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Background: The phase 3 GIMEMA-MMY-3006 trial, which compared bortezomib, thalidomide, and dexamethasone (VTD) combination therapy with thalidomide and dexamethasone (TD) as induction therapy before and consolidation therapy after double autologous haematopoietic stem-cell transplantation (HSCT) for newly diagnosed multiple myeloma, showed the superiority of the triplet regimen over the doublet in terms of increased complete response rate and improved progression-free survival. We report the results from the final analysis of the study. Methods: In this randomised, open-label, phase 3 study, patients aged 18–65 years with previously untreated symptomatic multiple myeloma and a Karnofsky Performance Status of 60% or higher were enrolled at 73 centres in Italy. Patients were randomised (1:1) by a web-based system to receive three 21-day cycles of thalidomide (100 mg daily orally for the first 14 days and 200 mg daily thereafter) plus dexamethasone (total 320 mg per cycle; 40 mg on days 1–2, 4–5, 8–9, and 11–12 in the VTD regimen, and 40 mg on days 1–4 and 9–12 in the TD regimen), either alone (TD group) or with bortezomib (1·3 mg/m2 intravenously on days 1, 4, 8, and 11; VTD group). After double autologous HSCT, patients received two 35-day cycles of either the VTD or TD regimen, according to random assignment, as consolidation therapy. The primary outcome was the rate of complete response and near complete response after induction (already reported). In this updated analysis we assessed long-term progression-free survival and overall survival (secondary endpoints of the study) with an extended 10-year median follow-up, and analysed the variables influencing survival. Analysis was by intention to treat. The study is registered with ClinicalTrials.gov, NCT01134484. Findings: Between May 10, 2006, and April 30, 2008, 480 patients were enrolled and randomly assigned to receive VTD (241 patients) or TD (239 patients). Six patients withdrew consent before start of treatment. 236 (99 [42%] women) in the VTD group and 238 (102 [43%] women) in the TD group were included in the intention-to-treat analysis. The data cutoff date for this analysis was May 31, 2018. Median follow-up for surviving patients was 124·1 months (IQR 117·2–131·7). The 10-year progression-free survival estimate for patients in the VTD group was 34% (95% CI 28–41) compared with 17% (13–23) for the TD group (hazard ratio [HR] 0·62 [95% CI 0·50–0·77]; p

Interactions of malignant multiple myeloma (MM) plasma cells with the microenvironment control MM plasma-cell growth, survival, drug-resistance and dissemination. As microvascular density increases in the bone marrow in MM, we investigated whether bone marrow MM endothelial cells control disease progression via the junctional adhesion molecule-A (JAM-A). Membrane and cytoplasmic JAM-A levels were upregulated in MM endothelial cells in 111 patients with newly diagnosed MM and in 201 with relapsed/refractory MM compared to the levels in patients with monoclonal gammopathy of undetermined significance and healthy controls. Elevated membrane expression of JAM-A on MM endothelial cells predicted poor clinical outcome. Mechanistically, addition of recombinant JAM-A to MM endothelial cells increased angiogenesis, whereas inhibition of this adhesion molecule impaired angiogenesis and MM growth in two-dimensional and three-dimensional in vitro cell cultures and chorioallantoic membrane assays. To corroborate these findings, we treated MM-bearing mice with a JAM-A-blocking monoclonal antibody and demonstrated impaired MM progression, corresponding to decreased MM-related vascularity. These findings support the concept that JAM-A is an important mediator of MM progression through facilitating MM-associated angiogenesis. Elevated JAM-A expression on bone marrow endothelial cells is an independent prognostic factor for the survival of both patients with newly diagnosed MM and those with relapsed/refractory MM. Blocking JAM-A restricts angiogenesis in vitro, in utero and in vivo and represents a suitable druggable molecule to halt neo-angiogenesis and MM progression.

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.

The treatment of hairy cell leukemia (HCL) has considerably changed over years. Purine analogues, namely cladribine, now represent the treatment of choice. One hundred and eighty-four patients were followed between 1986 and 2018 and treated according to era-specific guidelines. Responses were classified by combining Consensus Resolution criteria and marrow immunohistochemistry. Patients were grouped according to the number of treatment lines they received. Patients treated first line responded in 86% of cases, with complete response (CR) in 44% of cases. Response rates remained high throughout the first four lines (84%, 81%, 79% for the second line onward, with CR in 38%, 37%, 15% of cases respectively). One hundred and twenty-two patients received cladribine as first line treatment, with a response rate of 86% and a CR rate of 54%. Among the 66 CR patients, 45 (68%) have never received further therapy: 11 patients are in continuous CR between 5 and 10 years after treatment, 14 between 10 and 20 years and three patients at more than 20 years. Median time-to-next treatment (TTNT) for frontline cladribine-treated patients was 8.2 years: partial responders had a significantly shorter median TTNT than CR patients (5.3 years vs median not reached at 25.8 years, p< 0.001). Patients with HCL require subsequent lines of therapy in more than 50% of cases. Purine analogues allow significant response rates when applied first line and upon retreatment. Some patients may enjoy long lasting treatment-free intervals after one course of cladribine.

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 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.

Personalized treatment is an attractive strategy that promises increased efficacy with reduced side effects in cancer. The feasibility of such an approach has been greatly boosted by next-generation sequencing (NGS) techniques, which can return detailed information on the genome and on the transcriptome of each patient's tumor, thus highlighting biomarkers of response or druggable targets that may differ from case to case. However, while the number of cancers sequenced is growing exponentially, much fewer cases are amenable to a molecularly-guided treatment outside of clinical trials to date. In multiple myeloma, genomic analysis shows a variety of gene mutations, aneuploidies, segmental copy-number changes, translocations that are extremely heterogeneous, and more numerous than other hematological malignancies. Currently, in routine clinical practice we employ reduced FISH panels that only capture three high-risk features as part of the R-ISS. On the contrary, recent advances have suggested that extending genomic analysis to the full spectrum of recurrent mutations and structural abnormalities in multiple myeloma may have biological and clinical implications. Furthermore, increased efficacy of novel treatments can now produce deeper responses, and standard methods do not have enough sensitivity to stratify patients in complete biochemical remission. Consequently, NGS techniques have been developed to monitor the size of the clone to a sensitivity of up to a cell in a million after treatment. However, even these techniques are not within reach of standard laboratories. In this review we will recapitulate recent advances in multiple myeloma genomics, with special focus on the ones that may have immediate translational impact. We will analyze the benefits and pitfalls of NGS-based diagnostics, highlighting crucial aspects that will need to be taken into account before this can be implemented in most laboratories. We will make the point that a new era in myeloma diagnostics and minimal residual disease monitoring is close and conventional genetic testing will not be able to return the required information. This will mandate that even in routine practice NGS should soon be adopted owing to a higher informative potential with increasing clinical benefits.

mRNA vaccines BNT162b2 and mRNA1273 are highly effective in preventing SARS-CoV-2 infection and mortality in healthy adults. However, their immunogenicity in immunocompromised Multiple Myeloma (MM) patients is less clear. We performed an observational prospective study of 96 MM patients (pts) treated at our centre, aimed at assessing the humoral and cell-mediated immune (CMI) response following the full immunization schedule. To this aim, we measured serum levels of neutralizing IgG anti Spike-protein (IgG anti S-RBD) at 1, 3, 6, 9 and 12 months after the 2 nd dose of vaccination, using the electrochemiluminescence (ECLIA) platform (Elecsys® Anti-sars-Cov-2 ECLIA assay) and evaluated CMI response in terms of pts with a SARS-CoV-2 specific IFNγ T cell response by IGRA (Interferon-Gamma Release Assays) test at 3 and 12 months after 2 nd dose. A concentration level of IgG anti S-RBD ≥0.80 U/ml was considered a seropositive result. Herein, we report preliminary data on the development of humoral response in 96 MM pts who reached the first study timepoint (1 month after 2 nd dose), compared to 54 health-care workers as controls. At vaccination, the median age of the 96 patients (51 males/45 females) was 66.5 (range 47-83) years. The median number of previous lines of therapy was 1 (range 1-11) and only 7 (7.3%) pts were not receiving active treatment. 44.8% (n=43) pts had relapse/refractory MM. Among 72 (75%) transplant-eligible pts, 63 patients had previously received autologous stem-cell transplantation (ASCT) with a median time between ASCT and vaccine of 31 (range 3-274) months. 70 (72.9%) pts had received immunomodulatory drugs (IMIDs) containing regimens, 30 (31.3%) proteasome inhibitors (PIs), 11 (11.5%) IMIDs + PIs, 32 (33.3%) anti-CD38 monoclonal antibodies (anti-CD38 moAbs). 33 (34.4%) pts were in lenalidomide (R) maintenance therapy. At vaccination, 67 (68.8%) pts were in VGPR or higher, 18 (18.7%) in PR and 11 (11.3%) in SD or PD. Immunoparesis (≥1 uninvolved Ig below lower level limit) was observed in 78 (88.6%) pts, of whom 59 (67.1%) showed a reduction of two Ig classes. All pts had completed the 2 planned doses of BNT162b2 (40.6%) or mRNA1273 (59.4%) vaccine 3 or 4 weeks apart, respectively. Control cohort (n=54; median age of 51 [range 40-66] years) received mRNA vaccine during the same period. People with previous SARS-CoV-2 infection (positive IgG anti S-RBD or anti-nucleocapsid N antibody titer before vaccines) were excluded from the analysis. At 1 month post 2 nd dose, (median 30 days, IQR 28-32) seropositive response rate to vaccination was 91.7% (n=88) for MM pts vs 100% for controls, p=0.05; the median IgG anti S-RBD titer was 435 (range 0.4-2500) vs 1040.5 U/ml (range 160-2500), respectively; p=0.008. No difference in the rate of seropositive response between those who received the 2 type of vaccines was found (p=0.09). Pts with response level ≥ CR had a median antibody (Ab) titer (1242 U/ml, range 0.4-2500) significantly higher than those with ≤CR (221.5 U/ml, range 0.4- 2500), p Pts receiving PI (median Ab titer 156; range 0.4- 2500) and anti-CD38 MoAbs (median titer 265 U/ml; 0.4- 2500) containing regimens had a lower Ab titer than all the other pts (p=0.003 and p A distribution analysis of the Ab titer revealed a significant correlation between better humoral response and hematological response ≥ CR (p Multivariate analysis confirmed ≥ CR [OR 2.54, 95% CI 93-756], being in first line treatment [OR 2.10, CI 22-722] and R maintenance therapy [OR 4.53, CI 484-1233] as independent predictors of better humoral response at 1 month after 2 nd vaccine dose. In conclusion, mRNA vaccines provided a high seropositivity rate in pts in active MM treatment, with a better humoral response in pts achieving CR, those who received ASCT and receiving R maintenance. Immunoparesis was confirmed to be an unfavourable factor for the development of humoral response, as well as treatment with anti-CD-38 moAbs. Disclosures Mancuso: Celgene: Honoraria; Takeda: Honoraria; Sanofi: Honoraria; Amgen: Honoraria; Janssen: Honoraria. Zamagni: Takeda: Honoraria; Amgen: Honoraria; Bristol-Myers-Squibb: Honoraria; Janssen: Honoraria. Pantani: Amgen: Honoraria; Janssen: Honoraria. Rocchi: Amgen: Honoraria; GalxoSmithKline: Honoraria; Janssen: Honoraria. Rizzello: Amgen: Honoraria; GlaxoSmithKline: Honoraria; Sanofi: Honoraria. Tacchetti: Amgen: Honoraria; BMS/Celgene: Honoraria; Janssen: Honoraria; Takeda: Honoraria; AbbVie: Honoraria; Sanofi: Honoraria; GlaxoSmithKline: Honoraria; Oncopeptides: Honoraria. Zinzani: JANSSEN-CILAG: Other: Advisory board, Speakers Bureau; MSD: Consultancy, Other: Advisory board, Speakers Bureau; SANDOZ: Other: Advisory board; TG Therapeutics: Other: Advisory board, Speakers Bureau; GILEAD: Other: Advisory board, Speakers Bureau; SERVIER: Other: Advisory board, Speakers Bureau; BMS: Other: Advisory board, Speakers Bureau; CELLTRION: Other: Advisory board, Speakers Bureau; TAKEDA: Other: Advisory board, Speakers Bureau; ROCHE: Other, Speakers Bureau; EUSAPHARMA: Consultancy, Other, Speakers Bureau; KYOWA KIRIN: Other, Speakers Bureau; Incyte: Other, Speakers Bureau; NOVARTIS: Consultancy, Other, Speakers Bureau; ADC Therap.: Other; Beigene: Other, Speakers Bureau; VERASTEM: Consultancy, Other: Advisory board, Speakers Bureau. Cavo: Takeda: Consultancy, 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; Adaptive Biotechnologies: Consultancy, Honoraria; Novartis: Honoraria; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GlaxoSmithKline: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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.

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.

Lynch syndrome (LS) is associated with germline mutations in one of the mismatch repair genes or EPCAM. The majority of the causative alterations are point mutations. Large genomic rearrangements represent only 5–20%. Hypothetically, the allelic imbalance, like the loss of heterozygosity, may be another high penetrance risk factor. We describe the case of a patient who developed 5 tumors during her lifetime and with a family history characterized by a high frequency of tumors associated with LS. The proband was tested for mutations and copy number alterations with a panel of hereditary cancer genes and by SNP array. She showed a 187 Kb duplication including EPCAM and the first 7 exons of MSH2, plus two loss of heterozygosity (LOHs) in chromosome 20 and one in chromosome X which include many tumor suppressor genes. We found a novel large EPCAM-MSH2 duplication associated with LS and the presence of LOHs in regions containing numerous tumor suppressors, raising the hypothesis that these alterations could contribute to cancer susceptibility. Our results underline the importance to deepen the knowledge of molecular mechanisms in order to determine the role in cancer predisposition of novel genetic alterations.

There is an increasing clinical interest in the measure and achievement of minimal residual disease (MRD) negativity in the bone marrow of Multiple Myeloma (MM) patients, as defined equally either by Multicolor Flow Cytometry (MFC) or by Next Generation Sequencing (NGS) technologies. At present, modern technologies allow to detect up to one on 104 or on 105 or even on 106 cells, depending on their throughput. MFC approaches, which have been progressively improved up to the so-called Next Generation Flow (NGF), and NGS, which proved clear advantages over ASO-PCR, can detect very low levels of residual disease in the BM. These methods are actually almost superimposable, in terms of MRD detection power, supporting the lack of unanimous preference for either technique on basis of local availability. However, some technical issues are still open: the optimal assay to use to detect either phenotype (e.g., next generation multidimensional flow cytometry, imaging) or genotype aberrations (e.g., ASO-RQ PCR, digital droplet PCR, NGS) and their standardization, the sample source (BM or peripheral blood, PB) and its pre-processing (red-cell lysis vs. Ficoll, fresh vs. frozen samples, requirement of CD138+ cells enrichment). Overall, MRD negativity is considered as the most powerful predictor of favorable long-term outcomes in MM and is likely to represent the major driver of treatment strategies in the near future. In this manuscript, we reviewed the main pitfalls and caveats of MRD detection within bone marrow in MM patients after front-line therapy, highlighting the improving of the currently employed technology and describing alternative methods for MRD testing in MM, such as liquid biopsy.

// 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.

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.

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.

Background Several new drugs are approved for treatment of patients with multiple myeloma (MM), but no validated biomarkers are available for the prediction of a clinical outcome. We aimed to establish whether pretreatment blood and bone marrow plasma concentrations of major cytokines and angiogenic factors (CAFs) of patients from a phase 3 trial of a MM treatment could have a prognostic and predictive value in terms of response to therapy and progression-free and overall survival and whether these patients could be stratified for their prognosis. Methods Blood and bone marrow plasma levels of Ang-2, FGF-2, HGF, VEGF, PDGF-β, IL-8, TNF-α, TIMP-1, and TIMP-2 were determined at diagnosis in MM patients enrolled in the GIMEMA MM0305 randomized controlled trial by an enzyme-linked immunosorbent assay (ELISA). These levels were correlated both reciprocally and with the type of therapy and patients’ characteristics and with a group of non-MM patients as controls. Results No significant differences were detected between the blood and bone marrow plasma levels of angiogenic cytokines. A cutoff for each CAF was established. The therapeutic response of patients with blood plasma levels of CAFs lower than the cutoff was better than the response of those with higher levels in terms of percentage of responding patients and quality of response. Conclusion FGF-2, HGF, VEGF, and PDGF-β plasma levels at diagnosis have predictive significance for response to treatment. The stratification of patients based on the levels of CAFs at diagnosis and their variations after therapy is useful to characterize different risk groups concerning outcome and response to therapy. Trial registration Clinical trial information can be found at the following link: NCT01063179 Electronic supplementary material The online version of this article (10.1186/s13045-018-0691-4) contains supplementary material, which is available to authorized users.

Multiple myeloma (MM) is often associated with renal insufficiency (RI) which adversely influences the prognosis. Several studies demonstrated that bortezomib can improve both renal function and outcome. We prospectively evaluated 21 newly diagnosed MM patients with severe renal impairment secondary to tubular-interstitial damage, most of them due to myeloma kidney, who were primarily treated with bortezomib-based therapy combined with high cut-off hemodialysis (HCOD). The median serum creatinine level at baseline was 6.44 mg dL(-1) and calculated median estimated glomerular filtration rate (eGFR), according to Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) creatinine equation, was 8 mL/min/1.73 m(2) . Serum free light chain (sFLC) median concentration was 6,040 mg L(-1) . Post induction and best stringent complete response rates were 19 and 38%, respectively. Responses were fast, occurring within a median of 1.4 months. The combination of bortezomib and HCOD led to a prompt and remarkable (>90%) decrease in sFLC levels. Sixteen patients (76%) became dialysis independent within a median of 32 days. With a median follow up of 17.2 months, the 3-year PFS and OS were 76 and 67%, respectively. No early deaths were observed. This study demonstrates that incorporation of bortezomib into induction therapy combined with HCOD is a highly effective strategy in rescuing renal function and improving outcomes in patients with MM and RI.

Precise assessment of response to therapy is of high importance in every phase of multiple myeloma (MM). In addition to the well-established role of monoclonal protein for clinical monitoring, several methods of minimal residual disease evaluation, both inside and outside the bone marrow (BM), are to date available. Next generation flow cytometry and sequencing are probably the best approaches at the BM level, being highly sensitive and uniformly applied. FDG PET/CT is the best imaging technique for evaluating and monitoring response to therapy outside the BM. Functional whole-body MRI techniques (DCE and DWI) seem promising for response evaluation and need further studies. Standardization of most of these techniques is in progress.

Thal-dex (TD) is an effective therapy for advanced MM. We evaluated TD as salvage treatment of MM patients at first relapse. Thal was given at a daily dose of 100 or 200 mg until progression. Dex was administered 160 mg/month. One hundred patients were enrolled. First line therapy included ASCT (72%) and conventional CHT (28%). Fifty-nine percent received a fixed thal dose of 100 mg/day. The most frequent adverse events were constipation (42%), peripheral neuropathy (58%, 5% grade 3), bradycardia (20%), skin rash (11%), and VTE (7%). Discontinuation of thal due to adverse events was recorded in eight patients. On ITT, 46% of patients achieved at least a PR. Median DOR was 28 months, median time to next therapy was 15.5 months. Median OS, TTP, and PFS were 43, 22, and 21 months, respectively. TTP and PFS were significantly longer for patients with at least PR to TD. TD was an effective salvage treatment for MM patients at first relapse, as demonstrated by durable disease control and prolonged OS. TD was well tolerated, as reflected by the long stay on treatment without disease progression (median 25 months) and a low discontinuation rate due to toxicity (8%).

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

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.

The treatment of hairy cell leukemia (HCL) has deeply changed over years. Purine analogs, namely cladribine (2CdA) now represent the treatment of choice. The BRAF V600E mutation is now regarded as the pathogenic event. One hundred and eighty-four patients were followed between 1986 and 2018 and treated according to era-specific guidelines. This is the largest monocentric series reported. Responses were classified by combining Consensus Resolution criteria and marrow immunohistochemistry. Patients were grouped according to the number of treatment lines they received (table). Ten patients treated with frontline 2CdA and in complete response (CR) for at least 5 years were tested for the presence of the BRAF V600E mutation in peripheral blood by droplet digital PCR as a molecular marker for active disease. Patients treated first line responded in 86% of cases, with 44% CR. Response rates remained high throughout the first 4 lines (84%, 81%, 79% for the second line onward, with CR in 38%, 37%, 15% of cases respectively), although decreasing progressively with the number of treatments received. One hundred and twenty-two patients received 2CdA as first line treatment, with a response rate of 86% and a CR rate of 54%. Among the 66 CR patients, 45 (68%) have never received further therapy: 11 patients are in continuous CR between 5 and 10 years after treatment, 14 between 10 and 20 years and 3 patients at more than 20 years. Median time-to-next treatment (TTNT) for patients after receiving 2CdA was 8.2 years: partial responders had a significantly shorter median TTNT than CR patients (5.3 years versus median not reached at 25.8 years, p=0.0001) (figure). Seven patients in CR for more than 5 years after front line 2CdA were BRAF V6500E negative in peripheral blood. One of these displayed disease recurrence and required further treatment roughly 2 years later. Three patients were positive for the BRAF V600E mutation at 6.5, 8.4 and 13.7 years after treatment and developed an overt disease relapse between 4 months and 2 years. Patients with HCL require subsequent lines of therapy in more than 50% of cases. Purine analogs allow significant response rates when applied first line and upon retreatment. Some patients may enjoy long lasting responses after one course of 2CdA and display no evidence of BRAF V600E mutation in peripheral blood. A PCR-based evaluation of the allelic burden in peripheral blood may provide information regarding disease activity over time. Figure Disclosures Cavo: Celgene, Janssen, Amgen, BMS, Abbvie, Takeda: Honoraria; Janssen, Celgene: Other: Travel Accommodations; Janssen, Celgene: Speakers Bureau; Janssen, Celgene, Amgen, Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zinzani:TG Therapeutics: Honoraria, Speakers Bureau; Roche: 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; Immune Design: 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; Servier: 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; MSD: Consultancy, Honoraria, 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; Sanofi: Consultancy; Celltrion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen-Cilag: 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; Portola: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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.

Multiple myeloma (MM) plasma cell (MMPC) interactions with the microenvironment control MMPC growth, survival, drug-resistance and intra- and extramedullary dissemination. Dissemination of MMPCs through bone marrow niches and in extra-medullary sites is an active process of invasion involving bone marrow endothelial cells, multiple adhesion molecules and chemokine receptors. Since enhanced angiogenesis characterizes MM, we investigated whether junctional adhesion molecule-A (JAM-A) mediated interactions between MM bone marrow endothelial cells (MMECs) and MMPCs impact disease progression. To this end, we analyzed JAM-A expression levels in MMECs of 312 MM patients in two independent cohorts with flow cytometry, namely 111 newly diagnosed (NDMM) and 201 relapsed/refractory (RRMM) and compared them to 36 monoclonal gammopathy of undetermined significance (MGUS) and healthy subjects. To corroborate our data and investigate at a gene-expression level the prognostic value of deregulated genes (FDR Surface protein expression of JAM-A on MMECs predicted poor overall survival (OS) in NDMM (not reached (NR) vs. 78 months univariate hazards ratio-HR=9.14, 95% CI 2.8-29.76, P Ensuing functional shRNA knockdown of JAM-A reduced MM invasion (P Collectively, our findings pinpoint JAM-A as a key player propagating a vicious cycle of MMECs and MMPCs interaction; the expression of JAM-A and the related adhesion pathways can prognostically stratify patients in the late disease stages impacting two main MM progression processes: angiogenesis and extra-medullary dissemination. Therefore, we propose JAM-A as a promising MM biomarker and novel therapeutic target in advanced disease. Disclosures Klapper: Roche, Takeda, Amgen, Regeneron: Honoraria, Research Funding. Rosenwald:MorphoSys: Consultancy. Cavo:celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; 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; 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; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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.

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.

The aim of the present study was to compare thalidomide-dexamethasone (Thal-Dex) and vincristine-doxorubicin-dexamethasone (VAD) as primary therapy in preparation for autologous peripheral blood stem-cell (PBSC) transplantation for multiple myeloma (MM). For this purpose, we performed a retrospective matched case-control analysis of 200 patients who entered 2 consecutive studies from 1996 to 2004 and received Thal-Dex (n = 100) or VAD (n = 100) administered for 4 months before collection of PBSCs and autologous transplantation. Matching criteria included age, clinical stage, and serum β2-microglobulin levels. In comparison with VAD, Thal-Dex resulted in a significantly higher response rate (52% versus 76%, respectively; P < .001) and effected more profound reduction in myeloma cell mass of both immunoglobulin G (IgG; P = .02) and IgA (P = .03) type. More frequent toxicities included nonfatal deep vein thrombosis with Thal-Dex (15%) and granulocytopenia with VAD (12%). In each of the 2 treatment groups, 91% of patients proceeded to PBSC mobilization. The median number of collected CD34+ cells was 7.85 × 106/kg in the Thal-Dex group and 10.5 × 106/kg in the control group. Thal-Dex may be considered an effective and relatively well-tolerated oral alternative to the more complex VAD regimen as front-line therapy for MM patients who are candidates for subsequent autologous transplantation.

Abstract 805FN2 Background. Achievement of CR is generally associated with improved clinical outcomes for patients (pts) with MM and represents a primary endpoint of current clinical trials. The GIMEMA Italian Myeloma Network designed a phase 3 study to demonstrate that the triplet VTD regimen was superior over a doublet such as thalidomide-dexamethasone (TD) as induction therapy prior to double ASCT for newly diagnosed MM. On an intention-to-treat basis, the rate of complete or near complete response (CR/nCR) was 31% for the 236 pts on VTD induction therapy, while it was 11% (p Methods. For this purpose, in a molecular substudy to the main clinical study we assessed the ability of gene expression profile (GEP) to predict attainment of CR/nCR in 122 pts enrolled in the VTD arm of the study. Their characteristics at baseline, including cytogenetic abnormalities, were comparable with those of the whole population of 236 pts. Highly purified CD138+ plasma cells were obtained at diagnosis from each of these pts and were profiled for gene expression using the Affymetrix U133 Plus2.0 platform. In order to build a low-dimensional signature with optimal performance, genomic data were analyzed with an original algorithm that exploits quadratic discriminant analysis with a bottom-up approach that builds N-gene signatures starting from two-dimensional signatures. Gene models were applied to test datasets to predict achievement of either CR/nCR or less than nCR, and classification performances were validated by a leave-one-out crossvalidation procedure. Results. Thirty four pts out of the 122 (28%) who were included in the present analysis achieved a CR/nCR, while the remaining 88 patients failed this objective. The molecular approach described above allowed to identify several gene signatures among which we choose a 163-gene signature that provided a predictive capability of 79% sensitivity, 87% specificity, 71% positive predictive value (PPV) and 92% negative predictive value (NPV). These expression values were used in an unsupervised hierarchical clustering to stratify the population of 122 profilated pts into 3 well defined subgroups. Seventy nine pts were included in subgroup A, while the remaining 43 pts were included in either subgroup B (n=22) or subgroup C (n=21). Notably, 19 out the 34 CR/nCR pts (56%) clustered in subgroup B, whereas the remaining 15 pts were randomly distributed within subgroup A. Analysis of demographic and disease characteristics of the pts belonging to the 3 major subgroups, revealed that in subgroup B the frequencies of pts carrying del(13q) (78%) or del(17p) (22%) or with an IgA isotype (54%) were significantly higher in comparison with the corresponding values found in subgroup A (47%, 4%, and 10%, respectively) and subgroup C (38%, 10%, and 5%, respectively). In order to obtain a more feasible set of genes predictive of CR/nCR, several smaller signatures originating from the 163-gene signature were further analyzed by means of the same algorithm described above. The best predictive capability was obtained with a 41-gene signature that provided 88% sensitivity, 97% specificity, 91% PPV and 95% NPV. A GeneGo ® network analysis of genes included in the signatures showed that the most relevant network nodes included tumour suppressor genes (FBXW7 and MAD), genes involved in inflammatory response (TREM1 and TLR4) and genes involved in B cell development (IKZF1, IL10 and NFAM1). Genes included in the signatures do not gather in specific chromosomes, thus confirming the absence of bias on selection of signatures genes, potentially due to prevalence of MM typical chromosomal aberrations. Conclusions. GEP analysis of a subgroup of pts who received VTD induction therapy allowed to provide a 41-gene signature that was able to predict attainment of CR/nCR and, conversely, failure to achieve at least nCR in 91% and 95% of cases, respectively. These favorable results might represent a first step towards the possible application of a tailored therapy based on the single patient's genetic background. Supported by: Fondazione Del Monte di Bologna e Ravenna, Ateneo RFO grants (M.C.) BolognAIL. Disclosures: Bringhen: Celgene: Honoraria; Janssen-Cilag: Honoraria; Novartis: Honoraria; Merck Sharp & Dhome: Membership on an entity's Board of Directors or advisory committees. Offidani:Janssen: Honoraria; Celgene: Honoraria.

Abstract 1429 The development of resistance against chemotherapy remains one of the major challenges in the clinical management of leukemia. The Sonic Hedgehog (Hh) pathway is an essential regulator of multidrug resistance (MDR) in leukemia by exerting it's effect on leukaemia stem cells (LSC). PF-04449913 is a selective and potent small molecule inhibitor of the Hh pathway and leukemia self-renewal and is currently being evaluated in Phase I clinical trials. In order to evaluate the activity of PF-0444913 in overcoming the drug resistance of leukemia stem cells, we studied leukemia stem cell population (CD34+ subpopulation) collected before and after 28 days treatment in a phase I dose escalation protocol (Clinical Trial Gov. NTC00953758) enrolling selected hematological malignancies including Myelofibrosis (MF), MDS, Chronic Myeloid Leukemia (CML), Chronic Myelomonocytic leukemia (CMML) and Acute Myeloid Leukemia (AML) patients. We were able to collect and separate highly purified (98%) bone marrow hematopoietic progenitor cells (CD34+ populations) in 5 AML, 1 MF and 2 CML patients, by immunomagnetic separation, and analyze them for gene expression profile (GEP) using Affimetrix HG-U133 Plus 2.0 platform. We have observed that 1197 genes were differentially expressed between CD34+ cells collected before and after 28 days of PF-04449913 dose finding oral therapy. Among these genes, we demonstrated a down regulation of Bcl2 (fold change −1.03004; p value= 0.01), ABCA2 (fold change −1.08966; p value=0.03), Bcl2l13 (fold change −1,04259; p-value=0,027642), Bcl2l2 (fold change −1,17214; p-value=0,000768), Casp4 (fold change −1,06551; p-value=0,032428), Casp7 (fold change −1,01569; p-value=0,006688), Casp10 (fold-change −1,3076; p-value=0,050431), ABCF1 (fold change −1,04999; p-value=0,07213). On the contrary, ABCB1 (fold change 1,46592) and ABCG2 (fold change −1,16103) are respectively up and down regulated, with a not statistically significant p-value (0,35375 and 0,288194 respectively). Bcl2 (B-cell lymphoma 2), Bcl2l2 (Bcl2-like protein 2) and Bcl2l13 (Bcl2-like 13) are the founding members of the Bcl-2 family of apoptosis regulator proteins. Recent studies showed that Hh signals upregulate Bcl2 to promote cellular survival. Casp 4,7,10 (Caspases, or c ysteine-asp artic proteases) are a family of cysteine proteases that play essential roles in apoptosis, necrosis, and inflammation. ABCA2 (ATP-binding cassette sub-family A member 2), ABCF1 (ATP-binding cassette sub-family F member 1), ABCB1 (ATP-binding cassette sub-family B member 1, MDR1), ABCG2 (ATP-binding cassette sub-family G member 2) belong to the superfamily of adenosine triphosphate-binding cassette (ABC) transporters. ABC proteins transport various molecules across extra- and intracellular membranes. One mechanism of MDR is the increased expression of ABC drug transporters that mediate energy-dependent transport of drugs out of the cells against a concentration gradient, resulting in low intracellular drug concentrations. This is a common finding in LSC, and represents an important clinical problem for disease eradication. Furthermore, we evaluated Gli1, Gli2 and Smo expression by GEP, comparing data before and after 28 days of treatment with PF-04449913 and, as expected, we observed a down regulation of Gli1 (fold change −1.0775), Smo (fold change −1.07702), and an up regulation of Gli2 (fold change 1.08191). Our results suggest that PF-04449913 is able to revert MRD mechanisms of LSC by a strong down regulation of genes (Bcl-2, Bcl-2l13, Bcl-2l2, ABCA2, and ABCF1), which are critical for chemoresistance in acute and chronic leukemia patients. Therefore, the combination of PF-04449913 with Tyrosine Kinase inhibitors or conventional chemotherapy could represent a valid new therapeutic approach in these haematological malignancies. Acknowledgments. Work supported by Pfizer, European LeukemiaNet, FIRB 2008, PRIN 2009, AIRC, AIL, COFIN, University of Bologna and BolognAIL. Disclosures: Levin: Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Courtney:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Baccarani:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jamieson:Wintherix: Equity Ownership; Pfizer Oncology: Research Funding; Celgene: Research Funding; Novartis: Honoraria. Cortes:Novartis: Consultancy; Novartis: Research Funding; BMS: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. McLachlan:Pfizer: Employment. Van Arsdale:Pfizer: Employment. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria.

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.

Background. The incorporation of tyrosine kinase inhibitors (TKIs) in treatment schemes of Ph+ ALL has remarkably improved survival. In adult patients with Ph+ ALL, ponatinib in combination with chemotherapy showed a 3-year event-free survival rate of 69%, a 3-year overall survival (OS) of 83%, and a higher rate of response when compared with dasatinib plus chemotherapy. However, in unfit or elderly ALL patients, TKIs combined with chemotherapy are associations with higher toxicity. Therefore, we examined the efficacy and safety of steroids plus ponatinib alone for the treatment of elderly or unfit patients with Ph+ ALL in a multi-center Phase II prospective clinical Italian trial, GIMEMA LAL1811 (EudraCT number 2012-002761-35). Methods. From March 2014 to December 2016, we enrolled 44 patients with untreated Ph+ ALL, ≥ 60 years or unfit (i.e. for intensive chemotherapy and stem cell transplantation). Two out of 44 patients were not elegible for the study. Patients received oral administration of 45 mg/day of ponatinib for 8 consecutive courses of 6 weeks (w). Steroids were administered from day -14 to day 29 during course 1. Intrathecal therapy with methotrexate, cytarabine and dexametasone was performed every 28 days for central nervous system (CNS) disease prophylaxis. In patients with CNS disease at diagnosis, intrathecal therapy was administered twice a week until complete remission. Dose reduction of ponatinib was allowed for adverse events. Patient samples were obtained at diagnosis and at every course, BCR-ABL mutational analisys and BCR-ABL/ABL ratio by quantitative real time PCR was performed. Complete molecular response (CMR) was defined as BCR-ABL/ABL ratio below 0.01 or undetectable, and with a sensitivity of at least 30,000 molecules of ABL. Results. Forty-two patients were eligible for the study. Median age was 68 years (range 27-85). Nine out of 42 patients were At week 24, 15/42 patients still received 45 mg of ponatinib daily, only 4/42 patients permanently withdrew study drug. During the study, 75 adverse events (AE) were reported; 36 of the 75 AEs were considered related to ponatinib. Twenty-six of the 75 AEs were considered serious (SAE); 13/26 SAEs were considered related to ponatinib. A death was suspected to be related to ponatinib. We performed BCR-ABL mutational analysis in 22 patients at diagnosis, and 15 patients at 24w. T315L (abundance 100%) was detected in a patient relapsed during ponatinib therapy. We could not identify the emergency of other mutations. Conclusions. Ponatinib and steroid show a high efficacy in newly diagnosed unfit/elderly Ph+ ALL patients. Toxicities were manageable and cardiovascular AEs were limited. In the small cohort of patients relapsed in the study, relapse mechanisms were unclear; only one patient had evidence of mutations that caused resistance to ponatinib. The fast and deep reduction of the disease burden in the majority of patients, the ability of ponatinib to prevent the emergence of clones harboring BCR-ABL mutations, and the synthetic lethality with steroids on the BCR-ABL, FLT3, HCK, CDK6, MCL1 pathway could explain the therapeutic effectiveness. Acknowledgments. GIMEMA, ELN, AIL, AIRC, Regione-Università 2010-12, FP7 NGS-PTL, HARMONY, Fondazione del Monte BO e RA. Disclosures Soverini: Bristol-Myers Squibb: Consultancy; Incyte Biosciences: Consultancy; Novartis: Consultancy. Bocchia: Novartis: Other: Travel grant; Celgene: Other: Travel grant; Roche: Other: Travel grant; Jansen: Other: Travel grant. Cuneo: Abbvie: Honoraria, Other: Advisory Board; Janssen: Honoraria, Other: Advisory Board; Gilead: Honoraria, Other: Advisory Board; Roche: Honoraria, Other: Advisory Board. Bonifacio: Pfizer: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees. Falini: Roche: Research Funding. Galieni: Takeda: Other: Advisory Board; Abbvie: Other: Advisory Board. Foà: Sandoz: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; janssen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Baccarani: Pfizer: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Incyte ARIAD: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau.

Introduction: The phase 3 GIMEMA-MMY-3006 trial comparing bortezomib-thalidomide-dexamethasone (VTD) versus thalidomide-dexamethasone (TD) as induction therapy before, and consolidation after, double autologous stem cell transplantation (ASCT) for newly diagnosed multiple myeloma (MM) provided the first demonstration of increased CR rate, the primary study endpoint, and prolonged PFS with VTD (Cavo M et al, Lancet 2010). However, updating trial results with longer follow-up than earlier reported is needed to assess the effects of treatment interventions on OS and to identify factors predicting for favorable long term outcomes. Aims: We performed a post-hoc analysis of that study to evaluate long term results and construct a prognostic index of survival. Methods: 474 patients were enrolled, 236 randomized to VTD and 238 to TD. Median follow-up for surviving patients was 124 months (IQR: 117-131). Analyses were performed on an intention-to-treat basis. Semi-parametric Cox regression analysis was used to construct the prognostic index. To assess the evolution of prognosis over time, conditional survival CS(t|s) estimate for PFS was calculated as the probability of surviving without progression a further 2 (t) years (yrs) after having already survived s yrs. Results: Estimates of PFS and OS at 10 yrs for the VTD arm were 34% (HR=0.62; 95% CI=0.50-0.77; p Conclusions: With a follow up of 10 yrs, the final analysis of the GYMEMA MMY-3006 trial comparing VTD versus TD showed a persistent PFS benefit translating into extended OS for the VTD arm. A prognostic model based on cytogenetic, ISS stage and achievement of CR, identified three risk groups with statistically different long-term survival probabilities. Both IR and HR groups significantly benefited from VTD. A PFS time of 78 months predicted for long term survival outcomes in the LR and IR groups. Disclosures Tacchetti: Celgene: Honoraria; Janssen: Honoraria; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Di Raimondo:Takeda: Honoraria, Research Funding; Celgene: Honoraria. Zamagni:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: 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; BMS: 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. Bringhen:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Amgen: Honoraria, Other: Advisory Board; Janssen: Honoraria, Other: Advisory Board; Takeda: Consultancy. Offidani:Celgene: Honoraria, Other: Advisory Board; Janssen: Honoraria, Other: Advisory Board; Amgen: Honoraria, Other: Advisory Board; Bristol-Myers Squibb: Honoraria, Other: Advisory Board; Takeda: Honoraria, Other: Advisory Board. Montefusco:Celgene: Other: Advisory Board; Amgen: Other: Advisory Board; Janssen: Other: Advisory Board. Cavo:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Adaptive Biotechnologies: 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; GlaxoSmithKline: 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.

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.

Purpose Imatinib mesylate is a potent inhibitor of BCR-ABL, the constitutively active tyrosine kinase protein critical for the pathogenesis of chronic myeloid leukemia. Patients and Methods We reviewed 284 patients with late chronic-phase Philadelphia chromosome (Ph) –positive chronic myeloid leukemia treated with imatinib 400 mg daily after interferon-α failure. In a retrospective study, we evaluated the pattern and rapidity of the response to imatinib, comparing the cytogenetic and molecular responses, progression-free and overall survival rates in patients who obtained a complete cytogenetic response within 1 year of treatment (early responders), and in patients where a complete cytogenetic response was detected after 12 months (late responders). Results After 3 or 4 years of treatment, the molecular response of the late cytogenetic responders was similar to that of the early cytogenetic responders. At 36 months of treatment the amount of residual disease measured by standardized quantitative reverse-transcriptase polymerase chain reaction was 0.00047 in late responders versus 0.00022 in early responders, and at 48 months it was 0.00019 versus 0.00026 (median values, P value = nonsignificant). The estimated 4-year progression-free survival rate was 88% for early responders and 100% for late responders, while the estimated 4-year overall survival rates were 92% and 100% for early and late responders, respectively. Conclusion The sensitivity and the response (cytogenic and molecular) to imatinib may require 1 year or more. Long-term follow-up results continue to improve in terms of rates and durability of the complete cytogenetic response, major or complete molecular response, and progession-free and overall survival.