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Because metaphase cytogenetic studies in multiple myeloma (MM) are hampered by a low proliferative activity of myeloma cells in vitro, interphase cytogenetics by means of fluorescence in situ hybridization (FISH) should improve the detection of chromosomal abnormalities in MM. We therefore investigated chromosomal aneuploidy in 36 patients with MM using interphase FISH and alpha-satellite DNA probes for chromosomes 1, 3, 7, 8, 11, 12, 16, 17, 18, and X. By FISH, myeloma cells from 32 patients (88.9%) were aneuploid for at least one of the chromosomes examined. In 24 patients (66%), aberrations of > or = 3 chromosomes were observed. Aneuploidy was predominantly characterized by a gain of chromosome numbers, with involvement of chromosomes 3, 7, and 11 occurring in > 50% of patients. Loss of a centromeric signal suggesting monosomy was most frequently observed for chromosomes 17 (22.2% of patients) and X (monosomic in 42.3% of female patients, but loss of chromosome X was never observed in males, P < 0.05). Dual-color FISH studies provided evidence for marked heterogeneity of aneuploid cells in 8 patients (22.8%). Occurrence of chromosomal aneuploidy was independent of stage and pretreatment status. Gain of chromosome 3 was significantly correlated with an IgA paraprotein (P < 0.05). In 12 patients, the direct comparison of metaphase cytogenetics and FISH showed that FISH detected aneuploidy of chromosomes in 9 patients that was missed by metaphase analysis. In conclusion, interphase FISH, by which chromosomal aneuploidy was detected in almost 90% of patients with MM, represents an approach for evaluating the clinical significance of specific chromosomal abnormalities in MM.