International consensus
on serum free light
chain analysis
Alex Legg PhD
Scientific Affairs Manager
The Binding Site
Distributor in Poland BIOKOM
beata.olsz@biokom.com.pl
Serum free light chain immunoassay
Heavy
chain
Light
chain
Kappa
Hidden
surface
Exposed
surface
Lambda
Serum free light chain immunoassay
Heavy
chain
Light
chain
Kappa
Hidden
surface
Exposed
surface
Lambda
Light chain concentration (mg/L)
Sensitivity of light chain analysis techniques
1000
SPE
100
10
CZE
sIFE
UPE
Normal range in serum
1
sFLC
uIFE
Renal Metabolism of FLC
FLC - 25 kDa
FLC - 50 kDa
Glomerulus
40-60 kDa pores
10-30g/day
reabsorption
& breakdown
Urine
Renal Metabolism of FLC
FLC - 25 kDa
FLC - 50 kDa
Glomerulus
40-60 kDa pores
Renal metabolism results in
sFLC half-life of 2-6 hours
This is in contrast to intact
immunoglobulins which
have half-lives of 5-21 days
10-30g/day
reabsorption
& breakdown
Urine
Light Chain Multiple Myeloma
Normal sera
Kappa LCMM
Lambda LCMM
Renal impairment
(CKD2)
Hutchison, unpublished data; Bradwell, Lancet 2003; 361: 489-491
LCMM – Renal Threshold
3000
1
2000
0.5
1000
0
9
Time (months)
Urine Kappa BJP
(g/24hr)
0.2
0.15
3000
0.05
1000
0
0
2
15
Patient 3
sFLC
24 hr urine BJP excretion
3
5
9 12 13
Patient 2
HDC
ASCT
0.2
0
Time (months)
100
0.5
Time (months)
4000
2000
3
1
1
0.1
2
200
Patient 1
Severe
osteolytic
lesions
1
1.5
11
Urine Kappa BJP
(g/24hr)
6
300
2
0
0
1
2.5
400
Serum Kappa FLC
(mg/L)
1.5
Urine Kappa BJP
(g/24hr)
4000
Serum Lambda FLC
(mg/L)
2
Thalidomide
3
0.15
800
600
0.1
400
0.05
200
0
Serum Kappa FLC
(mg/L)
5000
HDC
ASCT
Serum Kappa FLC
(mg/L)
Urine Lambda BJP
(g/24hr)
2.5
0
1
2
3
4
Time (months)
5
Patient 4
Alyanakian Am J. Hematol. 2004; 75: 246 – 248
Nonsecretory Multiple Myeloma
10000
VAD
Kappa sFLC (mg/L)
ABCM
HDM/ PBSCT
1000
100
NR
10
0
0
10
20
30
40
50
60
70
80
Number of weeks since presentation
90
100
Intact Immunoglobulin MM
No correlation between Ig’ and sFLC
Serum  FLC (mg/L)
100000
n = 120 IgG MM patients
10000
1000
100
10
1
0
20
40
60
80
100
Total IgG (g/L)
Mead et al. Br J Haematol 2004;126 : 348 – 54
Rapid evaluation of response
to chemotherapy
Dispenzieri et al.
• Retrospective analysis of ECOG trial E9486
“FLC response after 2 months of
• VBMCP  IFN or  cyclophosphamide
therapy was superior to early
• 399 patients
M-protein measurement to predict
• Assessed sFLC and M-protein responses
overall response.”
to therapy
Dispenzieri et al. Blood 2008; 111: 4908 - 4915
Light chain escape
“Rising monoclonal free light chain
production at relapse without increased
monoclonal intact immunoglobulin”
Drayson et al.
• Myeloma IX trial
• Estimate incidence: 5% for IgG MM
15% for IgA MM
Drayson, M.T., et al., Clin Lymphoma Myeloma, 2009. February: 346a.
6xMP
PD
7xMP
No therapy
HD-Dex
HD-Mel
Tx
700
20
18
16
sFLC (mg/L)
600
14
500
12
IgAλ paraprotein
Serum lambda FLC
Creatinine
400
10
8
300
6
200
4
100
2
BM infiltration rate
100%
9/01
10/02
4/03
10/03
4/04
12/04
0
1/05
2/05
Date
Kϋhnemund, A., et al. J Cancer Res Clin Oncol, 2009. 135(3): p. 477-84
IgA (g/L), creatinine (mg/dL)
800
Dual plasma cell subsets
Stain with:
Plasma cell
Populations:
anti- IgG
Anti-Kappa
IgG
anti-BrDU
 FLC
Ayliffe Haematologica 2007; 92: 1135 - 1138
IgA (g/L)
Serum kappa FLC
(mg/L)
Model of light chain escape
Time
International guidelines for sFLC
analysis in MM and related disorders
• Assessment of response
• Serial sFLC should be routinely performed in:
• Oligosecretory MM/ NSMM
* • AL amyloidosis
* • LCDD (personal experience of authors)
• Periodic urine or sFLC assessment for LCE
Dispenzieri et al Leukemia (2009) 23, 215–224
sFLC identify residual disease
Serum λ FLC (mg/L)
1000
Normal sera
100
IFE negative MM
Normal /
10
Abnormal /
1
1
10
100
Serum κ FLC (mg/L)
1000
International guidelines for sFLC
analysis in MM and related disorders
• Assessment of response
• All MM patients to define a stringent CR
CR
Stringent CR
Negative S/U IFE
Negative S/U IFE
Normal sFLC ratio
BM plasma cells ≤ 5% Absence of clonal cells in BM
Dispenzieri et al. Leukemia 2009: 23, 215–224
Durie et al., Leukemia, 2006. 20, 1467-73
Screening for
Monoclonal Gammopathies
• Serum electrophoresis
→ Monoclonal intact immunoglobulins
• Urine electrophoresis
→ Monoclonal free light chains
Can the serum FLC assay replace
urine electrophoresis?
Screening for monoclonal gammopathy
sFLC + CZE
• 9 additional B-cell disorders identified from
1003 consecutive unknown samples
Bakshi et al. Am J Clin Path 2005; 124: 214-218
Adding the serum FLC assay to screening protocol
increased tumour detection rate by 56%
sFLC + SPE
• 8 additional monoclonal gammopathies
identified from 923 unknown samples
Hill et al. Clin Chem 2006; 52: 1743-1748
Replacement of urine tests with sFLC
• 428 urine samples positive by uIFE
• On paired serum samples:
Serum electrophoresis + sFLC missed only 2
urine positive light chain MGUS patients
No significant pathology was missed
Katzmann et al. Mayo Clin Proc 2006; 81: 1575 - 1578
International guidelines for sFLC
analysis in MM and related disorders
• Screening
– Recommended in combination with serum electrophoresis
– sFLC can replace 24 h urine IFE*
*For AL screening, 24 h urine IFE still recommended
Dispenzieri et al. Leukemia 2009: 23, 215–224
Serum FLCs are prognostic in:
• MGUS progression
• Myeloma outcome
• Smouldering MM progression
• Plasmacytoma progression
• AL amyloidosis outcome
• B-CLL outcome
• Waldenstrom’s outcome
Risk of progression 1% per year
MGUS
?
WM
IgM
lymphoma
CLL
AL
amyloid
Solitary
plasmacytoma
MM
LCDD
Follow up MGUS patients: How frequently?
Monoclonal Gammopathy of
Undetermined Significance (MGUS)
Risk Factors for progression:
• Serum M protein >15g/L
• Serum M protein NOT IgG
Kyle R. NEJM 2002; 346: 564-569
Monoclonal Gammopathy of
Undetermined Significance (MGUS)
Risk Factors for progression:
• Serum M protein >15g/L
• Serum M protein NOT IgG
•sFLC ratio
Kyle R. NEJM 2002; 346: 564-569
Rajkumar Blood 2005; 106: 812-817
60
(/ = <0.26 or >1.65)
Normal FLC ratio
(/ = 0.26 – 1.65)
10
20
30
40
50
Abnormal FLC ratio
0
Cumulative probability of progression (%)
MGUS progression
0
5
10
15
20
25
30
Years
Rajkumar Blood 2005; 106: 812-817
MGUS risk stratification model incorporating
M-protein size, type and FLC ratio
Risk of
progression
No. of
abnormal risk
factors
No.
patients
Absolute risk
of progression
at 20 years*
Low
0
449
2%
Low-Intermediate
1
420
10%
High-Intermediate
2
226
18%
High
3
53
27%
* Accounting for death as a competing risk
Rajkumar Blood 2005; 106: 812-817
FLC-MGUS may evolve into LCMM
-16
-8
0
Years prior to diagnosis of MM
No M-protein by sIFE, SPEP or sFLC
sFLC positive
sFLC and sIFE positive
Weiss et al. , Blood ePub Feb 20, 2009
IgA paraprotein
Serum kappa FLC
Creatinine
PD
DTC
No therapy
5
VAD
HD-Mel
18
Tx
4
HD-Mel
Tx
3.5
14
3
12
2.5
10
2
8
1.5
6
1
20%
0.5
BM infiltration rate
16
4
2
60%
0
0
4/04
7/04
10/04
12/04
1/05
2/05
3/05 7/05
Date
Kϋhnemund, A., et al. J Cancer Res Clin Oncol, 2009. 135(3): p. 477-84
IgA (g/L), creatinine (mg/dL)
4.5
sFLC (g/L)
20
International guidelines for sFLC
analysis in MM and related disorders
• Prognosis
– sFLC should be measured at diagnosis for all patients
with MGUS, SMM or MM, solitary plasmacytoma and
AL amyloidosis
Dispenzieri et al. Leukemia 2009: 23, 215–224
Summary of IMWG sFLC Guidelines
Intact Ig
MM
Screening
Light
Non
chain secretory
MM
MM
+ serum electrophoresis
Prognosis
Monitoring
(Oligosecretory)
(LCE)
(sCR)
Dispenzieri, A., et al. Leukemia, 2009. 23(2): p. 215-24
Kyle, R.A. and S.V. Rajkumar. Leukemia, 2009. 23(1): p. 3-9
Any Questions?
• alex.legg@bindingsite.com
Distributor in Poland BIOKOM
beata.olsz@biokom.com.pl
Serum free light chain
immunoassay platforms
BECKMAN COULTER
IMMAGE, IMMAGE 800
BINDING SITE
SPAPLUS
OLYMPUS
AU400 / 640 / 2700 / 5400
ROCHE
MODULAR P
Cobas Integra 400 / 800
Cobas c501
ROCHE (Hitachi)
911, 912 , 917
SIEMENS (Bayer)
ADVIA 1650 / 1800 / 2400
SIEMENS (Dade Behring)
BNII, Prospec
Normal

5.0 10.0
50.0
500.0

0.5 1.0
Relative Risk of Progression
MGUS progression
0.01
0.10
0.26
1.0 1.65
10.00
100.00
Free Light Chain Ratio
Rajkumar Blood 2005: 106: 812-817
Multiple myeloma survival
risk factors*
0
1
2
3
Overall survival %
100
80
60
med. survival
51 m
39 m
30 m
22 m
40
20
0
0
2
4
6
8
10
12
14
Years
* sFLC / <0.03 or >32, albumin < 35 g/L, 2MG ≥ 3.5 mg/L
Snozek, C.L., et al., Leukemia, 2008. 22:1933-7
Risk stratification summary:
Risk Factors
No.
Serum FLC ratio
Other risk factors
MGUS
3
<0.26 or >1.65
M-protein size
M-protein type
SPB
2
<0.26 or >1.65
M-protein size
SMM
3
<0.125 or >8
% BM plasma cells
M-protein size
MM
3
<0.03 or >32
Albumin
2-microglobulin
Renal Metabolism of FLC
FLC - 25 kDa
FLC - 50 kDa
Glomerulus
40-60 kDa pores
10-30g/day
reabsorption
& breakdown
Urine
Light chain escape
“Rising monoclonal free light chain
production at relapse without increased
monoclonal intact immunoglobulin”
Kühnemund et al:
• Largest case series of LCE
• Estimate incidence is 2.5% MM at relapse
• 5/10 cases of LCE had renal impairment
Kuhnemund, A., et al., J Cancer Res Clin Oncol, 2009. 135, 477-84
Light chain escape
IgA paraprotein
Serum lambda FLC
Velcade
5000
4000
25
3000
20
15
2000
10
1000
5
0
0
100
200
300
IgA paraprotein (g/L)
Serum lambda FLC
(mg/L)
30
0
400
Time (days)
Courtesy of Effie Liakopoulou, Christies Hospital
Prevalence of MGUS
Prevalence of MGUS (%)
10
8
6
4
2
0
50
60
70
80
90
Age (years)
Kyle NEJM 2006, 354: 1362 – 1369