ALBERT

Description: Blastic plasmacytoid dendritic cell neoplasm is a clonal disease derived from precursors of plasmacytoid dendritic cells (pDC). It is a rare neoplasm involving the skin which may or may not be associated from the outset with a leukemic component. The disease invariably progresses to aggressive leukemic dissemination, leading to a differential diagnosis with acute leukemia. In 2004, we set up a French network to recruit biological data at diagnosis. Diagnosis was according to recommendations (Swerdlow et al, 2008), with, in addition, a mandatory panel of pDC markers (Garnache-Ottou et al, 2009) detected by flow cytometry or by immunohistochemistry on infiltrated blood, bone marrow or cutaneous lesions. In total, 109 cases of BPDCN were included in 35 hospitals (2000-2013). BPDCN is more prevalent in men (sex ratio 4.4/1) and in elderly subjects (median age: 63 years; 7 patients were

Description: Background: Clonal heterogeneity of the AML blast cell population has been well documented using next generation sequencing (NGS) strategies. This heterogeneity extends to the presence of LSC fraction among the leukemic population: a dynamic compartment evolving to overcome cell selection pressure during disease progression and its treatment including allogeneic Hematopoietic Stem Cell Transplantation (HSCT). However, it remains difficult to discriminate between LSC and nHSC or other myeloid progenitors as MPP, LMPP or GMP. Here we present how to combine FLOWSom and KALUZA software to obtain robust identification among tumoral and normal progenitors. We compared the flowLSC Profile with stemness 17 genes score to identify patients with adverse outcome. Methods: 11 AML patients from ALFA07-01 clinical trial diagnosed in Lyon University Hospital between 2009-2010 were included in this study. The quantification of CD34+CD38- fraction at diagnosis from total bulk leukemia was made as previously described. To better discriminate between LSC and nHSC we designed MFC panel based on 2-tube antibody combinations to identify the pattern of LSC in the CD34+CD38- cell compartment. CD34/CD38/CD45/CD117« backbone » was used in both tubes, completed by lineage defined markers CD7/CD56/CD19/CD13/CD33/ and/or LSC identified markers CD123 +/- CD90/CD45RA, CLL1, TIM3, CD97. Results: As seeing in Table Nr 1, between 11 AML patients we observed a strong correlation identifying patients with adverse prognostic using previously published threshold by Flow (〉 1% CD34+CD38- from bulk leukemia cells). 4 from 11 patients showed a discrepancy between GES 17 LSC and Flow CD34+CD38-: one from four patients (Nr 3) was classified as "high" by Flow (high CD34+ expression) and "low" by GES LSC 17 and three other patients (Nr 8, 10, 11) were classified inversely as "low" by Flow (

Description: Abstract 5013 Immunomodulatory drugs represent a major therapeutic advance in the treatment of patients with multiple myeloma. While these agents appear to exert various effects on the microenvironment, including effect on immune cells and angiogenesis, a direct effect on the tumor cells themselves is also likely. To describe and compare the effect of the three clinically available agents (thalidomide, lenalidomide, pomalidomide) we analyzed the gene expression profiles of fresh human myeloma cells exposed to thalidomide, lenalidomide or pomalidomide, using high density DNA arrays. Fresh human myeloma samples were obtained from bone marrow aspirates of patients with myeloma, and myeloma cells were immunopurified using anti CD138 magnetic beads. Purified myeloma cells (1.106 cells/ml) were incubated for 24 hours in RPMI 1640 medium supplemented with 10% fetal calf serum under each of the four following conditions: 1) DMSO; 2) thalidomide 40 microM; 3) lenalidomide 1 microM; 4) pomalidomide 100 nM. These levels are achievable in the plasma of MM pts. Pangenomic array experiments were performed usingWhole Human Genome 4 × 44K Agilent one-color microarrays. Data were normalized using the quantile normalization method. Samples were analysed for differentially expressed genes, taking into account both the level of significance and the fold-change. Ten evaluable samples were processed. Exposure to thalidomide, lenalidomide and pomalidomide induced differential expression of 36, 50 and 75 genes, respectively, in comparison to DMSO-exposed controls, the total list including 101 genes. Twelve of these were found to be differentially expressed after exposure to all of the three agents, including trophoblast glycoprotein, WAS protein family member 1, dickkopf homolog 1, pentraxin-related gene, CD28, interleukin 12B, tissue factor pathway inhibitor 2, phospholipase A2, dehydrogenase/reductase (SDR family) member 9, hypothetical LOC145788 and betacellulin. These commonly altered genes could be mechanistically involved in themultiple activities of these agents in multiple myeloma or may represent epiphenoma mechanistically unrelated to drug-induced cell death. Genes differentially expressed between the treatment with each of these agents could be indicative of the different and non-overlapping actions these agents have in multiple myeloma. An example of this is the recent demonstration that pomalidomide is clinically active in lenalidomide refractory patients. These results suggest that exposure to IMIDs induce various intracellular signalization pathways in myeloma cells which might be involved in the cytotoxic activity of these compounds. Disclosures: Dumontet: Celgene: Research Funding.

Description: Introduction: Despite recent improvements in the treatment of adult acute myeloid leukemia (AML), many patients still fail to achieve complete remission (CR) or relapse early after response to initial induction chemotherapy. Outcome of patients with primary refractory/relapsed (R/R) acute myeloid leukemia (AML) remains dismal. Nevertheless, most of these patients undergo salvage chemotherapy. Gemtuzumab ozogamicin (GO) is an antibody-targeted chemotherapy agent consisting of a humanized murine anti-CD33 monoclonal antibody linked to calicheamicinϒ1, a potent antitumor antibiotic. Binding of GO induces cell death in CD33-positive cells by internalization of the calicheamicin drug and toxin release intracellulary leading then to then cleavage of double-stranded DNA. In relapsed AML, GO used alone with an unfractionated dose of 9 mg/m² on days 1 and 14 resulted in 13% of CR and a median OS of 5 months. Combined with intermediate-dose cytarabine and mitoxantrone, unfractionated dose of GO gave 50% of CR in patients with R/R AML, and a 2-year OS of 41%. However, early toxic deaths were reported related to sinusoidal obstruction syndrome (SOS). These results were confirmed by subsequent studies evaluating unfractionated GO combined with various agents in patients with R/R AML. The main objective of the present retrospective study was to evaluate the efficacy of the combination of fractionated GO with intermediate-dose cytarabine and daunorubicin in very high-risk patients with AML, and its potential use as bridge to transplant in this patient population. Methods: Herein we present a retrospective monocentric study of 24 very high-risk AML patients who received a combination of fractionated gemtuzumab ozogamicin (GO) with intermediate-dose cytarabine and daunorubicin as salvage therapy. Results: Median age was 55.3 years. Diagnoses were secondary AML for 33% of them. Seven patients had favorable, 8 had intermediate-1 or intermediate-2 and 6 unfavorable-risk AML according to the European LeukemiaNet prognostic index. Complete remission rate was achievied in 50% of cases (46% in refractory and 55% in relapsed AML) without excessive toxicity. Only allogeneic hematopoietic stem cell transplantation provided a benefit in this patient cohort with a one-year overall survival of 50.7% versus 18.1% in the absence of transplantation. Patients treated with reduced intensity conditioning (RIC) showed a higher survival as compared to those undergoing myeloablative conditioning regimen. CD33 expression was not associated with survival. In multivariate analysis in a model including the age older than 60 years, allo-HSCT, refractory AML and achievement of CR after GO, only the performance of allo-HSCT predicts patients' outcome with a Hazard Ratio (HR) of 0.25 (95% CI 0.07-0.86), p=0.02. Conclusions: Our data suggest that salvage therapy with fractionated GO combined with intermediate-dose cytarabine and daunorubicin in very high-risk patients may serve as a potential bridge therapy to RIC transplant. Data from studies published after withdraw of GO from the market, of which our present study, suggest that the licence status of GO might be reviewed, at least for certain subtypes of patients and certain situations of which R/R AML patients. Disclosures Nicolini: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria; Ariad pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Salles:Gilead: Honoraria, Research Funding; Roche/Genentech: Consultancy, Honoraria, Research Funding; Mundipharma: Honoraria; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Thomas:Pfizer: Consultancy.

Description: High dose melphalan is an essential component in the treatment of patients with advanced multiple myeloma. However little is known regarding genetic polymorphisms possibly correlated with patient outcome in this setting. To identify polymorphisms predictive of survival and/or toxicity we performed a SNP analysis in 83 patients having received high dose melphalan for multiple myeloma. All patients had received one or two courses of high dose melphalan with stem cell reinfusion. DNA was obtained from peripheral blood mononuclear cells and analyzed for 230 SNPs in genes selected for their role in drug metabolism, DNA repair, cell cycling or apoptosis. There were 48 males and 35 females and the median age was 58 years. The average number of VAD courses received before high dose melphalan was 4 (3–6). 49 patients received two high dose melphalan courses. The best response to melphalan was CR in 22 pts, very good response in 14 pts, partial response in 37 patients and stable or progressive disease in 10 patients. 37 patients had experienced disease progression and 8 patients had died at last follow-up. 29 patients experienced high grade (III-IV) mucositis after high dose melphalan. The median freedom from progression was 40 months. CYP2E1 polymorphism 7632 was correlated with freedom from progression (FFP) after high dose melphalan (p=0.02). SULT1A2 704 polymorphism was correlated both with FFP (p=0.002) and with overall survival (p=0.001). Occurrence of severe mucositis after high dose melphalan therapy was correlated with XRCC1 polymorphism 1198 (24% vs 51% of high grade mucositis) as well as ATM polymorphisms. These results suggest that SNPs of genes involved in drug metabolism or repair could be used to distinguish patients subgroups with different toxicity/efficacy profiles after high dose melphalan. OS according to SULT1A2 N235T OS according to SULT1A2 N235T

Description: Abstract 1485 Aim: Myeloablative chemotherapy followed by autologous PBSCT remains one treatment strategy in adult AML patients. Relapse has been shown higher for those who received the highest CD34+ PB doses. Although highly active against the leukemic bulk, intensive chemotherapy often spare the hardier leukemia stem cells (LSCs) responsible for relapse. Detection of MRD in harvest products may reflect inadequate in vivo purging at least in part responsible for relapses. Although recent data have challenged the CD34+CD38− phenotype of LSCs, this cell population remains generally considered enriched for LSCs. In this setting, MRD remaining during CR should be relatively enriched in CD34+CD38− leukemic cells and their persistence should correlate with disease recurrence. Methods: CD34+ cells were harvested after CR achievement in 123 AML patients [median age: 53 y (25–72)] treated by induction chemotherapy in our Institution between 10/1994 and 04/2003. Patients were included in different clinical trials planning autologous SC harvest in CR and autologous SCT in absence of donor or allogeneic SCT indication. Seventy-one of them received effectively autologous PBSCT. Harvests performed in 15 normal donors were used as controls. CD34/CD38 cell profile was analyzed in harvests in one single tube by multicolor flow cytometry using multiple MoAbs. The gating strategy was based on CD45low/SSC and CD34+CD45low cell populations from total FSC/SSC viable cells. Three populations of CD34+ were distinguished: CD34+CD38–; CD34+CD38low; and CD34+CD38+. Results: Patients from the entire cohort with higher percentage of CD34+ cells (cut-off level: 1%) in PBSC products were associated with shorter EFS [median: 5.6 months (3-y EFS: 13%) vs 13.6 (37%); p=0.0005] and OS [median: 10 months (3-y OS: 19%) vs 23.4 (47%); p=0.004]. This was also the case when analyzing only patients who received autologous SCT: [median EFS: 5 months (3-y EFS: 13%) vs 22.2 (48%); p=0.0006, and median OS: 9.1 months (3-y OS: 21%) vs 43.3 (57%); p=0.001]. Among CD34+ populations, only CD34+CD38– had a prognostic impact on EFS and OS. At a cut-off level of 0.9%, median EFS was 8.2 months (3-y EFS: 29%) for those with higher percentage vs 91.9 (62%) for those with lower percentage and median OS was 14.2 months (3-y OS: 36%) vs 95.4 (69%) respectively for the entire cohort. These results were confirmed in patients undergoing autologous SCT: median EFS was 7.3 months (3-y EFS: 31%) vs 91.1 (70%) (p= 0.05), and median OS was 14.4 months (3-y OS: 39%) vs 94.6 (80%). CD34+CD38low and CD34+CD38+ populations did not show any prognostic impact. Harvests from AML patients were divided into 3 groups: Group A: 51 patients with CR duration 1 y; and Group C: 50 patients without relapse. Harvests from 15 normal donors (Group D) were used as controls. Significant differences were only observed when comparing Group A and Group D for total CD34+ cells (mean ± SEM 2.5 ± 0.5 vs 1.2 ± 0.3; p 〈 0.05) and CD34+CD38– (4.5 ± 0.7 vs 2.3 ± 0.5; p 〈 0.05). To confirm the prognostic value of CD34+CD38–, 19 patients (Group 1) with evidence of leukemic contamination in harvests (abnormal cytogenetics at presentation found in aphereses) were compared with 22 patients (Group 2) without evidence of contamination (abnormal cytogenetics at presentation not found in aphereses). Median EFS was 10.1 months (3-year EFS: 45%) in Group 2 vs 6.3 (13%) in Group 1 (p=0.01), and median OS was 36.6 months (3-year OS: 55%) vs 10.8 (23%), respectively (p=0.03). Harvests from 15 normal donors (Group 3) served as controls. Significant differences were noted in harvest products regarding CD34+CD38– between Group 1 and Group 3 (mean ± SEM 6.0 ± 1.5 vs 2.3 ± 0.5; p = 0.04) and Group 1 and Group 2 (6.0 ± 1.5 vs 2.4 ± 0.5; p = 0.03), while there were no differences between Group 2 and controls. There were no significant differences between groups regarding CD34+CD38low and CD34+CD38+. We also measured MFI of CD13, CD33, CD123, CD117 in CD34+ subpopulations. Phenotypes were compared among the different groups. Conclusions: Higher proportions of CD34+CD38− in apheresis products appear to reflect inadequate in vivo purging and distinguish samples as enriched in ‘leukemic cells’ from those with lower CD34+CD38− as largely constituted of ‘normal cells’. This could serve as detection of MRD and help to identify samples associated with high-risk of relapse after autologous SCT. Disclosures: No relevant conflicts of interest to declare.

Description: High dose melphalan is an essential component in the treatment of patients with advanced multiple myeloma. However few data are available regarding genetic polymorphisms possibly correlated with patient outcome or toxicity in this setting. To identify polymorphisms predictive of survival and/or toxicity we performed a retrospective single nucleotide polymorphism (SNP) analysis using APEX technology in 169 patients having received high dose melphalan for multiple myeloma. All patients had received one or two courses of high dose melphalan with stem cell reinfusion. DNA was obtained from peripheral blood mononuclear cells and analyzed for 230 SNPs in genes selected for their role in drug metabolism, DNA repair, cell cycling or apoptosis. There were 108 males and 61 females and the median age was 55 years. The average number of VAD courses received before high dose melphalan was 4 (3–6). 58 patients (34%) received two high dose melphalan courses. A complete/very good response was observed in 43 of 103 evaluable patients (42%). Forty patients among 107 evaluable (37%) experienced high grade (III–IV) mucositis after high dose melphalan. The median freedom from progression was 45 months and the overall survival was 91 months. SNPs in SULT1A1, MDR, ERCC5, GSTP1, NQO1, CYP1A2, CCND1, PARP, RAD51, DRD4, P53 or associated proteins were found to be correlated with response to VAD therapy. SNPs in GRPR, ERCC5, GSTM3, GSTP1, XRCC1, ERCC4, CYP1B1, BARD1, MDR1, P53BP1, TERT, DRD4 were found to be correlated with response to high dose melphalan. Polymorphisms in CYP2E1, CYP2C9, CYP1A1, CYP1B1, TMPT, OGG1, PCNA, DRD2, XPC were correlated with disease progression while polymorphisms in ERCC1, DRD4, CYP2D6, CYP2C9, CYP1A1, CYP1A2, CYP1B1, ALDH, BARD1, BRCA1, MYH, TP53BP1 were correlated with death. Polymorphisms in XRCC1, SNP103, CYP2C9, CYP2C19, BRCA1, BRCA2, NBS1, CDKN1A were correlated with severe mucositis. These results suggest that SNPs of genes involved in drug metabolism or repair could be used to distinguish patients subgroups with different toxicity/efficacy profiles after high dose melphalan.

Description: Abstract 2336 Poster Board II-313 The principal aim of our study was to determine the level of CXCR4 expression in lymphocyte cells and CD34+ mobilized cells, SDF1 polymorphism and SDF-1 plasma levels in CLL. The secondary objective was to check if there is a correlation of these variables with PBSC mobilisation, disease characteristics and outcome. We studied 73 CLL patients enrolled in SFGM-TC & FCLLG phase III randomized trial. Patients received 3 mini-CHOP followed by PBSC mobilisation and then 3 courses of Fludarabine. Patients were randomised between autologous HSCT versus “watch and wait” for responders and between autologous HSCT versus Fluda+cyclophosphamide in non-responders after 2 DHAP. After induction, there were 20 CR, 28 PR, 10 SD and 5 PD. Thirty-two patients were mobilized, the median number of collected CD34+ cells was 3.5×106/kg [0-53.6] with a median of 15% of lymphocyte infiltration in the final product. At last follow up 63 patients are alive and 10 died. The CXCR4 expression in CLL cells (n=73) and in CD34+ mobilized cells (n=32) was evaluated by multicolour flowcytometry analysis, CXCL12 G801A gene polymorphism by PCR-RFLP and SDF-1 plasma levels by ELISA Quantikine SDF-1alpha kit. There was an heterogeneous expression of CXCR4 in CLL cells at diagnosis and patients were clustered into 3 groups depending of ratio MFICXCR4 vs IgG1 isotype control: 37 (51%) patients with low CXCR4 rMFI (median=39 [range 8-86]), 23 (31%) patients with intermediate CXCR4 rMFI (median 148 [range 94-216]) and 13 (18%) patients with high CXCR4rMFI (mediane 286 [range 220-534]). Concerning CXCL12-3'A polymorphism study, 5 (7%) patients showed A/A, 16 (22%) G/A and 52(71%) G/G genotype. The median of SDF1 plasma level was 1520 pg/ml (1150.4-4907.2). We did not find any correlation neither between CXCR4 rMFI in CLL cells and CXCL12-3'A polymorphism (p=0.99) nor between CXCR4 rMFI and SDF1 plasma levels (p=0.73) but CXCR4 rMFI in CD34+ mobilized cells was correlated with CXCL12-3'A polymorphism (p=0.02). In addition, we didn't find any significant correlation between Binet stage, disease status after induction and CXCR4 rMFI in CLL cells. Among the 32 mobilized patients, the median of CXCR4 rMFI in CLL cells was 124 (11-534) and 19.5 (3-38) in CD34+ mobilized cells; the CXCL12-3'A polymorphism showed 2 A/A, 7 G/A and 23 G/G. We didn't find any correlation between CXCR4 rMFI in CLL cells and CXCR4 rMFI in CD34+ mobilized cells. Among the potential impacting factors on CD34+ cell mobilization, we didn't find any correlation with age (p=0.99), lymphocyte infiltration (p=0.36), CXCL12-3'A polymorphism (p=0.8) and plasma levels SDF1 (p=0.12). There was a significant impact of disease status (p=0.006) and a negative trend of CXCR4 rMFI (p=0.07). In conclusion, the CXCR4 rMFI was very heterogeneous among our population. There was no correlation between this variable and CXCL12-3'A polymorphism. We showed a significant correlation between CD34+ mobilization and disease status, also a trend of CXCR4 rMFI. The CXCL12-3'AA genotype was associated with lower expression of CXCR4 in CD34+ mobilised cells and probably a good mobilisation; higher expression of CXCR4 in CLL cells at diagnosis was significantly correlated with poor CD34+mobilisation. Disclosures: No relevant conflicts of interest to declare.