Abstract

Immunoglobulin free light chains (FLCs) are the precursors of amyloid fibrils in primary amyloidosis (AL). We studied the relationship between FLC levels and clinical features in 730 patients with newly diagnosed AL. The plasma cell clone was λ in 72% patients, and κ in 28% patients. κ-AL had more GI tract and liver involvement, where as renal involvement was more with λ-AL. While the overall survival (OS) was similar for κ and λ-AL, the median OS for those without an identifiable serum heavy chain was significantly shorter (12.6 vs 29.9 months; P = .02). The OS was shorter among those with a higher dFLC (involved FLC−uninvolved FLC; κ > 29.4 mg/dL or λ > 18.2 mg/dL using median for cutoff); 10.9 vs 37.1 months; P < .001. In multivariate analysis, dFLC was independent of other prognostic factors. The type of light chain impacts the spectrum of organ involvement and the FLC burden correlates with survival in AL.

Introduction

Primary systemic or light-chain amyloidosis (AL) is characterized by multiorgan deposition of amyloid fibrils derived from immunoglobulin free light chains (FLCs), either κ or λ.1-5  The introduction of a nephelometric FLC assay (Freelite) has enabled quantification of circulating FLCs.6-12  FLC assay, used along with serum and urine protein electrophoresis and immunofixation, significantly improves the detection of monoclonal proteins in AL.13  The FLC assay by measuring the amyloid precursor protein provides us a unique opportunity to study disease biology. We undertook this study in a large cohort of patients with long follow-up, to better define the impact of the FLC measurements on clinical characteristics and survival.

Methods

The current study included patients with biopsy proven AL seen at Mayo Clinic between 1980 and 2006, who had FLC measurements within 90 days of diagnosis performed as part of clinical evaluation or subsequently on stored serum. Proof of a clonal plasma cell process, either by presence of monoclonal protein (on serum or urine protein electrophoresis or immunofixation or serum FLC assay) or presence of clonal marrow plasma cells, was required. Of the 1938 patients seen during this period, 730 (38%) satisfied the criteria. The study was conducted with approval from Mayo Clinic Institutional Review Board.

Major organ (cardiac, hepatic, or renal) involvement was defined as previously described. Renal, cardiac or hepatic involvement required a positive biopsy of the respective organ or 24-hour urine protein excretion > 0.5 g/d, an interventricular septal thickness > 12 mm, or an alkaline phosphatase > 1.5× normal, respectively. We used decreased serum carotene as a marker for intestinal involvement and resultant malabsorption.

Serum FLC quantitation was carried out as previously described using the Freelite FLC assay (The Binding Site Limited). The clonal light-chain is considered the “involved” FLC (iFLC) and the other is referred to as the “uninvolved” FLC (uFLC), with the numerical difference between the 2 denoted by dFLC.

The χ2 and Fisher exact tests were used to compare differences between nominal variables and the Mann-Whitney U test or Kruskal-Wallis test for continuous variables. Kaplan-Meier analysis was used for analyzing overall survival (OS), and survival curves were compared using the log-rank test.14  Curves were generated with all patients surviving beyond 10 years censored at that time. Multivariate analysis was performed using the Cox Proportional Hazards model.15 

Results and discussion

The median age was 63.3 years (range, 32-90 years) with 463 (63%) males; and the estimated median follow-up from diagnosis was 58.4 months with 212 patients (29%) alive at the time of analysis. The baseline laboratory and clinical features are described in Table 1. The κ/λ FLC ratio was abnormal (< 0.26 or > 1.65) in 644 patients (88%), consistent with previous reports comparing the FLC assay to electrophoretic tests in serum and urine.7,9-11  Based on immunofixation, marrow immunohistochemistry or FLC assay, the clonal light-chain was determined to be λ in 528 (72.3%) patients, unlike in myeloma where κ is more often (60%) the clonal light chain.9,16  Also in contrast to myeloma, only 366 (51.3%) of the 714 patients with immunofixation results, had a detectable heavy chain. The median iFLC and dFLC was higher for κ-AL (31.4 and 29.4 mg/dL, respectively) compared with 19.4 and 18.2 mg/dL, respectively, for the λ-AL. This is in contrast with myeloma where the median involved κ and λ concentrations were 37.1 and 71.3 mg/dL, respectively in one large study.16  This is likely a reflection of the higher tumor burden and the higher prevalence of renal insufficiency in the κ-AL patients (Table 1).

Table 1

Baseline characteristics and relationship between immunoglobulin light-chain type/levels and organ involvement

Variable All patients Light-chain type
 
dFLC
 
κ-restricted patients λ-restricted patients P High (> 19.6 mg/dL) Low (≤ 19.6 mg/dL) P 
Median κ FLC, mg/dL (range) 1.6 (0.032-1360) 31.4 (0.3-1360) 1.2 (0.032-13) NA    
Median λ FLC, mg/dL (range) 11.4 (0.06-2480) 1.3 (0.06-24) 19.4 (0.9-2480) NA    
κ:λ ratio abnormal, n (%) 644 (88) 191 (95) 453 (86) < .001    
Median dFLC, mg/dL (range) 19.6 (0.01-2478) 29.4 (0.01-1359) 18.2 (0.03-2478) < .001 56.2 (19.8-2478) 7.8 (0.01-19.6) NA 
Median plasma cell % (range) 8 (0-95) 9.5 (1-95) 8 (0-90) .04 10 (2-95) 6 (0-60) < .001 
Heart, n (%) 445 (65) 109 (60) 331 (67) NS 244 (71) 196 (59) < .001 
    Septum > 12 mm 423 (63) 104 (58) 316 (65) .09 233 (69) 187 (57) .001 
    EF < 50% 151 (23) 34 (19) 117 (23) NS 100 (30) 51 (16) < .001 
    cTnT > 0.035 ng/mL 265 (44) 60 (38) 205 (47) .05 154 (53) 111 (36) < .001 
    NT-ProBNP > 332 pg/mL 345 (70) 96 (71) 249 (70) NS 244 (71) 196 (59) < .001 
Liver, n (%) 129 (20) 57 (31) 72 (16) < .0001 65 (20) 64 (20) NS 
    Bilirubin > 1.5 mg/dL 63 (10) 23 (13) 40 (9) NS 40 (12.5) 23 (7) .03 
    Alk Phos > 1.5 × ULN 118 (18) 51 (28) 67 (14) .0002 59 (18) 59 (18) NS 
Median serum carotene (range) 126 (12-662) 113 (12-370) 130 (23-662) .0006 107 (12-363) 141 (36-662) < .001 
Kidney, n (%) 385 (55) 80 (41) 35 (60) < .0001 158 (45) 227 (64) < .001 
    Creatinine > 1.5 mg/dL 160 (24) 62 (33) 98 (21) .0012 93 (28) 67 (20) .02 
    Urine albumin > 3 g/d 198 (29) 35 (19) 163 (33) .0003 79 (23) 119 (34) .007 
    Serum albumin < 3.5 g/dL 575 (80) 142 (74) 433 (83) .08 279 (78) 296 (83) .07 
Variable All patients Light-chain type
 
dFLC
 
κ-restricted patients λ-restricted patients P High (> 19.6 mg/dL) Low (≤ 19.6 mg/dL) P 
Median κ FLC, mg/dL (range) 1.6 (0.032-1360) 31.4 (0.3-1360) 1.2 (0.032-13) NA    
Median λ FLC, mg/dL (range) 11.4 (0.06-2480) 1.3 (0.06-24) 19.4 (0.9-2480) NA    
κ:λ ratio abnormal, n (%) 644 (88) 191 (95) 453 (86) < .001    
Median dFLC, mg/dL (range) 19.6 (0.01-2478) 29.4 (0.01-1359) 18.2 (0.03-2478) < .001 56.2 (19.8-2478) 7.8 (0.01-19.6) NA 
Median plasma cell % (range) 8 (0-95) 9.5 (1-95) 8 (0-90) .04 10 (2-95) 6 (0-60) < .001 
Heart, n (%) 445 (65) 109 (60) 331 (67) NS 244 (71) 196 (59) < .001 
    Septum > 12 mm 423 (63) 104 (58) 316 (65) .09 233 (69) 187 (57) .001 
    EF < 50% 151 (23) 34 (19) 117 (23) NS 100 (30) 51 (16) < .001 
    cTnT > 0.035 ng/mL 265 (44) 60 (38) 205 (47) .05 154 (53) 111 (36) < .001 
    NT-ProBNP > 332 pg/mL 345 (70) 96 (71) 249 (70) NS 244 (71) 196 (59) < .001 
Liver, n (%) 129 (20) 57 (31) 72 (16) < .0001 65 (20) 64 (20) NS 
    Bilirubin > 1.5 mg/dL 63 (10) 23 (13) 40 (9) NS 40 (12.5) 23 (7) .03 
    Alk Phos > 1.5 × ULN 118 (18) 51 (28) 67 (14) .0002 59 (18) 59 (18) NS 
Median serum carotene (range) 126 (12-662) 113 (12-370) 130 (23-662) .0006 107 (12-363) 141 (36-662) < .001 
Kidney, n (%) 385 (55) 80 (41) 35 (60) < .0001 158 (45) 227 (64) < .001 
    Creatinine > 1.5 mg/dL 160 (24) 62 (33) 98 (21) .0012 93 (28) 67 (20) .02 
    Urine albumin > 3 g/d 198 (29) 35 (19) 163 (33) .0003 79 (23) 119 (34) .007 
    Serum albumin < 3.5 g/dL 575 (80) 142 (74) 433 (83) .08 279 (78) 296 (83) .07 

The reference range for κFLC is 0.33-1.94 mg/dL, for λFLC is 0.57-2.63 mg/dL, and for the κ:λ ratio is 0.26-1.65.

NA indicates not applicable; and NS, not significant.

Our study highlights interesting associations between the clinical features and the type and levels of FLC as shown in Table 1. Overall, cardiac, renal, and liver involvement was seen in 65%, 55%, and 20% of patients, respectively. Patients with κ-AL were more likely to have liver and GI tract involvement. While the proportion of patients with nephrotic range proteinuria was higher among λ-AL patients, the proportion with serum creatinine > 1.5 mg/dL was higher among κ-AL patients. No relationship was found between frequency of cardiac involvement and type of light chain.

To assess the relationship between FLC burden and clinical features, patients were divided into a high (> 19.6 mg/dL) and low (<= 19.6 mg/dL) FLC group, using the median. Patients with high dFLC had more frequent and severe cardiac involvement with lower ejection fraction, and higher levels of cardiac biomarkers troponin T and NT-ProBNP, consistent with previous reports (Table 1).9  Similarly, higher dFLC was associated with more severe GI and renal involvement.

Previous studies have demonstrated prognostic value for FLC in different plasma cell disorders including MGUS, myeloma, and amyloidosis.17,18  In AL, high baseline FLC level was associated with poor outcome in patients undergoing stem cell transplant and a reduction in FLC was associated with improved outcome.19-21  One study did not find a prognostic value for baseline FLC levels, but was limited by small patient numbers.11  In the current study, the median overall survival (OS) for the 86 patients with a normal κ/λ FLC-ratio (no clonal excess of light chain) was 63.6 months compared with 16.2 months for the remaining 644 patients (P < .001; Figure 1A). In terms of the FLC burden, the median OS for patients with high dFLC was 10.1 months compared with 38.2 months for those with low dFLC (P < .001; Figure 1B). Given the significantly different median value for the dFLC between κ-AL and λ-AL patients, we repeated the analyses using the respective medians for determining the high and low groups (29.4 mg/dL for κ-AL patients, 18.2 mg/dL for λ-AL patients). The results were similar; the median OS among patients with a high dFLC was 10.9 months compared with 37.1 months (P < .001) (Figure 1C). Given that treatments for AL have changed in recent years, we repeated the analysis using the more recent group of patients (1998-2006). The results were similar with a median OS for the high dFLC group of 11 months compared with 66 months for the rest (P < .001). In a multivariate model including NT-ProBNP, troponin T, number of organs involved, ventricular septal thickness, ejection fraction (EF), circulating plasma cells, and serum uric acid level, dFLC was an independent predictor of survival. The impact of elevated free light chains on survival likely represents the increased availability of precursor light chain for amyloid fibril formation. Interestingly, this may also explain the poor prognosis seen with t(11;14) in AL as translocations involving the heavy chain locus are associated with higher FLC levels.22,23 

Figure 1

Relationship between overall survival outcome and serum free light- chain measurements. (A) Comparison of OS between patients with an abnormal FLC (κ/λ) ratio to those with a normal ratio. The median OS for the 86 patients with a normal κ/λ FLC ratio (no clonal excess of light chain) was 63.6 months (95% confidence interval[CI]; 39, 92) compared with 16.2 months (95% CI; 12, 19) for the remaining 644 patients (P < .001). (B) Comparison of OS from diagnosis between patients with a high dFLC using the same cutoff for κ-AL and λ-AL patients (19.6 mg/dL). The median OS for patients with a high dFLC was 10.1 months (95% CI; 7, 13) compared with 38.2 months (95% CI; 29, 51) for those with a low dFLC (P < .001). (C) Comparison of OS from diagnosis between patients with a high dFLC using different cutoffs (individual median values) for κ-AL (29.4 mg/dL) and λ-AL patients (18.2 mg/dL) and low dFLC. The median OS among patients with a high dFLC was 10.9 months (95% CI; 8, 13) compared with 37.1 months (95% CI; 27, 47; P < .001).

Figure 1

Relationship between overall survival outcome and serum free light- chain measurements. (A) Comparison of OS between patients with an abnormal FLC (κ/λ) ratio to those with a normal ratio. The median OS for the 86 patients with a normal κ/λ FLC ratio (no clonal excess of light chain) was 63.6 months (95% confidence interval[CI]; 39, 92) compared with 16.2 months (95% CI; 12, 19) for the remaining 644 patients (P < .001). (B) Comparison of OS from diagnosis between patients with a high dFLC using the same cutoff for κ-AL and λ-AL patients (19.6 mg/dL). The median OS for patients with a high dFLC was 10.1 months (95% CI; 7, 13) compared with 38.2 months (95% CI; 29, 51) for those with a low dFLC (P < .001). (C) Comparison of OS from diagnosis between patients with a high dFLC using different cutoffs (individual median values) for κ-AL (29.4 mg/dL) and λ-AL patients (18.2 mg/dL) and low dFLC. The median OS among patients with a high dFLC was 10.9 months (95% CI; 8, 13) compared with 37.1 months (95% CI; 27, 47; P < .001).

We also examined if the type of light chain influenced outcome and found no relationship; median OS was 18.4 months for κ-AL patients compared with 19 months for λ-AL patients (P = .2). However, the fact that outcomes were similar despite lower levels of iFLC and dFLC for λ-AL patients suggest that λ-light chains might be more “amyloidogenic.” Interestingly patients without an identifiable heavy chain had an inferior survival, 12.6 compared with 29.3 months for those with a heavy chain identified (P = .02). It is important to note that patients without a heavy chain also had higher dFLC (25.5 vs 15.3 mg/dL; P < .001), which likely impacted the extent of organ deposition and outcome. However, in a multivariate model including dFLC and the presence/absence of heavy chain, both were independently prognostic for survival. Unbound light chains may be inherently more prone to undergo misfolding into an amyloid configuration and might explain this finding.

In conclusion, the results of this study provide several valuable observations. The association between type of light chain and organ involvement provides unique insights and can potentially improve our understanding of biology. Finally, it provides an assessment of the prognostic value of FLC measurements in AL paving the way for its incorporation in future risk stratification models.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Acknowledgments

This work was supported in part by Hematologic Malignancies Program, Paul Calabresi K12 grant (S.K.) and grants CA62242, CA93842, and CA10080 from the National Cancer Institute, National Institutes of Health, and the Department of Health and Human Services.

National Institutes of Health

Authorship

Contribution: S.K. designed the study, analyzed the data, and wrote the paper; R.J.C. performed the FLC assays; and A.D., J.A.K., D.R.L., C.L.C., S.R.Z., M.Q.L., S.R.H., F.K.B., N.L., M.R.-A., R.A.K., S.V.R., and M.A.G. were involved in the writing and reviewing of the manuscript.

Conflict-of-interest disclosure: A.D. has received honorarium from Binding Site. The remaining authors declare no competing financial interests.

Correspondence: Shaji Kumar, MD, Division of Hematology, Mayo Clinic, 200 First St SW, Rochester, MN 55905; e-mail: kumar.shaji@mayo.edu.

References

References
1
Gertz
 
MA
Lacy
 
MQ
Dispenzieri
 
A
Hayman
 
SR
Amyloidosis.
Best Pract Res Clin Haematol
2005
, vol. 
18
 
4
(pg. 
709
-
727
)
2
Gertz
 
MA
Comenzo
 
R
Falk
 
RH
, et al. 
Definition of organ involvement and treatment response in immunoglobulin light chain amyloidosis (AL): a consensus opinion from the 10th International Symposium on Amyloid and Amyloidosis, Tours, France, April 18-22, 2004.
Am J Hematol
2005
, vol. 
79
 
4
(pg. 
319
-
328
)
3
Kyle
 
RA
Gertz
 
MA
Primary systemic amyloidosis: clinical and laboratory features in 474 cases.
Semin Hematol
1995
, vol. 
32
 
1
(pg. 
45
-
59
)
4
Skinner
 
M
Cohen
 
AS
Amyloidosis: clinical, pathologic, and biochemical characteristics.
Monogr Pathol
1983
, vol. 
24
 (pg. 
97
-
119
)
5
Kyle
 
RA
Bayrd
 
ED
“Primary” systemic amyloidosis and myeloma. Discussion of relationship and review of 81 cases.
Arch Intern Med
1961
, vol. 
107
 (pg. 
344
-
353
)
6
Abraham
 
RS
Katzmann
 
JA
Clark
 
RJ
Bradwell
 
AR
Kyle
 
RA
Gertz
 
MA
Quantitative analysis of serum free light chains. A new marker for the diagnostic evaluation of primary systemic amyloidosis.
Am J Clin Pathol
2003
, vol. 
119
 
2
(pg. 
274
-
278
)
7
Katzmann
 
JA
Clark
 
RJ
Abraham
 
RS
, et al. 
Serum reference intervals and diagnostic ranges for free kappa and free lambda immunoglobulin light chains: relative sensitivity for detection of monoclonal light chains.
Clin Chem
2002
, vol. 
48
 
9
(pg. 
1437
-
1444
)
8
Katzmann
 
JA
Kyle
 
RA
Benson
 
J
, et al. 
Screening panels for detection of monoclonal gammopathies.
Clin Chem
2009
, vol. 
55
 
8
(pg. 
1517
-
1522
)
9
Bochtler
 
T
Hegenbart
 
U
Heiss
 
C
, et al. 
Evaluation of the serum-free light chain test in untreated patients with AL amyloidosis.
Haematologica
2008
, vol. 
93
 
3
(pg. 
459
-
462
)
10
Akar
 
H
Seldin
 
DC
Magnani
 
B
, et al. 
Quantitative serum free light chain assay in the diagnostic evaluation of AL amyloidosis.
Amyloid
2005
, vol. 
12
 
4
(pg. 
210
-
215
)
11
Morris
 
KL
Tate
 
JR
Gill
 
D
, et al. 
Diagnostic and prognostic utility of the serum free light chain assay in patients with AL amyloidosis.
Intern Med J
2007
, vol. 
37
 
7
(pg. 
456
-
463
)
12
Bradwell
 
AR
Carr-Smith
 
HD
Mead
 
GP
, et al. 
Highly sensitive, automated immunoassay for immunoglobulin free light chains in serum and urine.
Clin Chem
2001
, vol. 
47
 
4
(pg. 
673
-
680
)
13
Katzmann
 
JA
Abraham
 
RS
Dispenzieri
 
A
Lust
 
JA
Kyle
 
RA
Diagnostic performance of quantitative kappa and lambda free light chain assays in clinical practice.
Clin Chem
2005
, vol. 
51
 
5
(pg. 
878
-
881
)
14
Kaplan
 
E
Meier
 
P
Non-parametric estimation from incomplete observations.
J Am Stat Assoc
1958
, vol. 
53
 (pg. 
457
-
481
)
15
Cox
 
D
Regression models and life tables.
J R Stat Soc
1972
, vol. 
34
 (pg. 
187
-
202
)
16
Snozek
 
CL
Katzmann
 
JA
Kyle
 
RA
, et al. 
Prognostic value of the serum free light chain ratio in newly diagnosed myeloma: proposed incorporation into the international staging system.
Leukemia
2008
, vol. 
22
 
10
(pg. 
1933
-
1937
)
17
Rajkumar
 
SV
Kyle
 
RA
Therneau
 
TM
, et al. 
Presence of monoclonal free light chains in the serum predicts risk of progression in monoclonal gammopathy of undetermined significance.
Br J Haematol
2004
, vol. 
127
 
3
(pg. 
308
-
310
)
18
Dispenzieri
 
A
Kyle
 
RA
Katzmann
 
JA
, et al. 
Immunoglobulin free light chain ratio is an independent risk factor for progression of smoldering (asymptomatic) multiple myeloma.
Blood
2008
, vol. 
111
 
2
(pg. 
785
-
789
)
19
Dispenzieri
 
A
Lacy
 
MQ
Katzmann
 
JA
, et al. 
Absolute values of immunoglobulin free light chains are prognostic in patients with primary systemic amyloidosis undergoing peripheral blood stem cell transplantation.
Blood
2006
, vol. 
107
 
8
(pg. 
3378
-
3383
)
20
Lachmann
 
HJ
Gallimore
 
R
Gillmore
 
JD
, et al. 
Outcome in systemic AL amyloidosis in relation to changes in concentration of circulating free immunoglobulin light chains following chemotherapy.
Br J Haematol
2003
, vol. 
122
 
1
(pg. 
78
-
84
)
21
Sanchorawala
 
V
Seldin
 
DC
Magnani
 
B
Skinner
 
M
Wright
 
DG
Serum free light-chain responses after high-dose intravenous melphalan and autologous stem cell transplantation for AL (primary) amyloidosis.
Bone Marrow Transplant
2005
, vol. 
36
 
7
(pg. 
597
-
600
)
22
Bryce
 
AH
Ketterling
 
RP
Gertz
 
MA
, et al. 
Translocation t(11;14) and survival of patients with light chain (AL) amyloidosis.
Haematologica
2009
, vol. 
94
 
3
(pg. 
380
-
386
)
23
Kumar
 
S
Zhang
 
L
Dispenzieri
 
A
, et al. 
Relationship between elevated immunoglobulin free light chain and the presence of IgH translocations in multiple myeloma.
Leukemia
2010
, vol. 
24
 
8
(pg. 
1498
-
1505
)