OBJECTIVES:To compare the efficacy of venetoclax-azacitidine (VEN-AZA) with AZA in the real-life for patients with first relapsed or refractory acute myeloid leukaemia (R/R AML).METHODS:We retrospectively analysed R/R AML patients treated with VEN-AZA at the Institut Paoli Calmettes between September 2020 and February 2022. We compared them to a historical cohort of patients treated with AZA between 2010 and 2021.RESULTS:Thirty-five patients treated with VEN-AZA were compared with 140 patients treated with AZA. There were more favourable cytogenetics (25.7% vs. 8.6%; p = 0.01) and less FLT3-ITD mutated AML (8.8% vs. 25.5%; p = .049) in the VEN-AZA group. The overall 30-day mortality rate was 7.4% and the overall 90-day mortality was 20%, with no difference between the groups. The complete remission rate was 48.6% in the VEN-AZA group versus 15% (p < .0001). The composite complete response rate was 65.7% in the VEN-AZA group versus 23.6% (p < .0001). OS was 12.8 months in the VEN-AZA group versus 7.3 months (p = 0.059). Patients with primary refractory AML, poor-risk cytogenetics, prior hematopoietic stem-cell transplantation (HSCT) and FLT3-ITD mutated AML had lower response and survival rates.CONCLUSION:VEN-AZA was associated with a better response rate and a longer survival than AZA monotherapy in AML patients who relapsed after or were refractory to intensive chemotherapy.

Background: Venetoclax (VEN) belongs to a novel BH3-mimetic class of small molecules that selectively targets BCL-2, activating the apoptosis effectors BAX and BAK to drive mitochondrial outer membrane permeabilization, cytochrome c release and cell death. Combination of VEN and the hypomethylating agent azacitidine (AZA) has deeply changed the paradigm of treatment of newly diagnosed (ND) AML patients ineligible for high dose chemotherapy because of older age or comorbidities. There is scarce evidence for the utilization of VEN-AZA for relapsed or refractory (R/R) AML, a category of patients classically associated with an extremely poor outcome. Aims: The objective of our study was to describe a R/R AML cohorts of AML patients treated with VEN-AZA in our institution and to compare the clinical and molecular characteristics predicting response in R/R AML versus ND AML Methods: This retrospective study included consecutive patients treated with VEN-AZA for R/R AML and ND AML. Patients received AZA at standard dose of 75 mg/m2 QD for seven days and VEN was administrated either at 400 mg or at 100 mg when associated with strong CYP3P450 A3 inhibitors after three days ramp up. Response was determined using the ELN 2017 criteria. The ORR was defined as the combination of complete response (CR), CR with incomplete hematologic recovery (CRi), and morphologic leukemia-free state (MLFS). Results: We compared the outcome of 39 R/R AML and 38 concomitant ND AML patients treated in our institution between Jan. 2020 and Dec. 2021. The median age was 69 (22-86) and 73 (61-81) in the R/R and ND groups, respectively. Thirty-five percent of patients had MRC-AML. Adverse cytogenetics was found in 36% of patients in the R/R group and 59% of patients in the ND group. Most frequent mutations were ASXL1, RUNX1, TET2, IDH1/2 and TP53 found in 33%, 33%, 28%, 24% and 22% of patients, respectively. Overall response rate was lower in R/R AML (37% versus 56%) including 13% CR, 8% CRi, 3% PR and 13% MLFS in the R/R AML group and 32% CR, 13% CRi and 13% MLFS in the ND AML group. Adverse cytogenetics was associated with treatment failure only in the R/R group (Relative Risk=0.10, p=0.005). ASXL1, IDH1/2 and SFSR2 mutations were associated with a trend in a higher response rate in the R/R group. Median overall survival (OS) were 5.9 months in the R/R group and 9.4 months in the ND group. In the R/R group, median OS were 2.2 months in the adverse cytogenetics group versus 8.7 months in the intermediate cytogenetics group (p=0.02). Median leukemia-free survival of responding patient was not different between the two groups (9 months), indicating that VEN-AZA can be efficient as a salvage treatment for selected R/R AML patients. Summary/Conclusion: We described one of the largest series of R/R AML patients treated wiht VEN-AZA. By a doing a direct comparison between R/R AML and ND AML treated concomitantly, we found that adverse cytogenetics was associated with treatment failure only in the R/R group suggesting that this subgroup of patients should not be treated with VEN-AZA. Further analyses including more patients are needed to determine which subgroup may benefit from the VEN-AZA as a salvage treatment.

Although complete remission (CR) is achieved in 50 to 70% of older fit patients with acute myeloid leukemia (AML), consolidation therapy in this age group remains challenging. In this retrospective study, we aimed to compare outcome in elderly patients treated with different post-remission modalities, including allogenic and autologous hematopoietic stem cell transplantation (HSCT), intensive chemotherapy, and standard-dose chemotherapy (repeated 1 + 5 regimen). We collected data of 441 patients ≥ 60 years in first CR from a single institution. Median age was 67 years. Sixty-one (14%) patients received allo-HSCT, 51 (12%) auto-HSCT, 70 (16%) intensive chemotherapy with intermediate- or high-dose cytarabine (I/HDAC), and 190 (43%) 1 + 5 regimen. Median follow-up was 6.5 years. In multivariate analysis, allo-HSCT, cytogenetics, and PS had a significant impact on OS and LFS. In spite of a more favorable-risk profile, the patients who received I/HDAC had no significantly better LFS as compared with patients treated with 1 + 5 (median LFS 8.8 months vs 10.6 months, p = 0.96). In transplanted patients, median LFS was 13.3 months for auto-HSCT and 25.8 months for allo-HSCT. Pre-transplant chemotherapy with I/HDAC had no effect on the outcome. Toxicity was significantly increased for both transplanted and non-transplanted patients treated with I/HDAC, with more units of blood and platelet transfusion and more time spent in hospitalization, but no higher non-relapse mortality. This study shows that post-remission chemotherapy intensification is not associated with significantly better outcome as compared with standard-dose chemotherapy in elderly patients for whom, overall results remain disappointing.

Targeted next-generation sequencing (tNGS) and ex vivo drug sensitivity/resistance profiling (DSRP) have laid foundations defining the functional genomic landscape of acute myeloid leukemia (AML) and premises of personalized medicine to guide treatment options for patients with aggressive and/or chemorefractory hematological malignancies. Here, we have assessed the feasibility of a tailored treatment strategy (TTS) guided by systematic parallel ex vivo DSRP and tNGS for patients with relapsed/refractory AML (number NCT02619071). A TTS issued by an institutional personalized committee could be achieved for 47/55 included patients (85%), 5 based on tNGS only, 6 on DSRP only, while 36 could be proposed on the basis of both, yielding more options and a better rationale. The TSS was available in <21 days for 28 patients (58.3%). On average, 3 to 4 potentially active drugs were selected per patient with only five patient samples being resistant to the entire drug panel. Seventeen patients received a TTS-guided treatment, resulting in four complete remissions, one partial remission, and five decreased peripheral blast counts. Our results show that chemogenomic combining tNGS with DSRP to determine a TTS is a promising approach to propose patient-specific treatment options within 21 days.

Due to the lack of specific clinical and biological features, M6a-acute erythroid leukemia (M6a-AEL), defined as an erythroid/myeloid type of acute leukemia, is no longer a distinct entity in the last classification of myeloid neoplasms by the World Health Organization (WHO).1 The diagnosis of M6a-AEL was previously made if a proliferation of erythroid precursors 50% with a myeloblast count 20% when counted as a percentage of non-erythroid cells, was found in the bone marrow.2 In 2016, revision of the WHO classification, the denominator used for calculating the blasts percentage was changed from non-erythroid cells to all nucleated cells. Consequently, M6a-AELs are now either myelodysplastic syndromes (MDSs) if the percentage of myeloblasts is 20% of non-erythroid cells but <20% of all nucleated cells or acute myeloid leukemia (AML) if the percentage of myeloblasts is 20% of all nucleated cells. As for any other AMLs prior therapy, recurring WHO cytogenetic abnormalities, and criteria for AML with myelodysplasia-related changes (AML-MRC) have to be taken into consideration for classification.

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR(1)-MR(4)), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR(4.5) level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR(4.5) sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.

Tyrosine kinase inhibitor (TKI)-based targeted therapy has significantly modified the outcome for patients with chronic myeloid leukemia (CML) in chronic phase. However, resistance remains a major concern in blastic phase of CML and in Philadelphia chromosome positive B-cell acute lymphoblastic leukemia (Ph+ B-ALL). Second- and third-generation TKIs have been developed to overcome resistance to first generation drugs, but selecting the appropriate drug has become a challenge. Various tests are available to determine a patient’s disease status in CML including the mechanisms of resistance when involved, but clinical experience is limited in ALL, especially those with poorly defined ABL1 rearrangements. Here, we report a case of ALL associated with a t(1;9)(q24;q34) RCSD1-ABL1 rearrangement. We show how ex vivo drug response profiling (DRP) may help choose among various therapeutic options.

Fil: Antony Debre, I.. Albert Einstein College of Medicine; Estados Unidos. Universite de Paris XI; Francia

Recent advances in myeloproliferative neoplasms (MPN) have highlighted the prevalence of mutations in the calreticulin gene (CALR), bringing a major new actor in these disorders. CALR mutations were reported in 25% of ET and in 35% of MF patients, which were non-mutated for JAK2 and MPL. CALR mutations lead to a frame-shift generating a common 36 amino acids C-terminal end and loss of the KDEL motif. Two variants account for 85% of the CALR mutations in ET and PMF: type 1, a 52-bp deletion and type2, a 5-bp insertion.

JAK2 V617F point mutation is now well recognized in patients with chronic myeloproliferative neoplasms (MPNs). However, its frequency in Algerian patients is still unknown. We determined the JAK2 V617F mutation in blood samples from 344 patients from five hematology departments across West Algeria, using amplification refractory mutation system-polymerase chain reaction (ARMSPCR). The JAK2 V617F mutation was identified in 157/344 patients (45.63%), 80/98 polycythemia vera patients (81.6%), 44/75 essential thrombocythemia patients (58.6%), 6/13 primary myelofibrosis patients (46.2%), 21/87 with suspected MPN (12.9%), and 2/32 secondary thrombocytosis patients (6.2%), but was not detected in 28 patients with secondary erythrocytosis. Detection of JAK2 V617F mutation is an essential molecular tool to establish myeloproliferative neoplasm diagnosis, which allowed us to adjust the diagnosis for 6 patients and should be systematically included into the initial evaluation of patients suspected of MPNs in Algeria.

PcG methylation of the HIST1 cluster defines an epigenetic marker of acute myeloid leukemia

Bivalent H3K4me3 and H3K27me3 chromatin domains in embryonic stem cells keep active developmental regulatory genes expressed at very low levels and poised for activation. Here, we show an alternative and previously unknown bivalent modified histone signature in lineage-committed mesenchymal stem cells and preadipocytes that pairs H3K4me3 with H3K9me3 to maintain adipogenic master regulatory genes (Cebpa and Pparg) expressed at low levels yet poised for activation when differentiation is required. We show lineage-specific gene-body DNA methylation recruits H3K9 methyltransferase SETDB1, which methylates H3K9 immediately downstream of transcription start sites marked with H3K4me3 to establish the bivalent domain. At the Cebpa locus, this prevents transcription factor C/EBPβ binding, histone acetylation, and further H3K4me3 deposition and is associated with pausing of RNA polymerase II, which limits Cebpa gene expression and adipogenesis.

The Nucleoporin 98 gene (NUP98) is a promiscuous gene implicated in chromosomal aberrations in hematopoietic disorders. NUP98 encodes a 98-kDa protein of the nuclear pore complex that regulates nucleocytoplasmic transport of protein and RNA. Twenty-eight different NUP98 partner genes have been identified across various human hematological malignancies,1, 2 many of which encode for homeodomain (HD) transcription factors and chromatin-modifying factors. We report here two new oncogenic fusions for NUP98, involving the homeobox gene genetic screened homeobox 2 (GSX2, formerly Gsh2) and the putative zinc-finger transcription factor gene ALL-1 fused gene from chromosome 10 (AF10)/MLLT10. Patient 1 is a 57-year-old woman with a post-myeloproliferative neoplasm M4 acute myeloid leukemia (AML) associated with a t(4;11)(q12;p15) translocation and trisomy 8. Patient 2 is an 83-year-old man who developed an atypical myelodysplastic syndrome resembling chronic myelomonocytic leukemia associated with a t(10;11)(p12;p15) translocation. Molecular cytogenetic techniques demonstrated the NUP98-GSX2 and NUP98-AF10 fusions for the t(4;11) and the t(10;11) respectively (not shown). Reverse transcriptase (RT)-PCR using primers located within NUP98, GSX2 and AF10 exons validated the presence of the NUP98–GSX2 and NUP98–AF10 fusion transcripts in patient samples (Supplementary Figure 1). Nucleotide sequencing showed an in-frame fusion of the NUP98 exon 12 with GSX2 exon 2, predicting a putative chimeric protein of 59 kDa that joins the Gly-Leu-Phe-Gly (GLFG) repeats of the amino-terminal part of NUP98 at the carboxy (C)-terminal part of GSX2 containing a HD (Figure 1). Sequence analysis of the NUP98–AF10 fusion transcript showed an in-frame fusion of NUP98 exon 11 with AF10 exon 15, predicting a 1027-amino-acid protein with the NUP98 GLFG repeats fused to the C-terminal part of the AF10 protein including the octapeptide motif-leucine-zipper (OM-LZ) domain and the glutamine-rich (Q-rich) sequence of AF10, but without the nuclear localization signal (NLS) region. Figure 1 Schematic representation of the native or chimeric AF10 and GSX2 and NUP98 proteins. RNP-BD, ribonucleoparticle binding domain; PHD, plant homeodomain; Q-rich, glutamine-rich region. Both AF10 and GSX2 genes have been involved in human hematological malignancies. GSX2 is a brain-specific class II homeobox gene of the Antennapedia family that regulates the development of mouse embryonic telencephalon.3 It has been associated with acute leukemia through its involvement in the recurrent t(4;12)(q11q12;p13) translocation.4 AF10 belongs to a family of proteins that includes AF17 and BR140 characterized by the presence of a C-terminal OM-LZ domain. AF10 is considered a putative transcription factor, binding DNA through an AT hook motif and interacting with the SWI/SNF chromatin remodeling complex.5 Through its OM-LZ domain, AF10 interacts with the histone methyltransferase hDOT1L that methylates the lysine 79 residues of histone H3 (H3K79),6, 7 a mark associated with an open-chromatin configuration. In hematological malignancies, AF10 is fused to the mixed lineage leukemia (MLL) gene by the t(10;11)(p12;q23) translocation8 and to the clathrin assembly lymphoid myeloid leukemia (CALM) gene by the t(10;11)(p12;q14) translocation.9 The transformation potential of the two NUP98 fusions was investigated by transducing murine primary bone marrow (BM) hematopoietic progenitors, defined as lineage-negative cells, by murine stem cell virus (MSCV) retroviral vectors containing different versions of Flag-tagged NUP98–GSX2 and NUP98–AF10 sequences as reported.10 Cells were transduced with MSCV expressing the native NUP98–GSX2 and NUP98–AF10 fusions, fusions deleted for the conserved functional domains (NUP98–GSX2-⊗HD and NUP98–AF10-ΔOM-LZ) or with the empty MSCV. Transduced cells were seeded in methylcellulose medium for serial plating assays. We observed that NUP98–GSX2-transduced progenitors formed numerous colonies after the fourth round of replating, whereas control cells were not replated after the third round (Figure 2a). Cytological analysis of the colonies showed that cells expressing NUP98–GSX2 exhibit a blast morphology, whereas empty MSCV-transduced progenitors formed monocytic and mast cell colonies (not shown). In contrast, NUP98–GSX2-⊗HD-transduced progenitors showed no proliferative advantage and were not able to form colonies after the second replating, thus illustrating that the transforming effect of the NUP98–GSX2 fusion requires the GSX2 HD. Immunofluorescence analysis showed a marked nuclear presence for the NUP98–GSX2 protein, whereas NUP98–GSX2-⊗HD was located both in the nucleus and in the cytosol with a diffuse staining pattern (Supplementary Figure 2). These data demonstrated that the NUP98–GSX2 fusion encodes a nuclear protein with a GSX2 HD-dependent oncogenic capacity. Progenitors transduced with NUP98–AF10 were able to produce a significantly increased number of colonies up to the third round of plating, contrary to the NUP98–AF10-ΔOM-LZ-transduced cells, which did not generate a significant number after the second round of plating (Figure 2a). Thus, expression of NUP98–AF10 is sufficient to induce a proliferative advantage in progenitor BM cells and this proliferative effect depends on the integrity of the OM-LZ region. CALM–AF10 (as well as some MLL fusions) participates in gene deregulation by virtue of hDOT1L recruitment at target gene loci, in particular for certain HOXA genes. As its transforming potential is linked to the integrity of the OM-LZ domain, we hypothesized that the oncogenic capacity of NUP98–AF10 is linked to HOXA gene deregulation by a process similar to that involved in MLL–AF10 and CALM–AF10 fusions. We performed quantitative RT-PCR to measure the levels of the HOXA5, HOXA7, HOXA9, HOXA10 transcripts and that of the HOX cofactor MEIS1 transcript in patient 2's (NUP98–AF10 fusion) BM cells (Figure 2b) (no material was available for patient 1). Expression analyses were performed in parallel in samples from patients with AML1-ETO-positive leukemia (n=2) as a negative control for HOXA expression, and in samples from patients with MLL–AF10 (n=1)- and NUP98–HOXA9 (n=2)-positive leukemia as positive controls. In patient 2's BM cells, we observed an elevated expression of HOXA5, HOXA7, HOXA9 and MEIS1 as in leukemic cells with MLL–AF10 and NUP98–HOXA9 fusions. HOXA10 was specifically overexpressed in the MLL–AF10 sample. No HOXA cluster gene expression was observed in AML1-ETO BM cells. In murine models, NUP98 fusions are often associated with upregulation of HOXA cluster and MEIS1 genes in blast cells.11, 12, 13 Thus, NUP98–AF10-transduced BM cells were engrafted into sublethally irradiated mice. Mice transplanted with cells transduced with empty (n=10) or NUP98–AF10 MSCV vectors (n=5) remained free of hematological disease up to 12 months after transplantation (not shown), contrary to those receiving cells transduced with the NUP98–HMGB3 fusion.10 Altogether, our results indicate that contrary to the MLL–AF10 and CALM–AF10 fusions,14, 15 NUP98–AF10 has only weak oncogenic power. This may be explained by the lack of an exclusively nuclear localization for NUP98–AF10 due to the absence of the NLS of AF10 in the chimeric protein. Figure 2 (a) Serial colony-plating assay of bone marrow progenitors transduced by wild-type, NUP98, NUP98–GSX2, NUP98–AF10 or empty vector. The average colony numbers±s.d. values are shown for three independent experiments. (b) Quantitative ... In conclusion, the identification of AF10 and GSX2 as new NUP98 partner genes in hematological malignancies strengthens the predominance of homeobox and chromatin-modifier genes as NUP98 partners and the deregulation of HOXA cluster genes as an oncogenic mechanism in several NUP98-associated leukemia.