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Keywords:

  • paraproteinaemias;
  • amyloidosis;
  • chromosome abnormalities;
  • benign;
  • monoclonal gammopathies;
  • multiple myeloma

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Primary systemic amyloidosis (AL) is a plasma cell disorder characterized by deposition of monoclonal light chains in different organ systems. Although multiple and complex numerical chromosomal abnormalities have been described in patients with multiple myeloma, it is currently unknown whether such changes occur in systemic amyloidosis.

Bone marrow samples from 21 patients with AL were studied by standard cytogenetics and interphase fluorescence in situ hybridization (FISH) for the presence of numerical chromosomal abnormalities. We tested for six chromosomes (7, 11, 9, 15, 18 and X) using centromere-specific probes. The monoclonal plasma cells were identified by simultaneous fluorescent staining of the monotypic cytoplasmic immunoglobulin. We compared these results with those obtained from 19 patients with monoclonal gammopathy of undetermined significance (MGUS) and normal controls.

Multiple numerical chromosomal abnormalities were detected in AL by interphase FISH, including trisomy of chromosomes 7 (42%), 9 (52%), 11 (47%), 15 (39%), 18 (33%) and X (13% in women and 54% in men). Monosomy of chromosome 18 was seen in 72% of cases. Previous exposure to alkylator therapy did not appear to correlate with these abnormalities. No significant difference was observed in the prevalence of these abnormalities between AL and MGUS.

Multiple chromosomal numerical abnormalities were detected by interphase FISH analysis in patients with AL, especially monosomy of chromosome 18. Aneuploidy in the monotypic plasma supports a neoplastic nature for the disorder.

Primary systemic amyloidosis (AL) is a disorder of clonal plasma cells with a median survival of approximately 1.5 years ( Gertz & Kyle, 1994). Plasma cell clonality is demonstrated by the presence of monoclonal serum proteins and a monotypic plasma cell population in the bone marrow. However, it is at present unclear whether AL shares some of the features of other plasma cell malignant diseases, such as chromosomal abnormalities. In contrast to multiple myeloma, AL causes morbidity and mortality by production and deposition of the amyloid fibrils, not by the tumour burden imposed on the host ( Gertz & Kyle, 1994).

Multiple and complex chromosomal abnormalities have been described in patients with multiple myeloma ( Dewald et al, 1985 ; Gould et al, 1988 ; Weh et al, 1993 ; Lai et al, 1995 ; Sawyer et al, 1995 ; Zandecki et al, 1996 ). Cytogenetic analysis of plasma cells in patients with AL has not been reported. In this study we sought to determine if the bone marrow plasma cells of patients with AL have the same numerical chromosomal abnormalities as those reported in multiple myeloma and monoclonal gammopathy of undetermined significance (MGUS).

The use of standard cytogenetic analysis in the evaluation of AL is hampered by the small number of monotypic plasma cells, which in turn have a very low proliferative rate. Sensitive techniques to study interphase cells, such as fluorescence in situ hybridization (FISH), have been used to evaluate aneuploidy in multiple myeloma and MGUS ( Drach et al, 1995a , b; Zandecki et al, 1995 ), and similar applications are therefore possible in AL. However, the identification of plasma cells is subjective and depends on morphologic features and observer experience. To overcome this difficulty we improved the standard FISH technique by incorporating simultaneous fluorescent staining of cytoplasmic immunoglobulin (cIg) ( Ahmann et al, 1998 ). With this technique it is possible to identify plasma cells and perform cell-specific FISH analysis.

As a result of this study we can report that numerical chromosomal abnormalities, as determined by interphase FISH, are present in AL. We compare these results with those of standard cytogenetic analysis and interphase FISH in patients with MGUS and in normal controls. Knowledge of these chromosomal abnormalities enhances our understanding of the biology and pathophysiology of the disease and could provide clues to the necessary steps of disease pathogenesis.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Patient samples

We obtained bone marrow from 14 normal controls, 19 patients with MGUS, and 21 patients with AL. All patients and controls gave written informed consent for bone marrow aspiration before the time of routine procurement of clinical samples. We obtained Institutional Review Board approval and conducted the research in accordance with the Declaration of Helsinki for research with human subjects. AL was diagnosed when tissue diagnosis was consistent with amyloid deposition along with the clinical spectrum of the disease. This included detection of a monoclonal protein (in serum or urine) or a monoclonal plasma cell population in the bone marrow (immunohistochemistry or flow cytometry).

Standard cytogenetic analysis

Bone marrow specimens were processed by both direct and short-term culture techniques, as described previously ( Spurbeck et al, 1988 ). Each of our patients had at least 20 QFQ-banded (Q bands using fluorescence by quinacrine mustard) or GTL-banded (G bands using trypsin and Leishman's stain) metaphases analysed. Representative karyotypes were prepared from at least two metaphases from each clone. Each karyotype was described according to the International System for Human Cytogenetics Nomenclature ( Mitelman, 1995).

Fluorescence in situ hybridization and cIg staining

Bone marrow samples were drawn into a tube containing heparin and centrifuged on a Ficoll-Paque gradient (Pharmacia-Biotech, Sweden) (density 1.076) to enrich for mononuclear cells. Cytospin 3 (Shandon) slides were prepared and allowed to dry overnight. The slides were then fixed in 95% ethanol for 5 min at room temperature, allowed to dry, and aged in a 37°C oven for 1–6 h. The Cytospin slides were denatured in 91% formamide at 37°C for 5 min, dehydrated in a series of ethanol (70%, 85% and 100%) for 1 min each, and air dried. The α-satellite DNA probes were direct-labelled with either SpectrumOrange or SpectrumGreen (Vysis, Downers Grove, Ill.) and centromere specific for chromosomes 7, 9, 11, 15, 18 and X. The probes for these chromosomes were chosen because abnormalities of these chromosomes were previously described in multiple myeloma ( Drach et al, 1995a , c; Tabernero et al, 1996 ). 8 μl of chromosome enumeration probe buffer (Vysis), 1 μl of each chromosome enumeration probe (Vysis), and 1 μl of double-distilled water were mixed, denatured for 5 min in a 70°C bath, and then quenched on ice for 2 min. The probe mixture, containing two probes (one green and the other orange) with combinations of 7 and 11, 15 and 18 and X and 9, was placed on three separate slides. The slides were then cover-slipped, sealed with rubber cement, and allowed to hybridize in a 37°C humidified chamber overnight.

After hybridization, the slides were washed in 65% formamide at 37°C for 15 min, exposed to 2 × standard saline citrate for 8 min at 37°C, and washed at room temperature for 2 min with 2 × standard saline citrate and 0.1% nonidet phosphate 40. The slides were then stained with either goat anti-human kappa or lambda light chain conjugated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) (Vector Labs, Burlingame, Calif.) for 30 min at room temperature. After incubation the slides were washed twice in 1 × phosphate-buffered detergent. To enhance the intensity of AMCA staining, an anti-goat immunoglobulin conjugated with AMCA was added for 30 min at room temperature, and the slides were washed twice more in phosphate-buffered detergent. Finally, Vectashield (Vector Labs) was applied, and the slides were cover-slipped.

Statistical considerations

We intended to count 100 cIg-positive (plasma cell- or AMCA-positive) and 100 cIg-negative cells for each probe in all patients. Two observers scored the slides independently, and the mean of their results was used. A pair of chromosomes was considered evaluable if at least 20 plasma cells could be scored. Thus, for all chromosomes studied in a given patient sample, at least 20 observations were evaluable. The Cytospins were scored using a Zeiss or a Leitz epifluorescence microscope with a fluoroisothiocyanate, Texas red, or propidium iodide dual-pass filter and a DAPI ultraviolet filter (Chromotechnology, Brattleboro, Vt.). This procedure retained the characteristic plasma cell morphology along with the cytoplasmic blue fluorescent cIg staining and permitted simultaneous scoring of the centromere FISH signals (Fig 1). Images were stored and printed by the Vysis Smartcapture imaging system.

A patient was said to have trisomy or monosomy when the percentage of plasma cells exceeded the upper limit of a 95th percentile of normals. We used gender-specific 95th percentiles for the evaluation of gain or loss of chromosome X. The 95th percentiles were calculated on the basis of the number of cIg-negative cells in patients and cIg-positive cells in controls. The intraclass correlation coefficient statistic was used to assess interobserver variability. Distribution statistics were used to describe the percentage of plasma cells with the numerical abnormality. The main objective of our study was to document whether numerical chromosomal abnormalities were present or absent. Rank-sum tests were used to compare proportions among groups. The correlation of the chromosomal findings with clinical outcome was beyond the scope of this study.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Patient demographics

For patients with AL the median age was 62 years (range 38–82). Of the 21 patients, eight were female (38%) and 13 were male (62%). Of all the patients, nine (43%) had received previous chemotherapy for AL. One patient (5%) had associated multiple myeloma. Immunoelectrophoresis or immunofixation of the serum or urine demonstrated a monoclonal spike in 20 patients (95%): lambda type in 15 (75%) and kappa in five (25%). The remaining patient had a monoclonal plasma cell population in the bone marrow, as determined by immunohistochemistry and tissue biopsy proof of amyloidosis. For patients with MGUS, the median age was 66 years (range 36–83). Among these 19 patients, eight were female (42%) and 11 were male (58%). Bone marrow samples from controls were collected at the time of harvesting for allogeneic bone marrow donation ( 1 Table I).

Table 1. Table I. Patient demographic variables among three patient groups. MGUS, monoclonal gammopathy of undetermined significance; NA, not applicable.* The patient had a monoclonal plasma cell population in the bone marrow, as determined by immunohistochemical staining and positive tissue deposition with amyloid.Thumbnail image of

Standard cytogenetic analysis

Standard cytogenetic analysis was performed in 21 patients with AL, and adequate metaphases were obtained in 20 (95%). Results were normal in 18. In one patient the sole cytogenetic abnormality was absence of chromosome Y. In another patient, who had previously been exposed to alkylator therapy, the following translocation was found: 46,XX,+der(1;7)(q10;p10),−7[10]/46,XX[10]. Standard cytogenetic analysis was performed in 19 patients with MGUS, and adequate metaphases were obtained in all 19. The only cytogenetic abnormality detected was absence of chromosome Y in one patient.

Interphase FISH analysis

Monotypic plasma cells had easily identifiable trisomic or disomic FISH signals. The percentage of cells with trisomy and monosomy for each chromosome and condition is shown in 2 Tables II and III and Fig 2. There was no suggestion of trisomy or monosomy in the cIg-negative cells for any of the six chromosomes tested. In all patient samples studied at least one chromosomal abnormality was detected.

Table 2. Table II. Proportion of patients with trisomy by interphase fluorescence in situ hybridization. AL, systemic amyloidosis; MGUS, monoclonal gammopathy of undetermined significance. Comparison to normals with trisomy (=5%): *P < 0.001; †P = 0.02; ‡P geqslant R: gt-or-equal, slanted 0.05. Thumbnail image of
Table 3. Table III. Proportion of patients with monosomy by interphase fluorescence in situ hybridization. AL, systemic amyloidosis; MGUS, monoclonal gammopathy of undetermined significance. Comparison to normals with monosomy (=5%): *P < 0.001; †P < 0.01; ‡P = 0.04; §P geqslant R: gt-or-equal, slanted 0.05. Thumbnail image of
image

Figure 2. Fig 2. Dot plot graphs of the percentage of plasma cells with trisomy (above) and monosomy (below) for the different chromosomes in patients with systemic amyloidosis (▵) and monoclonal gammopathy of undetermined significance (○).

Download figure to PowerPoint

Multiple numerical abnormalities were detected in the cIg-positive cells for the chromosomes tested in patients with AL. Trisomy was found for chromosomes 7 (42%), 9 (47%), 11 (52%), 15 (39%) and 18 (33%) (all with P < 0.001). Gain of an extra copy of chromosome X was significant in men (53%) (P < 0.001) but not in women (28.6%) (P = 0.5631). Monosomy was observed for chromosomes 7 (47%), 11 (42%), 18 (72%) (P < 0.001) and 9 (19%) (P = 0.035). Loss of signal for chromosome X was seen in 12.5% of women (P = 0.5631) but not in men (0%). Scoring for a given chromosome was not achieved in zero to three patient samples for every set studied.

Coexistence of multiple chromosomal abnormalities in a given patient sample was common. For instance, most patients with trisomy for chromosome 9 were also trisomic for chromosome 7. Because of the sample size, no specific interrelations among chromosomal abnormalities were studied. Furthermore, in several patient samples, both monosomy and trisomy coexisted for a given chromosome. For example, with chromosome 18, three patients had both monosomy and trisomy in different plasma cell clones.

No significant difference was identified in the proportion of trisomy or monosomy between AL and MGUS (P > 0.05) ( 2 Tables II and III and Fig 2).

Comparison between methods

None of the numerical abnormalities detected by interphase FISH in both AL and MGUS were observed with conventional cytogenetic analysis. This study was not designed to look for structural abnormalities (i.e. translocations) with the FISH technique.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

In this study we have shown that patients with AL frequently have numerical chromosomal abnormalities, as determined by interphase FISH. This is an unprecedented finding and further supports the neoplastic nature of the monotypic plasma cell population in the bone marrow of AL, albeit with an atypical clinical behaviour. Similarly, we have shown that numerical chromosomal abnormalities are also found in patients with MGUS.

Plasma cells in patients with multiple myeloma frequently display complex chromosomal abnormalities, especially in the advanced stages of the disease ( Dewald et al, 1985 ; Gould et al, 1988 ; Weh et al, 1993 ; Drach et al, 1995 b, c; Laiet al, 1995 ; Smadja et al, 1995 ; Tricot et al, 1995 ; Zandecki et al, 1995 , 1996; Tabernero et al, 1996 ). With conventional cytogenetics, 30–50% of patients have these abnormalities ( Dewald et al, 1985 ). When more sensitive techniques, such as interphase FISH, are used, however, the proportion is significantly higher ( Drach et al, 1995a , c; Tabernero et al, 1996 ). We tested the hypothesis that numerical abnormalities would be seen in patients with AL. Our results support the idea that numerical chromosomal abnormalities are common in AL and that interphase FISH analysis is a more powerful technique for the analysis of these plasma cell disorders. None of the numerical chromosomal abnormalities detected by FISH were detected by conventional cytogenetics. In multiple myeloma, we recently found that when conventional cytogenetics detects numerical chromosomal abnormalities, the percentage of plasma cells abnormal by interphase FISH is high (>80%) (unpublished observations).

We tested for the six chosen chromosomes because they have been frequently reported as abnormal in patients with multiple myeloma ( Drach et al, 1995a , c; Tabernero et al, 1996 ). We found a high incidence of abnormalities in both AL and MGUS. Among all abnormalities, the high prevalence of monosomy of chromosome 18 in AL stood out. Although this abnormality is also observed in multiple myeloma, it occurs in a much lower proportion (6–11%) ( Drach et al, 1995c ; Tabernero et al, 1996 ). The significance of this observation is not known.

Certain oncogenes located in the trisomic chromosomes could be present in excess dosage and contribute to the pathogenesis of the disease; however, this seems unlikely. Alternatively, genomic instability resulting from loss of check mechanisms could be reflected as aneuploidy in the monoclonal plasma cells.

Although some of the patients had previously received alkylator therapy, most had not. Furthermore, when we analysed for the numerical abnormalities by stratification according to exposure to previous treatment, there was no suggestion of therapy-related aneuploidy ( 4 Table IV). The exception was that trisomy for chromosome 7 appeared to be more common in the patients treated with alkylating agents. Thus, most of these abnormalities seem not to be therapy-related.

Table 4. Table IV. Proportion of patients with primary systemic amyloidosis who had numerical chromosomal abnormalities according to previous treatment with an alkylating agent. * Chi-square testing comparing proportions between treated patients and those not previously treated.Thumbnail image of

Although labelling index and beta2-microglobulin ( Gertz et al, 1989 , 1990) are laboratory prognostic tools in AL, better prognostic factors are needed for the evaluation of these patients. As in multiple myeloma, the finding of specific chromosomal abnormalities could have prognostic significance. Tricot et al (1995 , 1997) analysed the prognostic value of cytogenetics in patients with multiple myeloma treated with high-dose chemotherapy and autotransplants. They found that the translocations, abnormalities of chromosome 11q, and deletions of chromosome 13 (the so-called unfavourable cytogenetics) were associated in an independent manner with adverse outcome. Some of these regions contain oncogenes or tumour suppressor genes capable of promoting cell cycle progression.

In AL the morbidity and mortality are secondary to the amyloid deposition in target organs and not secondary to cell replication. It is conceivable, then, that the genes involved in cell cycle regulation and growth control are not as important as those involved in the regulation of protein synthesis by the plasma cells. Because of this, the cytogenetic abnormalities described in multiple myeloma may not be of prognostic significance in patients with AL.

To better understand the pathogenesis of AL, more detailed studies of the chromosomal and molecular alterations in the monotypic plasma cells are needed. It has been suggested that translocations involving the 14q32 region are very common in multiple myeloma ( Chesi et al, 1996 ; Nishida et al, 1997 ), and it would be of interest to find out if similar structural abnormalities are frequent in AL.

In summary, trisomies involving chromosomes 7, 9, 11, 15, 18 and X are common in AL, but monosomy of chromosome 18 stands out and was observed in a large proportion of patients. Further studies to elucidate the pathogenic significance of these abnormalities and their prognostic significance are warranted.

Footnotes
  1. Nothing in this publication implies that Mayo Foundation endorses any products of companies mentioned in this article.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Mr G. J. Ahmann and Drs P. R. Greipp and R. A. Kyle are supported in part by research grant CA62242 from National Institutes of Health. Dr S. M. Jalal is supported in part by a research grant from Vysis Inc., Downers Grove, Ill. Dr G. W. Dewald is supported in part by research grants from Oncor Inc., Gaithersburg, Md., and Vysis Inc., Downers Grove, Ill.

References

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  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References
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