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© 2000 American Society for Clinical Oncology Predictive Role of Interphase Cytogenetics for Survival of Patients With Multiple MyelomaFrom the First Department of Internal Medicine, Divisions of Clinical Oncology, and Hematology and Hemostesiology, and Departments of Dermatology and Pathology, University of Vienna; Department of Internal Medicine I With Medical Oncology, Wilhelminenspital; Third Department of Internal Medicine and Ludwig-Boltzmann-Institute for Hematology and Leukemia Research, Hanuschspital, Vienna, Austria. Address reprint requests to Johannes Drach, MD, University of Vienna, First Department of Internal Medicine, Division of Clinical Oncology, Währinger Gürtel 18-20, A-1090 Vienna, Austria; email johannes.drach{at}akh-wien.ac.at
PURPOSE: Recent metaphase cytogenetic studies suggested that specific chromosomal abnormalities are of prognostic significance in patients with multiple myeloma (MM). Because the true incidence of chromosomal abnormalities in MM is much higher than that detected by metaphase analysis, we used interphase fluorescence in situ hybridization (FISH) to determine the prognostic value of specific chromosomal aberrations. PATIENTS AND METHODS: Bone marrow plasma cells from 89 previously untreated patients with MM were studied consecutively by FISH to detect the deletions of 13q14, 17p13, and 11q and the presence of t(11;14)(q13;q32). FISH results were analyzed in the context of clinical parameters (response to treatment and survival after conventional-dose chemotherapy), and a multivariate analysis of prognostic factors was performed. RESULTS: By FISH, the deletion of 13q14 occurred in 40 patients (44.9%), deletion of 17p13 in 22 (24.7%), and 11q abnormalities in 14 (15.7%; seven with t(11;14)). Deletions of 13q14 and 17p13 were associated with poor response to induction treatment (46.9% v 77.3% in those without deletions, P = .006 and 40.0% v 73.2%, P = .008, respectively) and short median overall survival (OS) time (24.2 v 88.1 months, P = .008 and 16.2 v 51.3 months, P = .008, respectively). Short median OS time was also observed for patients with 11q abnormalities (13.1 v 41.6 months, P = .02). According to the number of unfavorable cytogenetic features (deletion of 13q14, deletion of 17p13, and aberrations of 11q) that were present in each patient (0 v 1 v 2 or 3), patients with significantly different OS times could be discriminated from one another (102.4 v 29.6 v 13.9 months, P < .001, respectively). CONCLUSION: For patients with MM who were treated with conventional-dose chemotherapy, interphase FISH for 13q14, 17p13, and 11q provides prognostically relevant information in addition to that provided by standard prognostic factors. This observation may be considered for risk-adapted stratifications of MM patients in future clinical trials.
THE CLINICAL COURSE of patients with multiple myeloma (MM) may be highly variable, and survival times range between only a few months and several years.1 This heterogeneity has stimulated clinically oriented research to develop prognostic models that allow a more precise estimate of a patient’s prognosis. This development has become particularly relevant in view of the availability of different therapeutic options, ie, conventional-dose chemotherapy and high-dose therapy followed by autologous stem-cell support. Although the latter approach has recently been demonstrated to improve response rates, disease-free survival, and overall survival (OS),2,3 it is not yet clear which patients with MM benefit most from myeloablative therapy that is administered as frontline treatment. Currently available independent prognostic factors include clinical staging according to Durie and Salmon,4 plasma-cell proliferation,5,6 and plasma-cell morphology,7-9 as well as the serum levels of beta-2-microglobulin (B2M),5,10,11 of C-reactive protein (CRP),11 and of lactate dehydrogenase (LDH)12; each parameter, however, has its limitations.13 Most recently, there is increasing evidence for an important role of cytogenetics in MM: Tricot et al14,15 reported that patients with MM had short event-free survival and OS times in the presence of partial or complete loss of chromosome 13 and/or abnormalities that involve chromosome 11q. Of note, these patients were uniformly treated with tandem high-dose chemotherapy followed by autotransplantation. Other conventional cytogenetic studies failed to detect such prognostic implications of abnormal cytogenetics in MM,16,17 but this result may be related to difficulties in obtaining metaphases from myeloma cells. Interphase cytogenetics has been shown to be advantageous for the detection of specific chromosomal abnormalities in MM because interphase analysis is informative in all patients with newly diagnosed MM. This leads to the observation that virtually all myelomas are cytogenetically abnormal.18-21 In the context of cytogenetics and prognosis in MM, it is of particular note that deletions of 13q were again shown to have an unfavorable prognostic variable when detected by interphase fluorescence in situ hybridization (FISH).22,23 In addition, interphase FISH identified deletions of 17p that involved the p53 gene locus as being chromosomal abnormalities in MM that were independently correlated with short survival time after conventional-dose chemotherapy.24 Taken together, the results from both the metaphase and interphase cytogenetic studies support the hypothesis that specific chromosomal aberrations are of major prognostic relevance in MM. Therefore, we performed a comprehensive interphase FISH analysis of the deletion of 13q14, deletion of 17p13, and abnormalities of 11q in 89 consecutive patients with newly diagnosed MM. We aimed at (1) defining the predictive role of these chromosomal abnormalities for response to treatment and survival, (2) investigating the independent significance of FISH results and standard prognostic factors in a multivariate analysis, and (3) establishing a prognostic model for patients with MM based on cytogenetic abnormalities.
Patients Eighty-nine patients with previously untreated MM were studied consecutively. Pertinent demographic features are summarized in Table 1. The induction treatment of 76 patients consisted of conventional-dose chemotherapy, including melphalan-based regimens (melphalan and prednisone; vincristine, melphalan, cyclophosphamide, and prednisone ± carmustine) in 64 patients, VAD (vincristine, doxorubicin, and dexamethasone) in 11 patients, and pulsed high-dose dexamethasone in one patient. Response to treatment was evaluated using recently published consensus criteria.25 The reasons for not receiving chemotherapy were early death (three patients) or MM at stage I without symptoms and with no evidence of progressive disease (10 patients).
Cells Bone marrow (BM) aspirates were obtained from the posterior iliac crest or sternum and collected in a heparinized syringe. All procedures were performed for diagnostic purposes, and informed consent according to institutional guidelines was obtained for the use of an aliquot of the specimens for research purposes. After dilution with phosphate-buffered saline, mononuclear BM cells were separated by density-gradient centrifugation over Ficoll-Hypaque (density = 1.077; Sigma Inc, St Louis, MO). Mononuclear cells were washed twice with phosphate-buffered saline, treated with Carnoy’s fixative (methanol and glacial acetic acid [3:1, vol/vol]), and stored at -20°C or -80°C. Specimens that were used as controls consisted of peripheral blood samples (n = 5) of healthy individuals and of BM samples from patients with Hodgkin’s disease, non-Hodgkin’s lymphoma, or solid tumors without histologic evidence of BM involvement (n = 4).
Interphase FISH Studies
Hybridization was performed as detailed in a previous report.27 At least 200 cells with the morphologic appearance of plasma cells were scored with a Zeiss Axioplan-2 immunofluorescence microscope equipped with a triple band pass filter (Zeiss, Jena, Germany). Slides that met the criteria of high hybridization efficiency (
Cutoff levels for the distinction between background and true aneuploidy were derived from experiments with the control specimens. The frequency of normal cells that exhibited apparent abnormalities was recorded; cutoff levels for true aneuploidy, which were set at the mean plus 3 SDs of nondisomies observed in normal cells, were as follows: deletion of 13q14, 9.3%; deletion of 17p13, 8.6%; deletion of 11q23, 4.4%; gain of 11q23, 4.8%; t(11;14), 8.9%; gain of chromosome 11 centromeric probe, 1.3%; and gain of chromosome 17 centromeric probe, 3.3%. Metaphase cytogenetic studies were performed as previously described.24
Statistical Analysis
FISH Results in 89 Patients With MM As summarized in Table 2, a deletion of 13q14 was present in 40 patients (44.9%), a deletion of 17p13 in 22 patients (24.7%), and 11q abnormalities in 14 patients (15.7%). In this series of patients with previously untreated MM, deletions of 13q14 and 17p13 were found to be monoallelic, which is in agreement with our previous experience.23,24 With respect to abnormalities of chromosome 11q by interphase FISH, five patients had a deletion of 11q23, whereas a deletion of 11q13 was never observed. A fusion signal of 11q13 and 14q32, which indicated the presence of a t(11;14) (Fig 1), was observed in seven patients (7.9%). Two patients had a gain of hybridization signals, with the 11q13- and 11q23-specific probes, in the absence of a gain of 11 centromere, suggesting other structural aberrations that involved the long arm of chromosome 11.
In 37 patients (41.6%), there was no evidence of a deletion of 13q14, deletion of 17p13, or an abnormality of 11q by interphase FISH. Thirty-two patients (37.6%) exhibited concomitant gains of all chromosome-11 probes (centromere, 11q13, and 11q23), which indicates the presence of a trisomy 11. Trisomy 17 by FISH, which was defined as a concomitant gain of chromosome-17 centromere and 17p13, was observed in nine patients (10.1%). When all types of abnormalities were considered (both structural aberrations, as detailed above, and trisomies 11 and 17), there were only 16 patients (18.0%) with normal hybridization patterns of chromosomes 11, 13, and 17 by FISH.
Comparison of Metaphase Cytogenetics and Interphase FISH
Response to Treatment and Survival Seventy-six patients who received conventional-dose chemotherapy as induction treatment were assessable for the analysis of response to treatment. Objective responses were observed in 49 patients (64.5%), with partial remission in 46 (60.5%) and complete remission in three (3.9%). However, response rates were significantly lower if a deletion of 13q14 (46.9%; P = .006) or a deletion of 17p13 (40.0%; P = .008) was present (Table 2). Although response rates were also lower in patients with 11q abnormalities (53.8%), this difference was below the level of statistical significance (P = .40). Regarding the OS time of all 89 patients as determined from the time of diagnosis (Table 2), the median OS time was significantly shortened for patients with deletion of 13q14 (24.2 months; P = .008), deletion of 17p13 (16.2 months; P = .008), or 11q aberrations (13.1 months; P = .02). Survival time was not influenced by the presence of trisomy 11 (median, 40.1 v 41.5 months in those without trisomy 11; P = .42) or trisomy 17 (median, 40.1 v 38.7 months in those without trisomy 17; P = .70). On the basis of these observations, we analyzed whether different groups of patients could be discriminated from one another according to the number of unfavorable cytogenetic features (deletion of 13q14, deletion of 17p13, or aberration of 11q) that were present in each patient. Three groups of patients with significantly different OS times were identified (P < .001) (Fig 2): low-risk patients (n = 37; 41.1%) with absence of all unfavorable features (median OS time, 102.4 months), intermediate-risk patients (n = 31; 34.8%) with one unfavorable cytogenetic abnormality (median Os time, 29.6 months), and high-risk patients (n = 21; 23.6%) with two to three unfavorable chromosomal aberrations (median OS time, 13.9 months). All three patients who achieved complete remission belonged to the low-risk group, whereas patients who achieved no objective response were predominantly among the high-risk patients (no response in 28.6% of these patients).
Multivariate Analysis of Prognostic Factors Having demonstrated that deletions of 13q14 and 17p13 as well as aberrations of 11q were associated with shortened OS time, we next analyzed whether these chromosomal abnormalities were of independent prognostic significance in MM. Besides the findings by interphase FISH (each chromosome was considered separately), the following variables were included in the analysis of prognostic factors: age, stage according to Durie and Salmon,4 type of paraprotein (immunoglobulin A v non–immunoglobulin A), extent of histologic BM involvement, and BM plasma-cell morphology,28 as well as levels of B2M, CRP, LDH, plasma-cell proliferation (Ki-67 growth fraction), albumin, hemoglobin, platelets, creatinine, and calcium. On Cox stepwise regression analysis, deletion of 13q14 (P = .02), B2M level (P = .01), stage (P = .03), and, with borderline significance, age (P = .05) were independent prognostic parameters for survival. When interphase FISH results were collectively considered as numbers of unfavorable cytogenetic features (Fig 2), the two most important independent parameters that were predictive for shortened survival time were unfavorable cytogenetics (P = .02) and B2M level (P = .05); in this analysis, stage (P = .04) and age (P = .05) were also independent prognostic variables.
We also determined whether any prognostic factor or combination of parameters could further contribute to a definition of risk groups in the 89 patients with MM. From this analysis, it became evident that knowledge of chromosome-13 status and of serum levels of B2M provides powerful prognostic information on MM (Fig 3): patients with normal 13q14 and low B2M levels (
Our study represents a comprehensive interphase FISH analysis of specific chromosomal abnormalities (deletions of 13q14 and 17p13 as well as 11q aberrations) in 89 patients with MM that addresses the question of the clinical and prognostic significance of these aberrations in MM. In this population of patients who were treated with conventional-dose chemotherapy, deletions of 13q14 and 17p13 were significantly correlated with poor response, and all three abnormalities were predictive for short OS time. When patients were grouped according to the number of unfavorable chromosomal abnormalities present in each patient, a prognostic model discriminating low-risk, intermediate-risk, and high-risk patients was established. Besides B2M level, chromosomal aberrations were strong independent parameters for shortened survival time, as shown by multivariate analysis. In the presence of unfavorable cytogenetics, other prognostic factors such as CRP level, plasma-cell proliferation, and plasma-cell morphology,5,7,8,11,28 which were all important in the univariate analysis, were no longer of independent significance. Because interphase FISH is rapid and informative in all patients with newly diagnosed MM, we propose to include FISH analyses in the initial diagnostic procedures of patients with MM. Even if a comprehensive FISH analysis, such as the one in our study, is not available, knowledge of the 13q14 status, which is the most informative chromosomal region in MM as of yet, will provide powerful prognostic information in addition to that provided by standard parameters. This fact is demonstrated in the analysis presented in Fig 3, which shows different survival times of MM patients that depend on the serum levels of B2M and on chromosome 13q14 status. Thus, in the context of clinical trials, a risk-oriented analysis of subpopulations of patients can be performed that will possibly lead to the identification of therapeutic approaches that are beneficial for patients with particular MM entities. Specific chromosomal aberrations, particularly monosomy13 and deletion of 13q, were linked with prognosis in previous karyotypic studies of MM,14,15 but conventional cytogenetics underestimates the true incidence of these abnormalities in MM.21 Again, this was evident in the present study, in which the lack of metaphases from myeloma cells precluded the detection of chromosomal aberrations in 10 of 16 patients. In the 20 patients with informative results from both metaphase and interphase analyses, the results were consistent overall and, in particular, with respect to chromosome 13q. However, some chromosomal abnormalities seem to be difficult to detect by either method: as already reported previously,24 some deletions involving 17p may be small and thus remain unrecognized on metaphase chromosomes from myeloma cells. On the other hand, it is possible that interphase FISH analysis fails to detect abnormalities observed on metaphase chromosomes (11q deletions in patients no. 3 and 9) (Table 3), a possibility that requires further investigation. It should also be mentioned that FISH provides information only about specific target regions, whereas conventional cytogenetics and, with some limitations, new molecular cytogenetic techniques29-31 give a complete overview of chromosomal aberrations present in the malignant clone. In MM, interphase FISH may thus be particularly useful for the detection of specific chromosomal aberrations with known clinical and prognostic significance. The major new finding of the present study is that, despite the frequent detection of unfavorable cytogenetic abnormalities by interphase FISH, particularly the deletion of 13q14, chromosomal aberrations are still significant independent variables for shortened survival time. Collectively, our data suggest that the inactivation of tumor suppressor genes may play an important role in the development and progression of MM. Regarding 13q14, it is not yet clear whether the rb-1 gene or any other gene locus may be the chromosomal region of a putative myeloma tumor suppressor gene. A recent study suggested that a region on 13q14 distal to the rb-1 gene may be commonly deleted in a biallelic fashion,32 an observation that is reminiscent of the findings in studies of B-cell chronic lymphocytic leukemia.33,34 11q also represents a chromosomal region that is found to be recurrently aberrant in lymphoproliferative disorders. Deletions that involved band 11q22.3 to q23.1 were observed in patients with B-cell chronic lymphocytic leukemia, and this genomic region harbors the ATM (ataxia-teleangiectasia mutated), RDX (radixin), and FDX 1 (ferredoxin 1) genes.35 It is possible that these genes are also affected by the deletions observed in patients with MM, because in our patients all deletions were detected with a probe specific for the MLL gene (mapping to 11q23), but never with a probe hybridizing to a proximal 11q locus (11q13, cyclin D-1). Target genes on 11q may, however, be heterogenous, because t(11;14)(q13;q32) commonly results in the fusion of the BCL-1 locus, although the break points in MM may be different from those observed in mantle-cell lymphoma. Results presented in this study have been obtained in a patient population that was treated by conventional-dose chemotherapy. Considering results from recent clinical trials that demonstrated improved response rates and prolonged survival time after high-dose chemotherapy with autotransplantation,2,3 it will be of particular clinical significance to determine the outcome of the different cytogenetic risk groups after high-dose chemotherapy. Because deletion of 13q and 11q aberrations were unfavorable cytogenetic features in patients who were treated by tandem autotransplantation,14,15 it may be possible that the prognosis of such cytogenetically defined high-risk patients may not be substantially improved by high-dose therapy. In this case, innovative therapeutic concepts need to be developed for this particular type of MM with poor prognosis.
Supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (grant no. P12432-MED) and the ICP-Program (Molecular Medicine) of the Austrian Ministry for Research and Transport, Vienna, Austria.
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Copyright © 2000 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
two signals for the reference probe in at least 90% of cells) were independently evaluated by two to four investigators (R.K., N.Z., J.A., and L.R.). In assays that were aimed at the detection of a deletion, only cells with 
2 test, t test, and the nonparametric Kruskal-Wallis test. The survival time, which was measured from the date of diagnosis, was calculated using Kaplan-Meier estimates, and differences between survival curves were analyzed by means of the Breslow and Mantel-Cox tests. Multivariate analysis of the survival time results was performed using the Cox proportional hazards regression model. Stepwise evaluation of the maximum-partial likelihood ratio was used to identify prognostic factors. 
4mg/L) have superior survival time (median OS time, 102.4 months) compared with patients with one (either elevated B2M level or deletion of 13q14) or two risk features (median OS time, 45.5 and 10.9 months, respectively; P < .001). 
