[CANCER RESEARCH 55, 3854-3859, September 1, 1995] Multiple Myeloma: High Incidence of Chromosomal Aneuploidy as Detected by Interphase Fluorescence /// Situ Hybridization Johannes Drach,1 Judith Schuster, Hadwiga Nowotny, Jutta Angerler, Friederike Rosenthal, Michael Fiegl, Christian Rothermundt, Andrea Gsur, Ulrich Jäger,Renate Heinz, Klaus Lechner, Heinz Ludwig, and Heinz Huber First Department of Internal Medicine, Division of Clinical Oncology [J. D., J. A., F. R., M. F., C. R.. A. G., H. H.I and Division of Hematology and Hemosiesiolog\ [U. J., K. L.\, University of Vienna, Währinger Cartel 18-20, A-1090 Vienna: Ludwig Bollynann-lnstitule far Leukemia Research and Hematology. Hanusch Hospital ¡H.N.. R. H.¡. Vienna; and First Department of Internili Medicine with Oncolog\. Wilhelminenspital. Vienna [J. S., H. L], Austria ABSTRACT because previous cytogenetic studies suggested preferential involve ment of these chromosomes in karyotypic abnormalities in MM Because metaphase cytogenetic studies in multiple myeloma (MM) are (1-14). In cases with successful karyotyping, we compared FISH hampered by a low proliferative activity of myeloma cells in vitro,inter- results with those obtained by conventional cytogenetics. We also phase cytogenetics by means of fluorescence in situ hybridization (FISH) should improve the detection of chromosomal abnormalities in MM. We investigated whether there was any correlation between specific therefore investigated chromosomal aneuploidy in 36 patients with MM chromosomal aberrations and clinical parameters. using interphase FISH and a-satellite DNA probes for chromosomes 1, 3, 7, 8, 11, 12, 16, 17, 18, and X. By FISH, myeloma cells from 32 patients MATERIALS AND METHODS (88.9%) were aneuploid for at least one of the chromosomes examined. In 24 patients (66%), aberrations of a3 chromosomes were observed. Aneu Patients. Thirty-sixpatientsdiagnosedwithovertMMwerestudied(Table ploidy was predominantly characterized by a gain of chromosome num 1). BM aspirates were obtained from the posterior iliac crest or sternum and bers, with involvement of chromosomes 3,7, and 11 occurring in >50% of collected in a heparinized syringe. All procedures were done for diagnostic patients. Loss of a centramene signal suggesting monosomy was most purposes, and informed consent after institutional guidelines was obtained for frequently observed for chromosomes 17 (22.2% of patients) and X the use of an aliquot of the specimens for research purposes. One patient with iimmusonìifin 42.3% of female patients, but loss of chromosome X was advanced MM also had an extramedullary manifestation of the disease, which never observed in males, P < 0.05). Dual-color FISH studies provided was surgically removed and treated with extensive mechanical disintegration evidence for marked heterogeneity of aneuploid cells in 8 patients to obtain a single cell suspension. Material used for control hybridizations (22.8%). Occurrence of chromosomal aneuploidy was independent of included peripheral blood from 3 healthy volunteers and BM aspirates from a stage and pretreatment status. Gain of chromosome 3 was significantly BM transplant donor and from 4 patients with localized solid tumors without correlated with an IgA paraprotein (/' < 0.05). In 12 patients, the direct BM involvement. comparison of metaphase cytogenetics and FISH showed that FISH de FISH Studies. Ficoll-Hypaque-separatedmononuclearcells were washed tected aneuploidy of chromosomes in 9 patients that was missed by twice with PBS and treated with Carnoy's fixative (methanol-glacial acetic metaphase analysis. In conclusion, interphase FISH, by which chromo acid, 3:1, v/v). Interphase FISH was performed using biotin- and digoxigenin- somal aneuploidy was detected in almost 90% of patients with MM, labeled a-satellite DNA probes (all obtained from Oncor, Gaithersburg, MD) represents an approach for evaluating the clinical significance of specific specific for chromosomes 1 (DIZ3), 3 (D3ZI), 7 (D7ZÃŒ),8(D8Z1), 11 chromosomal abnormalities in MM. (DllZl), 12 (DÃŒ2Z3),16(D12Z2), 17 (D17Z1), 18 (D18Z1), and X (DXZ1). For additional FISH studies in patients 10 and 22, DNA probes being specific for chromosomes 13 and 21 (directly labeled with SPECTRUM-orange) were INTRODUCTION obtained from Imagenetics (Framingham, MA). FISH was performed accord Cytogenetic studies in MM2 have been hampered by the low ing to the protocol by Pinkel et al. (20) with minor modifications (23). Cells proliferative rate of myeloma cells in vitro. Previous studies using were analyzed under a fluorescence microscope (Olympus AN-3), and hybrid ization signals were evaluated in 200-800 cells. Microphotographswere taken metaphase cytogenetics reported often complex numeric and struc tural chromosomal abnormalities in 30-40% of patients with MM with Kodak Ektachrome 1600 color slide films (Eastman Kodak, Rochester, (1-14). However, by using DNA flow cytometry, DNA-aneuploid NY). Cytogenetic Analysis. MononuclearcellsuspensionsafterFicoll-Hypaque stemlines were demonstrated in up to 80% of patients with MM; DNA density separation were cultured at 2 X 106/ml in a-MEM:McCoy's 5A hyperdiploidy was the most common observation (15-19). These medium (1:1, v/v) supplemented with 20% heat-inactivated fetal calf serum. results suggest that chromosomal abnormalities in MM occur much Parallel cultures were set up with the addition of granulocyte macrophage- more frequently than is detected by conventional cytogenetics. colony-stimulating factor (100 units/ml) plus interleukin 6 (5000 units/ml) and The use of DNA specific probes and the technique of FISH enables 10% B- and T-cell growth supplement (Origen) for 24, 48, and 72 h. Harvest us to study chromosomal abnormalities in interphase nuclei (20, 21). ing and spreading of metaphases for staining was carried out according to Interphase FISH has been shown to be particularly advantageous in standard techniques. The culture with the best metaphases was selected for the analysis of chromosomal aberrations of differentiated cells such as staining by the tristain method according to Schweizer (27. 28). Twenty metaphases were evaluated for each patient using the semi-automated Gene- in chronic lymphocytic leukemia, where trisomy 12 was detected 2-3 times more often by FISH than by conventional cytogenetics (23-25), vision 221 karyotyping system (Applied Imaging). Chromosomal abnormali ties were defined and described according to the International System for and in MM (26, 27). We therefore used interphase FISH and a-sat- Cytogenic Nomeclature (29). ellite DNA probes for chromosomes 1, 3, 7, 8, 11, 12, 16, 17, 18, and Enrichment of B-B4 Positive Myeloma Cells. The mAb B-B4 (Immuno X to investigate the actual incidence of chromosomal abnormalities in Quality Products, Groningen, the Netherlands) and an immunomagnetic sep 36 patients with MM. We decided to focus on these chromosomes aration method (30, 31) were used for the enrichment of myeloma cells. Ten U.1of undiluted B-B4 (0.2 mg/ml) were added per IO6BM mononuclear cells, and the cell suspension was incubated for 30 min at 4°C.Cellswere washed Received 3/23/95; accepted 6/30/95. The costs of publication of this article were defrayed in part by the payment of page twice with PBS supplemented with 0.5% BSA, followed by incubation with rat charges. This article must therefore be hereby marked advertisement in accordance with antimousc IgGl tagged with magnetic microbeads (Miltenyi Biotec, Bergisch- 18 U.S.C. Section 1734 solely to indicate this fact. Gladbach, Germany) for 15 min at 4°C.Cellswere again washed and then 1To whom requests for reprints should be addressed. 2 The abbreviations used are: MM, multiple myeloma; BM, bone marrow; FISH, layered on a MiniMACS separation column (Miltenyi Biotec) following the manufacturer's instructions. B-B4-positive cells were removed from the col- fluorescence in situ hybridization. 3854 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1995 American Association for Cancer Research. FISH IN MULTIPLE MYELOMA Table 1 Characteristics of Patients Feature No. of patients Sex Male 9 Female 27 Age (yr) 42-83 (median, 64) Stage (Durie and Salmon) I 7 n 9 m 20 Type of paraprotein IgG 22 IgA 12 Bence-Jones only 2 Treatment No prior treatment 24 Previous chemotherapy 12 Time from diagnosis 9-126 months (median, 30) ß-2microglobulin <6 21 >6 8 Not evaluated 7 umn and washed with 0.5% PBS-BSA. Purity of the plasma cell fraction was evaluated by morphology on Giemsa-stained cytospin preparations. RESULTS Controls. Normal peripheral blood (3 samples) and BM (5 sam ples) specimens were used to determine background levels for cells with 1 and >3 domains, respectively, for each DNA probe in normal hematopoietic cells. Cutoff levels for the diagnosis of aneuploidy were defined by the mean plus 3 SDs of the frequency of these cells in normal specimens (these results are part of Tables 3 and 4). Chromosomal Aneuploidy in Myeloma Cells as Detected by Interphase FISH. After hybridization, sufficient morphological de tails were preserved to identify myeloma cells as large cells with faint fluorescence of the cytoplasm and large eccentric nuclei (Fig. 1). Cells with the typical appearance of mature myeloid cells were scored separately, and deviations from the normal controls were not observed in any of the cases (data not shown). Thus, these cells were excluded from the final analysis, and percentages of cells with chromosomal aneuploidy by FISH represent plasma cells and cells with a lymphoid appearance. Thirty-six myeloma specimens (35 BM aspirates, 1 extra-medullary lesion) were analyzed with a-satellite DNA probes specific for chro Fig. 1. FISH studies of myeloma cells. A, hybridization with an a-satellitc DNA probe mosomes 1, 3, 7, 8,11,12, 16, 17, 18, and X. Aneuploidy for at least for chromosome 7 (biotin-labcled, detection with streptavidin-FITC: DNA counterstain 1 of these chromosomes was found in 32 of 36 patients with MM with propidium iodide) revealed three hybridization signals. Note the preserved morpho (88.9%; Table 2). In 24 of 36 patients (66.6%), aneuploidy of 3 or logical features of the plasma cell (eccentric nucleus, autofluorescence of the cytoplasm).