¹Servicio de Hematología, Hospital Universitario de Salamanca and Centro de Investigación del Cáncer (CIC), Universidad de Salamanca-CSIC, Spain

²Hospital General de Segovia, Spain

Correspondence to: J San Miguel Izquierdo, Servicio de Hematología, Hospital Universitario de Salamanca, Paseo San Vicente 58-182, 37007 Salamanca, Spain; Fax: 34 923 294624

To analyze the genomic differences between multiple myeloma (MM) and plasma cell leukemia (PCL), a total of 30 cases were studied by comparative genomic hybridization (CGH). In five cases with a low proportion of plasma cells (PC) in bone marrow, an enrichment of PC was performed by using immunomagnetic beads conjugated with the monoclonal antibody B-B4. In 24 out of the 25 MM (96%) and in all five PCL (100%) patients DNA copy number changes were identified by CGH analysis; in the MM case without chromosomal imbalances, the immunomagnetic enrichment of PC had failed. The most recurrent changes in MM patients were gains at chromosomes 15q (48%), 11q (44%), 3q (40%), 9q (40%) and 1q (36%). By contrast, all PCL patients showed gains in 1q. Losses of chromosomal material were significantly more frequent in PCL than in MM patients (P = 0.03): losses on 13q in 80% of PCL vs 28% of MM; and on chromosome 16 in 80% vs 12%, respectively. In addition, PCL patients showed losses of 2q and 6p that were not present in MM. The CGH data show differences in chromosomal imbalances between MM and PCL. Leukemia (2001) 15, 840-845.

myeloma; plasma cell leukemia; CGH; cytogenetics

Introduction

Multiple myeloma (MM) is considered a clonal disorder of B cells at a last stage of differentiation (plasma cells). The low proliferative index of plasma cells (PC) reduces the number of analyzable metaphases and hampers cytogenetic studies.¹ In order to improve the detection of chromosomal abnormalities different cytokines have been used, however the incidence of abnormal karyotypes remains below 60%.^2,3,4,5 Comparative genomic hybridization (CGH) is a molecular cytogenetic technique that allows analysis of chromosomal copy number changes without the requirement of tumoral metaphases.⁶ Accordingly, CGH would be particularly useful for identifying gains and losses of DNA sequences in tumors with a low proliferative index, such as MM. Three recent studies, including small series of patients (8, 25 and 24 cases, respectively) have shown that chromosomal imbalances are present in most MM patients.^7,8,9 A comparison between the abnormalities detected by cytogenetics and CGH has only been performed in one of these studies, showing a good level of concordance in 50% of cases.⁸ Primary plasma cell leukemia (PCL) is a rare form of PC neoplasia, characterized by the presence of an increased number of circulating PC and an aggressive clinical course. Recently, our group has shown a very high incidence of chromosome 13 monosomies in PCL,¹⁰ but information on the overall chromosomal changes in PCL is still scant.¹¹ Moreover, to the best of our knowledge CGH has not been used to compare the changes present in PCL and MM patients. In a previous study CGH has been applied to analyze both MM and PCL, but differences have not been explored.⁷ In the present study we have analyzed by CGH, 25 patients diagnosed with MM and five with PCL.

Materials and methods

Patients

A total of 30 patients, 25 with MM and five with primary PCL were included in the study. The diagnosis of MM was based on criteria from the Chronic Leukemia-Myeloma Task Force;¹² for the diagnosis of PCL more than 2 ´ 10⁹/l blood PC were required. Median age was 70 years (range: 45-90 years). Most cases (73%) had an advanced clinical stage according to Durie and Salmon's clinical staging system.¹³ Bone marrow plasma cell infiltration ranged from 14% to 93% with a median infiltration of 51%.

PC enrichment

In five cases (Nos 5, 6, 8, 10 and 22) with a low proportion of PC in bone marrow (less than 35%), an enrichment of PC was performed by using immunomagnetic separation with the monoclonal antibody BB4 conjugated with immunomagnetic beads.¹⁴ In the first four cases (Nos 5, 6, 8 and 10) an enrichment of more than 60% PC was achieved while in the last case (No. 22) the enrichment of PC failed.

Cytogenetic analysis

IL-4 stimulated and unstimulated cultures were carried out as previously described.⁵ G-banding by standard procedures was used. Karyotypes are described according to the ISCN.¹⁵ Karyotypes of cases Nos 1, 3, 4, 5 and 6 have been published before (Table 1).⁵

Comparative genomic hybridization

Tumor DNA was isolated from bone marrow in all patients and reference DNA was obtained from peripheral blood lymphocytes of healthy donors (same sex as patients). The phenol-chloroform method was used for DNA extraction according to standard procedures.¹⁶ CGH was performed as described by Lichter et al.¹⁷ Tumor DNA (test DNA) was labeled with biotin-16-dUTP (Boehringer Mannheim, Mannheim, Germany) and normal DNA (reference DNA) was labeled with digoxigenin-11-dUTP (Boehringer Mannheim) by a standard nick translation reaction. Equal amounts (1 g) of labeled tumor and normal DNAs, and 70 g of unlabeled human Cot-1 DNA (GIBCO/BRL, Gaithersburg, MD, USA) were cohybridized to slides with human metaphase chromosome spreads. Tumor and normal DNA were detected by avidin-fluorescein isothiocyanate (FITC) and rhodamine-conjugated anti-digoxigenin, respectively. Image acquisition was performed with an epifluorescence microscope (Olympus BX60, Hamburg, Germany) equipped with a cooled charge-coupled device (CCD) camera. Calculation of the tumor DNA to normal DNA fluorescent ratios along the length of each chromosome was performed by means of an automated CGH software package (Applied Imaging, Sunderland, UK). Ratio values obtained from at least 10 metaphase cells for each case were averaged. Ratio values above 1.25 and below 0.75 were considered to represent chromosomal gain and loss, respectively. Over-representations were defined as high-level amplifications when the profiles exceeded the cut-off value of 1.5. Chromosomal gains exceeding 1.5 involving the whole chromosome or large areas of a chromosomal arm were not considered as high-level DNA amplification. Negative control experiments were performed using differentially labeled male vs male DNA, and female vs female DNA. Additional control experiments included the interchange of the digoxigenin-dUTP and biotin-dUTP labels between normal and tumor.

Statistical methods

To estimate the statistical significance of differences observed between mean values for PCL and MM patients for continuous variables, the Mann-Whitney U test was used with SPSS statistical software (SPSS Inc., Chicago, IL, USA).

Results

In 24 out of the 25 MM (96%) and in all five PCL (100%) patients, chromosomal imbalances were identified by CGH analysis (Table 1). The only MM case without changes (No. 22) had a low proportion of PC (15%) and the enrichment by immunomagnetic beads failed. Overall, a total of 214 DNA copy number changes were detected with a median of seven abnormalities per case (range: 1-28): 156 gains, 54 losses and four high-level amplifications (Table 1). Upon comparing the incidence of genetic changes in MM and PCL this was higher in the primary PCL group (mean of 12.2 ± 10.4) than it was in MM patients (mean of 6.1 ± 4; P > 0.05) (Figure 1). In 28 patients (93%) gains of chromosomal material were observed. In MM group, the most common gains of chromosomal material involved chromosomes 15q (48%), 11q (44%), 3q (40%), 9q (40%) and 1q (36%). By contrast, all PCL patients showed gains in 1q. In 19 cases (63%) there was a loss of chromosomal material. In 10 of the 19 cases with losses only one chromosome was affected. PCL patients showed a significantly higher proportion of losses of chromosomal material (mean of 4.8 ± 3.4) than MM patients (mean of 1.2 ± 1.5; P = 0.028) (Figure 1). Although the most frequent losses both in MM and in PCL affected 13q and 16, the incidence of these under-representations were higher in PCL than in MM (80% in PCL vs 28% in MM for losses on 13q, and 80% in PCL vs 12% in MM for losses on 16). Moreover, losses involving 2q and 6p were only present in PCL. High-level DNA amplifications were identified in three different regions of the genome in MM patients (9q34, 11q14-22, and Xq24-26) and in one region in the five PCL patients (8q24). Examples of the CGH profiles are given in Figure 2.

Considering both MM and PCL, CGH analysis allowed the delimitation of minimal overlapping regions (consensus regions) on each of the chromosomes most frequently involved. In the group of gains the commonly over-represented regions were delineated to 1q11-41, 3p21, 3q25-27, 5q31-32, 7q22, 8q24, 11q13-22, 15q15-21 and 15q24. For the group of losses, 2 consensus regions could be delimited in 13q31-32 and 16q13-21 (Figure 1).

Seventeen of 25 patients (68%) in which G-banding analyses were available showed clonal chromosomal abnormalities. CGH identified genetic changes in seven cases with unsuccessful or normal karyotypes. In 10 cases, a good correlation between CGH and cytogenetics was found (Table 1).

Discussion

In the present study, we have detected DNA copy number changes in 96% of MM and 100% of PCL cases by CGH. Interestingly, the four MM cases with a low infiltration in which an enrichment of PC was achieved, showed abnormalities. By contrast, the only patient (No. 22) with a low plasma cell infiltration, in which the enrichment of PC was unsuccessful, did not display any change by CGH. The incidence of chromosomal changes detected by CGH in this study is slightly higher than the proportion reported by Cigudosa et al⁸ (70%), but it is concordant with two other reports.^7,9 The lower incidence found in the previous study⁸ may be due to the fact that some of the cases had bone marrow PC infiltration below 30% and according to our experience, enrichment in PC may be very important for CGH analysis when the bone marrow infiltration is low. In fact, the use of immunomagnetic bead enrichment in cases with <30% PC would allow the detection of genomic imbalances by CGH in most MM patients.

Our study shows that patients with PCL displayed interesting differences compared to myeloma patients. Thus, the number of copy alterations is higher in PCL than in MM patients, which may reflect an increased genetic instability in aggressive forms of tumors that have an apparently common target neoplastic cell. A similar finding has been observed in mantle cell lymphomas (MCL), where blastoid variants have a higher number of chromosomal imbalances than typical variants.^18,19,20 In line with this observation, in some solid tumors the average number of copy alterations correlates with tumor aggressiveness, progression and prognosis.²¹ Interestingly, PCL cases had a mean number of losses of chromosomal material significantly higher than those of MM cases. This finding is consistent with DNA content studies by flow cytometry: PCL display lower DNA content than MM patients.¹⁰ Gains on 1q were found in all five PCL cases while in only 36% of MM patients. Extra copies of 1q are widely reported in neoplasia and have been considered as secondary aberrations associated with tumor progression and advanced disease.^22,23,24 The higher incidence of gains on 1q in PCL compared to MM may be related to the more advanced and aggressive stage of PCL. Similarly, losses on 13q were more frequent in PCL than in MM group, which is consistent with the data reported by our group by using fluorescence in situ hybridization (FISH) technique in PCL.¹⁰ The high incidence of 13q losses detected in the PCL group is similar to the frequency of deleted 13 found in plasmacytomas by Aalto et al.⁹

Overall, the most frequent genetic imbalance in our series of 30 patients was the gain of material on 15q (47%) with two minimum overlapping areas at 15q15-21 and 15q24. The potential target genes in these regions are not known. Gains involving chromosome 15 have not been described as the most common aberration in MM patients, when either CGH,^7,8,9 classical cytogenetics^1,25 or the recently developed multicolor spectral karyotyping (SKY)^26,27 are used. However, in myeloma cell lines a high proportion of gains in chromosome 15 has been identified by CGH (62%).⁷ In accordance with other studies, the most recurrent losses involved chromosome 13. A consensus area was delineated to 13q31-32 suggesting the presence of potential tumor suppressor genes located telomeric to RB, DBM and BRCA2 genes, involved in the pathogenesis of MM.^28,29,30

Finally, we have analyzed the correlation between the results of G-banding and CGH analysis. CGH showed genomic imbalances in seven cases with unsuccessful or normal cytogenetics. Moreover, CGH studies allowed us to detect losses on chromosome 13, an abnormality associated with a poor outcome,³¹ not identified by classical cytogenetics in five cases. The discrepancies may be explained by the capability of CGH to detect chromosomal imbalances in the whole tumor that may not be present in the proliferating malignant clone in the culture used for cytogenetic analysis. These results support the interest in performing CGH on MM and PCL cases with normal or unsuccsesful cytogenetics. In some hematological diseases, like acute myeloblastic leukemia, a better correlation between CGH and G-banding results has been found.³² However, the complexity of karyotypes in MM (high numbers of numerical and structural chromosome aberrations and intratumor heterogeneity) would explain such discrepancies between CGH and G-banding. In fact, this situation resembles that found in solid tumors in which there is also an important clonal heterogeneity and frequent differences between these two techniques.³³

In summary, our CGH data show the existence of marked differences in chromosomal imbalances between MM and PCL, which may help to explain the different clinical course of these disorders. In addition, our study shows the importance of performing an enrichment of tumor cells in MM cases with low PC infiltration in order to avoid false negative results. Finally, CGH allowed us to delineate consensus regions in 15q15-21 and in 15q24 that are frequently gained in PC dyscrasias, as well as deletions of 13q31-32.

Acknowledgements

This work was partially supported by grants from the Spanish FIS (97/1248; 98/1161 and 00/1089). The authors thank ME Fernández, P Fernández, MA Hernández and M Anderson for technical assistance.

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Figure 1  Summary of chromosomal imbalances detected by CGH in 25 patients with MM (a) and in five patients with PCL (b). Lines on the right side of the idiograms indicate gain of chromosomal material. Lines on the left side indicate loss of chromosomal material. Thick bars represent chromosome gains exceeding 1.5 in a large chromosome region. High level amplifications are represented as solid squares.

Figure 2  Examples of fluorescence ratio profiles. The ratios of tumor/normal fluorescence are plotted along the ideograms. The central line indicates a ratio value of 1.0; lines on the left side indicate ratio values of 0.75 and 0.5, respectively; lines on the right side indicate ratio values of 1.25 and 1.5, respectively. n = number of chromosomes analyzed for calculating the respective average ratio profile. The following genetic changes are observed: (a) Gain of 1q (case 12); (b) gain of chromosome 7 (case 3); (c) gain of chromosome 15 (case 6); (d) amplification of Xq24-26 (case 17); (e) loss of 12p13 (case 24); (f) loss of chromosome 13 (case 2); (g) loss of 16q12-21 (case 29).

Table 1  Genomic changes in patients with MM and PCL