Abstract

Recent observations indicate that chromosome aberrations are important prognostic factors in patients with multiple myeloma (MM) treated with high-dose chemotherapy. Nevertheless, the inherent problems of conventional cytogenetics have hampered the systematic evaluation of this parameter in series of patients treated with conventional chemotherapy. Fluorescence in situ hybridization (FISH) analysis is an attractive alternative for evaluation of numerical chromosomal changes. In the present study, we analyze the relationship between aneuploidies of 15 different chromosomes assessed by FISH and prognosis in a series of 63 patients with MM treated with conventional chemotherapy. After a median follow-up of 61 months (range, 6 to 109), 49% of patients are still alive with a median survival of 33 months. The overall incidence of numerical chromosome abnormalities was 70%. This incidence significantly increased when seven or more chromosomes were analyzed (53 patients), reaching 81%. Trisomies of chromosomes 6, 9, and 17 were associated with prolonged survival (P = .033, P = .035, and P = .026, respectively); by contrast, overall survival (OS) was lower in cases with monosomy 13 (as assessed by deletion of Rb gene,P = .0012). From the clinical point of view, loss of Rb gene was associated with a poor performance status; low hemoglobin levels; high creatinine, C-reactive protein, and lactic dehydrogenase serum levels; high percentage of bone marrow plasma cells (BMPC); extensive bone lytic lesions; and advanced clinical stage. Other chromosome abnormalities such as trisomy of chromosome 9 and 17 were associated with good prognostic features including high hemoglobin levels, early clinical stage, β2microglobulin less than 6 μg/mL, and low percentage of BMPC. A multivariate analysis for OS showed that S-phase PC greater than 3% (P = .010) and β2microglobulin serum levels greater than 6 μg/mL (P = .024), together with monosomy of chromosome 13 (P = .031) and nontrisomy of chromosome 6 (P = .048) was the best combination of independent parameters for predicting survival in patients with MM. According to these results, chromosomal analysis is of great use in patients with MM at diagnosis to have a correct prognostic evaluation for clinical decision making.

THE SURVIVAL OF patients with multiple myeloma (MM) ranges from a few months to more than 10 years (median, 2.5 to 3 years). For the last 30 years, investigators have attempted to identify clinical and laboratory features affecting survival of patients with MM. At present, β2microglobulin and C-reactive protein serum levels, together with the proliferative activity of plasma cells (labeling index or proportion of S-phase plasma cells [PC]) are considered to be the best prognostic indicators for predicting survival.1-6 Recently, a poor prognostic value has been shown in patients with monosomy and/or deletions of chromosome 13 treated with high-dose chemotherapy.7,8 Nevertheless, the availability of cytogenetic information in MM has long been hampered by the low mitotic activity of the myelomatous PC as well as by their poor growth in cell culture, which make it difficult to obtain metaphases from neoplastic cells for conventional chromosome analysis. Cytogenetic studies have shown a 20% to 50% incidence of abnormal karyotypes in patients with MM.9-12 This incidence increases up to 60% when bone marrow (BM) cells are incubated with interleukin-3 (IL-3) and IL-6.13 However, it is important to note that the majority of these studies have failed to obtain metaphases in many patients; therefore, it is not possible to say whether or not they have an abnormal karyotype.9-12 Procedures that can be applied to both metaphase cells and interphase nuclei, such as the flow cytometric measurement of cell DNA content and fluorescence in situ hybridization (FISH), are very well suited to the analysis of cytogenetic abnormalities in tumor cells. Studies using FISH and conventional cytogenetics simultaneously have shown that FISH can detect chromosome abnormalities which are hidden to metaphase cytogenetic analysis.14 Thus, FISH studies have shown an incidence of aneuploidy in 67% to 90% of patients14,15—higher than that obtained by conventional cytogenetics (20% to 50%). Despite these limitations, cytogenetic abnormalities, and particularly deletions of chromosome 13 and abnormalities involving the 11q chromosome, have been proven to be associated with poor survival9,10 in patients undergoing autologous transplantation.7,8 However, until now, the prognostic influence of chromosome aberrations detected by FISH in patients treated with conventional chemotherapy has not been explored.

In the present study, we analyze the relationship between aneuploidy of 15 different chromosomes assessed by FISH and prognosis in a series of 63 untreated patients with MM. Deletion of chromosome 13 was found to be an independent prognostic factor, associated with a significantly poor outcome, whereas trisomies of chromosomes 6, 9, 11, and 17 were related to a better prognosis.

MATERIALS AND METHODS

Patients.

Sixty-three previously untreated patients with MM diagnosed according to the criteria of the Chronic-Leukemia-Myeloma Task Force were included in the study.2 The mean age of the series was 66 ± 8 years (range, 46 to 82). According to Durie & Salmon's clinical staging system, the patients were distributed as follows: stage I, 5.5%; stage II, 32.7%; stage III, 61.8%.2 The monoclonal component was IgG in 50% of cases, IgA in 28%, IgD in 3%, and Bence Jones in the remaining 19% of patients. In one case no monoclonal serum protein was found. The serum monoclonal light chain was Κ in 58% and λ in 42%. Forty patients were men and 23 were women. Three patients presented plasmocytomas at diagnosis. All patients were treated according to the protocols of the PETHEMA (Spanish Cooperative Group for the Treatment of Malignant Hemopathies) group.16 

Immunophenotypic identification of PC.

The percentage of BMPC was assessed by two different observers on May-Grünwald-Giemsa stained smears, the mean value being 49% ± 25%. The immunophenotypic identification of PC was based on their strong reactivity for the CD38 (Leu17; Becton Dickinson, San José, CA) and CD138 (Imico, Madrid, Spain) monoclonal antibodies (MoAbs) whose specificity has been described elsewhere.17-19 The immunophenotypic characterization of the PC was performed by direct immunofluorescence using the following MoAbs: Leu19 (CD56), Leu16 (CD20), antiHLA-DR (HLA DR), LeuM7 (CD13), LeuM1 (CD15; Becton Dickinson), FMC56 (CD9; FMC, Adelaide, Australia), and C-kit (CD117; Imico), together with the previously mentioned CD38 and CD138 reagents. For data acquisition and analysis, a FACScan flow cytometer (Becton Dickinson), equipped with an argon ion laser tuned at 488 nm and 15 mW was used. Results were stored and analyzed for at least 10,000 cells per test, using the LYSYS-II and PAINT-A-GATE PRO software programs (Becton Dickinson), respectively.17,18 

FISH studies.

FISH analysis of numerical chromosome abnormalities was performed on erythrocyte-lysed whole BM samples obtained before cytotoxic treatment, according to previously described methods.15-18 Samples were hybridized as previously described with either a biotinylated, fluoresceinated, or digoxigenin-labeled alpha-satellite DNA probes20-22 specific for either the centromeric or the pericentromeric regions of the following chromosomes: 1 (pUC1.77; Boehringer Mannheim), 3 (pAE0.68; Boehringer Mannheim), 6 (D6Z1; Oncor, Gaithersburg, MD), 7 (pZ7.6B; Boehringer Mannheim), 8 (pZ8.4; Boehringer Mannheim), 9 (D9Z1; Oncor), 10 (CEP10; Vysis, Framingham, MA), 11 (CEP11; Vysis), 12 (D12Z3; Oncor), 15 (pMC15; Boehringer Mannheim), 17 (pZ17-1.6A; Boehringer Mannheim), 18 (pZXba; Boehringer Mannheim), X (pDMX1; Boehringer Mannheim), and Y (pHY2.1; Boehringer Mannheim). In addition, a locus-specific DNA probe for the Rb gene sequence in chromosome 13 was used (LSI13; Vysis). To probe the efficacy of the hybridization for chromosome 13, cohybridization was simultaneously performed using a chromosome 10 probe; only cells with clear signals for the control probe (chromosome 10) were scored. The number of hybridization spots were evaluated using a DMRB fluorescence microscope (Leitz, Wetzlar, Germany) equipped with a 100× oil-objective, which was used for counting hybridization spots per cell in at least 200 cells per sample. In all slides analyzed, the number of unhybridized cells in the areas assessed was lower than 1% and only those spots with a similar size, a strong intensity, and a round shape were counted. The mean percentage of trisomic/monosomic cells in control samples ranged between 0% to 2% for trisomies and 0% to 5% for monosomies.15 

A patient was considered to carry a numerical chromosomal abnormality for a certain chromosome when the percentage of cells displaying an abnormal number of spots was at percentages higher than the mean value plus two SDs of the percentages obtained for that specific chromosome in normal controls.

The expected amount of total DNA cell content according to the FISH results was assessed by the chromosome index calculated as previously reported.15 

Flow cytometry DNA measurements.

DNA measurements by flow cytometry were performed as previously described.15,18,22 Briefly, BM cells were incubated for 15 minutes with 10 μL of the GR7A4 (CD38) and BB4 (CD138) MoAbs, washed once (5 minutes, 1,900 rpm) in phosphate-buffered saline, and incubated for another 15 minutes with an MoAb anti-mouse Igs (F[ab′]2 fragments; Dakopatts, Copenhagen, Denmark). Afterwards, 2 mL of ammonium chloride were added and cells were incubated in the dark for 10 minutes. After lysing the erythrocytes, cells were washed once in 1 mL of sodium citrate buffer and resuspended in 200 μL of the same buffer. Then, 1.5 mL of solution containing RNAse and Nonident P40 were added and the cells were incubated for 10 minutes. Finally, 1.5 mL of solution containing propidium iodide (PI) was added and another incubation period was performed for at least 15 minutes in the dark.

In all cases, measurements were performed within 1 hour on a FACScan flow cytometer (Becton Dickinson) using the Lysis II software program (Becton Dickinson), for at least 10,000 cells per sample. The electronics of the instrument were adjusted so that the modal channel for the G0/G1 diploid nuclei was 200 (fluorescence scaled from channel 0 to 1023). Fluorescence compensation between fluorescein isothiocyanate (FITC) and PI was established using a mixture of PI-stained chicken erythrocyte nuclei and FITC-labeled beads (CALIBRITE beads; Becton Dickinson). The percentage of CD38/CD138 strong positive PC was calculated after gating out cell doublets on a FL2A/FL2W dot plot using the PAINT-A-GATE PRO software (Becton Dickinson).

The criteria for flow cytometry DNA aneuploidy was defined by the presence of two distinct peaks of G0/G1-phase cells in the DNA/propidium iodide histogram. The positivity for the CD38/CD138 MoAbs was used to identify which of the G0/G1 peaks corresponded to myelomatous PC (CD38/CD138 strong positive events) and the normal residual hemopoietic cells (CD38/CD138 negative or dim/intermediate positive events). The analysis of the cell cycle was performed on CD38/CD138 strong positive gated events using the MOD FIT software (Verity Software Home, Topsham, ME).

Statistical methods.

To estimate the significance of the differences observed between means, the Student's t-test was used (t-test, SPSS). The Chi-square test (SPSS, chi-square) was used for dichotomic variables. Overall survival (OS) curves were plotted according to the method of Kaplan and Meier, and compared using the Mantel-Cox, Peto-Prentice, and Breslow tests.

The different clinical and biological characteristics were considered individually for their relationship with OS by univariate tests (t-test, Chi-square, correlation, and nonparametric tests, SPSS). Subsequently, a multivariate analysis—stepwise regression—(regression, SPSS)23,24 was performed to examine the simultaneous effect of the different variables. Variables considered for possible inclusion in the regression analysis were those displaying a significant association with survival in the univariate analysis (P < .05) or for which prior studies had suggested a possible prognostic value. The stepwise regression method was discontinued when the P value for entering an additional factor was greater than .05. The model was tested both by including the variables in a continuous fashion (continuous model) and by grouping them into categories (dichotomous model).

RESULTS

After a median follow-up of 61 months (range, 6 to 109), 49% of patients remain alive, with a median survival of 33 months (range, 12 days to 109 months).

The overall incidence of aneuploidies assessed by FISH analysis in patients with MM included in the present study was 70%. This incidence increased to 81% when only those cases in which seven or more chromosomes were analyzed (53 patients). Overall, trisomies were more frequent than monosomies (84% v 16%). Table1 shows the distribution of numerical chromosomal abnormalities according to each of the 15 chromosomes analyzed in the present study. Chromosome 9 (55.8%), chromosome 1 (44.9%), and chromosome 15 (43.7%) were the most frequently altered. Of the remaining chromosomes analyzed, most displayed trisomies either as the only chromosomal abnormality detected (chromosomes 3, 6, 7, 10, 12, and 17) or the most frequent one (chromosomes 8, 11, and 18). In contrast, monosomies were the only numerical abnormality detected for chromosome 13 (33.3%). There was a frequent association between chromosome abnormalities. Thus, trisomy 6 was significantly associated with gains of chromosomes 7 (P = .0001), 9 (P = .001), 10 (P = .0002), and 17 (P = .005). In addition, abnormalities of chromosomes 1, 7, and 11 were associated with those of chromosomes 3 (P = .0005), 6 (P = .0002), and 17 (P = .0006), respectively.

Table 1.

Incidence of Numerical Chromosome Changes in Patients With MM According to the Different Chromosomes Analyzed

Chromosome AbnormalityChromosome
1 3 6 7 8 910 11 12 13 15 17 18 Xm Xf
Trisomy  19/49  10/30  15/55  14/47  3/39  29/52 3/38  13/40  1/39   13/32  10/46   9/35  1/39 
 38.8  33.3  27.3  29.8  7.7  55.8  7.9 32.5  2.6   40.6  21.7  25.7  4.5 
Monosomy   3/49     2/39    1/40   16/48   1/32    2/35   6/19 
 6.1     5.1    2.5   33.3  3.1  5.7   31.5  
 
Total  22/49 10/30  15/55  14/47  5/39  29/52  3/38  14/40 1/39  16/48  14/32  10/46  11/35  1/39  6/19 
 44.9  33.3  27.3  29.8  12.8  55.8  7.9 35  2.6  33.3  43.7  21.7  31.4  4.5 31.5 
Chromosome AbnormalityChromosome
1 3 6 7 8 910 11 12 13 15 17 18 Xm Xf
Trisomy  19/49  10/30  15/55  14/47  3/39  29/52 3/38  13/40  1/39   13/32  10/46   9/35  1/39 
 38.8  33.3  27.3  29.8  7.7  55.8  7.9 32.5  2.6   40.6  21.7  25.7  4.5 
Monosomy   3/49     2/39    1/40   16/48   1/32    2/35   6/19 
 6.1     5.1    2.5   33.3  3.1  5.7   31.5  
 
Total  22/49 10/30  15/55  14/47  5/39  29/52  3/38  14/40 1/39  16/48  14/32  10/46  11/35  1/39  6/19 
 44.9  33.3  27.3  29.8  12.8  55.8  7.9 35  2.6  33.3  43.7  21.7  31.4  4.5 31.5 

Results expressed as number of abnormalities found/number of cases studied (percentage below). Chromosome Y was normal in all men studied (n = 36).

Abbreviations: Xm, chromosome X in men; Xf, chromosome X in women.

Concerning the clinical implications of numerical chromosome abnormalities, it was found that monosomy of chromosome 13 was significantly associated with a poor performance status; low hemoglobin levels; high creatinine, C-reactive protein, and lactic dehydrogenase (LDH) serum levels; high percentage of BMPC; extensive bone lytic lesions; and advanced stage. This relationship with poor prognostic features was confirmed in the survival analysis (see below). Other chromosome abnormalities such as trisomies of chromosome 9 and 17 were associated with good prognostic features including high hemoglobin levels, early clinical stage, β2microglobulin less than 6 μg/mL, and low percentage of BMPC. Other associations between numerical chromosome abnormalities and clinical characteristics are listed in Table2.

Table 2.

MM Relationship Between the Presence of Numerical Chromosome Abnormalities and Clinico-Biological Characteristics of the Disease

Chr Age PS Hb Ca Creat Bone Stageβ2M Crp LDH PC BJ Resp
 1 —  —  —  —  —  —  —  —  ↑ —  —  —  Better  
 3  —  —  —  — ↓  ↓  —  —  —  —  —  —  —  
 6  —  —  —  ↓  —  — —  —  ↓  —  —  —  —  
 7  — —  —  —  —  —  Early  —  —  — —  —  —  
 8  —  —  —  —  — —  —  —  —  —  —  Absent  —  
 9 —  —  —  ↓  —  —  —  ↓  — —  —  —  —  
10  —  —  —  — —  —  —  —  —  —  —  —  — 
11  —  —  —  —  ↓  —  —  — —  —  —  —  —  
12  —  —  — —  —  —  —  —  —  —  —  —  —  
13  —  ↑  ↓  —  ↑  ↑ Advanced  —  ↑  ↑  ↑  —  —  
15 —  —  —  —  —  —  —  —  — —  —  Present  —  
17  —  —  ↑  — —  ↓  Early  ↓  —  —  ↓  —  — 
18  —  —  —  —  ↓  —  —  — —  —  ↓  —  —  
X  —  —  — —  —  —  —  —  —  —  —  —  — 
Chr Age PS Hb Ca Creat Bone Stageβ2M Crp LDH PC BJ Resp
 1 —  —  —  —  —  —  —  —  ↑ —  —  —  Better  
 3  —  —  —  — ↓  ↓  —  —  —  —  —  —  —  
 6  —  —  —  ↓  —  — —  —  ↓  —  —  —  —  
 7  — —  —  —  —  —  Early  —  —  — —  —  —  
 8  —  —  —  —  — —  —  —  —  —  —  Absent  —  
 9 —  —  —  ↓  —  —  —  ↓  — —  —  —  —  
10  —  —  —  — —  —  —  —  —  —  —  —  — 
11  —  —  —  —  ↓  —  —  — —  —  —  —  —  
12  —  —  — —  —  —  —  —  —  —  —  —  —  
13  —  ↑  ↓  —  ↑  ↑ Advanced  —  ↑  ↑  ↑  —  —  
15 —  —  —  —  —  —  —  —  — —  —  Present  —  
17  —  —  ↑  — —  ↓  Early  ↓  —  —  ↓  —  — 
18  —  —  —  —  ↓  —  —  — —  —  ↓  —  —  
X  —  —  — —  —  —  —  —  —  —  —  —  — 

Abbreviations: Chr, chromosome; PS, performance status according to the Eastern Cooperative Oncology Group scale; Hb, hemoglobin levels; Ca, calcium levels; Creat, creatinine levels; Bone, lytic bone lesions; Stage, Durie & Salmon clinical stage; β2M, β2 microglobulin serum levels; Crp, C-reactive protein levels; PC, percentage of BM plasma cells (morphology); BJ, Bence Jones protein; Resp, response to first line of treatment; —, no relationship; ↑, higher in cases with the chromosome abnormality; ↓, lower in cases with the chromosome abnormality (with statistical significance [P < .05]).

As far as the response to treatment was concerned, no major differences were observed regarding the presence of specific abnormalities with the exception of a lower rate of responses in patients with abnormalities of chromosome 1 (47% in trisomic cases v 78% in cases without trisomy 1; P = .04).

OS was superior in patients with trisomies of chromosomes 6, 9, and 17 compared with that of cases without trisomy (P = .033,P = .035, and P = .026, respectively; Table3 and Figs 1 and2); by contrast, OS was lower in cases with monosomy of chromosome 13 (P = .0012; Table 3 and Fig 2). In addition, all clinical and biological disease characteristics were considered individually for their relationship with OS by univariate tests. The variables for which a statistically significant relationship with OS was found are summarized in Table 4.

Table 3.

Univariate Analysis of the Prognostic Value for the OS of the Different Chromosome Abnormalities

Chromosome N Events Survival (mo) PUnivariate
1  Trisomy  19  11  29  .49 
 Nontrisomy  30  15  34  
3  Trisomy  10  35  .84  
 Nontrisomy  20  11  39  
Trisomy  15  8  70  .033* 
 Nontrisomy  40 16  33  
7  Trisomy  14  6  46  .11 
 Nontrisomy  33  20  32  
8  Trisomy  3  39  .86  
 Nontrisomy  36  20  33  
Trisomy  29  13  46  .035* 
 Nontrisomy  23 13  26  
10  Trisomy  3  0  —  .15 
 Nontrisomy  35  24  33  
11  Trisomy  13  70  .060  
 Nontrisomy  27  14  33  
12 Trisomy  1  1  22  .21  
 Nontrisomy  38  22 34  
13  1 signal  16  9  14  .0012* 
 Nontrisomy  32  13  60  
15  Trisomy  13  34  .99  
 Nontrisomy  19  8  60  
17 Trisomy  10  3  103  .026* 
 Nontrisomy  36 22  33  
18  Trisomy  9  6  60  .50 
 Nontrisomy  26  14  35  
X  Monosomy 6  4  29  .75  
 Nontrisomy  13  9  35  
Chromosome N Events Survival (mo) PUnivariate
1  Trisomy  19  11  29  .49 
 Nontrisomy  30  15  34  
3  Trisomy  10  35  .84  
 Nontrisomy  20  11  39  
Trisomy  15  8  70  .033* 
 Nontrisomy  40 16  33  
7  Trisomy  14  6  46  .11 
 Nontrisomy  33  20  32  
8  Trisomy  3  39  .86  
 Nontrisomy  36  20  33  
Trisomy  29  13  46  .035* 
 Nontrisomy  23 13  26  
10  Trisomy  3  0  —  .15 
 Nontrisomy  35  24  33  
11  Trisomy  13  70  .060  
 Nontrisomy  27  14  33  
12 Trisomy  1  1  22  .21  
 Nontrisomy  38  22 34  
13  1 signal  16  9  14  .0012* 
 Nontrisomy  32  13  60  
15  Trisomy  13  34  .99  
 Nontrisomy  19  8  60  
17 Trisomy  10  3  103  .026* 
 Nontrisomy  36 22  33  
18  Trisomy  9  6  60  .50 
 Nontrisomy  26  14  35  
X  Monosomy 6  4  29  .75  
 Nontrisomy  13  9  35  

*P < .05.

Presence of monosomy X in women.

Fig. 1.

Survival curves of patients with MM according to the number of copies of chromosomes 9 and 17 per nuclei.

Fig. 1.

Survival curves of patients with MM according to the number of copies of chromosomes 9 and 17 per nuclei.

Fig. 2.

Survival curves of patients with MM according to the number of copies of chromosomes 6 and 13 (deletion of Rb) per nuclei.

Fig. 2.

Survival curves of patients with MM according to the number of copies of chromosomes 6 and 13 (deletion of Rb) per nuclei.

Table 4.

Univariate and Multivariate Analysis of the Prognostic Value of the Different Clinico-Biological Characteristics in MM With Respect to OS

Variable Percentage of Cases Median OS (mo)P Value Univariate Analysis P Value Multivariate Analysis
S-phase PC  
 <3%  68  39 <.0001  .010  
 ≥3%  32  15  
β2microglobulin  
 <6 mg/L  69  44  .0030  .024 
 ≥6 mg/L  31  15  
Chromosome 13  
 Monosomy  33 14  .0012  .031  
 Normal  67  60  
Chromosome 6 
 Trisomy  27  70  .0330  .048  
 Nontrisomy  73 33  
C–reactive Protein  
 <6 mg/dL  77  32  .0026 NS  
 ≥6 mg/dL  23  11  
ECOG  
 <2  48  39 .0040  NS  
 ≥2  52  16  
Calcium  
 <11.5 mg/dL  87  36  <.0001  NS  
 ≥11.5 mg/dL  13  
Creatinine  
 <2 mg/dL  78  36  <.0001  NS 
 ≥2 mg/dL  22  9  
Albumin  
 ≤3.5 mg/dL  57 39  .0198  NS  
 >3.5 mg/dL  43  22  
Clinical Stage  
 I & II  38  60  .0010  NS  
 III  62 19  
LDH  
 <320 U/mL  65  34  .0295  NS 
 ≥320 U/mL  35  16  
DNA index  
 >1  62  39 .0088  NS  
 ≤1  38  15  
Hemoglobin  
 ≤9.5 mg/dL  54  60  .0123  NS  
 >9.5 mg/dL  46  19 
Chromosome 9  
 Trisomy  56  46  .0350  NS 
 Nontrisomy  44  26  
Chromosome 17  
 Trisomy  22 103  .0260  NS  
 Nontrisomy  78  33   
Variable Percentage of Cases Median OS (mo)P Value Univariate Analysis P Value Multivariate Analysis
S-phase PC  
 <3%  68  39 <.0001  .010  
 ≥3%  32  15  
β2microglobulin  
 <6 mg/L  69  44  .0030  .024 
 ≥6 mg/L  31  15  
Chromosome 13  
 Monosomy  33 14  .0012  .031  
 Normal  67  60  
Chromosome 6 
 Trisomy  27  70  .0330  .048  
 Nontrisomy  73 33  
C–reactive Protein  
 <6 mg/dL  77  32  .0026 NS  
 ≥6 mg/dL  23  11  
ECOG  
 <2  48  39 .0040  NS  
 ≥2  52  16  
Calcium  
 <11.5 mg/dL  87  36  <.0001  NS  
 ≥11.5 mg/dL  13  
Creatinine  
 <2 mg/dL  78  36  <.0001  NS 
 ≥2 mg/dL  22  9  
Albumin  
 ≤3.5 mg/dL  57 39  .0198  NS  
 >3.5 mg/dL  43  22  
Clinical Stage  
 I & II  38  60  .0010  NS  
 III  62 19  
LDH  
 <320 U/mL  65  34  .0295  NS 
 ≥320 U/mL  35  16  
DNA index  
 >1  62  39 .0088  NS  
 ≤1  38  15  
Hemoglobin  
 ≤9.5 mg/dL  54  60  .0123  NS  
 >9.5 mg/dL  46  19 
Chromosome 9  
 Trisomy  56  46  .0350  NS 
 Nontrisomy  44  26  
Chromosome 17  
 Trisomy  22 103  .0260  NS  
 Nontrisomy  78  33   

Abbreviations: ECOG, Eastern Cooperative Oncology Group scale; NS, not significant (P > .05).

A multivariate analysis for OS was performed using all variables with significant prognostic influence on univariate analysis (Table 4). This analysis showed that S-phase PC greater than 3% (P = .010) and β2microglobulin serum levels greater than 6 μg/mL (P = .024), together with monosomy of chromosome 13 (P = .031) and nontrisomy of chromosome 6 (P = .048) was the best combination of independent parameters for predicting survival in patients with MM. Although trisomy of chromosome 17 was associated with a better outcome, its impact on OS in the multivariate analysis was on the limit of the statistical significance (P = .06).

DISCUSSION

In the present study we have explored the clinical impact of aneuploidies for 15 different human chromosomes assessed by FISH in a series of 63 consecutive patients with MM. The presence of monosomy 13 was associated with poor prognostic features and a shorter survival, whereas the existence of trisomy for chromosomes 6, 9, and 17 was associated with a better outcome.

The favorable prognostic impact observed in patients with trisomy of chromosomes 6, 9, and 17 would be in accordance with the longer median survival observed in patients with MM displaying DNA hyperploidy (DNA index > 1) by conventional flow cytometry5,25,26 and would also support the high incidence of hyperploidy observed in patients with monoclonal gammopathy of undetermined significance.27 Although the favorable prognosis of hyperploidy has often been reported, the biological mechanisms responsible for this positive influence are yet to be clarified. Nevertheless, it could be speculated that the DNA damage occurring in these patients is partially offset by an increase in the number of copies of tumor suppressor genes located in those specific chromosomes. This could be the case of the p16 and p15 tumor suppressor genes, placed at 9p21,28,29 which have recently been considered to have an important role in MM because of the high frequency of hypermethylation of their CpG islands in this disease.30 The products of both genes are capable of inhibiting the interaction between CDK4/6 and D1 cyclins, and as a consequence, cells are arrested at the G1-phase of the cell cycle. If these genes are hypermethylated in MM, their function would be hampered, thus allowing clonal plasma cells to enter S-phase. However, the presence of trisomy of chromosome 9 would provide the presence of a normal extra copy of the p16 and p15 genes that would increase the probability of a nonhypermethylated gene copy continuing to work normally despite the hypermethylation of other copies; this would help to explain the better survival observed in MM cases with trisomy 9. A similar hypothesis could be used to explain the favorable outcome observed in patients with MM with trisomy 17, because the p53 tumor suppressor gene is located in this chromosome at 17p13.31-34 In a similar way, recent findings in endometrium carcinoma or melanoma suggest the presence of tumor suppressor genes in chromosome 6.35-37 The loss of heterozygosity at 6q25 has been found in several solid tumors such as ovarian, breast, kidney, or mesothelial cancers.35-37 This would suggest the presence of tumor suppressor genes at this level.

Tricot et al8 reported that monosomy 13 is associated with poor outcome in patients treated with high-dose chemotherapy. In the present study conducted in patients treated with conventional chemotherapy it was also confirmed that monosomy 13, as assessed by deletion of the Rb gene, has an adverse prognostic influence. Moreover, this chromosomal deletion retained the independent value on multivariate analysis. The Rb tumor suppressor gene located at chromosome 13 plays a central role in the control of the cell cycle, because its phosphorylation by cyclin-dependent kinases relieves the G1 block and promotes the transition of cells into S-phase.38-43 

According to these results, chromosomal analysis is of great use in patients with MM at diagnosis to have a correct prognostic evaluation for clinical decision making. Because FISH analysis is a reliable technique that may afford accurate information on numerical changes, overcoming the problems of conventional cytogenetics, FISH evaluation of chromosomes 6, 9, 13, and 17 should be recommended in all patients with MM at diagnosis.

In summary, this report shows the high prognostic value of the FISH analysis to evaluate the presence of aneuploidies in patients with MM. In addition, our findings show a potential important role of the p16/CDK4/Rb axis in the pathogenesis of the MM and suggest the existence of new tumor suppressor genes, placed at chromosome 6, that could be involved in the disease.

Supported in part by a grant from the Spanish Fondo de Investigaciones Sanitárias (95/1475) and Dirección General de Investigación Cientifica y Tecnológica (PB93-0614). J.A. was supported by a grant from the Spanish Fondo de Investigaciones Sanitárias de la Seguridad Social (97/3537).

Address reprint requests to J.F. San Miguel, MD, PhD, Servicio de Hematologı́a, Hospital Universitario de Salamanca, Paseo de San Vicente, 58-182, 37007, Salamanca, Spain.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

REFERENCES

1
Bataille
R
Boccadoro
M
Klein
B
Durie
B
Pileri
A
C-reactive protein and β2microglobulin produce a simple and powerful myeloma staging system.
Blood
80
1992
733
2
San Miguel
JF
Garcı́a-Sanz
R
González
M
Moro
MJ
Hernández
JM
Ortega
F
Borrego
D
Carnero
M
Casanova
F
Jiménez
R
Portero
JA
Orfão
A
A new staging system for multiple myeloma based on the number of S-phase plasma cells.
Blood
85
1995
448
3
Durie
BGM
Young
LA
Salmon
SE
Human myeloma in vitro colony growth: Interrelationship between drug sensitivity, cell kinetics, and patient survival duration.
Blood
61
1983
929
4
Boccadoro M, Massaia M, Dianziani U, Pileri A: Multiple myeloma: Biological and clinical significance of bone marrow plasma cell labeling index. Haematologica (Pavia) 72:171, 1987
5
Garcı́a-Sanz
R
Orfão
A
González
M
Moro
MJ
Hernández
JM
Ortega
F
Borrego
D
Carnero
M
Casanova
F
Jiménez
R
Portero
JA
San Miguel
JF
Prognostic implications of DNA aneuploidy in a series of 156 untreated MM patients.
Br J Haematol
90
1995
106
6
Greipp
PR
Lust
JA
O'Fallon
M
Katzmann
JA
Witzig
TE
Kyle
RA
Plasma cell labeling index and β2-microglobulin predict survival independent of thymidine kinase and C-reactive protein in multiple myeloma.
Blood
81
1993
3382
7
Barlogie
B
Jagannath
S
Vesole
DH
Naucke
S
Cheson
B
Mattox
S
Bracy
D
Salmon
S
Jacobson
J
Crowley
J
Tricot
G
Superiority of tandem autologous transplantation over standard therapy for previously untreated multiple myeloma.
Blood
89
1997
789
8
Tricot
G
Barlogie
B
Jagannath
S
Bracy
D
Mattox
S
Vesole
DH
Naucke
S
Sawyer
J
Poor prognosis in multiple myeloma is associated only with partial or complete deletions of chromosome 13 or abnormalities involving 11q and not with other karyotype abnormalities.
Blood
86
1995
4250
9
Dewald
GW
Kyle
RA
Hicks
GA
Greipp
PRP
The clinical significance of cytogenetic studies in 100 patients with multiple myeloma, plasma cell leukemia or amyloidosis.
Blood
66
1985
380
10
Durie
BGM
Cellular and molecular genetics features of myeloma and related disorders.
Hematol Oncol Clin North Am
6
1992
463
11
Sawyer
JR
Waldron
JA
Jagannath
S
Barlogie
B
Cytogenetic findings in 200 patients with multiple myeloma.
Cancer Genet Cytogenet
82
1995
41
12
Laı̈
JL
Zandecki
M
Mary
JY
Bernardi
F
Izydorczyk
V
Flactif
M
Morel
P
Jouet
JP
Bauters
F
Facon
T
Improved cytogenetics in Multiple myeloma.
Blood
9
1995
2490
13
Cuneo
A
Balsamo
R
Roberti
MG
Bardi
A
Piva
N
Balboni
M
Bigoni
R
Rigolin
GM
Castoldi
G
Interleukin-3 plus interleukin-6 may improve chromosomal analysis of multiple myeloma: Cytological and cytogenetic evidence in thirty-four patients.
Cancer Genet Cytogenet
90
1996
171
14
Drach
J
Schuster
J
Nowotny
H
Angerler
J
Rosenthal
F
Fiegl
M
Rothermundt
C
Gsur
A
Jager
U
Heinz
R
Multiple myeloma: High incidence of chromosomal aneuploidy as detected by interphase fluorescence in situ hybridization.
Cancer Res
55
1995
3854
15
Tabernero
D
San Miguel
JF
Garcı́a-Sanz
R
Nájera
L
Garcı́a-Isidoro
M
Pérez-Simón
JA
González
M
Wiegant
J
Raap
AK
Orfão
A
Incidence of chromosome numerical changes in multiple myeloma. Fluorescence in situ hybridization analysis using 15 chromosome-specific probes.
Am J Pathol
149
1996
153
16
Bladé
J
San Miguel
JF
Alcalá
A
Maldonado
J
Sanz
MA
Garcı́a-Conde
J
Moro
MJ
Alonso
C
Besalduch
J
Zubizarreta
A
Besses
C
González Brito
G
Hernández-Martı́n
J
Fernández-Calvo
J
Rubio
D
Ortega
F
Jiménez
R
Colominas
P
Faura
MV
Font
L
Tortosa
J
Domingo
A
Fontanillas
M
Rozman
C
Estapé
C
Alternating combination VCMP/VBAP chemotherapy versus melphalan/prednisone in the treatment of multiple myeloma: A randomized multicentric study of 487 patients.
J Clin Oncol
11
1993
1165
17
San Miguel
JF
González
M
Gascón
A
Moro
MJ
Hernández
JM
Ortega
F
Jiménez
R
Guerras
L
Romero
M
Casanova
F
Sanz
MA
Sánchez
J
Portero
JA
Orfão
A
Immunophenotype heterogeneity of multiple myeloma: Influence on the biology and clinical course of the disease.
Br J Haematol
77
1992
185
18
Orfão
A
Garcı́a-Sanz
R
López-Berges
MC
Vidriales
MB
González
M
Caballero
MD
San Miguel
JF
A new method for the analysis of plasma cell DNA content in multiple myeloma samples using a CD38/propidium iodide double staining technique.
Cytometry
17
1994
332
19
Garcı́a-Sanz
R
González
M
Orfão
A
Moro
J
Hernández
M
Borrego
D
Carnero
M
Casanova
F
Bárez
A
Jiménez
R
Portero
JA
San Miguel
JF
Analysis of natural killer associated antigens in peripheral blood and bone marrow of multiple myeloma patients and prognostic implications.
Br J Haematol
93
1996
81
20
Wiegant
J
Ried
T
Nederlof
PM
Van der Ploeg
M
Tanke
HJ
Raap
AK
In situ hybridization with fluoresceinated DNA.
Nucleic Acids Res
19
1991
3237
21
Arnoldus
EPJ
Noordemeer
IA
Peters
ACB
Boormolen
JHC
Bots
TAM
Raap
AK
Van der Ploeg
M
Interphase cytogenetics of brain tumors.
Gene Chromosom Cancer
3
1991
101
22
Vindelov
LL
Christehnsen
IBJ
Jensen
G
Nissen
NI
Limits of detection of nuclear DNA abnormalities by flow cytometric DNA analysis. Results obtained by a set of methods for sample storage, staining and internal standardization.
Cytometry
3
1983
332
23
Cox DR, Oakes D: Analysis of Survival Data. London, UK, Chapman and Hall, 1981
24
Dixon WJ: Biomedical-Data Package: Statistical Software. Berkley, CA, University of California, 1983
25
Morgan
RJ
Gonchoroff
NJ
Katzmann
JA
Witzig
TE
Kyle
RA
Greipp
PR
Detection of hypodiploidy using multi-parameter flow cytometric analysis: A prognostic indicator in multiple myeloma.
Am J Hematol
30
1989
195
26
Smith
L
Barlogie
B
Alexanian
R
Biclonal and hypodiploid multiple myeloma.
Am J Med
80
1985
841
27
(abstr, suppl 1)
San Miguel
JF
Almeida
J
Ocqueteau
M
Orfão
A
Detection of two subpopulations of plasma cells in MGUS patients: Utility in the differential diagnosis with MM.
Blood
88
1996
640a
28
Nobori
T
Miura
K
Wu
DJ
Lois
A
Takabayashi
K
Carson
DA
Deletions of the cyclin-dependent kinase 4 inhibitor p16INK4 In multiple human cancers.
Nature
368
1994
753
29
Kamb
A
Gruis
NA
Weaver-Feldhaus
J
Liu
Q
Harshman
K
Tavtigian
SV
Stockert
E
Day RS
III
Johnson
BE
Sckolnik
MH
A cell cycle regulator potentially involved in genesis of many tumor types.
Science
264
1994
436
30
Ng
MHL
Chung
YF
Lo
KW
Wickham
NWR
Lee
JCK
Huang
DP
Frequent hypermethylation of p16 and p15 genes in multiple myeloma.
Blood
89
1997
2500
31
Miller
C
Mohandas
T
Wolf
D
Prokocimer
M
Rotter
V
Koeffler
PH
Human p53 localized at short arm of chromosome 17.
Nature
319
1986
783
32
Isobe
M
Emanuel
BS
Givol
D
Oren
M
Croce
CM
Localization of gene for human p53 to band 17p13.
Nature
320
1986
84
33
Prokocimer
M
Rotter
V
Structure and function of p53 in normal cells and their aberrations in cancer cells: Projection on the hematologic cell lineages.
Blood
84
1994
2391
34
Corradini
P
Inghirami
G
Astolfi
M
Ladetto
M
Voena
C
Ballerini
P
Gu
W
Nilsson
K
Knowles
D
Boccadoro
M
Pileri
A
Dalla-Favera
R
Inactivation of tumor-supressor genes, p53 and Rb1, in plasma cell dyscrasias.
Leukemia
8
1994
758
35
Foulkes
WD
Ragoussis
J
Stamp
GW
Allan
GJ
Travsdale
J
Frequent loss of heterozygosity on chromosome 6 in human ovarian carcinoma.
Br J Cancer
67
1993
551
36
Tibiletti
MG
Bernasconi
B
Taborelli
M
Furlan
D
Fabbri
A
Franchi
M
Taramelli
R
Trubia
M
Capella
C
Involvement of chromosome 6 in endometrial cancer.
Br J Cancer
75
1997
1831
37
Sheng
ZM
Marchetti
A
Buttitta
F
Champeme
MH
Campani
D
Bistocchi
M
Lidereau
R
Callahan
R
Multiple regions of chromosome 6q affected by loss of heterozygosity in primary human breast carcinomas.
Br J Cancer
73
1996
144
38
Dao
DD
Sawyer
JR
Epstein
J
Hoover
RG
Barlogie
B
Tricot
G
Deletion of the retinoblastoma gene in multiple myeloma.
Leukemia
8
1994
1280
39
Zandecki
M
Façon
T
Preudhomme
C
Vanrumbeke
M
Vachee
A
Quesnel
B
Lai
JL
Cosson
A
Fenaux
P
The retinoblastoma gene (Rb-1) status in multiple myeloma: A report on 35 cases.
Leuk Lymphoma
18
1995
497
40
Juge-Morineau
N
Mellerin
MP
Francois
S
Rapp
MJ
Harousseau
JL
Amiot
M
Bataille
R
High incidence of deletion but infrequent inactivation of the retinoblastoma gene in human myeloma cells.
Br J Haematol
91
1995
664
41
Maestro
R
Piccinin
S
Doglioni
C
Gasparotto
D
Vukosavljevic
T
Sulfaro
S
Barzan
L
Boiocchi
M
Chromosome 13q deletion mapping in head and neck squamous cell carcinomas: Identification of two distinct regions of preferential loss.
Cancer Res
56
1996
1146
42
Cooney
KA
Wetzel
JC
Merajver
SD
Macoska
JA
Singleton
TP
Wojno
KJ
Distinct regions of allelic loss on 13q in prostate cancer.
Cancer Res
56
1996
1142
43
Liu
Y
Szekely
L
Grander
D
Soderhall
S
Juliusson
G
Gahrton
G
Linder
S
Einhorn
S
Chronic Lymphocytic leukemia cells with allelic deletion at 13q14 commonly have one intact Rb-1 gene: Evidence for a role of an adjacent locus.
Proc Natl Acad Sci USA
90
1993
8697