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 (IgVL) 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 VλVI germ line gene were more likely to present with dominant renal involvement, whereas those with clones derived from the 1c, 2a2, and 3r Vλ genes were more likely to present with dominant cardiac and multisystem disease. Patients withVκ clones were more likely to have dominant hepatic involvement and patients who met the Durie criteria for myeloma (38%, 23 of 60) were more likely to present with dominant cardiac involvement independent of germ line gene use. In the in vitro model, unlike all other AL light chains tested, λVI light chains formed amyloid rapidly both with and without amyloid-enhancing factor. These data support the hypothesis that germ line gene use and plasma cell burden influence the organ tropism of AL.

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 λ isotype) is deposited in key viscera by an unknown mechanism.3-10 At diagnosis, AL usually displays a tropism for one organ system or another, resulting in various dominant symptomatic clinical presentations. For example, patients with dominant cardiac involvement may present with right-sided heart dysfunction and may have electrocardiograms showing low precordial voltage and a pattern of myocardial infarction in the absence of coronary artery disease (pseudoinfarct pattern). Patients with renal involvement often present with proteinuria in the nephrotic range, whereas those whose peripheral nervous system is involved present with orthostasis or sensorimotor polyneuropathy.10 These distinctive features—fibrillar deposits, λ predominance, and the tropism of organ involvement—remain unexplained.

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 (IgVL;VL = Vλ andVκ). In a clonal B-cell disorder, these sequences provide a signature for the clone. Identification of clonal Ig VL genes with the polymerase chain reaction (PCR) has proven useful in the study of B-cell disorders and has permitted the compilation of comprehensive directories of IgVλ and Vκ germ line genes.11-14 Using these databases, the clonal IgVL genes of patients with B-cell disorders can be assigned germ line genes and assessed for homology to germ line sequences. In addition, investigators have determined the relative contributions of germ line genes to the circulating B-cell pool in healthy individuals (the normal expressed repertoire).15-19 By such approaches, a germ line gene rarely used in the normal repertoire may be shown to be used preferentially in a particular B-cell disorder.20 

In this report we identify clonal Ig VLgenes 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 IgVL germ line gene use and clonal plasma cell burden.

Patients and plasma cell disease

The patients with AL were evaluated for the extent of amyloid-related organ involvement and for dominant organ involvement by standard criteria as previously described.22-24 Briefly, at presentation patients were categorized according to clinical manifestations as having renal, cardiac, hepatic, or neuropathic dominant organ involvement. Patients with more than one of these features were categorized according to the most prominent and symptomatic organ involvement. Other manifestations, such as soft-tissue involvement, were defined based on tissue biopsy results or pathognomonic physical findings (eg, shoulder-pad sign). Plasma cell disease was evaluated as previously described,23 and clonal plasma cell disorders were categorized as meeting or not meeting diagnostic criteria for myeloma using the Durie major and minor criteria as described.21 

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 IgVLgenes

Bone marrow aspirates were obtained from patients with biopsy-proven AL who gave written informed consent under institutional review board–approved protocols. Marrow cells were treated with ammonium chloride to lyse red blood cells, washed, pelleted, and frozen as previously described.25,26For preparation of complementary DNA (cDNA), total RNA was extracted from 107 marrow cells using TRIzol (Gibco-BRL, Gaithersburg, MD). Preparations of cDNA were made with Superscript II reverse transcriptase (Life Technologies, Grand Island, NY) as previously described.25,26 The cDNA was amplified by PCR using 5′ oligonucleotide FR1 primers specific for the expressed Ig VL subtypes with aCκ or pan-Cλprimer.27 Each specimen was subject to multiple amplifications and bands were selected for cloning from 3 separate PCR experiments. The amplified material from the PCR reactions was prepared in a TA Cloning Kit (Invitrogen, San Diego, CA). Twelve colonies representative of the 3 separate reactions were picked for plasmid isolation and screened for appropriately sized inserts byEcoR1 digest and electrophoresis. Five to 10 inserts were sequenced backward and forward at core facilities. A clonal IgVL was identified provided that one gene was distinctly overrepresented in each patient and that the gene was present in a minimum of 3 inserts. Because the FR1 primers routinely introduce minor sequencing errors, primers forVL leader (L) regions were designed and additional PCR amplifications with L-CLprimers were performed to sequence FR1 correctly (Table1, Figure1). Genes with correctly sequencedFR1 regions were analyzed for homology to germ line donor sequence and evidence of somatic hypermutation.

Fig. 1.

Light-chain variable region gene, protein, and PCR.

(A) The upper sketch shows the Leader,VL (variable), JL(joining), and CL (constant) gene segments as found in an expressed gene, and the various sets of primers that may be designed to amplify the variable region gene or portions of it. The lower sketch shows a schema of the light-chain protein and its regions. Framework regions (FR1-4) provide structure and usually have few amino acid replacements due to somatic mutation. Complementarity-determining regions (CDR1-3) provide sites for antigen binding and have frequent amino acid mutations due to somatic mutation. The constant region (C) also provides structure. Note that theVJ junction comprises the middle of the CDR3 region. (B) A PCR gel is shown of the same VλVI light-chain cDNA amplified with different sets of primers. Lanes 1 through 4 show segments of different length amplified with primers to different portions of the gene: lane 1, L-3′ CLprimers → 708 bp; lane 2, L-5′ CLprimers → 435 bp; lane 3, FR1-5′ CL primers → 378 bp; and lane 4, CDR1-CDR3 primers → 231 bp. PCR with unique CDR1-CDR3 primers is useful for detecting minimal residual disease and clonotypic contamination of stem cell components. MX indicates size markers as shown in legend; B, no cDNA blank; bp, base pairs.

Fig. 1.

Light-chain variable region gene, protein, and PCR.

(A) The upper sketch shows the Leader,VL (variable), JL(joining), and CL (constant) gene segments as found in an expressed gene, and the various sets of primers that may be designed to amplify the variable region gene or portions of it. The lower sketch shows a schema of the light-chain protein and its regions. Framework regions (FR1-4) provide structure and usually have few amino acid replacements due to somatic mutation. Complementarity-determining regions (CDR1-3) provide sites for antigen binding and have frequent amino acid mutations due to somatic mutation. The constant region (C) also provides structure. Note that theVJ junction comprises the middle of the CDR3 region. (B) A PCR gel is shown of the same VλVI light-chain cDNA amplified with different sets of primers. Lanes 1 through 4 show segments of different length amplified with primers to different portions of the gene: lane 1, L-3′ CLprimers → 708 bp; lane 2, L-5′ CLprimers → 435 bp; lane 3, FR1-5′ CL primers → 378 bp; and lane 4, CDR1-CDR3 primers → 231 bp. PCR with unique CDR1-CDR3 primers is useful for detecting minimal residual disease and clonotypic contamination of stem cell components. MX indicates size markers as shown in legend; B, no cDNA blank; bp, base pairs.

Close modal

Sequence analysis of VLgenes and proteins

Sequence alignment analyses were performed by GenBank BLAST (Basic Local Alignment Search Tool) search and germ line gene counterparts were assigned by V-BASE (Ig variable region gene database,http://www.mrc-cpe.cam.ac.uk/imt-doc/vbase-home-page.html) sequence directory comparison based on maximum homology of the nucleotide sequences.28,29 Homology to germ line sequence was calculated for complete VL genes excluding nucleotides associated with the VJ junction (codons 95 and 96) and FR4. All AL gene sequences were submitted to GenBank. Where indicated, protein sequences were deduced and analyzed for characteristics of amyloid-associated light chains.8 30 

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 Table2. Fifty-two percent had dominant renal and 25% dominant cardiac amyloid. Seventy-five percent had λ clones and 38% had clonal plasma cell disorders that met criteria for myeloma.

AL Ig VLgerm 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 IgVL gene amplified, requiring sequencing of additional inserts to identify the predominant clone (ratios of 8:1, 4:1, and 6:2). The VλI,VλVI , andVκI subtypes provided germ line donors for 75% of the clones, whereas the remainder derived from germ line donors of the VλII andVλIII subtypes (Table3).

In the Vλ cases, there was preferential germ line gene utilization. The 1c gene was used in 8 of 15VλI cases, 2a2 in 6 of 7VλII cases, 3r in 7 of 8VλIII cases, and the 6a gene in all 18 VλVI cases. In the normal expressed repertoire, 7% to 8% of light chains are derived from1c, 20% to 35% from 2a2, 7% to 8% from3r, and < 5% from 6a.17,18 Light chains of the rare VλVI subtype have been found frequently in AL.20,26 In theVκ cases, there was also preferential utilization. The relatively rare LFVK431 germ line gene was used in 4 cases and the more common O18-O8 gene in 7.20 All of the Vκ genes used by the κ clones in this series were members of the VκIsubtype. In normal usage, genes of theVκIII subtype dominate.20 

AL organ system tropism

Data on germ line gene use, myeloma, and organ disease are summarized in Table 3. The 1c, 2a2, and3r 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 the6a donor and dominant renal involvement is striking and a comparison of the frequency of dominant renal involvement in6a patients versus all others achieves significance (P << .01, χ2 = 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, χ2 = 6.79, df = 1, relative risk = 3.22, 95% CI, 1.26-8.23). In addition, 3 of 12 patients with clones had dominant hepatic involvement as compared to only 2 of 48 patients with Vλ clones (P < .05, Fisher exact test, 2-tailed; relative risk = 6.0, 95% CI, 1.13-32.0).

AL VLgene 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 (VλI = 3,VλIII = 2,VλVI = 3,VκI = 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, theVλVI AL genes were more homologous to germ line than the other AL Vλ genes.

Fig. 2.

Percent homology to germ line for commonly used AL

IgVL genes. The percent homology (%) is a reflection of the frequency with which individual nucleotides in an Ig gene have been mutated from germ line. Percents were determined for individual AL Ig VL genes correctly sequenced with L-CL primers. Aggregate results are depicted by germ line subgroup; medians and ranges for specific subgroups are shown on the chart and P values of Mann-Whitney comparisons in the table. The 6a genes are the most homologous to germ line and the 3r genes the least. The difference between 6a versus 2a2 or 3r Vλ genes achieves statistical significance. Because the 6a donor is not a common contributor to the normal expressed repertoire, the difference in homology with genes of other Vλ subgroups may reflect a normal process that distinguishes infrequently used germ line genes but may also reflect the propensity of 6a clones to produce amyloid due to germ line-encoded features. It may also reflect a difference in the origins of AL clones. For example, one might in theory see such a contrast if 6a clones were derived from de novo postgerminal center B cells, whereas clones of other Vλdonors were derived from memory B cells subject to additional circuits through the germinal center.

Fig. 2.

Percent homology to germ line for commonly used AL

IgVL genes. The percent homology (%) is a reflection of the frequency with which individual nucleotides in an Ig gene have been mutated from germ line. Percents were determined for individual AL Ig VL genes correctly sequenced with L-CL primers. Aggregate results are depicted by germ line subgroup; medians and ranges for specific subgroups are shown on the chart and P values of Mann-Whitney comparisons in the table. The 6a genes are the most homologous to germ line and the 3r genes the least. The difference between 6a versus 2a2 or 3r Vλ genes achieves statistical significance. Because the 6a donor is not a common contributor to the normal expressed repertoire, the difference in homology with genes of other Vλ subgroups may reflect a normal process that distinguishes infrequently used germ line genes but may also reflect the propensity of 6a clones to produce amyloid due to germ line-encoded features. It may also reflect a difference in the origins of AL clones. For example, one might in theory see such a contrast if 6a clones were derived from de novo postgerminal center B cells, whereas clones of other Vλdonors were derived from memory B cells subject to additional circuits through the germinal center.

Close modal

AL VL protein analysis

The protein sequences were deduced for ALVL genes derived from the 1c,2a2, 3r, 6a, O18-O8, andLVFK431 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).8,33Amino 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 Vκ and Vλ 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 (λVI = 3, λII = 2, κI = 1, κII = 1). Three were controls; 2 were light chains from patients with myeloma with nonamyloid renal disease (λI = 1, λII = 1) and one a κI 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 λVI light chains tested in this model system formed more amyloid than all other AL light chains and, unlike all other AL light chains, the λVI 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 λVI but not other types of light chains.

GenBank accession numbers

VλI = AF124163-70, AF320833-4, AF320843-4,AF115347, AF054641, AF115350.

VλII = AF124171-6, AF320831.

VλIII = AF124177-9, AF124186, AF320832, AF115354,AF054648.

VλVI = AF124180-90, AF320837-9, AF320841-2, AF115357,AF054649, AF115358.

κI = AF121191-99, AF320835, Bankit 391247, AF054658

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 VλVI clonal disease are more likely to have dominant renal involvement, whereas those with otherVλ clones often have dominant cardiac amyloid and multisystem disease. And patients with clones are more likely to have dominant hepatic involvement, an association that has been linked by Stevens and others to a possible contribution ofN-glycosylation of kappa light chains to the propensity to form amyloid (Table 4).30 In addition, patients with AL with myeloma are more likely to have dominant cardiac amyloid independent of germ line gene use. The association between increased plasma cell burden and cardiac amyloid is consistent with a recent report in which the degree of plasma cell clonality and marrow plasma cell burden were shown to confer a poor prognosis in AL.35 

These results also support the claim of preferential germ line gene use in AL, based both on the well-described preponderance of λ clones and on the frequent use of genes such as 1c, 3r, 6a, andLFVK431 that are uncommon in the normal expressed repertoire. Moreover, given the range of homologies to germ line sequences, these results support the claim that AL clones originate from postgerminal center B cells subject to prior antigenic challenge, as depicted in a recent analysis of the sequences of 14 AL genes.36 Furthermore, the striking association of6a VλVI clonal plasma cell disease and renal amyloidosis is given additional credibility by the results of in vitro testing in a human renal mesangial cell model. There is specificity to this association; it is clearly apprehended but remains unexplained.

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 Vλ genes, and between dominant renal involvement and the 6a gene, also contain prognostic significance. That is, links are likely to exist between germ line gene use and overall survival, as well as between germ line gene use and survival after stem cell transplant, as we have recently suggested.26 

With respect to the technique of clonal gene identification, RT-PCR was used to amplify clonal light-chain genes from bone marrow cDNA usingFR1-CL primers and strict rules developed for identification of candidate cloned genes. Clonal ALVL 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 byFR1 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 advancedglycation 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 the6a and the 2a2 or 3r clones. It is unknown whether the difference is typical of germ line genes such as6a 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 IgVL 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 VλVI germ line gene and dominant renal disease is supported by data from an in vitro assay using human renal mesangial cells. The specificity of the association justifies our current effort to understand its physicochemical basis in order to develop pharmaceutical approaches that may impede amyloid deposition and the progression of disease.

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.

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.

1
Falk
R
Comenzo
RL
Skinner
M
The systemic amyloidoses: recent advances in diagnosis and treatment.
N Engl J Med.
337
1997
898
909
2
Gillmore
J
Hawkins
PN
Pepys
MB
Amyloidosis: a review of recent diagnostic and therapeutic developments.
Br J Haematol.
99
1997
245
256
3
Randall
RE
Williamson
WE
Mullinax
F
Tung
MY
Still
MJ
Manifestations of systemic light chain deposition.
Am J Med.
60
1976
293299
4
Arbustini
E
Merlini
G
Gavazzi
A
et al
Cardiac immunocyte-derived (AL) amyloidosis: an endomyocardial biopsy study in 11 patients.
Am Heart J.
130
31
1995
528
536
5
Gallo
G
Goni
F
Boctor
F
et al
Light chain cardiomyopathy: structural analysis of the light chain tissue deposits.
Am J Pathol.
148
5
1996
1397
1406
6
Shirahama
T
Cohen
AS
High resolution electron microscopic analysis of the amyloid fibril.
J Cell Biol.
131
1967
1373
1375
7
Schiffer
M
Molecular anatomy and pathologic expression of antibody light chains.
Am J Pathol.
148
1996
1339
1344
8
Stevens
FJ
Myatt
EA
Chang
CH
et al
A molecular model for self-assembly of amyloid firbrils from immunoglobulin light chains.
Biochemistry.
34
1995
10697
10702
9
Schormann
N
Murell
JR
Liepnieks
JJ
Benson
MD
Tertiary structure of an amyloid immunoglobulin light chain protein: a proposed model for amyloid fibril formation.
Proc Natl Acad Sci U S A.
92
1995
9490
9494
10
Kyle
RA
Gertz
MA
Primary systemic amyloidosis: clinical and laboratory features in 474 cases.
Semin Hematol.
32
1995
45
59
11
Korsmeyer
SJ
Arnold
A
Bakhshi
A
et al
Immunoglobulin gene rearrangement and cell surface antigen expression in acute lymphocytic leukemias of T cell and B cell origins.
J Clin Invest.
71
1983
301
313
12
Griesser
H
Tkachuk
D
Reis
DM
Mak
TW
Gene rearrangements and translocations in lymphoproliferative diseases.
Blood.
73
1989
1402
1415
13
Billadeau
D
Blackstadt
M
Greipp
P
et al
Analysis of B-lymphoid malignancies using allele-specific polymerase chain reaction: a technique for sequential quantitation of residual disease.
Blood.
78
1991
3021
3029
14
Zachau
HG
The immunoglobulin kappa locus or what has been learned from looking closely at one-tenth of a percent of the human genome.
Gene.
135
1993
167
173
15
Klein
R
Zachau
HG
Expression and hypermutation of human immunoglobulin kappa genes.
Ann NY Acad Sci.
764
1995
74
83
16
Cannell
PK
Amlot
P
Attard
M
Hoffbrand
AV
Foroni
L
Variable kappa gene rearrangement in lymphoproliferative disorders and analysis of V-kappa gene usage, VJ joining and somatic hypermutation.
Leukemia.
8
1994
1139
1145
17
Ignatovich
O
Tomlinson
IM
Jones
PT
Winter
G
The creation of diversity in the human immunoglobulin V-lambda repertoire.
J Mol Biol.
267
1997
69
77
18
Farner
NL
Dorner
T
Lipsky
PE
Molecular mechanisms and selection influence the generation of the human VλJλ repertoire.
J Immunol.
162
1999
2137
2145
19
Tomlinson
IM
Cox
JPL
Gherardi
E
Lesk
AM
Chothia
C
1995. The structural repertoire of the human V-kappa domain.
EMBO J.
4
1995
4628
4638
20
Solomon
A
Frangione
B
Franklin
EC
Preferential association of the V λ VI subgroup of human light chains with amyloidosis AL.
J Clin Invest.
70
1982
453
457
21
Durie
BGM
Staging and kinetics of multiple myeloma.
Semin Oncol.
13
1986
300
309
22
Skinner
M
Anderson
JJ
Simms
R
et al
Treatment of 100 patients with primary amyloidosis: a randomized trial of melphalan, prednisone, and colchicine versus colchicine alone.
Am J Med.
100
1996
290
298
23
Comenzo
RL
Vosburgh
E
Falk
RH
et al
Dose-intensive melphalan with blood stem-cell support for the treatment of AL amyloidosis: survival and responses in 25 patients.
Blood.
91
1998
3662
3670
24
Kyle
RA
Gertz
MA
Greipp
P
et al
A trial of three regimes for primary amyloidosis: colchicine alone, melphalan and prednisolone, and melphalan, prednisolone and colchicine.
N Engl J Med.
336
1997
1202
1207
25
Comenzo
RL
Michelle
D
LeBlanc
M
et al
CD34-selected mobilized blood autografts in AL amyloidosis: rationale and application.
Transfusion.
38
1998
60
69
26
Comenzo
RL
Wally
J
Kica
G
et al
Clonal immunoglobulin light chain variable region germline gene use in AL amyloidosis: association with dominant amyloid-related organ involvement and survival after stem cell transplantation.
Br J Haematol.
106
1999
744
751
27
Welschof
M
Terness
P
Kolbinger
F
Zewe
M
Dubel
S
Amino acid sequence based PCR primers for amplification of rearranged human heavy and light chain immunoglobulin variable region genes.
J Immunol Methods.
179
1995
203
214
28
Altschul
SF
Madden
TL
Schaffer
AA
et al
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res.
25
1997
3389
3402
29
Kabat
EA
Wu
TT
Perry
HM
Gottesman
KS
Foeller
G
Sequences of Proteins of Immunological Interest.
5th ed.
1991
National Institutes of Health
Bethesda, MD
30
Stevens
FJ
Four structural risk factors identify most fibril-forming kappa light chains.
7
2000
200
211
Int J Exp Clin Invest.
Amyloid
31
Tagouri
YM
Sanders
PW
Picken
MM
Siegal
GP
Kerby
JD
Herrera
GA
In vitro AL-amyloid formation by rat and human mesangial cells.
Lab Invest.
74
1996
290
302
32
Isaac
J
Kerby
JD
Russell
WJ
Dempsey
SC
Sanders
PW
Herrera
GA
In vitro modulation of AL-amyloid formation by human mesangial cells exposed to amyloidogenic light chains.
5
1998
238
246
Int J Exp Clin Invest.
Amyloid
33
Herrera
GA
Russell
WJ
Isaac
J
et al
Glomerulopathic light chain-mesangial cell interactions modulate in vitro extracellular matrix remodeling and reproduce mesangiopathic findings documented in vivo.
Ultrastruct Pathol.
23
1999
107
126
34
Kiselevsky
R
Boudreau
L
Kinetics of amyloid deposition, I: the effects of amyloid enhancing factor and splenectomy.
Lab Invest.
48
1983
53
59
35
Perfetti
V
Colli Vignarelli
M
Anesi
E
et al
The degrees of plasma cell clonality and marrow infiltration adversely influence the prognosis of AL amyloidosis patients.
Haematologica.
84
1999
218
221
36
Perfetti V, Ubbiali P, Colli Vignarelli M, et al. Evidence that amyloidogenic light chains undergo antigen-driven selection. Blood. 1998;2948-2954.
37
Comenzo
RL
Autologous hematopoietic stem cell transplantation for AL amyloidosis. In Thomas ED, Blume KG, Forman SJ, eds. Hematopoietic Cell Transplantation.
2nd ed.
1999
1014
1028
Blackwell
New York, NY
38
Comenzo
RL
Hematopoietic cell transplantation for primary systemic amyloidosis: what have we learned.
Leuk Lymphoma.
37
2000
245
258
39
Gertz
MA
Lacy
MQ
Dispenzieri
A
Myeloablative chemotherapy with stem cell rescue for the treatment of primary systemic amyloidosis: a status report.
Bone Marrow Transplant.
25
2000
465
470
40
Helms
LR
Wetzel
R
Specificity of abnormal assembly in immunoglobulin light chain deposition disease and amyloidosis.
J Mol Biol.
257
1996
77
86
41
Yan
SD
Zhu
H
Zhu
A
et al
Receptor-dependent cell stress and amyloid accumulation in systemic amyloidosis.
Nat Med.
6
2000
643
651
42
Rajewsky
K
Clonal selection and learning in the antibody system.
Nature.
381
1996
751
758
43
Arpin
C
Dechanet
J
Van Kooten
C
et al
Generation of memory B cells and plasma cells in vitro.
Science.
268
1995
720
722

Author notes

Raymond L. Comenzo, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021; e-mail:comenzor@mskcc.org.

Sign in via your Institution