Search

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.

BRAFV600E mutation is the pathogenic driver of hairy cell leukemia (HCL) found in the vast majority of cases both at onset and during recurrences. The identification of the mutated allele in blood and marrow correlates with the presence of neoplastic cells and can be considered a marker of active disease. Likewise, the absence of themutation after treatment may indicate a state of deep response. The BRAFV600E burden was measured by droplet digital polymerase chain reaction (ddPCR) and expressed as fractional abundance in 35 HCL patients at different stages of disease (onset, relapse, complete response [CR] after treatment, long-term remission) in peripheral blood and/or bone marrow (when available). Mean values of fractional abundance for patients at diagnosis, relapse and response, respectively, were 12.26%, 16.52% and 0.02% in peripheral blood and 23.51%, 13.96% and 0.26% in bone marrow. Four patients out of 6 evaluated at response were molecularly negative for BRAFV600E in peripheral blood. Mean fractional abundance in peripheral blood tested in 14 patients with long lasting CR was 0.05%, and 10 patients were BRAFV600E negative. These preliminary results suggest that ddPCR permits to assess the active tumor burden in HCL at different disease phases and support the hypothesis that some patients in CR qualify for a molecular CR.

DNA microarrays and RNA-based sequencing approaches are considered important discovery tools in clinical medicine. However, cross-platform reproducibility studies undertaken so far have highlighted that microarrays are not able to accurately measure gene expression, particularly when they are expressed at low levels. Here, we consider the employment of a digital PCR assay (ddPCR) to validate a gene signature previously identified by gene expression profile. This signature included ten Hedgehog (HH) pathways’ genes able to stratify multiple myeloma (MM) patients according to their self-renewal status. Results show that the designed assay is able to validate gene expression data, both in a retrospective as well as in a prospective cohort. In addition, the plasma cells’ differentiation status determined by ddPCR was further confirmed by other techniques, such as flow cytometry, allowing the identification of patients with immature plasma cells’ phenotype (i.e., expressing CD19+/CD81+ markers) upregulating HH genes, as compared to others, whose plasma cells lose the expression of these markers and were more differentiated. To our knowledge, this is the first technical report of gene expression data validation by ddPCR instead of classical qPCR. This approach permitted the identification of a Maturation Index through the integration of molecular and phenotypic data, able to possibly define upfront the differentiation status of MM patients that would be clinically relevant in the future.

Background MM is a hematological malignancy always evolving from pre-malignant stages, with progressive increase of genomic complexity. MM is characterized by a large abundance of copy number alterations (CNA); many of them, regarded as “driver”, stack up progressively from early tumor stages, causing biological changes that give rise to tumor hallmarks and malignant phenotypes. The combined application of whole genome analysis and mathematical models allows to deeply describe these alterations and to infer their order of acquisition during oncogenesis from their clonality levels, assuming that clonal ones are more ancestral than subclonal. Aims: (1) To define the temporal order of acquisition of CNA, leading to the onset of symptomatic MM and (2) to define a scoring model able to stratify patients (pts) according to the ancestrality of the alterations observed in their genomic landscape. Methods Genomic data collected from a total of 1384 newly diagnosed MM pts were included in the study: SNPs array data were collected from 514 pts of our Institution (BO dataset); in 870 pts, WES data were downloaded from CoMMpass study. CN calls and clonality levels were harmonized by an analysis pipeline including ASCAT, GISTIC v2 and custom R scripts. Timing estimates were obtained with BradleyTerry2 package. Survival analysis were performed on R. Results A full call-set of CNAs was obtained by harmonizing BO and CoMMpass datasets. The clonality information was first extrapolated from the whole call-set, to define the temporal order of acquisition of non-primary CNAs. CNAs were then accurately ranked, by using the obtained timing estimates, characterized by a quite narrow confidence interval. Of interest, chr 1q gains and chr 13q losses were frequently clonal and ranked as ancestral events, whereas chr 17p losses were late occurring events. By weighting the CNAs carried by any given pts at diagnosis with their relative timing estimate in a combinatorial process, an Ancestrality Index (AI) was defined for each pts (median AI=3.4, IQR=1.7-6.0). The AI was found to be significantly associated with progression free (PFS) and overall survival (OS) (p3.4 (i.e. with a more “ancestral” profile) had a worse outcome as compared to the rest of pts (OS 40% vs 58%, PFS 42% vs 56%, at a median follow up of 92m and 34m, p Conclusions By means of whole genome analysis and dataset harmonizing, the temporal order of acquisition of MM CNAs has been confidently described. A score reflecting the disease ancestrality of MM pts at diagnosis was generated and associated to survival outcomes. Overall, these findings support the evidence that MM pts at diagnosis carrying an excess of ancestral alterations, expected to likely be drivers, are prone to have a dismal prognosis.

Introduction Multiple Myeloma (MM) is a genetically complex disease, characterized by the recurrence of several chromosomal aberrations, which impair the disease prognosis. The use of genome-wide technologies has recently highlighted the existence of Complex Chromosomal Events (CCEs), caused by distinct phenomenons: Chromothripsis (CT), caused by single-step genomic events, and Stepwise Events (SE), consequence of multiple, small and sequential genomic events, occurring throughout subsequent cell cycles. The prognostic impact of CT in MM has not yet been fully elucidated. Aims of our study were: (1) to detect CCEs in MM, with a focus on CT, by using an original and reliable bio-informatic algorithm, (2) to characterize the genetic and genomic context of CT and (3) to correlate the presence of CTwith pts prognosis. Methods A total of 488 newly diagnosed MM patients (pts) have been included in the study. Genomic data have been obtained by SNPs arrays on bone marrow (BM) aspirates CD138+ enriched cell fractions; data were analysed by Affymetrix’s programs and R-scripts. Results An original algorithm able to discriminate among the 2 different CCEs (CT and SE),was set up and tested, by implementing the most commonly reported guidelines for CCEs identification with knowledges on MM-specific highly heterogeneous genomic context. CCEs were detected in 174 pts (36% with at least one CCE): in particular, 46/174 pts (26%) carried CT. CT can affect any chromosome, yet showing significant associations with the following genomic regions: chr1p (p=8.37E-15), chr2q (p=4.27E-8), chr11q (p=6.99E-5) and chr22q (p=5.15E-7). Pts carrying CT were more likely to carry also IgH translocations associated to bad prognosis (p=0.002, HR 3.4) and TP53 deletions (p=1.16E-5, HR=6.26). The presence of any CT event conferred hazard ratio of 1.52 (p=0.019) and 1.68 (p=0.019) to pts’ progression-free (PFS) and overall survival (OS), respectively, independently from the presence of both TP53 deletion and translocation t(4;14), as evaluated in a multivariated model. An association between CT events and XBP1 gene deletion was observed; since XBP1 expression has been correlated to an effective proteasome inhibitor (PI) therapy response, CT events particularly impacted survival expectancies of PI-treated pts, whose PFS and OS were significanlty worse than those of other pts (p=0.0098 and =0.023, respecitvely). Finally, the same CT events acquired at diagnosis were observed in those 4/55 pts, whose BM aspirates were analysed also at relapse, thus suggesting that, once occurred, CT might have a driver role for disease progression. Conclusion Our results showed that CT impact MM pts prognosis, independently from the genomic region affected and the pts’ genomic backgroung. Acknowledgment AIRC_IG2014, RF-2016.

Carfilzomib–lenalidomide–dexamethasone (KRd) has been approved for the treatment of relapsed/refractory multiple myeloma (RRMM). We conducted a retrospective analysis of 197 RRMM patients (pts) between January 2016 and March 2018 in six Italian hematologic centers, with the aim to evaluate efficacy and safety of KRd in real-life. At KRd initiation 27% carried high risk cytogenetic abnormalities (HRCA) [del17p and/or t(4;14) and/or t(14;16)], median number of prior lines of therapy was 2 (1–8), nearly all pts (96%) received prior bortezomib (18% refractory) while 45% were exposed to lenalidomide (R; 22% refractory). At the median of 12.5 months, 52% of the pts had discontinued treatment, mainly (66%) for progression. Main grade 3–4 adverse events were neutropenia (21%), infections (11%), and hypertension (6%). Overall, the response rate was 88%. The median progression-free survival (PFS) was 19.8 months and 1-year overall survival (OS) rate was 80.6%. By subgroup analysis, extended PFS and OS were observed for pts who received ≤2 prior lines of therapy (HR = 0.42, p < 0.001 and HR = 0.35, p = 0.001, respectively), not refractory to prior R (HR = 0.37, p < 0.001, and HR = 0.47, p = 0.024), without HRCA (HR = 0.33, p = 0.005 and HR = 0.26, p = 0.016) and achieving ≥ very good partial response (VGPR; HR = 0.17, p < 0.001 and HR = 0.18, p < 0.001). In conclusion, KRd demonstrated to be effective in RRMM pts treated in real-world setting, without new safety concerns. Better survival outcomes emerged for pts with ≤2 prior lines of therapy, achieving at least a VGPR, and without HRCA.

No abstract available.

In multiple myeloma, novel treatments with proteasome inhibitors (PIs) and immunomodulatory agents (IMiDs) have prolonged survival but the disease remains incurable. At relapse, next-generation sequencing has shown occasional mutations of drug targets but has failed to identify unifying features that underlie chemotherapy resistance. We studied 42 patients refractory to both PIs and IMiDs. Whole-exome sequencing was performed in 40 patients, and RNA sequencing (RNA-seq) was performed in 27. We found more mutations than were reported at diagnosis and more subclonal mutations, which implies ongoing evolution of the genome of myeloma cells during treatment. The mutational landscape was different from that described in published studies on samples taken at diagnosis. The TP53 pathway was the most frequently inactivated (in 45% of patients). Conversely, point mutations of genes associated with resistance to IMiDs were rare and were always subclonal. Refractory patients were uniquely characterized by having a mutational signature linked to exposure to alkylating agents, whose role in chemotherapy resistance and disease progression remains to be elucidated. RNA-seq analysis showed that treatment or mutations had no influence on clustering, which was instead influenced by karyotypic events. We describe a cluster with both amp(1q) and del(13) characterized by CCND2 upregulation and also overexpression of MCL1, which represents a novel target for experimental treatments. Overall, high-risk features were found in 65% of patients. However, only amp(1q) predicted survival. Gene mutations of IMiD and PI targets are not a preferred mode of drug resistance in myeloma. Chemotherapy resistance of the bulk tumor population is likely attained through differential, yet converging evolution of subclones that are overall variable from patient to patient and within the same patient.

BACKGROUND: Multiple Myeloma (MM) maintenance therapy is a low intensive, prolonged treatment, commonly administered to newly diagnosed patients (pts) at the end of front-line regimens. Lenalidomide (LEN) is considered the best available maintenance option for MM, the actual benefits or disadvantages of a LEN-based maintenance and its potential role as “selective pressure” on MM sublcones are still unclear. AIM: In this study we estimated the role of LEN maintenance therapy in eliciting genomic changes in a cohort of MM pts homogeneously up-front treated. PATIENTS-METHODS: Whole genome Copy Number Alterations (CNA) was obtained by SNPs array in 54 pts samples collected both at diagnosis(D) and at first relapse(R). Pts had an high-risk (HR) disease, defined by a median TTP of 29m. A custom gene-level CN calling algorithm was set up, to compute the evolution of every gene CN value and the genomic evolutive trajectories associated to changes of these values. High-risk genomic loci were defined using GISTIC to derive target genomic relevant for MM biology. After PIs induction therapy, 31/54 pts were treated with HD chemotherapy followed by either single or double ASCT; LEN maintenance therapy was then offered both to 20/31 auto-transplanted and to 6/23 not auto-transplanted pts. RESULTS: Three main evolutive trajectories (linear L, drift D, and branching B) were defined according to the CN changes' direction, reflecting a putative positive, negative, or both positive and negative selective pressure, respectively. A fourth, stable (S) trajectory was also observed, characterized by the absence of CN changes. Overall, 29, 15 and 10 pts relapsed with B/D, L and S pattern, respectively; at R, all LEN-treated pts changed their sub-clonal architecture: a B/D evolutive pattern characterized 70% of pts. By contrast, genome remained mostly stable in 61% of not-treated pts. We then focused on CN changes of specific chromosomal regions and/or genomic loci identified as high-risk, whose prognostic role has been already established in MM (i.e. TP53, CDKN2C, CKS1B). When present at D, these CNA tended to persist throughout the disease course, regardless of whether pts received or not maintenance. The emersion of any of these CNAs at R was widely observed both in pts receiving or not maintenance, whereas a negative selective pressure over them was more likely to occur in pts receiving maintenance, as compared to the others (50% vs 11% of B/D trajectories in LEN-treated vs not-treated pts, respectively). Strikingly, in LEN-treated pts, the extension of both TTP and OS was favored by the extinction and/or negative selection (B/D patterns) of the HR CNAs, and shortened by their stability or positive selection (L/S patterns) (median TTP 46 vs 32m HR=3.6, p=0.01; median OS 111 vs 63m HR 5.7, p=0.04 in B/D vs L/S pts, respectively). On the contrary, the absence of maintenance selective pressure seemed to affect neither the evolution trajectory, nor the clinical course of not-treated pts. CONCLUSION: The extinction of sub-clones carrying HR lesions in pts receiving maintenance therapy is likely to be associated to the negative selection exerted by the therapy. This might explain the extended survival of these pts. On the contrary, the subclones of pts not receiving maintenance might randomly evolve, due to the absence of a specific selective pressure. Citation Format: Andrea Poletti, Vincenza Solli, Marina Martello, Enrica Borsi, Lucia Pantani, Agboyi Lakpo, Silvia Armuzzi, Luca Nunzio Cifarelli, Elena Zamagni, Paola Tacchetti, Serena Rocchi, Katia Mancuso, Ilaria Rizzello, Giulia Marzocchi, Nicoletta Testoni, Luca Dozza, Maria Teresa Petrucci, Anna Marina Liberati, Giuseppe Rossi, Mario Boccadoro, Michele Cavo, Carolina Terragna. Negative selective pressure exerted by maintenance therapy promotes the extinction of sub-clones carrying high-risk lesions in multiple myeloma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2700.

Background The introduction of novel agents into the therapeutic algorithm of Multiple Myeloma (MM) has led to remarkable improvements in the rate of Minimal Residual Disease (MRD) negative status. Sustained MRD negativity is a robust prognosticator and a driver of treatment strategies. Therefore, it is expected that MRD molecular tracking would become critical in the management of MM, and could be included in daily clinical practice. Aim To evaluate the performances of next generation sequencing (NGS), implemented as best practice in MM MRD daily evaluation. Methods A cohort of 166 newly diagnosed transplant-eligible and -ineligible MM pts were screened to define the ID clonotypes. MRD was firstly assessed at achievement of at least a very good partial response during maintenance, and thereafter once a year. In high-risk pts, MRD was also detected between 1° and 2° autologous stem-cell transplantation (ASCT). The ID clonotypes screening has been performed by NGS, using an assay covering IgH and Igk genes (Invivoscribe®). The MRD tracking has been done both by conventional ASO-qPCR and by NGS. Data were analyzed by Lymphotrack Dx and MRD software (Invivoscribe®). Results The ID clonotypes screening was successful in 158/166 pts (95%), even though a small proportion of pts resulted polyclonal (14/158). Overall, 63, 27 and 54 pts had IgH, Igk and IgH/k ID clonotypes, respectively, mainly restricted to the VH3 (47%) family genes. MRD assessment was performed by NGS in 124/144 pts (overall, 96 MRD samples for 64 pts monitored to date), with 10-5 sensitivity in most cases (75/96). 44 pts were assessed during maintenance and 23 achieved MRD-negativity. On the contrary, 20 pts were evaluated between 1° and 2° ASCT, resulting MRD-positive in all cases. Overall, few FU samples (13/96) were reported as “positive-not-quantifiable” (PNQ), since had residual disease levels of 10-7, much lower than the internal control. In a subset of 20 pts with trackable IgH/Igk sequences, pts-specific assays were designed, according to the EuroMRD guidelines. In these pts, MRD was tested both by ASO-qPCR (including a standard curve and healthy donor cells, to define the sensitivity and the specificity, respectively) and by NGS. Overall, ASO-qPCR allowed the tracking of a single MRD clone (with up to 10-4 sensitivity) in 12/20 pts (60%), whereas NGS was feasible in all of them, confirming the ASO-qPCR results, yet with higher sensitivity and >95% confidence. Moreover, in 2 cases, NGS allowed to disclose ASO-qPCR PNQ results due to nonspecific amplification, thus unveiling MRD negativity with 10-5 sensitivity. Conclusion NGS has been successfully implemented as best practice in the management of MM pts, showing the superiority of this method over ASO-qPCR conventional approach. The ID clonotype was defined in almost all pts included in the workflow, thus allowing their MRD molecular monitoring, according to the clinical requirements. Acknowledgment AIRC IG2018-22059

Background Multiple Myeloma (MM) is a plasma cell (PC) disorder characterized by the presence of multiple lytic lesions at the time of diagnosis. Recently, cell-free DNA (cfDNA) has proven to resume the heterogeneity of spatially distributed clones. However, the potential of cfDNA to track the evolutionary dynamics and the heterogeneity of MM, possibly anticipating the emergence of therapy resistant residual cells, remains to be confirmed. Aim of this study is to evaluate cfDNA at diagnosis and during follow-up in order to compare this approach with both bone marrow (BM) molecular and whole-body imaging residual disease assessment. Methods A total of 97 patients (pts) were screened at baseline and during follow-up with 18F-FDG PET/CT and NMR, and molecularly assessed by Ultra Low Pass-Whole Genome Sequencing (ULP-WGS). ULP-WGS was used to characterize both the neoplastic PC clone from BM (gDNA) and the cfDNA from peripheral blood. Data were analysed by ichorCNA and Clonality R packages. Results Overall, cfDNA tumour fraction (TF) at diagnosis was significantly lower as compared to gDNA TF [median (M) TF: 3.0%vs.74.4%, respectively]. Nevertheless, high cfDNA TF levels (49/97 pts = 50%, M cfDNA TF: 10%, range : 3.0-40.6%) correlated with high gDNA TF levels (M gDNA TF: 84%, range: 5.9-95.2%). Similarly, a significant correlation was shown between cfDNA TF and the BM plasma cells clone throughout disease progression, highlighting a parallel dynamics of tumour burden from SMM to MM to +3m post-induction (cfDNA TF vs. % CD138+/CD38+ BM cells: 1.5 vs 1.0; 3.0 vs. 2.3; 1.2 vs. 0.01, respectively, p: 0.008). Pts with high cfDNA TF were likely to have more sites of extramedullary disease (EMD) at PET-CT, a higher number of PET lesions and higher tumour metabolic activity, as compared to pts with low TF (EMD 8/49, 17% vs. 2/49, 3.4%; M n. PET lesions: 8 vs. 2; SUVmax: 12.9 vs. 3.9). Similarly, bone involvement as detected by MRI, was more evident in pts with high cfDNA TF (M n. focal lesions: 6 vs. 1). More interestingly, after 3 cycles of induction therapy, imaging and cfDNA TF dynamics were concordant: indeed, in those cases where FDG activity and focal lesions still persisted (4/10=40%: SUVmax 5.8; >2 PET lesions), cfDNA TF as well was still detectable (>1 TF).Finally, the comparison between cfDNA and BM genomic profiles showed an overall concordance of the copy number alterations (CNAs) landscape in most patients (83/97; 86.4%), whereas three patients (10/97; 10,3%) displayed a different genomic profile in cfDNA, as compared to BM. Conclusions although only few cases showed genomic evidence of spatial heterogeneity, in pts with high cfDNA TF, imaging data overall suggested a propensity to a metastatic spread of the disease. Future studies will be addressed to exploit the use of cfDNA in disease monitoring. Acknowledgements AIRC IG2018-22059.

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.

Growing evidence highlights the endocannabinoid (EC) system involvement in cancer progression. Lipid mediators of this system are secreted by hematopoietic cells, including the ECs 2-arachidonoyl-glycerol (2AG) and arachidonoyl-ethanolamide (AEA), the 2AG metabolite 1AG, and members of N-acylethanolamine (NAE) family&mdash, palmitoyl-ethanolamide (PEA) and oleoyl-ethanolamide (OEA). However, the relevance of the EC system in myeloproliferative neoplasms (MPN) was never investigated. We explored the EC plasma profile in 55 MPN patients, including myelofibrosis (MF, n = 41), polycythemia vera (PV, n = 9), and essential thrombocythemia (ET, n = 5) subclasses and in 10 healthy controls (HC). AEA, PEA, OEA, 2AG, and 1AG plasma levels were measured by LC&ndash, MS/MS. Overall considered, MPN patients displayed similar EC and NAE levels compared to HC. Nonetheless, AEA levels in MPN were directly associated with the platelet count. MF patients showed higher levels of the sum of 2AG and 1AG compared to ET and PV patients, higher OEA/AEA ratios compared to HC and ET patients, and higher OEA/PEA ratios compared to HC. Furthermore, the sum of 2AG and 1AG positively correlated with JAK2V617F variant allele frequency and splenomegaly in MF and was elevated in high-risk PV patients compared to in low-risk PV patients. In conclusion, our work revealed specific alterations of ECs and NAE plasma profile in MPN subclasses and potentially relevant associations with disease severity.

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.

// Carolina Terragna 1 , Daniel Remondini 2 , Marina Martello 1 , Elena Zamagni 1 , Lucia Pantani 1 , Francesca Patriarca 3 , Annalisa Pezzi 1 , Giuseppe Levi 2 , Massimo Offidani 4 , Ilaria Proserpio 5 , Giovanni De Sabbata 6 , Paola Tacchetti 1 , Clotilde Cangialosi 7 , Fabrizio Ciambelli 8 , Clara Virginia Vigano 9 , Flores Angela Dico 1 , Barbara Santacroce 1 , Enrica Borsi 1 , Annamaria Brioli 1 , Giulia Marzocchi 1 , Gastone Castellani 2 , Giovanni Martinelli 1 , Antonio Palumbo 10 , Michele Cavo 1 1 ”Seragnoli” Institute of Hematology, Department of Experimental, Diagnostic and Specialty Medicine (DIMES), Bologna University School of Medicine, Bologna, Italy 2 Department of Physics and Astronomy (DIFA), Bologna University, Bologna, Italy 3 Clinica Ematologica, DISM, University of Udine, Udine, Italy 4 Clinica di Ematologia, A.O.U. Ospedali Riuniti di Ancona, Ancona, Italy 5 U.O Oncologia Medica, Ospedale di Circolo e Fondazione Macchi, Varese, Italy 6 Ematologia Clinica, A.O.U. Ospedali Riuniti, Trieste, Italy 7 Hematology Division UTMO, Azienda “Ospedali Riuniti Villa Sofia-Cervello” Presidio Ospedaliero V.Cervello, Palermo, Italy 8 S.C.Oncologia Medica, A.O. Sant’Antonio Abate, Gallarate, Varese, Italy 9 Unita Operativa di Ematologia, Istituti Ospitalieri di Cremona, Cremona, Italy 10 Myeloma Unit, Division of Hematology, University of Torino, A.O.U. Citta della Salute e della Scienza di Torino, Torino, Italy Correspondence to: Michele Cavo, e-mail: michele.cavo@unibo.it Keywords: multiple myeloma, gene expression profile, SNPs, VTD, complete response Received: August 06, 2015 Accepted: September 16, 2015 Published: November 09, 2015 ABSTRACT The prime focus of the current therapeutic strategy for Multiple Myeloma (MM) is to obtain an early and deep tumour burden reduction, up to the level of complete response (CR). To date, no description of the characteristics of the plasma cells (PC) prone to achieve CR has been reported. This study aimed at the molecular characterization of PC obtained at baseline from MM patients in CR after bortezomib-thalidomide-dexamethasone (VTD) first line therapy. One hundred and eighteen MM primary tumours obtained from homogeneously treated patients were profiled both for gene expression and for single nucleotide polymorphism genotype. Genomic results were used to obtain a predictor of sensitivity to VTD induction therapy, as well as to describe both the transcription and the genomic profile of PC derived from MM with subsequent optimal response to primary induction therapy. By analysing the gene profiles of CR patients, we identified a 5-gene signature predicting CR with an overall median accuracy of 75% (range: 72%–85%). In addition, we highlighted the differential expression of a series of genes, whose deregulation might explain patients’ sensitivity to VTD therapy. We also showed that a small copy number loss, covering 606Kb on chromosome 1p22.1 was the most significantly associated with CR patients.

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.

High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a standard frontline therapy for multiple myeloma (MM). Therapeutic options for patients with relapsed MM after ASCT include novel agents in different combos, salvage ASCT (sASCT), and allogeneic transplant, with no unique standard of care. We retrospectively analyzed 66 MM patients who relapsed after up-front single or double ASCT(s) and received novel agent-based sASCT at five Italian centers. Median event-free survival from up-front ASCT(s) to first relapse (EFS1) was 44 months. Seventy-three percent of patients received sASCT at first disease progression. Re-induction regimens were bortezomib based in 87% of patients. Response to re-induction therapy included complete response (CR) 18%, ≥ very good partial response (VGPR) 48%, and overall response rate (ORR) 83%. Response to sASCT included CR 44%, ≥ VGPR 77%, and ORR 94%. With a median follow-up of 24 months after sASCT, 39 patients experienced disease progression. Median EFS from sASCT (EFS2) was 17 months. Median overall survival from ASCT (OS1) and sASCT (OS2) was 166 and 43 months, respectively. EFS2 and OS2 were significantly shorter in patients with EFS1 ≤ 24 months, in patients who did not receive sASCT at first disease progression and in patients with extramedullary disease (EMD). In multivariate analysis, EFS1 ≤ 24 months was associated with shorter EFS2 and OS2, EMD was associated with shorter EFS2, and

A subanalysis of the GIMEMA-MMY-3006 trial was performed to characterize treatment-emergent peripheral neuropathy (PN) in patients randomized to thalidomide-dexamethasone (TD) or bortezomib-TD (VTD) before and after double autologous transplantation (ASCT) for multiple myeloma (MM). A total of 236 patients randomized to VTD and 238 to TD were stratified according to the emergence of grade ≥2 PN. Gene expression profiles (GEP) of CD138+ plasma cells were analyzed in 120 VTD-treated patients. The incidence of grade ≥2 PN was 35% in the VTD arm and 10% in the TD arm (P

There has been a fast progress in clinical use of antibody-based immunotherapy, given the superior efficacy commonly achieved in clinic and the limited toxicity. However, personalization of treatment remains of major importance, both to achieve better clinical performance for monotherapy and to identify the best combinations on a patient-by-patient basis. Predicting patient's response is complex due to the need to characterize both tumor response and immunologic mechanisms possibly activated by the therapy, including antibody dependent cellular cytotoxicity (ADCC). We present the Inter-Cell Networking Profiling (ICNP), a novel analytical method enabling a comprehensive and precise characterization of the modulatory effect of immunotherapies on immune-tumor cell interactions. ICNP works on the Open Microwell (OMW) microfluidic system which recreates 20,000 unique cell clusters from ex-vivo patient samples and exposes them to anticancer drugs. We validated the ICNP using multiple myeloma (MM) patient samples to characterize the efficacy of Daratumumab, an anti-CD38 antibody (Ab). Bone marrow samples in EDTA were collected from 11 MM patients. 8 samples were processed by Ficoll-Paque, preserving the original composition of effector (E) and target (T) cells, i.e. NK and plasma cells respectively. 3 samples were processed to obtain co-cultures of WBC depleted of plasma cells (which include NK cells) and U-266 MM cell line. NK and plasma cells were stained with anti-CD16/CD56 and anti-CD138 fluorescently-labeled Abs, respectively. Propidium Iodide (PI) was used as death marker. A statistical model was created to project the optimal experimental setup (E:T ratio, cell concentration) to maximize the co-localization of E/T cells in the 20,000 microwells of the OMW platform. After seeding, cells were incubated with Daratumumab 10µg/mL or no drug and analyzed through fluorescence time lapse microscopy for up to 12 hours. ICNP analysis first separates microwells with both E/T cells in close proximity from those not featuring both cell types (Fig 1A). Then, anti-CD38 efficacy is evaluated in microwells with E/T co-localization, thus implementing a miniaturized ADCC assay (Fig 1B). At the same time, spontaneous NK activity is measured in microwells with E/T co-localization and no drug, while direct effect of the drug on target cells can be measured in microwells with T but not E cells. We first validated our statistical model of co-localization in microwells against the actual number of wells with E/T co-localization (correlation R2: 0.79-0.97, n=5). Then, we characterized the immune composition of 8 MM patients samples with the OMW, finding that E/T cell fractions were in the ranges 5-21% (E) and 1-28% (T). Interestingly, according to our statistical model, co-localization occurs in at least 1% of microwells for all the 8 samples, making ICNP applicable with good statistical significance in the OMW system on ex-vivo clinical samples without any pre-enrichment. Finally, ICNP was evaluated on 4 cases (3 obtained by mixing patient's WBC with U-266 MM cell line and 1 complete MM patient sample). We measured the effect of anti-CD38 Ab using i) the standard approach, considering all microwells regardless of the co-presence of E and T; ii) ICNP approach, considering only microwells featuring E/T co-localization. Results show that drug effect is much evident with ICNP in all the 4 cases with an average increase in target cell death of 40%, indicating a higher sensitivity of this approach than the averaged analysis (Fig 1C, right). Importantly, in one case, the standard analysis did not identify significant differences between anti-CD38 treated and control cells, that could instead be observed with ICNP (p ICNP proved to enable a comprehensive profiling of the immune system by evaluating in one test the immune composition and the fitness of immune cells, both native and drug-treated. These results open the opportunity to develop functional precision medicine approaches for predicting patient's response to drugs with immune-mediated mechanisms of action. AB and RR equally contributed Figure 1 Disclosures Bettelli: CellPly.S.r.l.: Employment. Ruggiano:Cellply S.r.l.: Employment. Bocchi:CellPly S.r.l.: Employment. Rocchi:CellPly.S.r.l.: Employment. Faenza:CellPly S.r.l.: Employment. Zamagni:Janssen: Honoraria, Other: Advisory board, Speakers Bureau; Amgen: Honoraria, Other: Advisory board, Speakers Bureau; BMS: Honoraria, Other: Advisory Board, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Sanofi: Honoraria, Other: Advisory Board, Speakers Bureau; Celgene Corporation: Honoraria, Other: Advisory board, Speakers Bureau. Bettelli:CellPly S.r.l.: Employment. Rambelli:CellPly S.r.l.: Employment. Guadagnuolo:CellPly S.r.l.: Employment. Pecorari:CellPly S.r.l.: Employment. Giulianelli:CellPly S.r.l.: Employment. Pisani:CellPly S.r.l.: Employment. Biscarini:CellPly S.r.l.: Employment. Cavo:amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Guerrieri:CellPly S.r.l.: Equity Ownership. Bocchi:CellPly S.r.l.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Introduction The differentiation status plasticity of Multiple Myeloma (MM) plasma cells (PCs) is an adaptive strategy that might confer a specific fitness to tumour cells, enabling their interaction with an evolving microenvironment. Therefore, proliferation rates of MM clones are not constant, and the immunophenotypic profile of the most skilled MM PCs might be the expression of specific genetic and genomic programs, that emerge under the therapeutic pressure and promote tumour development. However, the genomic background that supports any diverse plasma cell differentiation phenotypes has not yet been inferred. Aim To correlate the genetic and genomic background with the immunophenotypic profile of MM clones at diagnosis, in order to stratify patients (pts) according to a Maturation Index, and ultimately to evaluate the impact of this stratification on the disease outcome. Patients and Methods 117 newly diagnosed MM (NDMM) pts, mostly treated up-front with a proteasome inhibitor (PI) -based treatment, were included in the study. For each pts, both neoplastic PCs and CD19+ B cells compartments were characterized by 8-color multi-parameter flow cytometry analysis, combining CD138-PE, CD38-PE-Cy7, CD56-APC-Cy7, CD20-APC, CD19-FITC, CD27-APC, CD45-PerCp, CD28-APC, CD117-FITC, CD28-APC, CD81-APC, IgK-APC and IgL-FITC. In a subgroup of pts, both a SNP array profile for copy number alterations (CNAs) and a 25-genes targeted mutational panel were assessed in CD138+ PCs. A custom ddPCR assay was also employed to evaluate through a 10-Hh gene signature the self-renewal status of PCs. Results In order to define a Maturation Index, pts were evaluated at different levels of heterogeneity for: a) differential expression of CD19/CD81 markers; b) level of chromosomal instability (CIN) and point mutations; d) self-renewal status. Based on the co-expression of CD19 and CD81 markers, pts were stratified in 3 different subgroups, recapitulating a progressive PC maturation process: the most immature, which included pts with CD19+/CD81+ PCs (18/117 = 15%), the intermediate, including CD19-/CD81+ PCs (42/117 = 36%) and the most mature, including CD19-/CD81- PCs (57/117 = 49%). The advanced PC differentiation status characterizing this latter subgroup was further confirmed by the high expression of CD28 and CD44, and the reduced expression of CD20, CD27 and CD45, commonly associated with less mature stages of the disease (p CIN was defined according both to the total CNAs count and to the number of chromosomes' breakpoints (BPs). Pts with an advanced differentiation status displayed a higher number of total CNAs and BPs (median tot. CNAs = 550 vs. 105, and median BPs nr. = 18.5 vs. 8 in CD19-81- and CD19+81+ pts, respectively, p Finally, a more mature PC clone was significantly related to a higher prevalence of unfavourable features at baseline (e.g. ≥ 3 PET lesions, ≥ 100 k/l ratio, ISS III disease stage p Conclusions A Maturation Index defined NDMM pts with an advanced differentiation status both at immunophenotypic and molecular level, finding lastly associated with a prevalence of poor prognostic features. Chromosomal instability, together with cellular phenotypic plasticity, represents an important, yet poorly defined, mechanism by which MM clones accelerate their own evolution and survival. Acknowledgements: AIRC_IG2014-15839, RF-2016-02362532. Disclosures Mancuso: Celgene: Honoraria, Speakers Bureau; Amgen: Honoraria; Takeda: Honoraria; Janssen: Honoraria. Zamagni:Sanofi: Honoraria, Other: Advisory Board, Speakers Bureau; Celgene Corporation: Honoraria, Other: Advisory board, Speakers Bureau; Janssen: Honoraria, Other: Advisory board, Speakers Bureau; Amgen: Honoraria, Other: Advisory board, Speakers Bureau; BMS: Honoraria, Other: Advisory Board, Speakers Bureau; Takeda: Honoraria, Speakers Bureau. Tacchetti:Oncopeptides: Honoraria, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; BMS: Honoraria; Celgene: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Janssen: Honoraria. Zinzani:CELGENE: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PORTOLA: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ROCHE: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GILEAD: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; JANSSEN-CILAG: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SERVIER: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SANDOZ: Membership on an entity's Board of Directors or advisory committees; IMMUNE DESIGN: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; EUSAPHARMA: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; KYOWA KIRIN: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SANOFI: Consultancy; MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CELLTRION: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; VERASTEM: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Cavo:janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

In Multiple myeloma (MM) no treatment has a curative potential and even complete response to novel agents such as proteasome inhibitors (PIs) and immunomodulatory agents (IMiDs) are followed by relapse over time. Next generation sequencing (NGS) has showed how MM at diagnosis is defined by several somatic mutations, but only few drivers, even fewer "druggable" mutations, and many found at a subclonal level. At relapse, targeted studies have shown occasional mutations in drug target genes but the genomic and transcriptomic determinants of chemoresistance in MM remains elusive. We selected 42 MM patients refractory to both lenalidomide and PIs. Whole exome sequencing was performed in 40 of them, and RNAseq in 27. Clinical annotation was available for all patients. Standard analysis pipelines where applied to analyze mutations, copy number alterations (CNAs), mutational signatures, gene expression and expressed mutations. Patients received a median of 3 lines of treatment, with median overall survival of 14.6 months from sampling. We found a median of 77.5 mutations per patient, which is more than what reported at diagnosis (Bolli et al, Nature Communications 2014;5:2997). 100% of samples showed evidence of subclonality, and 37% of them exhibited a higher number of subclonal than clonal variants. Therefore, even at this advanced stage the MM genome is evolving and is composed of different subclones that may display different chemosensitivity. The mutational landscape was also different. TP53 mutations were the second most common after KRAS (20% and 17.5%, respectively). Interestingly TP53 mutations all clustered in patients receiving bortezomib as the last line of treatment. Only 2 patients showed a CRBN mutation, both subclonal. Combining mutations and CNA analysis, the TP53 pathway was the most frequently inactivated (45% of patients). Altogether, mutations or deletions of genes in the CRBN E3 ubiquitin ligase complex were found in 32.5% of patients, while proteasomal subunit genes were infrequently hit. Refractory cases were also uniquely characterized by a novel signature linked to exposure to alkylating agents, whose activity was more pronounced after high-dose melphalan suggesting a mutagenic effect of the drug on residual cells at the time of transplant. Whether this has any pathogenetic role on the disease course remains to be elucidated. RNAseq analysis did not show any influence of treatment or mutational data on the clustering of samples, which was mainly influenced by karyotypic events. The main cluster was composed by non-hyperdiploid patients with both amp(1q) and del(13): these showed CCND2 and MCL1 upregulation, the latter representing a marker of venetoclax resistance and novel target of experimental treatments. Only 26.3% of mutations were expressed, and this correlated with the clonality level of the mutation. However, most mutations in driver genes were expressed, with the notable exception of those causing nonsense mediated decay. Overall, classical high-risk features or CRBN pathway mutations were found in 65% of the cohort. However, only amp(1q) predicted survival in our cohort. The lack of prognostic value of high-risk lesions is likely explained by a higher prevalence of such features in double-refractory stages. Our data suggest that gene mutation is not a preferred mode of evolution of drug resistance in MM. Chemoresistance of the bulk tumor population is likely attained though differential, yet converging evolution of different subclones that are overall highly variable from patient to patient and within the same patient. Disclosures Kastritis: Prothena: Honoraria; Genesis: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria. Dimopoulos:Sanofi Oncology: Research Funding. Cavo:bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Corradini:Janssen: Honoraria, Other: Travel Costs; Takeda: Honoraria, Other: Travel Costs; Jazz Pharmaceutics: Honoraria; Gilead: Honoraria, Other: Travel Costs; Daiichi Sankyo: Honoraria; Celgene: Honoraria, Other: Travel Costs; Amgen: Honoraria; AbbVie: Consultancy, Honoraria, Other: Travel Costs; KiowaKirin: Honoraria; Kite: Honoraria; BMS: Other: Travel Costs; Sanofi: Honoraria; Servier: Honoraria; Roche: Honoraria; Novartis: Honoraria, Other: Travel Costs. Bolli:Celgene: Honoraria; Novartis: Honoraria; Gilead: Other: travel expenses.

Multiple Myeloma (MM) maintenance therapy is a low intensive, prolonged treatment, commonly administered to newly diagnosed patients (pts) at the end of fixed-time, front-line regimens. Lenalidomide (LEN) is considered the best available maintenance option for MM able to prolong pts survival. However, the actual benefits or disadvantages of a LEN-based continuous maintenance and its potential role as selective pressure able to induce genomic changes are still unclear. The identification of specific therapy-driven modifications has been so far prevented by the scarce number of homogeneously-treated cohorts of pts with paired samples (diagnosis, D and relapse, R). In this study we estimated the role of LEN maintenance therapy in eliciting genomic changes in a cohort of MM pts homogeneously up-front treated with PI-based regimens. Whole genome Copy Number Aberrations (CNAs) landscape was obtained by SNPs array in 54 pts whose BM-CD138+ were collected both at D and at first R. Pts had an high-risk (HR) disease, as defined both by the presence of HR cytogenetic aberration at baseline in 81% of pts and by the 29 m median TTP. RawCopy 1.10 was used for the segmentation of SNPs array signals; samples' purity was adjusted by using manually reviewed ASCAT solutions. A custom gene-level CN calling approach was set up, to match every gene's CN value at D and R, thus generating genomic evolutive patterns based on changes of these values. Finally, high-risk genomic loci were computed using GISTIC 2.0_v7 to derive focal regions with oncogene and/or tumor suppressor genes relevant for MM biology. After induction therapy, 31/54 pts were treated with HD chemotherapy followed by either single or double ASCT; LEN maintenance therapy was then offered both to 20/31 auto-transplanted and to 6/23 not auto-transplanted pts. Three main evolutive trajectories (linear L, drift D, and branching B) were defined according to the CN changes' direction, reflecting a putative positive, negative, or both positive and negative selective pressure, respectively. A fourth, stable (S) trajectory was also observed, characterized by the absence of CN changes between D and R. Overall, 29, 15 and 10 pts relapsed with B/D, L and S pattern, respectively; at R, all LEN-treated pts quantitively and qualitatively changed their sub-clonal architecture: a B/D evolutive pattern characterized 70% of pts. By contrast, genome remained mostly stable in 61% of not-treated pts. We then focused on CN changes of specific chromosomal regions and/or genomic loci, whose prognostic role has been already established in MM (i.e. CN losses of TP53 on chr17p, RB1 on chr13q, CYLD on chr16q, CDKN2C and FAM46C on chr1p; CN gain of CKS1B on chr1q). When present at D these CNAs tended to persist throughout the disease course regardless of whether pts received or not maintenance. The emersion of any of these CNAs at R was widely observed both in pts receiving or not maintenance, whereas a negative selective pressure over them was more likely to occur in pts receiving maintenance, as compared to the others (50% vs 11% of B/D trajectories in LEN-treated vs not-treated pts, respectively). Strikingly, in LEN-treated pts, the extension of both TTP and OS was favored by the extinction and/or negative selection of the HR CNAs (B/D patterns), and shortened by their stability or positive selection (L/S patterns) (median TTP 46 vs 32 m HR=3,6 CI 1,2-10,6, p=0.01; median OS 111 vs 63 m HR 5,7 CI 1,1-32,4, p=0.04 in B/D vs L/S pts, respectively). On the contrary, the absence of maintenance selective pressure seemed to affect neither the evolution trajectory, nor the clinical course of not-treated pts (fig 1). The extinction of sub-clones carrying HR lesions in pts receiving maintenance therapy is likely to be associated to the negative selection exerted by the therapy itself. This might explain the extended survival of pts receiving maintenance and relapsing with B/D evolution patterns, as compared to that of pts whose genomic landscape either tended to remain stable or let the prevalent emersion of HR genomic features. On the contrary, the sub-clonal architecture of pts not receiving maintenance seemed to randomly evolve, due to the absence of a specific therapy-related selective pressure. Overall, information on the genomic changes occurring throughout the disease course might be relevant for the recognition of pts most benefitting from a continuous therapy. Thanks to AIRC_IG2014-15839, RF-2016-02362532 Disclosures Zamagni: Takeda: Honoraria, Speakers Bureau; Sanofi: Honoraria, Other: Advisory Board, Speakers Bureau; Janssen: Honoraria, Other: Advisory board, Speakers Bureau; Amgen: Honoraria, Other: Advisory board, Speakers Bureau; BMS: Honoraria, Other: Advisory Board, Speakers Bureau; Celgene Corporation: Honoraria, Other: Advisory board, Speakers Bureau. Tacchetti:BMS: Honoraria; Takeda: Honoraria, Speakers Bureau; Janssen: Honoraria; Oncopeptides: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau. Mancuso:Celgene: Honoraria, Speakers Bureau; Janssen: Honoraria; Takeda: Honoraria; Amgen: Honoraria. Petrucci:Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Liberati:Incyte: Consultancy; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Bristol & Mayer: Honoraria; Janssen: Honoraria; Celgene: Honoraria. Rossi:Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria. Boccadoro:Bristol-Myers Squibb: Honoraria, Research Funding; Mundipharma: Research Funding; Sanofi: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie: Honoraria. Cavo:amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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.

Multiple myeloma (MM) is a malignant disorder of post-germinal center B cells, characterized by the clonal proliferation of malignant plasma cells (PCs) within the bone marrow (BM). The reciprocal and complex interactions that take place between the different compartments of BM and the MM cells result in tumor growth, angiogenesis, bone disease, and drug resistance. Given the importance of the BM microenvironment in MM pathogenesis, we investigated the possible involvement of Hypoxia-Inducible transcription Factor-1 alpha (HIF-1α) in the PCs-bone marrow stromal cells interplay. To test this hypothesis, we used EZN-2968, a 3rd generation antisense oligonucleotide against HIF-1α, to inhibit HIF-1α functions. Herein, we provide evidence that the interaction between MM cells and BM stromal cells 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 BM stromal cells were able to revert the anti-proliferative effect induced by EZN-2968. Moreover, we observed a down-modulation of cytokine-induced signaling cascades and a reduction of MM cells adhesion capability to the extracellular matrix proteins in EZN-2968-treated samples. Taken together, these results strongly support the concept that HIF-1α plays a critical role in the interactions between bone BM cells and PCs in Multiple Myeloma.

We retrospectively investigated the role of serial serum-free light chain (sFLC) evaluations in 150 multiple myeloma (MM) patients treated with first-line bortezomib-based regimens. Baseline sFLC ratio (sFLCR) identified three groups of patients - normal, lightly abnormal (

Despite significant improvement in outcomes have been observed for multiple myeloma (MM) patients over the past 10-15years, mainly due to the introduction of novel agents targeting the tumor clone and the bone marrow microenvironment, treatment of refractory and/or relapsed (RR) disease remains a challenge, particularly for patients who have failed prior bortezomib- and lenalidomide-based therapies. More recently, new drugs with different mechanisms of action, including second generation proteasome inhibitors, third generation immunomodulatory drugs, histone deacetylase inhibitors and monoclonal antibodies, have been developed and are under investigation, further increasing treatment options for RRMM patients. Overall, novel agent-based triplet combinations demonstrated superior response rates and prolonged disease control when compared with two-drug regimens in several randomized clinical trials, without adding any relevant additional toxicity. Salvage triplet therapies are likely to play a key role in overcoming drug-resistance and hold promise to further improve long-term outcomes of RRMM patients.

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.

We performed a molecular study aimed at identifying a gene expression profile (GEP) signature predictive of attainment of at least near complete response (CR) to thalidomide-dexamethasone (TD) as induction regimen in preparation for double autologous stem cell transplantation in 112 younger patients with newly diagnosed multiple myeloma. A GEP supervised analysis was performed on a training set of 32 patients, allowing to identify 157 probe sets differentially expressed in patients with CR versus those failing CR to TD. We then generated an eight-gene GEP signature whose performance was subsequently validated in a training set of 80 patients. A correct prediction of response to TD was found in 71 % of the cases analyzed. The eight genes were downregulated in patients who achieved CR to TD. Comparisons between post-autotransplantation outcomes of the 44 non-CR-predicted patients and of the 36 CR-predicted patients showed that this latter subgroup had a statistically significant benefit in terms of higher rate of CR after autotransplant(s) and longer time to progression, event-free survival, and overall survival. These results can be an important first step to identify at diagnosis those patients who will respond more favourably to a particular treatment strategy.

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by accumulation of malignant plasma cells (PCs) within the bone marrow (BM). The PCs are in close contact with stromal cells, which secrete growth factors and cytokines, promoting tumor cell growth and survival. Despite the availability of new drugs with immunomodulatory properties targeting the neoplastic clone and its microenvironment, MM is still an incurable disease, with patients experiencing subsequent phases of remission and relapse, eventually leading to disease resistance and patient death. It is now well established that the MM BM microenvironment is hypoxic, a condition required for the activation of the hypoxia-inducible factor 1 alpha (HIF-1α). It has been shown that HIF-1α is constitutively expressed in MM even in normoxic conditions, suggesting that HIF-1α suppression might be part of a therapeutic strategy. Constitutively activated HIF-1α enhances neovascularization, increases glucose metabolism, and induces the expression of antiapoptotic proteins. HIF-1α is thought to be one of the most important molecular targets in the treatment of cancer, and a variety of chemical inhibitors for HIF-1α have been developed to date. This review examines the role of HIF-1α in MM and recent developments in harnessing the therapeutic potential of HIF-1α inhibition in MM.