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Biotechnical Methods Section (BTS)

Ploidy, as detected by fluorescence in situ hybridization, defines different subgroups in multiple myeloma


Ploidy appears as an important parameter in both the biology and the clinical evolution of multiple myeloma. However, its evaluation requires either a successful karyotyping (obtained in 30% of the patients) or a DNA index calculation by flow cytometry (not routinely performed in myeloma). We validated a novel method based on interphase fluorescence in situ hybridization that can be utilitized to analyze almost all the patients. The method was very specific and sensitive for the detection of hyperdiploidy. Extended studies showed that most recurrent 14q32 translocations occur in nonhyperdiploid clones, and that deletions of chromosome 13 were less frequently observed in hyperdiploid clones (48 vs 66%). Further large studies are ongoing to evaluate the prognostic value of ploidy in myeloma.


Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells (PC) usually within the bone marrow. Owing to a heterogeneous bone marrow infiltration and the low proliferation of malignant PC, cytogenetics is a difficult art in MM. Most series have revealed abnormal karyotypes in about one-third of the patients.1, 2, 3, 4, 5, 6 In the other two-thirds of the patients, karyotypes are either normal or unsuccessful with normal metaphases corresponding to cycling normal bone marrow myeloid cells. Despite this difficulty generating metaphases within the malignant clone, a few large cytogenetic studies have been reported enabling the characterization of chromosomal aberration patterns in MM. In contrast to acute leukemias, karyotypes in MM are usually complex displaying both numerical and structural chromosomal changes. These two types of abnormalities grossly divide MM into two groups: one with mostly numerical gains and few structural changes (the hyperdiploid group), and the other with many chromosomal rearrangements and sometimes loss of chromosomes (the nonhyperdiploid group, including the pseudodiploid and hypodiploid karyotypes). The two types of MM are approximately equally distributed.1, 2, 3, 4, 5, 6, 7

This cytogenetic classification is clinically valuable since patients with hyperdiploidy seem to present a better outcome than nonhyperdiploid patients.7, 8, 9, 10 However, this prognostic value has been demonstrated only in a few retrospective studies based on the analysis of karyotypes. Since cytogenetics is informative in only one-third of the patients, the prognostic significance of hyperdiploid vs nonhyperdiploid status in patients with uninformative karyotypes is not known. To address this issue, other techniques have to be used that are not based on the requirement of clonal metaphases. Flow cytometry has been proposed by several authors in order to evaluate the DNA index (DI).11, 12, 13 Nevertheless, no large study analyzing its prognostic value has been reported. Since bone marrow samples are often poor in MM and molecular cytogenetics is currently routinely performed at diagnosis, we investigated the ability of fluorescence in situ hybridization (FISH) to assess hyperdiploidy. In order to validate this approach, we compared FISH and DI analyses in a series of 205 patients with MM.

Patients, materials and methods

Patients and PC sorting

In all, 205 patients with MM were analyzed. All the patients were enrolled in one of the Intergroupe Francophone du Myélome (IFM) therapeutic trials and gave written informed consent for the study. Patients with at least one million purified PC (see below) were included in this study. There were 96 females and 109 males with a median age of 61 years (range=37–74). A measure of 1–3 ml of heparinized bone marrow mononuclear cells were separated using Ficoll–Hypaque. PC were then sorted using anti-CD138-coated magnetic beads according to the manufacturer's instructions (Miltenyi Biotec, Paris, France). PC purity was evaluated on Giemsa-stained cytospined cells.

Probe selection

In order to choose adequate probes for the assessment of hyperdiploidy, we started by systematically reviewing the cytogenetic literature focusing on the largest series.5, 7, 14, 15, 16, 17, 18, 19In these reports, we specifically analyzed karyotypes with more than 48 chromosomes (with high hyderdiploidy) searching for the more frequently gained chromosomes. Eight large published series have been reviewed constituting 173 hyperdiploid karyotypes.5, 7, 14, 15, 16, 17, 18, 19 The most frequently gained chromosomes were chromosomes 9 (156/173), 15 (142/173), 19 (136/173), 5 (128/173), 11 (118/173), 3 (115/173), 7 (93/173), and 21 (75/173). We then examined what would be the most sensitive and specific combination of three probes for the detection of hyperdiploidy. The analysis showed that the combination of chromosomes 5, 9, and 15 is the best compromise between specificity and sensitivity. These three chromosomes were gained together in 97 of the 173 hyperdiploid karyotypes, and two of them were gained in 60 other cases. In contrast, none of the 197 analyzed nonhyperdiploid karyotypes presented a gain of the three chromosomes, and only four displayed a gain of two of them. Thus, with a FISH definition of hyperdiploidy based on the gain of at least two of the three chromosomes, 16 of the 173 hyperdiploid cases would have been missed (false negative rate=9%) and four of the 197 nonhyperdiploid cases would have been misclassified (false positive rate=2%). Based on this analysis, we developed a specific probe cocktail for hyperdiploidy evaluation containing the LSI® D5S721/D5S23 labeled with SpectrumGreen™, CEP®9 (alpha satellite) labeled with SpectrumAqua™, and CEP®15 (alpha satellite, D15Z4) labeled with SpectrumOrange™.

DI and FISH experiments

After purification, PC were stained with 40 μg/ml propidium iodide (Sigma) in phosphate-buffered saline containing 0.1% Triton X-100 and 0.1% sodium citrate for 30 min at room temperature. The samples were run in duplicates where one sample contained the internal control (peripheral blood mononuclear cells obtained from a normal volunteer donor) and the other did not. Flow cytometry analysis was performed on a FACSCalibur using CELLQuest Pro software (BD-Bioscience). Data were gated on the FL2-Area vs FL2-Width dot plot to exclude doublets and aggregates, and a minimum of 20 000 gated cells were collected per suspension. The DI was calculated as described20 (ratio of the mean or peak of sample G0/G1 population divided by the mean or peak of diploid reference cells). DNA diploid PC were defined as containing a single G0/G1 population presenting a DI=1.0 (Figure 1).

Figure 1

(a) A typical profile corresponding to a patient with a diploid clone. Only one peak is observed. (b) The same patient hybridized with the ploidy probe. Two green, two red, and two white signals corresponding to chromosomes 5, 9, and 15, respectively, are observed. (c) A typical profile obtained in a hyperdiploid patient. The first peak corresponds to the normal cells, whereas the second peak corresponds to the myeloma cells. The DI was calculated to 1.32. (d) The same patient hybridized with the ploidy probe, showing three signals with each probe, and thus confirming hyperdiploidy.

For FISH analysis, 0.6 million PC were fixed in methanol/acetic acid (3/1 vol) and dropped on slides. FISH experiments were performed according to standard Abbott/Vysis protocols. Microscope analysis was performed by two examiners using specific filters (for SpectrumAqua™, SpectrumGreen™ and SpectrumOrange™, Abbott, Rungis, France). In all, 100 nuclei were scored by each examiner, and hyperdiploidy was assessed when at least two of the three probes were in more than two copies in at least 20% of the nuclei. Probes specific for t(4;14)(p13;q32), t(11;14)(q13;q32), and del(13) (LSI D13S319) were furnished by Abbott/Vysis and used according to standard recommendations.

Results and discussion

Results are summarized in Table 1. Hyperdiploidy was arbitrarily fixed for DI higher than 1.04, and tetraploidy for DI higher than 1.6. Based on DI, 91 patients were diploid (only one patient with a DI=1.03), 104 patients were hyperdiploid (median DI=1.24, range=1.05–1.5), seven patients were hypodiploid (median DI=0.95, range=0.78–0.98), and only three patients were classified as tetraploid. In 12 patients (6%), two or even three different peaks were observed, corresponding to the presence of two or three clones. Thus, hyperdiploidy was assessed in 51% of the patients, in tight agreement with the conventional cytogenetics literature showing hyperdiploidy in 50–55% of the patients. The two methods of ploidy assessment were highly correlated. This was especially true for the identification of hyperdiploidy, which was the goal of this study (Figure 1). One expected exception to this correlation was observed for the evaluation of hypodiploidy. Flow cytometry is known to be insensitive for hypodiploidy detection. This is especially the case in MM where deep hypodiploidy is rather uncommon. Moreover, the probes selected for this assay have been chosen for hyperdiploidy detection and correspond to chromosomes that are almost never lost in hypodiploid cases. Thus, this lack of sensitivity for hypodiploidy detection is not problematic as these cases would be included in the nonhyperdiploid group.

Table 1 Correlations between flow cytometry and FISH experiments

In contrast, FISH was highly sensitive for the detection of hyperdiploidy. Only six cases with low hyperdiploidy by DI (indexes=1.05–1.10) were not detected by FISH. These results are concordant with the cytogenetic literature, which showed that most hyperdiploid karyotypes are characterized by the gain of at least two of these three chromosomes. Interestingly, these six false negative patients presented with t(11;14). Most cases with t(11;14) are pseudodiploid or have a low hyperdiploidy with gain of unusual chromosomes. This study also shows that hyperdiploid cases detected by karyotyping are not biased by the high proliferation rate, but reflect hyperdiploidy per se.

All 205 patients have also been analyzed for t(4;14)(p16;q32), t(11;14)(q13;q32), and del(13q14). Translocation t(11;14) was detected in 52 patients (25% of the series). This incidence is higher than previously reported by us and others,21, 22, 23 possibly related to the size of the series or to some bias induced by the selection of cases with at least 106 purified PC. Although not reported previously, patients with t(11;14) may have a higher bone marrow plasmacytosis. However, as previously reported by others, t(11;14) was especially observed in nonhyperdiploid patients (diploidy in 42 cases, range=1–1.03, hypodiploidy in three cases, range=0.85–0.95, tetraploidy in one patient, DI=1.69, and hyperdiploidy in seven patients, range=1.05–1.20).23 Translocation t(4;14) was observed in 26 cases (13% of the series). Most t(4;14)-positive cases were also in the nonhyperdiploid group (17 diploid cases, two hypodiploid cases, and seven hyperdiploid cases, range=1.07–1.32). Thus, as described previously, the two most frequent 14q32 translocations clustered in the nonhyperdiploidy group (87% for t(11;14) and 73% for t(4;14)).24 The rare t(11;14)-positive cases falling in the hyperdiploidy group presented a low hyperdiploidy (median=1.08, range=1.05–1.2), whereas the median DI was 1.19 (range=1.07–1.32) in the t(4;14)-positive hyperdiploid cases.

Del(13) was observed in 116 patients (57%). Of the 104 hyperdiploid patients, 50 (48%) did present del(13) vs 67 of the 101 nonhyperdiploid patients (66%). Even though this correlation is statistically significant (P<0.02), these results seem slightly different from those previously published, which showed a much higher significant association between del(13) and hypodiploidy.7 We confirm here the strong association between t(4;14) and del(13).22 The del(13) was observed in 21/26 of the t(4;14)-positive patients (81%). In contrast, no association was seen with t(11;14) and del(13) as reported previously.

In conclusion, this study utilizes a novel FISH test to separate myeloma into hyperdiploid and nonhyperdiploid groups. The nonhyperdiploid group is tightly associated with recurrent 14q32 translocations and to a lesser degree with del(13). As this test is performed in interphase, it is independent of the proliferation rate. This test will permit the evaluation of the prognostic value of ploidy in myleoma without the need for karyotyping and is currently being used for the patients enrolled in the IFM 99 therapeutic trials. These studies should enable to address the issue of the true prognostic value of ploidy per se. Actually, the reports addressing these questions have been performed by conventional cytogenetics, thus in patients with a high proliferation. Whether the prognostic value is associated mostly to ploidy or mostly to proliferation is an interesting question.


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This study has been supported by a grant from the Ligue contre le Cancer, and by a Programme Hospitalier de Recherche Clinique.

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Correspondence to H Avet-Loiseau.

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Wuilleme, S., Robillard, N., Lodé, L. et al. Ploidy, as detected by fluorescence in situ hybridization, defines different subgroups in multiple myeloma. Leukemia 19, 275–278 (2005).

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  • myeloma
  • FISH
  • ploidy
  • DNA index
  • cytogenetics

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