ALBERT

Description: ATP binding cassette (ABC) transporters are a superfamily of highly conserved membrane proteins that transport a wide variety of substrates across cell membranes and confer drug resistance against a wide range of chemotherapeutic agents. We recently found that WT1, which is regularly overexpressed in AML and interact with the splicing machinery, modifies the splicing of ABC transporters A2, A3, A5, and C2. For ABCA3, WT1 knock-down in three AML cell line coupled with Affymetrix HTA2 exon arrays analysis confirmed by exon-specific PCR revealed that WT1 influences the skipping of exon 19. ABCA3 belongs in the ABC subclass and induces a significant reduction in cytotoxicity observed following exposure to DNR, mitoxantrone, etoposide, Ara-C and vincristine. The ABCA3 domain encoded by exon 19 (amino acid 805-847) is localized at the junction of the first nucleotide-binding domain and the second transmembrane domain, and is involved in ATP hydrolysis. In silico, skipping of exon 19 deletes a sequence of 32 amino acids rich in positively charged residues and is thereby assumed to increase drug efflux through increased ATP hydrolysis. The effects of the skipping of exon 19 on chemoresistance and DNR efflux are currently investigated while for the present study, we hypothesized that skipping of exon 19 of ABCA3 might negatively influence outcome in AML patients. Analyzing 132 bone marrow AML samples harvested at diagnostic confirmed the statistically significant correlation between WT1 expression and ABCA3 splicing in vivo (p

Description: Abstract 1307 Background. Mutation of MYD88 gene has recently been identified in activated B-cell like diffuse B-cell lymphoma, and enhanced JAK STAT and NF-kB signalling pathways. Whole exome sequencing study in Waldenstrom macroglobulinemia (WM) suggested a high frequency of MYD88 L265P mutation in WM. Although the genetic background is not fully deciphered in WM, the role of NF-kB and JAK STAT pathways has been demonstrated in WM; which underlying mechanisms of deregulation remain to be elucidated. We aimed to analyze MYD88 mutation in exon 5 and to characterize the clinical significance of this genetic alteration in 67 WM. Method. 67 patients (42 males, 25 females) diagnosed with WM were included in this study, along with 9 patients with chronic lymphocytic leukemia (CLL), 4 multiple myeloma (MM) and 9 marginal zone lymphoma (MZL) were also studied. Patients were untreated at time of BM collection and gave informed consent prior to research sampling. Clinical features, immunophenotypic markers using flow cytometry (Matutes score panel, CD38, CD138, CD27, CD80), conventional cytogenetic, FISH and SNP array data (n = 46) were analysed. B cells from bone marrow and T cells from blood were isolated respectively using B cell isolation kit and Pan T isolation kit (Myltenyi Biotech). For DNA sequencing of exon 5 of MYD88, the exon 5 of MYD88 gene was amplified from genomic DNA by PCR. The purified PCR products were directly sequenced in both directions using BigDye® Terminator Cycle Sequencing Kit (Applied Biosystems, CA, USA) and analyzed on the Applied Biosystems 3130xl Genetic Analyzer. Data were analyzed with SeqScape software version 2.5 (Applied Biosystems). Results. MYD88 L265P mutation (MYDmut) was observed in 79% of patients, including homozygous mutation in two patients (3%). MYD88 mutation was not identified in T lymphocytes isolated from 4 WM patients that confirmed MYD88 mutation was acquired in the tumoral cells. We haven't observed any other mutation on exon 5. We then sought for other mechanisms of MYD88 gene alteration, such as copy number alteration (CNA) and copy neutral –loss of heterozygosity (CN-LOH) also considered as an acquired UPD (uniparental disomy) at MYD88 locus. We found an UPD at MYD88 locus in solely one patient (2%), and haven't identified any deletion at 3p22. On the contrary, we observed a gain on chromosome 3 at 3p22 locus (including MYD88 gene) in 7/57 (12%) patients. Taking together, we identified alteration of the MYD88 locus in 85% of patients with WM, by either gain-of-function mutation (79%) or CNA (12%). Interestingly, we found gain on chromosome 3 more frequently in the MYDwildgroup than in the MYDmutgroup (p=0.02). Twenty one percent of the patients with WM had no mutation of MYD (MYDwild), and were characterized with a female predominance, a splenomegaly, gain of chromosome 3 and CD27 expression. We did not observed difference in terms of survival according to the MYD88 mutation status. MYD88 mutation was not related to deletion 6q, gain of 4, deletion 11q, deletion 17p, deletion 13q14 in our study. Interestingly, deletion 7q, a frequent cytogenetic aberration in marginal zone lymphoma, was rare in our series (4/57; 7%) and was independent of MYD88 mutation status (2 in the MYDwild and 2 in the MYDmut) (p=ns). No MYD88 L265P mutation was observed in CLL and MM. In MZL, 1/9 patient without M monoclonal component had a MYDL265p mutation. Conclusion. These results confirm a high frequency of MYD88 L265P mutation in WM that may become a useful biomarker for diagnostic in WM and may help better understand the physiopathogeny of WM. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2392 In humans, the majority of all protein-coding transcripts contain introns that are removed by mRNA splicing carried out by spliceosomes. Mutations in the spliceosome machinery have recently been identified using whole-exome/genome technologies in myelodysplastic syndromes (MDS) and in acute myeloid leukemia (AML). In MDS the frequency of somatic spliceosomal mutations (SSM) range from 1–3% for U2AF1 in RARS/RCMD-RS to more than 70% for SF3B1 in ARSI. These values are significantly lower in AML whereas AML cells cumulate numerous splicing defects. Beside SSMs, one can propose that alternative splicing (AS) might be disturbed by other processes such as abnormal protein-protein interactions. DEK and WT1 are 2 oncogenes overexpressed in most patients with AML. They physiologically influence AS through physical interactions with the heterodimer U2AF1/U2AF2 involved in the recognition of splice acceptor site by the splicing machinery. It is therefore possible that the leukemogenic overexpression of DEK or WT1 might deregulate AS in AML cells, even in the absence of SSM. Here we show that DEK and WT1 affect AS in AML cells. Exon expression profiling was performed in triplicate with MOLM13, KASUMI and KG1 AML cells stably knocked down or not for DEK and WT1 through shRNA. The efficiency of shRNA-mediated silencing was confirmed by western blot and total RNA was analyzed using the Exon microarray platform GeneChip Human Exon 1.0 ST (Affymetrix). Microarray data were cross-compared between cell lines and only statistically significant modifications (p

Description: Context. The prognostic value of gene mutations in older AML patients (pts) treated intensively remains unclear. Only one study has explored the role of mutation patterns determined by NGS in older AML pts prospectively treated with various chemotherapies in years 2000-2010 (Eisfeld Leukemia 2018). Methods. Pts older than 60y enrolled in the ALFA-1200 trial (NCT01966497) between 09/2012 and 06/2016 were sequenced with a 37-gene myeloid panel. Pts received one 7+3 course followed by 2 intermediate-dose cytarabine courses. Pts with non-favorable risk were eligible for allogeneic stem cell transplantation (SCT). Variable selection for multivariate analyses was performed by lasso penalized regression including age, gender and log(WBC) as covariates. Results. Sequencing was done in 471 (93%) of the 509 enrolled pts. Median age and WBC count were 68y and 5.3x109/L, respectively (resp). CR (including CRp) was achieved in 341 (72%) pts and 90 underwent RIC-SCT in first CR. With a median follow-up of 25.4 months, median OS was 20.7 months. Pts had a median of 3 mutations (range 1-10). The 17 mostly frequently mutated genes (≥5% of pts, by decreasing frequency: DNMT3A, NPM1, TET2, ASXL1, FLT3, SRSF2, IDH2, RUNX1, NRAS, IDH1, STAG2, BCOR, TP53, PTPN11, U2AF1, EZH2 and KRAS) were retained for prognostic analyses. Genes belonging to a common pathway (eg. NRAS and KRAS) may have divergent prognostic values, preventing biology-informed grouping of mutations. Cytogenetic risk (derived from ELN 2017, Döhner Blood 2017, not considering gene mutations) was favorable (fav), intermediate (int), adverse (adv) and missing in 3%, 72%, 18% and 7% resp. Because of the few pts with fav cytogenetics in our cohort, pts were further grouped into non-adv and adv cytogenetics. CR rates and median OS were 75.6% vs 56.6% and 24.8 vs 9.5 months in pts with non-adv and adv cytogenetics, resp (both p

Description: Mutations of the CCAAT/enhancer binding protein alpha (CEBPA) gene have been associated with a favorable outcome in patients with acute myeloid leukemia (AML), but mainly in those with a normal karyotype. Here, we analyzed the impact of associated cytogenetic abnormalities or bad-prognosis fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) in 53 patients with CEBPA+ de novo AML treated in the Acute Leukemia French Association trials. We found that only those with a normal karyotype and no FLT3-ITD displayed the expected favorable outcome. In this context, relapse-free, disease-free, and overall survival were significantly longer than in corresponding patients without the CEBPA mutation (P = .035, .016, and .047, respectively). This was not observed in the context of an abnormal karyotype or associated FLT3-ITD. Furthermore, after adjustment on age, trial, and mutation type, these features were independently predictive of shorter overall survival in the subset of patients with CEBPA+ AML (multivariate hazard ratio = 2.7; 95% confidence interval, 1.08-6.7; and 2.9; 95% confidence interval, 1.01-8.2; with P = .034 and .05, for abnormal karyotype and FLT3-ITD, respectively).

Description: Introduction Therapy decision in young adults with acute myeloid leukemia (AML) is mostly guided by pre-therapeutic risk assessment based on cytogenetical and molecular characterizations. Mutations in the nucleophosmin 1 (NPM1 m) gene represent one of the most common gene mutations, found in around one third of AML and associated with normal karyotype in 85% of cases. Because of their homogeneous mutation pattern and their clonal stability, NPM1 mutations are an effective tool for monitoring MRD. The aim of this study was to assess the prognostic impact of post-induction NPM1 MRD in homogeneously treated patients and to address the question of whether NPM1 MRD may be used as a predictive factor of allogeneic stem cell transplantation (SCT) benefit in this subgroup of patients. Materiel and methods Among 196 NPM1 m patients treated in the ALFA-0702 trial, 172 achieved complete remission (CR). A MRD was available in 152 patients, on peripheral blood (PB) in 135 patients and on bone marrow (BM) in 135 patients. MRD levels were reported as the normalized values of NPM1 m copy number/ABL copy number x 100 (%). Patients were also screened for FLT3-TKD, FLT3-ITD, CEBPA, DMT3A, IDH1, IDH2, WT1 and TET2 mutations. Patients that did not belong to ELN-favorable group were eligible for allogeneic SCT (N=71). Results The median follow-up was 3.5 years (95%CI: 3.1-3.9). The karyotype was normal in 122 patients (80.3%), and a FLT3-ITD was found in 59/150 (39.3%) with a median FLT3-ITD allelic ratio of 0.385 (range: 0.02-8.0). Fifty out of the 71 patients eligible for SCT were allografted in first CR. The 3-year CIR and OS censored at SCT were 29.3% (95%CI: 21.5-39.4) and 78.6% (95%CI: 69.3-85.5) respectively. At diagnosis, NPM1 baseline levels did not differ between PB and BM (p=.20). However, after induction, the median MRD log reduction was 4.5 in PB (range: 1.7-5.8) and 3.8 in BM (range: 0.01-5.5) (p50 G/L) or FLT3-ITD had higher NPM1 baseline but also PB-MRD levels. However, no correlation between PB-MRD log reduction and age, WBC, karyotype, or any of the gene mutations was observed. Patients who did not achieve a 4-log reduction in NPM1 PB-MRD were exposed to a higher risk of relapse and had a shorter survival (3-year CIR censored at ASCT: 65.8% vs. 20.5%, SHR 5.83, 95%CI: 2.78-12.23, p

Description: Abstract 3377 It is generally accepted that the BCR-ABL oncoprotein transformes haematopoietic stem cell and initiates chronic myeloid leukemia (CML). However, leukemogenesis is a complex process, and genomic heterogeneity of the chronic phase (CP) of the disease has been reported. At the molecular level, this intrinsic heterogeneity could support a causative link with the varying response to treatment and disease progression. Genetic analysis of candidate genes in myeloid malignancies reported mutations of the ten-eleven translocation 2 (TET2), the isocitrate deshydrogenase (IDH) 1 and IDH2, and the additional sex combs like 1 (ASXL1) genes in myeloproliferative, acute myeloid and myelodysplasic neoplasms. Similarly, we can stipulate that these candidate genes may contribute to phenotypic heterogeneity of CML. To investigate whether TET2, IDH1, IDH2 and ASXL1 defect could represent a significant event in CML, we selected 91 CML patients (pts) treated with imatinib (IM) at first line and presenting five profiles of IM response at time of the analysis: 1) 25 pts in major molecular response (MMR) at 12 months of IM; 2) 11 pts in CCR but presenting additional Philadelphia (Ph) negative clonal evolution; 3) 20 pts in partial cytogenetic response at 18 months of IM, referred as primary resistant (R1); 4) 20 pts in acute transformation 4 to 72 months after onset IM; and 5) 15 pts relapsing in CP of the disease, referred as secondary resistant (R2). The search for mutation was performed by sequencing the entire TET2 coding region (11 exons), the IDH1 and IDH2 exon 4 and the ASXL1 exon 12. Analysis of paired samples from CML diagnosis, time of IM response and, when available, CCR revealed: 1) 2 pts (2.2%) in acute transformation presenting 3 TET2 stop mutations not located within conserved region (del at A2079, substitution T4893A - both also been detected at diagnosis -, and del at C4851 which has not been detected at diagnosis, even by mutation-specific ASO-PCR); 2) no IDH1 and IDH2 mutation; and 3) 8 pts (8.7%) presenting ASXl1 stop mutations at diagnosis. Among them, 3 pts (two ins at G646 and one ins at V751) have reached MMR without detected mutations at this time; one R1 pt presenting ins at G646 had major cytogenetic response with 5% Ph+ cells but the mutation was not found at this time and the pt have progressed to MMR 9 months later; one pt with 23 bp del at R634 has evolved in acute transformation with detected mutation at this time; and 3 R2 pts presenting either 4 bp del at S895, del at R860 or 2 pb ins at A752 have lost CCR associated with lost of hematologic response in one case. In this later group of 3 pts, except for del at R860, all ASXL1 mutations were found in samples at time of relapse. We therefore conclude that, contrary to what has been reported in other myeloid malignancies, TET2, IDH1 and IDH2 are not commonly acquired in CML and may not represent a major genetic event in CML transformation. However, ASXL1 alteration seems to be an early event in CML leukemogenesis but does not seem to participate in the disease transformation. Disclosures: No relevant conflicts of interest to declare.

Description: Background 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.

Description: Abstract 4171 NF1 acts as a tumor-suppressor gene by encoding neurofibromin1, a GTPase-activating protein (GAP) inhibiting Ras signaling pathway. Germline mutations or microdeletions of NF1 are responsible for neurofibromatosis type 1, and the somatic loss of the remained wild-type allele lead to malignant tumors or juvenile myelomonocytic leukemia (JMML). Furthermore, several studies revealed heterozygous somatic deletions of the 17q11.2 region including NF1 in adult myeloid malignancies. The reported frequencies of this abnormality varied between 2.6% and 11% in AML and this variation can be attributable to heterogeneity or size of the analysed cohorts. Previously, we analyzed 131 de novo AML cases (AML3 excluded) by Agilent™ 105K microarrays. 6/131 cases (4.6%) showed somatic deletions in 17q11.2, including a small minimal deleted region of 300 kb comprising the entire NF1 gene. To further investigate the incidence of NF1 deletion in de novo AML, 354 additional patients were therefore screened for the deletion by quantitative real-time PCR (Primers and TaqMan-based probe Hs 01778367_cn from Applied Biosystems), and FISH (NF1/MPO probe KBI-40144 from Kreatech) was performed to confirm the loss of NF1 copy number. Altogether, heterozygous NF1 deletion was observed in 17/485 (3.5%) de novo AML. Clinico-biological data were available from 14 NF1 deleted patients and 380 non-deleted patients included in the ALFA-9801 and 9802 French Trials. There were no significant differences between the 2 groups in age, sex ratio, leukocytosis, FAB classification of AML, mutational status of FLT3, NPM1, CEBPα and IDH. Interestingly, NF1 deletion was significantly correlated with unfavourable cytogenetic (50% vs 18%, p=0.008) and especially with monosomal karyotype (29% vs 9%, p=0.03). However, no statistical significant differences were observed for complete remission rate, relapse risk 3 years after diagnosis and 3-years overall survival. Screening for bi-allelic inactivation by sequencing the remained allele in NF1 deleted patients is in progress. We next evaluated NF1 gene expression for 93 patients of our cohort (3 with NF1 deletion and 90 without) by Affymetrix U133 Plus 2.0 microarrays. The 3 NF1 deleted patients revealed a significant reduced mean of NF1 expression level. Interestingly, about 10% of the NF1 non-deleted patients presented a similar decrease in NF1 expression rate. This suggests that mechanisms for transcriptional regulation (such as mutations or epigenetic silencing of NF1) may also contribute to AML pathogenesis. In conclusion, NF1 deletions occur in only 3.5% of de novo AML and are associated with unfavourable cytogenetic. This relatively low frequency of NF1 deletion can however be counterbalanced by others alterations acting at the transcriptional level and this remains to be investigated. Disclosures: No relevant conflicts of interest to declare.