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Correlation Between Serum Levels of Free Light Chain and Phenotype of Plasma Cells in Bone Marrow in Primary AL Amyloidosis

Posted on: Thursday, 14 July 2005, 03:01 CDT

Keywords: Flow cytometry, free light chain, plasma cell, AL amyloidosis

Abbreviations: CD, cluster of differentiation; FLC, free light chain; mAb, monoclonal antibody; MGUS, monoclonal gammopathy of undetermined significance

Abstract

To investigate whether there is a correlation between subtypes of plasma cells in the bone marrow and the production of M-protein, flow cytometry and serum free light chain (FLC) analyses were carried out in 17 patients with primary systemic AL amyloidosis (mean age, 59.9 8.8 years) and controls with M-protein (MGUS controls, n = 6) and without it (negative controls, n=9. The patients showed a significantly higher value in the serum predominant FLC:serum creatinine ratio (43.8 63.2) and CD38^sup ++^ CD19^sup -^CD56^sup +^ subpopulation (monoclonal plasma cells) (2.57 5.35%) than either the negative (p < 0.0005 and p < 0.001, respectively) or MGUS controls (p < 0.05). With respect to maturation of plasma cells in the bone marrow, the intermediate (MPC- 1^sup +^ CD45^sup -^CD49e^sup -^) and mature (MPC-1^sup +^ CD45^sup +^ CD49e^sup -^) subtypes were significantly higher (49.2 23.2%, p < 0.005) and lower (27.6 21.3%, p < 0.005) in the patients than in the negative controls, respectively. The serum predominant FLC:serum creatinine ratio was elevated in parallel with an increase in CD38^sup ++^ CD19^sup -^CD56^sup +^ and MPC-1^sup +^ CD45^sup - ^CD49e^sup -^ cells and a decrease in mature subtypes (MPC-1^sup +^ CD45^sup +^ CD49e^sup -^ and MPC-1^sup +^ CD45^sup +^ CD49e^sup +^ cells), There was a significantly positive correlation between the serum predominant FLC:serum creatinine ratio and either CD38^sup ++^ CD19^sup -^CD56^sup ^+ (r=0.510, p < 0.05) or MPC-1^sup +^ CD45^sup - ^CD49e^sup -^ cells (r = 0.481, p < 0.05). In primary AL amyloidosis M-protein is probably produced by increased monoclonal plasma cells in the bone marrow, particularly by the intermediate subpopulation with a phenotype of MPC-1^sup +^ CD45^sup -^CD49e^sup -^.

Introduction

AL amyloidosis is an intractable disorder with progressive dysfunction in multiple visceral organs, mainly in the heart and kidneys. This disease is characterized by extracellular deposition of amyloid derived from immunoglobulin light chains, and the precursor protein designated as M-protein is usually present in serum and/or urine, particularly in the systemic type [1,2]. M- protein has, therefore, been considered to be a key marker for diagnosis and treatment of AL amyloidosis. To detect M-protein in multiple myeloma and AL amyloidosis, a sensitive nephelometric assay for free light chains (FLCs) has recently become usable instead of serum protein electrophoresis and immunofixation, particularly as a therapeutic marker [3-7]. This assay is specific for κ- and λ-type FLCs, and does not recognize light chains bound to immunoglobulin heavy chains [4]. With respect to sensitivity and quantification the FLC analysis is superior to the conventional methods mentioned above.

As another therapeutic marker of AL amyloidosis monoclonal plasma cells in the bone marrow can also be employed. In the previous report we demonstrated that these cells could consistently be detected in patients with AL amyloidosis by CD 19 and CD56 in combination with CD38 on flow cytometry, and were classified into immature, intermediate and mature stages according to the expression of CD45, CD49e and MPC-I, even though the smear specimens of the bone marrow showed a small number of plasma cells with morphologically normal appearance [8]. In addition, we showed that these monoclonal plasma cells rapidly decreased in accordance with disappearance of M-protein after intensive chemotherapy [9]. To investigate whether there is a correlation between the subtype of plasma cells in the bone marrow and the production of Mprotein, we performed flow cytometry and serum FLC analysis in patients with primary systemic AL amyloidosis.

Patients and methods

Patients

We studied 17 patients with primary AL amyloidosis who were referred to our hospital between April 2000 and October 2003 (11 men and 6 women; age range, 44-71 years; mean, 59.9 8.8 years). None of the patients had received any medical treatment for plasma cell dyscrasia prior to being referred to us. The diagnosis of this type of amyloidosis was based on two criteria: apparent deposition of amyloid with evidence of AL type in at least one biopsy site, and no associated disorder possibly underlying the amyloidosis. To detect amyloid deposition, conventional alkaline Congo red staining was performed in several biopsied tissues such as the kidney and the gastroduodenal and rectal mucosa, and in at least one of these specimens immunohistochemical staining was carried out using different antibodies to ALκ, ALλ, amyloid A, transthyretin and β2 microglobulin in order to confirm it as AL type [1O]. Multiple myeloma was excluded by serum M-protein lower than 3.0 g/ dl, plasma cells less than 10% of the total number of nucleated cells in the bone marrow and no related organ or tissue impairment according to the diagnostic criteria proposed by the International Myeloma Working Group [U]. The percentage of plasma cells in the bone marrow was examined on smear specimens treated with Wright- Giemsa staining. To identify M-protein in serum and urine, immunofixation was performed in all patients using a commercially available kit (Titan Gel IFE kit, Helena Laboratories, Saitama, Japan), and other information was obtained from their medical records. At study entry the extent of amyloidrelated organ involvement was evaluated in each patient according to clinical criteria proposed by Comenzo et al. [12].

Bone marrow aspirates from six patients with monoclonal gammopathy of undetermined significance (MGUS) lacking amyloid deposition (three with neuropathy and one each with parkinsonism, systemic sclerosis and Crow-Fukase syndrome; 2 men and 4 women; age range, 43 to 79 years; mean, 62.8 15.3 years) and 9 patients with other diseases (three with systemic lupus erythematosus, two each with rheumatoid arthritis and chronic inflammatory demyelinating polyneuropathy, and one each with Sjogren's syndrome and Raynaud's phenomenon; five men and four women; age range, 29-78 years; mean, 58.6 21.1 years) were also processed for flow cytometry as MGUS and negative controls, respectively. Negative controls showed no M- protein in either serum or urine on immunofixation.

Flow cytometry

Flow cytometry was performed as described elsewhere [8,9]. Briefly, cells were isolated from heparinized bone marrow aspirates using erythrocyte lysis buffer (Becton Dickinson, San Jose, CA, USA). They were resuspended at 5 10^sup 6^/ml, and an aliquot of 200 1 was put into a 10 ml tube. Twenty l of each appropriate monoclonal antibody (mAb) were then added to this tube, and incubated at 4 C in the dark for 30 min. The following mAbs were employed for flow cytometry: fluorescein isothiocyanate-conjugated mAb to CD38 (HIT2, Pharmingen, Marseille, France), phycoerythrin- conjugated mAbs to CD49e (VC5, Pharmingen), CD56 (B 159, Pharmingen) and MPC-I (JIMRO, Takasaki, Japan), PerCP-conjugated mAb to CD45 (2Dl, Becton Dickinson) and allophycocyanin-conjugated mAb to CD 19 (J4.119, Immunotech, Marseille, France). The labelled cells were analyzed by threeor four-color flow cytometry using FACSCalibur (Becton Dickinson). The gate was set on plasma cells, and 2 10^sup 4^ cells were analyzed to determine the percentages of cells positive for each mAb.

Serum FLC assay

Blood was taken from all subjects at the same time as the bone marrow aspiration, and after separation serum was cryopreserved at - 8OC with sodium azide as preservative until the assay. FLCs were measured in stored serum samples using a commercially available kit based on a latex-enhanced immunoassay (The Binding Site, Birmingham, UK) on a Behring BN II nephelometric analyzer (Dade Behring, Deerfield, IL, USA). The assay utilizes antibodies against FLC epitopes that are hidden in whole immunoglobulin molecules, and normal values in healthy subjects are 3.3-19Λ mg/1 in K, 5.7- 26.3 mg/1 in λ, and 0.26-1.65 in the &955; ratio. In each subject both K- and -type FLCs were determined, and the higher one was regarded as the serum predominant FLC. Serum creatinine was determined simultaneously at sampling, and the serum predominant FLC:serum creatinine ratio was calculated in each subject.

Statistics

To determine statistically significant differences among negative and MGUS controls and AL amyloidosis, Mann-Whitney's U test was employed for the serum predominant FLQserum creatinine ratio and phenotypes of plasma cells in the bone marrow. Correlation coefficient test was used for detection of a significant relationship between the serum predominant FLC:serum creatinine ratio and phenotypes of plasma cells. The results represent the mean standard deviation where applicable, and a p-level less than 0.05 was considered to be statistically significant. Commercially available statistics software was used for data analysis (StatView for Macintosh, Abacus Concepts, Berkeley, CA, USA).

Results

Clinical profiles of the patients are summarized in Table I. All patients showed plasma cells less than 10% on smear specimens of the bone ma\rrow except for case 16. In case 16, however, no increase in serum levels of immunoglobulin or related organ impairment was seen throughout the course of illness. In case 17, amyloid in tissues was identified as ALic-type protein by immunohistochemistry, although no M-protein could be detected in either serum or urine. Results of the serum FLC analysis in the patients are also demonstrated in Table I. All patients showed an increase in either κ- or λ-type FLCs except for case 8. The FLCs predominantly increased in serum completely coincided with the types of immunoglobulin light chain shown by immunofixation and/ or the immunohistochemical analysis of amyloid deposits. Immunofixation demonstrated no Mprotein in serum in cases 13, 15 and 17, while the serum FLC analysis showed positive results in all of these patients.

The statistical analysis of serum FLC and the phenotype of plasma cells are summarized in Table II. The serum predominant FLC:serum creatinine ratio showed a significantly higher level in either the patients (43.8 63.2) or the MGUS controls (5.62 4.55) than in the negative controls (2.29 0.52) (p < 0.0005 and p < 0.005, respectively). There was also a statistically significant difference in the serum predominant FLC: serum creatinine ratio between the patients and the MGUS controls (p < 0.05).

On flow cytometry the CD38^sup ++^ population is identified as plasma cells [13]. CD38^sup ++^ CD19^sup +^ CD56^sup -^ cells (polyclonal population) showed no significant difference between the patients (0.36 0.33%) and either the negative (1.57 2.11%) or MGUS controls (0.37 0.27%), while CD38^sup ++^ CD19^sup -^CD56^sup +^ cells (monoclonal population) showed a significantly higher level in the patients (2.57 5.35%) than in either the negative (0.06 0.03%, p < 0.001) or MGUS controls (0.10 0.07%, p < 0.05). According to antigens expressed on the cell surface, plasma cells are classified into three subtypes on flow cytometry: immature cells; MPC-l^sup - ^CD45^sup +^ CD49e^sup -^ or MPC-1^sup -^CD45^sup -^CD49e^sup -^, intermediate cells; MPC-I^sup +^CD45^sup -^CD49e^sup -^, and mature cells; MPC-I^sup +^CD45^sup +^ CD49e^sup -^ or MPC-I^sup +^CD45^sup +^ CD49e^sup +^ [13-17]. Intermediate and mature plasma cells were predominantly seen in 9 and 6 patients, respectively. In the immature subtypes, MPC-TCD45^sup +^ cells were significantly lower in the patients (4.30 4.25%) than in MGUS controls (13.5 12.6%, p < 0.05). The intermediate subtype (MPC-1^sup +^ CD45^sup - ^CD49e^sup -^) showed a significantly elevated level in the patients (49.2 23.2%) over the negative controls (16.9 10.7%, p< 0.005), whereas no significant difference was seen between the patients and the MGUS controls (27.0 20.6%). In the mature subtypes, MPC-1^sup +^ CD45^sup +^ CD49e^sup -^ cells were significantly decreased in the patients (27.6 21.3%) compared with the negative controls (55.9 14.8%, p < 0.005). Another mature subtype, CD45 + MPC-l^sup +^CD49e^sup +^ cells, showed no significant differences between the patients and either the negative or MGUS controls.

The correlation between phenotypes of plasma cells and the serum predominant FLC: serum creatinine ratio is demonstrated in Figure 1. In parallel with an increase in CD38^sup ++^ CD19^sup +^ CD56^sup - ^ and CD38^sup ++^ CD19^sup -^CD56^sup +^ cells, the serum predominant FLC:serum creatinine ratio was decreased and increased, respectively. There was a significantly positive correlation between the serum predominant FLC:serum creatinine ratio and either CD38^sup ++^ CD19^sup -^CD56^sup +^ cells (Figure IA, r= 0.510, p < 0.05) or the intermediate subtype (MPC-l^sup +^CD45^sup -^CD49e^sup -^) (Figure 1B, r= 0.481, p; < 0.05). Neither the immature (MPC- TCD45^sup +^ and MPC-1^sup -^CD45^sup -^) nor mature (MPC-I^sup +^CD45^sup +^ CD49e^sup -^ and MPC-I^sup +^ CD45^sup +^ CD49e^sup +^) subtypes showed a significant relationship between the percentage of these subpopulations and the serum predominant FLC:serum creatinine ratio.

Discussion

All patients were diagnosed as having primary systemic AL amyloidosis at enrollment based on immunohistochemical characterization of amyloid deposits and intensive survey of visceral organs. Possible association of multiple myeloma was excluded from the diagnosis in all patients by serum immunoglobulin lower than our criteria and absence of related organ impairment, although one patient showed bone marrow plasmacytosis of more than 10% of total nucleated cells. In another patient no M-protein could be detected in either serum or urine on immunofixation, but FLC analysis showed a high serum level with an abnormal &955; ratio.

Table 1. Clinical profiles of the patients and free light chain in serum.

Figure 1. The serum predominant FLC: serum creatinine ratio showed a significantly positive correlation with either CD38^sup ++^ CD19^sup -^CD56^sup +^ (A, r= 0.510, p < 0.05) or MPC-1^sup +^CD45^sup -^CD49e^sup -^ cells (B, r=0.481, p < 0.05).

In monoclonal gammopathies such as AL amyloidosis and multiple myeloma, protein electrophoresis and immunofixation have been used for detection of M-protein in serum and/or urine. At diagnosis, however, serum electrophoresis is not able to identify M-protein in more than half of all patients with AL amyloidosis, and quantification is usually imprecise or impossible because the concentration is too low in many patients [2]. Immunofixation is more sensitive for detecting M-protein in serum, but the results are not quantitative. In contrast to multiple myeloma in which M- protein is typically 10- to 100-fold more abundant, these conventional techniques are inadequate for evaluating minute changes in M-protein in AL amyloidosis. In this study, we employed the FLC analysis which has appeared in the last 5 years in order to detect M- protein in serum [3-7], as it is much more sensitive in detecting M- protein than conventional methods. Serum protein electrophoresis and immunofixation typically have detection limits of 500-2000 mg/1 and 150-500 mg/1, respectively, whereas the serum FLC analysis usually shows a sensitivity of less than 5 mg/1 [7]. According to a recent report FLC analysis could identify serum monoclonal protein in more than 98% of patients with AL amyloidosis [7]. Also in the present study, immunofixation was not able to detect M-protein in sera from three patients, all of whom showed elevated levels of serum FLCs with abnormal &955; ratios. Although the antibodies in the assay cannot distinguish monoclonal FLCs from low-level polyclonal ones that exist in the healthy population, a relative excess of κ- or λ-type FLCs correctly identified the amyloidogenic FLC class in each patient in this study. Another reason we used FLC analysis is that M-protein can be quantitatively detected by this method. To investigate which subpopulation of plasma cells contributes to the production of M-protein in AL amyloidosis, precise and quantitative detection of serum FLC was thought to be necessary. In patients with renal dysfunction, as in those with systemic AL amyloidosis, serum FLCs can probably be overestimated because their concentration depends on renal function [4,7,18]. As a marker of production of the amyloidogenic FLC, therefore, we employed the serum predominant FLC: serum creatinine ratio in this study [4].

The patients showed a significantly higher value in the serum predominant FLC:serum creatinine ratio and CD38^sup ++^ CD19^sup - ^CD56^sup +^ subpopulation indicating monoclonal plasma cells than either the negative or MGUS controls. With respect to maturation of plasma cells in the bone marrow, the intermediate (MPC-1^sup +^CD45^sup -^CD49e^sup -^) and mature (MPC-1^sup +^CD45^sup - ^CD49e^sup -^) subtypes were significantly higher and lower in the patients than in the negative controls, respectively. In parallel with an increase in CD38^sup ++^CD19^sup -^CD56^sup +^ and MPC- 1^sup +^CD45^sup -^CD49e^sup -^ cells and a decrease in mature subtypes, the serum predominant FLC:serum creatinine ratio was elevated. There was a significantly positive correlation between the serum predominant FLC: serum creatinine ratio and either CD38^sup ++^CD19^sup -^CD56^sup +^ or MPC-1^sup +^CD45^sup -^CD49e^sup -^ cells. These results suggest that the serum FLC in AL amyloidosis may be produced mainly by increased monoclonal plasma cells in the bone marrow, particularly by the intermediate subpopulation with the phenotype of MPC-1^sup +^CD45^sup -^CD49e^sup -^. In multiple myeloma, no report has so far described such a correlation between serum levels of M-protein and phenotypes of plasma cells. With respect to the production of M-protein, intermediate cells in the bone marrow might play a key role in the pathogenesis of primary systemic AL amyloidosis. In addition, 90% of the patients showed a predominant increase in either intermediate or mature plasma cells in the bone marrow. This result might reflect a low level of monoclonal plasma cells in the bone marrow in primary systemic AL amyloidosis, since the intermediate and mature subtypes slowly proliferate with no response to interleukin-6 [15,16,19].

Table II. Free light chain in serum and phenotype of plasma cells in the bone marrow.

In conclusion, M-protein can quantitatively and reliably be detected in serum using FLC analysis, which is more sensitive than serum protein electrophoresis and immunofixation. Monoclonal plasma cells were significantly increased in the bone marrow, and the serum predominant FLC:serum creatinine ratio showed a positive correlation with the intermediate subtype. In primary systemic AL amyloidosis, therefore, this subpopulation might mainly contribute to the production of M-protein. To confirm this hypothesis, serial studies of flow cytometry and serum FLC analysis are required before and after treatment in a larger set of patients.

Acknowledgment\s

The authors are grateful to Dr T. Ishihara, Department of Pathology, Yamaguchi University School of Medicine, for his help with immunohistochemical staining of the biopsy specimens. This work was supported by a grant from the Intractable Disease Division, the Ministry of Health and Welfare, Amyloidosis Research Committee, Japan.

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YASUHIRO SHIMOJIMA1, MASAYUKI MATSUDA1, TAKAHISA GONO1, WATARU ISHII1, TOMOHISA FUSHIMI1, YOSHINOBU HOSHII2, TOSHIYUKI YAMADA3, & SHU-ICHI IKEDA1

Third Department of Medicine, Shinshu University School of Medicine, Matsumoto, 2 First Department of Pathology, Yamaguchi University School of Medicine, Yamaguchi, and ''Department of Clinical Pathology, Juntendo University School of Medicine, Tokyo, Japan

Correspondence: Dr Masayuki Matsuda, Third Department of Medicine, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan. Tel: 81 263 372673. Fax: 81 263 373427. E- mail: matsuda@hsp.md.shinshu-u.ac.jp

Copyright CRC Press Mar 2005

Source: Amyloid

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