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Accumulating data have shown that molecular aberrations have the potential to trigger the development of acute leukemia, and that the routine application of novel molecular biology technologies has facilitated the development of investigational drugs which target driver genetic mutations [...]

The optimal stem cell (SC) mobilization strategy for patients with multiple myeloma (MM) remains a matter of debate. Possible approaches include low or high doses of cyclophosphamide (Cy), other chemotherapeutic agents, or granulocyte colony-stimulating factor (G-CSF) alone. The scope of the study was to compare low-dose Cy plus G-CSF versus intermediate-high-dose Cy plus G-CSF versus G-CSF alone for SC mobilization in MM, in terms of efficacy and safety. We retrospectively analyzed 422 MM patients undergoing SC mobilization in 6 Italian centers, including 188 patients who received low-dose Cy (LD-Cy group, defined as 2 g/m2), 163 patients who received intermediate-high-dose Cy (HD-Cy group, defined as ≥ 3 g/m2), and 71 patients who received G-CSF alone (G-CSF group). The median peak of circulating CD34+ cells was 77/µL in the LD-Cy group, 92/µL in the HD-Cy group, and 55/µL in the G-CSF group (P = .0001). The median amount of SCs collected was 9.1 × 106/kg, 9.7 × 106/kg, and 5.6 × 106/kg in the 3 groups, respectively (P = .0001). The rate of mobilization failure (defined as failure to collect ≥2 × 106/kg) was 3.7% in the LD-Cy group, 3.4% in the HD-Cy group, and 4.3% in the G-CSF group (P = .9). The target SC dose of at least 4 × 106/kg was reached in 90.4%, 91.1%, and 78.6% of the patients in these 3 groups, respectively (P = .014). The "on demand" use of plerixafor was higher in the G-CSF group (76%) compared with the LD-Cy group (19%) and the HD-Cy group (6%). In multivariate analysis, G-CSF mobilization and previous use of melphalan or radiotherapy were independently associated with failure to collect the target SC dose of ≥4 × 106/kg. No impacts of age, blood counts, or previous treatment with lenalidomide, bortezomib, or carfilzomib were observed. Our results suggest that LD-Cy may be considered for successful SC mobilization in patients with MM.

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.

Plerixafor inhibits CXCR4, thus inducing the mobilization of hematopoietic stem/progenitor cells in lymphoma and multiple myeloma (MM) patients eligible for autologous stem cell transplantation (ASCT). However, the kinetics of plerixafor-induced mobilization of lymphocyte subsets is poorly known. Here, we evaluated the graft content, the engraftment, and the immunological reconstitution of MM patients receiving plerixafor. Thirty-seven patients undergoing one or tandem ASCT were enrolled. After mobilization with cyclophosphamide plus G-CSF, plerixafor was added at hematological recovery regardless of CD34(+) cell count. We evaluated the number of CD34(+), CD34(+)/CD38(-), CD3(+), CD4(+), CD8(+), CD19(+), CD56(+)/CD3(-), CD4(+)/CD25(+)/FOXP3(+), and CD138(+)/CD38(+) cells on each apheresis. Hematological and immunological recovery were determined at 30 days, 3, 6, 9, and 12 months after ASCT. Overall, 34/37 patients mobilized a median of 10.1 × 10(6) CD34(+) cells/Kg (IQ 7.7-13.4). Patients with

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.

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.

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

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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%).

Venous thromboembolism (VTE) is a disease with a high prevalence in elderly people, affecting > 5% of the population > 65 years of age. Cancer patients have a 4.3-fold higher incidence of thrombotic complications, due to multiple risk factors that are not always related to the disease. Among hematologic malignancies, multiple myeloma (MM) confers a high risk of developing such complications, with a VTE rate of nearly 10%. Multiple factors are involved in MM-related VTE, such as increased blood viscosity, high levels of immunoglobulin, procoagulant activity of monoclonal protein, and inflammatory cytokines. Since the introduction of the immunomodulatory derivatives (IMiDs) thalidomide and lenalidomide in the therapeutic armamentarium of MM, VTE has emerged as one of the leading complications, in particular in patients with newly diagnosed MM. In this setting, IMiDs-based treatments are associated with rates of VTE reaching values up to 14 to 26%, particularly when dexamethasone or chemotherapy are added. The optimal prophylaxis for patients receiving these antiangiogenetic agents is still a matter of debate. Due to the lack of prospective randomized clinical trials, different studies have used various anticoagulant prophylaxes, including fixed low-dose warfarin (1 mg or 1.25 mg), therapeutic doses of warfarin (international normalized ratio between 2.0 and 3.0), low molecular weight heparin, or low-dose aspirin. As yet, no study has clearly demonstrated a significant superiority of one prophylactic regimen in comparison with the others. Further investigation and more randomized clinical trials are needed to define the best thromboprophylaxis.

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.

Bortezomib (bort)-dexamethasone (dex) is an effective therapy for relapsed/refractory (R/R) multiple myeloma (MM). This retrospective study investigated the combination of bort (1.3 mg/m(2) on days 1, 4, 8, and 11 every 3 weeks) and dex (20 mg on the day of and the day after bort) as salvage treatment in 85 patients with R/R MM after prior autologous stem cell transplantation or conventional chemotherapy. The median number of prior lines of therapy was 2. Eighty-seven percent of the patients had received immunomodulatory drugs included in some line of therapy before bort-dex. The median number of bort-dex cycles was 6, up to a maximum of 12 cycles. On an intention-to-treat basis, 55 % of the patients achieved at least partial response, including 19 % CR and 35 % achieved at least very good partial response. Median durations of response, time to next therapy and treatment-free interval were 8, 11.2, and 5.1 months, respectively. The most relevant adverse event was peripheral neuropathy, which occurred in 78 % of the patients (grade II, 38 %; grade III, 21 %) and led to treatment discontinuation in 6 %. With a median follow up of 22 months, median time to progression, progression-free survival (PFS) and overall survival (OS) were 8.9, 8.7, and 22 months, respectively. Prolonged PFS and OS were observed in patients achieving CR and receiving bort-dex a single line of prior therapy. Bort-dex was an effective salvage treatment for MM patients, particularly for those in first relapse.

Multiple myeloma (MM) is a malignant disorder of post-germinal center B cells, characterized by the clonal proliferation of malignant plasma cells (PCs) within the bone marrow (BM). The reciprocal and complex interactions that take place between the different compartments of BM and the MM cells result in tumor growth, angiogenesis, bone disease, and drug resistance. Given the importance of the BM microenvironment in MM pathogenesis, we investigated the possible involvement of Hypoxia-Inducible transcription Factor-1 alpha (HIF-1α) in the PCs-bone marrow stromal cells interplay. To test this hypothesis, we used EZN-2968, a 3rd generation antisense oligonucleotide against HIF-1α, to inhibit HIF-1α functions. Herein, we provide evidence that the interaction between MM cells and BM stromal cells is drastically reduced upon HIF-1α down-modulation. Notably, we showed that upon exposure to HIF-1α inhibitor, neither the incubation with IL-6 nor the co-culture with BM stromal cells were able to revert the anti-proliferative effect induced by EZN-2968. Moreover, we observed a down-modulation of cytokine-induced signaling cascades and a reduction of MM cells adhesion capability to the extracellular matrix proteins in EZN-2968-treated samples. Taken together, these results strongly support the concept that HIF-1α plays a critical role in the interactions between bone BM cells and PCs in Multiple Myeloma.

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

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

Incorporation of novel agents into auto-SCT for patients with multiple myeloma has led to improvement in their outcomes. However, the effects of new drugs, either single or combined, on PBSC mobilization have not been fully evaluated, particularly in phase 3 clinical studies. We analyzed the impact of two novel agent-based induction treatments in patients enrolled in the GIMEMA MMY-3006 study comparing bortezomib, thalidomide and dexamethasone (VTD) versus thalidomide and dexamethasone (TD) in preparation for double auto-SCT. Results showed that a short-term induction therapy with VTD did not adversely affect CD34(+) cell yields as compared with TD (9.75 vs 10.76 × 10(6) CD34(+) cells/kg, P=0.220). For poor mobilizers (

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

Lok et al.1 recently reported in this Journal on the efficacy and toxicity of subcutaneous (sc) bortezomib given at a reduced dose in combination with thalidomide and dexamethasone (sc vTD) as induction therapy before, and as consolidation after, autologous stem-cell transplantation (ASCT) in 31 patients with newly diagnosed multiple myeloma (MM). The treatment plan included four cycles of induction therapy and two cycles of consolidation, both comprising sc bortezomib (1.0 mg/m2 twice weekly), thalidomide (100 mg per day) and dexamethasone (total dose 320 mg per cycle on the first two cycles and 160 mg per cycle on the subsequent two cycles of the induction phase). The rate of at least very good partial response (VGPR) after induction therapy was 52% for all patients and increased up to 73% among patients who actually received consolidation therapy. The incidence of treatment-emergent peripheral neuropathy (PN) after the induction phase (16% grade 1–2, including 3% grade 2) was lower than that previously reported by the same group after four cycles of either vTD at the same doses but using intravenous (iv) bortezomib (53% all grades, including 14% grade 2 or higher) or iv standard dose bortezomib (1.3 mg/m2) and dexamethasone (VD) (70% all grades, 34% >grade 2, 11% grade 3–4).2 We report, herein, on the outcomes of 22 newly diagnosed, ASCT-eligible, MM patients who were programmed to receive at our center four 21-day cycles of sc bortezomib (1.3 mg/m2 twice weekly) plus dexamethasone (total dose 320 mg per cycle) and either thalidomide (100 mg per day) (sc VTD: 13 patients) or cyclophosphamide (500 mg/m2 on Days 1 and 8 per cycle) (sc VCD: 9 patients). All patients were prospectively evaluated for efficacy and toxicity of sc bortezomib-based induction therapy. Presence of higher than grade 1 PN at diagnosis was an exclusion criterion for enrollment. Symptoms and signs of PN were carefully assessed at every programmed clinical appointment and were graded according to National Cancer Institute’s Common Toxicity Criteria (NCI CTCAE) v.3.0. No electrophysiological study was performed. Base-line patients’ characteristics and treatment response are summarized in Tables 1 and ​and2.2. A median of four cycles was administered. The overall response rate among all patients was 95%, including 27% stringent complete response (sCR) and 77% VGPR or better. Treatment-emergent grade 1–3 PN was observed in 14 patients (64%), including 18% (4 patients: 2 treated with VCD and 2 with VTD) grade 2 and 14% (3 patients: 2 receiving VTD and one VCD) grade 3. In one of these 3 patients, treatment was discontinued after the third cycle. Among the 7 patients with grade 2–3 PN, the median time from start of induction therapy to the first onset of PN was 72 days (range 48–109). In 3 patients, symptoms and signs of PN (grade 2 in 2 cases and grade 3 in the remaining one) emerged after the induction phase was completed, more specifically 103, 106 and 109 days after starting induction therapy. With appropriate treatment modifications,3,4 symptoms and signs of PN resolved or improved to grade 1 in 9 out of 14 (64%) patients within a median time of 94 days. Table 1. Base-line patients’ characteristics. Table 2. Efficacy and neurological toxicity of sc VTD or VCD induction therapy. Data reported here raise several considerations, but should be interpreted with caution due to the limited number of patients. The high overall response rate of 95%, including 27% sCR and 77% VGPR or better, further supports the conclusion that the efficacy of sc bortezomib reported in the relapsed/refractory setting5 is retained when the drug is administered in patients with newly diagnosed MM.1 Although cross-trial comparison is inadequate due to heterogeneities in the design of the studies and patients’ characteristics, it is likely that the discrepancy in the rates of high-quality responses between our series and that reported by Lok et al.1 reflects the lower activity of reduced-dose bortezomib used in the French study, with the possible contribution of the lower total dose of dexamethasone given in the third and fourth cycles of the induction phase. Differences between the two studies with respect to the doses of sc bortezomib incorporated into induction therapy may only explain in part the controversies about the frequency and severity of treatment-induced PN. Additional factors potentially compromising a meaningful interpretation of our data, as well as of those reported by others,1,6,7 include: i) the retrospective nature of several analyses which were performed either on patients who, due to their age, were eligible or ineligible to receive ASCT or on patients who had plasma cell dyscrasias other than MM, such as systemic amyloidosis;6,7 ii) the possible different methodological approaches used to assess PN. In our study, clinical evaluation of PN was performed at baseline, at the beginning of each cycle of induction therapy, before each dose of bortezomib, and every 21–28 days after the last dose of bortezomib was given. Neurological monitoring was continued until ASCT was received and allowed us to register 3 patients who suffered from late emergence of neurological toxicity, after induction therapy was completed. Although worsening of taxane-induced neurotoxicity was observed even after treatment was stopped,8 to the best of our knowledge, a similar phenomenon has not been reported for bortezomib. As far as this last issue is concerned, it is important to highlight that all the patients described here had a late emergence of previously undetected, typical bortezomib-induced PN (BiPN) and that in each of them any additional cause potentially contributing to the development of neurotoxicity was excluded. With the limits of the small sample size of patients analyzed, the 32% rate of grade 2 or higher PN is very similar to values previously reported after four cycles of iv VD or VTD incorporating standard-dose bortezomib.2,9,10 Knowledge of the mechanisms underlying BiPN has remained limited for many years.11 Recently, new insights into a better understanding of the pathogenesis of BiPN have been provided by analyses of gene expression profiles and single nucleotide polymorphisms of MM plasma cells.12–14 Results of these studies, performed in patients treated in Western countries but whose ethnicity was not detailed, showed that differently expressed genes involved in drug-induced apoptosis, DNA repair, inflammatory pathways, and development and function of nervous system are related to a different risk for, and time to onset of, BiPN. In addition, the patient’s inherited genetic background might also contribute to the individual risk for neurological toxicity.12,13 The possibility that patient- and disease-related genetic profiles might have been differently expressed in our series of patients compared to those reported by Lok et al.,1 thus partly contributing to the controversies observed in terms of frequency, severity and time to onset of PN, cannot be confirmed or ruled out. In conclusion, our data support the efficacy of sc bortezomib when incorporated into induction therapy for newly diagnosed, ASCT-eligible, MM patients. On the other hand, no conclusions about the different rate and severity of sc versus iv BiPN can be drawn. The possibility of late emergence of PN, even after an uneventful induction phase, should be taken into consideration and alert the physician to the need to continue close neurological monitoring after sc bortezomib-based induction therapy has been discontinued. Results of ongoing studies that aim to prospectively evaluate the efficacy and toxicity of sc bortezomib as part of first-line therapy will hopefully clarify the controversies discussed here.

Extramedullary disease (EMD) is an uncommon manifestation of multiple myeloma (MM). The incidence ranges from 2% to 20%, with higher frequency in later phases of the disease. Positron emission tomography with computed tomography (PET/CT) proved to be accurate in the detection of EMD. We report here on a massive EMD progression of MM in an elderly patient with cytogenetically high-risk disease that was treated up-front with novel agents. The extramedullary spread was huge, involving both common and atypical sites. The diagnosis of EMD was based on findings of PET/CT at the time of biochemical relapse, before the progression of MM became clinically evident. Considering PET/CT results in the restaging of MM patients at relapse may help to possibly detect, as in the present case, unsuspected and uncommon sites of EMD. This finding might ultimately affect prognosis and therapeutic decisions. Early and prompt diagnosis of EMD by novel imaging techniques, such as PET/CT, may be the first step toward improved management of patients with this rare manifestation of MM.

Background Multiple Myeloma (MM) is a genetically complex disease. In MM, prevalent chromosomal numerical and structural aberrations are used to cluster patients (pts) into subtypes, frequently displaying distinct clinical behaviors. Among the umpteen chromosomal aberrations described so far in MM, the TP53 deletion on chromosome (chr) 17p13 defines a pts group with a particularly poor prognosis, even if its prevalence at diagnosis is quite low. Overall, the TP53 tumor-suppressor gene is mutated or functionally inactivated in most human cancers. Tumors that retain wild-type p53 frequently harbor defects either in the pathways that allow for p53 stabilization in response to stress, or in the effectors of p53 apoptotic activity. One of the most potent inhibitor of p53 is MDM4, which is often amplified in several types of tumors. The MDM4 locus is located on chr1q32.1, a region frequently amplified in MM. Aim Aim of the present work was to investigate the possibility that both TP53 deletion (del) and MDM4 amplification (amp) might affect similar pathways, thus finally leading to a poor prognosis MM pts carrying at diagnosis at least one of them. Pts and methods Eighty-nine pts treated with bortezomib-thalidomide-dexamethasone (VTD) as induction therapy prior to, and as consolidation after, double ASCT were analyzed at diagnosis by means of unpaired analysis of copy number alterations (CNA) (Affymetrix 6.0 SNP array) and gene expression profile (GEP) (Affymetrix U133 Plus2.0 array); in twenty-one pts carrying MDM4 amplification and for whom samples were available, the p53 activity was explored both by analyzing the TP53 mutational rate by deep sequencing (Roche GS Junior 454) and by evaluating the p53 activity by immunoblotting assays of MDM4, p-p53 and p53. Genomic results were evaluated in the clinical context. Results The CNA analysis showed a 482 Kb minimal deleted region on chr17p13, including TP53, in 8/89 pts (8,9%) and a 1.1 Mb minimal amplified region on chr1q32.1 including MDM4 in 27/89 pts (30,3%). The GEP analysis performed at diagnosis in TP53 del and MDM4 amp pts generated two lists, including genes either differentially expressed among pts carrying or not amplified MDM4 (5840 probes sets, corresponding to 3841 genes, p In order to verify whether the presence of MDM4 amp might be related to a decreased p53 function, we analyzed 21 pts carrying MDM4 amp for their p53 activity, as evaluated by p-p53 immunoblotting assays, and we showed the absence of p-p53 protein in 60% of them. Finally, we analyzed the TP53 mutational rate, as detected by deep sequencing of exons 4-11, in 21 pts carrying MDM4 amp and we showed the presence of point mutations in 15 of them, with mutated reads frequencies ranging from 1.03% to 16.9% (median coverage for each amplicon = 1000 reads). We lastly analyzed the prognostic relevance of p53 pathway impaired function; to this purpose, we stratified pts into two subgroups according to the presence of MDM4 amp and/or TP53 del (group A, 34 pts, or 38%) or the absence of both these abnormalities (group B, 55 pts, or 62%). Baseline clinical characteristics were homogeneous, except for a higher rate of IgA isotype in group A. On the contrary, groups A and B resulted clearly imbalanced with respect to the genomic background: indeed, the t(4,14) frequency, as well as the average number of CNAs were overall higher in group A as compared to group B (38% vs. 14% t(4;14) positive, p=0.0002 and 165 vs 103 CNAs, p = 0.03). Despite the initially slightly higher response rate after VTD induction therapy of group A, as compared to group B (38% vs 20% ³near complete response), the presence of TP53 del and/or MDM4 amp correlated with shorter median PFS (44.05 months vs. undefined, p=0.003) and OS (66.6 vs undefined, p=0.0006). Of note, the poorer impact associated with MDM4 amplification was retained also in the absence of TP53 deletion (PFS: 46.45 months vs undefined, p=0.009). Conclusions The results overall suggest that the involvement of the p53 pathway alteration in MM might be wider than expected, possibly due to the activation of negative regulators of p53. Disclosures: Zamagni: Celgene: Honoraria; Janssen-Cilag: Honoraria. Cavo:Celgene: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Millennium: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Background Multiple Myeloma (MM) is a biologically complex disease, whose genetic plasticity favors the coexistence of genetically heterogeneous subclones, selected in a Darwinian fashion throughout the disease course. Based on a disease evolution model, it is likely that therapy acts like an evolutionary bottleneck, applying a selective pressure on the clones' genomic background. However, a formal correlation between the modulation of intra-clonal heterogeneity and the treatment has yet to be demonstrated. Aim To explore the existence of different clonal evolution patterns in MM, eventually driven by therapeutic selective pressure, we longitudinally analyzed a cohort of patients (pts) by using a high-throughput technology, able to finely dissect the genomic intra-clonal changes occurring in each pts, across disease progression. Patients and methods Thirty-three pts with symptomatic MM were included in this study. Most pts (28) were up-front treated with regimens including a proteasome inhibitor (PI), either in combination with an immunomodulator (IMiD) (thalidomide or lenalidomide) or with cyclophosphamide. The remaining pts (5) were up-front treated with IMiDs-based regimens, not including PI. For each pts, paired bone marrow samples were collected both at diagnosis and at relapse. SNPs array analyses were performed on the CD138+ enriched cell fractions (Affymetrix 6.0 and CytoscanHD) and data were analyzed with ChAS v3.1 and Nexus software, to obtain Copy Number Alterations (CNAs) results. Results: The genomic landscape's modulations were evaluated in details in each pair of samples included in the study: by one side, by monitoring the variations of macro CNAs types; by the other, by focusing on changes of CNAs frequencies, as observed in 27 genes of interest (selected according to their recognized role in MM pathogenesis). Both approaches were consistent in highlighting three major evolution patterns: in 7/33 (21%) pts, the genomic background at relapse was almost identical to that of diagnosis, suggesting that no evolution affected the diagnosis' genomic architecture. In 13/33 (39%) pts, an overall increase in the frequencies of the same CNAs as observed at diagnosis was detected at relapse, suggesting a linear evolution of the diagnosis' predominant clone. Finally, in 13/33 (39%) pts, either increased or decreased frequencies of several CNAs, as well as several differences in the CNAs type's prevalence were observed in samples collected at relapse, as compared to those collected at diagnosis, suggesting a branching evolution of sub-clones not-detectable at diagnosis. Of interest, even if an overall increase in the median number of CNAs was observed in the CD138+ cell fractions collected from the whole population of newly diagnosed and relapsed pts (226 vs 507, respectively) - supported by acquisition of CNAs either commonly described as secondary genomic events (i.e. del17p13, amp1q21, del1p23), or associated to the resistance to bortezomib (i.e. del8p21) - nevertheless any peculiar CNAs resulted significantly prevalent in the three identified subgroups of pts. The subgroup of pts who underwent a branching evolution was characterized by a high rate (92%) of achievement of VGPR or better quality of response to upfront therapy, regardless of incorporation of PI into induction regimens. On the contrary, although pts who experienced either linear or no evolution were mostly treated with highly effective PI-based triplet combinations (19/20 pts), the rate of VGPR in this subgroup was only 20% and PR or SD were observed in 9 and 7 pts, respectively. Finally, the median time to first progression of this subgroup of pts was significantly shorter as compared to that of pts with branching evolution (24 vs 35 months, range 4-41 and 7-123 months, respectively, p=0,01). Conclusion The genomic architecture of a subgroup of relapsed MM pts, who were up-front responsive to new drugs-based combination therapies, resulted overall different from that of diagnosis, suggesting a branching evolution of the disease, sustained by the shrinking of the most prevalent clone (therapy-sensitive), as well as by the expansion of subclones (therapy-resistant) not already evident at diagnosis. This observation raises the question whether re-treatment of relapsed pts should be appropriate in the case of branching evolution. Acknowledgments: AIRC (MC), Fondazione Berlucchi (CT), FUV (EB). Disclosures Cavo: Takeda: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

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

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

Monoclonal Ig deposition disease (MIDD) is a broad and uncommon entity, including light chain deposition disease (LCDD), light- and heavy-chain deposition disease and heavy-chain deposition disease.1 Overall, the kidney is the major target organ, the clinical picture being most frequently characterised by nephrotic syndrome, hypertension and renal failure.2 Light-chain Fanconi syndrome due to proximal tubular involvement may also occur, and typically manifests with type II renal tubular acidosis, hypophosphatemia, glycosuria and hypouricemia. Heart, liver and other organs are less frequently involved.3, 4 In approximately one-third of patients with MIDD the underlying clone of monoclonal plasma cells cannot be routinely detected.2 However, recent availability of serum free light chain (FLC) assay has allowed to demonstration of the presence of increased monoclonal FLC in virtually all patients.5 Treatment strategies in these patients have been highly variable, ranging from steroids with or without chemotherapy2 to high-dose therapy followed by autologous stem-cell transplantation (ASCT).5, 6 We retrospectively analyzed the outcomes of 8 consecutive patients who were admitted to our Institute from 1993 to 2006 and received a diagnosis of MIDD, as confirmed by kidney biopsy. Additional investigations to confirm the diagnosis included light microscopy, congo red staining, immunofluorescence with anti-κ and anti-λ antibodies and EM; immunohistochemistry with antibodies directed against plasma cell-associated Ags was performed, when appropriate.

No abstract available.

We prospectively analyzed the prognostic relevance of positron emission tomography–computed tomography (PET/CT) at diagnosis, after thalidomide-dexamethasone (TD) induction therapy and double autotransplantation (ASCT) in 192 newly diagnosed multiple myeloma (MM) patients. Presence at baseline of at least 3 focal lesions (FLs; 44% of cases), a standardized uptake value (SUV) > 4.2 (46%), and extramedullary disease (EMD; 6%) adversely affected 4-year estimates of progression-free survival (PFS; ≥ 3 FLs: 50%; SUV > 4.2: 43%; presence of EMD: 28%). SUV > 4.2 and EMD were also correlated with shorter overall survival (OS; 4-year rates: 77% and 66%, respectively). Persistence of SUV > 4.2 after TD induction was an early predictor for shorter PFS. Three months after ASCT, PET/CT was negative in 65% of patients whose 4-year rates of PFS and OS were superior to those of PET-positive patients (PFS: 66% and OS: 89%). In a multivariate analysis, both EMD and SUV > 4.2 at baseline and persistence of fluorodeoxyglucose (FDG) uptake after ASCT were independent variables adversely affecting PFS. PET/CT involvement at diagnosis, after novel agent-based induction and subsequent ASCT is a reliable predictor of prognosis in MM patients. This study is registered at www.clinicaltrials.gov as NTC01341262.

Allogeneic stem cell transplantation (allo-SCT), which provides a tumor-free graft, is an alternative approach to autologous transplantation for multiple myeloma (MM) that offers the possibility of cure through a graft vs myeloma effect (GVM). However, the inherent non relapse mortality (NRM) and the high post-transplant relapse rate are the major shortcomings of this strategy which continues to have a controversial role in MM treatment. To highlight the long-term clinical outcomes of allo-SCT, we performed a retrospective analysis on 102 patients (pts) with MM who received at our Institution either a myeloablative (MA) (74 pts) or a non-myeloablative (NMA) (28 pts) conditioning regimen between 1990 and 2014. The MA regimen consisted in low dose TBI and cyclophosphamide (cyclo) ± melphalan (mel) or busulfan-cyclo. The NMA regimen was mel-fludarabine. Graft versus host disease (GVHD) prophylaxis consisted in cyclosporine + methotrexate (83%) or mycophenolate (17%), with the addition of thymoglobulin for unrelated or related female vs male recipient donors. The median age was 42 yrs (IQR 38-47), 60% pts were male. The hematopoietic cell donors were sibling in 77 pts and unrelated in 25, while the source of stem cells was peripheral blood in 65 and bone marrow in 37 pts. Fifty-six pts received allo-SCT as first-line therapy, 31 as second-line and 13 as third or fourth-line. Median time from diagnosis to allo-SCT was 22 months. Response status at the time of transplant was at least PR in 63% of the pts, including ≥ VGPR in 36% and CR in 12%. Overall, the response rate after allo-SCT was as follows: CR 58%, VGPR 19%, PR 18%. Median CR duration was 10 years (44% at 15 years). The incidence of acute grade II-IV and III-IV GVHD was 25% and 15%, respectively. The incidence of all grades chronic cGVHD was 48%, grade ≥2: 24%, with a median onset time of 178 days. By competitive risks analysis, the cumulative incidence of cGVHD at 1 and 3 years was 20% and 32%, respectively. On univariate analysis, the gender combination of female donor-male patient resulted in significantly higher incidence of cGVHD (sub hazard ratio, SHR, 2.3, P= 0.03). The cumulative incidence of NRM was 6.6% at 100 days, 11% at 1 year and 14.4% at 3 years, with lack of statistically significant relationship with the conditioning regimen. By univariate analysis, < PR prior to allo-SCT (SHR 2.8) and all grades cGVHD (SHR 4.6) were significantly associated with an increased NRM. By competitive risks analysis, the cumulative incidence of relapse was 50% at 5 years, 58% at 10 years and 59% at 15 years. On univariate analysis, sibling donor (SHR 0.54, P=0.047), all grades cGVHD (SHR 0.5, P=0.011) and ≥VGPR after allo-SCT (SHR 0.38, P=0.001) were significantly associated with extended time to progression. All grades cGVHD (SHR 0.43, P=0.005) and ≥VGPR after allo-SCT (SHR 0.36, P= 0.001) were independent predictors for a lower risk of progression on multivariate analysis. With a median follow-up of 13 years, overall survival (OS) was 43% at 5 years and 34% at 10 years. In univariate analysis, low ISS stage at diagnosis, sibling donor, absence of female donor-male patient gender combination, ≥VGPR prior to allo-SCT and first-line allo-SCT were significantly related to OS. Multivariate analysis confirmed an OS benefit for having a sibling donor (HR 0.30, P In conclusion, this retrospective analysis performed with an extended follow-up of 13 years shows that a fraction of MM pts can be long-term survivors after allo-SCT, one third of them being potentially cured. The best outcomes were obtained when allo-SCT was applied in pts in an early phase of their disease and with a small residual tumor size. The major challenge, both with MA and NMA allo-SCT, was the relatively high post-transplant relapse rate. The presence of cGVHD was protective for the risk of progression, supporting the role of GVM, but increased the risk of NRM. New approaches aimed at modulating cGVHD are warranted. In addition, incorporation of novel agents before and after allo-SCT to increase the rate and duration of high-quality responses, as well as identification of those patients mostly benefiting from this procedure, will likely contribute to improve long-term outcomes. Disclosures Zamagni: Celgene Corporation: Honoraria, Speakers Bureau; Janssen Pharmaceuticals: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Cavo:Sanofi: Consultancy, Honoraria; Onyx: Honoraria; Jansenn: Consultancy, Honoraria; Millenium Pharmaceuticals: Honoraria; Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; BMS: Honoraria.

INTRODUCTION In newly diagnosed Multiple Myeloma (MM) patients (pts), Copy Number (CN) losses of chromosome 17p13, carrying the TP53 tumor-suppressor gene, are strong predictors of poor outcomes. On the contrary, the prognostic relevance of TP53 mutations at the onset of the disease is less clear, due to the very limited frequency of clonal lesions, as revealed by Sanger sequencing. To address this poorly investigated issue, we used an ultra-deep sequencing (UDS) approach to characterize the TP53 structural architecture in both newly diagnosed and relapsed MM pts and to assess the prognostic role and evolution over time of small TP53 mutated sub-clones. SAMPLES AND METHODS A cohort of 99 newly diagnosed MM pts treated up-front with bortezomib-based regimens and autologous stem cell transplantation, was included in this molecular study. In 29 cases, samples were collected both at diagnosis and at relapse(s). DNA was obtained from CD138+ highly purified plasma cells. TP53 gene mutational status was analysed by using an amplicon-targeted UDS approach (GSJ, 454 Roche Life Sciences). In order to discriminate between low frequency sub-clonal TP53 variants and sequencing errors, sequencing raw data were filtered according to cut-off values based on different ranges of sequences' coverage depth. Additional filters were also applied, based on both quality and biological cut-offs, to obtain a final confident list of variants. Analysis of CN alterations (CNAs) was performed by SNPs array and results analysed with ChAS software. RESULTS With a median coverage of 1386X, a list of 129 correctly called TP53 variants (either missense, or nonsense or splice ones), including 20 INDELs, was detected. Only deleterious and N/A variants (according to SIFT classification) were included in the list. Most newly diagnosed MM pts (55%) carried at least one TP53 sub-clonal variant (on average 1.08 variants per pts), with 45/99 (45%) carrying non-mutated TP53. Pts carrying TP53 sub-clonal variants bared also TP53 CN hemizygous losses (20%), CKS1B gains (56%) and cdkn2c losses (14%). According to TP53 sub-clonal mutational load, pts were stratified in two sub-groups, including 28 pts with ≥2 (high load) and 71 with The clinical impact of the TP53 sub-clonal mutational load, as well as of variants recurrence, was evaluated in 90/99 MM (median follow up = 70 months). Results of statistical analysis are summarized in Table 1. Pts carrying either high TP53 sub-clonal mutational load or RVs had significantly shorter OS and OS after relapse, as compared to the others, while no difference between these two groups was seen regarding PFS and TTP. Multivariate analysis showed that high TP53 mutational load, as well as the presence of TP53 RVs, both resulted independent factors adversely affecting OS and OS after relapse (Table 2). Of note, none of the detected genomic aberrations significantly influenced the response to front-line induction therapy. The distribution of both TP53 sub-clonal variants and genomic CNAs was overall modified in samples collected at relapse(s): 90% of relapsed pts carried at least one sub-clonal variant (on average 1.63 variants per pts) with 3/29 (10%) relapsed MM carrying non-mutated TP53. Moreover, 5 different sub-clonal lesions proved a linear increment of both TP53 VAFs (from 29.4% to 54.6%; from 7.8% to 12.4%; from 0.5% to 4.3%) and TP53 CN loss smooth signal (from 7% to 89% and from 50% to 100%), as evaluated in longitudinally collected samples. CONCLUSIONS The UDS analysis of TP53 coding sequence in newly diagnosed MM highlighted for the first time a high rate of variants, recurring with a wide range of frequencies among samples. The increased number of TP53 sub-clonal variants per pts in samples collected at relapse(s), compared to that seen at the onset of the disease, suggests a sub-clonal dynamics over time. This finding might explain the adverse impact of high TP53 sub-clonal mutational load and TP53 RVs on OS, due to a shorter OS after relapse. Acknowledgments: Roche Diagnostics for applicationsupport in the realization of this project. Disclosures Zamagni: Celgene Corporation: Honoraria, Speakers Bureau; Janssen Pharmaceuticals: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Martinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Ariad: Consultancy; AMGEN: Consultancy; ROCHE: Consultancy; Pfizer: Consultancy; MSD: Consultancy. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.

RS-001 Hedgehog Pathway Over Expression Identifies Myeloma Patients With Worse Prognosis M. Martello, D. Remondini, A. Pezzi, E. Borsi, M. Procacci, A.F. Dico, B. Santacroce, L. Pantani, E. Zamagni, P. Tacchetti, S. Rocchi, B.A. Zannetti, G. Marzocchi, N. Testoni, G. Martinelli, M. Cavo, C. Terragna Institute of Hematology “Seragnoli”, Experimental, Diagnostics and Specialty Medicine DIMES, University of Bologna; Physics and Astronomy Department DIFA, University of Bologna

AML is a heterogeneous disease with a variety of structural and numerical chromosomal and genetic alterations that provided important prognostic information capable to guide therapy and predict outcome. The reported TP53 mutation rate in AML is low (2.1%). By contrast, the incidence of FLT3 disruption is frequent (20-30%) and is an independent predictor of unfavourable outcome of acquired clinical resistance to FLT3 inhibitors. TP53 mutations in AML with a complex karyotype (CK) is higher (69-78%), but few data are available for association between the most common AML associated lesions such as FLT3, NPM1, DNMT3A, IDH2 and TP53 mutation or CK. Aims: To investigate the frequency, the types of mutations, the associated cytogenetic, the molecular abnormalities, the correlation with known molecular alterations (FLT3, NPM, etc.) and the prognostic role TP53 mutations in adult AML pts. Patients and Methods: 886 AML patients were analysed for cytogenetic and for a panel of genetic alterations (FLT3, NPM, WT1, DNMT3A, IDH1-2 etc). Of these, 200 adult AML pts were also examined for TP53 mutations using several methods, including Sanger sequencing, NGS and HiSeq 2000 platform (38/200) and were correlated with cytogenetic analysis. Results: 55 pts (27.5%) showed 3 or more chromosome abnormalities (CK-AML), 83 (41.5%) presented one or two cytogenetic abnormalities (other-AML) and 43 pts (21.5%) have normal karyotype (nK). In 19 cases the karyotype was not available. Sanger sequencing analysis detected TP53 mutations on 29 patients with 36 different types of mutations (32 deleterious point mutations; 4 deletions); seven pts (4%) have 2 mutations. Mostly (23/29) of the TP53 mutated pts (79.3%) had CK while only 6/29 (21%) mutated pts have “no CK”. Overall, between pts with CK, TP53 frequency is 41.8% (P>0,0001). 120 pts were analysed for concomitant presence of TP53 mutations and FLT3/NPM1 disruption/mutation: revealing a significant relation between pts with FLT3-ITD or NPM1 and TP53 wild-type (p = 0.043 and 0.022 respectively). As for clinical outcome alterations of TP53 were significantly associated with poor outcome (OS and EFS p WES analysis done in 38 pts (33 TP53 wt and 5 pts TP53 mutated) revealed no genes exclusively mutated in the 5 TP53 mutated pts. Conclusions: Our data demonstrated that TP53 mutations occur in 14.5% of AML with a higher frequency in the subgroup of CK-AML (p Citation Format: Anna Ferrari, Cristina Papayannidis, Elisa Zuffa, Carmen Baldazzi, Antonella Padella, Eugenia Franchini, Ilaria Iacobucci, Stefania Paolini, Viviana Guadagnuolo, Margherita Perricone, Valentina Robustelli, Claudia Venturi, Maria Chiara Abbenante, Sarah Parisi, Chiara Sartor, Francesca Volpato, Federica Cattina, Giorgia Simonetti, Maria Chiara Fontana, Maria Teresa Bochicchio, Federica Frabetti, Elisa Lani, Katia Mancuso, Beatrice Zannetti, Simona Luatti, Emanuela Ottaviani, Nicoletta Testoni, Giovanni Martinelli. TP53 mutations are mutually exclusive with FLT3 and NPM mutations in AML patients and are strongly associated with complex karyotype and poor outcome. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4906. doi:10.1158/1538-7445.AM2015-4906

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

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

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

PET/CT is a reliable technique for assessing skeletal involvement in MM and a valuable tool at the onset of the disease for predicting outcomes in those patients who are eligible to subsequently receive autologous stem cell transplantation (ASCT). However, the prognostic role of PET/CT after treatment and/or during the follow up of the disease, as well as in the non ASCT setting, still remains less defined. To address these issues, we retrospectively analyzed 282 symptomatic MM pts, with a median age of 58 yo, (range 22-83), who were diagnosed and treated in a single institution between 2002 and 2012, and were followed for a median of 66 months. Treatment included ASCT in 70%, novel agents in 77% and was bortezomib-based in 37% of the cases. All the pts were studied with PET/CT at baseline, then every 12-18 months during follow-up, and at the time of each subsequent relapse; for 189 of them PET/CT scans at baseline and 3 months after the end of first line treatment were available. Bone marrow involvement was described as negative, diffuse or focal. The number of focal lesions (FLs), their associated standardized uptake value (SUVmax) and presence of extra-medullary disease (EMD) were recorded. Forty two percent of the pts at diagnosis had > 3 FLs and in 50% of them SUVmax was > 4.2; EMD was present in 5% of the cases. On multivariate analysis, these 3 variables adversely affected PFS and OS, and retained prognostic relevance independently of the treatment received (including or not ASCT, bortezomib- or non-bortezomib-based). On multivariate analysis, ISS stage 3, presence of >3 FLs at PET/CT and failure to achieve CR during or after first line treatment were the leading factors independently associated with shorter PFS and OS. These 3 variables enabled the definition of a scoring system, based on the number of risk factors simultaneously present (score 0: none of the 3 adverse factors, 31% of the pts; score 1: only one out of the 3, 37%, score 2+: 2 or 3 factors, whatever of them, 32% ), that predicted for PFS and OS. More specifically, median PFS was 36 months (mos) for pts with score 0, 58 mos for score 1 and 74 mos for score 2+ (P=0.000). OS was also significantly influenced by the number of adverse factors, with a progressive increase in hazard ratios. A similar stratification into 3 prognostic groups was obtained when SUVmax > 4.2 and presence of EMD replaced FLs>3 within the scoring system. After treatment, PET/CT negativity (PET-CR) was observed in 70% of the pts, while conventionally-defined CR was achieved in 53% of them. Attainment of PET-CR favorably influenced PFS and OS, both in uni and multivariate analyses. Notably, 29% of the pts who achieved CR according to conventional criteria still had positive PET/CT scans: their median PFS was 50 mos as compared with 90 mos for those pts who also achieved PET-CR (P= 0.01) (fig. 1). OS was significantly inferior, as well, for pts not achieving PET-CR, with 6-year estimate of 65% in comparison to 90% for PET negative pts (P=0.0035) (fig. 1). On multivariate analysis, PET-CR was an independent factor predicting for prolonged PFS (P= 0.004) and OS (P= 0.02) within the conventionally-defined CR group. Sixty three percent of the pts experienced relapse or progression, after a median of 56 mos from the end of first-line treatment. In 37% of them, progression was only serological, both serological and skeletal in 48%, only skeletal in 15% and in 12% of these latter patients it was exclusively detected by systematic PET/CT during the follow-up (no pain or pathological fractures). A logistic regression analysis of baseline and post treatment features revealed that persistence of SUVmax > 4.2 after the end of first line treatment was independently associated with exclusive PET/CT progression. In conclusion, PET/CT was confirmed as a reliable predictor of outcome in newly diagnosed MM pts, whatever the treatment. PET/CT combined with ISS stage and attainment of CR to first line therapy was able to split pts in different prognostic groups. Importantly, PET/CT contributed to a more careful and deep evaluation of CR, going beyond the conventionally defined level, and should thus be recommended as a complementary tool to define “true” CR. Finally, in pts with a persistent high glucose metabolism after first line treatment, PET/CT can be recommended during the follow-up, in order to point out possible progression, not otherwise identifiable. Disclosures: Zamagni: Celgene: Honoraria; Janssen-Cilag: Honoraria. Cavo:Celgene: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Millennium: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.

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

Multiple Myeloma (MM) is an incurable hematologic malignancy characterized by the accumulation of malignant plasma cells. Dysregulation of MYC by rearrangement or translocation are common somatic events described either in early or late stage of the disease, and transcriptional profiling of MYC pathway activation is observed in more than 60% of MM cell lines. Hypoxia Inducible Factor-1α (HIF-1α) overexpression has been described in several MM cell lines and in about 30% of MM patients samples. In solid tumours, deregulation of c-MYC has been associated with HIF-1α up-regulation. In the present study we explored the interaction between c-MYC and HIF-1α in a panel of MM cell lines. We had previously shown that treatment with EZN2968, an antisense oligonucleotide against HIF-1α, resulted in a significantly reduction of HIF-1α protein level as soon as 24h and lasted over 96h. To confirm the inhibition of HIF-1α activity, MM1S cells were treated with EZN2968 for 24h, lysed, co-precipitated with p300, and incubated with anti-HIF-1α antibody. We showed that HIF-1α was no longer associated p300 in EZN-treated compared to untreated samples, suggesting an inhibitory effect of HIF-1α activity. We next observed that treatment with EZN2968 induced a progressive accumulation of cells in S-phase with concomitant reduction of G2/M phase. By western blot analysis, we observed that p21, cell cycle check point negatively regulated by c-MYC, was up-regulated in treated samples. We further verified the effect of HIF-1α inhibition on c-MYC protein level by western blotting. After treatment with EZN2968, c-MYC protein expression was reduced in a time dependent manner, suggesting that c-MYC protein level is associated with inhibition of HIF-1α. To examine whether HIF-1α and c-MYC regulate each other promoter activity, we performed Chromatin Immunoprecipitation (ChIP) assays with HIF-1α or c-MYC antibodies. HIF-1A and MYC promoter amplification signals, were present in the controls samples, and decreased after EZN2968 exposure. Recently, it has been shown that SIRT1, a transcription factor involved in a development and cellular stress responses, can modulate HIF-1α and c-MYC activity. By Immunoblotting assay, we observed that SIRT1 physically interacts with both proteins and that, after 24h of exposure to EZN2968, c-MYC and HIF-1α were strongly associated to SIRT1. These results were also confirmed at the transcriptional level, by ChIP assay using an anti-SIRT1 antibody. After 24h of treatment with EZN2968, we observed a significant increase of HIF-1A and MYC promoter amplification signals in treated compared to untreated samples, suggesting that SIRT1 recruitment at both promoters is dependent on HIF inhibition. We showed that in MM cells the expression of HIF-1α and c-MYC are linked and mediated by SIRT1 deacetylase protein. The data suggests a new regulatory mechanism for controlling c-MYC and HIF-1α activity by SIRT1. Supported by PRIN. Citation Format: Enrica Borsi, Giulia Perrone, Carolina Terragna, Marina Martello, Manuela Mancini, Elisa Leo, Elena Zamagni, Paola Tacchetti, Annamaria Brioli, Lucia Pantani, Beatrice Zannetti, Giovanni Martinelli, Michele Cavo. SIRT regulates the molecular interaction between c-MYC and HIF-1α in multiple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3415. doi:10.1158/1538-7445.AM2013-3415

Abstract 594 Survival of patients with multiple myeloma (MM) has been extended with the introduction of autologous stem cell transplantation (ASCT). More recently, availability of highly effective novel agents has further improved patient outcomes. However, it is still the matter of debate whether a proportion of patients treated with ASCT can enjoy a long term survival, while sustaining prolonged high quality response. To address this issue and to identify those variables which were related to long-term survival, we performed a post-hoc analysis of two large prospective clinical trials of ASCT in newly diagnosed MM patients, the first one comparing single versus double ASCT and the second one incorporating thalidomide-dexamethasone (TD) into double ASCT. A total of 321 patients were randomly assigned in the first study to receive either a single or double ASCT, as previously described (Cavo M et al, JCO 2007). Three hundred and fifty seven patients were enrolled in the subsequent multicenter phase 2 study incorporating TD from the outset until the second ASCT; details of the protocol were previously reported (Cavo et al, J. Clin. Oncol 2009). Results were updated as of 30 March 2012 and compared with those previously reported. All the analyses were performed on an intention-to-treat basis. After a median follow-up of 61 months for the entire treatment population of the first study, PFS remained significantly longer with tandem versus single ASCT (median 37 vs 25 months, P= 0.012), while OS was similar in the two groups (median 71 vs 67 months). 47% and 33% of the patients in the double and single ASCT group achieved a CR+nCR (P= 0.008). Overall, in 24% and 11% of the patients, CR+nCR was sustained for more than 5 and 10 years, respectively. In a multivariate Cox regression analysis, best response (CR+nCR) ever achieved was the most important variable significantly extending PFS (P= 0.003) and OS (P=0.050); random assignment to double ASCT was an additional variable predicting for prolonged PFS(P= 0.026). After a median follow-up of 84 months from starting TD in the second study, median values of PFS and OS were 47.2 and 109.6 months, respectively. The final rate of CR+nCR was 34%, which was maintained for a median of 53 months. Overall, in 42.1% and 9.1% of the patients CR+nCR was sustained for more than 5 and 8 years, respectively. On multivariate analysis, failure to ever achieve at least CR+nCR, low Hb, high β2-m and t(4;14)±del(17p) were found to be independent variables predicting for poorer outcomes. In particular, a shorter OS was seen for patients ever lacking high-quality responses (HR: 0.35, 0.23–0.54, p Overall, 23% and 20% of patients in the first and second study were alive over 10 or 8 years, respectively (long-term survivors). Median PFS of long-term survivors in the 2 studies were 74 and 87.7 months, respectively, versus 25 and 37 months for the rest of the population (P= 0.0000). Median duration of CR+nCR were 70 and 78 months in the long-term survivors group for the first and second study, respectively, in comparison with 21 and 49 months in the remaining patients (P In conclusion, although the comparison between TD incorporated into ASCT and ASCT without thalidomide was not directly addressed by this analysis, TD + ASCT was associated with extended PFS and OS. Approximately 20% of the patients undergoing up-front ASCT can achieve long term survival (8–10 years from start of treatment), with 33% of them remaining relapse free. Attainment of sustained high-quality responses was the leading independent variable predicting for long-term OS. Prolonged survival after relapse was a contributing factor to long-term OS. Disclosures: Off Label Use: One of the 2 protocols discussed includes the use of thalidomide as induction prior to ASCT.

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

The p53 tumor suppressor pathway is tightly kept in check or completely silenced in cancer cells. A potent inhibitor of p53 is MDM4, which is located on chr.1q: it is critical for the control of p53 activity during the response to stress and is often amplified in several types of cancer. Since both del(17p) and amp(1q) identify a subgroup of high-risk MM pts, even when the novel agents are part of up-front treatment strategy, we molecularly analyzed a subgroup of MM patients treated with bortezomib-thalidomide-dexamethasone (VTD) and autologous stem cell transplantation (ASCT) in order to investigate mechanisms which might be activated in myeloma plasma cells to direct and/or indirect limit the p53 function. CD138+ plasma cells obtained at diagnosis from 61 pts, subsequently treated with VTD and ASCT were analysed by means of GEP and unpaired analysis of CNA (Affymetrix U133 Plus2.0 and 6.0 SNP arrays). Nineteen out of 61 pts (31%) carried a minimal amplification region on chr.1q, harboring MDM4. Eight out of 61 pts (13%) carried a 482 Kb minimal deletion region on chr.17 harboring TP53. To explore the involvement of the p53 pathway in MM, pts were stratified according to the presence of amplified MDM4 and/or deleted p53 (group A, 24 pts) or the absence of both these abnormalities (group B, 37 pts). Baseline clinical characteristics were homogeneous, except for a higher rate of ≤ 3 bone lesions in pts of group A. The rate of best complete or near complete response was 40% in group A and 14% in group B (p = 0.04). With a median follow-up of 36 months, the risk of progression was 46% for pts in group A and 19% for those in group B (p = 0.03). The average number of aberrations per group was overall higher in group A as compared to group B (165 vs. 103 CNAs, p =0.03). A comparison of expression profiles of the two subgroups of pts highlighted a deregulated expression of genes involved in the mechanisms of response to genotoxic stress, i.e. of damage sensor genes (ATM, RAD9, RAD 50, ATRip), of damage signal mediator genes (H2AFX, 14-3-3), of genes involved in regulation of cell proliferation (CDK6, CDC25, CCND2) and of anti-apoptotic genes (BCL2, p73) (one-way ANOVA plus multiple-test correction with FDR Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1873. doi:1538-7445.AM2012-1873

Abstract 1821 Introduction: PN is an important complication of MM and its incidence has been further increased after the introduction of the novel agents thalidomide and bortezomib. In a phase 3 trial comparing TD with VTD as induction therapy prior to and consolidation therapy after double autologous stem-cell transplantation for previously untreated MM patients, the VTD arm was significantly superior over TD in terms of improved rates of complete or near-complete response (CR/nCR) (the primary study endpoint) and progression free survival (PFS). Toxicity of VTD and TD regimens, including PN, was a secondary study endpoint. Methods: We performed a subanalysis of the study to assess the frequency, reversibility, risk factors and molecular markers associated with treatment-emergent PN. PN was graded by use of National Cancer Institute's Common Toxicity Criteria (NCI CTCAE) version 3.0. Since grade 1 PN could be misinterpreted and does not interfere with the daily activities, only patients who developed PN of at least grade 2 were evaluated. A total of 474 patients (of whom, 236 randomized to the VTD arm and 238 to TD) were stratified according to the development or not of grade ≥2 neurological adverse events (NAEs). Gene expression profiles (GEP) of pre-treatment CD138+ bone marrow plasma cells (BMPCs) were analyzed in a subset of 127 VTD-treated patients for whom biological samples taken at diagnosis were adequate for genomic analysis. GEP experiments were performed using the Affymetrix HG-U133 Plus 2.0 platform and class comparison of groups of array was done with one-way ANOVA Partek Genomic Suite (version 6.4). Results: Occurrence of PN throughout the entire treatment program was significantly higher in the VTD arm compared with TD. In particular, the rate of grade ≥2 PN was 35% vs 10% (p Conclusions: Although VTD incorporated into double ASCT was associated with a higher incidence of grade ≥2 PN compared with TD, the probability of complete resolution or improvement to at least grade 1 was comparable in both VTD- and TD-treated groups. Importantly, NAEs did not adversely affect the rate of CR/nCR, and TTP and PFS. No relationship between development of PN and both patient demographics and disease characteristics was observed. Conversely, GEP analysis of BMPCs from patients with VTD-induced PN showed the significant deregulated expression of genes involved in the nervous system function. Disclosures: Off Label Use: Bortezomib and Thalidomide as induction therapy prior to and consolidation therapy after double autologous stem-cell transplantation in newly diagnosed multiple myeloma. Tosi:Janssen: Honoraria; Celgene: Honoraria; Novartis: Honoraria. Baccarani:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Mayers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Boccadoro:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria. Cavo:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: 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.

Abstract 369 F-18-fluorodeoxyglucose positron emission tomography integrated with computed tomography (FDG-PET/CT) has been reported to be a careful technique for a widespread screening of myelomatous lesions, both medullary and extramedullary, at the onset of multiple myeloma (MM). By the opposite, the role of FDG-PET/CT in assessing and monitoring response to therapy and the prognostic value of this imaging technique is less explored. Aim of the present study was to prospectively evaluate the prognostic significance of FDG-PET/CT at diagnosis and after therapy in an homogeneous population of patients with newly diagnosed MM who received up-front single or double autologous stem cell transplantation (ASCT). By study design, all patients were studied with FDG-PET/CT at baseline, 3 months after ASCT, every year during the maintenance/follow-up phase and at time of relapse. Bone marrow involvement was described as negative, diffuse or focal. The number of focal lesions (FL), as well as their size and associated standardized uptake values (SUV) were recorded. Extramedullary disease, if present, was described by location, size, number of lesions and SUV. A total of 146 patients who actually received the planned treatment program and were followed for a median of 39 months were analyzed. Their median age was 58 years. ISS stage was as follows: 48% stage I, 31% stage II, 14% stage III. Six per cent of the patients had renal failure, related to the presence of hypercalcemia at diagnosis. Del (13q), t(4;14) and del(17p), detected by FISH, were present in 47%, 25% and 10% of the patients, respectively. Induction treatment prior to ASCT included an IMiD in 55% of the patients, bortezomib in 34% and chemotherapy in 11% of the cases. The best overall response rate, including CR and at least VGPR, was 56% and 82%, respectively. Median PFS of the entire population was 55 months and the 5-year projected OS rate was 85%. At baseline, 22% of the patients had a negative PET/CT, 27% presented less than 3 FL and 51% 3 or more FL or a diffuse bone marrow uptake of FDG. In 44% of the cases the SUV was low (< 3.9) while in 56% was 4 or more. In 6% of the patients extramedullary disease was present. No significant differences between patients with positive or negative PET/CT were found regarding baseline characteristics and response to therapy. The entity of PET/CT involvement at baseline influenced clinical outcomes. In particular, patients with more than 3 FL and/or SUV > 3.9 had a significantly shorter OS in comparison with patients with FL < 3 and/or SUV < 3.9 (5-year projected OS: 92% vs 75%, respectively, P=0.04). After 3 months from ASCT, PET/CT was negative in 65% of the patients and remained positive, either unchanged or with a reduction in the SUV, in 35% of them. A correlation between PET/CT findings and response to ASCT was evident; in particular, among patients with negative PET/CT the rate of CR and VGPR was significantly higher than among patients with positive PET/CT (CR: 68% vs 38%; P= 0.001) (VGPR: 90% vs 76%; P= 0.008). Complete FDG suppression at PET/CT after ASCT conferred superior PFS and OS than persistence of FL (PFS: median 58 vs 45 months, respectively, P=0.05; 5-year projected OS: 92% vs 79%, respectively, P=0.004). In conclusion, involvement of PET/CT at diagnosis in terms of number of FL and intensity of tumour metabolism (SUV) predicted survival outcome. Complete FDG suppression after ASCT was strictly related with achievement of high-quality response and was prognostically relevant for durable disease control and longer OS. Disclosures: Cavo: Jansen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, no; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, no; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, no.

The availability of novel drugs such as immunomodulatory drugs, IMiDs, and proteasome inhibitors, PI, for the front line treatment of Multiple Myeloma (MM) has dramatically changed patients’ outcomes, significantly prolonging progression free survival (PFS) and translating into an extended overall survival (OS). Despite these impressive results, concerns have been raised regarding the possibility that IMiDs (lenalidomide and thalidomide) might increase the risk of developing SPMs, especially when associated with alkylating agents such as melphalan (MEL). It has long been known that the use of MEL can result in an increased incidence of SPMs, and this risk seems to be increased by the association with IMiDs. By the opposite, the use of PI in newly diagnosed MM (NDMM) patients (pts) was not associated with an increased risk of SPMs in a phase III study aimed at comparing BOR, MEL and prednisone (MP) vs MP.[1] Despite these results, the full impact of an upfront treatment containing both a PI and an IMiD in association with high dose MEL still has to be investigated. To address this issue, we have evaluated the incidence of SPMs in pts enrolled in the GIMEMA 26866138-MMY-3006 multicentre phase III study aimed at comparing BOR, thalidomide and dexamethasone (VTD) versus TD as induction before, and consolidation after, a double course of high dose MEL. The trial, conducted in Italy from 2006 to 2008, enrolled 480 transplant eligible NDMM, of which 474 received assigned treatment. Data on the incidence of SPMs are available for 299 pts (63%, 148 VTD and 151 TD). With a median follow up of 73 months, 25/299 pts (8%) developed a SPM: 7 (2%) SPMs were hematologic and 18 (6%) were non hematologic. The median time from trial entry to development of the SPM was 36 months (range 8.4-69.0). The number of pts developing a SPM was lower in VTD arm (5%) compared to TD arm (11%, p=0.068). Among pts developing a SPM, the proportion of solid malignancies was similar between treatment arms (75% and 71% in VTD and TD, respectively). Similarly, the percentages of hematologic SPMs was 25% for VTD pts and 29% for TD. On the overall population, the incidence rate (IR) of developing a SPM was 1% at 1 year and 9.9% at 6 years (yrs). This incidence was significantly lower for patients randomised to VTD compared with patients receiving TD (6% vs 13% at 6 yrs, p=0.037). When looking at the IR of solid tumours we noticed that only 5% of VTD-treated patients developed a solid SPM, as compared to 9.6% in TD; similarly, less hematologic SPMs were observed in the BOR arm (1% vs 4% at 6 yrs for VTD and TD, respectively). When the analysis was performed according to SPMs type, no statistical significance could be demonstrated. Our data compare favourably with data previously reported on the incidence of developing SPMs in NDMM treated with BOR frontline in association with MEL. With a follow up of 6 years, we were able to confirm that treatment with PI is associated with a low risk of developing SPM. Interestingly our data suggest that treatment with PI might decrease the risk of developing a SPM compared to IMiDs based treatment. Randomised trials with PI and IMiDs treatment should prospectively evaluate this issue. References 1. San Miguel, J.F., et al., Persistent overall survival benefit and no increased risk of second malignancies with bortezomib-melphalan-prednisone versus melphalan-prednisone in patients with previously untreated multiple myeloma. J Clin Oncol, 2013. 31(4): p. 448-55. Disclosures Musto: Janssen: Honoraria; Celgene: Honoraria. Cavo:Onyx: Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Millenium Pharm: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau.