Barbara Santacroce

Opposite activation of the Hedgehog pathway in CD138+ plasma cells and CD138−CD19+ B cells identifies two subgroups of patients with multiple myeloma and different prognosis

Hyperactivation of the Hedgehog (Hh) pathway, which controls refueling of multiple myeloma (MM) clones, might be critical to disease recurrence. Although several studies suggest the Hh pathway is activated in CD138− immature cells, differentiated CD138+ plasma cells might also be able to self-renew by producing themselves the Hh ligands. We studied the gene expression profiles of 126 newly diagnosed MM patients analyzed in both the CD138+ plasma cell fraction and CD138−CD19+ B-cell compartment. Results demonstrated that an Hh-gene signature was able to cluster patients in two subgroups characterized by the opposite Hh pathway expression in mature plasma cells and their precursors. Strikingly, patients characterized by Hh hyperactivation in plasma cells, but not in their B cells, displayed high genomic instability and an unfavorable outcome in terms of shorter progression-free survival (hazard ratio: 1.92; 95% confidence interval: 1.19–3.07) and overall survival (hazard ratio: 2.61; 95% confidence interval: 1.26–5.38). These results suggest that the mechanisms triggered by the Hh pathway ultimately led to identify a more indolent vs a more aggressive biological and clinical subtype of MM. Therefore, patient stratification according to their molecular background might help the fine-tuning of future clinical and therapeutic studies.

Treatment optimization for Multiple Myeloma: schedule-dependent synergistic cytotoxicity of pomalidomide and carfilzomib in an in vitro and ex-vivo model

Proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs), significantly enhanced the depth of response and prolonged the survival of Multiple Myeloma (MM) patients, although not all patients respond favourably to treatment. Optimization of treatment schedules and dosages of IMiDs and PIs might lead to improved treatment efficacy. In this study we aimed at exploring the optimal schedule of IMiDs and PIs in both in vitro models, including bone marrow (BM) microenvironment simulation, and an ex-vivo model using patient-derived BM samples. MM cells were exposed to IMiDs and PIs either simultaneously or sequentially. Using the median effect method of Chou Talalay, we evaluated the combination indices for simultaneous and sequential treatment schedules. We demonstrated schedule-dependent synergistic cytotoxicity for the combination of IMiDs and PIs and a maximal apoptosis was observed in IMiDs pre-exposure schedule. The superior activity of this schedule was maintained even in BM microenvironment models, and was further confirmed using patient-derived samples. Our data overall suggest that the administration of IMiDs before PIs can improve treatment efficacy. Clinical trials are needed to investigate the most effective schedule, which could be to start the administration of IMiDs before PIs to increase cells killing.

Abstract 3189: The alternate activation of hedgehog pathway, either in CD138+ or in CD138-CD19+ multiple myeloma primary cells, impacts on disease outcome

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-Universita 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.

Abstract 3582: Chromothripsis in AML patients: A new mechanism of cancer initiation and progression

Introduction: Genomic rearrangements can drive the development of cancer through different mechanisms: chromothripsis, a catastrophic mechanism of genomic instability, could be relevant for hematological disease. Aim: To discover the mechanisms underlying the pathogenesis of Acute Myeloid Leukemia (AML), we studied chromothripsis in our cohort of patients (pts). Methods: We perform SNP Array 6.0 or Cytoscan HD Array (Affymetrix) in a cohort of 104 AML pts at diagnosis. SNP Array data were analyzed by Nexus Copy Number™ v7.5 (BioDiscovery). Results: Seven/104 pts (6.7%) showed chromothripsis events involving different chromosomes (8, 17, 11, 5 and 16). These pts had median age of 68.5 years (range 56-76), complex karyotype and high risk disease according to ELN definition. Among the pts showing chromothripsis, we compared chromosomic abnormalities of pts with de novo (5/7) and secondary (2/7) AML. De novo AML showed a prevalence of trisomy of chromosome 8 in a non-statistical way (4/5 vs 0/2), due to the low number of cases. However, we identified significant differences in the pattern of genes altered in the two groups. De novo AML had copy number gain of PEX1, ANK1, NCOA2, ESRP1, TPD52, ESRP1, ZFPM2 and MITF (p Conclusion: Our data suggest that different pathways and genomic alterations are involved in chromothripsis events in de novo and secondary AML, which could be explained by the repeated rounds of stress underwent by leukemic cells in secondary AML cases. Cytoskeleton and microtubules formation pathways appear to be the main cellular processes implicated in chromothripsis genesis, while alterations of histone acetyltransferase, immune response and antigen presentation pathways could sustain leukemic cells after chromothripsis. Acknowledgement: ELN, AIL, AIRC, PRIN, progetto Regione-Universita 2010-12(L. Bolondi), FP7 NGS-PTL project. Citation Format: Maria Chiara Fontana, Viviana Guadagnuolo, Cristina Papayannidis, Giovanni Marconi, Giorgia Simonetti, Antonella Padella, Marco Manfrini, Barbara Santacroce, Margherita Perricone, Silvia Lo Monaco, Emanuela Ottaviani, Simona Soverini, Michele Cavo, Giovanni Martinelli. Chromothripsis in AML patients: A new mechanism of cancer initiation and progression. [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 3582.

Abstract 5022: The selection of TP53 sub-clonal variants over time identifies MM patients with adverse clinical outcome

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.

Abstract 1521: High-throughput molecular profiling of Multiple Myeloma clonotypic CD19+ B cells highlights pathways potentially involved in the disease endurance

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