Bone marrow-derived clonal plasma cells, as found in systemic amyloidogenic light chain–associated (AL) amyloidosis, are presumed to be the source of light chains that deposit as fibrils in tissues throughout the body. Paradoxically, people with this disorder, in contrast to multiple myeloma, often have a low percentage of such cells, and it is unknown whether this relatively sparse number can synthesize enough amyloidogenic precursor to form the extensive pathology that occurs. To investigate whether another hematopoietic organ, the spleen, also contains monoclonal light chain–producing plasma cells, we have immunostained such tissue from 26 AL patients with the use of antiplasma cell, antifree κ and λ, and anti-VL subgroup-specific monoclonal antibodies (mAbs). In 12 cases, there was statistically significant evidence of a monoclonal population bearing the same κ or λ isotype as that within the bone marrow and identical to the amyloid. Our studies have shown that the spleen may be another source of amyloidogenic light chains.

Systemic amyloidogenic light chain–associated (AL) amyloidosis is characterized by the deposition in the heart, kidneys, liver, nerves, and other organs or tissues of κ or λ light chain–related fibrils.1  These molecules are the products of plasma cells deemed monoclonal based on the finding of a predominance of κ+ or λ+ cytoplasmic immunoglobulin (Ig), and the presence of such cells in bone marrow is one of the diagnostic hallmarks of this disorder. In comparison with multiple myeloma, patients with AL amyloidosis typically have a relatively low number of plasmacytes in this site, that is, less than 5% to 10%2 ; thus, it is not known whether this relatively sparse population secretes sufficient amounts of amyloidogenic precursor to account for the extensive deposits that can occur throughout the body. To address this question, we have determined whether another hematopoietic organ, namely the spleen, also contains monoclonal light chain–producing plasma cells. We now report the results of immunophenotypic analyses that used monoclonal antibodies (mAbs) specific for κ and λ free light chains (FLCs),3  as well as reagents reactive with the major Vκ and Vλ gene families.4  Here we showed that the spleens of 4 of 8 ALκ and 8 of 18 ALλ patients had a statistically significant preponderance of plasma cells with a light chain isotype identical to that expressed by the bone marrow–derived plasma cells and/or the amyloid deposits

Patient population

The 26 patients included in this study had a diagnosis of systemic AL amyloidosis (manifested primarily by renal, cardiac, or neurologic dysfunction) based on the presence of a serum or urinary monoclonal Ig; an abnormal serum FLC κ/λ ratio, as determined by our mAb-based enzyme-linked immunosorbent assay (ELISA)5 ; or identification of the light chain nature of amyloid extracted from autopsy-derived tissue, as documented by amino acid sequencing and/or mass spectrometry.6,7  The study was approved by the University of Tennessee Medical Center's Institutional Review Board, and informed consent was obtained in accordance with the Declaration of Helsinki.

Immunohistochemistry

Four-micrometer-thick sections, cut from formalin-fixed, paraffin-embedded blocks of spleen, were deparaffinized and subjected to antigen retrieval by exposure in a 90°C water bath for 30 minutes to a Dako Target Retrieval Solution containing citrate buffer, pH 6.0 (Dako Cytomation, Carpenteria, CA), followed by cooling at room temperature for 20 minutes. The tissue was immunostained with a commercial antiplasma cell antibody (Dako); our murine mAbs Fκ-C3 and Fλ-G9, which react only with free κ or λ light chains, respectively3 ; and reagents specific for the major VL subgroups (Vκ1, 2, 3, and 4; Vλ1, 2, 3, 6, and 8).4  Immunoreactivity was visualized with the use of a streptavidin-biotin detection system (BioGenex, San Ramon, CA) and color was developed with diaminobenzidine (DAB; Vector Laboratories, Burlingame, CA). The slides were counterstained with hematoxylin (Gill #3, Sigma-Aldrich, St Louis, MO). The number of reactive plasma cells was enumerated in 15 high-power fields using a Leitz DMRB microscope (Vashaw Scientific, Norcross, GA) fitted with a 40×/0.75 dry objective and a 1.6× magnifying lens. The results were averaged and the statistical significance determined by the t test. Cytospin preparations of bone marrow obtained at the time of diagnosis also were evaluated with the same antibodies by methods detailed elsewhere.8 

The predominant site of deposition and the VL nature of the amyloid in all 26 patients are given in Table 1. Immunocytochemical analyses of bone marrow–derived specimens obtained at the time of diagnosis were performed in 19 cases in which clonal populations of plasma cells were found, as evidenced by their reactivity with either the antifree κ or λ reagent, as well as a particular anti-VL subgroup mAb. In each instance, there was complete concordance between the chemical nature of the AL amyloid and the predominant plasma cell immunophenotype. Further, a striking and statistically significantly similar population was found in the spleens from 12 patients (κ1, 4; λ1, 3; λ2, 2; λ6, 2; and λ8, 1), as illustrated for cases 8 (ALκ) and 7 (ALλ; Figure 1). Even in 7 patients in whom there was no κ or λ predominance and who had more than 2 plasma cells per high power field, the presence of such cells having the same cytoplasmic VL phenotype as that of the amyloid was evidenced by their staining with specific anti-VL mAbs (Table 1). Bone marrow specimens taken within 2 months of death from 6 patients also were evaluated; in the 5 who had statistically significant clonal splenic plasma cells, comparable results were found in the marrow. In the 6th case, as in the spleen, there was no clonal predominance.

Table 1

Number of plasma cells per high-power field immunoreactive with free κ and λ immunoglobulin light chains in sections of spleen from patients with AL amyloidosis

CaseSiteImmunophenotype
Splenic plasma cells
Serum*
ALMarrow plasma cellsSpleen plasma cellsFree κFree λPFree κ, μg/mL§Free λ, μg/mL§Free κ/λ ratio
λ6 λ6 λ6 0.6 ± 0.2 87.1 ± 7.2 <.001 1.57 396.2 0.004 
λ1 λ1 λ1 7.4 ± 1.1 15.2 ± 1.1 <.001 8.96 525.8 0.017 
κ1 κ1 κ1 102.1 ± 3.7 9.0 ± 1.7 <.001 n.d. n.d. n.d. 
N, LU λ6 λ6 λ6 12.8 ± 1.6 28.3 ± 1.5 <.001 12.6 277.8 0.045 
λ2 λ2 λ2 7.7 ± 0.7 15.3 ± 1.0 <.001 n.d. n.d. n.d. 
κ1 n.d. κ1 4.9 ± 0.8 0.5 ± 0.3 <.001 634.4 14.5 43.8 
K, TH λ1 λ1 λ1 1.1 ± 0.3 16.1 ± 1.6 <.001 22.6 71.7 0.32 
κ1 κ1 κ1 15.4 ± 1.4 1.7 ± 0.4 <.001 332.8 43.7 7.62 
H, T λ1 λ1 λ1 6.3 ± 0.8 11.1 ± 1.0 <.001 46.1 258.3 0.18 
10 L, N κ1 κ1 κ1 5.7 ± 1.7 0.3 ± 0.2 <.01 738.3 50.1 14.7 
11 λ8 λ8 λ8 5.5 ± 0.8 9.5 ± 0.9 <.01 17.5 471.7 0.04 
12 L, S, A λ2 λ2 λ2 0.7 ± 0.3 2.2 ± 0.3 <.01 n.d. n.d. n.d. 
13 K, H, S λ3 λ3 λ3 1.9 ± 0.4 2.4 ± 0.5 n.s. 11.6 120.2 0.10 
14 H, K λ2 λ2  0.3 ± 0.2 0.1 ± 0.1 n.s. 36.4 329.0 0.11 
15 κ4 κ4 — 0.2 ± 0.1 0.3 ± 0.2 n.s. 103.4 123.9 0.83 
16 S, L λ1 n.d. — 0.3 ± 0.2 1.1 ± 0.6 n.s. n.d. n.d. n.d. 
17 λ1 λ1 λ1 18.1 ± 1.6 14.5 ± 1.2 n.s. 15.0 844.3 0.02 
18 λ2 λ2 — 0.1 ± 0.1 0.3 ± 0.1 n.s. 21.9 320.8 0.07 
19 λ3 λ3 λ3 2.1 ± 0.4 1.6 ± 0.3 n.s. 36.2 221.8 0.16 
20 K, H λ3 λ3 — 0.2 ± 0.1 0.0 n.s. 8.9 106.9 0.08 
21 λ2 λ2 λ2 1.7 ± 0.7 1.6 ± 0.5 n.s. 48.8 279.4 0.17 
22 L, S κ3 n.d. κ3 3.6 ± 0.5 4.5 ± 0.9 n.s. n.d. n.d. n.d. 
23 λ3 n.d. — 0.1 ± 0.1 1.3 ± 0.3 n.s. n.d. n.d. n.d. 
24 K, S, LN κ1 n.d. κ1 2.9 ± 0.7 2.5 ± 0.6 n.s. n.d. n.d. n.d. 
25 K, L, S λ2 n.d. — 0.3 ± 0.2 0.6 ± 0.2 n.s. n.d. n.d. n.d. 
26 κ3 n.d. κ3 8.7 ± 0.8 9.9 ± 1.4 n.s. n.d. n.d. n.d. 
Normal     5.0 ± 0.6 3.5 ± 0.9 n.s. 4.2-13.0 16.4-127.3 0.07-0.28 
CaseSiteImmunophenotype
Splenic plasma cells
Serum*
ALMarrow plasma cellsSpleen plasma cellsFree κFree λPFree κ, μg/mL§Free λ, μg/mL§Free κ/λ ratio
λ6 λ6 λ6 0.6 ± 0.2 87.1 ± 7.2 <.001 1.57 396.2 0.004 
λ1 λ1 λ1 7.4 ± 1.1 15.2 ± 1.1 <.001 8.96 525.8 0.017 
κ1 κ1 κ1 102.1 ± 3.7 9.0 ± 1.7 <.001 n.d. n.d. n.d. 
N, LU λ6 λ6 λ6 12.8 ± 1.6 28.3 ± 1.5 <.001 12.6 277.8 0.045 
λ2 λ2 λ2 7.7 ± 0.7 15.3 ± 1.0 <.001 n.d. n.d. n.d. 
κ1 n.d. κ1 4.9 ± 0.8 0.5 ± 0.3 <.001 634.4 14.5 43.8 
K, TH λ1 λ1 λ1 1.1 ± 0.3 16.1 ± 1.6 <.001 22.6 71.7 0.32 
κ1 κ1 κ1 15.4 ± 1.4 1.7 ± 0.4 <.001 332.8 43.7 7.62 
H, T λ1 λ1 λ1 6.3 ± 0.8 11.1 ± 1.0 <.001 46.1 258.3 0.18 
10 L, N κ1 κ1 κ1 5.7 ± 1.7 0.3 ± 0.2 <.01 738.3 50.1 14.7 
11 λ8 λ8 λ8 5.5 ± 0.8 9.5 ± 0.9 <.01 17.5 471.7 0.04 
12 L, S, A λ2 λ2 λ2 0.7 ± 0.3 2.2 ± 0.3 <.01 n.d. n.d. n.d. 
13 K, H, S λ3 λ3 λ3 1.9 ± 0.4 2.4 ± 0.5 n.s. 11.6 120.2 0.10 
14 H, K λ2 λ2  0.3 ± 0.2 0.1 ± 0.1 n.s. 36.4 329.0 0.11 
15 κ4 κ4 — 0.2 ± 0.1 0.3 ± 0.2 n.s. 103.4 123.9 0.83 
16 S, L λ1 n.d. — 0.3 ± 0.2 1.1 ± 0.6 n.s. n.d. n.d. n.d. 
17 λ1 λ1 λ1 18.1 ± 1.6 14.5 ± 1.2 n.s. 15.0 844.3 0.02 
18 λ2 λ2 — 0.1 ± 0.1 0.3 ± 0.1 n.s. 21.9 320.8 0.07 
19 λ3 λ3 λ3 2.1 ± 0.4 1.6 ± 0.3 n.s. 36.2 221.8 0.16 
20 K, H λ3 λ3 — 0.2 ± 0.1 0.0 n.s. 8.9 106.9 0.08 
21 λ2 λ2 λ2 1.7 ± 0.7 1.6 ± 0.5 n.s. 48.8 279.4 0.17 
22 L, S κ3 n.d. κ3 3.6 ± 0.5 4.5 ± 0.9 n.s. n.d. n.d. n.d. 
23 λ3 n.d. — 0.1 ± 0.1 1.3 ± 0.3 n.s. n.d. n.d. n.d. 
24 K, S, LN κ1 n.d. κ1 2.9 ± 0.7 2.5 ± 0.6 n.s. n.d. n.d. n.d. 
25 K, L, S λ2 n.d. — 0.3 ± 0.2 0.6 ± 0.2 n.s. n.d. n.d. n.d. 
26 κ3 n.d. κ3 8.7 ± 0.8 9.9 ± 1.4 n.s. n.d. n.d. n.d. 
Normal     5.0 ± 0.6 3.5 ± 0.9 n.s. 4.2-13.0 16.4-127.3 0.07-0.28 

H indicates heart; L, liver; LN, lymph node; LU, lung; K, kidney; I, intestine; TH, thyroid; T, tongue; A, adrenal; n.d., not determined; and n.s., not significant.

*

Specimens obtained less than 1 month before death, except in cases 9 and 21 (taken 3 and 4 months before death, respectively).

Principal site of amyloid deposition based on postmortem examination.

Determined by chemical analyses of amyloid extracts.

§

Values derived from a mAb-based enzyme-linked immunosorbent assay (ELISA).

No clonal plasma cells detected.

Figure 1

Photomicrographs of clonal plasma cells present in the spleens of patients with systemic ALκ (case 8) and ALλ (case 7) amyloidosis. The primary antibodies are as indicated (avidin biotin complex [ABC] technique; secondary antibody, biotinylated mouse IgG). Original magnification ×640. Photographs were taken with a SPOT RT color camera using SPOT RT software, Version 3.0 (Diagnostic Instruments, Sterling Heights, MI).

Figure 1

Photomicrographs of clonal plasma cells present in the spleens of patients with systemic ALκ (case 8) and ALλ (case 7) amyloidosis. The primary antibodies are as indicated (avidin biotin complex [ABC] technique; secondary antibody, biotinylated mouse IgG). Original magnification ×640. Photographs were taken with a SPOT RT color camera using SPOT RT software, Version 3.0 (Diagnostic Instruments, Sterling Heights, MI).

Close modal

Serum samples obtained less than 1 month before death from 15 subjects (and, in 2 cases, 3 and 4 monhs before death) were available for FLC assay. In 7 of 11 cases with a statistically significant population of splenic plasma cells, the FLC κ/λ ratios were concordantly abnormal. Among the remaining 8, where a clonogenic population was not detected, these values were abnormal in 2.

On the basis of the results of these studies, it was shown that in AL amyloidosis, the spleen (in addition to the bone marrow) can contain a population of clonal plasmacytes that produce amyloidogenic light chains. Experimental data suggest that these cells presumably arise from bone marrow–derived transitional B lymphocytes that home to the spleen and there mature into low-level native follicular (or marginal zone) B cells.9-11  Their further development within germinal centers and evolution into plasma cells results from exposure to an antigenic stimulus that leads to transcriptionally regulated clonal expansion and production of somatically mutated antibody molecules. This terminally differentiated population, which contains long-lived plasma cells, resides in splenic and (predominantly) bone-marrow survival niches.12,13 

At present, therapeutic options for patients with AL amyloidosis are limited to antiplasma cell chemotherapy given in conventional amounts or high doses combined with autologous stem cell transplantation (or other “antimyeloma” regimens).14-22  The rationale for this approach is to reduce the synthesis of amyloidogenic precursor light chains,23  although the sensitivity of clonogenic bone marrow versus splenic plasma cells to such agents remains to be determined.

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.

This work was supported in part by US Public Health Service Research Grant CA10056 from the National Cancer Institute (Bethesda, MD), the Aslan Foundation (Knoxville, TN), the Swedish Research Council (Stockholm, Sweden), and the European Union Sixth Framework Programme (Brussels, Belgium). P.W. was a Harry and Elsa Jiler American Cancer Society International Visiting Scientist. A.S. is an American Cancer Society Clinical Research Professor.

National Institutes of Health

Contribution: A.S. and D.T.W. wrote the paper; S.D.M. and C.W. performed the studies; and P.W. wrote the paper and evaluated the data.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Alan Solomon, MD, University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, TN 37920; e-mail: asolomon@utmck.edu.

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