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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

Background: The prognostic utility of minimal residual disease (MRD) analysis in multiple myeloma (MM) patients has been well described in the last few years. The role of prolonged maintenance therapy even in persistent MRD negative patients is still unclear. The aim of this study is to evaluate the role of MRD by allelic-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR) as predictor of progression-free survival (PFS) in newly diagnosed MM (NDMM) patients receiving Lenalidomide maintenance after frontline treatment. Patients and Methods: NDMM patients enrolled in the RV-MM-EMN-441 (NCT01091831) and the RV-MM-COOP-0556 (EMN02/HO95 MM) phase III trials achieving ≥ very good partial response (VGPR) after consolidation/intensification were included in the pooled MRD molecular analysis. After induction therapy, patients in the RV-MM-EMN-441 study were randomized to Cyclophosphamide-Lenalidomide-Dexamethasone (CRD) or Autologous Stem Cell Transplantation (ASCT); patients in the RV-MM-COOP-0556 were randomized to Bortezomib-Melphalan-Prednisone (VMP) vs ASCT (Gay F et al Lancet Oncol 2016, Cavo M et al J Clin Oncol 34, 2016 abstr 8000). All patients received Lenalidomide maintenance until progression or intolerance. MRD analysis was performed on bone marrow (BM) aspirates after intensification/consolidation, after 6 courses of maintenance and then every 6 months until clinical relapse. Patient-specific IgH rearrangements were amplified and directly sequenced from genomic DNA at diagnosis. IgH-based MRD detection by ASO-RQ-PCR was performed using an AbiPrism7900HT.MRD analysis was interpreted following the Euro-MRD guidelines(van der Velden VH et al. Leukemia 2007). Molecular-CR (m-CR) was defined as two consecutive negative MRD results by ASO-RQ-PCR with minimal sensitivity of 10−5. PFS was analyzed using the Kaplan-Meier method, curves were compared with the log-rank test. Multivariate Cox model was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). Results: a total of 105 patients entered the molecular MRD pooled study: a specific IgH molecular marker was identified in 73 patients (70%), 32 (30%) did not obtain a successful sequencing. Median age was 57 years (37-65); 30 (41%) patients had International Staging System (ISS) stage I, 33 (45%) stage II and 10 (14%) stage III. FISH risk profile was standard in 43 (59%) patients, high in 24 (33%) and not available in 6 (8%). Thirty-eight (52%) patients did not receive ASCT consolidation and 35 (48%) underwent ASCT. After consolidation/intensification 33/73 (45%) patients achieved m-CR: 19/35 (54%) ASCT patients and 14/38 (37%) no ASCT patients. The impact of m-CR on outcome after consolidation was explored: after a median follow-up of 44 months, median PFS was 48.8 months versus not reached in no m-CR vs m-CR patients, respectively (p=0.01). Lenalidomide maintenance further improved depth of MRD response: 11/40 (27%) MRD positive patients after consolidation obtained a m-CR during maintenance and a median of 2 natural logarithms of tumor burden reduction was recorded. In multivariable Cox analysis the risk of progression/death was higher for ISS stage II/III versus I (HR, 2.91, CI: 1.01-8.41, p=0.048), high-risk FISH versus standard-risk (HR, 2.23 CI: 0.81-6.10, p=0.12), age > 60 years versus ≤60 years (HR: 3.55, CI: 1.26-10.04, p=0.017) and patients who did not achieve m-CR during treatment versus patients who did (HR, 7.65 CI: 2.77-21.11, p We identified a very high risk group defined by high risk FISH at diagnosis and persistent MRD positivity, with a median PFS of 29.4 months (figure1). Conclusions: MRD status by ASO-RQ-PCR is a predictor of outcome significantly superior to standard risk factors in NDMM patients and the achievement of m-CR seems to overcome the high risk FISH status in PFS analysis. Molecular CR rate and reduction of tumor burden obtained after consolidation can be enhanced with Lenalidomide maintenance. Based on these preliminary results, the assessment and monitoring of MRD should be suggested as a better prognostic indicator than CR, and the potential role of a MRD-guided therapy should be investigated in future prospective trials. Figure 1 PFS of patients stratified by MRD status (molecular-CR vs no molecular-CR) and FISH (high risk vs standard risk) Figure 1. PFS of patients stratified by MRD status (molecular-CR vs no molecular-CR) and FISH (high risk vs standard risk) Disclosures Oliva: Celgene: Honoraria; Takeda: Honoraria; Amgen: Honoraria. Larocca:Amgen, Celgene, BMS, Janssen-Cilag: Honoraria. Offidani:Janssen: Honoraria; Celgene: Honoraria, Research Funding. Palumbo:Janssen Cilag: Honoraria; Takeda: Employment, Honoraria. Boccadoro:Sanofi, Celgene, Amgen, Janssen, Novartis, Abbivie, BMS: Honoraria; Celgene, Janssen, Amgen, BMS, Mundipharma, Novartis, Sanofi: Research Funding.

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.

Background. Recently, the somatic MYD88L265P mutation 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, MYD88L265P might 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 MYD88L265P allele-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 MYD88L265P screening; 2) to evaluate whether MYD88L265P might be a suitable marker for MRD monitoring in WM. Methods. Bone marrow (BM) and PB samples were collected at diagnosis and during follow-up from a local series of patients affected by WM, IgM MGUS and IgG-secreting lymphoplasmacytic lymphoma (LPL), as well as samples from healthy subjects and MM were used as negative controls. Genomic (gDNA) and cell-free DNA (cfDNA) were extracted as recommended (Qiagen). MYD88L265P was assessed on 100 ng of gDNA by ASqPCR as previously described [Xu 2013] and by ddPCR, using a custom dual labelled probe assay (Bio-Rad). When available, 50 ng of cfDNA were tested for MYD88L265P, only by ddPCR. ddPCR was performed on 20 µl of reaction at 55°C for 40 cycles, run on QX100 droplet reader and analyzed by QuantaSoft v1.6.6 (Bio-Rad). MYD88L265P ASqPCR level was estimated as described [Treon 2012]. ΔCT Results. Once the ddPCR assay was optimized, the sensitivity of MYD88L265P ddPCR was compared to ASqPCR on a ten-fold serial dilution standard curves built with a 70% MYD88L265P mutated WM sample, previously identified by Sanger sequencing [Treon 2012]. Whereas ASqPCR confirmed the reported sensitivity of 1.00E−03, ddPCR reached a sensitivity of 5.00E−05. Thereafter, overall 105 samples (48 BM, 57 PB, 52 diagnosis and 53 follow up) from 58 patients (49 WM, 5 IgM MGUS and 4 LPL) as well as 20 controls (15 healthy subjects and 5 MM) were tested by both methods. 32/33 (97%) diagnostic BM scored positive for MYD88L265P by both ddPCR and ASqPCR (being the only one negative a WM), while ddPCR, was able to detect more mutated cases, than ASqPCR, among diagnostic PB samples: 15/19 (79%) vs 9/19 (47%) (Table1). Moreover, to investigate whether the MYD88L265P ddPCR tool could be used for MRD detection we compared it to the standardized IGH-based MRD. An IGH-based MRD marker was found in 40/53 (75%) patients (37 WM and 3 LPL). Five Patients, so far analyzed, with baseline and follow up samples (18 BM, 5 PB) showed highly superimposable results between the two methods. Finally, pivotal results on cfDNA from 10 patients showed higher median levels of MYD88L265P mutation in plasma if compared to PB. Conclusions. We developed a new tool for diagnosis and MRD monitoring in WM, showing that: 1) ddPCR is a highly sensitive tool for MYD88L265P detection, especially useful in low infiltrated samples, like PB; 2) MYD88L265P can be effectively and easily used for MRD monitoring in WM, achieving similar results to standardized IGH-based MRD; 3) cfDNA recovered from plasma might be an attractive alternative for MYD88L265P detection, deserving further investigation. Methodological validation against IgH-based MRD detection and Flow cytometry and correlations with clinical impact are currently ongoing on external samples series. Table 1.PATIENTSWM (45)LPL (2)IgM MGUS (5)TISSUEBMPBBMPBBMPBSAMPLES31141114MYD88L265P ddPCR/ASqPCR30/3011/71/10/01/14/2 TABLE 1. MYD88L265P mutation detection in diagnostic samples: ddPCR vs ASqPCR Disclosures 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: In the recent years, numerous studies based on multicolor flowcytometry have analyzed the different subpopulations of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) (Manz MG et al, PNAS 2002; Majeti R et al, Cell Stem Cell 2007): the common myeloid progenitors (CMPs: Lin-CD34+CD38+CD45RA-CD123+), the granulocyte-macrophage progenitors (GMPs: Lin-CD34+CD38+CD45RA+CD123+) and the megakaryocyte-erythroid progenitors (MEPs: Lin-CD34+CD38+CD45RA-CD123-) constitute the progenitor compartment, while the hematopoietic stem cells (HSCs: Lin-CD34+CD38- CD45RA-CD90+), the multipotent progenitors (MPPs: Lin-CD34+CD38- CD45RA-CD90-) and the lymphoid-myeloid multipotent progenitors (LMPPs: Lin-CD34+CD38- CD45RA+CD90-) represent the more immature HSPCs. In animal models, the progenitor compartment includes short-term repopulating cells, leading to the hematological recovery in the first 5 weeks after transplantation, whereas the stem cell compartment comprehends the long-term repopulation cells, responsible for the long-term hematological recovery. However, very little is known about the different subpopulations of HSPCs among peripheral blood (PB) CD34+ in basal state and after mobilization for harvest and transplantation. Our study was conducted to analyze PB CD34+ cells from healthy volunteers and from hematological patients during CD34+ cells mobilization. Our main aim was to understand if the proportions of different HSPCs among PB CD34+ cells were similar to those found in BM and whether the mobilizing regimens employed in chemo treated patients differently affected CD34+ cells subfractions in PB. METHODS: multicolor flowcytometry was used to analyze CD34+ cells from 4 BM samples and 9 PB samples from healthy volunteers and 32 PB samples from hematological patients prior CD34+ cells harvesting. RESULTS: Percentages of CD34+ cells subpopulations were different in basal PB compared to the BM: indeed, CMPs, GMPs and MEPs constituted respectively 27.6% ± 9.5, 23.8% ± 7.2 and 27.6% ± 16.2 of BM CD34+ cells and 47.8% ± 9.5, 10.3% ± 6.9 and 16.1% ± 7.6 of the total PB CD34+ cells. HSCs constituted 2.1% of BM and 1.5% of PB CD34+ cells. The differences between BM and circulating CMPs and GMPs were significant (p No differences in subpopulations proportions were shown comparing G-CSF mobilized and basal PB CD34+ cells. Interestingly, the 2 patients mobilized with AMD3100 (the inhibitory molecule for CXCR4) showed a higher percentage of GMPs (33.8% and 37.8% versus the average 16.3% ± 9.8 in G-CSF mobilized samples) and a lower fraction of CMPs (29.5% and 41.6% versus the average 58% ± 12 in G-CSF mobilized samples). In order to understand this result, we looked then at the CXCR4 mean fluorescence intensity among the progenitor subsets: GMPs showed significantly higher levels of this molecule compared to CMPs and MEPs. Regarding the mobilizing chemotherapy regimens, CMPs percentages were higher (61.1% versus 49.1%, p: 0.038) and GMPs’ were significantly lower (11.1% versus 27.6%, p A linear positive correlation between the number of mobilized CD34+ cells and the number of mobilized CMPs, GMPs, and MEPs was observed indicating that the proportions of different HSPCs did not significantly change among high- and low-mobilizers. There were no correlations between the number of mobilized subpopulations and leucocytes, hemoglobin and platelets levels. CONCLUSIONS: Our data displayed the heterogeneity of HSPC compartment between PB and BM. Many factors could contribute to this variegated scenario. These mechanisms comprehension can help us to choose the most suitable chemotherapy and cytokine administrations in order to improve clinical outcomes as infections complications, length of aplasia and transfusion requirements during an hematopoietic stem cell transplantation. Disclosures 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.