ALBERT

Description: Multiple myeloma (MM) is proposed to consist of two main pathogenetic groups. While hyperdiploidy (HD) is characterized by multiple trisomies of odd-numbered chromosomes (i.e. 3, 5, 9, 11, 15, and 19), non-hyperdiploid MM (NHD) show frequently one of the several recurrent IgH-translocations. The aim was to compare HD versus NHD by gene expression profiling (GEP). CD138-positive multiple myeloma cells from 74 newly diagnosed MM patients (42 GEP training group (TG), 32 GEP validation group (VG)) were purified by autoMACS-sorting. Sorted cells were analyzed by interphase-FISH with probes specific for 6q21, 8p21, 9q34, 11q23, 13q14, 15q22, 17p13, 19q13 and translocations t(4;14) and t(11;14). HD and NHD were defined by using a copy number score (CS), which was calculated by subtracting the number of probes indicating losses from the number of probes detecting additional copies (CS 〉0: HD; CS ≤0: NHD). GEP was performed with Affymetrix DNA-microarrays. Nearest shrunken centroid classification (NSCC) was used to discriminate the different groups, using GCRMA-normalized gene expression values. The prediction error was estimated by means of nested cross-validation using 10 repetitions of 10-fold cross-validation within the training set and separately calculated by use of the NSCC classifier of the training set to predict the validation set. Goeman’s global test was used to check the influence of ribosomal protein expression between HD and NHD. In the TG, both HD and NHD were found in 21 patients. The VG comprised 13 patients with NHD and 19 patients with HD. NSCC resulted in a predictor for HD versus NHD of 81 probe sets with a cross-validated misclassification rate of 14.2% for the TG and 26.5% for the VG. Three of the top ten genes were ribosomal proteins, overexpressed in patients with HD. Goeman’s global test further showed that ribosomal proteins are overexpressed in HD (TG: p

Description: BACKGROUND. Angiogenesis is a hallmark of active multiple myeloma. However, two etiologic hypotheses have been proposed: an angiogenic switch (i.e. differential or de novo expression of pro/antiangiogenic genes in MM), and, alternatively an effect of increased plasma cell number. AIM of this study was to investigate the angiogenic signature of multiple myeloma cells (MMC), normal bone marrow plasma cells (BMPC), the bone marrow microenvironment (BMME) and cellular subfractions therein. PATIENTS AND METHODS. 128 newly diagnosed MM-patients (65 training (TG) / 63 independent validation group (VG)) and 14 normal donors (ND) were included. Bone marrow aspirates were CD138-purified by activated magnetic cell sorting. Whole bone marrow (n=49) and FACSAria sorted subfractions thereof (n=5) were investigated. RNA was in-vitro transcribed and hybridised to Affymetrix HG U133 A+B GeneChip (TG) and HG U133 2.0 plus arrays (VG). Expression data were gcrma-normalised and the empirical Bayes algorithm used. p-Values were adjusted using the Benjamini-Hochberg method (Bioconductor). iFISH was performed on purified MM-cells using probesets for chromosomes 1q21, 9q34, 11q23, 11q13, 13q14, 15q22, 17p13, 19q13, 22q11 and the translocations t(4;14) and t(11;14). HGF expression was verified by real time RT-PCR and western blotting. Based on Medline review, we established a list of 89 pro- and 56 antiangiogenic genes and investigated their expression according to the stage of disease: BMPC vs. MGUS, SD stage I (asymptomatic myeloma) vs. SD stage II/III (symptomatic myeloma requiring therapy). RESULTS. BMPC express pro- (e.g. VEGFA) and antiangiogenic genes (e.g. TIMP2). Only one pro-angiogenic gene (hepatocyte growth factor, HGF) is significantly overexpressed in MMC compared to BMPC. HGF has previously been linked with myeloma progression and induction of angiogenesis. Six antiangiogenic genes (TIMP2, SERPINF1, COL18A1, PF4, THBS1, CXCL14) are downregulated in MMC compared with BMPC. Compared to healthy donors, the BMME of MM shows a significant downregulation of PLAU (urokinase, antiangiogenic) and upregulation of TNF(proangiogenic). CONCLUSION. Upregulation of HGF-expression, downregulation of TIMP2, SERPINF1, COLA18A1, PF4, THBS1 and CXCL14 expression in MMC as well as downregulation of PLAU and upregulation of TNFα in the BMME seem to indicate an “angiogenic switch”. However, given the relatively low number of differentially expressed genes (7/145) and the expression of angiogenic genes by BMPC, an effect caused by an increasing number of plasma cells might be evenly important.

Description: AIM. Expression changes of D-type cyclins are thought to be an early event in the genesis of Multiple Myeloma and are associated with distinct cytogenetic aberrations. These aberrations appear with different percentages (“clonal” or “subclonal”) in a given patient. We assessed whether the height of CCND expression assessed by gene expression profiling and quantitative RT-PCR (qRT-PCR) correlates with the presence of clonal or subclonal aberrations of 11q13, t(11;14) and t(4;14). PATIENTS AND METHODS. 128 newly diagnosed MM-patients (65 training (TG)/63 independent validation group (VG)) and 14 normal donors (ND) were included. Bone marrow aspirates were CD138-purified by activated magnetic 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 and CCND2 expression was verified by real time RT-PCR and western blotting. iFISH was performed on purified MM-cells using probesets for chromosomes 1q21, 9q34, 11q23, 11q13, 13q14, 15q22, 17p13, 19q13, 22q11 and the translocations t(4;14) and t(11;14). Clonal aberrations were defined as being present in 〉60%, subclonal aberrations in 20 to 60% of MMC in a given patient. Expression data were gcrma normalised and a Kruskal-Wallis rank sum test used (Bioconductor). RESULTS. 11q13+. CCND1 (208711_s_at, 208712_at) is significantly higher (p

Description: The simultaneous quantification of thousands of genes in gene expression profiling (GEP) on DNA chips is part of the whole-genome sequencing revolution. Affymetrix(R) chip technology provides both a quantitative fluorescence signal and a decision of absent or present gene expression (absent or present call) based on signed-rank algorithms applied to several hybridization repeats of each gene spread on a single chip. To avoid an empirical normalization between chips of the same experiment, we developed an analysis of GEP based on Affymetrix present or absent calls. Bone marrow aspirates from newly-diagnosed multiple myeloma (MM) patients were purified with CD138 automated magnetic cell sorting. Amplified RNA was run on U133A+B Affymetrix DNA microarrays for a first set of 65 patients, or U133Plus2 for a second cohort of 40 patients. Scan files were transferred to an Oracle(R) data base and analyzed with web-oriented scripts for both unsupervised and supervised non-parametric analysis on either the fluorescence signal or the Affymetrix call. To build a multiclass call-based predictor, the observed distribution of present call of each probeset was first compared between predetermined sample groups using a chi2 test and probesets were kept only if above a threshold compatible with further analysis and calculation time (usually 100 to 1000 genes). The power of a probeset list to classify the different groups (number of presence/number of probesets) was then evaluated for each sample of a group and compared to each sample of all other groups by calculating the reduced deviation (RD) in paired comparisons and evaluating the overall number of non significant comparisons (NS) with a chosen precision, the sum of the reduced deviations divided by the square root of the probeset number (f, independent of the list size), and the smallest RD (RDmin). The minimum predictive probeset list was obtained by deleting each probeset one after the other, and computing NS, f and RDmin from the remaining probeset list. If either NS is reduced, or both NS unchanged and f increased, or together NS and f unchanged and RDmin either increased or higher than precision, the probeset is left out and the process run again on the shortened list until no more leave-outs are possible. This method was successfully validated by determining a 22-gene sex predictor with the 65 patient series that made it possible to classify gender with no error in the 40 patient validation group. Partial loss of chromosome Y was confirmed in 3 male MM patients by short tandem repeat analysis. Significant predictors could not be generated with randomly selected patient groups. Validation was also successful with P

Description: Using Affymetrix microarrays, we identified the expression of the CD200 gene in multiple myeloma cells (MMC) of 112 patients with newly-diagnosed multiple myeloma (MM). The CD200 gene was either absent or present (Affymetrix call) in 22% and 78% of MMC, respectively. CD200 was not expressed by CD14 monocytes, CD15 polynuclear cells and CD3 T cells that were purified from the bone marrow of 5 newly-diagnosed patients. It is also not expressed in 7 osteoclast samples. BM stromal cells from 5 patients with MM expressed CD200, but at a 3.9 fold lower median signal compared to that in CD200present MMC (P = .04). CD200 is a membrane glycoprotein that imparts an immunoregulatory signal through CD200R, leading to the suppression of T-cell-mediated immune responses. Patients with CD200absent MMC have an increased event free survival (24 months) compared to patients with CD200present MMC (14 months), after high-dose therapy and stem cell transplantation. In a Cox-proportional-hazard model, the absence or presence of CD200 expression in MMC is predictive for EFS for patients independently of ISS stage or B2M serum levels. Thus, CD200 is an independent prognosis factor for patients with MM that could represent a new therapeutic target in MM.