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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.
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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.
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.