Molecular and genetic events associated with the transition from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) are still poorly characterized. We investigated serial bone marrow specimens from 11 patients with MGUS who eventually progressed to MM (MM post-MGUS) by interphase fluorescence in situ hybridization for immunoglobulin heavy-chain gene (IgH) translocations and chromosome 13q deletions (del(13q)). In nine patients, IgH translocations were present both in MGUS and MM post-MGUS plasma cells, including three t(11;14)(q13;q32) and one t(4;14)(p16;q32), which was observed already 92 months prior to MM. Similarly, all five MM patients with del(13q) had this aberration already at the MGUS stage. Two patients without IgH translocation and del(13q) had chromosomal gains suggesting hyperdiploidy, but IgH translocations and/or del(13q) did not emerge at MM post-MGUS. IgH translocations and del(13q) are early genetic events in monoclonal gammopathies, suggesting that additional events are required for the transition from stable MGUS to progressive MM.
Multiple myeloma (MM) may be preceded by a premalignant condition termed monoclonal gammopathy of undetermined significance (MGUS). Among individuals with MGUS, the probability of progression to MM, amyloidosis, or another B-cell lymphoproliferative disease has been reported to be 1% per year.1 However, genetic and molecular events associated with the transition from MGUS to MM are still unknown. Recurrent chromosomal aberrations have been detected at all stages of monoclonal gammopathies,2, 3 and they have been used to define a multistep model of the progression from a normal plasma cell through MGUS to MM.4, 5 Specifically, primary translocations involving the immunoglobulin heavy-chain locus (IgH) and deletions of chromosome 13q (del(13q)) have been shown to occur in MGUS plasma cells.6, 7, 8, 9, 10 Existing data suggest that IgH translocations are primary chromosomal events in a substantial proportion of patients with monoclonal gammopathies; however, the role of del(13q) in the progression of MM is still a matter of debate.3 In order to address this issue, we investigated IgH translocations and del(13q) in serial bone marrow specimens from patients who were diagnosed with MGUS and eventually progressed to symptomatic MM.
Patients and methods
Patients and cells
Among 31 patients with MM developing from a pre-existing MGUS (MM post-MGUS), we identified 11 patients in whom bone marrow samples were available both from the time of MGUS and after progression to MM. MGUS had been diagnosed according to standard criteria1, 11 including disease stability over a period of at least 12 months. All cases of MM post-MGUS were characterized by a sudden development of MM, that is, within 6 to 12 months corresponding to the time between the last follow-up and recognition of MM. The median time between diagnosis of MGUS and progression to MM was 66 months (range, 37–94 months). The median age at the time of MM was 69 years (range, 48–76 years); the paraprotein was IgG in seven patients and IgA in four patients.
Interphase fluorescence in situ hybridization (FISH) studies
To detect translocations of the IgH gene locus at 14q32, differentially labeled probes bracketing this locus and specific for VH and CH regions (purchased from Vysis Inc., Downers Grove, IL, USA) were used. To identify the two most common primary IgH translocations, probes localizing to the chromosomal loci involved in the translocation were utilized: For the t(11;14)(q13;q32), the probe set from Vysis was purchased, and the t(4;14)(p16.3;q32) was detected with probes previously described and kindly provided by Rafael Fonseca (Mayo Clinic, Rochester, MN, USA).6, 12 The status of chromosome 13q14 was determined by the retinoblastoma gene-1 (rb-1) probe, and numerical abnormalities were evaluated by the respective pericentromeric probes (Vysis).
FISH studies were always combined with cytoplasmic staining of kappa or lambda light chains (cIg) according to the paraprotein in order to correlate the chromosomal finding with the clonal plasma cell population. Cells fixed in Carnoy's solution were stained with anti-human kappa or lambda antibody, followed by the hybridization procedure with the FISH probes as described.13 Whenever possible, 100 cIg-positive plasma cells were evaluated by fluorescence microscopy. Cells with separation of the VH/CH signals were considered as having an IgH translocation; the presence of a fusion signal with the translocation-specific probes was interpreted as evidence for a t(11;14) and t(4;14), respectively. Signals of the rb-1 probe were evaluated as described.14
Results and discussion
Serial studies were performed in 11 patients to compare the chromosomal pattern of MGUS plasma cells with that of the plasma cells after progression to MM (Table 1). In nine of the 11 patients, there was evidence for an IgH translocation: In patients 1–5, the abnormality was designated as a t(14q) (IgH translocation with an unknown partner chromosome) because hybridization results were negative for t(11;14)(q13;q32) and t(4;14)(p16;q32). Patients 6–8 were characterized by a t(11;14)(q13;q32), and patient 9 had a t(4;14)(p16;q32). All IgH translocations were present not only in MM post-MGUS but also in MGUS plasma cells; moreover, with the exception of patient 2, the abnormalities were detected in the majority of cIg-positive plasma cells. In five of the patients with an IgH translocation (patients 3, 4, 5, 8 and 9), a del(13q) was observed; the abnormality was present in the plasma cells both at the time of MGUS and MM post-MGUS. All other patients were consistently normal for chromosome 13q14 at both evaluation time points.
Patients 10 and 11 did not have any IgH translocation, neither at MGUS nor at MM post-MGUS. Similarly, chromosome 13q14 was found to be disomic. Plasma cells from these two patients obviously corresponded to the hyperdiploid variant of MM12 because gains of chromosomes 3, 7, 9 and 11 were found at MM post-MGUS. At MGUS, cells were available to study only one of these numerical abnormalities, and gain of chromosome 11 was observed at MGUS as well. When numerical abnormalities were tested among patients with an IgH translocation±del(13q14), disomic results were obtained in all evaluations (see Table 1).
Results of this comparative analysis of serial bone marrow samples indicate that chromosomal abnormalities, in particular IgH translocations and del(13q), observed at MM post-MGUS were already present in the MGUS plasma cells 37–94 months prior to the development of MM. Previous studies have suggested that IgH translocations occur early in the clonal development of monoclonal gammopathies, possibly as an immortalizing event,4 because similar frequencies of IgH translocations were described in MGUS and MM.6, 7, 9 The situation was less clear with respect to del(13q), since del(13q) was reported to occur at a substantially lower incidence in MGUS as compared to MM and hence was suggested to represent a progression event.7, 9 Other reports show a near identical prevalence of del(13q) in MGUS and MM.6, 8 The present study demonstrates that in all five cases with a del(13q) the MGUS plasma cells already carried the abnormality, usually in a large proportion of plasma cells. In the remaining cases with a normal status of 13q in MGUS plasma cells, there was no evidence for evolution of a del(13q) at the time of MM post-MGUS. Our observations are therefore in support of the notion that del(13q) is an initiation rather than a progression event in monoclonal gammopathies.
Patient 9 was the only case in this series in whom a t(4;14)(p16.3;q32) was detected. This abnormality is observed at a lower frequency in MGUS (<10%)6, 7 compared to MM (about 15%), where it is also associated with short survival.15, 16, 17 It has therefore been suggested that t(4;14) leads to a rather rapid development of MM due to the aggressive nature of this entity, with a lower probability of detecting the t(4;14) during a MGUS phase. In our patient, it was therefore rather unexpected to observe the t(4;14) already 94 months prior to MM in the majority of MGUS plasma cells. If confirmed also in other patients, this would imply that a t(4;14) by itself is insufficient to cause further progression to MM. The same can be assumed for del(13q), which was also present in this patient's plasma cells. In this particular case, the t(4;14) may have preceded the occurrence of the del(13q), because the frequency of MGUS plasma cells carrying the t(4;14) was substantially higher (72.6%) compared to that of the MGUS plasma cells with del(13q) (30.8%; see Table 1). The t(4;14) plus del(13q) clone must have undergone preferential expansion over time due to similar frequencies of both abnormalities at the time of MM post-MGUS.
Numerical chromosomal abnormalities, usually reflecting hyerdiploidy, are known recurrent aberrations in MGUS and MM.3, 18, 19, 20 Among the cases tested in this series, there was no apparent change between MGUS and MM post-MGUS, again suggesting lack of an imminent association of numerical chromosomal gains with the MGUS to MM transition.
It can be assumed that considerable time may be required for a particular plasma cell clone to expand in order to develop from MGUS to the stage of MM. Despite the chromosomal abnormalities observed in the MGUS plasma cells of patients examined in this investigation, MGUS remained stable for years before the emergence of progressive MM requiring therapy. The serial determinations reported here therefore suggest that IgH translocations as well as del(13q) do not immediately precede the transition from stable MGUS to progressive MM. We rather hypothesize that presence of such chromosomal aberrations in MGUS plasma cells may facilitate the emergence of other molecular events (eg ras mutations and dysregulation of other oncogenes,2, 3 epigenetic changes,21 genes implicated in various signaling pathways22) that eventually lead to progression to MM. Prospective follow-up of individuals with MGUS will be required to resolve the issue of whether or not time to progression from MGUS to MM may be different depending on the primary chromosomal event(s). It is hoped that these studies may ultimately lead to the definition of reliable predictive factors to identify patients at risk for development of MM.
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This work was supported in part by grants from the Austrian National Bank (Jubiläumsfonds Project # 7730), CLEXO (Center of excellence in clinical and experimental oncology) and UNRUHE Privatstiftung.
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