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  • 1
    In: European Journal of Immunology, Wiley, Vol. 44, No. 10 ( 2014-10), p. 3068-3080
    Abstract: Acute myeloid leukemia (AML) cells are killed by allogeneic NK cells. However, autologous NK cells from AML patients express decreased levels of activating receptors, and show reduced cytotoxicity. Here, we investigated how interactions between NK and AML cells might cause loss of NK‐cell activity in patients. Our results show that AML cell lines and primary blasts alter the NK‐cell phenotype, reducing their cytotoxic potential upon prolonged contact. Downregulation of NK‐cell‐activating receptors was contact‐dependent and correlated with conjugate formation. Time‐lapse imaging of HL60 AML cell line and NK‐cell interactions showed a high proportion of noncytolytic contacts. Studies of NK‐cell immunological synapses revealed a defect in lytic synapse formation. Namely, despite correct F‐actin and LFA‐1 recruitment, polarization of lytic granules toward primary blasts or AML cell lines was reduced. The NK–AML cell line synapses showed impairment of CD3ζ recruitment. Attempts to correct these synapse defects by cytokine stimulation of NK cells improved conjugate formation, but not granule polarization. Pretreatment of AML cell lines with the immunomodulating molecule lenalidomide significantly enhanced granule polarization. We speculate that combining immunomodulatory drugs and cytokines could increase AML cell sensitivity to autologous NK cells and reinforce the activity of allogeneic NK cells in adoptive immunotherapy.
    Type of Medium: Online Resource
    ISSN: 0014-2980 , 1521-4141
    URL: Issue
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    Language: English
    Publisher: Wiley
    Publication Date: 2014
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  • 2
    In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 1353-1353
    Abstract: Acute myeloid leukemia (AML) with t(8;21) chromosomal translocation, leading to the RUNX1-RUNX1T1 fusion, belong to the favorable risk AML subset. However, relapse incidence may reach 30-40% in these patients. Minimal residual disease monitoring (MRD) based on the quantification of RUNX1-RUNX1T1 fusion transcript by real-time quantitative PCR (RQ-PCR) has been reported to be an independent prognostic factor for the risk of relapse. The specificity of the RUNX1-RUNX1T1 fusion and the high sensitivity of RQ-PCR techniques have made RUNX1-RUNX1T1 an ideal marker to assess treatment response in t(8;21) AML. Undetectable MRD could mean either that tumor cells persist in a latent state without RNA expression or that MRD level is below the sensitivity threshold. Studies in chronic myeloid leukemia showed that BCR-ABL DNA was still detectable in patients in long-term complete molecular response with undetectable BCR-ABL fusion transcript. Using a similar approach, we investigated the use of RUNX1-RUNX1T1 DNA as a MRD marker in t(8;21) AML, instead of RUNX1-RUNX1T1 mRNA. This approach allows linking results directly to the amount of leukemic cells, since each leukemic cell contains one copy of the RUNX1-RUNX1T1 sequence, while the level of RUNX1-RUNX1T1 mRNA may vary from a patient to another. Methods This study focuses on 17 patients with t(8;21) AML included in the CBF-2006 trial and for whom frozen material was available for further molecular analysis. Bone marrow and blood samples were collected at AML diagnosis and during follow-up, as defined in the CBF-2006 trial. Eight patients relapsed during follow-up and 9 were still in complete remission at the end of the study. Interestingly, 3 patients relapsed with a previously undetectable MRD (in blood and bone marrow samples). First, we identified the breakpoints in the RUNX1 and RUNX1T1 genes for each patient using long-range PCR approaches, coupled with next-generation sequencing (NGS) on Personal Genome Machine™ (PGM). The stability of the RUNX1-RUNX1T1 rearrangement at relapse was checked by Sanger sequencing. Then, we performed quantification of RUNX1-RUNX1T1 DNA by RQ-PCR using Taqman technology. For each patient, a primer pair and a probe were designed using the patient's unique RUNX1-RUNX1T1 breakpoint sequence. The forward and reverse primers were located in RUNX1 and RUNX1T1 genes, respectively, and the probe was located at the RUNX1-RUNX1T1 junction. Calibration curves were established using 10-fold dilutions of the diagnostic DNA of each patient in normal control DNA. Results were given as a ratio of chimeric DNA amount in the follow-up sample to chimeric DNA amount at diagnosis. Results Chromosomal breakpoints were located in RUNX1 intron 5 for all patients. RUNX1T1 breakpoints were located in intron 1b for 15 patients, and in intron 1a for 2 patients (Fig. 1). Quantification failed for 1 patient which was further leave up. Between 2 and 7 follow-up samples were studied for the other patients (median 4.5). DNA monitoring was strongly correlated with RNA monitoring (Fig. 2). Sensitivity threshold, determined by the lowest diagnostic sample dilution which gives a signal, was 10-5 for 7 patients, 10-4 for 6 patients, and only 5.10-4 for 3 patients. MRD was detectable in 31 samples and undetectable in 30 samples by both methods, whereas MRD was detectable only on RNA in 7 samples, probably because of a lack of sensitivity of the RQ-PCR assay. Interestingly, RUNX1T1-RUNX1 DNA was detected in 3 samples from 2 patients who relapsed and for whom RUNX1T1-RUNX1 transcript was undetectable, despite a good RNA quality. Conclusions Overall, RUNX1-RUNX1T1 MRD levels on DNA and RNA were quite similar. The level of mRNA expression did not seem to change during follow-up when compared with the amount of DNA. MRD monitoring on genomic DNA is a useful method, but with sensitivity variations depending on the patient's breakpoint sequence and the efficiency of the RQ-PCR assay. DNA has potential advantages: it is more stable than RNA and a best substrate for collection, processing, transport and storage. Additionally, interpretation of the results is easier because it is closely related to the number of leukemic cells. However, this method greatly increases complexity, time of implementation, and cost of monitoring MRD, which limits its interest in routine practice. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2013
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  • 3
    In: Blood, American Society of Hematology, Vol. 124, No. 9 ( 2014-08-28), p. 1445-1449
    Abstract: ASXL2 was mutated in 22.7% (25/110) of adult and pediatric t(8;21)/RUNX1-RUNX1T1 acute myeloid leukemia patients. ASXL2 mutations are mutually exclusive with ASXL1 mutations and occur in t(8;21) but not inv(16)/t(16;16) or RUNX1-mutant AML.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
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  • 4
    In: Frontiers in Immunology, Frontiers Media SA, Vol. 4 ( 2013)
    Type of Medium: Online Resource
    ISSN: 1664-3224
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2013
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  • 5
    In: American Journal of Hematology, Wiley, Vol. 89, No. 6 ( 2014-06), p. 610-615
    Abstract: Although acute myeloid leukemia (AML) with t (8;21) belongs to the favorable risk AML subset, relapse incidence may reach 30% in those patients. RUNX1‐RUNX1T1 fusion transcript is a well‐established marker for minimal residual disease (MRD) monitoring. In this study, we investigated the feasibility and performances of RUNX1‐RUNX1T1 DNA as MRD marker in AML with t (8;21). In 17/22 patients with t (8;21)‐positive AML treated in the French CBF‐2006 trial, breakpoints in RUNX1 and RUNX1T1 were identified using long‐range PCR followed by next‐generation sequencing. RUNX1‐RUNX1T1 DNA quantification was performed by real‐time quantitative PCR using patient‐specific primers and probe. MRD levels were evaluated in 71 follow‐up samples from 16 patients, with a median of four samples [range 2–7] per patient. RUNX1 breakpoints were located in intron 5 in all cases. RUNX1T1 breakpoints were located in intron 1b in 15 cases and in intron 1a in two cases. RUNX1‐RUNX1T1 MRD levels measured on DNA and RNA were strongly correlated ( r  = 0.8, P   〈  0.0001). Discordant MRD results were observed in 10/71 (14%) of the samples: in three samples from two patients who relapsed, RUNX1‐RUNX1T1 was detectable only on DNA, while RUNX1‐RUNX1T1 was detectable only on RNA in seven samples. MRD monitoring on genomic DNA is feasible, but with sensitivity variations depending on the patient breakpoint sequence and the qPCR assay efficiency. Although interpretation of the results is easier because it is closely related to the number of leukemic cells, this method greatly increases time, cost and complexity, which limits its interest in routine practice. Am. J. Hematol. 89:610–615, 2014. © 2014 Wiley Periodicals, Inc.
    Type of Medium: Online Resource
    ISSN: 0361-8609 , 1096-8652
    URL: Issue
    Language: English
    Publisher: Wiley
    Publication Date: 2014
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  • 6
    In: British Journal of Haematology, Wiley, Vol. 165, No. 5 ( 2014-06), p. 722-725
    Type of Medium: Online Resource
    ISSN: 0007-1048 , 1365-2141
    URL: Issue
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    Language: English
    Publisher: Wiley
    Publication Date: 2014
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  • 7
    In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 2360-2360
    Abstract: Background CEBPA mutations are found in approximately 10% of acute myeloid leukemia (AML). Recent studies revealed an association between CEBPA and GATA2 mutations. GATA2 gene encodes a transcription factor involved in hematopoiesis. In two recent studies (Grossmann et al., BJH 2013; Fasan et al., Leukemia 2013), GATA2 mutations appeared to be associated specifically with double CEBPA mutations and improved overall survival (OS). In contrast, another study failed to show any impact on OS (Green et al., BJH 2013). The aim of our study was to investigate the prognostic significance of GATA2 mutations in a large cohort of patients with CEBPA-mutated AML. Patients and methods We studied a cohort of 128 patients with CEBPA-mutated AML treated with intensive chemotherapy. The entire coding region of CEBPA, NPM1 exon 12, and GATA2 exons 4 to 6 (that encode the two multifunctional zinc finger domains of the protein) were screened on genomic DNA by PCR and direct Sanger sequencing. FLT3 internal tandem duplications (FLT3-ITD) were screened on genomic DNA by PCR and fragment analysis. Additionally, transcriptome analysis was performed with Affymetrix HG U133 Plus 2.0 array in pre-treatment samples from 72 patients for which RNA was available. Results Median age at AML diagnosis in the whole cohort was 50 years (range, 3-80). Almost all patients belonged to the intermediate cytogenetic risk-group (n=117), of which 90 had a normal karyotype. The remaining patients had favorable (n=1) or adverse cytogenetics (n=4). CEBPA mutations were distributed as follows: 29 single-mutated (sm) cases with N-terminal (N-ter) mutations, 12 sm cases with C-terminal (C-ter) mutations, 80 double-mutated (dm) cases with both N-ter and C-ter mutations, 2 cases with homozygous N-ter and 5 cases with homozygous C-ter mutations. GATA2 mutations were found in 29/128 patients (23%) and were significantly associated with CEBPA dm cases (4/41 sm vs 25/87 dm: Fisher's exact test p=0.022). NPM1 mutations were detected in 12 patients. They were specifically associated with CEBPA sm cases (12/40 sm vs 0/78 dm, p 〈 0.001) and mutually exclusive with GATA2 mutations (12/93 GATA2 wild-type vs 0/25 GATA2 mutated cases, p 〈 0.001). In contrast with previous studies, FLT3-ITD and GATA2 mutations did co-occur in our cohort, with 3 GATA2 mutants identified in 10 FLT3-ITD positive patients. Transcriptome analysis revealed that GATA2 mutations were not associated with a specific gene expression signature. As previously described, we found that CEBPA dm AML harbored a specific gene expression profile distinct from CEBPA sm cases. Since AML with homozygous CEBPA mutations were found to have a similar gene expression signature as CEBPA dm AML, we decided to pool them together for prognostic analysis. Overall, complete remission was achieved in 113 patients (88%), of whom 36 relapsed (estimated cumulated incidence of relapse [CIR] at 3 and 5 years, 39%). Neither age nor karyotype or gene mutations (including NPM1, FLT3, GATA2 and type of CEBPA mutation) significantly influenced CIR in multivariate analysis. Five-year OS was estimated at 58% in the whole cohort, with longer OS in cases with normal karyotype (p=0.05) and double CEBPAmutations (sm vs dm, p=0.03). In multivariate analysis, younger age (p=0 .020) and normal karyotype (p=0.029) remained the only factors significantly associated with a longer OS. Conclusions This study confirmed the strong association between GATA2 mutations and CEBPA double mutations, in line with previous studies. However, we did not find any prognostic impact of GATA2 mutations in our cohort of CEBPA-mutated AML. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2014
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  • 8
    In: American Journal of Hematology, Wiley, Vol. 88, No. 4 ( 2013-04), p. 306-311
    Abstract: Germline heterozygous alterations of the tumor‐suppressor gene neurofibromatosis‐1 ( NF1 ) lead to neurofibromatosis type 1, a genetic disorder characterized by a higher risk to develop juvenile myelomonocytic leukemia and/or acute myeloid leukemia (AML). More recently, somatic 17q11 deletions encompassing NF1 have been described in many adult myeloid malignancies. In this context, we aimed to define NF1 involvement in AML. We screened a total of 488 previously untreated de novo AML patients for the NF1 deletion using either array comparative genomic hybridization (aCGH) or real‐time quantitative PCR/fluorescence in situ hybridization approaches. We also applied massively parallel sequencing for in depth mutation analysis of NF1 in 20 patients including five NF1 ‐deleted patients. We defined a small ∼0.3 Mb minimal deleted region involving NF1 by aCGH and an overall frequency of NF1 deletion of 3.5% (17/485). NF1 deletion is significantly associated with unfavorable cytogenetics and with monosomal karyotype notably. We discovered six NF1 variants of unknown significance in 7/20 patients of which only one out of four disappeared in corresponding complete remission sample. In addition, only one out of five NF1 ‐deleted patients has an acquired coding mutation in the remaining allele. In conclusion, direct NF1 inactivation is infrequent in de novo AML and may be a secondary event probably involved in leukemic progression. Am. J. Hematol. 88:306–311, 2013. © 2013 Wiley Periodicals, Inc.
    Type of Medium: Online Resource
    ISSN: 0361-8609 , 1096-8652
    URL: Issue
    Language: English
    Publisher: Wiley
    Publication Date: 2013
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  • 9
    In: Blood, American Society of Hematology, Vol. 118, No. 21 ( 2011-11-18), p. 4132-4132
    Abstract: Abstract 4132 Introduction: Although precise matching of the donor/recipient pairs has been made easier by HLA typing at the allelic level, several issues with respect to unrelated transplantation remain to be addressed. In particular, the impacts of allelic HLA matching in patients with Acute Myeloid Leukemia (AML) and myelodysplasic syndrome (MDS) who receive allogeneic Peripheral Blood Stem cells (PBSC) after a reduced intensity conditioning (RIC) regimen is still unclear. In the present study, we aim to compare the impact of the donor type in this setting: HLA identical sibling versus HLA matched 10/10 (high resolution) unrelated donor (MUD). Method and transplantation characteristics: From 01/2001 to 12/2010, 108 consecutive patients with AML (n=63) and MDS (n=45) received PBSC after RIC in our center, either from HLA identical sibling (n=69) or MUD (n=39). Conditioning regimen was fludarabine based in 95% of patients and GvHD prophylaxis consisted in cyclosporine plus mycophenolate in 79% of patients. Engraftment, acute and chronic graft-versus-host disease (GvHD), transplantation-related mortality (TRM), relapse rate and overall survival (OS) at 3 years were compared according to type of donor: HLA identical sibling donor and MUD. Disease characteristics: WHO classification for MDS at time of hematopoietic stem cell transplantation (HSCT) was RAEB1 (24%), RAEB2 (36%), MDS transformed into secondary AML (20%), CMML2 (9%), RA (4%), or other (7%). Disease risk was assumed by cytogenetic (MRC for AML, IPSS for MDS) and EBMT score (good risk: CR1 for AML or MDS or untreated MDS, intermediate risk: CR2 for AML, CR2 or partial remission for MDS, poor risk: all other status). Cytogenetic (no missing data) was poor, intermediate or good for 21, 74 and 5% of AML and 24, 36 and 40% of MDS, respectively. EBMT score at time of HSCT was poor, intermediate or good for 29, 7, 64% of MDS and 11, 21, 68% of AML, respectively. Results of the comparison: Patients characteristics according to type of donor were similar for age (median 57 years), gender and disease distribution. Particularly, disease risks were comparable in 2 groups. Conversely, conditioning regimen (more ATG in MUD: 69 vs. 43%, p=0.016), donor age (younger for MUD: 30 vs. 52 years, p 〈 0.0001) and number of CD34+ cells infused (higher in MUD: 7 vs. 6.5 × 106/kg, p=0.022) were different. The median follow-up was 36 months (range 2 to 72). All patients engrafted. The cumulative incidence of acute GvHD was 40% with HLA matched sibling donor and 44% for MUD (p=0.58). The cumulative incidence of chronic GvHD at 3 years was 49% with HLA matched sibling donor and 45% with MUD (p=0.66). No risk factor was associated with acute GvHD but chronic GvHD was less frequent in patients with AML vs. MDS (41% vs. 59%, p=0.077) and in those patients who received ATG in conditioning regimen (54% vs. 43%, p=0.067). During follow-up, 47 patients died. The 3-year cumulative incidence of TRM was 17% and 22% with HLA matched sibling donor and MUD, respectively (p=0.55). Adjusting for age, MDS was the only factor increasing TRM (HR 3.4; 95% CI 1.2 to 9.5; p=0.02). The 3-year cumulative incidence of relapse was 46% with HLA matched sibling donor and 30% with MUD (p=0.28) knowing that there was no difference between both groups regarding disease risk (cytogenetic and EBMT score). The 3-year OS was 44% with HLA matched sibling donor (95%CI: 33–61) and 50% with MUD (95%CI: 35–71) (Figure 1). Disclosures: Fenaux: Celgene: Honoraria, Research Funding. Peffault de Latour:Alexion: Consultancy, Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
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  • 10
    In: Blood, American Society of Hematology, Vol. 120, No. 22 ( 2012-11-22), p. 4317-4323
    Abstract: IPH2101 is an anti-killer inhibitory receptor (anti-KIR) mAb that can block KIR-mediated inhibition of natural killer (NK) cells to enhance cytotoxicity against acute myeloid leukemia blasts. We have conducted a phase 1 study of IPH2101 in elderly patients with acute myeloid leukemia in first complete remission. Patients received escalating doses (0.0003-3 mg/kg) of IPH2101 following a 3 + 3 design. Safety, toxicity (primary end points), pharmacokinetics, outcome, and immunologic correlates were evaluated. Twenty-three patients (median age, 71 years), were enrolled. Adverse events were mild and transient, consisting mainly of infusion syndrome and erythema. The maximum tolerated dose was not reached, although full KIR saturation ( 〉 90%) was sustained for more than 2 weeks at 1 and 3 mg/kg. There was a clear correlation between mAb exposure and KIR occupancy. Neither hematologic toxicity nor significant changes in the numbers and distribution of lymphocyte subsets, NK cell receptor expression, or in vitro cytotoxicity were seen. At the highest dose levels (0.3, 1, and 3 mg/kg), transient increases in TNF-α and MIP-1β serum concentrations and NK cell CD69 expression were observed. Overall and relapse-free survival in the present study compared favorably to reports in comparable patient populations. We conclude that IPH2101 administration is safe and can block KIR for prolonged periods of time with limited side effects. Registered with the European Union Drug Regulating Authorities Clinical Trials (EUDRACT) as 2005-005298-31.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
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    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2012
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    detail.hit.zdb_id: 80069-7
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