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Supplementary Tables 1 and 2. Supplementary Table 1 describes Summary of FDG PET/CT positivity criteria Supplementary Table 2 contains Univariate analysis of baseline and post-treatment variables adversely affecting progression-free survival and overall survival

Purpose: To evaluate the role of 18F-FDG PET/CT in 282 symptomatic multiple myeloma patients treated up-front between 2002 and 2012.Experimental Design: All patients were studied by PET/CT at baseline, during posttreatment follow-up, and at the time of relapse. Their median duration of follow-up was 67 months.Results: Forty-two percent of the patients at diagnosis had >3 focal lesions, and in 50% SUVmax was >4.2; extramedullary disease was present in 5%. On multivariate analysis, ISS stage 3, SUVmax >4.2, and failure to achieve best complete response (CR) were the leading factors independently associated with shorter progression-free survival (PFS) and overall survival (OS). These 3 variables were used to construct a prognostic scoring system based on the number of risk factors. After treatment, PET/CT negativity (PET-neg) was observed in 70% of patients, whereas conventionally defined CR was achieved in 53%. Attainment of PET-neg favorably influenced PFS and OS. PET-neg was an independent predictor of prolonged PFS and OS for patients with conventionally defined CR. Sixty-three percent of patients experienced relapse or progression; in 12%, skeletal progression was exclusively detected by systematic PET/CT performed during follow-up. A multivariate analysis revealed that persistence of SUVmax >4.2 following first-line treatment was independently associated with exclusive PET/CT progression.Conclusions: PET/CT combined with ISS stage and achievement or not of CR on first-line therapy sorted patients into different prognostic groups. PET/CT led to a more careful evaluation of CR. Finally, in patients with persistent high glucose metabolism after first-line treatment, PET/CT can be recommended during follow-up, to screen for otherwise unidentifiable progression. Clin Cancer Res; 21(19); 4384–90. ©2015 AACR.

Purpose: To evaluate the role of 18F-FDG PET/CT in 282 symptomatic multiple myeloma patients treated up-front between 2002 and 2012. Experimental Design: All patients were studied by PET/CT at baseline, during posttreatment follow-up, and at the time of relapse. Their median duration of follow-up was 67 months. Results: Forty-two percent of the patients at diagnosis had >3 focal lesions, and in 50% SUVmax was >4.2; extramedullary disease was present in 5%. On multivariate analysis, ISS stage 3, SUVmax >4.2, and failure to achieve best complete response (CR) were the leading factors independently associated with shorter progression-free survival (PFS) and overall survival (OS). These 3 variables were used to construct a prognostic scoring system based on the number of risk factors. After treatment, PET/CT negativity (PET-neg) was observed in 70% of patients, whereas conventionally defined CR was achieved in 53%. Attainment of PET-neg favorably influenced PFS and OS. PET-neg was an independent predictor of prolonged PFS and OS for patients with conventionally defined CR. Sixty-three percent of patients experienced relapse or progression; in 12%, skeletal progression was exclusively detected by systematic PET/CT performed during follow-up. A multivariate analysis revealed that persistence of SUVmax >4.2 following first-line treatment was independently associated with exclusive PET/CT progression. Conclusions: PET/CT combined with ISS stage and achievement or not of CR on first-line therapy sorted patients into different prognostic groups. PET/CT led to a more careful evaluation of CR. Finally, in patients with persistent high glucose metabolism after first-line treatment, PET/CT can be recommended during follow-up, to screen for otherwise unidentifiable progression. Clin Cancer Res; 21(19); 4384–90. ©2015 AACR.

Introduction: plasticity is a hallmark of Multiple Myeloma (MM) clone(s), where both quiescent MM cells, acting as tumor-initiating cells, and proliferative MM cells, able to invade and disseminate, might co-exist. Aim of the study is to stratify patients (pts) according to both the level of chromosomal instability (CIN) and their plasma cells (PCs) differentiation stages, and to evaluate the impact of this stratification on the disease outcome. Patients & Methods: 145 newly diagnosed MM pts were included in the study. Whole-genome copy number alterations (CNAs) were analysed by SNP array both in the CD138+PCs and CD19+B-cells. In each pts, both the CD138+/CD38high PCs and CD19+B-cells compartments were characterized by 6-color multi-parameter flow cytometry analysis, combining CD138-PE, CD38-PE-Cy7, CD20-APC, CD19-APC-Cy7, CD27-FITC, CD45-FITC, CD28-APC, CD44-FITC, CD54-APC, CD81-PerCP-Cy5.5, CD56-APC. Results: According to the CD138+ PCs’ CIN, as described both by the total amount of CNAs and by the portion of genome changed (GC), three major pts subgroups were identified: the most representative, including 21/64 pts, was characterized by a higher CIN (median CNAs: 550 %GC ≥ 25) as compared to the others (intermediate and low CIN, median CNAs: 220 10≤ GC%≥25). Hyperdyploidy, but also high-risk features [i.e. 17p del (TP53)] mainly characterized CD138+ PCs with high CIN. On the contrary, in the same pts, the CD19+B-cells display a quite simple karyotype with very few microalterations (>50kb), mostly involved in the signal transduction pathway (loss on KRAS, chr12p12.1 and on SIRPB1, chr20p13). According to the the co-expression of CD19/CD81, describing the MM clone(s) differentiation status, PCs with a high level of CIN resulted more mature CD19-/CD81-. Both the high expression of CD28 and CD44 and the reduced expression of CD20, CD27 and CD45 confirmed the advanced differentiation status. Finally, although baseline clinical features of pts with more mature, genomically instable PCs, are associated to bad prognosis (e.g. PET lesions, k/l ratio, ISS III, β2-microglobulin; p Conclusion: High level of genomic complexity correlates with advanced PCs differentiation stages, and this is lastly associated with a prevalence of poor prognosis features. Both CIN and phenotypic pliancy represent important, yet poorly defined, mechanisms by which MM clone(s) accelerate their own evolution and survival. Acknowledgements: AIRC, AIL, HARMONY, Berlucchi Citation Format: Marina Martello, Rosalinda Termini, Enrica Borsi, Vincenza Solli, Andrea Poletti, Lucia Pantani, Serena Rocchi, Katia Mancuso, Elena Zamagni, Paola Tacchetti, Mario Arpinati, Gabriella Chirumbolo, Nicoletta Testoni, Giulia Marzocchi, Giovanni Martinelli, Michele Cavo, Carolina Terragna. Higher levels of genomic complexity correlates with an advanced plasma cell differentiation status in newly diagnosed multiple myeloma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 473.

BACKGROUND MM patients (pts) are characterized by a prevalence of aneuploidies and Copy Number Alterations (CNAs) broadly scattered over the whole genome. Along with translocations and/or single nucleotide variants, they account for the MM-distinctive genomic heterogeneity, which characterizes the onset of symptomatic MM and identifies each pts. AIM. To deeply explore the genomic landscape of newly diagnosed, homogeneously treated MM pts, to assess the prognostic relevance of whole genome aneuploidies, CNAs, and structural aberrations. PATIENTS AND METHODS 344 highly purified BM CD138+ samples were profiled by SNPs array. R scripts were employed to analyse genomic data. Pts, enrolled in the EMN02 phase III trial, were randomized to receive VMP (121 pts) or high-dose MEL and autologous stem cell transplantation (223 pts), after bortezomib-based induction. At a median follow-up of 46 months (m), the estimated PFS and OS rates were 55% and 83%, respectively. RESULTS To evaluate the contribution of each genomic variables to the overall pts variability, a Principal Component Analysis of the whole genome’s numerical and structural aberrations was performed, highlighting that a “common ground” of at least 4 chromosomal aberrations (hyperdiploidy, H, 13qCN loss, IgH-t and 1qCN gains) was sufficient to describe and resume the MM overall genomic heterogeneity. Therefore, since 13qCN loss and 1qCN gains co-segregate, 3 pts sub-types might be identified, partially overlapping, even though well-defined by the presence of either H, or IgH-t or 13qCN loss and 1qCN gain. Outcomes of the latter MM sub-type was explored. Pts carrying 13qCN loss and 1qCN gain were characterized by the presence of several small deletions in MM-critical genes (YAP1, TRAF3, CYLD), and by the markedly de-regulated expression of CCND2 and CCND1 (as evaluated by the analysis of CoMMpass-derived RNAseq expression dataset). Their 46-m PFS and OS estimates were shorter as compared to that of patients carrying either just one or any of these CNAs (PFS: 39%, 44% and 73%, respectively; p=0.0001. OS: 68%, 83% AND 91%; p=0.0001). PFS and OS hazard ratios (HR) of pts carrying 13qCN loss and 1qCN gain (PFS: 1.76; OS: 2.24) were comparable to that of pts carrying either del(17p) (PFS: 2.1; OS: 2.1) or t(4;14) (PFS: 1.80; OS: 1.7), resulting as an independent factor predicting both PFS and OS in a Cox multivariate analysis. Survival data were confirmed on additional SNPs array data derived from 151 unrelated MM pts not included in the EMN02 trial, as well as on 700 pts, whose CNAs data were extrapolated from the MMRF CoMMpass Study dataset. CONCLUSIONS We propose a simple way to stratify MM pts, according to the detection of 2 CNAs, which is able to select pts with high (25%) and with low (40%) risk of progression and/or death, allowing to predict the clinical outcome in most MM pts. Supported by AIRC, Fond. del Monte Bo-Ra, Fond. Berlucchi, BO-AIL, Harmony Citation Format: Carolina Terragna, Marina Martello, Andrea Poletti, Vincenza Solli, Enrica Borsi, Rosalinda Termini, Lucia Pantani, Elena Zamagni, Giulia Marzocchi, Paola Tacchetti, Nicoletta Testoni, Serena Rocchi, Luca Cifarelli, Luca Dozza, Giovanni Martinelli, Michele Cavo. Chromosomal instability and bad prognosis both connote a multiple myeloma (MM) sub-type carrying 13qCN loss and 1qCN gain [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 470.

The inter- and intra-patients (pts) genetic heterogeneity of MM is the result of a competition between subclones harboring different genomic background, under the selective pressure of both microenvironment constrains and therapeutic treatment. Aim: to describe the clonal architecture changes driven by PI-based regimens in a cohort of homogeneously treated MM pts, by means of a genome-wide approach The study included 50 MM pts up-front treated with PI-based regimens. SNPs array data, derived from the BM CD138+ enriched cell fractions, as collected both at diagnosis (D) and at relapse (R), were analyzed with TAPS and Raw Copy R packages. In addition novel and personalized R-based scripts were designed, aimed at the detailed description of the whole genome CN changes. To be able to analyze the whole genome's changes between D and R, we first translated the signal derived from each of the 2,7x106 markers of the array (Cytoscan HD, Affymetrix) into gene-specific signals, therefore being able to describe CN changes of 20,172 genes. To highlight specific chromosomal regions affected by PI-based selective pressure, we than categorised the overall genomic CN changes, assuming that a limited number (9) of alternative genes' transitions might be observed between D and R. We therefore obtained dynamic profiles describing in details each genes' CNAs transitions, thus being able to highlight the following chromosomal regions particularly involved: chr 1p, 2p, 5, 15p, 19q (negative selection/extinction against CNs gains); chr 2q, 4 and 1p (positive selection/emersion of CNs losses and gains); chr13 and chr19p (mostly stable CN losses and gains). Each gene's CNAs transitions were also evaluated per pts, in order to describe the pts-specific evolutionary trajectories, thus highlighting an extremely high heterogeneity among pts, which might be roughly resumed in four main clonal evolution patterns: 32% of pts showed a stable, whereas 68% a branching evolution pattern. Among the branching patterns, three different behaviours were shown: 8% of pts showed a prevalent positive selection of CNAs, 24% showed a prevalent extinction of CNAs, whereas the remaining showed a blend of both behaviours. Of note, any correlation was observed between the presence of particular CNAs and the propensity to particular evolution patterns. Nevertheless, pts with stable evolution patterns had a slightly shorter median TTP, as compared to pts with branching patterns (22 vs 30 months, range 4-122 and 5-65, respectively). We concluded that the selective pressure of PI-based regimens mostly causes branching trajectories, even if any peculiar CNAs seem to guide at baseline this process. Overall, the clonal evolution patterns in MM remain still unpredictable, being probably driven by a combination of different factors, not yet fully depicted. Thanks to AIRC (MC), FB and FM (CT), AILBologna. Citation Format: Carolina Terragna, Andrea Poletti, Vincenza Solli, Marina Martello, Barbara Santacroce, Rosalinda Termini, Enrica Borsi, Chiara Benni, Lucia Pantani, Elena Zamagni, Paola Tacchetti, Giovanni Martinelli, Michele Cavo. Whole-genome analysis of CNAs identifies four main evolution trajectories in multiple myeloma (MM) patients front-line treated with PI-based regimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2176.

The pliancy of Multiple Myeloma (MM) plasma cells differentiation status acts as adaptive strategy to exogenous stress (e.g. in response to therapy). However, the genomic background that supports any diverse plasma cell differentiation phenotype has not yet been inferred. Aim: to correlate the genomic background with the phenotypic plasticity of MM clone(s) at diagnosis, in order to stratify patients (pts) according to both the level of chromosomal instability (CIN) and their PC differentiation stages, and ultimately to evaluate the impact of this stratification on the disease outcome. 64 newly diagnosed pts were included in the present study. Most pts (54/64) received a PI-based treatment as front-line therapy. Each patient was characterized by 6-color multiparametric flow citometry analysis, combining CD138-PE, CD38-PE-Cy7, CD20-APC, CD19-APC-Cy7, CD27-FITC, CD45-FITC, CD28-APC, CD44-FITC, CD54-APC, CD81-PerCP-Cy5.5, CD56-APC and SHH-PE, as functional marker of Hedgehog pathway activation (Miltenyi Biotech). Whole CNAs characterization of CD138+ purified BM PCs was carried out by SNPs array ( Cytoscan HD ,Affymetrix). According to the detected CIN, as described both by total CNAs and portion of genome changed (GC), 3 major subgroups were identified: the first one with high CIN (21 pts; medium tot. CNAs = 550, % GC ≥ 25%); the second one with a intermediate CIN (25 pts; medium tot. CNAs = 315, % GC = 10-25%) and the third one with low CIN (18 pts; medium tot. CNAs = 105, % GC ≤ 10%). As expected, in pts with high CIN, hyperdiploidy explains more than a quarter of unstable genome; however, they were also characterized by a higher prevalence of high-risk features, such as 1p deletion (FAF1), 16q deletion (WWOX, FANCA) and 17p deletion (TP53), which were almost exclusively associated to high CIN pts (p A detailed immunophenotypic analysis of the three subgroups of pts showed that high CIN background mainly characterized mature PCs (17/21 = 81%), as described by: a) a significant deregulation of both CD19 and CD81; b) a higher expression of CD28 and CD44; c) a reduced expression of CD20, CD27 and CD45. Finally, the presence of more mature PCs with high CIN characterizes pts carrying baseline clinical features associated to bad prognosis (e.g. PET lesions, k/l ratio, ISS III, β2-microglobulin; p In conclusion, a high level of genomic complexity correlates with an advanced PCs differentiation stages in newly diagnosed MM patients; this is associated with a prevalence of poor prognosis features. Chromosomal instability, together with cellular phenotypic pliancy, represents an important, yet poorly defined, mechanism by which MM clone(s) accelerate their own evolution and survival. Acknowledgements: AIRC, AIL, Fond. Berlucchi. Citation Format: Marina Martello, Rosalinda Termini, Barbara Santacroce, Enrica Borsi, Vincenza Solli, Chiara Benni, Andrea Poletti, Lucia Pantani, Beatrice Zannetti, Serena Rocchi, Elena Zamagni, Paola Tacchetti, Francesca Ulbar, Mario Arpinati, Gabriella Chirumbolo, Nicoletta Testoni, Giulia Marzocchi, Giovanni Martinelli, Michele Cavo, Carolina Terragna. The pliancy of plasma cell differentiation status conceals a gradient of chromosomal instability in newly diagnosed multiple myeloma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3387.

Intro: Multiple Myeloma (MM) is a biologically complex disease, whose genetic plasticity favors the coexistence of genetically heterogeneous subclones, selected in a Darwinian fashion throughout the disease course. Therapy might represent a major selective pressure over the different subclones, thus supporting an evolutionary model of the disease. Aim: To explore the existence of different clonal evolution patterns in MM, eventually driven by therapeutic selective pressure. P&M The study included 33 pts with symptomatic MM, up-front treated either with combination regimens including a proteasome inhibitor (28), or with cyclophosphamide. For each pts, paired BM samples were collected both at diagnosis and at relapse. SNPs array analyses were performed on the CD138+ enriched cell fractions. Results: Two approaches were applied: a) monitoring the variations of macro CNAs; b) focusing on changes of CNAs frequencies, as observed in 27 genes of interest. Both approaches were consistent in highlighting three major evolution patterns: in 7/33 (21%) pts, the genomic background at relapse was almost identical to that of diagnosis. In 13/33 (39%) pts, an overall increase in the frequencies of the same CNAs as observed at diagnosis was detected at relapse. Finally, in 13/33 (39%) pts, either increased or decreased frequencies of several CNAs, as well as several differences in the CNAs type’s prevalence were observed at relapse, as compared to diagnosis. Of interest, even if an overall CNAs median number increase was observed from diagnosis to relapse (226 vs 507, respectively) - supported by acquisition of CNAs either commonly described as secondary genomic events (i.e. del17p13, amp1q21, del1p23), or associated to the resistance to bortezomib (i.e. del8p21) - any peculiar CNAs resulted significantly prevalent in the 3 identified subgroups of pts. A high rate (92%) of achievement of VGPR or better quality of response to upfront therapy characterized the third subgroup of pts, whereas the rate of VGPR in the remaining pts was only 20% and PR or SD were observed in 9 and 7 pts, respectively. Finally, the median time to first progression of this subgroup of pts was significantly shorter as compared to that of pts with branching evolution (24 vs 35 months, range 4-41 and 7-123 months, respectively, p=0,01). Conclusion: The genomic architecture of a subgroup of relapsed MM pts, up-front responsive to new drugs-based combination therapies, resulted overall different from that of diagnosis, suggesting a branching evolution of the disease, sustained by the shrinking of the most prevalent clone (therapy-sensitive), as well as by the expansion of subclones (therapy-resistant) not already evident at diagnosis. This observation raises the question whether re-treatment of relapsed pts should be appropriate in the case of branching evolution. Acknowledgements: AIRC (MC), Fondazione Berlucchi (CT), FUV (EB). Citation Format: Carolina Terragna, Marina Martello, Barbara Santacroce, Vincenza Solli, Lucia Pantani, Elena Zamagni, Paola Tacchetti, Beatrice Zannetti, Katia Mancuso, Giulia Marzocchi, Nicoletta Testoni, Gaia Ameli, Rosalinda Termini, Angela Flores Dico, Enrica Borsi, Giovanni Martinelli, Michele Cavo. A branching evolution model at relapse characterizes multiple myeloma patients who responded to upfront combination therapy including new drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3936. doi:10.1158/1538-7445.AM2017-3936

The iperactivation of Hedgehog (Hh) pathway, which controls the refuel of the tumor clone, might be critical to Multiple Myeloma (MM) recurrence. In order to dissect the role played by Hh pathway in different MM cells compartments, and to evaluate its clinical impact on patient outcomes, here we explore the transcriptomic and genomic profiles in both CD138+ plasma cells and CD138-19+ B progenitors cells obtained from newly diagnosed MM patients (pts), The study included a cohort of 126 pts, homogenously treated with bortezomib-based regimens and ASCT. DNA and RNA were obtained from both CD138+ plasma cell fraction and CD19+ B cells. Gene expression profiling (GEP) (HG U133 Plus 2.0) and genomic analysis (SNP 6.0) were performed on Affymetrix platform. Data were analysed by employing several software: dChip (GEP clustering), Ingenuity Pathway Analysis (GEP Enrichment) and Nexus (Copy number). By unsupervised hierarchical clustering, an Hh signature of 10 genes - SHH, IHH, DHH, SMO, PTCH1, PTCH2, SUFU, GLI1, GLI2 and GLI3 - was identified, and it was able to significantly cluster pts in two subgroups: cluster 1 (70 pts) and cluster 2 (56 pts. An overall significant activation of Hh pathway was shown in cluster 2, as compared to cluster 1. Of note, the Hh pathway was down regulated in CD19+ B cells obtained from pts included in cluster 2, while it was overexpressed in cluster 1 pts. Western blots on both cell fractions confirmed this opposite Hh genes behavior. A higher genomic instability (e.g. higher frequencies of both t(4;14) and del(17p)) was demonstrated in CD138+ cells from cluster 2 pts and, at least 5 known tumor suppressor genes, such as RB1, BRCA2, PDX1, FOXO1 and TP53 were affected. Conversely, cluster 1 pts were mainly characterized by hyperdiploid karyotypes. The more aggressive phenotype of cluster 2 pts was confirmed by an overall deregulation of cell adhesion processes (CD44, LIMS1, COL4A2, CTGF, COL1A1, FN1), increased proliferation (MYCBP, IL22, SDPR, SOX2, SOX6) and DNA repair mechanisms (SP1, SMARCD3, FOXA3). Hh pathway activation significantly influenced pts’ outcome, since those included in cluster 2 had a shorter PFS and OS compared to cluster 1. In fact, the 5-year PFS estimates were 31% vs 56% (p = 0.0062), whereas the OS probabilities were 66% and 83%, respectively (p = 0.0071). Of note, both hazard ratios for PFS and OS were doubled in pts included in cluster 2, as compared to pts included in cluster 1. Finally, multivariate analyses confirmed that being part of cluster 2 was an independent prognostic factor for both PFS and OS, along with del(17p) and ISS 3. Two alternate Hh-driven subtypes of MM might be identified at diagnosis, which correlated with pts outcomes. Stratification of pts according to their molecular background might help the fine-tuning of future clinical studies. Acknowledgements: FP7 NGS-PTL project, Progetto Regione-Università 2010/2012 L. Bolondi. Citation Format: Marina Martello, Daniel Remondini, Enrica Borsi, Mauro Procacci, Barbara Santacroce, Angela Flores Dico, Annalisa Pezzi, Elena Zamagni, Paola Tacchetti, Lucia Pantani, Giulia Marzocchi, Serena Rocchi, Katia Mancuso, Beatrice Anna Zannetti, Giovanni Martinelli, Michele Cavo, Carolina Terragna. The alternate activation of hedgehog pathway, either in CD138+ or in CD138-CD19+ multiple myeloma primary cells, impacts on disease outcome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3189.

Introduction In most human cancer p53 impairment is a driver event, which confers a survival advantage to affected cells. In Multiple Myeloma (MM), the role of p53 clonal aberrations (mainly 17p del) is well recognised, whereas the prognostic relevance of TP53 mutations is less clear, due to the very limited frequency of clonal lesions. Here we aim at characterizing by ultra-deep sequencing (UDS) the TP53 mutational state in both newly diagnosed and relapsed MM pts, to assess the prognostic role and evolution over time of small TP53 mutated sub-clones. Pts and methods A cohort of 99 newly diagnosed MM pts treated up-front with bortezomib-based regimens and ASCT was included in this study. In 29 cases, samples were collected also at relapse(s). DNA was obtained from CD138+ highly purified plasma cells and SNPs array profiled (Affymetrix). TP53 gene was analysed by amplicon-targeted UDS approach (GSJ, 454 Roche). A specific bioinformatics pipeline was set up to discriminate between low frequency TP53 variants and sequencing errors. Results With a median coverage of 1386X, 129 correctly called TP53 variants were detected. Most newly diagnosed MM pts (55%) carried at least one TP53 sub-clonal variant (on average 1.08 variants per pts).. According to TP53 sub-clonal mutational load, pts were stratified in two sub-groups, including 28 pts with ≥2 (high load) and 71 with Conclusion The TP53 UDS analysis in newly diagnosed MM highlighted for the first time a high rate of variants, recurring with a wide range of frequencies among samples. The increased number of TP53 sub-clonal variants per pts in samples collected at relapse(s), compared to that seen at the onset of the disease, suggests a sub-clonal dynamics over time. Acknowledgements: Roche Diagnostics, FP7 NGS-PTL project, Fondazione Berlucchi. Citation Format: Marina Martello, Daniel Remondini, Enrica Borsi, Barbara Santacroce, Mauro Procacci, Angela Flores Dico, Annalisa Pezzi, Elena Zamagni, Paola Tacchetti, Lucia Pantani, Giulia Marzocchi, Beatrice Anna Zannetti, Katia Mancuso, Serena Rocchi, Giovanni Martinelli, Michele Cavo, Carolina Terragna. The selection of TP53 sub-clonal variants over time identifies MM patients with adverse clinical outcome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5022.

The bulk of current knowledge on Multiple myeloma (MM) disease is mainly based on the molecular characterization of 138+ plasma cells, but we become aware that MM heterogeneity has more deepen roots. Recent studies support that Myeloma Propagating Cells (MPCs) might be responsible of relapses and they residing in the 138- compartment. In order to molecularly characterize mature and precursor cell fractions, we collected the 138+ and 138-19+27+ cell fractions from bone marrow (BM) and peripheral blood (PBL) of 50 newly diagnosed MM patients (pts). Clonogenic assays were performed by plating cell fractions of MM cell lines. The molecular characterization included: IgH rearrangement sequencing, analysis of genomic aberrations and gene expression profiling by Affymetrix 6.0 SNPs array and HG-U133 Plus 2.0 microarray. Clonogenic assays showed that 138- cells were able to form colonies more efficiently than 138+ cells. By VDJ gene rearrangement sequencing, a clonal relationship between the 138+ clone and precursor ones was confirmed. SNPs arrays showed that both BM and PBL 138+ cell fractions carried the same genomic macro-alterations. On the contrary, in the 138-19+27+ cell fractions from BM and PBL any macro-alteration was detected, whereas several micro-alterations unique of the memory B cells clone were highlighted. An enrichment analysis revealed that genes are involved in DNA repair mechanisms, transcriptional regulation, negative regulation of apoptosis and angiogenesis. Interestingly, KRAS, WWOX and XIAP genes are located in the amplified regions of immature cells. Moreover, we observed several LOH regions that covered important tumor suppressor genes, including TP53, CDKN2C and RASSF1A. The involvement of immature cells in MM pathogenesis was confirmed by the analysis of the 19+ cells’ transcriptome profiles. 20 MM 19+ cells samples (12 from PBL, 8 from BM) were compared both to their normal counterpart and to the mature 138+ cell fractions. In particular, unsupervised analysis by hierarchical clustering discriminated the differential expression of 11480 and 11360 probes in the PBL and BM 19+ clones, respectively (2; FDR = 0,05; p Interestingly, the protein homeostasis deregulation possibly caused by ER stress, resulted particularly evident in the BM 19+ cells (p = 7,25E-14; FDR = 2,98E-11); moreover, the down-regulation of genes related to the unfolded protein response (e.g. IRE1α and XBP1 FC = -18,0; -19,96. p Presented data support the emerging role of the immature cell compartment in the MM disease course, where the putative 19+ MPCs might be involved in the neoplastic clone supply. Citation Format: Marina Martello, Angela Flores Dico, Daniel Remondini, Barbara Santacroce, Enrica Borsi, Mauro Procacci, Lucia Pantani, Elena Zamagni, Paola Tacchetti, Anna Maria Brioli, Serena Rocchi, Giulia Marzocchi, Giovanni Martinelli, Michele Cavo, Carolina Terragna. High-throughput molecular profiling of Multiple Myeloma clonotypic CD19+ B cells highlights pathways potentially involved in the disease endurance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1521. doi:10.1158/1538-7445.AM2015-1521

TP53 is rarely reported as being affected either by deletions or mutations in MM, even if chr17p13 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 TP53 gene inactivation in newly diagnosed MM, assessing the mutational events’ frequency and their clinical impact. A cohort of 92 MM, receiving up-front velcade (vel)-based regimens, followed by autologous stem cell transplantation, was included in the study. TP53 gene mutational status was analysed by amplicon-targeted deep NGS approach. An upcoming NGS data analysis software was employed to detect vars (var) and to compare them to IARC database. p53 activity was evaluated by immunoblot assays. Ultra-deep TP53 coding sequence analysis (median depth: 1060) highlighted the presence of a median of 1.8 vars per samples in 73/92 (79%) MM: a total of 131 nucleotide substitutions emerged, with vars allele frequencies (VAF) ranging from 1 to 99% (median 1.3%). TP53 vars, were defined either neutral or deleterious mutations, according to their predicted effect at amino acid level. 36 cases carried 42 deleterious vars (VAF = 1.2%), 67% of which were predicted as non-functional at protein level. 9 samples carried 21 neutral vars (VAF = 1.1%), mainly affecting the DNA binding domain. 28 samples carried 68 SIFT unclassified vars, among which several polymorphisms were counted (VAF = 94.6%). SNPs arrays were performed on 83/92 samples: 14/83 (17%) carried a TP53 CN loss 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 var) or deleted TP53 (p = 0.006). By immunoblot assay we showed the absence of phosphorilation both in 3 TP53 deleted cases and in 3 MM carrying deleterious vars; on the contrary p-p53 was observed in 4 non-mutated cases. Clinical correlations were performed on 81 MM showing that the presence of either TP53 hemizygous deletions or at least one TP53 deleterious var was more likely associated to the response to vel-based induction therapy (frequencies of ≤partial response: 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%). In conclusion, the TP53 coding sequence analysis by ultra-deep NGS of a cohort of newly diagnosed MM highlighted an unexpected, still un-explored, high rate of TP53 vars. The impact of TP53 damage on MM disease course has to be confirmed in randomized clinical trial. Supported by Roche Diagnostics and FP7 NGS-PTL project Citation Format: Marina Martello, Giovanni Martinelli, Angela Flores Dico, Barbara Santacroce, Enrica Borsi, Mauro Procacci, Torsten Haferlach, Flavio Mignone, Igor Saggese, Elena Zamagni, Paola Tacchetti, Lucia Pantani, Anna Maria Brioli, Serena Rocchi, Annalisa Pezzi, Michele Cavo, Carolina Terragna. A long tail of sub-clonal TP53 mutations emerged by ultra-deep sequencing of newly diagnosed multiple myeloma (MM). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4248. doi:10.1158/1538-7445.AM2015-4248

Background Although the advances in Multiple Myeloma (MM) therapy, the disease remains incurable. The existence of Myeloma Propagating Cells (MPCs) is supposed to be one of the major causes of MM drug-resistance, leading to relapse. However, very little is known about the molecular characteristics of MPCs, even if some studies suggested that these cells have phenotypic characteristics resembling the memory B cells. Aim and methods In order to molecularly characterize the 138+ neoplastic clone and the memory B cells located both in bone marrow (BM) and in peripheral blood (PBL), we collected the 138+ 138- cell fractions of 50 newly diagnosed MM patients (pts). We isolated the B cell population and, when possible, the memory B cell clone. For each cell fraction we performed a VDJ rearrangement analysis. The complete set of genomic aberrations was evaluated by SNP Array 6.0 (Affymetrix). Gene expression profile HG-U133 Plus 2.0 microarray analyses (Affymetrix) were performed according to a standardized protocol. Results The VDJ rearrangement analyses confirmed the clonal relationship between the 138+ clone and the memory B cells clone. Both BM and PBL 138+ cell fractions showed exactly the same genomic macro-alterations. In the BM and PBL 138-19+27+ cell fractions any macro-alteration was detected, whereas several micro-alterations (range: 1-350 Kb) unique of the memory B cells clone, were highlighted. These micro-alterations were located out of any genomic variants region and are presumably associated to the MM pathogenesis, as confirmed by the presence of KRAS, WWOX and XIAP genes among the amplified regions. Moreover, the memory B cells were characterized by the presence of several LOH regions, including possibly involved tumor suppressor (TS) genes. To get insight into the biology of the clonotypic B cell population, we compared the gene expression profile of 7 MM B cells vs 5 donor B cells samples, thus showing a differential expression of 11480 probes (Fold Change: 2;-2; FDR: 0,05; p-value: Conclusions Data suggested that the MM 138+ clone might resume the end of the complex process of myelomagenesis, whereas the memory B cells have some intriguing micro-alterations and a specific transcriptional program, supporting the idea that these post germinal center cells might be involved in the transforming event that originate and sustain the neoplastic clone. Citation Format: Marina Martello, Carolina Terragna, Giovanni Martinelli, Flores Dico, Enrica Borsi, Elena Zamagni, Lucia Pantani, Paola Tacchetti, Beatrice Zannetti, Katia Mancuso, Serena Rocchi, Annamaria Brioli, Viviana Guadagnuolo, Michele Cavo. Gene expression profiling and copy number alterations of circulating clonotypic B cells of multiple myeloma newly diagnosed patients reveals pathways potentially involved in the development and in the disease persistence. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3884. doi:10.1158/1538-7445.AM2014-3884

Background. The TP53 tumor-suppressor gene is mutated or functionally inactivated in most human cancers. In MM, TP53 del on chr 17p13 defines a pts group with a particularly poor prognosis, even if its prevalence at diagnosis is quite low. One of the most potent inhibitor of p53 is MDM4, whose locus is on chr1q32.1, a region frequently amplified in MM. Aim. To investigate the possibility that both TP53 del and MDM4 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. 89 pts treated with bortezomib-thalidomide-dexamethasone (VTD) as induction therapy prior to, and as consolidation after, double ASCT were analyzed at diagnosis by unpaired analysis CNA (Affymetrix 6.0 SNP array) and GEP (Affymetrix U133 Plus2.0 array); in 21 pts carrying MDM4 amp the p53 activity was explored both by deep sequencing of TP53 (Roche GS Junior 454 platform) and by immunoblotting assays of MDM4, p-p53 and p53. Genomic results were evaluated in the clinical context. Results. CNA analysis showed a 482 Kb minimal del region on chr17p13, including TP53, in 8/89 pts (8,9%) and a 1.1 Mb minimal amp region on chr1q32.1 including MDM4 in 27/89 pts (30,3%). GEP analysis performed in TP53 del and MDM4 amp pts generated two lists, including genes differentially expressed among pts carrying or not either amp MDM4 (4211 probes sets, p In 21 pts carrying MDM4 amp the p53 activity was evaluated by p-p53 immunoblotting assays: the absence of p-p53 protein was shown in 60% of them. The TP53 mutational rate, as detected by deep sequencing of exons 4-11, was analyzed in 21 pts carrying MDM4 amp: point mutations were shown in 15 pts, with mutated reads frequencies ranging from 1.03% to 16.9% (median coverage for each amplicon = 1000 reads). The prognostic relevance of p53 pathway impaired function was evaluated by stratifying pts into two subgroups according to the presence or absence of MDM4 amp and/or TP53 del (group A: 34 pts, 38%, group B; 55 pts, 62%). Baseline clinical characteristics were homogeneous, whereas groups A and B resulted clearly imbalanced with respect to the genomic background: the t(4,14) frequency (38% vs. 14% positive, p=0.0002) and the average number of CNAs (165 vs 103, p = 0.03) were overall higher in group A. Despite the initially slightly higher response rate after VTD (38% vs 20% ≥near complete response), group A pts showed a shorter PFS (40.13 months vs not reached, p=0.001) and OS (66.6 vs not reached, p=0.0006). The poorer impact associated with MDM4 amp was retained also in the absence of TP53 del (PFS: 50.5 months vs not reached, p=0.01). Conclusions. The involvement of the p53 pathway alteration in MM might be wider than expected, possibly due to the activation of negative regulators of p53. Citation Format: Carolina Terragna, Marina Martello, Enrica Borsi, Lucia Pantani, Elena Zamagni, Paola Tacchetti, Annamaria Brioli, Beatrice Anna Zannetti, Flores Dico, Nicoletta Testoni, Giulia Marzocchi, Katia Mancuso, Serena Rocchi, Giovanni Martinelli, Michele Cavo. Impact of p53 impaired function on outcomes of multiple myeloma patients carrying deleted TP53 and/or amplified MDM4. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5595. doi:10.1158/1538-7445.AM2014-5595

Multiple Myeloma (MM) is a clonal B-cell malignancy characterized by accumulation of malignant plasma cells (PCs) within the bone marrow (BM) in close contact with stromal cells (SCs) which secrete growth factors and cytokines, promoting tumor cell growth and survival. The rapid progression of MM is dependent upon cellular interactions within the BM microenvironment, and novel agents targeting this interaction appear to be promising therapeutic strategies for the treatment of MM tumor expansion. Unlike most other organs, the BM microenvironment is physiologically hypoxic, a pre-requisite for normal BM hematopoiesis. It is well established that hypoxia is an important selective force in the evolution of tumor cells and a stabilization of HIF-1α protein has been documented in several human cancers. While the role of hypoxia in the pathogenesis of hematologic malignancies has yet to be elucidated, recent animal studies have shown that changes in oxygen levels within the BM microenvironment support the survival and expansion of MM cells. Furthermore, some drugs active in MM, such as Bortezomib and Lenalidomide, are believed to exert their effects in part by interfering with hypoxia-induced signaling cascades. Given the importance of the BM microenvironment in MM pathogenesis, we investigated the possible involvement of HIF-1α in the PCs-BMSCs interplay. To test this hypothesis, we used EZN-2968, a small 3rd generation antisense oligonucleotide against HIF-1α, to inhibit HIF-1α functions. We have already shown that EZN-2968 is highly specific for HIF-1α mRNA and it results in a long lasting and time dependent inhibition of HIF-1α protein level. Herein, we provide evidence that the interaction between MM cells and BMSCs is drastically reduced upon HIF-1α down-modulation. Notably, we showed that upon exposure to HIF-1α inhibitor, neither the incubation with IL-6 nor the co-culture with BMSCs were able to revert the anti-proliferative effect induced by EZN-2968. Moreover, we observed that EZN-2968 down-modulates cytokine-induced signaling cascades after a short incubation, and seems to induce a negative modulation of those transcripts previously shown to reflect the activation state of specific tumor cell pathways (cell proliferation and survival). This observation was also supported by gene expression profile experiments. One of the key finding of our study is that PC attachment to the extracellular matrix protein was markedly reduced in the presence of EZN-2968. The effects of HIF inhibition on MM cell adhesion are quite intriguing, since MM pathogenesis is dependent upon the interaction of MM cells with the SCs. Taken together, these results strongly support the concept that HIF-1α plays a critical role in the interactions between BMSCs and PCs in MM. We conclude that HIF inhibition may be an attractive therapeutic target for MM. Citation Format: Enrica Borsi, Giulia Perrone, Carolina Terragna, Marina Martello, Angela Flores Dico, Lucia Pantani, Annamaria Brioli, Giovanni Martinelli, Michele Cavo. HIF-1α inhibition blocks the cross talk between multiple myeloma plasmacells and tumour microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3608. doi:10.1158/1538-7445.AM2014-3608

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 phase 3 study demonstrated that VTD regimen was superior over thalidomide-dexamethasone (TD) as induction therapy prior to and as consolidation therapy after the double ASCT for newly diagnosed MM (rate of >nCR: 31% vs 11% (pnCR affected by VTD resulted in extended progression-free survival (PFS), prediction of CR by pharmacogenomic tools is likely to be an important goal to prospectively select those pts who are more likely to benefit from a given therapy. Methods. We assessed the ability of GEP to predict attainment of CR in 122 pts enrolled in the VTD arm of the study. CD138+ plasma cells were obtained at diagnosis from each pts and were profiled for their gene expression (Affymetrix U133 Plus2.0 platform). Results. 34/122 pts (28%) who were included in the present analysis achieved a ≥nCR after VTD induction therapy and were characterized by the differential expression of 2157 probesets (p≤0.01). According to the unsupervised hierarchical clustering, the population of 122 profilated pts resulted stratified into 2 subgroups, the first one including 79% and the second one 21% of ≥nCR pts. To obtain a classifier for response to VTD induction therapy, the differentially expressed genes was analyzed to identify a small group of predictor genes. The best predictive capability was obtained with a 41-gene classifier that provided 88% sensitivity, 97% specificity, 91% PPV and 95% NPV. A GeneGo® network analysis showed that the most relevant network nodes included tumour suppressor genes, genes involved in inflammatory response and genes involved in B cell development. To asses the relevance to know since diagnosis the sensitivity for a particular therapy, we evaluated the prognostic impact of the response prediction in our clinical context. Pts predicted to be responder to VTD induction therapy according to the 41-gene classifier were more likely to attain a ≥nCR after the consolidation therapy. The 3 years estimate of PFS of pts ≥nCR after the consolidation therapy was significantly higher as compared to pts who failed this objective (p=0.03). Conclusions. GEP analysis of a subgroup of pts who received VTD induction therapy provided a 41-gene classifier that was able to predict attainment of >nCR and, conversely, failure to achieve at least nCR in 91% and 95% of cases, respectively. These favorable results might represent a first step towards the possible application of a tailored therapy based on the single patient's genetic background. Supported by: Fondazione Del Monte di Bologna e Ravenna, Ateneo RFO grants (M.C.) BolognAIL. Citation Format: Carolina Terragna, Daniel Remondini, Marina Martello, Annalisa Pezzi, Francesca Patriarca, Anna Levi, Lucia Pantani, Daniela Donnarumma, Lorenzo Montanaro, Claudia Crippa, Sarah Bringhen, Alessandro Rambaldi, Massimo Offidani, Paolo Corradini, Franco Narni, Giuseppe Fioritoni, Alfonso Zaccaria, Luca Baldini, Tommaso Caravita, Giorgio La Nasa, Sergio Cortellazzo, Giovanni Martinelli, Michele Cavo. A 41-gene signature predicts complete response (CR) to Bortezomib-Thalidomide-Dexamethasone (VTD) as induction therapy prior to autologous stem-cell transplantation (ASCT) in multiple myeloma (MM). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2207. doi:10.1158/1538-7445.AM2013-2207

Background Although the advances in Multiple Myeloma (MM) therapy, the disease remains incurable. The existence of Myeloma Propagating Cells (MPCs) is supposed to be one of the major causes of MM drug-resistance, leading to relapse. However, very little is known about the molecular characteristics of MPCs, even if some studies suggested that these cells have phenotypic characteristics resembling the memory B cells. Aim and methods In order to molecularly characterize the CD138+ neoplastic clone and the memory B cells located both in BM and in PBL, we collected the CD138+ and CD138-19+27+ cell fractions from bone marrow (BM) and peripheral blood (PBL) of 50 newly diagnosed MM patients (pts), 7 MGUS pts and 15 relapsed pts. For several pts we collected buccal swab sample as a negative control. The complete set of genomic aberrations was evaluated by SNP Array 6.0 and copy number analysis was performed with Genotyping Console software and Partek Genomics. Results Both BM and PBL CD138+ cell fractions showed exactly the same genomic macro-alterations. In the BM and PBL CD138-19+27+ cell fractions any macro-alteration was detected, whereas several micro-alterations (range: 1-834 Kb) unique of the memory B cells clone, were highlighted. These micro-alterations were located out of any genomic variants region and are presumably associated to the MM pathogenesis. The micro-alterations involved HMGCLL1, DLGAP2 and RCOR3, PRR16, TSC1, ETS1, RBFOX1 in CD138-19+27+ derived from PBL and BM, respectively. These genes participated in cholesterol metabolism, embryonic development and transcriptional regulation. Interestingly, three focal micro-deletions (involving SKT, CES1P1, MIR650) were shared both by the PBL and the BM memory B cells clones. The MIR650 gene is located on chr22 in the immunoglobulin lambda gene locus, thus directly controlling its expression. By applying a more stringent analysis, the CD138-19+27+ cell fraction obtained from all pts analyzed showed a unique micro-deletion (410 Kb) on chr 14, involving JAG2, BRF1, PACS2, NUDT4 and BTBD6. This deletion has been already described as involved in a pediatric syndrome of chr.14q, and its presence is correlated to a variety of developmental disorders and mental retardation. Conclusions These data suggested that the MM CD138+ clone might resume the end of the complex process of myelomagenesis, proven by the presence of numerous macro-alterations, which might be probably due to an established genomic instability. In contrast, the memory B cells, which lack these macro-alterations, have some intriguing micro-alterations, supporting the idea that these post germinal center cells might be involved in the transforming event that originate the neoplastic clone. Work supported by European LeukemiaNet, AIRC, AIL, PRIN, University of Bologna and BolognAIL. Citation Format: Marina Martello, Carolina Terragna, Giovanni Martinelli, Enrica Borsi, Elena Zamagni, Lucia Pantani, Paola Tacchetti, Beatrice Zannetti, Katia Mancuso, Flores Dico, Michele Cavo. Genomic characterization of the putative myeloma stem cells clone reveals alterations possibly correlated with the origin of disease. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3749. doi:10.1158/1538-7445.AM2013-3749

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 up-regulation. In the present study we explored the interaction between c-MYC and HIF-1α in a panel of MM cell lines. We had previously shown that treatment with EZN2968, an antisense oligonucleotide against HIF-1α, resulted in a significantly reduction of HIF-1α protein level as soon as 24h 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, cell cycle check point negatively regulated by c-MYC, was up-regulated in treated samples. We further verified the effect of HIF-1α inhibition on c-MYC protein level by western blotting. After treatment with EZN2968, c-MYC protein expression was reduced in a time dependent manner, 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 decreased after EZN2968 exposure. Recently, it has been shown that SIRT1, a transcription factor involved in a development and cellular stress responses, 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 strongly 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 cells 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. Supported by PRIN. Citation Format: Enrica Borsi, Giulia Perrone, Carolina Terragna, Marina Martello, Manuela Mancini, Elisa Leo, Elena Zamagni, Paola Tacchetti, Annamaria Brioli, Lucia Pantani, Beatrice Zannetti, Giovanni Martinelli, Michele Cavo. SIRT regulates the molecular interaction between c-MYC and HIF-1α in multiple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3415. doi:10.1158/1538-7445.AM2013-3415

Hedgehog (Hh) pathway activation contributes to leukemia development and growth, and that targeted pathway inhibition is likely to offer an efficient therapeutic opportunity. PF-04449913, a Hh pathway inhibitor, is a new selective and potent inhibitor of leukemia self-renewal and is currently being evaluated in phase I clinical trials. In order to identify new potential clinical biomarkers for the PF-04449913, we studied CD34+ leukemia stem cell population (LSC) collected before and after 28 days treatment in a phase I dose escalation protocol (Clinical Trial Gov. NTC00953758) enrolling selected hematological malignancies. This experimental clinical trial enrolled Myelofibrosis (MF), MDS, blastic phases CML, chronic myelomonocytic leukemia (CMML) and AML patients (pts). We were able to collect and separate highly purified (98%) bone marrow CD34+ cells from 5 AML, 1 MF and 2 CML pts by immunomagnetic separation, and analysed them for gene expression profile using Affimetrix HG-U133 Plus 2.0 platform. 1197 genes were differentially expressed between CD34+ cells collected before and after 28 days of PF-04449913 dose finding oral therapy. Clustering of their expression profiles showed that mostly genes differentially expressed are mainly related to Hh signaling, this providing further evidences that PF-04449913 really therapeutically targets the Hh pathway. Regarding genes involved in Hh signaling pathway, Gas1 and Kif27 were strongly upregulated (fold change 1.0947 and 1.12757 respectively; p-value 0.01 and 0.02 respectively) in CD34+ LSC after 28 days exposure to PF-04449913 as compared to baseline, suggesting these two genes have potential as new biomarkers of activity. GAS-1 is a Sonic Hedgehog (Shh)-binding protein; it acts to sequester Shh and inhibit the Shh signalling pathway. Kif27 mainly acts as a negative regulator in the Hh signaling pathway, and inhibits the transcriptional activator activity of Gli1 by inhibiting its nuclear translocation. Other genes were differentially expressed after ‘ex- vivo’ treatment with PF-04449913 as compared to baseline: we observed a down regulation of Bcl2 (fold change -1.03004), ABCA2 (fold change -1.08966), LEF1 (fold change -1.28457), Gli1 (fold change -1.0775), Smo (fold change -1.07702), and an upregulation of Gli2 (fold change 1.08191). Conclusions: This data demonstrates that PF-04449913 specifically targets the Hh Pathway in CD34+ cells, suggesting that Hh inhibition may impair leukemia stem cell maintenance. In addition, we identify several new potential biomarkers (e.g. Gas1 and KIF27). Taken together, these data may be useful for pts selection strategies and subsequent eradication of the LSC. Acknowledgments: Work supported by Pfizer, European LeukemiaNet, FIRB 2006, AIRC, AIL, COFIN, University of Bologna and BolognAIL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 906. doi:1538-7445.AM2012-906

The p53 tumor suppressor pathway is tightly kept in check or completely silenced in cancer cells. A potent inhibitor of p53 is MDM4, which is located on chr.1q: it is critical for the control of p53 activity during the response to stress and is often amplified in several types of cancer. 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) and autologous stem cell transplantation (ASCT) in order to investigate mechanisms which might be activated in myeloma plasma cells to direct and/or indirect limit the p53 function. CD138+ plasma cells obtained at diagnosis from 61 pts, subsequently treated with VTD and ASCT were analysed by means of GEP and unpaired analysis of CNA (Affymetrix U133 Plus2.0 and 6.0 SNP arrays). Nineteen out of 61 pts (31%) carried a minimal amplification region on chr.1q, harboring MDM4. Eight out of 61 pts (13%) carried a 482 Kb minimal deletion region on chr.17 harboring 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, 24 pts) or the absence of both these abnormalities (group B, 37 pts). Baseline clinical characteristics were homogeneous, except for a higher rate of ≤ 3 bone lesions in pts of group A. The rate of best complete or near complete response was 40% in group A and 14% in group B (p = 0.04). With a median follow-up of 36 months, the risk of progression was 46% for pts in group A and 19% for those in group B (p = 0.03). 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). A comparison of expression profiles of the two subgroups of pts highlighted a deregulated expression of genes involved in the mechanisms of response to genotoxic stress, i.e. of damage sensor genes (ATM, RAD9, RAD 50, ATRip), of damage signal mediator genes (H2AFX, 14-3-3), of genes involved in regulation of cell proliferation (CDK6, CDC25, CCND2) and of anti-apoptotic genes (BCL2, p73) (one-way ANOVA plus multiple-test correction with FDR Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1873. doi:1538-7445.AM2012-1873

PF-04449913 is a selective and potent inhibitor of the Hh pathway and leukemia self-renewal and is currently being evaluated in Phase I clinical trials. We studied leukemia stem cell population (CD34+ subpopulation) collected before and after 28 days treatment in a phase I dose escalation protocol (Clinical Trial Gov. NTC00953758) enrolling selected hematological malignancies including MF, MDS, CML, CMML and AML. We collected and separated highly purified (98%) bone marrow hematopoietic progenitor cells (CD34+ populations) in 5 AML, 1 MF and 2 CML patients, by immunomagnetic separation, and analyzed them for gene expression profile (GEP) using Affimetrix HG-U133 Plus 2.0 platform. We have observed that 1197 genes were differentially expressed between CD34+ cells collected before and after 28 days of PF-04449913 dose finding oral therapy. We demonstrated a down regulation of Bcl2 (fold change –1.03004; p value= 0.01), ABCA2 (fold change –1.08966; p value=0.03), Bcl2l13 (fold change –1,04259; p-value=0,027642), Bcl2l2 (fold change –1,17214; p-value=0,000768), Casp4 (fold change –1,06551; p-value=0,032428), Casp7 (fold change –1,01569; p-value=0,006688), Casp10 (fold-change –1,3076; p-value=0,050431), ABCF1 (fold change –1,04999; p-value=0,07213). On the contrary, ABCB1 (fold change 1,46592) and ABCG2 (fold change –1,16103) are respectively up and down regulated, with a not statistically significant p-value. Bcl2 (B-cell lymphoma 2), Bcl2l2 (Bcl2-like protein 2) and Bcl2l13 (Bcl2-like 13) are the founding members of the Bcl-2 family of apoptosis regulator proteins. Recent studies showed that Hh signals upregulate Bcl2 to promote cellular survival. Casp 4,7,10 (Caspases) are a family of cysteine proteases that play essential roles in apoptosis, necrosis, and inflammation. ABCA2 (ATP-binding cassette sub-family A member 2), ABCF1 (ATP-binding cassette sub-family F member 1), ABCB1 (ATP-binding cassette sub-family B member 1, MDR1), ABCG2 (ATP-binding cassette sub-family G member 2) belong to the superfamily of adenosine triphosphate-binding cassette (ABC) transporters. One mechanism of MDR is the increased expression of ABC drug transporters, resulting in low intracellular drug concentrations. We evaluated Gli1 and Smo expression by GEP, comparing data before and after 28 days of treatment with PF-04449913 and we observed a down regulation of both genes (fold changes –1.0775 and –1.07702 respectively). PF-04449913 is able to revert MRD mechanisms of LSC by a strong down regulation of genes (Bcl-2, Bcl-2l13, Bcl-2l2, ABCA2, and ABCF1), which are critical for chemoresistance in acute and chronic leukemia patients. Acknowledgments. Pfizer, European LeukemiaNet, FIRB 2006, AIRC, AIL, COFIN, University of Bologna and BolognAIL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4619. doi:1538-7445.AM2012-4619

Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor that plays a critical role in survival and angiogenesis. In solid tumors, elevated expression of HIF-1α, 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 tumours, the role of HIF-1α in hematological malignancies is not completely known. In particular in multiple myeloma (MM) HIF-1α has been suggested to be constitutively expressed and HIF-1α 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 HIF-1α, as a molecular target in MM. We showed that the expression of HIF-1α in several MM cell lines is detectable in either normoxia or hypoxia conditions and is increased in the presence of growth stimuli (IL-6 and stroma cells). In vitro, EZN2968 induced selective and stable down-modulation of HIF-1α mRNA and protein expression, either in normoxia or hypoxia conditions. Immunofluorescence analysis confirmed the reduction of the protein after 24 hours of treatment, as well as a lower expression of VEGF. Clinically achievable doses of EZN2968 induced cell cycle arrest and cell death (10% to 60% of apoptotic cells compared to the controls after 24 to 96 hours, respectively) in MM cell lines. The activation of the apoptotic pathway was confirmed by western blot analysis at different time points. Moreover we observed an irreversible commitment to cell death after 48 hours of exposure. 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. EZN2968 showed moderate increase in efficacy in combination with conventional agents such as Melphalan, and immunomodulatory agents (Thalidomide and Lenalidomide). No significant effect on cell viability was observed on peripheral blood mononuclear cells and CD34+ cells from healthy donors treated with increasing doses of EZN2968. Evaluation of gene expression profiling modulation induced by EZN 2968 is ongoing. Our data suggest that HIF-1α inhibitor is an attractive therapeutic target for MM patients and provide the rationale for clinical evaluation of HIF inhibitor in combination with currently used MM agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B53.

Philadelphia-positive (Ph+) CML is generally regarded as a genetically heterogeneous disease and therapy with Bcr-Abl inhibitors results in high response rates. Nevertheless, resistance may develop, especially in high Sokal risk patients, and progression to BC still represents a major concern. The biological bases underlying high risk as well as the determinants of disease progression remain largely unknown. High-throughput technologies are nowadays available that allow to perform genome-wide studies with unprecedented informativity and resolution. We have integrated three such technologies - massively-parallel sequencing, gene expression profiling (GEP) by microarrays and high-resolution karyotyping by SNP-arrays - for a deeper characterization of a high-risk tyrosine kinase inhibitor-resistant patient at presentation and at the time of progression to BC. After having obtained written informed consent from her next of kin the samples collected at diagnosis, at the time of remission and at the time of progression were used for RNA and DNA extraction. Paired-end RNAseq was performed on an Illumina/Solexa Genome Analyzer. The number of 75bp-long sequence reads obtained was 40,193,384 (corresponding to 3.01 billion bases), 35,592,588 (2.7 billion bases), and 32,867,700 (2.5 billion bases) for diagnosis, remission and progression samples, respectively. Both custom scripts and published algorithms were used for read alignment and mapping against the human reference genome (NCBI build 36.1) and for subsequent single nucleotide variant (SNV) calling. Comparison of the SNVs identified in the diagnosis and relapse samples with the SNVs present in the remission sample was crucial to rule out all the inherited sequence variants besides known SNPs that were nonspecific of Ph+ leukemia cells. Twenty-nine mutations in the coding sequence of 28 genes were found at diagnosis (17 missense, 12 silent). Eighteen mutations (11 missense, 7 silent) in 18 additional genes were found to have emerged at the time of disease progression. In parallel, high-resolution ( Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1138.