ALBERT

Description: Abstract 1810 Background. The efforts to characterize the genomic background of Multiple Myeloma (MM) and to correctly stratify at diagnosis patients (pts) subsequently treated with novel drugs thoroughly meet the clinical requirement to handle highly powerful prognostic factors. On the other hand, the information obtained from each pts needs to be carefully interpreted, by considering the genomic background as a whole, since each aberration might represent the different expression of a common deregulated pathway. The TP53 deletion on chromosome (chr) 17p13 represents one of the genomic aberration most significantly associated with poor outcome in MM. Overall, the TP53 tumor-suppressor gene inactivation has a central role in the tumorigenesis, since p53 is the most frequently mutated protein in human cancers. The p53 pathway silencing might pass also through changes in the expression level or activation of p53 itself, regulated by several specific inhibitors and/or activators. One of the most potent inhibitor of p53 is MDM4, which is critical for control of p53 activity during response to stress and is often amplified in several types of tumors. The MDM4 locus is located on chr1q32.1, a region frequently amplified in MM. Aim. Since in MM the TP53 deletion on chr 17p13 is reported with low frequencies (7 to 11% at diagnosis) and since the vast majority of hemizygous TP53 deleted pts do not harbor mutations on the allele not affected by the deletion, we sought to investigate the frequency and the prognostic role of TP53 deletion and/or MDM4 amplification in newly diagnosed MM pts, assuming that both of these chromosomal aberrations might contribute to impaired p53 function. All pts have been treated with bortezomib-thalidomide-dexamethasone (VTD) as induction therapy prior to, and as consolidation after, double autologous stem-cell transplantation (ASCT). Pts and methods. Eighty-nine pts treated with VTD incorporated into double ASCT were analyzed at diagnosis by means of unpaired analysis of copy number alterations (CNA) (Affymetrix 6.0 SNP array), gene expression profile (GEP) (Affymetrix U133 Plus2.0 array) and Real-time PCR; the genomic results have been analyzed in the clinical context. Results. The CNA analysis showed that 9/89 pts (10%) carried a minimal deleted region of 482 Kb on chr17p13.1, including TP53, and that 27/89 pts (30,3%) carried a minimal amplified region of 1.1 Mb on chr1q32.1 including MDM4. Pts were stratified into two subgroups according to the presence of amplified MDM4 and/or deleted TP53 (group A, 34 pts, or 38%) or the absence of both these abnormalities (group B, 55 pts, or 62%). Baseline clinical characteristics were homogeneous, except for a higher rate of IgA isotype in group A. On the contrary, groups A and B were clearly imbalanced with respect to the genomic background: indeed, the t(4,14) frequency, as well as the average number of CNAs were overall higher in group A as compared to group B (38% vs. 14% t(4;14) positive, p=0.0002 and 165 vs 103 CNAs, p = 0.03). A GEP comparison among the two groups of pts highlighted an overall deregulation of pathways related to the cell cycle, the DNA damage repair and the cell adhesion and cytoskeleton remodeling, due to the differentiated expression of 627 probes-set in group A vs group B pts (false discovery rate

Description: Background. Array-based technology has been showing a great impact on clinical cancer cytogenetic, especially on genetically heterogeneous disease, such as MM, where relevant lesions might be the hallmarks of different patients’ subgroups, thus becoming of clinical relevance as well. We present herein the results of a molecular sub-study of the EMN02 phase III study (EMN02_HOVON95) which was designed to compare consolidation therapy Bortezomib, Melphalan and Prednisone versus upfront autologous stem cell transplantation, both applied after induction therapy with bortezomib-cyclophosphamide-dexamethasone (VCD). The sub-study was aimed at developing a comprehensive, high throughput genomic profile to be used to stratify uniformly treated MM patients according to their genomic background at baseline and to perform correlations with response to induction therapy. Patients and methods. Data obtained from 170 patients who consecutively entered the study and received three 21-day cycles of VCD induction therapy were analyzed. Baseline patients’ characteristics, including cytogenetic abnormalities, were comparable with those of 717 patients enrolled by participating Italian centres. Highly purified CD138+ bone marrow plasma cells were profiled by SNPs array (Affymetrix 6.0 and CytoScanHD® chip). ChAS (Affymetrix) and Nexus Copy NumberTM 7.5 (Biodiscovery) software were used to perform Copy Number Alterations (CNAs) analyses and clinical correlations, respectively. Results. After induction therapy, 66 out of 170 (38.8%) patients achieved a very good partial response (VGPR) or better, including 15 (8,8%) who attained a complete response (CR). On the contrary, 104/170 (61.1%) patients achieved

Description: Background Although remarkable advances have been achieved in MM therapy, mainly thanks to the introduction of novel-agent-based regimens, the disease remains incurable. Neoplastic CD138+ plasma cells are the hallmark of MM: both their expansion in the bone marrow (BM) and the production of monoclonal immunoglobulin (Ig) are responsible for the clinical manifestation of the disease. However, the existence of a Myeloma Propagating Cells (MPCs) has been proposed as a major cause of MM drug-resistance, leading to relapse. Several studies support the hypothesis that MPCs are phenotypically close to memory B cells residing in the CD138- compartment; however, very little is known concerning their molecular characteristics. Here we present an extensive molecular characterization of clonotypic CD19+ B cells clones obtained from newly diagnosed MM patients (pts), in order to recognize biological pathways possibly explaining the malignant clone’s persistence. Methods CD138+ and CD138- cell fractions were collected from BM and peripheral blood (PBL) of 50 newly diagnosed MM pts. CD19+ B cell and CD27+ memory B cell populations were isolated from CD138- cell fraction. Clonogenic assays were performed by plating cell fractions obtained from RPMI-8226 and NCI-H929 cell lines. The molecular characterization included: IgH gene rearrangement Sanger sequencing; analysis of the whole spectrum of genomic aberrations and gene expression profiling, by Affymetrix 6.0 SNPs array and HG-U133 Plus 2.0 microarray, respectively. Results Clonogenic assays showed that CD138- cells, plated on conditioned media, were able to form colonies after two weeks of culture more efficiently than CD138+ cells. By VDJ gene rearrangement sequencing, a clonal relationship between the CD138+ clone and the memory B ones was confirmed. SNPs arrays showed that both BM and PBL CD138+ cell fractions carried exactly the same genomic macro-alterations. On the contrary, in the CD138-19+27+ cell fractions from BM and PBL any macro-alteration was detected, whereas several micro-alterations (median number per sample: 32 amplifications and 16 losses, range: 8-122 Kb, average markers per region: 50) unique of the memory B cells clone were highlighted. An enrichment analysis revealed the involvement of genes affected by losses (17 genes) in both DNA repair mechanisms and transcriptional regulation and the involvement of genes affected by gains (46 genes) in both the negative regulation of apoptosis and the angiogenesis. Interestingly, KRAS, WWOX and XIAP genes, renown to be involved in MM pathogenesis, are located in the amplified regions in the immature cells. Moreover, several LOH regions were described, which covered at least 106 tumor suppressor genes involved in MM and leukemia (including TP53, CDKN2C and RASSF1A). Transcriptome profiles analysis of the CD19+ cell fractions highlighted pathways suggesting a possible involvement of immature cells in MM pathogenesis. The gene expression profiles of 20 MM CD19+ cells samples (12 from PBL, 8 from BM) were compared both to their normal counterpart and to the mature CD138+ cell fractions. In particular, unsupervised analysis by hierarchical clustering discriminated the differential expression of 11480 and 11360 probes in the PBL and BM CD19+ clones, respectively (2; FDR=0,05; p

Description: Abstract 2901 Hypoxia-inducible factor-1 alpha (HIF1 α) is a transcription factor that plays a critical role in survival and angiogenesis. In solid tumors, elevated expression of HIF-1 α, in response to hypoxia or activation of growth factor pathways, is associated with tumor proliferation, metastasis, and drug resistance and correlated with poor prognosis. In contrast to solid tumors, the role of HIF1 α in hematological malignancies is not completely known. In particular in multiple myeloma (MM) HIF1 α has been suggested to be constitutively expressed and HIF1 α knockdown cell lines have shown higher sensitivity to standard chemotherapy, suggesting a role in the pathophysiology of MM. In the present study, we explored the effect of EZN2968, an antisense oligonucleotide against HIF1 α, as a molecular target in MM. We showed, using real time PCR, and Western blotting analysis, that the expression of HIF1 α in several MM cell lines (MM1S, U266, OPM2, RPMI8226) is detectable under conditions of normoxia or hypoxia and is increased in the presence of growth stimuli (IL-6 and stroma cells). The immunofluorescence analysis suggested that the protein is ubiquitously present in both the cytosol and nucleus. To evaluate the specificity of the oligonucleotide for the target, we tested whether EZN2968 was able to induce a selective and stable down-modulation of HIF1 α mRNA and protein expression. We confirmed that the downmodulation was lasting in a long term culture experiment (up to 96 hours) either in normoxic or hypoxic conditions, and did not affect the expression of other family members of hypoxia inducible transcription factors (HIF2 α). We next explored the effects of EZN-2968 on the growth and survival of MM cells. Using an MTT colorimetric survival assay, we showed that, after 48 hours of culture in the presence of the HIF1 α inhibitor (20μM), MM1.S and U266 cell lines exhibited a reduction of 30% of viability compared to untreated cells, while RPMI8226 of 15%. AnnexinV/PI staining revealed that EZN-2968 (20μM) increased, after 48 hours of culture, the percentage of PI+ cells compared to the control, suggesting a disruption on membrane permeability. In addition, immunoblotting revealed PARP cleavage as early as 24 hours. Evaluation of cell cycle profile, by flow cytometric analysis, showed an increase of the sub-G0/G1 population from 3.5% to 30 %, after 48 hour of exposure to EZN-2968. To evaluate if the impact on cell viability was irreversible, we performed a cell death commitment assays. MM1S cells were incubated with EZN2968 (20 μM) for 24 to 96 hours, following incubation in drug-free medium for additional 24 to 72 hours. MTT colorimetric survival assay showed that EZN-2968 treatment for as early as 24h resulted in commitment to death in all cell lines tested. To evaluate the effect of microenvironment, MM cells treated with EZN2968 were exposed to IL-6 and stroma cells for additional 24 hours. EZN2968 overcame the proliferative effect induced by cytokines. We next evaluated the impact of EZN-2968 on purified CD138+ cells from MM patients with advanced MM. MTT colorimetric survival assay showed a reduction of cells viability of 30% after 24 hours of incubation. In addition we observed a low sensitivity of PBMCs and CD34+cells, derived from healthy donors, to EZN-2968 treatment suggesting that EZN-2968 has selective in vitro activity against MM cells. Evaluation of gene expression profiling modulation induced by EZN 2968 is on going. In summary, our results suggests that the inhibition of HIF1 α activity can be used as an attractive therapeutic target for MM patients and provide the rationale for clinical evaluation of HIF inhibitors. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 861 The high rate of complete response (CR) effected by novel agents as induction and consolidation/maintenance therapy in multiple myeloma (MM) has renewed interest in the evaluation of minimal residual disease (MRD) after these combined treatment strategies. For this purpose, a useful molecular marker is represented by the immunoglobulin heavy-chain (IgH) rearrangement which can be carefully detected by means of polymerase chain reaction (PCR). We designed a molecular sub-study to the phase 3 GIMEMA trial of bortezomib-thalidomide-dexamethasone (VTD) vs. thalidomide-dexamethasone (TD) incorporated into double autologous stem cell transplantation (ASCT) for newly diagnosed MM. By study design, patients randomized at diagnosis to VTD or TD as induction therapy before ASCT received two 35-day cycles of consolidation therapy with VTD or TD starting 3 months after ASCT(s), independently from prior response. Doses of study drugs were the following: V, 1.3 mg/m2 once weekly; T, 100 mg/d through d 1 to 70; D, 320 mg/cycle. Aim of the molecular study was to compare the activity of VTD consolidation with that of TD by qualitative and quantitative PCR analysis. Patients who were planned to receive consolidation with either VTD or TD and who were in confirmed CR or near CR before the start of consolidation therapy were eligible for entry into the molecular study. A qualitative and quantitative PCR analysis with patient-specific primers was performed on bone marrow samples collected at day 0 and at day +70 after the two preplanned cycles of either VTD or TD. At this time, 84 patients were included in the study; of these, 67 patients were analyzed for MRD detection based on the availability of: 1) a molecular marker (e.g. IgH rearrangement) assessed at diagnosis; 2) both pre- and post-consolidation bone marrow samples. According to randomization at diagnosis, 35 out of 67 patients received VTD consolidation, while TD consolidation was given to 32 patients. Qualitative PCR analysis performed on bone marrow samples collected at day 0 showed that MRD before the start of VTD consolidation therapy was undetectable in 15 out of 35 patients, or 43%; the corresponding value before the start of TD consolidation was 37.5% (or 12 out of 32 patients). In comparison with the pre-consolidation status, analysis of bone marrow samples collected at day +70 revealed an upgrade in PCR-negativity from 37.5% to 52% with TD consolidation and from 43% to 67% with VTD consolidation (p=0.05, according to McNemar's test). By using the Fisher's exact test, the proportion of patients with post-consolidation PCR-negativity was significantly higher with VTD vs. TD consolidation (p=0.05). These data were furtherly extended by a Real-time quantitative PCR analysis, which could be performed in 45 out of the 67 patients initially included in the study (22 in the TD arm and 23 in VTD). In comparison with residual tumor mass at day 0, quantitative PCR analysis of bone marrow samples collected at day +70 revealed a median 1 log reduction in tumor burden with TD consolidation vs. a median 5 log reduction with VTD consolidation. By using the Wilcoxon test, the overall reduction in residual tumor burden effected by VTD consolidation therapy was statistically significant (p=0.05). In conclusion, in comparison with TD consolidation, two 35-day cycles of consolidation therapy with VTD following double ASCT significantly increased the rate of molecular remissions and significantly reduced the burden of residual myeloma cells persisting after ASCT. Superior activity of VTD over TD consolidation was retained although prior exposure to the same regimen given as induction therapy in preparation for ASCT. Additional data on molecular follow-up analyses of bone marrow samples collected after day +70 and comparisons of the prognostic relevance of PCR-negativity with PCR-positivity, as well as of PCR-negativity induced by VTD with that induced by TD will be presented during the meeting. Supported by: Progetto di Ricerca Finalizzata Orientata (to M.C), BolognAIL, Fondazione del Monte di Bologna e Ravenna. Disclosures: Baccarani: NOVARTIS: Honoraria; BRISTOL MYERS SQUIBB: Honoraria. Cavo:jansen-cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, no; celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, no; novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, no.

Description: Introduction. The role of TP53 tumor-suppressor gene in mediating cellular basic tumor suppressive mechanisms is crucial: its utmost importance is underscored by a 50% mutational rate among most human cancers. In MM, TP53 is rarely reported as being affected either by deletions or mutations, even if chromosome (chr) 17p13 copy number (CN) loss defines a samples subgroup with a particularly poor prognosis. Here we aim at retrospectively analyzing by Next Generation Sequencing (NGS) the TP53gene inactivation in newly diagnosed MM, assessing the mutational events’ frequency and their clinical impact. Samples and methods. A cohort of 92 MM, receiving up-front velcade (vel)-based regimens, followed by autologous stem cell transplantation, was included in this molecular study. Response to therapy was evaluated according to the IMWG criteria. DNA was obtained from CD138+ highly purified plasma cells and profiled by SNPs array (Affymetrix). TP53gene mutational status was analysed employing an amplicon-targeted deep NGS approach (GSJ, 454 Life Sciences). A novel upcoming NGS data analysis software was employed to detect variants from raw sequencing data and to compare them to IARC database. As a validation, 30 randomly chosen samples have been re-sequenced. p53 activity was evaluated by p-p53 and p53 immunoblot assays. Results. Ultra-deep TP53coding sequence analysis (median depth: 1060) highlighted the presence of a median of 1.8 variants per samples in 73/92 (79%) MM: a total of 131 nucleotide substitutions emerged, with variants allele frequencies (VAF) ranging from 1 to 99% (median 1.3%). The polymorphism c.215C〉G (validated as SNP1042522) was the most prevalent detected variant, recurring in ~40% of the analysed samples. TP53variants were assessed according to the SIFT classification, in order to identify either neutral or deleterious mutations, on the basis of their predicted effect at amino acid level. 36 cases carried 42 deleterious variants (VAF=1.2%): they were mostly missense substitutions affecting mainly the DNA binding domain; most deleterious variants (67%) were predicted as non-functional at protein level. 9 samples carried 21 neutral variants (VAF=1.1%), which were mostly missense mutations, again mainly affecting the DNA binding domain. Finally, 28 samples carried 68 SIFT unclassified variants – among which several polymorphisms were counted – (VAF=94.6%). Of these, 29 were silent substitutions affecting the DNA-binding domain, 32 were missense variants affecting the SH3-like/Proline- rich domain. To assess the genomic background of deep-sequenced MM, SNPs arrays were performed on 83/92 samples: 14/83 (17%) carried a TP53 CN loss (1N) on chr17p13.1. The incidence of TP53 hemizygous deletion was higher among cases carrying deleterious mutations, as compared to cases carrying either neutral or unclassified mutations (25%, 12,5% and 12%, respectively). 2/17 non-mutated samples carried TP53 hemizygous deletion, as well. Interestingly, an Rb1 tumour suppressor gene CN loss on chr13q14.2 significantly characterized samples carrying either mutated (deleterious variant) or deleted TP53 (p=0.006). The activation state of p53 was evaluated by p-p53 immunoblotting assay, showing the absence of phosphorilation either in 3 TP53 deleted cases, or in 3 cases carrying deleterious variants; on the contrary p-p53 was observed in 4 non-mutated cases. Finally, clinical correlations were performed on 81 MM, for whom clinical data were available, showing that the presence of either TP53 hemizygous deletions or at least one TP53 deleterious variant was more likely associated to the response to vel-based induction therapy (frequencies of ≤partial response were 35% and 64% among samples carrying or not impaired TP53, respectively; p=0.05). Conversely, the frequency of progression events was slightly higher among MM carrying impaired TP53 (69% vs. 50%). Conclusions. The analysis by ultra-deep next generation sequencing of TP53 coding sequence in a cohort of newly diagnosed MM highlighted an unexpected, still un-explored, high rate of TP53 variants, recurring with a wide range of frequencies among samples. The impact of TP53 damage on MM disease course has to be confirmed in randomized clinical trial. Acknowledgments: Roche Diagnostics for supporting us in the realization of this project; ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Cavo:Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; BMS: Consultancy, Honoraria; Millenium: Consultancy, Honoraria; Onyx: Honoraria. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy; Roche: Honoraria.

Description: Abstract 574 Multiple Myeloma (MM) is an incurable hematologic malignancy characterized by the accumulation of malignant plasma cells. Dysregulation of MYC by rearrangement or translocation are common somatic events described either in early or late stage of the disease, and transcriptional profiling of MYC pathway activation is observed in more than 60% of MM cell lines. Hypoxia Inducible Factor-1α (HIF-1α) overexpression has been described in several MM cell lines and in about 30% of MM patients samples. In solid tumours, deregulation of c-MYC has been associated with HIF-1α upregulation: under physiologic conditions HIF-1α inhibits c-MYC activity by direct interaction and stimulation of a proteasome-dependent pathway. In the present study we explored the interaction between c-MYC and HIF-1α in a panel of MM cell lines (MM1S, OPM2, RPMI8226, U266). We had previously shown that treatment with EZN2968, a locked nucleic acid antisense oligonucleotide directed against HIF-1α, resulted in a significantly reduction of HIF-1α protein level after 24h of incubation. The reduction of HIF-1α was specific and lasted over 96h. To confirm the inhibition of HIF-1α activity, MM1S cells were treated with EZN2968 for 24h, lysed, co-precipitated with p300, and incubated with anti-HIF-1α antibody. We showed that HIF-1α was no longer associated p300 in EZN-treated compared to untreated samples, suggesting an inhibitory effect of HIF-1α activity. We next observed that treatment with EZN2968 induced a progressive accumulation of cells in S-phase with concomitant reduction of G2/M phase. By western blot analysis, we observed that p21 and p27, cell cycle check points negatively regulated by c-MYC, were up-regulated in treated samples. We further verified the effect of HIF-1α inhibition on c-MYC protein level by western blotting analysis. After treatment with EZN2968, c-MYC protein expression was reduced in a time dependent manner (c-MYC protein was almost undetectable after 72h of incubation), suggesting that c-MYC protein level is associated with inhibition of HIF-1α. To examine whether HIF-1α and c-MYC regulate each other promoter activity, we performed Chromatin Immunoprecipitation (ChIP) assays with HIF-1α or c-MYC antibodies. HIF-1A and MYC promoter amplification signals, were present in the controls samples, and increased after EZN2968 exposure, suggesting that these proteins can play a direct role in regulating each other's activity. Recently, it has been shown that SIRT1, a transcription factor involved in a development, cellular stress responses, and metabolism, can modulate HIF-1α and c-MYC activity. By Immunoblotting assay, we observed that SIRT1 physically interacts with both proteins and that, after 24h of exposure to EZN2968, c-MYC and HIF-1α were no longer associated to SIRT1. These results were also confirmed at the transcriptional level, by ChIP assay using an anti-SIRT1 antibody. After 24h of treatment with EZN2968, we observed a significant increase of HIF-1A and MYC promoter amplification signals in treated compared to untreated samples, suggesting that SIRT1 recruitment at both promoters is dependent on HIF inhibition. We showed that in MM cell lines the expression of HIF-1α and c-MYC are linked and mediated by SIRT1 deacetylase protein. The data suggests a new regulatory mechanism for controlling c-MYC and HIF-1α activity by SIRT1. The identification of a HIF-c- MYC–SIRT 1 interaction in MM cell lines suggests a novel therapeutic target for MM patients. Disclosures: Cavo: Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Millennium Pharmaceuticals: Consultancy.

Description: Abstract 3935 Background The p53 tumor suppressor pathway is tightly kept in check, or completely silenced in cancer cells. A potent inhibitor of p53 is represented by MDM4, which is critical for the control of p53 activity during the response to stress and is often amplified in several types of cancer. TP53 mutations are rare in newly diagnosed MM, while occur more frequently as late event in the course of the disease and are related to survival. Recently, the adverse prognostic impact of chr. 1q amplification, described in almost 40% of newly diagnosed MM pts, has been reported. The minimal amplified region on chr. 1q harbors MDM4. Since both del(17p) and amp(1q) identify a subgroup of high-risk MM pts, even when the novel agents are part of up-front treatment strategy, we molecularly analyzed a subgroup of MM patients treated with bortezomib-thalidomide-dexamethasone (VTD) incorporated into autologous stem cell transplantation, in order to investigate mechanisms which might be activated in myeloma plasma cells to direct and/or indirect limit the p53 function. Methods Thirty eight pts treated with VTD incorporated into autologous stem cell transplantation were analysed by means of gene expression profile (Affymetrix U133 Plus2.0 array) and unpaired analysis of copy number alterations (CNA) (Affymetrix 6.0 SNP array). Both GEP and SNP arrays experiments were performed on highly purified CD138+ bone marrow plasma cells obtained at diagnosis from each pts. The presence of CNAs in chr.1 and 17 was evaluated to identify pts carrying amp(1q) and del(17p). Results Eighteen out of 38 pts (42%) carried a minimal amplification region of 1,1 Mb on chr.1q, which harbors MDM4. Five out of 38 pts (13%) carried a minimal deletion region of 482 Kb on chr.17, which harbors TP53. To explore the involvement of the p53 pathway in MM, pts were stratified according to the presence of amplified MDM4 and/or deleted p53 (group A, 18 pts) or the absence of both these abnormalities (group B, 20 pts). Baseline clinical characteristics were homogeneous, except for a higher rate of ≥ 3 bone lesions in pts carrying amplified MDM4 and/or deleted p53. The rate of best complete or near complete response was 89% in group A and 75% in group B. With a median follow-up of 36 months, the risk of relapse or progression was 50% for pts in group A and 25% for those in group B. The average number of aberrations per group was overall higher in group A as compared to group B (165 vs. 103 CNAs, p =0.03); indeed, the presence of amplified MDM4 and/or deleted p53 was significantly associated with a list of 95 CNAs (clustered on chr. 1, 2, 6, 8, 11, 13, 16 and 18), which included del16q (with a minimal area of deletion including WWOX), observed in 39% vs. 5% cases from group A and B, respectively (p

Description: Abstract 805FN2 Background. Achievement of CR is generally associated with improved clinical outcomes for patients (pts) with MM and represents a primary endpoint of current clinical trials. The GIMEMA Italian Myeloma Network designed a phase 3 study to demonstrate that the triplet VTD regimen was superior over a doublet such as thalidomide-dexamethasone (TD) as induction therapy prior to double ASCT for newly diagnosed MM. On an intention-to-treat basis, the rate of complete or near complete response (CR/nCR) was 31% for the 236 pts on VTD induction therapy, while it was 11% (p

Description: Background Multiple Myeloma (MM) is a genetically complex disease. In MM, prevalent chromosomal numerical and structural aberrations are used to cluster patients (pts) into subtypes, frequently displaying distinct clinical behaviors. Among the umpteen chromosomal aberrations described so far in MM, the TP53 deletion on chromosome (chr) 17p13 defines a pts group with a particularly poor prognosis, even if its prevalence at diagnosis is quite low. Overall, the TP53 tumor-suppressor gene is mutated or functionally inactivated in most human cancers. Tumors that retain wild-type p53 frequently harbor defects either in the pathways that allow for p53 stabilization in response to stress, or in the effectors of p53 apoptotic activity. One of the most potent inhibitor of p53 is MDM4, which is often amplified in several types of tumors. The MDM4 locus is located on chr1q32.1, a region frequently amplified in MM. Aim Aim of the present work was to investigate the possibility that both TP53 deletion (del) and MDM4 amplification (amp) might affect similar pathways, thus finally leading to a poor prognosis MM pts carrying at diagnosis at least one of them. Pts and methods Eighty-nine pts treated with bortezomib-thalidomide-dexamethasone (VTD) as induction therapy prior to, and as consolidation after, double ASCT were analyzed at diagnosis by means of unpaired analysis of copy number alterations (CNA) (Affymetrix 6.0 SNP array) and gene expression profile (GEP) (Affymetrix U133 Plus2.0 array); in twenty-one pts carrying MDM4 amplification and for whom samples were available, the p53 activity was explored both by analyzing the TP53 mutational rate by deep sequencing (Roche GS Junior 454) and by evaluating the p53 activity by immunoblotting assays of MDM4, p-p53 and p53. Genomic results were evaluated in the clinical context. Results The CNA analysis showed a 482 Kb minimal deleted region on chr17p13, including TP53, in 8/89 pts (8,9%) and a 1.1 Mb minimal amplified region on chr1q32.1 including MDM4 in 27/89 pts (30,3%). The GEP analysis performed at diagnosis in TP53 del and MDM4 amp pts generated two lists, including genes either differentially expressed among pts carrying or not amplified MDM4 (5840 probes sets, corresponding to 3841 genes, p