Résumé
AIM was to test the new molecular classification of MM based on changes in global gene expression attributable to cytogenetic aberrations detected by interphase FISH (iFISH) in order to (i) predict EFS in a group of 100 MM-patients treated by high dose chemotherapy, and (ii) to investigate whether the classification represents an independent prognostic factor.
PATIENTS AND METHODS. Bone marrow aspirates from MM-patients and normal donors were CD138-purified by magnetic activated cell sorting. RNA was in-vitro transcribed and hybridised to Affymetrix HG U133 A+B GeneChip (TG) and HG U133 2.0 plus array (VG). CCND1, CCND2 and FGFR3 expression was verified by real time RT-PCR and western blotting. iFISH was performed on purified MM-cells using probes for chromosomes 11q23, 13q14, 17p13 and the IgH-translocations t(4;14)(p16;q32.3) and t(11;14)(q13;q32.3). Expression data were normalised (Bioconductor package gcrma), and nearest shrunken centroids (NSC) applied to calculate and cross validate a predictor on a training group (TG) of 40 patients in whom a comprehensive iFISH panel combined with data on CCND overexpression were available. The ExpressMiner tool of the HUSAR bioinformatics laboratory was used to analyze genes differentially expressed between GEP-defined groups. A log-rank test and a Cox proportional hazard model were used to test the influence of prognostic parameters in combination with the predicted groups.
RESULTS. Four groups were distinguished: (1.1) CCND1 (11q13) overexpression and trisomy 11q13, (1.2) CCND1 overexpression and translocations involving 11q13, i.e. t(11;14), (2.1) CCND2 overexpression without 11q13+, t(11;14), t(4;14), (2.2) CCND2 overexpression with t(4;14) and FGFR3 upregulation. A predictor of 6 genes correctly classified all 40 patients of the TG (estimated cross validated error rate 0%). An independent validation group (VG) of 65 patients was used. Distribution of clinical parameters (i.e. beta2M, Durie Salmon stages, ISS) was not significantly different between the 4 groups. The groups defined by the predictor have a significantly different EFS after autologous stem cell transplantation according to the GMMG-HD3 protocol (n=100; median: 26 /not reached /22 /6 months in groups 1.1 /1.2 /2.1 /2.2, respectively). A model testing the combination of the predicted group and B2M (above or below 3.5 mg/dl) showed a significant (p<0.006, log-rank-test) correlation with EFS. The distribution of del(13q14) (n=118) was (1.1) 34.0%, (1.2) 60.8%, (2.1) 46.4% and (2.2) 94%. The presence of a del(13q14) either in a subclone (<60% of analyzed nuclei) or major clone had no significant influence on EFS.
CONCLUSION. Gene expression and iFISH allow a molecular classification of MM which can be predicted by GEP. Groups in the classification have distinct gene expression patterns as well as statistically significant different EFS. GEP-defined groups and B2M represent independent prognostic parameters.