient for MM screen.
Prognosis:•
MGUS progression (rFLC, PEL, IFE)›
Risk stratification should help define MGUS monitoring strategies
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
Prognosis Cut-points for FLC Ratio Depends on Plasma Cell Proliferative
Disorder
Diagnosis rFLC
MGUS <0.26 or >1.65
Plasmacytoma <0.26 or >4.0
Smoldering myeloma <0.125 or >8
Symptomatic myeloma <0.03 or >32
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
IMWG Guidelines for quantitative FLC: Impact on the Laboratory
Diagnosis:•
Screening panel: serum PEL/IFE & FLC unless AL suspected. ›
Do we need IFE?
Serum PEL & FLC are sufficient for MM screen.
Prognosis:•
FLC is prognostic in every PCD studied (MGUS, SMM, MM, AL, solitary plasmacytoma), and baseline values should be measured:
›
MGUS progression (rFLC, PEL, IFE)▪
Risk stratification should help define MGUS monitoring strategies›
Smoldering myeloma progression (rFLC, PEL, %BMPC)
›
Symptomatic myeloma survival (rFLC, β2M, Alb)›
Plasmacytoma survival (rFLC, IFE at 1 yr)›
AL amyloidosis survival (dFLC)
Monitoring:
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Department of Laboratory Medicine and Pathology
Monoclonal IgG lambda protein
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Department of Laboratory Medicine and Pathology
CV of M-spike measurement vs. M-spike size
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
CV of Monoclonal Protein Measurements in Stable Patients
Serum IgG
[IgG quant]
Measurable Serum M-spike
[M-spike>10g/L]
Measurable Urine M-spike
[M-spike>200 mg/24 hr]
Measurable Serum iFLC[iFLC>100
mg/L]
CV 13.0%(n=148)
8.1% (n=90)
35.8% (n=25)
28.4 (n=52)
Katzmann, J. Clin Chem, 2011
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
Calculation of Biologic Variability
Serum M- spike(≥10 g/L)
Urine M- spike (≥200 mg/24hr)
Serum iFLC (≥100 mg/L)
Total CV 8.1% 35.8% 28.4%
Analytic CV 2.1% 4.5% 5.8%
Biologic CV 7.8% 35.5% 27.8%
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Department of Laboratory Medicine and Pathology
Decrease needed in each assay for significance at various probability thresholds
Test n Total CV P=0.05 P=0.90 P=0.95
Serum M-
spike >10g/L
90 8.1% 7.4% 17.2% 20.1%
IgG 148 13.0% 11.7% 26.1% 30.3%Urine M-spike >200 mg/24 hr.
25 35.8% 28.8% 56.5% 62.9%
iFLC >100mg/dL
52 28.4% 23.7% 48.3% 54.5%
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
IMWG Guidelines for quantitative FLC: Impact on the Laboratory
Diagnosis:•
Screening panel: serum PEL/IFE & FLC unless AL suspected. ›
Serum PEL & FLC are sufficient for MM screen.
Prognosis:•
FLC is prognostic in every PCD studied (MGUS, SMM, MM, AL, solitary plasmacytoma), and baseline values should be measured.
›
Risk stratification should help define MGUS monitoring strategies
Monitoring:•
Oligosecretory PCD (AL, NSMM, LCDD…) 50% change for partial response
›
Criteria may change such that urine M-spike or
serum FLC can be used to monitor
›
90% reduction = partial response
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
Problems for electrophoretic diagnosis and monitoring of plasma cell dyscrasias
Free light chains: identification & monitoring•
FLC quantitation
Monitoring very large M-spikes on agarose•
Ig and/or HLC quantitation
Monitoring small M-proteins in γ or β fractions•
HLC quantitation
Broadly migrating M-proteins : identification & monitoring
•
HLC quantitation
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
Comparison of Agarose M-spikes and Ig Nephelometry Quantitation
IgA IgG
IgM
Murray et al, Clin Chem 2009
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Department of Laboratory Medicine and Pathology
Large IgG M-proteins are Artificially Low by SPEP Resulting in High Albumin Quantitation
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Department of Laboratory Medicine and Pathology
Overestimation of IgM by Nephelometry
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
Quantitation of Large M-proteinsBiases exist between agarose electrophoresis
and nephelometryIgA has good agreementIgG artificially lower results by SPEPIgM artificially higher results by NEPH
Clinicians should be encouraged not to switch methods when monitoring M-protein.
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
How to monitor Small M-proteins?
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Department of Laboratory Medicine and Pathology
Small monoclonal IgG kappa protein
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Department of Laboratory Medicine and Pathology
Quantitation and monitoring of very small M-proteins: Fuzzy γ, elevated β
or α
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
Principle of Hevylite™ Assay
Epitopes span the junction between immunoglobulin heavy chain and light chain and therefore can quantitate each HL pair
[GK, GL, AK, AL, MK, ML].
HLC junction
The Binding Site
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
Potential of HL-pair Ratios for Monitoring Small M-proteins
MGUS Monoclonal
protein isotype
Number of cases
Percent abnormal HLC-pair
ratios IgG 726 45% IgA 117 89%
IgM 156 90%
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
Identification of broadly migrating M-proteins: Hevylite Reagents
PEL
G
A
M
K
L
Pretransplant August, 2006 August 2010
IFE normal
IgA 757 mg/dL
κ/λ
1.08
IgA κ
=
638 mg/dL [55-292]IgA λ
= 115 mg/dL [39-249]
AK/AL = 5.6 [0.7-2.2]GK/GL = 1.8 [1.2-3.6]
Increase in IgA is not polyclonal-
preferential expression of IgA κ
Donato et al, Clin Chem, 2011
© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.
Department of Laboratory Medicine and Pathology
MADDOG: Myeloma, Amyloid, MADDOG: Myeloma, Amyloid, Dysproteinemia Disease Oriented GroupDysproteinemia Disease Oriented Group