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© 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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Page 1: 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

Page 2: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

Department of Laboratory Medicine and Pathology

DISCLOSURES:

Travel expenses, The Binding Site, Ltd.TBS FLC & HLC immunoassay reagents at no cost for clinical studies

Page 3: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

Department of Laboratory Medicine and Pathology

Page 4: Jerry Katzmann

© 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

Page 5: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

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)

Page 6: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

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

Page 7: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

Department of Laboratory Medicine and Pathology

Serum & Urine Protein Electrophoresis and Immunofixation Electrophoresis

LCMM Serum LCMM Urine

Page 8: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

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

Page 9: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

Department of Laboratory Medicine and Pathology

Serum & Urine Protein Electrophoresis and Immunofixation Electrophoresis:

Primary Amyloid

AL, Serum AL, Urine

Page 10: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

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

Page 11: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

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

Page 12: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

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

Page 13: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

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

Page 14: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

Department of Laboratory Medicine and Pathology

Katzmann, J. Clin Chem, 2009

Page 15: Jerry Katzmann

© 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

Page 16: Jerry Katzmann

© 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:

Page 17: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

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

Page 18: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

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)

Page 19: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

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

Page 20: Jerry Katzmann

© 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

Page 21: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

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

Page 22: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

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

Page 23: Jerry Katzmann

© 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

Page 24: Jerry Katzmann

© 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

Page 25: Jerry Katzmann

© 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:

Page 26: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

Department of Laboratory Medicine and Pathology

Monoclonal IgG lambda protein

Page 27: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

Department of Laboratory Medicine and Pathology

CV of M-spike measurement vs. M-spike size

Page 28: Jerry Katzmann

© 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

Page 29: Jerry Katzmann

© 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%

Page 30: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

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%

Page 31: Jerry Katzmann

© 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

Page 32: Jerry Katzmann

© 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

Page 33: Jerry Katzmann

© 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

Page 34: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

Department of Laboratory Medicine and Pathology

Large IgG M-proteins are Artificially Low by SPEP Resulting in High Albumin Quantitation

Page 35: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

Department of Laboratory Medicine and Pathology

Overestimation of IgM by Nephelometry

Page 36: Jerry Katzmann

© 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.

Page 37: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

Department of Laboratory Medicine and Pathology

How to monitor Small M-proteins?

Page 38: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

Department of Laboratory Medicine and Pathology

Small monoclonal IgG kappa protein

Page 39: Jerry Katzmann

© 2010 Mayo Foundation for Medical Education and Research. All Rights Reserved.

Department of Laboratory Medicine and Pathology

Quantitation and monitoring of very small M-proteins: Fuzzy γ, elevated β

or α

Page 40: Jerry Katzmann

© 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

Page 41: Jerry Katzmann

© 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%

Page 42: Jerry Katzmann

© 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

Page 43: Jerry Katzmann

© 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


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