ALBERT

Description: Key Points Multiple myeloma cells inhibit myeloma-specific T cells through expression of carcinoembryonic antigen-related cell adhesion molecule-6.

Description: Identification of growth factors in neoplasias may be a target for future therapies by blocking either growth factor receptor interaction or the induced pathway. Using gene expression profiling, we identified overexpression of 2 receptors for a proliferation-inducing ligand (APRIL) and B-cell activating factor (BAFF) in malignant plasma cells compared with normal plasma cells. APRIL and BAFF are involved in a variety of tumor and autoimmune diseases, including B-cell malignancies. We confirmed the expression of BAFF and APRIL receptors (B-cell maturation antigen [BCMA], transmembrane activator and calcium modulator and cyclophilin ligand interactor [TACI], and BAFF-R) in a majority of 13 myeloma cell lines and in the purified primary myeloma cells of 11 patients. APRIL and BAFF were potent survival factors for exogenous cytokine-dependent myeloma cell lines and were autocrine growth factors for the RPMI8226 and L363 autonomously growing cell lines. These factors activated nuclear factor (NF)–κB, phosphatidylinositol-3 (PI-3) kinase/AKT, and mitogen-activated protein kinase (MAPK) kinase pathways and induced a strong up-regulation of the Mcl-1 and Bcl-2 antiapoptotic proteins in myeloma cells. BAFF or APRIL was also involved in the survival of primary myeloma cells cultured with their bone-marrow environment, and protected them from dexamethasone (DEX)–induced apoptosis. Finally, the serum levels of BAFF and APRIL were increased about 5-fold in patients with multiple myeloma (MM) as compared with healthy donors. Altogether, these data suggest that APRIL/BAFF inhibitors may be of clinical value in MM. (Blood. 2004;103:3148-3157)

Description: Background: In multiple myeloma (MM), deletion of chromosome (chr.) 13q and hypodiploidy are adverse prognostic factors. Prognostic evaluation is frequently based on interphase-fluorescence in-situ hybridization (FISH) with a single probe for chr. 13q14. Patients and methods: CD138-positive bone marrow cells from 97 patients with newly diagnosed MM (58 training group (TG) and 39 validation group (VG)) were enriched by magnetic-activated cell-sorting (median purity, 95%). Sorted cells were analyzed by interphase-FISH with probes for chr. 13q14, and additionally 9q34, 11q23, 19q13, t(11;14), and t(4;14). GEP was performed with Affymetrix U133A+B (TG) and HGU133-2.0plus (VG) microarrays. Nearest shrunken centroids classification (NSC) was applied to discriminate clones with FISH-detected del(13q14) vs. those without, using VSN-normalized gene expression values. Chromosomal localization of predictor genes was determined using the MapIt program, and functional relationship was established by Gene Ontology (GO) annotation and creation of GO-slims. Results: A deletion of chr. 13q14 was found in 27/58 patients (47%) of the training and in 21/39 (54%) of the validation group. Frequencies of trisomies were lower (9q: 48 vs. 74%; 11q: 41 vs. 74%; 19q: 44 vs. 84%) and of IgH translocations higher (48 vs. 16%) in patients with del(13q14). NSC resulted in a predictor for del(13q14) of 378 probe-sets with a cross-validated classification error rate of 22%; the VG is under statistical investigation. Of the predictor genes, 18% were localized on chr. 13 (distributed evenly from 13q12 to 13q33), followed by chr. 19, 11 and 3 (10/6/6%). In the 50 probe-sets with the highest scores, the most frequent localizations of the represented genes were chr. 19, 9, and 13 (12/8/6 of 50). In 8/8 incorrectly classified patients with del(13q14), at least 2 of 3 trisomies (9q, 11q, 19q) were present, hinting at hyperdiploidy. Only 1/5 incorrectly classified patients without del(13q14) harbored 3 trisomies. Biological functions (GO level 3) of predictor genes were related to protein and DNA metabolism (43%), cell growth/maintenance (27%), and cell communication (17%). The most frequent GO term for cellular component was ribosome (34%). Sixty-nine of 80 human ribosomal protein (RP) genes were represented in the predictor, and made up 33 of the top-50 probe-sets. RP genes were overexpressed in patients without del(13q14) compared to plasma cells from 7 normal donors. Expression levels of RPL12, RPLP2 and RPL13A (on chr. 9, 11 and 19) correlated with the respective chr. copy numbers. Conclusion: FISH-detected del(13q14) is associated with non-hyperdiploidy rather than defining an independent subentity of MM. Overexpression of RP-genes in malignancies has been linked to cell growth, disease progression and drug resistance. A possible pathogenetic role of the upregulation of virtually all ribosomal protein genes observed in del(13q)-negative/hyperdiploid MM clones has to be evaluated further.

Description: Introduction: The bone marrow microenvironnement (BMMe) play a significant role in the physiopathology of the multiple myeloma (MM). However, its abnormality still remains controversial. To address this question, we studied bone marrow mesenchymal stem cells (MSCs), the only long-lived cells of the BMMe. We compared, at a genomic and functional level, the MSCs isolated from patients with MM, to MSCs isolated from healthy subjects and those with monoclonal gammopathy of unknown significance (MGUS). Material and methods : Bone marrow samples from 26 MM patients, 7 MGUS patients and 11 healthy individuals were compared. The MSCs were selected by their adherence on plastic and were cultured in alpha-MEM medium + 10% SVF and antibiotics during 2 passages (primo-culture = P0 and first passage = P1). The gene expression profiling was carried out by Affymetrix GeneChip microarrays (U133 plus 2.0). The expression of interesting differentially expressed genes was validated by ELISA or qRT-PCR. The phenotype was studied by flow cytometry (CD45, CD90, CD73, CD13, CD14). The CFU-F frequencies in BM samples and in cell suspensions after P0 and P1 were studied as well as the cell productions after P0 and P1. The osteoblastic differentiation was evaluated both by alkaline phosphatase dosing and matrix mineralization quantification. We also carried out co-cultures of the MSCs with CD34+ cells to quantify their hematopoietic supportive potential. Finally XG1 and Molp-6, respectively stroma independent and stroma dependent cell lines, were co-cultured with MSCs to check the capacity of the MSCs to support malignant plasma cell growth. Results: Gene expression profile independently classified the MSCs in a normal and in a MM group. MGUS MSCs were interspersed between those 2 groups. 145 distinct genes were differentially expressed in MM and normal MSCs. Among them, 46% could be involved in tumor-microenvironment cross-talk. Known soluble factors involved in MM physiopathologic features, such as IL-6, IL-1ß, DKK1 and amphiregulin, were identified and new ones found. In particular growth and differentiation factor-15 (GDF-15), already described as a accurate biomarker of numerous tumours, was significantly overexpressed (p

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: The identification of novel tumor-associated antigens is critical for the development of immunotherapeutic strategies. Cancer-testis (CT) antigens represent attractive targets due to their restricted pattern of expression. More than 90 CT genes have been previously classified into four categories according to their expression profiles: testis-restricted (expression in testis and tumor samples only), “tissue restricted” (mRNA detected in 2 or fewer non-gametogenic tissues), “differentially expressed” (mRNA detected in three to six non-gametogenic tissues), and “ubiquitously expressed”. Among those, we previously reported that 18 CT genes were expressed by primary myeloma cells (MMC) of more than 10% of patients with multiple myeloma (MM). This study aimed at finding novel putative CT genes expressed in MM using cDNA microarray analysis and real-time RT-PCR validation. Gene expression profiles of 5 testis samples, 64 MMC, 7 normal memory B cell (MB), 7 normal bone marrow plasma cell samples and 23 normal tissue samples available on a public database were obtained using Affymetrix U133AB microarrays. Out of 45000 probe sets of Affymetrix U133 AB chips, we selected 16982 probe sets which had a “Present” Affymetrix Call in MMC of at least 6/64 patients and in 3/5 testis samples. In order to select genes with a similar pattern of expression than the known CT genes, we developed 4 independent filters making it possible to keep a high number of known CT genes while decreasing the total number of probe sets. Firstly, 2514 of 16982 probe sets had a ratio of the mean signal in MMC with a Present call / mean signal in MB 〉 2.5. Secondly, 541 of these 2514 probe sets had a Present call in less than 7 of the 23 normal tissues. Thirdly, 333 of these 541 probe sets had a ratio of the mean signal in MMC with a Present call / mean signal in MMC with an Absent call 〉 2.5. Fourthly, we removed genes whose expression profiles were discordant with different probe sets or discordant with data of the literature. The final probe set list contains 88 probe sets which include 13 of 18 known CT genes reported in MM, thus resulting in a 190-fold enrichment. The expression in 13 normal tissues and in MM samples of 21 out of these 75 putative novel CT genes was investigated by real time RT-PCR. Seven genes were ubiquitously expressed or poorly expressed in MMC samples and further deleted. According to the previously defined CT gene categories, we found one novel “testis-restricted” (TEX14), 8 “tissue-restricted” and 5 “differentially expressed” CT genes. Immunogenicity of one gene product - IGSF11 - was already demonstrated in other cancers by identifying a T-cell epitope. Two genes - NLGN4X and FAM133A - are located in X chromosome and 2 genes - CTNNA2 and FAM133A - are expressed only in brain and testis. In conclusion, by analyzing gene expression patterns with Affymetrix microarrays, we found 75 novel putative CT antigen candidates expressed in MMC of 10 to 100% of patients. Real time RT-PCR validation made it possible to confirm the CT status of 14 genes out of the 21 tested. Further studies are warranted to determine their immunogenicity.

Description: BACKGROUND. The proliferation-rate of primary myeloma cells is a strong adverse prognostic factor in various trials, but not routinely assessed, partially due to effort in obtaining it. AIM. As gene-expression profiling is increasingly considered a standard diagnostics in myeloma, we investigated the possibility to develop a prognostically relevant gene-expression based proliferation index (GPI). PATIENTS AND METHODS. Gene expression was determined by Affymetrix DNA-microarrays in 784 samples including two independent sets of 233 and 345 CD138-purified myeloma cells from previously untreated patients. The GPI was derived by selecting genes associated with proliferation (in terms of gene ontology) differentially expressed in proliferating malignant (human myeloma cell lines) and benign (plasmablastic) cells compared to non-proliferating, non-malignant cells (normal plasma cells and memory B-cells). The GPI comprises the sum of the expression values of 50 genes (ASPM, AURKA, AURKB, BIRC5, BRCA1, BUB1, BUB1B, CCNA2, CCNB1, CCNB2, CDC2, CDC20, CDC25C, CDC6, CDCA8, CDKN3, CEP55, CHEK1, CKS1B, CKS2, DLG7, ESPL1, GINS1, GTSE1, KIAA1794, KIF11, KIF15, KIF20A, KIF2C, KNTC2, MAD2L1, MCM10, MCM6, MKI67, NCAPD3, NCAPG, NCAPG2, NEK2, NPM1, PAK3, PCNA, PGAM1, PLK4, PTTG1, RACGAP1, SMC2, SPAG5. STIL, TPX2, ZWINT). Proliferation of primary myeloma cells was assessed by propidium iodinestaining (n=67). Chromosomal aberrations were assessed by comprehensive iFISH using a set of probes for the chromosomal regions 1q21, 6q21, 8p21, 9q34, 11q23, 11q13, 13q14.3, 14q32, 15q22, 17p13, 19q13, 22q11, as well as the translocations t(4;14)(p16.3;q32.3) and t(11;14)(q13;q32.3). RESULTS. In the two groups, 39 and 32 percent of primary myeloma cells show a GPI above the median plus three standard deviations of normal bone marrow plasma cells, respectively. The GPI is significantly higher in advanced- compared to early-stage myeloma (P=.001) and in patients harboring a gain of 1q21 (n=95, P

Description: The NF-κB pathway is involved in the physiological regulation of cell proliferation in many cell types as well as in the resistance of several malignancies to cell death. The pathophysiologic basis for multiple myeloma (MM) has been attributed to the dysregulation of various paracrine or autocrine growth factor loops and to perturbations in several signal transduction pathways including IKK/NF-κB. The aim of the present study was to investigate the effect of a pharmaceutical IKK2 inhibitor (IKK2-I), the anilinopyrimidine derivative AS602868 (Serono International SA), on the in vitro growth of human MM cell lines (HMCL) and primary MM cells. We evaluated the effect of AS602868 on the proliferation and the survival of 12 IL-6-dependent HCML and 2 autonomously growing HCML as well as on the survival of total bone marrow mononuclear cells from patients with newly diagnosed MM (n = 6) or with relapsing MM (n = 7). Results show that using HMCL or primary MM cells, AS602868 induces a clear dose-dependent inhibition of MM cell growth (the 50% inhibitory concentration (IC50) ranging from 0.28 to 8.3 μM, mean IC50 = 2.6 μM on HCML). It was shown using HMCL that the growth inhibition induced by AS602868 is the result of a simultaneous induction of apoptosis and inhibition of the cell cycle progression. Importantly, AS602868 does not alter the survival of other bone marrow mononuclear cells (CD138−) co-cultured with primary MM (CD138+) cells except on CD34+ hematopoietic stem cells. Interestingly, using gene expression profiling with Affymetrix microarrays on 13 HMCL, we show that the resistance (high IC50) to AS602868 inhibitor is strongly correlated to APRIL gene expression (r =.7603, p