Search

Background: Minimal residual disease (MRD) is one of the most relevant prognostic factors in patients with multiple myeloma (MM); however, the impact of maintenance therapy on MRD levels remains unclear. Among patients with newly diagnosed MM (NDMM) who received lenalidomide maintenance until they developed disease progression, the role of MRD status as a predictor of progression-free survival (PFS) was evaluated by multiparameter flow cytometry (MFC) and allelic-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR) analysis. Methods: Seventy-three patients with NDMM enrolled in the RV-MM-EMN-441 (clinical trials.gov identifier, NCT01091831) and RV-MM-COOP-0556 (clinicaltrials.gov identifier, NCT01208766; European Myeloma Network EMN02/HO95 MM Trial) phase 3 trials who achieved at least a very good partial response after intensification/consolidation were included. The median patient age was 57 years (interquartile range, 53-61 years), and all patients received lenalidomide maintenance until they developed progression. MRD was evaluated on bone marrow after intensification/consolidation, after 6 courses of maintenance, and every 6 months thereafter until clinical relapse using both ASO-RQ-PCR (sensitivity, 10 −5 ) and MFC (sensitivity, from 10 −4 to 10 −5 ). Results: After intensification/consolidation, 33 of 72 patients (46%) achieved a molecular complete response (m-CR), and 44 of 70 (63%) achieved a flow complete response (flow-CR). Almost 27% of patients who were MRD-positive after consolidation became MRD-negative during maintenance. After a median follow-up of 38 months, PFS was prolonged in patients who achieved negative MRD status during maintenance according to results from both ASO-RQ-PCR analysis (hazard ratio, 0.29; 95% confidence interval, 0.14-0.62; P =.0013) and MFC (hazard ratio, 0.19; 95% confidence interval, 0.09-0.41; P

We here describe a novel method for MYD88L265P mutation detection and minimal residual disease monitoring in Waldenstrom macroglobulinemia, by droplet digital polymerase chain reaction, in bone marrow and peripheral blood cells, as well as in circulating cell-free DNA. Our method shows a sensitivity of 5.00×10−5, which is far superior to the widely used allele-specific polymerase chain reaction (1.00×10−3). Overall, 291 unsorted samples from 148 patients (133 with Waldenstrom macroglobulinemia, 11 with IgG lymphoplasmacytic lymphoma and 4 with IgM monoclonal gammopathy of undetermined significance) were analyzed: 194 were baseline samples and 97 were followup samples. One hundred and twenty-two of 128 (95.3%) bone marrow and 47/66 (71.2%) baseline peripheral blood samples scored positive for MYD88L265P. To investigate whether MYD88L265P detection by droplet digital polymerase chain reaction could be used for minimal residual disease monitoring, mutation levels were compared with IGH-based minimal residual disease analysis in 10 patients, and was found to be as informative as the classical, standardized, but not yet validated in Waldenstrom macroglobulinemia, IGH-based minimal residual disease assay (r2=0.64). Finally, MYD88L265P detection by droplet digital polymerase chain reaction on plasma circulating tumor DNA from 60 patients showed a good correlation with bone marrow findings (bone marrow median mutational value 1.92×10−2, plasma circulating tumor DNA value: 1.4×10−2, peripheral blood value: 1.03×10−3). This study indicates that droplet digital polymerase chain reaction assay of MYD88L265P is a feasible and sensitive tool for mutation screening and minimal residual disease monitoring in Waldenstrom macroglobulinemia. Both unsorted bone marrow and peripheral blood samples can be reliably tested, as can circulating tumor DNA, which represents an attractive, less invasive alternative to bone marrow for MYD88L265P detection.

Background. Despite the improvement of therapeutic regimens, a relevant proportion of multiple myeloma (MM) patients (pts) experience early relapse (ER) [Majithia, Leukemia 2016] and represents an unmet medical need. It is therefore of high clinical interest to identify baseline factors that may predict ER. Aims To design models predictive of ER (defined as pts with a time-to-progression ≤18 or ≤ 24 months). To assess the accuracy of every model on an independent validation set. To build a score to predict ER. Methods Data were obtained from 2326 pts enrolled in 8 multi-center clinical trials: NCT01093196, NCT01346787, NCT01857115, NCT01190787, NCT00551928, NCT01091831, NCT01063179 and 2005-004730-41. Here, we included 14 baseline features (fts): age, creatinine, albumin (alb), b2microglobulin (b2m), bone marrow plasma cell (PCbm) were evaluated as continuous variables; free light chain (FLC, λ vs K), M-component subtype (IgA vs others), Revised International Staging System (R-ISS stage II/III vs I), lactate dehydrogenase levels >/≤ upper limit of normal (LDHULN), presence vs absence of chromosomal abnormalities detected by FISH [del17p, t(4;14), t(14;16), t(11;14)], and presence of plasmacytomas as categorical values. Trials were assigned to training and validation set to have a superimposed median (μe) age and follow-up in the two subsets. From the training set, a univariate analysis (UV) on outcome was performed according to both Chi-square and Kruskal tests, as appropriate. Features with p Results ER≤18 models. 10/14 fts were selected based on UV analysis: age, FLC, PCbm, del17p, t(4/14), t(14/16), alb and b2m, LDHULN and R-ISS stage. Pts with complete data were included in the training set (n=923; μe age =66 years [y]; ER=35%) and the validation set (n=313; μe age=67 y; ER=36%), respectively. In the MV incorporating the R-ISS, the R-ISS II/III vs I (OR=1.75, 95% CI:1.26-2.44) and increased PCbm (OR=1.05, 95% CI:1.02-1.08) increased the risk of ER. When the MV analysis was performed including single fts instead of R-ISS, increased PCbm (OR=1.05, 95% CI:1.02-1.08), λ FLC (OR=1.34, 95% CI:1.01-1.79), LDHULN (OR=1.80, 95% CI:1.16-2.81), presence of both del17q (OR=1.58, 95% CI:1.07-2.33) and t(4/14) (OR=2.01, 95% CI:1.36-3.01) increased the probability of ER; increased alb (OR=0.75, 95% CI:0.61-0.94), reduced the risk of ER (table 1). ER≤24 models. 8/14 fts were selected based on UV analysis: FLC, PCbm, del17p, t(4/14), alb and b2m levels, LDHULN and R-ISS stage. Pts with complete data were included in the training set (n=1009; μe age=67 y; ER=45%) and in the validation set (n=352; μe age=67 y, ER=45%). In MV analysis, including R-ISS, both R-ISS (OR=1.88, 95% CI:1.39-2.55) and PCbm (OR=1.04, 95% CI:1.02-1.06) impacted on outcome. When the MV analysis was performed including single fts, λ FLC (OR=1.31, 95% CI:1.01-1.69), PCbm (OR=1.04, 95% CI:1.02-1.07), del17p (OR=1.86, 95% CI:1.30-2.65) and t(4/14) (OR=2.00, 95% CI:1.38-2.88) retained their impact on outcome (table 1). Validation. Each MV model was tested on the validation set. Among the 4 MV models, the ER≤18 incorporating individual fts resulted in the highest AUC (0.66) and was therefore used to build up a prognostic score. Score. The ER score was calculated as 0.047 × PCbm + 0.589 × LDHULN + 0.459 × del17q (IF present) + 0.705 × t(4/14) (IF present) + 0.293 × FLC (IF FLC =λ) - 0.284 × alb. The ER score was calculated as 3 groups of pts with different risks of ER: L (42% of pts; risk of ER18=23%), M (33% of pts; risk of ER18=39%) and H (26% of pts; risk of ER18 =55%). Discussion. This is the first analysis proposing a score that includes standard baseline fts and aims at identifying pts at high risk of ER in the context of novel agent-based therapy. Based on our score, 26% of pts can be defined at high risk. To improve the clinical applicability, the construction of a simplified model with categorized variables is ongoing. Disclosures Petrucci: Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; 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. Offidani:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Di Raimondo:Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy. Liberati:Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Clinical trial support; Roche: Other: Clinical trial support; Novartis: Other: Clinical trial support; Janssen: Honoraria; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Clinical trial support; Celgene: Honoraria, Other: Clinical trial support; Bristol-Myers Squibb: Honoraria; Takeda: Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy. Omedé:Janssen: Membership on an entity's Board of Directors or advisory committees. Mannina:Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy. Caravita di Toritto:Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel and accomodation costs, Research Funding; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees, Other: Travel and accomodation costs; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Travel and accomodation costs; Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Patriarca:Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Benevolo:Novartis Pharmaceuticals: Consultancy. Belotti:Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Gaidano:AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astra-Zeneca: Consultancy, Honoraria; Sunesys: Consultancy, Honoraria. Hajek:Amgen: Honoraria, Other: Consultant or advisory relationship, Research Funding; Celgene: Honoraria, Other: Consultant or advisory relationship, Research Funding; AbbVie: Other: Consultant or advisory relationship; Bristol-Myers Squibb: Honoraria, Other: Consultant or advisory relationship, Research Funding; Novartis: Other: Consultant or advisory relationship, Research Funding; PharmaMar: Honoraria, Other: Consultant or advisory relationship; Takeda: Honoraria, Other: Consultant or advisory relationship, Research Funding; Janssen: Honoraria, Other: Consultant or advisory relationship, Research Funding. Spencer:Sanofi: Other: Consulting/advisory role; Specialised Therapeutics Australia: Consultancy, Honoraria; Amgen: Other: Consulting/advisory role, Research Funding; AbbVie: Other: Consulting/advisory role, Research Funding; Haemalogix: Other: Consulting/advisory role; Janssen Oncology: Other: Consulting/advisory role, Research Funding, Speakers Bureau; Servier: Other: Consulting/advisory role; Secura Bio: Other: Consulting/advisory role; Takeda: Other: Consulting/advisory role, Research Funding; Celgene: Other: Consulting/advisory role, Research Funding, Speakers Bureau. Sonneveld:Amgen: Honoraria, Research Funding; BMS: Honoraria; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Karyopharm: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; SkylineDx: Research Funding. Boccadoro:Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; AbbVie: Honoraria; Mundipharma: Research Funding; Sanofi: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Gay:AbbVie: Membership on an entity's Board of Directors or advisory committees; 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; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: The presentation includes discussion of off-label use of a drug or drugs for the treatment of multiple myeloma.

Background Multiple Myeloma (MM) is a neoplastic disorder characterized by clonal proliferation of malignant plasma cells (PCs). Flow cytometry is an essential tool to confirm diagnosis and evaluate minimal residual disease (MRD). This study aims at identifying new surface PC markers suitable for targeted therapy in MM and able to improve MRD detection. Methods The expression of 82 molecules provided by the “Ninth International Workshop on Leukocyte Antigens” was analyzed by flow cytometry in 5 MM cell lines and in 20 newly diagnosed MM (NDMM) patients. Based on the antigens expression and monoclonal antibody availability, CD150, CD48, CD229, CD352, CD319, CD272, CD86, CD200 and CD184 were subsequently tested in 24 NDMM, 8 relapsed MM (RMM), 6 plasma cell leukemia (PCL) and 13 healthy subjects. Results CD352 was less frequently expressed on NDMM than on healthy PCs; CD200 was more frequently expressed on NDMM than on RMM and healthy PCs. CD150, CD319, CD229, CD352 Mean Fluorescence Intensity (MFI) was lower in pathological than in healthy samples. The proportion of CD150-positive samples was lower in NDMM and RMM than in healthy subjects; CD86+ samples were less frequent in NDMM than in healthy subjects; CD200+ samples were more frequent in NDMM than in RMM and healthy subjects. Conclusions CD150, CD86 and CD200 can help to identify malignant PCs; CD272, CD319, CD229, CD48 are highly expressed on all PCs and could be considered for targeted therapy. All these antigens could be added to a routine panel for PCs identification and MRD evaluation. © 2015 International Clinical Cytometry Society

// Stefania Oliva 1, * , Manuela Gambella 1, * , Milena Gilestro 1 , Vittorio Emanuele Muccio 1 , Francesca Gay 1 , Daniela Drandi 2 , Simone Ferrero 2 , Roberto Passera 3 , Chiara Pautasso 1 , Annalisa Bernardini 1 , Mariella Genuardi 1 , Francesca Patriarca 4 , Elona Saraci 1 , Maria Teresa Petrucci 5 , Norbert Pescosta 6 , Anna Marina Liberati 7 , Tommaso Caravita 8 , Concetta Conticello 9 , Alberto Rocci 10 , Pellegrino Musto 11 , Mario Boccadoro 1 , Antonio Palumbo 1 , Paola Omede 1 1 Myeloma Unit, Division of Hematology, University of Torino, Azienda Ospeadliero-Universitaria Citta della Salute e della Scienza di Torino, Torino, Italy 2 Division of Hematology, Department of Molecular Biotechnologies and Health Sciences, University of Torino, Torino, Italy 3 Division of Nuclear Medicine, University of Torino, Azienda Ospedaliero-Universitaria Citta della Salute e della Scienza di Torino, Torino, Italy 4 Azienda Ospedaliera-Universitaria di Udine, DISM Universita di Udine, Udine, Italy 5 Division of Hematology, Department of Cellular Biotechnologies and Hematology, Sapienza University of Rome, Rome, Italy 6 Ematologia e Centro TMO, Ospedale Centrale Bolzano, Bozen, Italy 7 AO S.Maria di Terni, SC Oncoematologia, Terni, Italy 8 UOC Ematologia S.Eugenio ASL RM2 Roma, Rome, Italy 9 Divisione di Ematologia, Azienda Policlinico-OVE, Universita di Catania, Catania, Italy 10 Department of Haematology, Manchester Royal Infirmary, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK 11 Scientific Direction, IRCCS-CROB, Referral Cancer Center of Basilicata, Rionero in Vulture (Pz), Italy * Both authors share first authorship of the manuscript Correspondence to: Paola Omede, email: paolaomede@yahoo.com Keywords: myeloma, MRD, ASO-RQ-PCR, novel agents, flow cytometry Received: June 11, 2016 Accepted: September 21, 2016 Published: October 13, 2016 ABSTRACT We analyzed 50 patients who achieved at least a very good partial response in the RV-MM-EMN-441 study. Patients received consolidation with autologous stem-cell transplantation (ASCT) or cyclophosphamide-lenalidomide-dexamethasone (CRD), followed by Lenalidomide-based maintenance. We assessed minimal residual disease (MRD) by multi-parameter flow cytometry (MFC) and allelic-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR) after consolidation, after 3 and 6 courses of maintenance, and thereafter every 6 months until progression. By MFC analysis, 19/50 patients achieved complete response (CR) after consolidation, and 7 additional patients during maintenance. A molecular marker was identified in 25/50 patients, 4/25 achieved molecular-CR after consolidation, and 3 additional patients during maintenance. A lower MRD value by MFC was found in ASCT patients compared with CRD patients (p=0.0134). Tumor burden reduction was different in patients with high-risk vs standard-risk cytogenetics (3.4 vs 5.2, ln-MFC; 3 vs 6 ln-PCR, respectively) and in patients who relapsed vs those who did not (4 vs 5, ln-MFC; 4.4 vs 7.8 ln-PCR). MRD progression anticipated clinical relapse by a median of 9 months while biochemical relapse by a median of 4 months. MRD allows the identification of a low-risk group, independently of response, and a better characterization of the activity of treatments.

L’emoglobinuria parossistica notturna (EPN) e un disordine ematologico raro caratterizzato da anemia emolitica, trombosi e difetti dell’ematopoiesi. Tale patologia e dovuta a una mutazione somatica del gene PIG-A che comporta la mancata produzione del glicosil-fosfatidil-inositolo (GPI), a cui si ancorano proteine di membrana quali CD55 e CD59 che inibiscono l’attivazione del complemento su diversi tipi cellulari. Dal 2010 il nostro Laboratorio partecipa alla compilazione dell’archivio CLONEPnh. Integrando i dati in esso inseriti con quelli contenuti nell’archivio del nostro Laboratorio e stato valutato il cambiamento negli anni del numero di richieste di ricerca del clone EPN e, in relazione a esso, come e variato il numero dei nuovi cloni individuati. E stata effettuata un’analisi citofluorimetrica a sei colori su sangue periferico, di pazienti afferenti alla Divisione Universitaria di Ematologia dell’Azienda Ospedaliera Citta della Salute e della Scienza di Torino, utilizzando il FLAER (tossina batterica inattivata) e i seguenti anticorpi: CD45, CD14, CD33, CD66, CD55, CD59, CD235a. I campioni sono stati acquisiti con citofluorimetro FACSCantoII e analizzati con il software FACS Diva. I dati sono stati inseriti nell’archivio CLONEPnh ( www.CLONEPnh.com ). Dai dati inseriti nell’archivio CLONEPnh si evince che le richieste di ricerca di EPN sono state 13 nel 2010, 16 nel 2011, 28 nel 2012 e 12 nei primi sei mesi del 2013 e i cloni individuati sono stati, rispettivamente, 1, 0, 1 e 1. Un andamento simile, anche se con un numero decisamente minore di analisi richieste, e stato riscontrato anche negli anni precedenti (dal 2000) analizzando l’archivio del nostro Laboratorio. I nostri dati mostrano un aumento negli anni di analisi effettuate, a cui, pero, non e corrisposto un aumento dei cloni individuati. L’aumento delle richieste si puo giustificare considerando che la diagnosi di EPN e diventata prevalentemente citofluorimetrica e che le recenti linee guida consigliano, in pazienti con displasie midollari, di ricercare e monitorare cloni di modesta entita mediante la metodica ad alta sensibilita. Tuttavia, per ridurre il costo in termini di tempo e risorse, sarebbero auspicabili l’elaborazione e la validazione di protocolli citofluorimetrici piu rapidi con l’utilizzo di un pannello ridotto di reagenti.

Background: In multiple myeloma (MM), different clinical parameters and molecular prognostic factors can predict disease course and response to therapy. The classification of myeloma patients includes laboratory parameters associated with higher tumor activity, resistance to therapy and proliferative competence. Tumor circulating plasma cells (TCPC) in MM patients showed a strong correlation with a more aggressive disease. Aim: For the first time, we quantified the amounts of TCPC with single platform flow cytometric method and evaluated their relationship with patients' baseline characteristics and response to therapy before maintenance. Methods: Whole peripheral blood samples from 413 newly diagnosed MM patients ≤65 years enrolled in the UNITO-MM-01/FORTE trial were collected. Patients were randomized [1:1:1; stratification: International Staging System (ISS) and age] to ARM A: carfilzomib-cyclophosphamide-dexamethasone (KCyd) followed by melphalan 200 mg/m2 and autologous stem-cell transplantation (MEL200-ASCT) and consolidation with 4 KCyd; ARM B: carfilzomib-lenalidomide-dexamethasone (KRd) followed by MEL200-ASCT and 4 KRd; ARM C: 12 KRd cycles. Enrollment was completed in March 2017; data cut-off was November 30, 2018. For the single platform tube, the antibody combination CD38PC7/CD138PC5.5/ CD45KO/CD56PE/CD19PB was mixed with 100µL of EDTA peripheral blood, dispensed with reverse pipetting, and incubated for 15 min, added with 500µL of lysing solution and, after 15 min, 100µL of flow count fluorospheres were dispensed with reverse pipetting and cells acquired with Navios flow cytometer. Intracytoplasmic tube was set up to confirm the clonality of CPC. Results: Circulating plasma cells (CPC) were quantified in 413 samples, with median values of 0.03% (range: 0-51%) and 2.37/mm3 (range: 0-6272/mm3). White blood cells were 5710/mm3 (range: 1752-26102/mm3); total events acquired 1285000 (range: 40000-2000000); median CPC events were 58 (range: 0-441000); cellular events acquired were 190000 (range: 4428-1300000). In 390 out of 413 samples (94.4%), CPC were detected; 272 samples (66%) showed TCPC with a median of 1.24/mm3 (range 0.06- 6272/mm3). Patients were sorted according to different baseline characteristics and the medians of absolute TCPC were compared. The most statistically significant differences (p5.5mg/dL (16.47/mm3); lactate dehydrogenase (LDH) ≤upper limit of normal (ULN, 1.14/mm3) vs. >ULN (7.36/mm3); plasma cells (PC) in biopsy Finally, we compared the absolute number of TCPC and the quality of response at the end of consolidation therapy. Higher values of TCPC were related to worst response: Conclusions: Single-platform flow cytometry is a simple method to quantify TCPC, present in almost all peripheral blood from MM patients; a high number is related to poor clinical response to therapy and helps in identifying high-risk patients. Moreover, it allows the discrimination between normal and pathological plasma cell population in peripheral blood. However, a longer follow up is needed to evaluate how TCPC can affect survival in patients with MM. Disclosures Musto: Amgen: Honoraria; Celgene: Honoraria. Gay:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Amgen: 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; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Boccadoro:Sanofi: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; AbbVie: Honoraria; Mundipharma: Research Funding. Omedé:Janssen: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: This presentation includes discussion of off-label use of a drug or drugs for the treatment of multiple myeloma.

INTRODUCTION Post-transplant cyclophosphamide (PTCY) has expanded the application of haploidentical stem cell transplantation (haplo-HSCT). Thymic function may play a pivotal role in long-term clinical outcomes. OBJECTIVES To evaluate the kinetics of long-term immune thymus-dependent reconstitution after PTCY haplo-HSCT. METHODS Twenty-nine patients (median age 53) underwent T-cell replete haplo-HSCT with PTCY. Blood samples were collected before conditioning and at 1, 3, 6, 12, 18, 24 months after transplantation. Analyses of CD4 and CD8 T-cell subsets by flow-cytometry were correlated by generalized linear models with Real-Time PCR quantification of signal joint T-cell receptor excision DNA circles (sjTRECs), specific marker of naive T-cells thymopoiesis. A) Naive; b) central; c) memory; and d) revertant CD4 and CD8 T-cells were defined as follows: a) CD45RA+CD62L+; b) CD45RO+CD62L+; c) CD45RO+CD27-; and d) CD45RA+/45RO+, respectively. SjTRECs real-time PCR was performed on genomic DNA (100 ng) extracted from sorted CD4 and CD8 T-cells. RESULTS Following PTCY induced T-cell depletion, a constant gradual increase in absolute numbers of all CD4 and CD8 T cell subsets and of sjTRECs copies from the first month up to two years post-transplant was observed (Figure 1). Overall, at two years, CD4 and CD8 T-cell levels and sjTRECs levels were however lower than those observed in healthy donors. sjTRECs kinetics was associated with the increase in naive T-cells (overall, p CONCLUSIONS Active thymic function despite age-dependent involution, substantially contributes to T-cell reconstitution after haplo-HSCT. Chronic GVHD and older age are however significantly correlated with lower thymic activity. Overall, lower production of sjTRECs after haplo-HSCT as compared after HLA identical sibling HSCT may partly be due to a higher degree of “mismatching” of MHC molecules during thymic re-education.

Multiple Myeloma (MM) is a neoplastic disorder characterized by clonal proliferation of malignant plasma cells (PCs). Flow cytometry is an essential tool to confirm diagnosis and evaluate minimal residual disease (MRD). This study aims at identifying new surface PC markers suitable for targeted therapy in MM and able to improve MRD detection.The expression of 82 molecules provided by the "Ninth International Workshop on Leukocyte Antigens" was analyzed by flow cytometry in 5 MM cell lines and in 20 newly diagnosed MM (NDMM) patients. Based on the antigens expression and monoclonal antibody availability, CD150, CD48, CD229, CD352, CD319, CD272, CD86, CD200 and CD184 were subsequently tested in 24 NDMM, 8 relapsed MM (RMM), 6 plasma cell leukemia (PCL) and 13 healthy subjects.CD352 was less frequently expressed on NDMM than on healthy PCs; CD200 was more frequently expressed on NDMM than on RMM and healthy PCs. CD150, CD319, CD229, CD352 Mean Fluorescence Intensity (MFI) was lower in pathological than in healthy samples. The proportion of CD150-positive samples was lower in NDMM and RMM than in healthy subjects; CD86+ samples were less frequent in NDMM than in healthy subjects; CD200+ samples were more frequent in NDMM than in RMM and healthy subjects.CD150, CD86 and CD200 can help to identify malignant PCs; CD272, CD319, CD229, CD48 are highly expressed on all PCs and could be considered for targeted therapy. All these antigens could be added to a routine panel for PCs identification and MRD evaluation.

Conflicting data have been reported about the frequency and function of regulatory T cells in multiple myeloma. Most studies have investigated peripheral blood rather than bone marrow Tregs and side-by-side comparisons with bone marrow from healthy donors have still not been made. In this study, we show that regulatory T-cells total count, subset distribution, and expression of chemokine receptors are similar in the bone marrow of myeloma patients and healthy donors. Regulatory T cells are not recruited by myeloma cells in the bone marrow and their counts are unaffected by the tumor burden and the disease status. The diversity of T-cell receptor repertoire is highly preserved ensuring broad reactivity and effective suppressor function. Our results indicate that regulatory T cells may not be the main players of immunological tolerance to myeloma cells under base-line conditions, but their fully preserved immune competence may promote their inadvertent activation and blunt T-cell driven anti-myeloma immune interventions even after myeloma cells have successfully been cleared by chemotherapy.

Introduction: Multiple myeloma (MM) is still an incurable disease and patients may relapse despite achievement of complete remission (CR). Minimal residual disease (MRD) assessment by multiparameter flow cytometry (MFC) on bone marrow (BM) is a sensitive diagnostic tool to measure response and is highly predictive of outcome in MM as previously reported. The aim of this study is to evaluate the role of MRD monitoring by MFC in MM patients receiving novel agents with or without autologous stem cell transplantation (ASCT) and to investigate the efficacy of treatments by using MRD-negativity as a deeper response criteria. Methods: The RV-MM-COOP-0556 (EMN02/HO95 MM) study is a phase III, randomized, trial including newly diagnosed MM patients ≤ 65 years. All subjects received 4 cycles of Bortezomib-Cyclophosphamide-Dexamethasone (VCD) induction, followed by Cyclophosphamide chemotherapy and subsequent stem cell mobilization and collection. Afterward, patients were randomized to receive 4 cycles of Bortezomib-Melphalan-Prednisone (VMP) vs one or two cycles of High-Dose-Melphalan (HDM) followed by ASCT. After intensification patients were secondly randomized to receive two cycles of consolidation with Bortezomib-Lenalidomide-Dexamethasone (VRD) vs no consolidation, followed by Lenalidomide maintenance in both arms. Patients who achieved CR/sCR according to IMWG criteria (Rajkumar et al. Blood 2011) after intensification/consolidation treatment, were eligible for the MRD sub-study. MRD analysis by MFC was performed on BM samples after intensification/consolidation, after 6 courses of maintenance, and thereafter every 6 months until progression, to detect monoclonal plasma cells (PCs). Here, we used a double antibody combination (CD138Fitc/CD20PerCp-Cy5.5/CD117APC/CD45APC-H7/CD38PE-Cy7; cyKappaFitc/cyLambdaPE/CD19PerCp-Cy5.5/CD56APC/CD45APC-H7/CD38 PE-Cy7): one tube was employed to obtain PCs quantification, the other one to validate PCs clonality. MRD-negativity was defined when Results: One hundred-eleven Italian patients (58 male/53 female) with a median age of 56 years (IQR 52-62) entered MRD sub-study. Sixteen (14%) were ISS stage III, 24 (22%) had high-risk cytogenetic profile and 10 (9%) had LDH levels higher than the upper normal limit. Forty-five patients (40%) received VMP as intensification and 66 (60%) underwent ASCT, thereafter 65 (58%) received VRD consolidation, 24 after VMP and 41 after ASCT. The median follow-up were 28.7 and 17 months from starting treatment and from MRD enrollment, respectively. After intensification/consolidation, 4 patients were not evaluable for MRD due to unsuitable samples, MRD negativity rate was 79% (85 out of 107 evaluable patients) and was independent from the intensification therapy: actually 50/63 patients who received ASCT and 35/44 patients who received VMP achieved MRD negativity. Within MRD-negative patients, 48/85 (56%) received VRD consolidation without major differences between VMP and ASCT. With the limitation related to the shorter follow-up, depth of response further improved during maintenance: 11/22 (50%) of MRD-positive patients became MRD-negative, independently from previous intensification therapy. Conclusions: MRD detection by MFC is a feasible technique in MM and allows to detect residual tumor cells among CR and sCR patients. Preliminary MRD results show that, in patients achieving CR, intensification with VMP or ASCT induced comparable rates of MRD-negativity and maintenance with Lenalidomide further improved depth of response in both arms. Longer follow-up is needed to correlate MRD status to prognosis and clinical outcome and to evaluate the role of maintenance therapy in increasing the quality of response. Disclosures Off Label Use: Use off-label of drugs for the dose and/or schedule and/or association . Gay:Sanofi: 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-Cilag: Honoraria. Larocca:Janssen-Cilag, Celgene: Honoraria. Caravita:Celgene: Honoraria. Gamberi:Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Rossi:Celgene: Research Funding. Offidani:Janssen-Cilag, Celgene, Sanofi, Amgen, Mundipharma: 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. Sonneveld:Janssen-Cilag, Celgene, Onyx, Karyopharm: Honoraria, Research Funding; novartis: Honoraria. Palumbo:Novartis, Sanofi Aventis: Honoraria; Celgene, Millennium Pharmaceuticals, Amgen, Bristol-Myers Squibb, Genmab, Janssen-Cilag, Onyx Pharmaceuticals: Consultancy, Honoraria.

Background. Minimal residual disease (MRD) detection by multi-parameter flow cytometry (MFC) and real time quantitative PCR (RQ-PCR) is highly predictive of outcome in multiple myeloma (MM) patients (pts). Less is known on the ability of maintenance therapy to modulate MRD levels. The primary end-point of this study was to monitor MRD during maintenance therapy and to evaluate the impact on outcome. Patients and Methods. In the RV-MM-EMN-441 study (NCT01091831), after induction and consolidation pts received maintenance therapy with either Lenalidomide alone (R) or Lenalidomide-Dexamethasone (RD) until progression. Pts achieving at least very good partial response (VGPR) after induction/consolidation treatment were eligible for the MRD sub-study. MRD analysis was performed on bone marrow (BM) samples at diagnosis, after consolidation, after 3 and 6 courses of maintenance, and thereafter every 6 months until progression. MFC complete remission (CR) was defined by Results. MRD sub-study included 50 pts with a median age of 57 years (range 40-65). After consolidation 34/50 (68%) pts achieved VGPR, 16/48 (32%) achieved CR according to IMWG criteria (Rajkumar et al. Blood 2011). After a median follow-up of 44 months, 22/50 (44%) progressed and 7/50 (14%) deaths were recorded, with a 4-year PFS of 49% and 4-year OS of 87%. In the MFC analysis 19/50 pts (38%) achieved MFC-CR after consolidation, while other additional 7/50 pts (14%) achieved MFC-CR during maintenance. Among pts who achieved MFC–CR (< 1E-04), 7/26 (27%) pts progressed after a median of 30 months; among those who did not reach MFC–CR, 15/24 (62%) pts progressed after a median of 26 months (p=0.009). In 18/19 pts MFC-progression anticipated clinical relapse of a median of 9 months. In 1/19 pts, clinical extra-medullary progression anticipated MFC-progression. A molecular marker was identified in 25/50 pts (50%); 4/25 pts (16%) achieved molecular-CR after consolidation, while other additional 3/25 pts (20%) achieved molecular-CR during maintenance. Among pts who achieved molecular–CR ( Conclusions. Lower MRD values, both with MFC and RQ-PCR procedures, predict better outcome. 30% of patients achieved MFC-CR (7/26) or molecular-CR (3/7) during maintenance therapy suggesting that MRD levels can be modified by maintenance. MRD progression anticipates clinical relapse of approximately 8 months. Disclosures 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.

Abstract 446 Background: The outcome of myeloma (MM) patients who are no longer responding to thalidomide, lenalidomide or bortezomib is poor, with a median event-free survival of 5 months and median overall survival (OS) of 9 months (Kumar SK et al, Leukemia 2012). The newer immunomodulatory drug pomalidomide, has shown significant activity in these clinical conditions. Aims: We assessed dosing, efficacy and safety of pomalidomide-cyclophosphamide-prednisone (PCP) in MM patients relapsed/refractory to lenalidomide. Methods: Pomalidomide was administered in doses ranging from 1 to 2.5 mg/day on days 1–28, cyclophosphamide at 50 mg every other day on days 1–28 and prednisone at 50 mg every other day on days 1–28 for 6 cycles, followed by maintenance therapy with pomalidomide-prednisone. Thromboprophylaxis with aspirin 100 mg/day or low-molecular weight heparin was recommended at physician's discretion. Results: The maximum tolerated dose (MTD) of pomalidomide was defined as 2.5 mg/day. Fifty-two patients were enrolled at the MTD and evaluated after completing at least 1 PCP cycle. Median age was 69 years (range 41–83). The median time from diagnosis to enrolment was 55 months (range 15–203). Best responses to PCP included 6% of complete response (CR), 19% of at least very good partial response (VGPR), 54% of at least partial response (PR) and 75% of at least minimal response (MR). Time to PR was rapid (median 1.8 months). After a median follow-up of 11 months (range 1–18), 1-year progression-free survival (PFS) and OS rates were 52% and 78%, respectively. PFS was not significantly different in patients with high-risk cytogenetic compared with patients with standard-risk disease and in patients younger or older than 75 years. Toxicities were primarily hematologic and included grade 4 neutropenia (13%) and thrombocytopenia (4%). At least grade 3 non-hematologic toxicities included infections (8%), rash (6%) and neurologic (6%). Thromboembolism occurred in 1 patient. Four patients discontinued treatment for toxicity (2 infections, 1 neurologic and 1 hepatic toxicity). Conclusions: PCP induced high response rates and prolonged PFS after prior exposure to lenalidomide and bortezomib (Table), without adding significant toxicity. PCP could be considered a valuable salvage option for pre-treated MM patients. Disclosures: Palumbo: Celgene: Consultancy, Honoraria. Larocca:Celgene: Honoraria. Sciacca:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Guglielmelli:Celgene: Honoraria. Giuliani:Celgene: Research Funding. Boccadoro:Celgene: Consultancy, Honoraria.