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260 patients uniformly treated according to the GEM

2000 protocol, which includes an induction phase with VBCMP/VBAD followed by autologous cell transplantation (ASCT). In addition, we have explored whether or not these cytogenetic subgroups display distinct clinical and biological disease characteristics. Patients and methods Patients Patients under the age of

70 years, with newly diagnosed MM, enrolled in the GEM

2000 Spanish protocol (six alternating cycles of VBCMP/VBAD followed by high-dose therapy C melphalan

200 mg/m2 supported by ASCT) were included in the study. The study was approved by the research ethics committees of all participating centers and written informed Received

5 July 2006;

revised

25 August 2006;

accepted

30 August 2006;

published online

5 October

2006 Correspondence: Professor JF San Miguel, Servicio de Hematolog? ?a, Hospital Universitario de Salamanca, Paseo San Vicente 58-182, Salamanca 37007, Spain. E-mail: [email protected]

13 These authors contributed equally to this work Leukemia (2007) 21, 143C150 &

2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu consent was obtained from all patients. In order to interpret accurately FISH analysis only those patients with BM PC in?ltration by ?ow cytometry above 10% were eligible for this study (n ? 260). The main clinical and laboratory characteristics of these patients are shown in Table 1. Forty-seven of

260 patients (18%) did not undergo ASCT because of comorbidity (20 cases), progression of the disease (13 cases), failure in mobilization (11 cases) or withdrawal of informed consent (three cases). The median overall survival (OS) for the whole group was

43 months (95% con?dence interval, 36C49), and the median follow-up for survivors was

34 months. At the time of study,

102 patients remained alive. FISH analysis Interphase FISH studies for the detection of IGH rearrangements were carried out by means of LSI IGH dual color, break apart rearrangement probe (Vysis, Downers Grove, IL, USA). Patients with IGH translocations were explored ?rstly for t(11;

14)(q13;

q32) (LSI IGH/CCND1, dual fusion translocation probe) (Vysis), and subsequently analyzed for t(4;

14)(p16;

q32) (4p C BAC clones L75b9, L190b4, L96a2, PAC 184d6C;

14q32C VH: cosmid yIgH6-9, CH: BAC B158 A2C) and ?nally for t(14;

16)(q32;

q23) (16q23 C BAC clones 356D21, 484H2,

10205 and 10206C). The probes corresponding to the last two translocations were kindly provided by Rafael Fonseca from the Mayo Clinic, Scottsdale, AZ, USA. The presence of 13q and 17p deletions were evaluated with a speci?c probe for RB -LSI

13 (RB1) (Vysis) and for P53 C LSI P53 (17p13.1) (Vysis), respectively. The interphase-FISH procedure has been described previously in detail.13 A total of

500 interphase nuclei were analyzed using the scoring criteria recommended by the manufacturer. Based on the results using these probes in

25 healthy controls, the cutoff point for the identi?cation of alteration was set at more than 8% cells with abnormal signal. Statistical analysis Statistical analysis were performed using SPSS statistical soft- ware version 11.5 (SPSS Inc., Chicago, IL, USA). The w2 and the Fisher'

s exact test were used to test associations between chromosomal abnormalities as well as between genomic changes and other categoric variables. For continuous variables, the Wilcoxon rank sum test and t-test were used. OS was calculated from the start of the initial treatment to the date of death or last visit. Time to progression (TTP) was estimated from the day of initiation of treatment to the date of relapse or disease progression. Survival curves were plotted by means of the KaplanCMeier method and the difference in survival curves was tested for statistical signi?cance using the log-rank test. P-values below 0.05 were considered to re?ect statistical signi?cance. Multivariate analysis of survival was performed using the Cox proportional hazards model (stepwise regression approach). Factors were retained in the model if they were statistically signi?cant at Pp0.05. Results Frequency of chromosomal abnormalities Chromosomal abnormalities explored by FISH were identi?ed in