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IMMUNOBIOLOGY
From the Division of Hematologic Oncology, Department
of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY; the
Hospital Clinic of Barcelona, Catalonia, Spain; and the Department of
Pathology, Medicine and Cell Biology and Anatomy, Louisiana State
University Health Sciences Center, Shreveport.
Primary systemic amyloidosis (AL) is a protein conformation
disorder in which monoclonal immunoglobulin light chains produced by
clonal plasma cells are deposited as amyloid in the kidneys, heart,
liver, or other organs. Why patients with AL present with amyloid
disease that displays such organ tropism is unknown. This study tested
the hypothesis that both the light-chain variable region (Ig
VL) germ line genes used by AL clones and the
plasma cell burden influenced AL organ tropism. To assess the renal
tropism of some light chains, an in vitro renal mesangial cell model of amyloid formation was used. With reverse transcription-polymerase chain
reaction, Ig VL genes were sequenced from 60 AL
patients whose dominant involved organs were renal (52%), cardiac
(25%), hepatic (8%), peripheral nervous system (8%), and soft tissue and other (7%). Patients with clones derived from the 6a
V Primary systemic amyloidosis
(AL) is a rare protein conformation and clonal plasma cell
disorder similar to multiple myeloma.1,2 In AL, fibrillar
material usually composed of the amino termini of immunoglobulin light
chains (often of the During early B-cell development, immunoglobulin germ
line genes rearrange with retention of selected genes, endowing each B
cell with one heavy-chain and one light-chain variable region germ line
gene to encode the hypervariable or complementarity-determining regions
(CDRs) of the immunoglobulin protein. In the antigen-dependent stage of
B-cell development, the rearranged germ line genes mutate at a much
higher rate than somatic genes, a process that results in unique
immunoglobulin variable region gene sequences (Ig
VL; VL = V In this report we identify clonal Ig VL
genes from patients with AL to test the hypothesis that the tropism of
organ involvement is a function of Ig VL germ
line gene use and plasma cell burden. We used the presence of myeloma
as defined by the Durie diagnostic criteria as a surrogate for plasma
cell burden and, for the sake of analysis, assumed that patients with
myeloma have a greater plasma cell burden and produce more
amyloid-forming light chains.21 In addition, we used an in
vitro renal mesangial cell model for amyloid formation to assess the
relative activities of AL light chains of different isotypes. Our
results demonstrate that Ig VL germ line gene
use in AL is preferential, involving several genes that make minimal
contributions to the normal repertoire, and that the tropism of organ
involvement in AL is significantly influenced by Ig
VL germ line gene use and clonal plasma cell burden.
Patients and plasma cell disease
Renal involvement was defined as proteinuria more than 0.5 g/d and
renal failure as dialysis dependence or creatinine clearance less than
10 mL/min. Cardiac involvement was defined as the presence of a mean
left ventricular wall thickness on echocardiogram more than 11 mm in
the absence of a history of hypertension or valvular heart disease, or
as the presence of unexplained low voltage (< 0.5 mV) on the
electrocardiogram. Patients who were New York Heart Association (NYHA)
class 1 with evidence of cardiac amyloid by echocardiogram or
electrocardiogram were categorized as having asymptomatic cardiac
involvement. Patients who were NYHA class 2 or higher with evidence of
cardiac involvement were categorized as having dominant cardiac
involvement. Neuropathic involvement was defined based on clinical
history, autonomic dysfunction with orthostasis, gastric atony by
gastric emptying scan, and abnormal sensory or motor findings on
neurologic examination. Hepatic involvement was defined as hepatomegaly
with an alkaline phosphatase level more than 200 U/L.
Specimen preparation and cloning of Ig
VL genes
Sequence analysis of VL genes and
proteins
Isolation of urinary immunoglobulin light chains and culture of human mesangial cells Urinary immunoglobulin light chains from patients with plasma cells diseases were purified and characterized as previously described.30-32 Human mesangial cells were obtained from kidneys procured for transplantation but not used or from normal areas of nephrectomy specimens, after obtaining written informed consent under protocols approved by the institutional review board. The cortex was dissected and glomeruli isolated as previously described.30-32 The pellets containing glomeruli were resuspended for culture in medium containing RPMI 1640 (Life Technologies) buffered with 12.5 mM HEPES (Sigma-Aldrich, St Louis, MO) at pH 7.4 and supplemented with 20% heat-inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT), penicillin/streptomycin, and 5 µg/mL bovine insulin. Cellular outgrowths were observed 3 to 5 days after attachment of glomeruli to culture plates. Once outgrowths were established, cells were trypsinized, passed through a 75-µm sieve to remove whole glomeruli, and replated on 100-mm tissue culture dishes. Mesangial cells overgrew epithelial cells and became confluent 3 to 4 weeks after plating, were maintained in culture, and were analyzed as previously described for muscle-specific actin, vimentin, factor VIII, and keratin.31-33 The presence of the first 2, and absence of the last 2, confirmed that the cells were a homogeneous population of mesangial cells. In addition, ultrastructural evaluation confirmed morphologic findings including myofilaments and attachment plaques, indicative of mesangial cells. Second-passage mesangial cells grown on coverslips were used for these experiments.Amyloid formation and evaluation Amyloid-enhancing factor (AEF) was extracted from murine spleens and purified.34 Three days before incubation with purified light chains, the fetal bovine serum concentration of the mesangial cell medium was reduced to 0.5%. Mesangial cell cultures were incubated with purified light chains with and without AEF as indicated for up to 96 hours in triplicate for each experimental situation as previously described.31-33 Then, mesangial cells on coverslips were fixed in 80% ethanol and stained with hematoxylin and eosin and, for amyloid, with Congo red and thioflavin-T. Congo red-stained sections were viewed in polarized light using a BH2 Olympus microscope (C-squared, Tamarac, FL). The sections stained with thioflavin-T were examined under fluorescent light using a BH2 microscope with UV light capabilities, a Schott 4G2 exciter filter, and a simple UV filter passing only visible light as a barrier filter. Presence of amyloid was evaluated on examination of sections stained with Congo red and thioflavin-T by counting the number of apple-green birefringent or strongly fluorescent complexes at × 10 in 10 fields in each coverslip and computing the average number of complexes. The counts were repeated 3 times for each experimental condition and performed by 2 independent observers. Results were graded as 0 to 3+ using the following criteria: 0 = no amyloid complexes, trace = questionable amyloid complexes, 1+ = 1 to 5 complexes, 2+ = 5 to 10 complexes, and 3+ = >10 complexes.Statistics Means, SDs, medians, and ranges were calculated, and tests for significance performed, with PRISM (Graph Pad, San Diego, CA).
Patients with AL and plasma cell disorders Clonal Ig VL germ line genes were identified in 60 patients with AL, representing 72% of cases in which reverse transcription-PCR (RT-PCR) was attempted (60 of 83). In the unsuccessful instances, either the material was too scant or the percent plasma cells too low. The characteristics of these 60 patients and the plasma cell disorders are shown in Table 2. Fifty-two percent had dominant renal and 25% dominant cardiac amyloid. Seventy-five percent had clones
and 38% had clonal plasma cell disorders that met criteria for myeloma.
AL Ig VL germ line genes In these 60 cases, a median of 5 identical sequences were cloned per gene (range, 3-7 sequences) and in only 3 cases was a second Ig VL gene amplified, requiring sequencing of additional inserts to identify the predominant clone (ratios of 8:1, 4:1, and 6:2). The V I,
VVI , and
V I subtypes provided germ line
donors for 75% of the clones, whereas the remainder derived from germ
line donors of the VII and
VIII subtypes (Table
3).
In the V AL organ system tropism Data on germ line gene use, myeloma, and organ disease are summarized in Table 3. The 1c, 2a2, and 3r germ line genes are associated with dominant cardiac and multisystem disease, whereas the 6a gene is associated with dominant renal disease. Indeed, the association between the 6a donor and dominant renal involvement is striking and a comparison of the frequency of dominant renal involvement in 6a patients versus all others achieves significance (P << .01, 2 = 12.61, degrees of
freedom [df] = 1, relative risk = 2.5, 95% confidence interval
[CI], 1.56-4.02). In contrast, a comparison of the frequency of
dominant cardiac involvement among 6a patients versus all
others does not (P = .19). A comparison of the frequency of dominant cardiac involvement among patients with myeloma (10 of 23)
versus those without myeloma (5 of 37) also achieves significance (P < .01, 2 = 6.79, df = 1, relative
risk = 3.22, 95% CI, 1.26-8.23). In addition, 3 of 12 patients with
V clones had dominant hepatic involvement as compared to
only 2 of 48 patients with V clones (P < .05, Fisher exact test, 2-tailed; relative
risk = 6.0, 95% CI, 1.13-32.0).
AL VL gene analysis Having identified germ line genes in 60 cases, marrow cDNA was then used in PCR with L-CL primers to amplify and sequence the correct FR1, as depicted in Figure 1. Eighty-five percent (51 of 60) were successfully amplified and sequenced directly from PCR tubes with this technique; the remaining 9 cases gave no identifiable amplicons and further cloning attempts were not pursued (VI = 3,
VIII = 2,
VVI = 3,
VI = 1). All 51 genes sequenced in this
fashion corresponded identically to their previously cloned
counterparts except for the differences in FR1 introduced by
the FR1 primers ( 3 nucleotides). Using these correctly
sequenced 51 AL VL genes, we determined the
percent homology of AL VL genes with germ line
sequences, as an indication of the degree of antigen-driven mutation.
All 51 genes showed evidence of likely prior antigenic challenge. The
median percent homology to germ line was 95.5% (range, 88.3%-98.9%).
Of note, as depicted in Figure 2, the
VVI AL genes were more homologous to germ
line than the other AL V genes.
AL VL protein analysis The protein sequences were deduced for AL VL genes derived from the 1c, 2a2, 3r, 6a, O18-O8, and LVFK431 germ line genes, and the deduced sequences were assessed for amino acid replacement mutations that have been associated with amyloid light chains and for mutations that result in the creation of sites for N-glycosylation (N-x-S/T).8,33 Amino acid positions are as designated by Kabat-Wu numbering.29 The results of this analysis are shown in Table 4 and the features of the replacement amino acids are indicated. Germ line-encoded residues that may play a role in amyloid formation are not included.8 In these V and V AL light chains, residues with frequent replacement
mutations are located along the protein surface in areas involved with
the binding of antigen such as positions 30 to 32, 50 to 52, and 93 to 96.
Amyloid formation in vitro Ten urinary immunoglobulin light chains were tested in vitro. Seven were from patients with biopsy-proven AL with renal disease including 4 of the 60 whose Ig VL genes were identified (VI = 3, II = 2, I = 1, II = 1). Three
were controls; 2 were light chains from patients with myeloma with
nonamyloid renal disease (I = 1, II = 1) and one a I light
chain from a patient with light-chain deposition disease. The results
of incubation in mesangial cell cultures with and without AEF at a
light chain concentration of 10 µg/mL are shown in Table 5. Of note,
all of the AL light chains and none of the controls formed amyloid when
incubated with AEF. In addition, 2 of the 3 VI light chains tested
in this model system formed more amyloid than all other AL light chains and, unlike all other AL light chains, the VI light chains formed similar amounts of amyloid with and without AEF. These differences highlight the propensity of mesangial cells to form amyloid without AEF
when incubated with VI but not other types of light chains.
GenBank accession numbers VI = AF124163-70, AF320833-4, AF320843-4,
AF115347, AF054641, AF115350.
V V
Note: Italicized accession numbers relate to sequences previously described by subtype only in reference 26.
In this report, we identify the immunoglobulin light-chain
variable region (Ig VL) germ line genes used by
the plasma cell clones of 60 patients with AL. We assess the plasma
cell disorders in these patients as meeting or not meeting the Durie
criteria21 for multiple myeloma and analyze the
amyloid-related organ disease in these patients as a function of both
Ig VL germ line gene use and plasma cell burden
(patients with or without myeloma). The hypothesis that germ line gene
use and plasma cell burden influence the tropism of AL organ
involvement is supported by this analysis. Patients with 6a
V These results also support the claim of preferential germ line gene use
in AL, based both on the well-described preponderance of It should be emphasized that these data are not free of selection bias. First, the patients tested were seen on referral to tertiary centers, possibly explaining the disproportionate number of male patients. Second, in many instances the patients were referred for consideration of stem cell transplantation and, therefore, represent a possibly younger and healthier segment of the AL patient population.37-39 Third, the patients also represent a portion of the AL population whose clonal immunoglobulin genes could be amplified and identified, for the 60 successful cases represent just over two thirds of the cases in which RT-PCR was attempted. This point is particularly relevant to claims made with respect to plasma cell burden. Fourth, we assume that plasma cell burden can be estimated using criteria designed to distinguish myeloma from monoclonal gammopathy of undetermined significance and that this distinction correlates with light-chain production.21 Nevertheless, concerns of bias duly noted, 2 significant associations
between clonal AL VL gene use and dominant organ
involvement emerge from this analysis. Indeed, because we evaluated AL
organ involvement by standard accepted criteria, it is important to note that the categories used for dominant organ involvement have been
shown to possess prognostic significance with respect to survival.10,22 Therefore, the respective associations
identified between dominant cardiac involvement and the 1c,
2a2, and 3r V With respect to the technique of clonal gene identification, RT-PCR was used to amplify clonal light-chain genes from bone marrow cDNA using FR1-CL primers and strict rules developed for identification of candidate cloned genes. Clonal AL VL genes were assigned germ line donors and, for purposes of further analysis, 85% of them (51 of 60) were successfully amplified a second time with Leader primers and sequenced directly to identify potential errors in FR1 introduced by FR1 primers. In these instances, this second round of amplification also served to confirm the identity of the cloned genes as clonal genes. In addition, the distribution of germ line genes and the percent homologies to germ line indicate that certain germ line genes likely possess intrinsic features predisposing to amyloid formation.8,40 The critical physicochemical aspects of the proteins encoded by preferentially used light-chain genes remain unexplained. Although the mechanisms underlying organ tropism also remain unexplained, our analysis based on plasma cell burden indicates that light-chain availability or concentration is likely to be an important variable because patients with AL with myeloma are more likely to have dominant cardiac amyloid independent of germ line gene. This conclusion fits with long-standing clinical observations and the results of a similar analysis.10,35 Recent evidence in support of a role for receptor-dependent cell stress in secondary amyloid formation may be relevant in this regard. A multiligand receptor in the immunoglobulin superfamily (RAGE or receptor for advanced glycation end-products) was shown to be up-regulated by amyloid-prone proteins and integral to amyloid deposition.41 It is possible that, in primary amyloidosis, light-chain concentration plays a role in up-regulating RAGE receptors on macrophages and mononuclear phagocytes in different organs and that up-regulation of RAGE receptors in relevant cardiac cells may require a higher concentration of light chains. Of more concrete relevance, however, the amino acid replacements we identified in deduced protein sequences (Table 4) are in positions that may be associated with amyloid formation although specific substitutions that play a causal role or contribute to light-chain instability have not yet been generically identified.8,30 Nevertheless, the presence of these replacements in positions along the protein surface further supports a molecular model of amyloid fibril formation that involves initial dimerization of VL molecules due to interactions among CDR residues.8 A specific role for receptor-ligand interactions involving RAGE or other cell surface receptors and this molecular model are not mutually exclusive in theory. The AL Ig VL genes we have identified are likely derived from postgerminal center B cells, as is indicated by the assessment of homology to germ line sequences. Furthermore, a difference is seen among the subtypes with respect to homology to germ line, as highlighted by the significant difference between the 6a and the 2a2 or 3r clones. It is unknown whether the difference is typical of germ line genes such as 6a that are rarely used in the expressed repertoire. Indeed, the development of the expressed repertoire is incompletely documented with respect to marrow plasma cell Ig VL gene expression. It is also possible that the less homologous subtypes represent clones derived from the memory B-cell pool; their emergence may involve antigenic challenge or persistence in ways not well appreciated, and may be related to repeat journeys through the germinal center resulting in several generations of mutations in sequence.42,43 Although the difference in homology may reflect the emergence of AL clones against the backdrop of such hypothetical sources, it more likely represents the inherent tendency of 6a clones to cause amyloid because of germ line-encoded features. Indeed, given the renal tropism of 6a clones and the in vitro data we offer, a specific receptor-ligand interaction is suggested. In conclusion, we report a series of 60 clonal Ig
VL gene sequences from patients with AL, the
largest series to date. We demonstrate that germ line gene use and
plasma cell burden contribute to the tropism of organ involvement, one
of the hallmarks of AL. Ig VL germ line donors
were associated with dominant hepatic, dominant cardiac and multisystem
disease, and dominant renal disease. Patients with AL with myeloma in
this series were at increased risk of developing cardiac amyloid
independent of germ line donor. Both germ line gene use and clonal
plasma cell burden contribute to the tropism of organ involvement
observed in AL. Of particular note, the specific association between
the 6a V
We thank Dr Stephen D. Nimer for his advice and continued support; and Joanne Santorsa, RN, and Dr Carl O'Hara for assistance obtaining specimens.
Submitted November 17, 2000; accepted March 28, 2001.
Supported by grants from the Food and Drug Administration (FD-R-001346-01), the Arthritis Foundation, and the National Blood Foundation. C.M. was supported by the Program Generalitat de Catalunya-Fulbright.
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 U.S.C. section 1734.
Reprints: Raymond L. Comenzo, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021; e-mail: comenzor{at}mskcc.org.
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R. L. Comenzo, P. Zhou, M. Fleisher, B. Clark, and J. Teruya-Feldstein Seeking confidence in the diagnosis of systemic AL (Ig light-chain) amyloidosis: patients can have both monoclonal gammopathies and hereditary amyloid proteins Blood, May 1, 2006; 107(9): 3489 - 3491. [Abstract] [Full Text] [PDF]
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R. S. Abraham, K. V. Ballman, A. Dispenzieri, D. E. Grill, M. K. Manske, T. L. Price-Troska, N. G. Paz, M. A. Gertz, and R. Fonseca Functional gene expression analysis of clonal plasma cells identifies a unique molecular profile for light chain amyloidosis Blood, January 15, 2005; 105(2): 794 - 803. [Abstract] [Full Text] [PDF]
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L. Novak, W. J. Cook, G. A. Herrera, and P. W. Sanders AL-amyloidosis is underdiagnosed in renal biopsies Nephrol. Dial. Transplant., December 1, 2004; 19(12): 3050 - 3053. [Abstract] [Full Text] [PDF]
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 G. Merlini and V. Bellotti Molecular Mechanisms of Amyloidosis N. Engl. J. Med., August 7, 2003; 349(6): 583 - 596. [Full Text] [PDF]
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P. Zhou, Y. Zhang, C. Martinez, N. Kalakonda, S. D. Nimer, and R. L. Comenzo Melphalan-mobilized blood stem cell components contain minimal clonotypic myeloma cell contamination Blood, July 15, 2003; 102(2): 477 - 479. [Abstract] [Full Text] [PDF]
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R. S. Abraham, S. M. Geyer, T. L. Price-Troska, C. Allmer, R. A. Kyle, M. A. Gertz, and R. Fonseca Immunoglobulin light chain variable (V) region genes influence clinical presentation and outcome in light chain-associated amyloidosis (AL) Blood, May 15, 2003; 101(10): 3801 - 3807. [Abstract] [Full Text] [PDF]
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A. Lebeau, E. Zeindl-Eberhart, E.-C. Muller, J. Muller-Hocker, P. R. Jungblut, B. Emmerich, and U. Lohrs Generalized crystal-storing histiocytosis associated with monoclonal gammopathy: molecular analysis of a disorder with rapid clinical course and review of the literature Blood, August 13, 2002; 100(5): 1817 - 1827. [Abstract] [Full Text] [PDF]
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V. Perfetti, S. Casarini, G. Palladini, M. C. Vignarelli, C. Klersy, M. Diegoli, E. Ascari, and G. Merlini Analysis of Vlambda -Jlambda expression in plasma cells from primary (AL) amyloidosis and normal bone marrow identifies 3r (lambda III) as a new amyloid-associated germline gene segment Blood, July 18, 2002; 100(3): 948 - 953. [Abstract] [Full Text] [PDF]
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R. L. Comenzo and M. A. Gertz Autologous stem cell transplantation for primary systemic amyloidosis Blood, May 29, 2002; 99(12): 4276 - 4282. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
fibrillar deposits, 
708 bp; lane 2, L-5' CL primers 