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Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3366-3371
Prognostic Value of Numerical Chromosome Aberrations in Multiple
Myeloma: A FISH Analysis of 15 Different Chromosomes
By
J.A. Pérez-Simón,
R. García-Sanz,
M.D. Tabernero,
J. Almeida,
M. González,
J. Fernández-Calvo,
M.J. Moro,
J.M. Hernández,
J.F. San Miguel, and
A. Orfão
From the Castellano-Leonés (Spain) Cooperative Group for the
Study of Monoclonal Gammopathies, Department of Hematology, University
Hospital of Salamanca, University of Salamanca; and the General Service
of Cytometry, University of Salamanca, Salamanca, Spain.
 |
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,  2microglobulin 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
2microglobulin 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.
| |
INTRODUCTION |
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, 2microglobulin 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 K in 58% and  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 analysisstepwise
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%). Table 1 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.
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Table 1.
Incidence of Numerical Chromosome Changes in Patients
With MM According to the Different Chromosomes Analyzed
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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,
2microglobulin less than 6 µg/mL, and low percentage
of BMPC. Other associations between numerical chromosome abnormalities
and clinical characteristics are listed in Table
2.
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Table 2.
MM Relationship Between the Presence of Numerical
Chromosome Abnormalities and Clinico-Biological Characteristics of the
Disease
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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; Table
3 and Figs 1 and 2); 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.
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| Fig 1.
Survival curves of patients with MM according to the
number of copies of chromosomes 9 and 17 per nuclei.
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| Fig 2.
Survival curves of patients with MM according to the
number of copies of chromosomes 6 and 13 (deletion of Rb) per nuclei.
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Table 4.
Univariate and Multivariate Analysis of the Prognostic
Value of the Different Clinico-Biological Characteristics in MM With Respect to OS
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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 2microglobulin 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.
| |
FOOTNOTES |
Submitted August 11, 1997;
accepted December 17, 1997.
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
Hematología, 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.
| |
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Abstract
Introduction
Abstract