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Proteasome inhibitors (PI) are extensively used for the therapy of multiple myeloma (MM) and mantle cell lymphoma. However, patients continuously relapse or are intrinsically resistant to this class of drugs. Here, to identify targets that synergize with PI, we carried out a functional screening in MM cell lines using a short hairpin RNA library against cancer driver genes. Isocitrate dehydrogenase 2 (IDH2) was identified as a top candidate, showing a synthetic lethal activity with the PI carfilzomib (CFZ). Combinations of US Food and Drug Administration–approved PI with a pharmacological IDH2 inhibitor (AGI-6780) triggered synergistic cytotoxicity in MM, mantle cell lymphoma, and Burkitt lymphoma cell lines. CFZ/AGI-6780 treatment increased death of primary CD138+ cells from MM patients and exhibited a favorable cytotoxicity profile toward peripheral blood mononuclear cells and bone marrow–derived stromal cells. Mechanistically, the CFZ/AGI-6780 combination significantly decreased tricarboxylic acid cycle activity and adenosine triphosphate levels as a consequence of enhanced IDH2 enzymatic inhibition. Specifically, CFZ treatment reduced the expression of nicotinamide phosphoribosyltransferase (NAMPT), thus limiting IDH2 activation through the NAD+-dependent deacetylase SIRT3. Consistently, combination of CFZ with either NAMPT or SIRT3 inhibitors impaired IDH2 activity and increased MM cell death. Finally, inducible IDH2 knockdown enhanced the therapeutic efficacy of CFZ in a subcutaneous xenograft model of MM, resulting in inhibition of tumor progression and extended survival. Taken together, these findings indicate that NAMPT/SIRT3/IDH2 pathway inhibition enhances the therapeutic efficacy of PI, thus providing compelling evidence for treatments with lower and less toxic doses and broadening the application of PI to other malignancies.

Before the introduction of new drugs, we designed a trial where treatment of newly diagnosed myeloma patients was based on the presence or absence of HLA-identical siblings. First-line treatments included a cytoreductive autograft followed by a nonmyeloablative allograft or a second melphalan-based autograft. Here, we report long-term clinical outcomes and discuss them in the light of the recent remarkable advancements in the treatment of myeloma. After a median follow-up of 7 years, median overall survival (OS) was not reached (P = .001) and event-free survival (EFS) was 2.8 years (P = .005) for 80 patients with HLA-identical siblings and 4.25 and 2.4 years for 82 without, respectively. Median OS was not reached (P = .02) and EFS was 39 months (P = .02) in the 58 patients who received a nonmyeloablative allograft whereas OS was 5.3 years and EFS 33 months in the 46 who received 2 high-dose melphalan autografts. Among patients who reached complete remission in these 2 cohorts, 53% and 19% are in continuous complete remission. Among relapsed patients rescued with “new drugs,” median OS from the start of salvage therapy was not reached and was 1.7 (P = .01) years, respectively. Allografting conferred a long-term survival and disease-free advantage over standard autografting in this comparative study.

Complete response (CR) was an uncommon event in elderly myeloma patients until novel agents were combined with standard oral melphalan-prednisone. This analysis assesses the impact of treatment response on progression-free survival (PFS) and overall survival (OS). We retrospectively analyzed 1175 newly diagnosed myeloma patients, enrolled in 3 multicenter trials, treated with melphalan-prednisone alone (n = 332), melphalan-prednisone-thalidomide (n = 332), melphalan-prednisone-bortezomib (n = 257), or melphalan-prednisone-bortezomib-thalidomide (n = 254). After a median follow-up of 29 months, the 3-year PFS and OS were 67% and 27% (hazard ratio = 0.16; P< .001), and 91% and 70% (hazard ratio = 0.15; P< .001) in patients who obtained CR and in those who achieved very good partial response, respectively. Similar results were observed in patients older than 75 years. Multivariate analysis confirmed that the achievement of CR was an independent predictor of longer PFS and OS, regardless of age, International Staging System stage, and treatment. These findings highlight a significant association between the achievement of CR and long-term outcome, and support the use of novel agents to achieve maximal response in elderly patients, including those more than 75 years. This trial was registered at www.clinicaltrials.govas #NCT00232934, #ISRCTN 90692740, and #NCT01063179.

Cyclooxygenase 2 (COX-2) is an inflammation-associated enzyme involved in the pathogenesis of many solid tumors, but little is known about its presence and role in hematologic neoplasms. Multiple myeloma (MM) is known to involve a deregulated cytokine network with secretion of inflammatory mediators. We thus decided to investigate the involvement of COX-2 in this neoplasm. Western blotting (WB) was used to evaluate 142 bone marrow (BM) specimens, including MM and monoclonal gammopathy of undetermined significance (MGUS). Selected cases under-went further evaluation by WB on purified CD138+cells, immunohistochemistry (IC), and real-time polymerase chain reaction (PCR) for mRNA expression. COX-2 was expressed in 11% (2 of 18) of MGUS specimens, 31% (29 of 94) of MM at diagnosis, and 47% (14 of 30) of MM with relapsed/refractory disease. COX-2 positivity was associated with a poor outcome in terms of progression-free (18 vs 36 months; P< .001) and overall survival (28 vs 52 months; P< .05). Real-time PCR showed COX-2 mRNA overexpression. IC and cell separation studies demonstrated COX-2 expression to be restricted to malignant plasma cells. This is the first report of the presence and prognostic role of COX-2 expression in MM. Future studies will assess COX-2 involvement in other hematologic tumors and its potential use as a therapeutic or chemo-preventive target in onco-hematology. (Blood. 2005; 105:4784-4791)

A clinical relationship between dose-intensity of melphalan and response rate has been demonstrated in multiple myeloma. Promising results have been reported after 200 mg/m2melphalan, especially in younger patients. It is uncertain whether 100 mg/m2melphalan (MEL100) can offer similar results in older patients. To address this issue, patients were treated with 2 or 3 MEL100 courses followed by stem cell support. Seventy-one patients (median age, 64 years) entered the protocol at diagnosis. Their clinical outcome was compared with that of 71 pair mates (median age, 64 years) selected from patients treated at diagnosis with oral melphalan and prednisone (MP) and matched for age and β2-microglobulin. Complete remission was 47% after MEL100 and 5% after MP. Median event-free survival was 34 months in the MEL100 group and 17.7 months in the MP group (P< .001). Median overall survival was 56+ months for MEL100 and 48 months for MP (P< .01). In a multivariate analysis, β2-microglobulin levels and MEL100 were independent risk factors associated with outcome: superior event-free and overall survival were observed in patients presenting low β2-microglobulin levels at diagnosis and receiving MEL100 as induction regimen. In conclusion, MEL100 was superior to MP in terms of complete remission rate, event-free survival, and overall survival.

Rossi: Gilead: Honoraria, Research Funding; Janssen: Honoraria; AbbVie: Honoraria. Gaidano:Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Massaia:Janssen: Other: advisory board; Roche: Other: advisory board, research support; Gilead: Other: advisory board. Boccadoro:Mundipharma: Research Funding; Abbivie: Honoraria; SANOFI: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; BMS: Honoraria, Research Funding; CELGENE: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Coscia:Gilead: Honoraria; Janssen: Honoraria; ROCHE: Honoraria, Other: Advisory board; Mundipharma: Honoraria; Karyopharm: Research Funding.

Boccadoro: CELGENE: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Abbivie: Honoraria; Mundipharma: Research Funding; SANOFI: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

Oliva: Amgen: Honoraria; Takeda: Honoraria; Celgene: Honoraria. Boccadoro:CELGENE: Honoraria, Research Funding; Mundipharma: Research Funding; Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Abbivie: Honoraria; SANOFI: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Hajek:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sonneveld:Celgene: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria, Research Funding. Palumbo:Takeda: Employment, Honoraria; Janssen Cilag: Honoraria.

Massaia: Janssen: Other: advisory board; Gilead: Other: advisory board; Roche: Other: advisory board, research support. Cavallo:JANSSEN: Honoraria; CELGENE: Honoraria; ONYX: Honoraria. Palumbo:Janssen Cilag: Honoraria; Takeda: Employment, Honoraria. Boccadoro:Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Mundipharma: Research Funding; Abbivie: Honoraria; SANOFI: Honoraria, Research Funding; BMS: Honoraria, Research Funding; CELGENE: Honoraria, Research Funding.

Bringhen: Mundipharma: Other: ADVISORY BOARD; Amgen: Other: ADVISORY BOARD; Janssen-Cilag: Honoraria; Celgene: Honoraria; BMS: Honoraria; Karyopharm: Other: ADVISORY BOARD. Massaia:Janssen: Other: advisory board; Roche: Other: advisory board, research support; Gilead: Other: advisory board. Palumbo:Janssen Cilag: Honoraria; Takeda: Employment, Honoraria. Boccadoro:CELGENE: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; SANOFI: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Mundipharma: Research Funding; Abbivie: Honoraria; Novartis: Honoraria, Research Funding; Amgen: Honoraria, Research Funding.

Marchetti: GILEAD: Consultancy, Research Funding; JANSSEN: Other: tavel, accomodation, expenses; SANOFI: Membership on an entity's Board of Directors or advisory committees; GILEAD: Other: teaching, Research Funding; NOVARTIS: Research Funding. Gaidano:MorphoSys; Roche; Novartis; GlaxoSmithKline; Amgen; Janssen; Karyopharm: Honoraria, Other: Advisory boards; Celgene: Research Funding. 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. Massaia:Gilead: Research Funding; Janssen: Honoraria; Roche: Honoraria. Coscia:Roche: Honoraria, Other: Advisory board; Mundipharma: Honoraria.

Luminari: Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees.

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.

Background. Recently, the somatic MYD88L265Pmutation has been found as the hallmark of Waldenström Macroglobulinemia (WM), being detectable in nearly 90% of cases, as well as in up to 50% of IgM MGUS, rarely in other non-Hodgkin lymphomas and never in multiple myeloma (MM). Beyond its potential diagnostic role, this mutation has been associated with tumor growth and therapy resistance. Moreover, MYD88L265Pmight represent an ideal marker for minimal residual disease (MRD) monitoring in a disease whose therapeutic scenario has been rapidly changing, with many new available and highly effective drugs (nucleoside analogues, proteasome and BTK-inhibitors). However, the current MYD88L265Pallele-specific quantitative PCR (ASqPCR) diagnostic tool lacks sensitivity (1.00E-03) and thus is not suitable for MRD. Moreover, is not useful to test peripheral blood (PB), that harbors low concentrations of circulating tumor cells (especially after immunochemotherapy), neither to assess cell-free DNA (cfDNA), usually present at very low amount in plasma. Therefore, our study aims: 1) to assess whether a highly sensitive tool as droplet digital PCR (ddPCR) might be helpful in MYD88L265Pscreening; 2) to evaluate whether MYD88L265Pmight be a suitable marker for MRD monitoring in WM.

No relevant conflicts of interest to declare.

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.

Boccadoro: Celgene: Honoraria; Janssen: Honoraria; Onyx: Honoraria. 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.

Palumbo: Bristol-Myers Squibb: Consultancy, Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Array BioPharma: Honoraria; Amgen: Consultancy, Honoraria; Sanofi: Honoraria. Boccadoro:Celgene: Honoraria; Janssen: Honoraria; Onyx: Honoraria.

Palumbo: Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Array BioPharma: Honoraria; Amgen: Consultancy, Honoraria; Sanofi: Honoraria; Genmab A/S: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Off Label Use: Use off-label of Carfilzomib (proteasome inhibitor).. Bringhen:Merck Sharp & Dohme: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Janssen and Cilag: Honoraria; Celgene: Honoraria; Onyx: Consultancy. Larocca:Janssen Cilag: Honoraria; Celgene: Honoraria. Cavallo:Onyx: Honoraria; Janssen-Cilag: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Boccadoro:Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: 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; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Gaidano:Onyx: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Sonneveld:Millenium: Honoraria, Research Funding; Onyx: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

Off Label Use: lenalidomide used as off-label. Ferrero:MUNDIPHARMA: Honoraria. Gay:Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Patriarca:Merck Sharp & Dohme: Honoraria; Janssen and Cilag: Honoraria; Celgene: Honoraria. Petrucci:CELGENE: Honoraria; JANSSEN-CILAG: Honoraria; SANOFI: Honoraria; BRISTOL MYERS SQUIBB: Honoraria. Caravita:Celgene: Honoraria. Di Raimondo:CELGENE: Honoraria; JANSSEN-CILAG: Honoraria. Musto:CELGENE: Honoraria; JANSSEN-CILAG: Honoraria. Boccadoro:Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: 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; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Palumbo:Array BioPharma: Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Genmab A/S: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Sanofi: Honoraria.

While international stage (ISS) and the presence of absence of cytogenetic abnormalities on FISH somewhat define the clinical risk of MM patients, additional biomarkers are necessary for more precise risk-based classification. Emerging studies have shown that circulating microRNAs (miRNAs) can be detected in patients with a variety of malignancies, including MM, and they could be non-invasive biomarkers. We measured serum miRNA levels of a large cohort of well-characterized previously untreated MM patients and correlated results with clinical outcome to test their prognostic impact. Methods and Patients To profile the expression of circulating microRNAs in the serum of MM patients, we performed NanoString-nCounter microRNA assays on samples obtained from a discovery cohort of 54 newly diagnosed MM patients enrolled on a randomized GIMEMA phase 3 study comparing Velcade-Melphalan-Prednisone-Thalidomide versus Velcade-Melphalan-Prednisone followed by maintenance with Velcade-Thalidomide. To further analyze the expression of the differentially expressed microRNAs, stem-loop-RT-PCR was performed on a validation cohort of 234 MM patients enrolled in the same trial. The prognostic significance of differentially expressed microRNAs were evaluated in relation to progression-free (PFS) and overall survival (OS) using univariate and multivariate Cox proportional hazards models. The utility of incorporating microRNA expression into a risk score with known risk factors – specifically ISS stage and the presence of del17, t(4;14) or t(14;16) by FISH – was also explored.Out of the 800 miRNAs evaluated, only 25 were detectable (≥100 counts) in at least 20% of the patients. The expression of these miRNAs were then measured in a validation set, but only 10 (miRs-92a, 21, 30a, 720, 451, 223, 126, 19b, 25 and miR-16) were validated to be differentially expressed. We found that levels of miR-16 and miR-25, used as continuous variables, had significant impact on OS duration: miR-16 (HR 0.87; p=0.019) and miR-25 (HR 0.81; p=0.0012) where low expression corresponded with worse survival. Based on these observations we generated a microRNA-based risk score which was significantly associated with OS duration (p=0.008). We then integrated this score with ISS stage and presence of high risk features by FISH, generating an integrated-microRNA risk score that was significantly associated with OS (p<0.0001), and was better than a risk score that combined ISS and FISH (p=0.014), see figure.Circulating miR-16 and miR-25 can risk stratify elderly, previously untreated, MM patients beyond ISS-stage and high risk genetic features. The opportunity to isolate circulating miRNAs allows us to sequentially sample cancer patients in a relatively non-invasive manner, opening new avenues of investigation for disease stratification and response to therapy.Bringhen: Onyx: Consultancy.

Elderly multiple myeloma (MM) patients are an heterogeneous population. Aging is associated with an increased frequency of co-morbidities, frailty and disability, with negative impact on treatment tolerance and outcome. A simple and reliable scoring system, based on geriatric assessment, has been developed to predict survival and used also to predict the risk of severe toxicities or treatment discontinuation in elderly newly diagnosed MM patients treated with lenalidomide-, bortezomib- or carfilzomib-based induction regimens.Patients with newly diagnosed MM, ineligible for high-dose therapy and autologous stem cell transplantation due to age (≥65 years) or coexisting co-morbidities, enrolled in 3 prospective multicenter trials, were included in the analysis. Up-front dose reductions were performed according to patients age (full doses for patients ≤75 years and reduced for patients >75 years). Details on treatment regimens and results of these studies have previously been reported (Gay F et al EHA 2013, Larocca A et al EHA 2013, Bringhen S et al EHA 2013). At diagnosis, a geriatric assessment had been performed, to assess co-morbidities, cognitive and physical conditions.869 patients were included in the analysis: 659 enrolled in the lenalidomide-based, 152 in the bortezomib-based and 58 in the carfilzomib-based trial. Median age was 74 years, and 44% of patients were older than 75 years. Median follow-up was 18 months.In univariable analysis, the risk of death was higher in patients aged 75-80 (Hazard Ratio, HR 1.37, p=0.11), and in patients older than 80 years (HR 2.75, p<0.001), compared to patients younger than 75 years. Performance status and gender did not significantly impact overall survival (OS). In a multivariable Cox model, an additive scoring system (range 0-5), based on age, co-morbidities, cognitive and physical conditions, was categorized to identify 3 groups: fit (score=0, 39%); unfit (score=1, 31%), and frail (score≥2, 30%). The 18-month OS was 92%, 88% and 73% in the three categories (fit, unfit and frail), respectively.In a Cox's model, including ISS, gender and performance status, the HR compared to the fit category was 1.4 (p=0.18) and 2.9 (p<0.001) in unfit and frail patients. The cumulative 6-month risk of serious adverse events or treatment discontinuation was 44%, 47% and 55% in fit, unfit and frail patients, respectively. The higher mortality rate in unfit and frail patients seems mainly due to higher cumulative incidence of grade ≥3 adverse events (in particular extra hematologic toxicities) causing subsequent treatment discontinuation.The use of a simple scoring system, based on geriatric assessment, allows the identification of groups of patients with different survival and risk of severe toxicities.Larocca: Celgene: Honoraria; Janssen-Cilag: Honoraria. Bringhen:Celgene: Honoraria; Janssen-Cilag: Honoraria; Novartis: Honoraria; Merck Sharpe & Dohme: Membership on an entity’s Board of Directors or advisory committees; Onyx: Consultancy. Gay:Celgene: Honoraria; Janssen-Cilag: Honoraria; Celgene: Membership on an entity’s Board of Directors or advisory committees; Byotest: Membership on an entity’s Board of Directors or advisory committees. Boccadoro:Celgene: Research Funding; Janssen-Cilag: Research Funding; Celgene: Consultancy; Janssen-Cilag: Consultancy; Celgene: Membership on an entity’s Board of Directors or advisory committees; Janssen-Cilag: Membership on an entity’s Board of Directors or advisory committees. Palumbo:Amgen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen Pharmaceuticals: Consultancy, Honoraria; Millenium: Consultancy, Honoraria; Onyx: Consultancy, Honoraria.

The quality of response and the residual disease after treatment are important prognostic factors in several hematological diseases including multiple myeloma (MM). Several papers demonstrated that the deeper the response after treatment, the longer the survival. However few data are available on the monitoring of minimal residual disease (MRD) during the maintenance therapy in transplant eligible MM patients.to evaluate the role of maintenance therapy in reducing MRD and the role of monitoring the response to predict clinical relapse.newly diagnosed MM patients enrolled in the RV-MM-EMN-441 trial (NCT01091831) and achieving at least a very good partial response (VGPR) after consolidation were included in the study. Patients received 4 Lenalidomide-Dexamethasone (RD) courses as induction, Cyclophosphamide to mobilize bone marrow stem cells (BMSC) and then were randomized to receive 6 cycles of Cyclophosphamide-Lenalidomide-Dexamethasone (CRD) or Autologous Stem Cell Transplantation (ASCT) with Melphalan 200 mg/m2. All patients received maintenance therapy with Lenalidomide (R) or Lenalidomide-Dexamethasone (RD) until relapse. MRD analysis was performed in a single laboratory (University of Turin, Italy) using flow cytometry according to European Myeloma Network guideline (Rawstron AC, Haematologica 2008). Samples of bone marrow (BM) were collected at diagnosis, after consolidation, after 3 and 6 courses of maintenance and then every 6 months until clinical relapse. The samples were considered MRD +ve if ≥ 0.01% of PC were detected. Immunophenotypic (IF) relapse was defined as an increase of ≥ 25% in the amount of malignant plasma cells in BM compared to the previous determination.Fifty patients (27 female/23 male) with a median age of 57 yrs (40-65) entered the study. According to ISS, 27 patients were stage I, 15 stage II and 8 stage III. Fish risk profile was standard in 31 patients, high in 11 and not available in 8. Twenty-five patients received CRD as consolidation and 25 underwent ASCT. The median follow-up was 28.6 months. After consolidation 16 (32%) patients achieve a complete response (CR) and 34 (68%) a VGPR. MRD was negative in 19/48 (40%) patients, of which 12 received ASCT (out of 23, 52%) and 7 received CRD (out of 25, 28%). Patients receiving ASCT showed a lower value of residual cells (median 0.08%, range 0 – 1.00) compared to patients receiving CRD (median 0.5%, range 0 – 2.9%, p=0.0134). The lower MRD value was achieved after consolidation in 31 patients (62%), after 3 courses of maintenance in 6 patients (12%) and after 6 or more courses of maintenance in 13 patients (26%). The increase in quality of response was observed primarily in patients receiving CRD: the average amount of residual plasma cells in bone marrow was 71/uL after induction, lowering to 51/uL after 6 and 12 courses of maintenance therapy. Nine patients clinically relapsed after an average time of 25.6 months from the beginning of the therapy and in all patients this was anticipated by immunophenotypic relapse.1) consolidation therapy with ASCT determines a deeper response compared to CRD; 2) maintenance therapy can improve the quality of response, in particular in patients not receiving ASCT; 3) Immunophenotypic relapse anticipate the clinical relapse. These results suggest the possible role of MRD monitoring to better assess the response to therapy also during maintenance and as marker of early relapse.Ladetto: Celgene: Research Funding, Speakers Bureau; Janssen Cilag: Research Funding, Speakers Bureau; Mundipharma: Research Funding, Speakers Bureau; Roche: Research Funding, Speakers Bureau; Amgen: Research Funding, Speakers Bureau. Boccadoro:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Palumbo:Amgen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Millenium: Consultancy, Honoraria; Onyx: Consultancy, Honoraria.

In mature lymphoid disorders, minimal residual disease (MRD) detection based on real time quantitative PCR (RQ-PCR) of immunoglobulin heavy chain gene rearrangement (IgH) has a well-established role in prognostic assessment, particularly in Mantle cell Lymphoma (MCL) and Multiple Myeloma (MM). RQ-PCR has excellent sensitivity and specificity but has a major limitation in its relative quantification nature, as it requires a reference standard curve usually built with dilutions of diagnostic tumor DNA or on plasmids containing the target rearrangement. Droplet Digital PCR (DD-PCR), applying the principle of limiting dilution of DNA and single molecule detection allows a reliable absolute quantification of target. In this study we compared IgH-based MRD detection by RQ-PCR and DD-PCR, to assess whether DD-PCR could achieve the same performances of RQ-PCR in the absence of the limitation mentioned above.

Minimal residual disease (MRD) by multiparameter flow-cytometry recently showed a promising role in predicting outcomes in patients with multiple myeloma. However, data on immunophenotypic response (IR) after allografting are lacking.

While international stage (ISS) and the presence of absence of cytogenetic abnormalities on FISH somewhat define the clinical risk of MM patients, additional biomarkers are necessary for more precise risk-based classification. Emerging studies have shown that circulating microRNAs (miRNAs) can be detected in patients with a variety of malignancies, including MM, and they could be non-invasive biomarkers. We measured serum miRNA levels of a large cohort of well-characterized previously untreated MM patients and correlated results with clinical outcome to test their prognostic impact.

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by the aberrant expansion of clonal plasma cells (PCs) within the bone marrow. Malignant PCs produce intact or partial monoclonal immunoglobulin (M protein) and cause organ damage. More than 20,000 new cases of multiple myeloma (MM) are diagnosed every year in the US with approximately 10,700 deaths occurring. The pathogenesis of MM is still largely unclear, but several reports suggest that interaction of tumor cells with the bone marrow microenvironment and microRNAs (miRNAs) deregulation may play a role in the etiology and progression of MM. miRNAs are small non-coding RNAs capable of regulating protein expression by binding to mRNA, and have been implicated in the development of MM. First identified inside cells, miRNAs can also be detected in body fluids, including serum and plasma, and may be a valid biomarker. Few studies have investigated the agreement between circulating miRNAs and intracellular myeloma PC miRNAs at diagnosis.Using Nano-String nCounter technology we first performed a screening analysis on serum samples obtained from MM patients and healthy controls. We identified a candidate set of miRNAs differentially expressed in the serum of MM patients. The levels of these miRNA markers were validated by RT-PCR in both serum and bone marrow PCs from the same cohort. Agreement of the quantitative miRNA marker levels between sample types was evaluated using intraclass correlation coefficients (ICC) (both for normalized and log2 measures).Thirty-nine MM patients (21 male, 18 female) with a median age of 72 years (range: 65 – 83) were included in the analysis. Most were ISS stage I or II (59% vs. 41% ISS stage III) and 39% were high risk according to FISH abnormalities – 21% of patients carried del17p, 24% t(4;14) and 5% t(14;16). Medians and ranges for lab markers were as follows: hemoglobin 10.0 g/dl (7.2 to 15.1), beta2-microglobulin 5.18 ug/ml (1.38 – 12.1), creatinine 0.94 mg/dl (0.65 – 2.49), CRP = 1.6 mg/dl (0.02 – 116.0). Nine age-matched healthy controls were also used for the analysis. After the screening analysis, the following miRNAs were differentially expressed between healthy subjects and MM patients in serum samples: miR-92a, miR-451, miR-19b, miR-21, miR-16, miR-25, miR-30a, and miR-126. There was no significant agreement or correlation between serum and myeloma cell samples using either untransformed as well as log2measures (all p>0.40) (Table 1).Our preliminary results suggest a difference between circulating miRNAs in myeloma patients from controls. This indicates that future studies are needed to better define the role of miRNAs in the peripheral blood as a prognostic and even diagnostic biomarker in myeloma. From our preliminary data it also appears that circulating miRNAs are not simply secreted into the peripheral blood by myeloma PCs as it seems that circulating miRNAs do not reflect those of myeloma PCs. Differential expression could be determined by other cells that can release and or modify their miRNA expression in response to MM. Ongoing studies are examining the origin and function of miRNAs in the peripheral blood.No relevant conflicts of interest to declare.

The prognosis of primary plasma cell leukemia (PPCL) remains poor. The “novel agents” have recently shown promising results in PPCL patients in case reports or small retrospective series. Here we describe the first prospective, multicenter, phase II clinical trial of PPCL, where lenalidomide in combination with low dose dexamethasone (Ld) was tested as initial therapy in newly diagnosed patients fulfilling the IMWG diagnostic criteria of PPCL.Ld regimen consisted of lenalidomide 25 mg/d for 21 days and oral dexamethasone 40 mg on days 1, 8, 15, and 22 for each 28-day cycle. After 4 cycles, responding patients not eligible for stem cell transplantation (SCT) continued until 8 cycles of full-dose Ld, followed by a maintenance dose of single agent lenalidomide equal to 10 mg/d on days 1–21 of each 28-day cycle. Patients responding after 4 cycles and eligible for SCT proceeded according to the treatment centre's transplant policy. Patients not responding after 4 cycles or progressing during this treatment were considered off-study. Appropriate dose reductions (in particular for patients with reduced renal function at baseline), double contraception methods and anti-thrombotic/anti-infective prophylaxis were recommended. The primary end-point was early response rate according to IMWG uniform response criteria. Secondary end-points were PFS, OS, feasibility and efficacy of SCT, safety.According to the Simon optimal two-stage adaptive design, twenty-three patients were enrolled in between March 2009 and May 2011. The male/female ratio was 1.1, and median age was 60 years (range 44–80). The median absolute number and percentage of circulating plasma cells were 4.280/μl (range 1.500–114.660) and 34% (range 21–90) respectively. Fifteen patients (65.2%) had abnormal renal function at presentation. Twenty-one patients (91.3%) were tested by FISH analysis and cytogenetic abnormalities were detected in all of them, del13q being the most frequently found (16 patients). Seventeen patients showed multiple chromosomal lesions. Involvement of chromosome 14 was observed in 18 patients, three of whom showed t(4;14). Chromosome 1q gain and del17p were detected in 10 and 7 patients, respectively. In the intention-to-treat (ITT) population, overall response rate (ORR) after at least one Ld cycle was 73.9% (17/23), with 8 patients (34.7%) achieving partial remission (PR), 5 (21.7%) very good PR (VGPR), 3 (13%) complete response (CR), and 1(4.3%) near CR (nCR) (VGPR or better: 39%). In the efficacy-evaluable (EE) population, 14 out of 15 patients who received the initially planned 4 Ld cycles (65.2% of the ITT population) responded (ORR 93.3%), achieving 5 PR (33.3%), 5 VGPR (33.3%), 1 near-CR (6.6%) and 3 CR (20%) (VGPR or better: 59.9%). The maintenance phase was reached and safely performed in 4 responding patients not eligible for SCT, 3 of whom relapsed after 2, 8 and 22 months, respectively. After Ld induction therapy, 8 patients received single (n. 4) or double (n. 4) autologous SCT (ASCT); another patient underwent a sequence of ASCT followed by non-myeloablative allogeneic SCT (AlloSCT). Six eligible patients did not receive ASCT frontline, due to initial Ld failure or adverse events; three of them underwent single ASCT (n. 2) or tandem ASCT/non-myeloablative AlloSCT (n. 1) after a bortezomib-based salvage therapy, achieving 2 CR and 1 PR. After a median follow-up of 23 months, median OS and PFS in ITT population were not reached and 22 months, respectively. All transplanted patients remained alive, although three of them relapsed and started salvage treatments; OS was 12 months in the 11 patients who did not receive ASCT (p < 0.001). The correspondent figures for PFS were 29 and 16 months, respectively (p < 0.01). Considering ITT population, multivariate analyses showed that SCT was positively correlated to both OS and PFS. There were 17 episodes of grade 3/4 non hematological toxicity, which occurred in 13 patients (5 infections, 3 renal, 3 metabolic, 2 gastro-intestinal, 2 skin, 1 fatigue, 1 thromboembolic), causing early interruption of Ld treatment in 4 patients. Grade 3/4 hematological toxicity (mainly neutropenia) occurred in 11 patients (47.8%).Ld may be a feasible and effective initial therapeutic option for PPCL, particularly in patients who receive ASCT after a short course of induction treatment.Musto: Celgene: Honoraria, Research Funding. Off Label Use: Lenalidomide as first line therapy of plasma cell leukemia. Petrucci:Celgene: Honoraria. Cascavilla:Celgene: Honoraria. Di Raimondo:Celgene: Honoraria. Caravita:Celgene: Honoraria. Morabito:Celgene: Honoraria. Offidani:Celgene: Honoraria. Bringhen:Celgene: Honoraria. Boccadoro:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Palumbo:Celgene: Consultancy, Honoraria.

No relevant conflicts of interest to declare.

In this study, a serological proteome analysis (SERPA) was applied for the first time to identify novel tumor-associated antigens (Ags) capable of eliciting humoral immune responses in patients with chronic lymphocytic leukemia (CLL). SERPA has been demonstrated to be a valuable method to identify tumor associated Ags in several human solid and hematological malignancies. The identification and characterization of circulating antibodies (Abs) and corresponding Ags in CLL can provide useful information to understand cell transformation, predict clinical outcome, and develop immune-based interventions.SERPA was performed in 21 untreated patients. Proteins extracted from purified CLL cells were separated by 2-D electrophoresis (2-DE) to obtain proteomic maps which were blotted with corresponding sera by Western Blot to reveal Ab-based reactivity with autologous proteins. To verify the CLL specificity of Abs recognition, 7 out of 21 maps were also probed with sera collected from 7 healthy donors (HD). For identification, Ag spots in WB were aligned with proteins in 2-DE maps. The protein spots corresponding to the assigned Ags were excised from the gel, trypsin digested and analyzed by peptide mass fingerprint by MALDITOF Mass Spectrometry (MS) with the software MASCOT. T cells from 6 CLL patients and 3 HD were stimulated with autologous ENOA-pulsed and control dendritic cells (DC) and evaluated by IFNγ ELISPOT assay. Ags surface expression was analyzed by flow cytometry. Statistical correlations were performed using t-test, Mann-Withney rank sum test and χ2-test.Sixteen out of 21 CLL sera (76%) were immunoreactive and produced a total number of 45 Ag spots, whereas HD sera produced only 3 spots (p<.03). Eleven out of 16 (69%) reactive CLL sera recognized from 2 to 6 different Ags in each individual patients. MS analyses led to the identification of 16 different Ags and many of them were recognized by sera from different patients. Forty-eight percent of CLL sera reacted against α-Enolase (ENOA), whereas none of HD sera was ENOA reactive. The IGHV mutational status was available in 19 CLL patients: 10 were mutated (M), while 9 were unmutated (UM). Interestingly, ENOA was recognized by sera from 7/10 M patients (70%), but only by sera from 3/9 UM patients (33%). Cytofluoroimetric analyses performed in 7 patients showed that ENOA was undetectable on viable CLL cells surface, whereas it was translocated on the membrane of apoptotic CLL cells.Statistical correlation analyses showed that immunoreactive CLL patients are characterized by an early stage of disease. Moreover, ENOA-reactive patients have a better preserved immune system because they have higher numbers of CD3+ (p=.02), CD3+/CD4+ (p=.03) and CD3+/CD8+ (p=.05) cells in the peripheral blood than ENOA-unreactive patients.We also investigated the possibility to induce ENOA-specific T-cell immune responses in 6 CLL patients. ENOA-pulsed DC induced IFNγ production in 4/6 patients (66%). The response was ENOA and CLL specific because: 1) it was not induced by unpulsed DC or DC pulsed with an irrelevant protein; 2) it was not induced when T cells from 3 HD were stimulated with autologous ENOA-pulsed DC. Interestingly, ENOA Abs were detectable by SERPA in 3 out of 4 (75%) patients with ENOA-induced T-cell responses, whereas they were undetectable in patients with unresponsive T cells. Correlations with the IGHV mutational status showed that all patients with ENOA-reactive T cells were M.These results indicate that ENOA is able to elicit specific humoral and cellular immune responses suggesting that this protein can be a promising biomarker and a potential target for immunotherapy in CLL.Massaia: Novartis Farma S.p.A: Honoraria, Research Funding.

Abstract 4204This icon denotes a clinically relevant abstract

In a multicenter phase 3 randomized trial, VMPT-VT was superior to VMP for response rates, progression-free survival and time to next treatment (Palumbo A, et al. J Clin Oncol 2010). Here we report an updated analysis on survival after 4 years of follow-up.Patients (N=511) were randomly assigned to receive nine 6-week cycles of VMPT-VT (induction: bortezomib 1.3 mg/m2, d 1, 4, 8, 11, 22, 25, 29, 32, cycles 1–4, d 1, 8, 22, 29, cycles 5–9; melphalan 9 mg/m2 d 1–4, prednisone 60 mg/m2, d 1–4, thalidomide 50 mg d 1–42; maintenance: bortezomib 1.3 mg/m2 every 14 days and thalidomide 50 mg/day up to 2 years) or VMP alone. After the inclusion of 139 patients, the protocol was amended: both VMPT-VT and VMP induction schedules were changed to nine 5-week cycles and bortezomib schedule was modified to weekly administration (1.3 mg/m2 d 1,8,15,22, all cycles).After a median follow-up of 47.2 months, median OS was not reached in the VMPT-VT arm and was 58.2 months in the VMP arm; 5-year OS rates were 59.3% and 45.9%, respectively (HR 0.74, p=0.04), with 26% reduced risk of death for patients receiving VMPT-VT (Figure-panel A). This benefit was more evident in patients younger than 75 years (5-year rates 67.8% for VMPT-VT vs 49.9% for VMP, HR 0.63, p=0.01, Figure-panel B) and in patients in complete response (CR) after induction (5-year rates 81.4% for VMPT-VT vs 48.2% for VMP, HR 0.38, p=0.006, Figure-panel C) while no significant differences were evident in patients with standard- or high-risk features detected by FISH (HR 0.99, p=0.99). A 1-year landmark analysis for patients completing induction was performed: the 4-year OS was 64.6% in the VMPT-VT group and 49.7% in the VMP group, with 33% reduced the risk of death for patients receiving VT maintenance (HR 0.67, p=0.02). Forty-nine percent of VMPT-VT and 70% of VMP patients relapsed and received subsequent salvage therapies; there was no difference in survival from relapse in the two groups (2-year OS rates 40.7% vs 50.2%,HR 1.11, p=0.54).The median duration of VT maintenance was 23.8 months. During VT maintenance 7% of patients experienced grade 3–4 peripheral neuropathy, 5% grade 3–4 hematological toxicity, 3% grade 3–4 infection and 12% discontinued due to adverse events. Second primary malignancies were reported in 7/254 patients in the VMPT-VT group and 7/257 patients in the VMP group. These corresponded to incidence rates of 0.9 and 1.05 per 100 patient-years, respectively, and were consistent with background incidence rates in the general population (aged 65–74 years 1.9, aged ≥ 75 years 2.3, SEER database).VMPT-VT significantly prolonged OS compared with VMP, especially in patients younger than 75 years and in patients achieving CR after induction. In patients 67–75 years of age, VMPT-VT reduced the risk of death by 37% and it should be considered a new standard of care.Palumbo: Celgene: Advisory Board, Advisory Board Other, Consultancy, Honoraria; Janssen: Advisory Board Other, Consultancy, Honoraria. Bringhen:Janssen: Honoraria; Celgene: Honoraria. Gentilini:Janssen: Honoraria; Celgene: Honoraria. Patriarca:Janssen: Honoraria. Guglielmelli:Janssen: Honoraria; Celgene: Honoraria. Musto:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Petrucci:Janssen: Honoraria; Celgene: Honoraria. Boccadoro:Janssen: Consultancy, Research Funding, Scientific Advisory Board Other; Celgene: Consultancy, Research Funding, Scientific Advisory Board, Scientific Advisory Board Other.

Patients with multiple myeloma (MM) relapsed/refractory after immunomodulatory drugs and bortezomib have limited treatment options. Recently, the combination pomalidomide-dexamethasone has led to at least partial response (PR) of 25–42% in relapsed/refractory MM and 32% in patients refractory to lenalidomide.In this study we evaluate the safety and efficacy of the combination pomalidomide-cyclophosphamide-prednisone (PCP) in patients with MM who received 1–3 lines of treatment and were relapsed/refractory to lenalidomide therapy.Between August 2010 and July 2011, 41 patients were enrolled; median age was 69 years (range 49–82); 23 patients relapsed after lenalidomide and 18 patients were refractory to lenalidomide. The first 24 patients entered the phase I of the study to define the maximum tolerated dose (MTD) of PCP: 4 dose levels of pomalidomide (1, 1.5, 2 and 2.5 mg/day, days 1–28) were tested in combination with cyclophosphamide (50 mg every other day, days 1–28) and prednisone (50 mg every other day, days 1–28) for six 28-day cycles. Thromboprophylaxis with aspirin (100 mg/day) was recommended, low-molecular weight heparin was given to high risk patients. Dose Limiting Toxicities (DLTs) were defined as: grade 4 neutropenia for more than 3 days, grade 4 thrombocytopenia, grade 3–4 neutropenic fever, any grade 3–4 non-hematologic toxicity. The MTD was achieved when 25% of patients experienced a DLT, using the Bayesian Continual Reassessment Method. In the phase II of the study, the Simon two-stage design was used and 17 additional patients were enrolled and received the MTD of pomalidomide.DLTs occurred in 1/4 patient who received pomalidomide 1.5 mg (grade 4 thrombocytopenia) and in 3/12 patients who received pomalidomide 2.5 mg (grade 3 neuropathy, grade 3 hepatic toxicity and grade 4 thrombocytopenia). The MTD was defined at 2.5 mg/day, with an estimated DLT probability of 0.258 (95% credibility interval: 0.101–0.468). 32 patients received at least one cycle of therapy and could be evaluated for efficacy and safety. At least PR was reported in 19/32 (59%) patients, including 2 complete response (CR), 5 very good partial response (VGPR), 12 PR. In patients refractory to lenalidomide, at least PR was reported in 11/15 (73%) patients, including 1 CR, 2 VGPR, 8 PR. Grade 4 hematologic toxicities were neutropenia (9%) and thrombocytopenia (9%). Grade 3–4 non-hematologic toxicity included infection (9%), rash (9%), neurologic (6%) and hepatic (3%) toxicities. Thromboembolism occurred in 1 patient.At least PR was achieved in 73% of patients refractory to lenalidomide; grade 4 neutropenia and/or thrombocytopenia were less than 10%. The combination pomalidomide (2.5 mg/day), cyclophosphamide (50 mg every other day), prednisone (50 mg every other day) showed high response rates with limited toxicities in patients relapsed/refractory to lenalidomide. Updated data will be presented at the meeting.Palumbo: Amgen: Honoraria; 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. Larocca:Janssen-Cilag: Honoraria. Guglielmelli:Janssen-Cilag: Honoraria; Celgene: Honoraria. Giuliani:Celgene: Research Funding; Novartis: Research Funding. Boccadoro:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Musto: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Petrucci:Celgene: Honoraria. Cascavilla:Celgene: Honoraria. Di Raimondo:Celgene: Honoraria. Caravita:Celgene: Honoraria. Morabito:Celgene: Honoraria. Offidani:Celgene: Honoraria. Bringhen:Celgene: Honoraria. Boccadoro:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Palumbo:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

No relevant conflicts of interest to declare.

Telomere length (TL) at diagnosis has been established as an independent outcome predictor in CLL (Rossi et al Leukemia 2009). However data on TL dynamics over time are scant and anedoctal. Aim of this study was to evaluate telomere dynamics in the natural history of CLL. This issue has been here addressed on a series of 88 CLL patients (pts).25 pts were assessed for TL at diagnosis and at relapse and 63 pts had two determinations during the “watch and wait” (WW) phase. The series was fully characterized in terms of Binet stage, ALC, CD38, ZAP-70, IGHV mutational status (IGHV-MS), stereotyped receptors, cytogenetics and detailed clinical history. LDH, B2-microglobulin, p53 mutations and CD49d were available in more than 70% of pts. Treatment-free survival (TFS) analysis was performed exclusively in pts undergoing kinetic evaluation during the WW phase. This population had a median follow-up of 73 months and a median TFS of 130 months. TL was analyzed as previously described (Rossi et al Leukemia 2009; Ladetto et al Blood 2004). Median time between TL determinations was 44 months (range 12–231). Telomere loss was calculated in terms of both absolute loss (AL) and yearly loss (YL). Continuous variables were compared by the Mann-Whitney test, while TFS by the stratified Kaplan-Meyer method.Telomeres were shorter at follow-up compared to baseline with a median loss of 651bp (range +493bp, −5874bp; p<0.001) (Fig 1A). AL and YL were greater in cases with higher baseline TL while those with short telomeres at diagnosis had only modest additional erosion (p=ns for pts in the 25th lowest percentile) (Fig 1B). Telomere loss over time was noticeable both in pts assessed at diagnosis and at relapse as well as in those assessed during the WW phase, but clearly inferior in the former subgroup (YL of −61bp, p<0.05 and −210bp, p<0.01, respectively), possibly due to the higher number of patients with short telomeres. AL and YL did not correlate with any available clinical or biological parameter, with the exception of a positive association with IGHV-MS (p<0.05). Pts with baseline TL shorter than the validated cut-off value of 5000bp (Rossi et al Leukemia 2009) were associated to an inferior TFS (median TFS 41 months vs 182 months; p<0.0001) as expected. Moreover also Binet status and IGVH-MS were predictive for TFS in this series. Surprisingly, also an YL above the median value (-210bp) appeared to be predictive for an inferior TFS (median TFS 82 months vs 182 months; p<0.05) (Fig 1C), despite being more common in pts with longer telomeres and VH-mutated IgH genes. Following stratification of pts according to baseline TL (< or > 5000bp), YL was predictive for TFS in both pts subgroups (Fig 1D i.e. baseline TL >5000bp; YL ≥ −210bp vs YL <-210bp: TFS 88 months vs not reached p<0.01. Figure 1E i.e. baseline TL <5000bp; YL ≥-210bp vs TL <210bp: median TFS 36 months vs 50 months, p<0.01).The results of the first systematic analysis on TL dynamics in CLL indicate the following: i) progressive telomere erosion occurs as part of the natural history of CLL; ii) telomere loss is more pronounced when baseline TL is higher; iii) accelerated telomeric loss associates to an inferior TFS. The results described in the present analysis corroborate basic studies suggesting that telomere disruption represents a critical step associated to CLL progression.No relevant conflicts of interest to declare.

There is extensive evidence from numerous studies in the transplant setting that achievement of complete response (CR) or at least very good partial response (VGPR) is significantly associated with prolonged progression-free survival (PFS) and overall survival (OS). In elderly myeloma patients CR was a rare event since new drugs has been added to standard melphalan-prednisone (MP). After the introduction of novel agents, CR represents an achievable goal, also outside of the transplant setting.to assess the impact of response to treatment on time-to-event parameters (PFS and OS) in elderly myeloma patients.We retrospectively analysed newly diagnosed myeloma patients, older than 65 years old, or younger but not eligible for high-dose chemotherapy and transplant. Patients were enrolled in 3 multicentre randomized European trials of the GIMEMA and Hovon groups, and were treated with MP (n=332), MP plus thalidomide (MPT, n=332), MP plus bortezomib (VMP, n=257) or MP plus bortezomib-thalidomide followed by bortezomib-thalidomide maintenance (VMPT-VT, n=254). PFS, OS and duration of CR were analysed by the Cox proportional hazards model, comparing the two arms by the Wald test and calculating 95% confidence interval (CI). Univariate and multivariate analyses were performed for the following variables: age at diagnosis (>75 vs. ≤75 yrs), International Staging System (ISS) stages, type of chemotherapy and best response achieved. Best response was treated as a time-dependent variable.A total of 1,175 patients, enrolled from November, 2001 to January, 2009, were retrospectively analysed. The best response to treatment was available in 1,136 patients: CR was reported in 195, VGPR in 212, PR in 397. Baseline characteristics according to best response achieved in patients who obtained CR, VGPR or PR were similar. Since response rates vary according to treatment regimens the proportion of patients who received MP, MPT, VMP, and VMPT-VT was different in the different response categories. After a median follow-up of 29 months, PFS was significantly higher in patients who achieved CR compared to those who obtained VGPR (HR 0.16; 95% CI 0.10–0.24; p<0.001) or PR (HR 0.07; 95% CI 0.04–0.13; p<0.001). The advantage in PFS translated into an advantage in OS: patients obtaining CR have a significantly prolonged OS than patients who achieved VGPR (HR 0.15; 95% CI 0.08–0.28; p<0.001) or PR (HR 0.08; 95% CI 0.04–0.16, p<0.001), (table). In multivariate analysis CR achievement was as an independent predictor of longer PFS and OS, regardless of age, ISS stage, and treatment administered. In patients > 75 years, both PFS and OS were shorter as compared to younger patients. Despite these differences, the impact of CR on outcome was identical. In the subgroup of patients > 75 years, PFS was significantly prolonged in patients who achieved CR, compared with those who obtained VGPR (HR 0.26; 95% CI 0.12–0.58, p = 0.001) or PR (HR 0.20; 95% CI 0.10–0.41, p < 0.001). Accordingly, OS was significantly higher in patients who achieved CR, compared with those who obtained VGPR (HR 0.13; 95% IC 0.03–0.58; p = 0.007), or PR (HR 0.12; 95% IC 0.03–0.51, p = 0.004), (table). No significant PFS differences between patients obtaining CR during the first 6 months of treatment or later were seen (HR 1.06; 95% IC 0.49–2.27; p=0.878). Similarly, no OS differences between these two groups were detected (p = 0.676). Duration of CR was comparable in patients who obtained CR during or after the first 6 months of treatment (HR 0.66; 95% CI 0.30–1.45; p = 0.305). Patients whose CR lasted more than 18 months have a significant OS benefit compared to patients who did not (p=0.006).These finding highlight the importance of CR, also outside of the transplant setting, regardless of age, ISS and treatment administered, and support the use of new drugs, also in patients older than 75 years, to achieve and maintain maximal response.Gay: Celgene: Honoraria. Bringhen:Calgene: Honoraria; Janssen-Cilag: Honoraria. Guglielmelli:Celgene: Honoraria; Janssen Cilag: Honoraria. Boccadoro:Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees. Sonneveld:Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson : Membership on an entity's Board of Directors or advisory committees. Palumbo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees.

The thymus is fundamental for the generation of T-cell diversity following allografting even though its function declines with age. Non-myeloablative conditionings have extended the eligible age for allografting to 65–70 yrs for patients with hematological cancers. The thymic generation of the TCR diversity occurs through the recombination of gene segments coding for the TCR alpha and beta chains generating by-products defined as signal joint TCR excision circles (sjTRECs). sjTRECs are extrachromosomal DNA fragments, most frequently found in naive T cell, that do not replicate with subsequent cell divisions. sjTRECs evaluation by quantitative PCR and the kinetics of naive and memory T cells by flow-cytometry were used to asses the thymic function. Moreover, TCR repertoire analysis of V-beta families was evaluated, in a subset of patients, by spectratyping up to 1 year post-transplant. Finally, TRECs values were compared with 67 paediatric patients (median age 9, r 1–19 years). Subpopulations studied included peripheral mononuclear cells (PMC), sorted CD4- and CD8 – T cells.Overall, 55 patients, median age 51 (r 34–64) years, conditioned with low dose TBI (200 cGy), with/without fludarabine, followed by G-CSF mobilised donor peripheral blood stem cell infusion from HLA identical siblings or unrelated donors, were evaluated at different time points: baseline, at 28, 56, 84, 100, 180 days, and at 1, 2, 3, 4, 5 years post-transplant. Naive CD4+CD62L+CD45RA+bright T cells and memory CD4+CD62L+CD45R0+bright T cells showed a gradual increase up to 3 years post-transplant with median values of 882/ul and of 532/ul respectively. Median values of sjTREC copies/100ng DNA in PMC increased 5 fold at 1 yr, 20 fold at 3 yrs and 75 fold at 5 yrs from 0.7 (pre-transplant), whereas median sjTREC copies/100ng DNA from sorted CD4+ cells (purity>95%) increased 7 fold at 1 yr and 15 fold at 5 yrs from 5 at 3 months post-transplant. A significant correlation was demonstrated between TREC values and CD4+CD62L+CD45RA+bright T cells (p<0.001). Importantly, a 60 y/o patient thymectomised 10 years before transplant showed no thymic output and a very poor recovery of the TCR repertoire. However, in the subset of studied patients, though slow, TCR repertoire of V-beta families required at least 1 year to clearly become polyclonal. sjTRECs levels in both sorted CD4- and CD8- T cells at 1 year were significantly higher in patients without chronic GVHD (p=0.0135 and 0.007). At 2 years post-transplant, sjTRECs levels were significantly higher in paediatric patients versus adults (median values 240 vs 56.9, p=0.001).Detectable thymic function persisted post-transplant in all patients except one who had previously been thymectomised. T cell reconstitution was somewhat slow especially during the first year post-transplant. The thymus may be an important target of chronic GVHD. Age played an important role in thymic output.Boccadoro: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Role and timing of allografting in myeloma are hotly debated. Before the introduction of new drugs, we carried out a trial where the treatment assignment was based only on the presence/absence of an HLA-identical sibling (Bruno et al, N Engl J Med 2007).Overall, 162/199 (81%) of patients with at least one sibling were HLA-typed. First-line treatments included induction with VAD-based regimens and a cytoreductive autograft, followed by a nonmyeloablative allograft (Tandem auto-allo) or a second melphalan-based autograft (Double-auto). We now report an update at a median follow up of 7.1 years.Response rates [complete (CR) and partial remissions (PR)] at the time and after the non-myeloablative allograft and at the time and after the second autograft did not differ between the two cohorts: 76% and 86%, and 76% and 91% respectively (p=1 and p=0,54). However, CR rate was significantly higher after the non-myeloablative allograft than after the second autograft: 55% versus 26% (p=0,0026). At a median follow up of 7.1 years (range 2.5 – 10.7+), by intention-to-treat analysis, median overall survival (OS) and event free survival (EFS) were significantly longer in patients with HLA-identical siblings (No.80) as compared with those without (No.82): not reached vs. 4.25 years (HR 0.51, CI 95% 0.34–0.76, p=0.001) and 2.8 vs. 2.4 years (HR 0.62, CI 95% 0.44–0.87, p=0.005). By multivariate analysis, independent of age, gender, myeloma protein isotype, Durie&Salmon stage, and disease status at the first autograft; the presence of an HLA-identical sibling was significantly associated with longer OS (HR 0.5, CI 95% 0.3–0.8, p=0.001) and EFS (HR 0.63, CI 95% 0.4–0.9, p=0.01). At a median follow up of 7.3 (range 5.4 – 10.7+ years), median OS was not reached in the 58 patients who received a non-myeloablative allograft and 5.3 years in the 46 who received a second high-dose melphalan autograft (HR 0.55, CI 95% 0.32–0.94, p=0.02), whereas EFS was 39 months and 33 months (HR 0.62, CI 95% 0.40–0.96, p=0.02) respectively. Cumulative incidence of transplant related mortality was 11% and 2% at 2 years respectively. At median follow-ups of 7.3 years from diagnosis (range 5.4 – 10.4+) and 6.5 years from the allograft (range 4.2 – 9.4+), and 7.4 years from diagnosis (range 5.6 – 10.7+) and 6.2 years from the second autograft (range 4.7 – 9.1+), 30/58 (52%) and 37/46 (80%) patients, respectively, were treated for disease relapse/progression. Salvage therapies included bortezomib- or thalidomide-containing regimens in most patients of both cohorts. After 1–3 lines of therapy, 22/30 (73%) had a response, including 5 CR and 17 PR, in the tandem auto-allo group, whereas 21/37 (54%) had a response, including 4 CR and 16 PR after the second autograft. Of note, at a median follow up of 3.9 years from the start of the first salvage therapy, OS was not reached and was 1.7 years in patients who had relapsed/progressed after the allograft and the second high-dose melphalan (HR 0.44, CI 95% 0.24–0.82, p=0.01) respectively.In this study, allografting conferred a long term survival advantage over standard autografting. Salvage therapy was associated with longer OS perhaps due to a synergistic effect between new drugs and residual graft-vs.-myeloma effects. In prospective clinical trials, the combination of graft-vs.-myeloma effects with “new drugs” should be explored and may increase the cure rate of myeloma patients.Bringhen: Celgene: Honoraria; Janssen-Cilag: Honoraria. Palumbo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees. Boccadoro:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Boccadoro: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Massaia: Novartis: Honoraria, Research Funding.

Multiple myeloma (MM) cells growth is sustained by several stimuli derived from surrounding cells of bone marrow (BM) microenvironment. Besides the increase in growth factors production, a constitutive activation of several pathways determining protection from apoptosis and growth advantage have been reported too. Aberrant activation of Met/HGF (Hepatocyte Growth Factor) pathway has been described in several tumors causing cell proliferation, cell migration and neoplastic angiogenesis. A qualitative analysis demonstrated that Met transcription is present in MM plasma cells and increase plasmatic levels of HGF has been related to a bad prognosis subgroup of patients. However a comprehensive investigation of quantitative analysis on Met expression, plasmatic HGF values and correlation with clinical outcome on a large amount of MM patients treated with novel agents is still missing.: to investigate the role of Met mRNA expression and HGF levels in a large panel of myeloma patients treated with novel drugs.one hundred and five samples of purified plasma cells derived from newly diagnosed myeloma patients have been included in this study. Fifty two patients received 9 courses of Velcade-Melphalan-Prednisone (VMP) as part of the VMP-VMPT trial (Palumbo A, 2009 ASH Meeting, abs 128) while fifty three patients have been included in the PAD-MEL100-LP-L trial (Palumbo A, JCO 2010). Met and HGF mRNA quantitative expression have been investigated on both purified plasma cells (CD138+ bone marrow fraction) and on bone marrow CD138 negative fraction. mRNA expression has been evaluated using a quantitative Real-Time PCR (qRT-PCR) on Abi Prism 7900 (Applied Biosystems) with a relative quantification based on ΔΔCt approach. JUM2 cell line was used as calibrator and Gus as housekeeping gene. HGF serum level has been evaluated on 76 of those patients too. ELISA assay has been employed to determine HGF serum value. On 51 samples with higher levels of mRNA Met expression, a FISH analysis reaching for Met amplification has been performed. Purified plasma cells were treated with a dual-color FISH assay using a MET/CEP7 probe cocktail (Cytocell, Cambridge, UK).Met mRNA expression was higher in CD138+ cells than in CD138- fractions (median 76,90 range 0,81-916,51 vs 11,03 range 0–243,88 respectively; p=0,0001). Similarly HGF mRNA expression was higher in CD138+ cells than in CD138- population (median 2,07 range 0–65,34 vs 0,49 range 0,11-3,22 respectively; p=0,03). Patients with high and low Met levels were divided using the median value of Met expression as cutoff. At a median follow up of 30 months, patients with low Met mRNA expression displayed a higher progression free survival (PFS) and overall survival (OS) compared with those with high Met levels (PFS 73% vs 42% respectively, p<0,001; OS 94% vs 78%, p=0,01). No differences in baseline albumin, β2-microglobulin, BM plasma cell infiltration and cytogenetic profile have been observed between patients with high and low Met mRNA levels. No differences in outcome were demonstrated when patients were divided using HGF mRNA level as parameter. No correlation between Met and HGF mRNA values have been found. Quantification of HGF levels in serum confirmed the increase of this growth factor in myeloma patients in comparison with age-matched healthy subjects (median 2082 pg/ml range 740–9000 vs median 1586 pg/ml, range 420–3079 respectively; p=0.03). FISH analysis revealed amplifications of Met gene in 20 samples out of 50 (40%). In 13 samples three copies of Met gene have been detected in more than 50% of cells, while in 7 samples 4 or more copies have been found in more than 50% of cells.1) high Met mRNA expression levels identify a group of patients with worse prognosis even when treated with novel drugs containing regiments; 2) Met amplification has been described for the first time in myeloma cells and can determine high Met mRNA levels characterizing a more aggressive disease.Cavallo: Celgene: Honoraria. Patriarca:Roche: Honoraria; Janseen-Cilag: Honoraria; Celgene: Honoraria; Merck: Membership on an entity's Board of Directors or advisory committees. Boccadoro:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janseen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Palumbo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janseen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees.

No relevant conflicts of interest to declare.

Boccadoro: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Massaia:Novartis: Honoraria, Research Funding.

Cavo: Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Off Label Use: Use of bortezomib-based treatment for newly diagnosed multiple myeloma. Petrucci:CELGENE: Honoraria; JANSSEN-CILAG: Honoraria. Boccadoro:NOVARTIS: Honoraria; CELGENE: Honoraria; JANSSEN-CILAG: Honoraria. Palumbo:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, no; Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, no.

Over-expression of the tyrosine kinase receptor Met has been reported in several solid tumors and increases cell proliferation, cell migration (scattering) and neoplastic angiogenesis. The Hepatocyte Growth Factor (HGF) is the only known Met ligand and the HGF/Met pathway seems to play a pivotal role in Multiple Myeloma (MM) pathogenesis. HGF is secreted by bone marrow stromal cells and Met is expressed at the surface of malignant plasma cells and endothelial cells. The phosphorylation of Met induced by HGF is a strong signal for cell growth and migration. The activity of the HGF/Met pathway and the heterogeneity of HGF/Met expression on MM cells suggest that the over-expression of Met could be a prognostic marker in MM patients.To investigate the role of HGF/Met pathway as prognostic marker predictive of response rate, progression-free survival (PFS) and overall survival (OS) in MM patients treated with novel agents.102 newly diagnosed MM patients received the PAD-Mel100-LP-L regimen (Palumbo A, Abs 159, ASH 2008): as induction, four 21-day PAD cycles (Bortezomib, Pegylated-lyposomal-doxorubicin, Dexamethasone); as transplantation, tandem Mel100 (Melphalan 100 mg/m2) followed by stem cell support; as consolidation four 28-day LP cycles (Lenalidomide plus Prednisone) followed by Lenalidomide alone as maintenance. Samples from 47 newly diagnosed patients enrolled in this study have been analyzed. On CD138+ and CD138- cells separated from bone marrow (BM) aspirate, HGF and Met mRNA levels have been evaluated using a quantitative Real-Time PCR (qRT-PCR) on Abi Prism 7900 (Applied Biosystems). The JUM2 cell line was used as a calibrator for relative quantification of mRNA using the σσCt approach and Gus has been used as housekeeping gene. The cut-off value of Met mRNA expression/percentage calibrator was selected according to Receiver Operating Characteristic (ROC) analysis.mRNA expression of Met was higher in CD138+ cells than in CD138- cells (median 103, range 2 – 586 vs median 31, range 0-243 respectively; p=0.002). Met mRNA expression was significantly higher in CD138+ cells of patients with suboptimal response (partial response PR, stable disease SD, progression disease PD), compared with patients achieving complete response (CR) or very good partial remission (VGPR) (median 140 range 27-586 vs median 36.5 range 1-163 respectively; p=0.0001) (FIG 1A). With a median follow-up of 18 months, the 2-year PFS was 54% in patients with high Met mRNA expression and 87% in patients with low Met mRNA expression (p=0.007, FIG 1B); 2-year OS was 72% in patients with high Met mRNA expression and 95% in patients with low Met mRNA expression (p=0.047, FIG 1C). Patients with high levels of Met mRNA displayed albumin and beta-2-microglobuline levels similar to that observed in patients with low levels of Met mRNA. The BM plasma cells infiltration was also comparable (median 61% vs 65%) within the two groups. A similar percentage of patients in both groups showed the following cytogenetic abnormalities: t(4;14), t(14;16), del17 and del13. Interestingly, the percentage of patients carrying t(11;14) was higher in the group of patients with low Met mRNA levels in comparison with patients with high Met mRNA levels (28% vs 6%). mRNA expression of HGF was similar in responders and non responders patients and did not predict PFS and OS.1) mRNA level of Met on CD 138+ cells is a biological marker predictive of response; 2) high level of Met mRNA at diagnosis delineates significantly inferior progression-free and overall survival in MM patients treated with both bortezomib and lenalidomide based regimens.Patriarca: Celgene: Honoraria. Caravita:Celgene: Consultancy. Ladetto:Celgene: Research Funding; Janssen-Cilag: Research Funding. Boccadoro:Celgene: Consultancy, advisory Committees, Research Funding; Janssen-Cilag: Consultancy, advisory Committees, Research Funding; Pharmion: Consultancy, advisory Committees, Research Funding. Palumbo:Celgene: Honoraria; Janssen-Cilag: Honoraria.