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

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

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.

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

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

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

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

// Valentina Salvestrini 1 , Stefania Orecchioni 2 , Giovanna Talarico 2 , Francesca Reggiani 2 , Cristina Mazzetti 3 , Francesco Bertolini 2 , Elisa Orioli 4 , Elena Adinolfi 4 , Francesco Di Virgilio 4 , Annalisa Pezzi 1 , Michele Cavo 1 , Roberto M Lemoli 5, * , Antonio Curti 1, * 1 Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy 2 European Institute of Oncology, Milan, Italy 3 Department Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy 4 Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy 5 Clinic of Hematology, Department of Internal Medicine (DiMI), University of Genoa, Genoa, Italy * These authors are contributed equally to this work Correspondence to: Valentina Salvestrini, email: valentin.salvestrin2@unibo.it Keywords: acute myeloid leukemia (AML), leukemic stem cell (LSC), P2X7R, ATP, apoptosis Received: May 24, 2016 Accepted: September 25, 2016 Published: December 13, 2016 ABSTRACT Recent studies have shown that high ATP levels exhibit direct cytotoxic effects on several cancer cells types. Among the receptors engaged by ATP, P2X7R is the most consistently expressed by tumors. P2X7R is an ATP-gated ion channel that could drive the opening of a non-selective pore, triggering cell-death signal. We previously demonstrated that acute myeloid leukemia (AML) cells express high level of P2X7R. Here, we show that P2X7R activation with high dose ATP induces AML blast cells apoptosis. Moreover, P2X7R is also expressed on leukemic stem/progenitor cells (LSCs) which are sensitive to ATP-mediated cytotoxicity. Conversely, this cytotoxic effect was not observed on normal hematopoietic stem/progenitor cells (HSCs). Notably, the antileukemic activity of ATP was also observed in presence of bone marrow stromal cells and its addition to the culture medium enhanced cytosine arabinoside cytotoxicity despite stroma-induced chemoresistance. Xenotransplant experiments confirmed ATP antineoplastic activity in vivo . Overall, our results demonstrate that P2X7R stimulation by ATP induced a therapeutic response in AML at the LSC level while the normal stem cell compartment was not affected. These results provide evidence that ATP would be promising for developing innovative therapy for AML.

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Attenuated polyposis could be defined as a variant of familial adenomatous polyposis (FAP) in which synchronous polyps of the large bowel range between 10 and 99. We analysed all cases of attenuated polyposis observed over the last 30 years with the objectives: (A) to classify the disease according to different type and proportion of polyps; (B) To ascertain the contribution of APC and MutYH genes; (C) to discover features which could arise the suspicion of mutations; (D) To obtain indications for management and follow-up. 84 individuals in 82 families were studied. Polyps were classified into four groups as adenoma, hyperplastic, other serrated lesions or others; APC and MutYH mutations were assessed. Mean age at diagnosis was 54 ± 14 years in men and 48 ± 13 in women (P = 0.005). Polyps were more numerous in women (37 ± 26 vs 29 ± 22). Sixty % of patients underwent bowel resection, mainly for cancer; the remaining were managed through endoscopy. A total of 2586 polyps were detected at diagnostic endoscopy: 2026 (80 %) were removed and analysed. Adenomas were diagnosed in 1445 (70 %), hyperplastic polyps in 541 (26 %), other serrated lesions in 61 (2.9 %). Adenomas and hyperplastic lesions were detected in the majority of patients. In 68 patients (81 %) in whom studies were executed, APC mutations were found in 8 and MutYH mutations in 10. Genetic variants were more frequent in women (12 vs 6, P = 0.039). Taking into consideration the prevalent (>50 %) histology and presence of mutations, patients could be subdivided into four groups: (1) APC mutated polyposis (AFAP), when adenomas were >50 % and APC mutations detected (no. 8, 10 %); (2) MutYH mutated polyposis (MAP), adenomas >50 % and biallelic MutYH mutations (no. 10, 12 %); (1) attenuated polyposis without detectable mutations, prevalence of adenomas, 48 cases (57 %); (1) hyperplastic-serrated polyposis, with prevalence (>50 %) of hyperplastic/other serrated lesions and no constitutional mutation (no. 18, 21 %). Aggregation of tumors, cancer in probands, distribution of polyps and other clinical characteristics showed no difference among the four groups. In conclusions, AFAP and MAP, the polyposis labeled by constitutional mutations, represented about 25 % of all attenuated polyposis. Mutation-associated cases showed an earlier age of onset of polyps and were more frequent in the female sex.

Colorectal mucosa is targeted by toxic agents, which can initiate or promote colon cancer. The mechanism of damage might be a focal irritation with loss of normal epithelial cell barrier function. Genetic alterations in tumors may also affect host inflammatory response. The aim of this study was to define the extent of inflammation in colorectal mucosa, along colorectal carcinogenesis, and in microsatellite stable and unstable colorectal carcinomas. We collected 103 samples of normal colorectal mucosa from 65 patients (35 with colorectal cancer or adenoma, 8 with inflammatory bowel diseases, and 22 controls with normal colonoscopy). We also examined 24 aberrant crypt foci, 14 hyperplastic polyps, 16 adenomas, and 67 samples of colorectal carcinoma. Immunohistochemistry was used to count myeloperoxidase (MPO)-positive cells (neutrophils and monocytes) in ×100 optical fields under a light microscope. Patients with colorectal tumors had a higher mean number of MPO-positive cells in normal mucosa than controls (mean ± SD, 2.7 ± 2.0 versus 1.4 ± 1.4; P = 0.017). MPO-positive cell number was tightly linked to dysplasia in aberrant crypt foci and adenomas, and it was higher in carcinomas microsatellite unstable than those microsatellite stable (21.6 ± 15.5 versus 11.9 ± 8.0; P < 0.01). MPO immunohistochemistry is a simple and reliable technique for the quantification of inflammation in colorectal mucosa., and it may be a potential marker of colorectal cancer risk. Microsatellite instability seems to influence host immune responses to colorectal carcinoma. These observations strongly support a key role of inflammation in colorectal carcinogenesis. (Cancer Epidemiol Biomarkers Prev 2008;17(9):2291–7)

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 (

Introduction The introduction of novel agents in the treatment of Multiple Myeloma (MM) led to a significant improvement in the quality of response, increasing the number of patients able to achieve a complete response (CR). Several studies showed that the achievement of CR improved survival, both in young and elderly patients with newly diagnosed MM (NDMM). In this study we investigated the impact of CR on survival obtained with either autologous stem cell transplantation or conventional chemotherapy in NDMM patients. Patients and Methods Data from NDMM patientsenrolled in 5 phase III Italian trials were analysed. Three trials included patients eligible for autologous stem cell transplantation (ASCT): RV-MM-209 (melphalan-prednisone-lenalidomide [MPR] vs high-dose melphalan [Mel200] and ASCT, followed by lenalidomide maintenance vs no maintenance), RV-MM-EMN-441 (cyclophosphamide-lenalidomide-dexamethasone vs Mel200-ASCT, followed by lenalidomide [R] versus lenalidomide-prednisone [RP] maintenance) and MM-BO2005 (bortezomib-thalidomide-dexamethasone vs thalidomide-dexamethasone as induction/consolidation, followed by dexamethasone maintenance). The two remaining studies included elderly patients ineligible for ASCT: GIMEMA-MM0305 (bortezomib-melphalan-prednisone-thalidomide followed by bortezomib-thalidomide maintenance vs bortezomib-melphalan-prednisone) and EMN01 (MPR vs cyclophosphamide-prednisone-lenalidomide vs lenalidomide-dexamethasone, followed by R vs RP maintenance). The primary objective of the study was the evaluation of the impact of CR on overall-survival (OS) and progression-free survival (PFS) and its relationship with age (young vs elderly patients) and type of treatment (ASCT vs conventional chemotherapy [CC])). Univariate and multivariate analyses of OS and PFS, including ISS, ASCT and type of novel agents used as induction treatment, were performed. Response was treated as a time-dependent variable. A landmark analysis was performed. Results 2439 NDMM patients were evaluated; the best response was available in 2359 patients. 656 patients achieved a CR or better, whereas 1353 patients achieved a very good partial response (VGPR) or a partial response (PR), and were included in the analysis. After a median follow-up of 44 months, the 5-year OS was 75% in CR patients as compared with 60% in VGPR/PR patients (HR 0.49, p Among CR patients, 383 were treated with ASCT and 273 with CC. A trend towards a better 5-year OS was reported in the ASCT group as compared with the CC group (79% vs 69%; HR 0.6, p=0.09; Figure 1). A significant PFS advantage was observed among CR patients treated with ASCT in comparison with those who received CC (median, 59 vs 47 months; HR 0.54, p=0.008; Figure 2). No significant differences were observed between young and elderly CR patients treated with CC in terms of 5-year PFS (43% vs 41%; HR 0.9, p=0.5) and 5-year OS (73% vs 69%; HR 1.07, p=0.8). In the multivariate analysis, ASCT confirmed to be an independent predictor of prolonged PFS in CR patients, with a trend towards longer OS, in comparison with CC. Conclusions ASCT induced deeper CR that translated into prolonged PFS and OS as compared with CC. No differences were noticed between young and elderly patients achieving a CR with CC. Disclosures Off Label Use: Use off-label of drugs for the dose and/or schedule and/or association. Larocca:Janssen-Cilag, Celgene: Honoraria. Offidani:Janssen-Cilag, Celgene, Sanofi, Amgen, Mundipharma: Honoraria. Bringhen:Merck Sharp & Dohme: Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy; Janssen-Cilag, Celgene, Novartis: Honoraria. Caravita:Celgene: Honoraria. Di Raimondo:Janssen-Cilag, Celgene: Honoraria. Gaidano:Celgene: Research Funding; Morphosys, Roche, Novartis, GlaxoSmith Kline, Amgen, Janssen, Karyopharm: Honoraria, Other: Advisory Boards. Petrucci:Celgene, Janssen-Cilag, Sanofi, Bristol-Myers Squibb: Honoraria. Ria:Italfarmaco: Honoraria; Novartis: Honoraria; Janssen-Cilag: Honoraria; Celgene: Honoraria. Hajek:Celgene, Amgen: Consultancy, Honoraria; Janssen-Cilag: Honoraria. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria. Boccadoro:Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees. Palumbo:Novartis, Sanofi Aventis: Honoraria; Celgene, Millennium Pharmaceuticals, Amgen, Bristol-Myers Squibb, Genmab, Janssen-Cilag, Onyx Pharmaceuticals: Consultancy, Honoraria.

Purpose To construct and validate among patients with multiple myeloma (MM) who were treated with intensive therapy a prognostic index of early MM progression–related death. Patients and Methods Patient-level data from the Intergroupe Francophone du Myélome (IFM) 2005-01 trial (N = 482) were used to construct the prognostic index. The event was MM progression–related death within 2 years from treatment initiation. The index was validated using data from three other trials: the Gruppo Italiano Malattie Ematologiche dell' Adulto (GIMEMA) 26866138-MMY-3006 trial (N = 480), the Programa para el Estudio de la Terapéutica en Hemopatía Maligna (PETHEMA)–GEMMENOS65 trial (N = 390), and the Hemato-Oncologie voor Volwassenen Nederland (HOVON) –65/German-Speaking Myeloma Multicenter Group (GMMG) –HD4 trial (N = 827). Results The risk of early MM progression–related death was related to three independent prognostic variables: lactate dehydrogenase (LDH) higher than than normal, International Staging System 3 (ISS3), and adverse cytogenetics [t(4;14) and/or del(17p)]. These three variables enabled the definition of an ordinal prognostic classification composed of four scores (0 to 3). Patients with a score of 3, defined by the presence of t(4;14) and/or del(17p) in addition to ISS3 and/or high LDH, comprised 5% (20 of 387 patients) to 8% (94 of 1,139 patients) of the patients in the learning and validation samples, respectively, and they had a very poor prognosis. When applied to the population of 855 patients who had received bortezomib-based induction therapy in the four trials, the prognostic classification was also able to segregate patients into four categories, with a very poor prognosis attributed to patients with a score of 3. Conclusion Our model allows the simple definition of a subgroup of MM patients at high risk of early MM progression–related death despite the use of the most modern and effective strategies.

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.

Introduction Three-drug induction regimens including a first or second generation proteasome inhibitor are a current standard of care for autologous stem-cell transplantation (ASCT)-eligible, newly diagnosed, multiple myeloma (MM) patients (pts). In the absence of prospective randomized studies comparing different induction regimens, it is difficult to recommend one treatment over another, and the choice is ultimately based upon physician’s preference or single center’s policy. Bortezomib-thalidomide-dexamethasone (VTD) has been recently approved by EMA as induction for previously untreated, ASCT-eligible, MM pts. Bortezomib combined with cyclophosphamide and dexamethasone (VCD) is an attractive alternative to VTD, although efficacy results have not been backed by phase III studies. The present study aimed to evaluate the differences in response rates and toxicity between VTD and VCD induction regimens in preparation for subsequent ASCT. Methods Two hundred and thirty six pts who were randomized to the VTD arm of the GIMEMA-MMY-3006 study were compared with an equal number of pair mates who received induction therapy with VCD as part of the EMN02 trial designed to prospectively compare ASCT up front vs bortezomib-melphalan-prednisone followed by ASCT at the time of relapse or progression. Both groups of pts were treated in Italian centers participating in the two phase III studies. Case matching was performed with respect to the following pt characteristics at baseline: age (± 2 years), ISS stage (1 vs 2 vs 3), t(4;14) (detected by FISH in ≥ 10% vs < 10% CD138+ bone marrow plasma cells) and del(17p) (≥ 20% vs Results On an intention-to-treat basis, the overall rate of partial response (PR) or higher, as established according to the IMWG criteria, was 93% (95% CI: 89-96%) with VTD and 84% (CI: 79-89%) with VCD (χ2test p value=0.003). In particular, the complete response (CR) rate was approximately three fold higher for pts randomized to VTD (19%, CI: 14-24%) as compared to those in the VCD group (7%; CI: 4-10%) (p Conclusions The triplet VTD induction therapy was associated with significantly higher CR and ≥ VGPR rates compared to VCD, confirming that VTD is one of the most active bortezomib-based induction regimens in preparation for ASCT. Grade ≥ 2 PN rate was similar between the two regimens, although VTD-induced grade ≥ 3 PN was higher with VTD. Data on the impact of VTD and VCD induction therapy on post-ASCT high-quality response rates and additional outcomes will be presented at the meeting. The superiority of VTD over VCD needs to be confirmed by prospective randomized studies, one of which is currently running in Europe. Disclosures Cavo: Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Millennium Pharm.: Consultancy, Honoraria; Bristol-Myers Squib: Consultancy, Honoraria; Onyx: Honoraria. Cavallo:Onyx: Honoraria; Janssen-Cilag: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Petrucci:Celgene, Janssen-Cilag, Sanofi, Bristol-Myers Squibb: Honoraria. Ballanti:Janssen: Honoraria. Palumbo:Sanofi Aventis: Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Genmab A/S: Consultancy, Honoraria; Array BioPharma: Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Sonneveld:Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Research Funding; Onyx: Consultancy, Research Funding.

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

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.

Although patients with Stage I colorectal cancer show an excellent prognosis, a few of them die of metastatic disease. In this subgroup of individuals, the search of occult metastasis might reveal that early dissemination of tumor cells could be the cause of cancer progression.Through a Cancer Registry, we selected all patients with Stage I disease who died of metastatic tumor; a total of 32 patients were identified and in 25 of them paraffin-embedded material was available. The group was matched to 70 Stage I patients with favorable prognosis (controls). In cases and controls resected lymph nodes were cut, and micrometastases were searched using pan-cytokeratin antibodies.Micrometastases were detected in 18 of 25 (72%) Stage I patients who died of the disease, while they were almost absent among controls (1 of 70, p0.001 by χ(2) test). Vascular invasion and tumor budding were more frequent among Stage I patients with an unfavorable prognosis than in controls. By regression analyses, micrometastases (HR 12.3, CI 4.8-32) and vascular invasion (HR 3.5, CI 1.4-8.5) maintained an independent association with prognosis (cancer-specific survival).Micrometastasis in the lymph nodes can be revealed in the majority of patients with early colorectal cancer who die of tumor progression, while they appear extremely rare in Stage I individuals with good prognosis. The selection of patients through histology (vascular invasion) and search of occult metastatic cells might represent a way to identify individuals who might benefit from adjuvant chemotherapy.

A general improvement of colorectal cancer prognosis has been observed. Reasons of this more favourable trend are diffusion of screening, advancements in molecular biology, new developments in chemotherapy and surgical techniques. Through the data of a colorectal cancer registry, we purposed to evaluate changes in surgical procedures for colorectal neoplasms and to analyse trends of perioperative mortality. Patients with colorectal cancer were registered from 1984 to 2004. The main surgical procedures were recorded and classified. Perioperative mortality was defined as death of patients within 1 month since the operation. Regression analysis showed an increase over time of right and left hemicolectomy. Both colectomy and endoscopic polypectomy showed significant rise over time. In contrast, abdominoperineal operations dropped during the study period. A similar decrease was observed for palliative surgery. Perioperative mortality declined from 7–11% to 3–6% of all operations; main factors associated with perioperative mortality were presence of comorbidities, increasing age and advanced stage. The better prognosis of patients with colorectal cancer was associated with changes of surgical techniques, with a tendency to prefer large operations over limited resections. Perioperative mortality showed a gradual decrease and is at present in the order of 3% to 6% of all operations.

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.

TP53 is rarely reported as being affected either by deletions or mutations in MM, even if chr17p13 copy number (CN) loss defines a samples subgroup with a particularly poor prognosis. Here we aim at retrospectively analyzing by Next Generation Sequencing (NGS) the TP53 gene inactivation in newly diagnosed MM, assessing the mutational events’ frequency and their clinical impact. A cohort of 92 MM, receiving up-front velcade (vel)-based regimens, followed by autologous stem cell transplantation, was included in the study. TP53 gene mutational status was analysed by amplicon-targeted deep NGS approach. An upcoming NGS data analysis software was employed to detect vars (var) and to compare them to IARC database. p53 activity was evaluated by immunoblot assays. Ultra-deep TP53 coding sequence analysis (median depth: 1060) highlighted the presence of a median of 1.8 vars per samples in 73/92 (79%) MM: a total of 131 nucleotide substitutions emerged, with vars allele frequencies (VAF) ranging from 1 to 99% (median 1.3%). TP53 vars, were defined either neutral or deleterious mutations, according to their predicted effect at amino acid level. 36 cases carried 42 deleterious vars (VAF = 1.2%), 67% of which were predicted as non-functional at protein level. 9 samples carried 21 neutral vars (VAF = 1.1%), mainly affecting the DNA binding domain. 28 samples carried 68 SIFT unclassified vars, among which several polymorphisms were counted (VAF = 94.6%). SNPs arrays were performed on 83/92 samples: 14/83 (17%) carried a TP53 CN loss on chr17p13.1. The incidence of TP53 hemizygous deletion was higher among cases carrying deleterious mutations, as compared to cases carrying either neutral or unclassified mutations (25%, 12,5% and 12%, respectively). 2/17 non-mutated samples carried TP53 hemizygous deletion, as well. Interestingly, an Rb1 tumour suppressor gene CN loss on chr13q14.2 significantly characterized samples carrying either mutated (deleterious var) or deleted TP53 (p = 0.006). By immunoblot assay we showed the absence of phosphorilation both in 3 TP53 deleted cases and in 3 MM carrying deleterious vars; on the contrary p-p53 was observed in 4 non-mutated cases. Clinical correlations were performed on 81 MM showing that the presence of either TP53 hemizygous deletions or at least one TP53 deleterious var was more likely associated to the response to vel-based induction therapy (frequencies of ≤partial response: 35% and 64% among samples carrying or not impaired TP53, respectively; p = 0.05). Conversely, the frequency of progression events was slightly higher among MM carrying impaired TP53 (69% vs. 50%). In conclusion, the TP53 coding sequence analysis by ultra-deep NGS of a cohort of newly diagnosed MM highlighted an unexpected, still un-explored, high rate of TP53 vars. The impact of TP53 damage on MM disease course has to be confirmed in randomized clinical trial. Supported by Roche Diagnostics and FP7 NGS-PTL project Citation Format: Marina Martello, Giovanni Martinelli, Angela Flores Dico, Barbara Santacroce, Enrica Borsi, Mauro Procacci, Torsten Haferlach, Flavio Mignone, Igor Saggese, Elena Zamagni, Paola Tacchetti, Lucia Pantani, Anna Maria Brioli, Serena Rocchi, Annalisa Pezzi, Michele Cavo, Carolina Terragna. A long tail of sub-clonal TP53 mutations emerged by ultra-deep sequencing of newly diagnosed multiple myeloma (MM). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4248. doi:10.1158/1538-7445.AM2015-4248

Background Although remarkable advances have been achieved in MM therapy, mainly thanks to the introduction of novel-agent-based regimens, the disease remains incurable. Neoplastic CD138+ plasma cells are the hallmark of MM: both their expansion in the bone marrow (BM) and the production of monoclonal immunoglobulin (Ig) are responsible for the clinical manifestation of the disease. However, the existence of a Myeloma Propagating Cells (MPCs) has been proposed as a major cause of MM drug-resistance, leading to relapse. Several studies support the hypothesis that MPCs are phenotypically close to memory B cells residing in the CD138- compartment; however, very little is known concerning their molecular characteristics. Here we present an extensive molecular characterization of clonotypic CD19+ B cells clones obtained from newly diagnosed MM patients (pts), in order to recognize biological pathways possibly explaining the malignant clone’s persistence. Methods CD138+ and CD138- cell fractions were collected from BM and peripheral blood (PBL) of 50 newly diagnosed MM pts. CD19+ B cell and CD27+ memory B cell populations were isolated from CD138- cell fraction. Clonogenic assays were performed by plating cell fractions obtained from RPMI-8226 and NCI-H929 cell lines. The molecular characterization included: IgH gene rearrangement Sanger sequencing; analysis of the whole spectrum of genomic aberrations and gene expression profiling, by Affymetrix 6.0 SNPs array and HG-U133 Plus 2.0 microarray, respectively. Results Clonogenic assays showed that CD138- cells, plated on conditioned media, were able to form colonies after two weeks of culture more efficiently than CD138+ cells. By VDJ gene rearrangement sequencing, a clonal relationship between the CD138+ clone and the memory B ones was confirmed. SNPs arrays showed that both BM and PBL CD138+ cell fractions carried exactly the same genomic macro-alterations. On the contrary, in the CD138-19+27+ cell fractions from BM and PBL any macro-alteration was detected, whereas several micro-alterations (median number per sample: 32 amplifications and 16 losses, range: 8-122 Kb, average markers per region: 50) unique of the memory B cells clone were highlighted. An enrichment analysis revealed the involvement of genes affected by losses (17 genes) in both DNA repair mechanisms and transcriptional regulation and the involvement of genes affected by gains (46 genes) in both the negative regulation of apoptosis and the angiogenesis. Interestingly, KRAS, WWOX and XIAP genes, renown to be involved in MM pathogenesis, are located in the amplified regions in the immature cells. Moreover, several LOH regions were described, which covered at least 106 tumor suppressor genes involved in MM and leukemia (including TP53, CDKN2C and RASSF1A). Transcriptome profiles analysis of the CD19+ cell fractions highlighted pathways suggesting a possible involvement of immature cells in MM pathogenesis. The gene expression profiles of 20 MM CD19+ cells samples (12 from PBL, 8 from BM) were compared both to their normal counterpart and to the mature CD138+ cell fractions. In particular, unsupervised analysis by hierarchical clustering discriminated the differential expression of 11480 and 11360 probes in the PBL and BM CD19+ clones, respectively (2; FDR=0,05; p Interestingly, the protein homeostasis deregulation possibly caused by ER stress, resulted particularly evident in the BM 19+ cells (p=7,25E-14; FDR= 2,98E-11); moreover, the down-regulation of genes related to the unfolded protein response (e.g. IRE1α and XBP1 FC=-18,0; -19,96. p Conclusions Presented data support the emerging role of the immature cell compartment in the MM disease course, where the MM CD138+ clone might resume the end of the complex process of tumorigenesis, whereas the putative CD19+ MPCs, by displaying peculiar genomic micro-alterations and a unique transcriptional profile, might be involved in the neoplastic clone supply. Supported by ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; Ariad: Consultancy. Cavo:BMS: Consultancy, Honoraria; Millenium: Consultancy, Honoraria; Onyx: Honoraria; Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

INTRODUCTION: Cytogenetic abnormalities (CA) are one of the major factor for risk stratification in Multiple Myeloma (MM). Approximately 50% of MM patients (pts) harbour a translocation involving the Immunoglobulin Heavy chain locus (IgH). Each translocation is associated with deregulation of a D group cyclin, either directly in t(11;14)(q13;q32) and t(6;14)(p21;q32) or indirectly in t(4;14)(p16;q32) and in MAF translocations: t(14;16)(q32;q23) and t(14;20)(q32;q12). Some translocations are recurrent but rare events, occurring in less than 5% of the pts: t(6;14) is considered a standard prognostic feature, whereas t(14;16) and t(14;20) are associated with bad prognosis. As a result of their low incidence, few data support their use of these abnormalities in the risk stratification. Currently, deletion(17)(p13) and gain(1)(q21) are linked with adverse clinical outcome and, recently, also deletion(1p) has been considered adverse prognostic factor. AIM: To assess the type and frequency of these rare IgH translocations and their association with other adverse genetic lesions and with clinical characteristics, in a large series of newly diagnosed MM pts. METHODS: A total of 682 pts newly diagnosed MM pts, enrolled in several prospective clinical trials of bortezomib-based induction therapy and subsequent autologous stem cell transplantation, including most recent EMN02 clinical trial, were analyzed. Probes for FISH analysis were used to detect t(4;14), t(6;14), t(11,14), t(14;16), t(14;20); deletion of 1p32, 13q14, 17p13; gain of 1q21; trisomy of chromosomes 5/9/15. RESULTS: On evaluable cases, the frequency of each IgH translocation, as detected by FISH was as follows: t(11;14) in 114/596 (19.1%), t(4;14) in 78/605 (12.9%), t(14;16) in 27/603(4.5%), t(14;20) in 9/591(1.5%) and t(6;14) in 7/587(1.2%). Every rare translocation was associated with deletions flanking the breakpoints, either as unique pattern or with classical translocation. The t(6;14) was closely associated with del(6)(p21)(6/7), del(13q)(4/7) and del(16)(q23)(3/7); additional gain 1q and deletion 17p was observed in a single case. All together, the high-risk (HR) translocations, t(14;16) and t(14;20), were detected in 36 pts: 63.9% were females and 36.1% males. The t(14;16) was frequently occurred with del(13q) (84.6%), gain(1q) (74.1%; 20/27) and del(16)(q23) (74.1%; 20/27), while t(14;20) was associated with del(13q) (88.9%; 8/9), gain(1q) (5/9; 55.6%) and del(20)(q12) (5/9; 55.6%). In selected cases, the presence of these additional abnormalities was shown also by SNPs arrays. Beyond del(13q), gain(1q) was the most frequent additional abnormality (69.4%; 25/36), occurring in 56.4% (44/78) of t(4;14) cases and in 20.2% (23/114) of t(11;14) cases (p Compared with pts without rare HR translocations, pts harbouring t(14;16) or t(14;20) showed a high probability to present with additional characteristics related to poor prognosis, including ISS stage 3 (42% vs 20%, p=0.002), Hb CONCLUSION: t(14;16)(q32;q23) and t(14;20)(q32;q12) are rare in newly diagnosed MM pts and are associated with an increased frequency of additional HR abnormalities, including gain(1)(q21), deletion(1)(p32) and deletion(17)(p13). Furthermore, they are related to features of aggressive disease, such as ISS stage 3, lower level of hemoglobin and lower platelet count. A longer follow-up is needed to point out their prognostic relevance of these abnormalities. Disclosures Cavallo: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria; Onyx: Honoraria. Petrucci:Bristol Meyer-Sqibb: Honoraria; Sanofi: Honoraria; Janssen-Cilag: Honoraria; Celgene: Honoraria. Palumbo:Amgen: Consultancy, Honoraria; Brystol-Myers Squibb: Consultancy, Honoraria; Array BioPharma: Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Millenium Pharmaceuticals, Inc.: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Onyx Pharmceuticals: Consultancy, Honoraria; Sanofi Aventis: Honoraria. Sonneveld:Celgene: Research Funding, Speakers Bureau; Millennium-Takeda: Research Funding; Onyx: Research Funding, Speakers Bureau; Janssen: Speakers Bureau. Cavo:Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Millennium Pharm.: Consultancy, Honoraria; Bristol-Myers Squib: Consultancy, Honoraria; Onyx: Honoraria.

Background: The phase 3 GIMEMA-MMY-3006 study comparing bortezomib-thalidomide-dexamethasone (VTD) vs. thalidomide-dexamethasone (TD) as induction therapy before, and consolidation after, double autologous stem-cell transplantation (ASCT) provided demonstration of superior CR/nCR rates after induction (the primary study endpoint) and across all subsequent treatment phases, a gain which translated into significantly longer PFS for patients randomized to the VTD arm (Cavo, Lancet 2010; Blood 2012). We herein report an updated analysis of the study with a focus on PFS2, time to second anti-myeloma therapy, treatment-free interval, post-relapse OS and long term outcomes. Methods: Overall, 474 patients were included in the trial. After a median follow up of 65 months, 251 patients (53%) have progressed and of these 221 (88%) had available data on salvage therapy after relapse. Results: On an intention-to-treat basis, median PFS was 57 months for patients randomized to the VTD arm as compared to 42 months for those assigned in the TD arm (HR 0.67; p=0.001). No statistically significant difference in 5-years estimates of OS was observed between VTD and TD groups (80% vs 73%). PFS2, defined as the time from initial randomization to second disease progression or death from any cause, was significantly longer for patients randomised to VTD than for those in the TD group (76% vs. 63% at 5 years, respectively; HR 0.64, p=0.009). Globally, 73% and 83% of patients in the VTD and TD arms required the start of salvage therapy due to symptomatic relapse. Median time to subsequent anti-myeloma therapy (defined as the interval between start of induction treatment and the first dose of second-line therapy) was significantly longer for patients assigned to VTD than for those who experienced relapse in the TD cohort (40 vs 31 months, p=0.014). Similarly, median treatment-free interval (defined as the time between last administration of front-line therapy and start of salvage treatment) was 26 months in VTD group vs 16 months in TD arm (p=0.016). Most of the patients received a novel agent-containing salvage therapy, while 18% was treated with conventional chemotherapy. As expected, a greater percentage of patients in the TD arm were treated with second-line bortezomib-based combinations in comparison with those relapsing in the VTD arm (72% vs 46%, respectively, p=0.001). Within the VTD group, no statistically significant difference in PFS2 was seen regarding the use of bortezomib or an IMiD as (part of) second-line therapy (64 vs 57 months, respectively). Clinical benefit from primary randomization to VTD vs TD was also observed in terms of second PFS (defined as the interval between first and second progression) (HR 0.61, p=0.032). The median OS after relapse was 36 months for both TD and VTD groups. No differences in post relapse OS were observed for patients primarily assigned to VTD or TD who received a subsequent bortezomib-based salvage therapy. Conclusion: With an extended follow-up of approximately 5 years, VTD was superior to TD in terms of extended PFS2, time to subsequent anti-MM therapy and treatment-free interval. PFS2 was significantly longer for patients randomized to VTD, with no difference regardless of the use of bortezomib or an IMiD as part of second-line therapy. This finding, along with similar post-relapse OS values across the two groups, suggest that induction and consolidation therapy with VTD did not select the emergence of bortezomib- resistant clones at the time of relapse. Disclosures Palumbo: Amgen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Array BioPharma: Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Sanofi Aventis: Honoraria. Cavo:Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Millennium Pharm.: Consultancy, Honoraria; Bristol-Myers Squib: Consultancy, Honoraria; Onyx: Honoraria.

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.

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.

Over the last years, incorporation of novel agents into autologous stem cell transplantation (ASCT) has improved markedly the outcomes of younger patients with newly diagnosed multiple myeloma (MM). Superior results with experimental treatments vs previous standards of care have been frequently reported after preliminary analyses and need to be confirmed with longer follow up. The randomized phase 3 GIMEMA-MMY-3006 study was designed to compare bortezomib-thalidomide-dexamethasone (VTD) vs thalidomide-dexamethasone (TD) as induction therapy before, and consolidation after, double ASCT. Data from the initial analysis, with a median follow up of 36 months, demonstrated that patients randomized to the VTD arm enjoyed superior complete/near complete response (CR/nCR) rates after both induction and consolidation therapy, and had a significantly longer PFS compared to those prospectively assigned to the TD arm. We performed an updated analysis of the study after a median follow up of 59 months and results are herein reported. A persistent TTP and PFS benefit with incorporation of VTD into ASCT was confirmed. On an intention-to-treat analysis of 236 patients randomized to the VTD arm, median TTP was 62 months and median PFS was 57 months. The median values for 238 patients randomly assigned to the TD arm were 45 months for TTP (HR=0.64, p=0.001) and 42 months for PFS (HR=0.66, p=0.001) (Fig. 1). With the longer follow up of this analysis, an initial divergence between OS curves could be appreciated after 4 years, although the difference was not yet statistically significant at 6 years (75% for VTD vs 69% for TD). Superiority of VTD over TD for TTP and PFS was retained across prespecified subgroups of patients with high risk and low risk disease. In particular, PFS benefit with VTD was seen for patients age >60 years (HR=0.62, p=0.013) and younger than 60 years (HR=0.70, p=0.026), with ISS stage 1 (HR=0.59, p=0.009) and ISS stage 2-3 (HR=0.69, p=0.018), and for those with t(4;14) and/or del(17p) (HR=0.43, p3.5 mg/L (HR=1.7, p Disclosures: Cavo: Bristol-Myers Squibb: 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; Millennium: 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; Celgene: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees. Tacchetti:Janssen and Celgene: Honoraria. Zamagni:Celgene: Honoraria; Janssen-Cilag: Honoraria. Caravita:Celgene: Honoraria, Research Funding. Brioli:Celgene: Honoraria.

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

Abstract 4210 In a previous analysis performed with a median follow-up of 36 months (mos), superior CR/nCR rates and extended PFS were demonstrated with bortezomib-thalidomide-dexamethasone (VTD) vs thalidomide-dexamethasone (TD) as induction therapy before, and consolidation after, double autotransplantation (ASCT) for newly diagnosed myeloma (MM). Here we report an updated analysis of that study, including a detailed analysis of outcomes after relapse or progression (R/P), an important issue for patients (pts) who received up-front a triplet novel agent-based therapy incorporated into ASCT. After a median follow-up of 52 mos, continued TTP and PFS benefits with VTD vs TD were demonstrated, while no difference in OS was seen between the two arms. Median PFS for pts randomized to the VTD arm was 56 mos vs 42 mos for those assigned to TD (HR=0.64, p=0.001). Similarly to PFS, median TTP for pts in the VTD group was longer than in the TD group (57 vs 45 mos, HR=0.63, p=0.0006), reflecting a 37% reduction in the risk of R/P with VTD. The probabilities of R/P in the VTD and TD arms were 41% vs 55% (p=0.002). In comparison with the VTD-treated group, a higher percentage of pts in the TD arm required the immediate start of salvage therapy after symptomatic R/P was documented (67% vs 82.5%). By the opposite, the fraction of pts with asymptomatic R/P that did not require any antimyeloma therapy until the cut-off date for the analysis was higher in the VTD arm compared with the TD-treated group (33% vs 17.5%, p=0.016). In these pts, the median interval from the date of R/P to the cut-off date for the analysis was 7 mos for the TD-treated group vs 17 mos for the VTD-treated group. Median time to subsequent antimyelomatherapy (defined as the interval between start of induction therapy and administration of the first dose of second-line therapy) was significantly longer for pts who experienced R/P in the VTD arm in comparison with pts who progressed in the TD arm (35 vs 29 mos, p=0.018). The associated interval between last administration of front-line therapy (e.g. consolidation for pts who received the entire treatment program or every other treatment before consolidation for those who discontinued earlier) and first administration of second-line therapy (salvage therapy-free interval) was 22.5 mos for the VTD-treated group vs 15 mos for the TD-treated group (p=0.009). Both bortezomib and lenalidomide were the most frequently used agents at the time of R/P (82% of all cases), while only 18% of pts were treated with conventional cytotoxic drugs. As expected, the majority of pts in the TD arm received a second-line therapy that included bortezomib (70%), while lenalidomide-dexamethasone was received by 10% of pts. By the opposite, in the VTD arm both bortezomib- and lenalidomide-based salvage therapies were equally distributed (41% vs 39%). The probability to achieve at least a partial response after second-line therapy including bortezomib was 60% for pts with prior exposure to VTD vs 63% for those randomized to TD. No difference in post-R/P OS was demonstrated between VTD-treated and TD-treated subgroups of pts who received bortezomib-based salvage therapy after R/P (2-yr estimates: 48% vs 53%, p=0.59). In the overall population, post-R/P OS was significantly shorter for pts who received salvage therapy with conventional cytotoxic drugs (2-yr estimate: 41%) in comparison with pts treated with novel agents, including bortezomib or lenalidomide (2-yr estimates: 52% and 53%, respectively) (p=0.0001). In conclusion, front-line therapy with VTD incorporated into ASCT was superior in comparison with TD plus ASCT in terms of extended TTP, PFS, time to subsequent antimyeloma therapy and salvage therapy-free interval. VTD-treated pts had a higher probability than TD-treated pts to experience long-lasting asymptomatic R/P not requiring second-line therapy. VTD-treated and TD-treated pts who subsequently received bortezomib-based salvage therapies had similar rates of response and post-R/P OS. This finding suggests that short-term exposure to VTD, as planned in our study, did not favor the selection of bortezomib-resistant clones, thus allowing to rechallenge bortezomib as (part of) second-line therapy after symptomatic R/P. Disclosures: Cavo: Janssen, Celgene, Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria. Di Raimondo:Celgene, Janssen: Honoraria, Speakers Bureau. Offidani:Janssen, Celgene: Honoraria. Caravita:Celgene, Janssen, Novartis, Genzyme: Honoraria. Boccadoro:Celgene, Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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 1871 Introduction: In a randomized phase 3 study, the use of VTD as induction therapy prior to and consolidation therapy following double ASCT increased the rate of complete or near complete response (CR/nCR) and extended PFS in comparison with TD given as induction and post-ASCT consolidation therapy in 474 newly diagnosed MM patients (Cavo et al, Lancet 2010). However, the specific impact of VTD consolidation on improved clinical outcomes was not defined. Methods: To address this issue, we performed a per-protocol analysis of 321 patients who received the entire treatment program, including the two pre-planned cycles of consolidation therapy with either VTD (160 of 236 patients, 68%) or TD (161 of 238 patients, 68%). By study design, two 35-d cycles of VTD (V 1.3 mg/m2 on days 1, 8, 15, and 22; T 100 mg/d on days 1–35; D 40 mg on the days of and after each V administration) or TD (at the same doses as in VTD) were given as consolidation therapy to patients randomly assigned to each of the two respective induction regimens. Patient and disease characteristics at baseline were comparable in the two groups of patients. Results: The rates of CR and CR/nCR were significantly higher after consolidation therapy with VTD compared with TD (CR: 61% vs 47%; p=0.012; CR/nCR: 73% vs 61%; p=0.020). The impact of VTD consolidation on post-ASCT enhanced rates of CR and CR/nCR was confirmed by the McNemar test (CR: p=0.0009; CR/nCR: p=0.004) which conversely failed to demonstrate a significant increase in the frequencies of CR (p=0.052) and CR/nCR (p=0.110) with TD consolidation therapy. The absolute probability of upgrading from less than CR before consolidation to CR after consolidation was 31% with VTD and 17% with TD (Pearson chi-square test: p=0.03). Ninety six percent of patients who upgraded from less than CR to CR after VTD consolidation were in nCR (44%) or VGPR (52%) before starting consolidation therapy. A landmark analysis (with the landmark set as the start of consolidation therapy) was performed to compare time to progression (TTP), PFS, and overall survival (OS) between treatment groups. With a median follow-up of 30 months, the estimated 3-year probability of relapse or progression was 38% with VTD and 52% with TD (p=0.039 by Kaplan-Meier analysis) (HR: 0.68, 95% CI: 0.47–0.98, p=0.041). PFS was significantly longer for patients receiving VTD consolidation than for those treated with TD (3-year estimates: 62% vs 46%; p=0.025) (HR: 0.66, 95% CI: 0.46–0.95, p=0.027), a gain particularly evident for patients who failed to achieve CR after ASCT (3-year PFS estimates: 66% vs 43%; HR: 0.54, 95% CI: 0.32–0.91, p=0.022). Superior PFS with VTD vs TD consolidation was retained across poor prognosis subgroups, including patients with t(4;14) and/or del(17q) (HR: 0.44, p=0.004), del(13q) (HR: 0.44, p=0.002), β2-microglobulin >3.5 mg/L (HR: 0.57, p=0.025), lactate dehydrogenase >190 U/L (HR: 0.57, p=0.005), or ISS stage 2 and 3 (HR: 0.57, p=0.021). In a multivariate regression analysis, the most important and independent variables positively correlated with PFS were VTD consolidation therapy (p=0.002), double ASCT (p=0.001), low β2-microglobulin (p Conclusions: In comparison with TD, post-ASCT consolidation therapy with the triplet VTD regimen significantly increased the rates of CR and CR/nCR, and extended landmarked TTP and PFS. Superior PFS with VTD consolidation was maintained across poor prognosis subgroups, including those with advanced ISS stage and/or high-risk cytogenetic profiles. In a multivariate regression analysis VTD consolidation was confirmed to be an independent variable favorably affecting PFS. The superior activity seen with VTD versus TD as induction therapy before ASCT was retained despite the same triplet regimen being used as post-ASCT consolidation. Disclosures: Cavo: 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; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria; Genzyme: Honoraria. Off Label Use: Bortezomib and Thalidomide as post autotransplantation consolidation therapy in myeloma. Patriarca:Schering-Plough: Honoraria; Celgene: Honoraria; Janssen: Honoraria; Roche: Honoraria. Petrucci:Celgene: Honoraria; Janssen: Honoraria. Di Raimondo:Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau. Palumbo:Merck: Honoraria; Janssen: 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; Amgen: Honoraria. Offidani:Celgene: Honoraria; Janssen: Honoraria. Baccarani:Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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.

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.