Entity Of Acute Myeloid Leukemia Research Articles

Advances in Acute Myeloid Leukemia Classification: A Comparative Analysis of Who-HAEM4R, Who-HAEM5, and International Consensus Classification

Introduction Two guidelines were recently published to refine the diagnostic criteria of acute myeloid leukemia (AML): the 5th edition of the World Health Organization Classification of Haematolymphoid Tumors (WHO-HAEM5, PMID: 35732831) and the International Consensus Classification (ICC; PMID: 35767897), and both expanded molecularly defined AML subtypes. In this study, we utilized a cohort of 1135 AML cases to examine their performance. Methods From Sep 1, 2018 to Dec 31, 2022, a consecutive cohort of 1135 cases diagnosed with de novo AML according to WHO-HAEM4R (PMID: 27069254) were enrolled, with a median follow-up of 20 months. All underwent high-throughput sequencing (PMID: 34135310, 29932212). Results When classified according to WHO-HAEM4R, 55% of the 1135 cases were diagnosed as AML with recurrent genetic abnormalities, 16% were AML with myelodysplasia-related changes (AML-MRC), 2% were therapy-related AML, 4% were AML with germline predisposition, and 24% fell into AML not otherwise specified (AML, NOS). Major changes in WHO-HAEM5 include the inclusion of AML subtypes of KMT2A rearrangement (KMT2A-r), MECOM-r, and NUP98-r; adding AML with other defined genetic alterations (AML-ODGA) as a basket entity; and annulling the entity of AML with mutated RUNX1 (AML-RUNX1). The entity of AML with CEBPA mutation (AML-CEBPA) has changed to include not only biallelic (biCEBPA) but also single bZIP (smbZIP-CEBPA) mutations. AML-MRC in WHO-HAEM4R is redefined as AML, myelodysplasia-related (AML-MR), with a mutation-based definition based on an 8-gene panel (without RUNX1). When comparing WHO-HAEM4R and WHO-HAEM5 (Fig. 1), AML with KMT2A-r increased from 2% to 10% because WHO-HAEM5 no longer restricts the partner genes of KMT2A. AML with NUP98-r and AML-ODGA in WHO-HAEM5 account for 6% and 4%, respectively. AML-MR increased from 12% AML-MRC to 21% benefited from the 8-gene panel-based definition. AML-RUNX1 has been annulled in WHO-HAEM5 and 63% cases of this subtype were reclassified as AML-MR, and 30 cases with RUNX1 mutations as the only basis for classification were reclassified as AML defined by differentiation. In total, AML without defining genetic abnormalities dropped from 24% to 11%. When comparing WHO-HAEM4R and ICC (Fig. 1), AML with mutated TP53 (AML-TP53) is a new category in ICC and accounted for 4% of AML, 79% of them were previously classified as AML-MRC in WHO-HAEM4R. AML-MR in WHO-HAEM5 is divided into two entities in ICC: AML with MR gene mutations (including RUNX1) and AML with MR cytogenetic abnormalities, comprising 13% and 5% of cases, respectively. ICC only includes in-frame bZIP CEBPA mutations. Thus 6 cases classified as AML-biCEBPA based on WHO-HAEM4R fell into AML, NOS under ICC since their CEBPA mutations were not in bZIP. Conversely, 21 cases with smbZIP-CEBPA and diagnosed as AML, NOS according to WHO-HAEM4R were classified as AML-CEBPA under ICC. Cases classified as AML-CEBPA based on WHO-HAEM5 tended to have a better overall survival (OS) than cases classified as AML-biCEBPA under WHO-HAEM4R because AML-smbZIP-CEBPA was added while cases with MR gene mutations which had a worse prognosis fell into AML-MR under WHO-HAEM5. Despite ICC only included in-frame bZIP-CEBPA mutations, the prognosis of AML-CEBPA diagnosed under ICC was not superior to that under WHO-HAEM5. This might be because cases with both MR cytogenetic abnormalities and CEBPA mutations were reclassified as AML-CEBPA under ICC's hierarchical principle. ICC distinguished AML with KMT2A::MLLT3 from AML with KMT2A-r, while WHO-HAEM5 only included AML with KMT2A-r entity. Survival analysis showed significantly better OS in AML with KMT2A::MLLT3 than AML with KMT2A-r. ICC separated AML-TP53 from AML-MR due to the notorious prognosis of TP53 defects, and cases classified as AML-TP53 showed the worst OS in this cohort. But prognostic analysis showed no difference between AML-TP53 and cases with concurrent TP53 mutation and other AML-defining genetic abnormalities. The rationality of considering AML-TP53 as a distinct entity remains debatable. Conclusions Both new classification systems consider genomic features more extensively and enhance the precise classification of AML. Discrepancies between WHO-HAEM5 and ICC, like classifying AML with RUNX1 mutations and the rationality of defining AML-TP53 as a unique entity, remain open questions that require further research for clearer answers.

Trinary Fusion with Ligand Binding Domain Castration Is Crucial in Rarg-Driven APL and Render Unresponsive to ATRA Via Allosteric Disability Mechanism

Introduction All-trans retinoic acid (ATRA) has been used with great success in acute promyelocytic leukemia (APL) cases with PML::RARA fusion gene (FG). There are still PML::RARA negative cases that manifest as APL, and they often carry FGs involved in RARA, RARB, or RARG. X::RARG positive APL ( RARG-APL) cases have been increasingly reported in recent years, thanks to the utilization of transcriptome sequencing (RNA-seq) and whole genome sequencing (WGS). Almost all evaluable RARG-APL cases were resistant to ATRA, and no mechanism has been elucidated. Intriguingly, cells transformed by artificial X::RARG were extremely sensitive to ATRA (PMID: 25510432), indicating intricate mechanisms unrevealed. This study first and systematically unveils the distinctive features and pivotal molecular mechanism of RARG-driven APL. Methods A total of 22 RARG-APL cases with RNA-seq and WGS data from 11 centers were enrolled (Fig. 1A). We used Arriba for routine FG calling and manually investigated the non-coding fusion sequence. Reverse transcription PCR (RT-PCR) and Sanger sequencing were used to validate the FGs and to determine whether the RARG 5′ and 3′ fusion events were located on the same cistron. The responsiveness of the fusion proteins to ATRA was evaluated using an optimized UAS/GAL4 reporter system. Results Among the cases enrolled, there were 15 males and 7 females, aged 0.9-69 (median 38) years. Morphological and immunophenotype analyses all showed features of APL. Routine FGs calling reported RARG fusion to a 5′ partner in each case, with variable RARG splicing sites (Fig. 1A). There were 7 RARG 5' partner genes, with CPSF6, HNRNPC, and NUP98 observed in multiple cases. In the 3 index cases, we identified that they all had RARG 3‘ fusion events besides the 5‘ one (Fig. 1A). RT-PCR confirmed that the 5′ and 3′ RARG fusion events were in the same cistron in each case. Such as, the fusion transcript is the insertion of RARG-e4_9 (e refers to exon) between NPM1-e4 and e11 in case #G1, rather than two separate transcripts of NPM1-e4:: RARG-e4 and RARG-9:: NPM1-e11. We named this novel form of tandem splicingtrinary fusion. Analysis of the 19 validation cases further confirmed that all RARG-APL cases had RARG 3′ fusion events (Fig. 1A). We also validated that the 5′ and 3′ RARG fusion events were in the same cistron in 3 more cases, of which archived cDNA were available. Remarkably, the 3' splicing sits of all cases were consistently at the end of RARG-e9, leading to RARG-e10 truncation. The RARG 3' partner gene was the same as its 5' partner in 11 cases. But in the other 11 cases, the RARG 3' partner was a transposon element (TE) sequence, the most common being a LINE-L2a (8 cases). Sequences analysis indicated that the committed locus of the involved LINE-L2a was at 4.2 Kbp upstream of the RARG gene. Gene expression analysis confirmed the aberrant activation of the involved TEs. All TEs involved in the RARG 3' fusions confer a poly_A signal sequence to the fusion transcript, which was essential for a mRNA. In-depth analysis indicated that TEs participate in the formation of RARG-FGs through a transposition mechanism rather than a translocation mechanism. RARG-e10 encodes helix 11_12 (H11_12) of its ligand binding domain (LBD), which plays a pivotal role in allosteric response to ATRA (PMID: 31178221). The trinary fusion protein with RARG LBD-H11_12 castration will lose responsiveness to ATRA via an allosteric disability mechanism (Fig. 1B, 1C). Experiments confirmed that cells transfected with X::RARG fusion with intact LBD responded well to ATRA, while fusion with LBD-H11_12 castration showed no response to physiological and pharmacological concentrations of ATRA (Fig. 1D, 1E). Conclusions This study clearly revealed that trinary fusion and LBD castration are crucial molecular etiologies in RARG-APL. The trinary fusion orchestrates the aberrant activation of RARG (5‘ splicing) and the complete allosteric disability in responding to ATRA (3‘ splicing). Thus, this study elegantly explains the mechanism of leukemogenesis and the extensive ATRA resistance of RARG-APL. The formation of trinary fusion requires two steps of genome splicing or transposition events, which explains the rarity of RARG-APL. This also clarifies that RARG-APL has a significant disparity in molecular mechanism from PML::RARA-positive APL, and we suggest that it should be considered as a separate entity of acute myeloid leukemia.

RUNX1 Mutation Does Not Significantly Impact the Outcome of Newly Diagnosedadult AML: A Retrospective Study of Chinese AML Patients

Background RUNX1 is one of the recurrent mutated genes in newly diagnosed acute myeloid leukemia (AML) with a frequency of 6%~15%. Although historically recognized as a provisional distinct entity, the AML subtype with RUNX1 mutations (AML- RUNX1mut) was eliminated from the 2022 WHO classification system. With the update of AML diagnosis and treatment protocol, different outcomes of AML- RUNX1muthave been reported. However, an in-depth study about the prognostic value of RUNX1 mutations, epecifically the classification change related to AML- RUNX1mut in a large well-annotated cohort is lacking. Methods The clinical data of 1065 AML (excluding acute promyelocytic leukemia and RUNX1 germline mutations) patients according to WHO 2016 and treated from January 2017 to December 2021 at Jiangsu Institute of Hematology were retrospectively collected and analyzed after informed consent. All AML diagnosis samples were analyzed by targeted Next Generation Sequencing (NGS) panel covering 172 frequently mutated genes in hematological malignancies. Demographic and clinical data, disease characteristics at diagnosis, first-line treatment and clinical outcome data were available for all patients. The survival time of allogeneic hematopoietic stem cell transplantation patients was censored at the time of stem cell transfusion. Cytogenetic and molecular characteristics were used to classify patients into ELN2017 and ELN2022 risk groups (excluding the effect of RUNX1 mutation). Results RUNX1 mutations were detected in 112 (10.5%) patients, namely, 66 (58.9%) male and 46 (41.1%) female. A total of 132 RUNX1 alterations were detected with a medianvariant allelic frequency of 40% (3%~93%). RUNX1 mutations were associated with older age (48.5 vs. 46.0 years old, P=0.087) and antecedent myeloid disorders (6.3% vs. 1.0%, P<0.001). One or more comutations were detected in 106 (94.7%) AML- RUNX1mut patients. The most frequent co-gene abnormalities were FLT3-ITD and DNMT3A mutations which represented the 23.2% and 18.7%. Mutations of CEBPA bZIP, NPM1 and translocations of t(8;21), t(16;16)/inv(16) were uncommon in AML- RUNX1mut with a frequency of 6.3%, 1.8%, 2.7%, 0%, respectively (Table 1). Compared with AML with wild-type RUNX1 (AML- RUNX1wt), AML- RUNX1mut patients had a lower complete remission rate (19.6% vs. 29.7%, P=0.034), lower overall response rate (ORR) (73.5% vs. 84.3%, P=0.006) and higher no response rate (26.5% vs. 15.5%, P=0.021) in the first induction chemotherapy. When comparing groups based on the ELN2017 risk stratification criteria, no significant difference in ORR was observed between RUNX1mut and RUNX1wt patients in the favorable (91.2% vs. 94.0%, P=0.572), intermediate (75.0% vs. 78.4%, P=0.570) and adverse (63.3% vs. 71.4%, P=0.370 ) subgroups (Table 2). The median duration of follow-up of the entire cohort was 8.7 months ( RUNX1wt: 8.4 months; RUNX1mut: 10.0 months). Comparing the two groups, patients with RUNX1 mutations had a lower 5-year overall survival (OS) rate (35.4% vs. 42.3%; HR=1.563; 95% CI: 1.091~2.237, P=0.026), but no significant difference was observed in the 5-year event-free survival (EFS) rate (40.8% vs. 35.4%; HR=1.084; 95% CI: 0.750~1.570, P=0.098) (Table 2). However, when patients were stratified according to the ELN2017 guidelines (excluding the effect of RUNX1 mutation), outcomes did not differ between patients withor without RUNX1 mutation in the favorable (OS: P=0.629, EFS: P=0.137), intermediate (OS: P=0.629, EFS: P=0.114) and adverse (OS: P=0.544; EFS: P=0.714) subgroups. Similar results were obtained according to the ELN2022 guidelines. In multivariate analysis, RUNX1 was not an independent prognostic factor for OS (HR=1.353, 95% CI: 0.922~1.985, P=0.122) and EFS (HR=1.422, 95% CI: 0.975~2.075, P=0.068), while significantly worse OS and EFS were observed in patients with mutated FLT3-ITD, TP53, and DNMT3A ( P-values were all less than 0.05). Conclusion RUNX1 mutations were rarely isolated genetic abnormities that conferred inferior outcomes. However, on the basis of ELN prognosis risk stratification, RUNX1mutation had no impact on the clinical outcomes of AML patients in stratified subgroups. RUNX1 was not an independent prognostic factor for survival. Overall, our findings agree with the updated WHO classification system for AML that AML- RUNX1mut should not be recognized as a distinct AML entity.

Acute Myeloid Leukemia (AML) Patients with Myelodysplasia (MDS) Related-Gene Mutations in First Complete Remission (CR1) Found to Have Benefit from Allogeneic Hematopoietic Stem Cell Transplant (HCT)

Introduction: Acute myeloid leukemia (AML) with myelodysplasia-related gene mutations is a new disease entity introduced in the International Consensus Classification of Myeloid Neoplasms 2022 (ICC 2022). Myelodysplasia-related gene mutations include ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, or ZRSR2 . Allogeneic hematopoietic stem cell transplant (HCT) represents a curative option for subgroups of AML patients, but not for all. In a prior study (Song et al.) reported the survival benefit of allogeneic HCT in 59 patients with AML with secondary type mutations (STM) including SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, and STAG2. Allogeneic HCT could abrogate adverse prognosis in AML with STMs, and reduce its mortality risk by 58% (Song et al. BMT 2022). In the present study, we aim to evaluate the frequency of this new disease entity of AML with MDS-related mutations and evaluate the prognostic impact of allogeneic HCT in patients with MDS-related mutations achieving CR1. For comparison of the benefit from HCT, the mantel-Byar test was applied to take into account time-dependent covariate of HCT in CR1 to avoid immortal bias. Patients and Methods: We conducted a retrospective study that included 1,228 adult patients who were diagnosed with AML from January 1997 to January 2020and achieved CR1 in 5 centers (Toronto, Vancouver, and Montreal, Canada; Hwasun and Seoul, South Korea). Next-generation sequencing was performed as their standard clinical practice using targeted sequencing panel in each institution. As defined in ICC 2022, AML with myelodysplasia-related gene mutations was defined as the case of AML with ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, or ZRSR2 mutation(s). We considered allogeneic HCT in CR1 as a time-dependent covariate and used a Mantel-Byar test to avoid immortal bias. We identified 278 pts (22.6%) who had at least one MDS-related gene mutation at diagnosis. Relapse-free survival (RFS) at 2 years was selected for the primary endpoint, defined as the time from CR1 to relapse or death from any cause. EZR software version 1.41 was used throughout the study for statistical analysis. Results: The median follow-up duration among survivors after achieving CR1 was 16.5 months (mo) (Range: 1.3-217.9 mo). Between the HCT vs non-HCT group, there were no differences in sex, cytogenetic risk or other somatic mutation except NPM1 mutation which was more frequently detected in non-HCT group (p <0.001). As expected, younger pts were more frequently noted in HCT group, p=0.001). First, considering HCT as non-time dependent covariate, we have compared RFS and OS between HCT vs non-HCT groups. The 1 year RFS rate was superior in the HCT group (77.7%) to that in non-HCT group (49.9%, p<0.0001; Fig.1A). A similar trend was noted for the 1-year OS rate presenting higher OS rate in the HCT group (83.8%) than in the non-HCT group (72.7%, p=0.0003). Next, when HCT in CR1 was taken into account as a time-dependent (td) covariate, Mantel-Byar analysis showed a consistent finding of superior RFS in HCT group to non-HCT group: time dependent covariate of HCT in CR1 was found to be favorable (Hazard ratio [(HR) of 0.45, (95% C.I. [0.31-0.65]; p=0.00003)] as shown in Fig.1B which is a Simon-Makuch plot. In multivariate analysis (MVA) for RFS, time-dependent covariate of HCT in CR1 retained its statistical significance, thus concluding that HCT in CR1 is an independent prognostic factor for RFS (HR: 0.44, 95% CI [0.29-0.68], p =0.0002) together with other variables found to be adverse on RFS including monosomal karyotype (p=0.03), IDH1 mutation (p=0.01), and age > 60 years (p=0.002); while CEBPA (p=0.02) and NPM1 (p=0.049) mutations were found to be favorable on RFS in MVA. Conclusion: In our cohort of 1228 AML patients, we have identified 278 AML patients with myelodysplasia-related gene mutations (22.6%). We have confirmed that allogeneic HCT in CR1 is strongly recommended in a subgroup of AML with myelodysplasia-related gene mutations based on the favorable impact of HCT in CR1 on RFS from the present study. This result supports the urgent referral of this patient group for allogeneic HCT in CR1.

A global study for acute myeloid leukemia with RARG rearrangement.

Acute myeloid leukemia (AML) with retinoic acid receptor γ (RARG) rearrangement has clinical, morphologic, and immunophenotypic features similar to classic acute promyelocytic leukemia. However, AML with RARG rearrangement is insensitive to alltrans retinoic acid (ATRA) and arsenic trioxide (ATO) and carries a poor prognosis. We initiated a global cooperative study to define the clinicopathological features, genomic and transcriptomic landscape, and outcomes of AML with RARG rearrangements collected from 29 study groups/institutions worldwide. Thirty-four patients with AML with RARG rearrangements were identified. Bleeding or ecchymosis was present in 18 (54.5%) patients. Morphology diagnosed as M3 and M3v accounted for 73.5% and 26.5% of the cases, respectively. Immunophenotyping showed the following characteristics: positive for CD33, CD13, and MPO but negative for CD38, CD11b, CD34, and HLA-DR. Cytogenetics showed normal karyotype in 38% and t(11;12) in 26% of patients. The partner genes of RARG were diverse and included CPSF6, NUP98, HNRNPc, HNRNPm, PML, and NPM1. WT1- and NRAS/KRAS-mutations were common comutations. None of the 34 patients responded to ATRA and/or ATO. Death within 45days from diagnosis occurred in 10 patients (∼29%). At the last follow-up, 23 patients had died, and the estimated 2-year cumulative incidence of relapse, event-free survival, and overall survival were 68.7%, 26.7%, and 33.5%, respectively. Unsupervised hierarchical clustering using RNA sequencing data from 201 patients with AML showed that 81.8% of the RARG fusion samples clustered together, suggesting a new molecular subtype. RARG rearrangement is a novel entity of AML that confers a poor prognosis. This study is registered with the Chinese Clinical Trial Registry (ChiCTR2200055810).

Biallelic disruption of DDX41 activity is associated with distinct genomic and immunophenotypic hallmarks in acute leukemia.

Inherited DDX41 mutations cause familial predisposition to hematologic malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), with the majority of DDX41 mutated MDS/AMLs described to date harboring germline DDX41 and co-occurring somatic DDX41 variants. DDX41-AMLs were shown to share distinguishing clinical features such as a late AML onset and an indolent disease associated with a favorable outcome. However, genotype-phenotype correlation in DDX41-MDS/AMLs remain poorly understood. Here, we studied the genetic profile, bone marrow morphology and immunophenotype of 51 patients with DDX41 mutations. We further assessed the functional impact of ten previously uncharacterized DDX41 variants of uncertain significance. Our results demonstrate that MDS/AML cases harboring two DDX41 variants share specific clinicopathologic hallmarks that are not seen in other patients with monoallelic DDX41 related hematologic malignancies. We further showed that the features seen in these individuals with two DDX41 variants were concordant with biallelic DDX41 disruption. Here, we expand on previous clinicopathologic findings on DDX41 mutated hematologic malignancies. Functional analyses conducted in this study unraveled previously uncharacterized DDX41 alleles and further illustrate the implication of biallelic disruption in the pathophysiology of this distinct AML entity.

Identification of a novel NPM1 mutation in acute myeloid leukemia.

e19019 Background: Nucleophosmin (NPM1) is predominant nucleolar localization. NPM1-mutant acute myeloid leukemia (AML) account for 25-35% of adult AML patients, has been defined as a distinct AML entity in the 2022 WHO classification. The majority of NPM1 mutations reported affect exon 12 (classical type A mutation), account for 75-80% of adult NPM1-mutated AML cases, which generate an extra nuclear export signal (NES) in the C-terminal leading to aberrant cytoplasmic localization. Similar aberrant cytoplasmic translocation has also been reported in NPM1 mutants that identified in exons 9 and 11. Identification of novel non-type A mutants is paramount for diagnosis, risk stratification, and prognosis for targeted population. Methods: Somatic mutation analysis is performed on 566 AML patients using next-generation sequencing (NGS). Immunofluorescence and immunohistochemistry were used to identify the cytological localization of mutant NPM1. HEK-293T cells transiently transfected with plasmids for ectopic expression of the GFP-mutated NPM1 fusion proteins were treated with the specific Crm1/XPO1 inhibitor leptomycin B and to evaluate the NES dependence of their subcellular localization. Results: We identified one AML patient with NPM1 mutant located on exon 5. This patient was a 59-year-old female with de novo AML, with an 18-nucletide in-frame insertion at position 405 (c.405_406insGCCCTGGAACTGGGGAAC, named MutSong) in the middle of exon 5 [variant allele fraction (VAF), 11.9%]. It generated a new NPM1 mutant protein (p.135insALELGN), and containing a leucine-rich NES. Notably, different with all NPM1 mutant in other exons reported so far, our exon 5 mutant retained the functional c-terminal but insert one leucine-rich NES, similar finding was recently reported in NPM1 mutants that identified in exon 5 but on different position. This NPM1 mutant in exon 5 resulted in cytoplasmic localization both by confocal and immunohistochemistry in AML patient sample. HEK-293T overexpressing the new GFP-NPM1 exon 5 fusion protein indicated the aberrant localization in the cytoplasm and partially in nucleoli. Moreover, NES-dependent cytoplasmic localization was inhibited by exportin-1 inhibitor leptomycin B, indicated that the novel NES generated by exon 5 mutant is responsible for cytoplasmic localization. This patient carried WT1, IKZF1, JAK2, and NUP98 mutations, and was intravenously treated with daunorubicin plus cytarabine for induction followed by 3 cycles of intermediate-dose cytarabine as consolidation. Then the patient received allogeneic hematopoietic stem cell transplantation (allo-HSCT). Unfortunately, the patient died of severe pulmonary infection and viral encephalitis 14 months after the initial remission, 7 months after allo-HSCT. Conclusions: Our finding of the novel NPM1 mutation in exon 5 supported that beside exon 12, exon 5 mutant is another NPM1 “born to be exported” mutant critical for leukemogenesis.

UBTF tandem duplications define a distinct subtype of adult de novo acute myeloid leukemia

Tandem duplications (TDs) of the UBTF gene have been recently described as a recurrent alteration in pediatric acute myeloid leukemia (AML). Here, by screening 1946 newly diagnosed adult AML, we found that UBTF-TDs occur in about 3% of patients aged 18–60 years, in a mutually exclusive pattern with other known AML subtype-defining alterations. The characteristics of 59 adults with UBTF-TD AML included young age (median 37 years), low bone marrow (BM) blast infiltration (median 25%), and high rates of WT1 mutations (61%), FLT3-ITDs (51%) and trisomy 8 (29%). BM morphology frequently demonstrates dysmyelopoiesis albeit modulated by the co-occurrence of FLT3-ITD. UBTF-TD patients have lower complete remission (CR) rates (57% after 1 course and 76% after 2 courses of intensive chemotherapy [ICT]) than UBTF-wild-type patients. In patients enrolled in the ALFA-0702 study (n = 614 patients including 21 with UBTF-TD AML), the 3-year disease-free survival (DFS) and overall survival of UBTF-TD patients were 42.9% (95%CI: 23.4–78.5%) and 57.1% (95%CI: 39.5–82.8%) and did not significantly differ from those of ELN 2022 intermediate/adverse risk patients. Finally, the study of paired diagnosis and relapsed/refractory AML samples suggests that WT1-mutated clones are frequently selected under ICT. This study supports the recognition of UBTF-TD AML as a new AML entity in adults.

Highlights from Recent Cancer Literature.

Highlights from Recent Cancer Literature.

Molecular cytogenetics in acute myeloid leukemia in adult patients: practical implications.

Throughout the last 50 years, cytogenetic analyses of pretreatment bone marrow and / or blood samples from patients diagnosed with acute myeloid leukemia (AML) revealed a large number of recurring chromosome aberrations, both structural and numerical. Using standard banding methods and, more recently, molecular cytogenetic techniques, such as fluorescence in situ hybridization, spectral karyotyping, multiplex fluorescence in situ hybridization and comparative genomic hybridization, cytogenetic investigations detect acquired abnormalities that, together with submicroscopic gene mutations and changes in gene expression, strongly influence the clinical features and prognosis of patients with AML. Selected reciprocal translocations and inversions and their molecular counterparts, as well as a number of unbalanced chromosome abnormalities are used, together with bone marrow morphology, immunophenotype, and clinical characteristics, to define separate AML entities in the World Health Organization Classification of Haematolymphoid Tumours. Moreover, cytogenetic findings (and specific gene mutations) are being used in geneticrisk classifications, such as the 2022 European LeukemiaNet classification. Such classifications divide patients into broad prognostic categories: favorable, intermediate, and adverse, which are useful in the management of adults with AML. In this article, I review the present data on recurrent chromosome rearrangements in AML and on correlations between cytogenetic findings and clinical features and treatment outcomes of adult patients diagnosed with AML.

Alternative chemotherapy for acute myeloid leukemia with Down syndrome

Abstract Myeloid leukemia with Down syndrome (ML-DS) is a unique entity of acute myeloid leukemia (AML) with superior treatment response and overall survival compared with children with AML. However, despite the advances in treatment approaches, ML-DS survival rates for children in low- and middle-income countries remain poor. In this article, we describe 3 cases of ML-DS, which were treated with a novel protocol using vincristine, cytarabine, and daunorubicin plus triple intrathecal drugs. All the 3 patients successfully finished the treatment, with 2 patients in complete remission until now. One patient died because of uncontrolled bleeding for 2 days after finishing the chemotherapy regimen. Our findings indicate that using our treatment protocol, ML-DS is treatable in limited-resource settings such as that in Manado, Indonesia.

EAPB0503, an Imidazoquinoxaline Derivative Modulates SENP3/ARF Mediated SUMOylation, and Induces NPM1c Degradation in NPM1 Mutant AML.

Nucleophosmin-1 (NPM1) is a pleiotropic protein involved in numerous cellular processes. NPM1 shuttles between the nucleus and the cytoplasm, but exhibits a predominant nucleolar localization, where its fate and functions are exquisitely controlled by dynamic post-translational modifications (PTM). Sentrin/SUMO Specific Peptidase 3 (SENP3) and ARF are two nucleolar proteins involved in NPM1 PTMs. SENP3 antagonizes ARF-mediated NPM1 SUMOylation, to promote ribosomal biogenesis. In Acute Myeloid Leukemia (AML), NPM1 is frequently mutated, and exhibits an aberrant cytoplasmic localization (NPM1c). NPM1c mutations define a separate AML entity with good prognosis in some AML patients, rendering NPM1c as a potential therapeutic target. SENP3-mediated NPM1 de-SUMOylation induces resistance to therapy in NPM1c AML. Here, we demonstrate that the imidazoquinoxaline EAPB0503 prolongs the survival and results in selective reduction in the leukemia burden of NPM1c AML xenograft mice. Indeed, EAPB0503 selectively downregulates HDM2 expression and activates the p53 pathway in NPM1c expressing cells, resulting in apoptosis. Importantly, we unraveled that NPM1c expressing cells exhibit low basal levels of SUMOylation paralleled with high SENP3 and low ARF basal levels. EAPB0503 reverted these molecular players by inducing NPM1c SUMOylation and ubiquitylation, leading to its proteasomal degradation. EAPB0503-induced NPM1c SUMOylation is concurrent with SENP3 downregulation and ARF upregulation in NPM1c expressing cells. Collectively, these results provide a strong rationale for testing therapies modulating NPM1c post-translational modifications in the management of NPM1c AML.

AML with germline DDX41 variants is a clinicopathologically distinct entity with an indolent clinical course and favorable outcome.

Germline DDX41 variants in myeloid neoplasms (MNs) are not uncommon, and we explored the prevalence and characterized the clinical and pathologic features in a cohort of 3132 unrelated adult MN patients. By targeted next-generation sequencing, we identified 28 patients (20 men and 8 women) with pathogenic germline DDX41 variants who developed acute myeloid leukemia (AML), in which only 3 (11%) had a family history (FH) of MNs. A subacute clinical course of cytopenia (mean duration of 11.2 months, range 0-72 months) prior to the initial AML diagnosis was accompanied by a low blast count (median at 30%, range 20-70%) in hypocellular marrows (93% of all patients), in vast contrast to the typical proliferative subtypes of AML in the elderly. Most patients had a normal karyotype (75%) and acquired a second DDX41 variant (69%). A favorable overall survival (OS) was observed in comparison to that of common subtypes of AML with wild-type DDX41 in age-matched patients. Our study demonstrated that the frequent germline pathogenic DDX41 variants characterized a clinically distinct AML entity. Features characteristic of DDX41-mutated AML include male predominance, often lack of FH, indolent course, low proliferative potential, frequent somatic DDX41 variants, and a favorable OS.

The correlation of next-generation sequencing-based genotypic profiles with clinicopathologic characteristics in NPM1-mutated acute myeloid leukemia

The purpose of this study was to analyze the association between next-generation sequencing (NGS) genotypic profiles and conventional clinicopathologic characteristics in patients with acute myeloid leukemia (AML) with NPM1 mutation (NPM1mut). We selected 238 NPM1mut patients with available NGS information on 112 genes related to blood diseases using the χ2 and Mann-Whitney U tests and a multivariable logistic model to analyze the correlation between genomic alterations and clinicopathologic parameters. Compared with the NPM1mut/FLT3-ITD(−) group, the NPM1mut/FLT3-ITD(+) group presented borderline frequent M5 morphology [78/143 (54.5%) vs. 64/95 (67.4%); P = 0.048], higher CD34- and CD7-positive rates (CD34: 20.6% vs. 47.9%, P < 0.001; CD7: 29.9% vs. 61.5%, P < 0.001) and a lack of favorable−/adverse-risk karyotypes (6.4% vs. 0%; P = 0.031). In the entire NPM1mut cohort, 240 NPM1 mutants were identified, of which 10 (10/240, 4.2%) were missense types. When confining the analysis to the 205 cases with NPM1mut insertions/deletions-type and normal karyotype, multivariable logistic analysis showed that FLT3-ITD was positively correlated with CD34 and CD7 expressions (OR = 5.29 [95% CI 2.64–10.60], P < 0.001; OR = 3.47 [95% CI 1.79–6.73], P < 0.001, respectively). Ras-pathway mutations were positively correlated with HLA-DR expression (OR = 4.05 [95% CI 1.70–9.63], P = 0.002), and KRAS mutations were negatively correlated with MPO expression (OR = 0.18 [95% CI 0.05–0.62], P = 0.007). DNMT3A-R882 was positively correlated with CD7 and HLA-DR expressions (OR = 3.59 [95% CI 1.80–7.16], P < 0.001; OR = 13.41 [95% CI 4.56–39.45], P < 0.001, respectively). DNMT3A mutation was negatively correlated with MPO expression (OR = 0.35 [95% CI 1.48–8.38], P = 0.004). TET2/IDH1 mutations were negatively correlated with CD34 and CD7 expressions (OR = 0.26 [95% CI 0.11–0.62], P = 0.002; OR = 0.30 [95% CI 0.14–0.62], P = 0.001, respectively) and positively correlated with MPO expression (OR = 3.52 [95% CI 1.48–8.38], P = 0.004). In conclusion, NPM1mut coexisting mutations in signaling pathways (FLT3-ITD and Ras-signaling pathways) and methylation modifiers (DNMT3A and TET2/IDH1) are linked with the expressions of CD34, CD7, HLA-DR and MPO, thereby providing a mechanistic explanation for the immunophenotypic heterogeneity of this AML entity.

Mutational Landscape and Clinical Outcome Associated with Different Rearrangements Involving 11q23/ MLL in De Novo Acute Myeloid Leukemia (AML)

Mutational Landscape and Clinical Outcome Associated with Different Rearrangements Involving 11q23/ MLL in De Novo Acute Myeloid Leukemia (AML)

Genomic Landscape of Splicing Factor Mutant Acute Myeloid Leukemia

Mutations in components of the RNA-splicing machinery are among the most prevalent genomic alterations in myelodysplastic syndromes (MDS, 45-85% based on disease subtype). With the advent of new powered genomic technologies, spliceosomal mutations have been also detected in acute myeloid leukemia (AML). AML cells have shown to express a large number of alternative spliced events with splicing factor (SF) mutations altering pre-mRNA splicing of downstream targets. How these mutations mediate, modify or support leukemogenic transformation is still unknown. One could think that indirectly the targets of SF alterations could be unveiled through detailed analysis of the phenotype of AML patients carrying those events. Therefore, we aimed to describe the frequency of mutations, clinical associations and molecular cooperative lesions of the most common SF genes (SF3B1, SRSF2, U2AF1, ZRSR2) in AML to speculate unique molecular combinations contributing to disease phenotypes. We collected data of targeted deep sequencing and whole exome sequencing of a cohort of 2188 AML cases from the Cleveland Clinic (n=855) and publicly available data. In total, we had 1636 primary (p), 433 secondary (s) and 119 (t) AML. Eighteen% of AML patients (n=388) carried a mutation in SF (SF mutant, SF-MT) while 83% were wild type (SF-WT AML, n=1800). More specifically, the frequency of SF-MT individuals was 14% in pAML (n=234) and tAML (n=17) while a significantly higher frequency was observed in sAML (32%, n=137) as compared to pAML + tAML (P&amp;lt;0.0001). Next, we compared the baseline, clinical and genomic characteristics of SF-MT vs SF-WT AML to uncover invariant phenotypic features and molecular associations that could potentially define SF-AML. SF-MT patients were significantly older than SF-WT AML [71 vs 60 years; P&amp;lt;0.0001] with an 83% having and age ≥60 years vs 56% in each group, respectively (P&amp;lt;0.0001). SF-MT AML were significantly of male predominance as compared to SF-WT AML (72 vs 50%, P&amp;lt;0.0001). Percentages of anemia and thrombocytopenia were similar in both groups. Median bone marrow blasts% was lower in SF-MT compared to SF-WT AML (49 vs 61; P&amp;lt;0.0001). In terms of cytogenetic profile, SF-mutant AML had lower odds of normal karyotype compared to SF-WT AML (50% vs 59%; P=0.002). Trisomy 8 was significantly frequent in SF-MT AML (15 vs 8%; P&amp;lt;0.0001) while -5/del(5q) was more common in SF-WT AML (10 vs 5%, P=0.008). The molecular profile of SF-MT AML was characterized by significantly frequent mutations in ASXL1 (23 vs 5%, P&amp;lt;0.0001), BCOR/L1 (10 vs 3%, P&amp;lt;0.0001), CBL (5 vs 2%, P=0.001), ETV6 (4 vs 1%, P&amp;lt;0.0001), IDH2 (15 vs 10%, P=0.006), IDH2R140 (13 vs 8%, P=0.0009), JAK2 (5 vs 1%, P&amp;lt;0.0001), KRAS (6 vs 3%, P=0.03), MECOM (4 vs &amp;lt;1%, P=0.02), RUNX1 (29 vs 7%, P&amp;lt;0.0001), STAG2 (12 vs 5, P&amp;lt;0.0001), TET2 (19 vs 11%, P&amp;lt;0.0001) while mutations in DNMT3A (15 vs 26%, P&amp;lt;0.0001), FLT3ITD (18 vs 10%, P=0.005), NPM1 (32 vs 11%, P&amp;lt;0.0001), PTPN11 (8 vs 4%, P=0.005), TP53 (8 vs 4%, P=0.002) shaped the SF-WT AML profile. Survival analyses showed that SF-MT AML had dismal survival outcome compared to SF-WT AML (17.4 vs 26.2 mo., P&amp;lt;0.0001). We then used multivariate cox proportional hazards to identify genomic features associated with survival in each group separately and found that favorable features included NPM1MT in both groups and biallelic CEBPA in SF-WT AML. On the other hand, adverse genomic lesions included complex karyotype, DNMT3AMT, FLT3ITD-MT and TP53MT in both groups, in addition to KRASMT and PHF6MT in SF-MT while ASXL1MT, KDM6AMT, RUNX1MT in SF-WT AML. The comparison of OS between SF-MT vs SF-WT AML for the association of a specific genomic lesion (cytogenetic/gene abnormality) revealed that mutations in DNTM3A (17.8 vs 21.6 mo., P=0.02), ETV6 (17.4 vs 32.1 mo., P=0.04), KRAS (11.9 vs 28.7, P&amp;lt;0.0001), NRAS (12.9 vs 26.5 mo., P=0.0005), cohesins (12.1 vs 29.3 mo., P=0.0002) and TET2 (13.4 vs 28.1 mo., P=0.006) significantly contributed to shortened survival and worse outcomes. In sum, we described the presence of SF mutations in AML and defined the phenotypic features of this subset and its genomic associations as compared to SF-WT AML. We further highlighted the unique genomic contributors associated with their survival outcomes. Perhaps, future larger scale studies of this AML group will delineate its potential to represent a unique well-defined entity of AML. Disclosures Maciejewski: Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

The Role of Forkhead Box Proteins in Acute Myeloid Leukemia.

Forkhead box (FOX) proteins are a group of transcriptional factors implicated in different cellular functions such as differentiation, proliferation and senescence. A growing number of studies have focused on the relationship between FOX proteins and cancers, particularly hematological neoplasms such as acute myeloid leukemia (AML). FOX proteins are widely involved in AML biology, including leukemogenesis, relapse and drug sensitivity. Here we explore the role of FOX transcription factors in the major AML entities, according to “The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia”, and in the context of the most recurrent gene mutations identified in this heterogeneous disease. Moreover, we report the new evidences about the role of FOX proteins in drug sensitivity, mechanisms of chemoresistance, and possible targeting for personalized therapies.

Clinical Characteristics and Outcome in IDH1/2 Mutant AML Patients - Analysis of 3898 Newly Diagnosed Patients with Acute Myeloid Leukemia

Clinical Characteristics and Outcome in IDH1/2 Mutant AML Patients - Analysis of 3898 Newly Diagnosed Patients with Acute Myeloid Leukemia

Genetic heterogeneity of cytogenetically normal AML with mutations of CEBPA

AbstractBiallelic mutations of the CCAAT/enhancer binding protein α (CEBPA) gene define a distinct genetic entity of acute myeloid leukemia (AML) with favorable prognosis. The presence of GATA2 and CSF3R mutations that are specifically associated with this subgroup but not mutated in all samples suggests a genetic heterogeneity of biCEBPA-mutated AML. We characterized the mutational landscape of CEBPA-mutated cytogenetically normal AML by targeted amplicon resequencing. We analyzed 48 biallelically mutated CEBPA (biCEBPA), 32 monoallelically mutated CEBPA (moCEBPA), and 287 wild-type CEBPA (wtCEBPA) patient samples from German AML Cooperative Group studies or registry. Targeted sequencing of 42 genes revealed that moCEBPA patients had significantly more additional mutations and additional mutated genes than biCEBPA patients. Within the group of biCEBPA patients, we identified 2 genetic subgroups defined by the presence or absence of mutations in chromatin/DNA modifiers (C), cohesin complex (C), and splicing (S) genes: biCEBPACCSpos (25/48 [52%]) and biCEBPACCSneg (23/48 [48%]). Equivalent subgroups were identified in 51 biCEBPA patients from the Cancer Genome Project. Patients in the biCEBPACCSpos group were significantly older and had poorer overall survival and lower complete remission rates following intensive chemotherapy regimens compared with patients in the biCEBPACCSneg group. Patients with available remission samples from the biCEBPACCSpos group cleared the biCEBPA mutations, but most had persisting CCS mutations in complete remission, suggesting the presence of a preleukemic clone. In conclusion, CCS mutations define a distinct biological subgroup of biCEBPA AML that might refine prognostic classification of AML. This trial was registered at www.clinicaltrials.gov as #NCT00266136 and NCT01382147.

The Mutational Patterns Associated with Cytogenetic Subsets of De Novo Acute Myeloid Leukemia (AML): A Study of 1603 Adult Patients (Pts)

The classification of AML is based on the morphology of leukemic blasts, their immunophenotypes, and the presence of specific chromosomal abnormalities and selected gene mutations. It is widely acknowledged that some gene mutations are enriched in certain cytogenetic subsets, in which they may serve as therapeutic targets. We created a comprehensive oncoprint of mutations associated with centrally reviewed cytogenetic findings in 1603 adults (1080 aged <60 years, and 523 aged ≥60 years) with de novo AML (excluding acute promyelocytic leukemia) who were treated on CALGB/ALLIANCE protocols. The pts were classified into 5 cytogenetic subgroups: (1) cytogenetically normal (CN); (2) with complex karyotype (CK); (3) with t(8;21)(q22;q22) or inv(16)(p13.1q22)/t(16;16)(p13.1;q22), i.e., core-binding factor (CBF)-AML; (4) with balanced rearrangements other than t(8;21) and inv(16)/t(16;16); (5) with unbalanced chromosomal abnormalities in non-complex karyotype.Using a targeted next-generation sequencing panel, we analyzed the mutational status of 80 cancer- and leukemia-associated genes. The mutated genes were assigned to 9 previously described by the Cancer Genome Atlas Research Network (N Engl J Med. 2013;368:2059) functional groups: chromatin remodeling, cohesin complex, kinases, methylation-related, NPM1, RAS pathway, spliceosome, transcription factors, and tumor suppressors. The mutational oncoprint revealed major differences in gene mutation frequencies among the 5 major cytogenetic groups.CN-AML pts had a broad mutational spectrum, involving all functional groups. Mutations in methylation-related genes (61% of pts), NPM1 (57%), and kinases (46%) dominated the mutation pattern.TP53 mutations were most common in pts with CK (38%). The second most common mutation, TET2, was detected only half as often (17%). The mutation patterns associated with "typical" (containing abnormalities resulting in loss of chromosome material from 5q, 7q and/or 17p), and "atypical" (without the aforementioned abnormalities) CKs differed. TP53 mutations were present in 52% of pts with typical but only in 5% of pts with atypical CKs (P<0.001). Conversely, the latter pts harbored mutations in another tumor suppressor gene, PHF6, more often (15% vs. 2%, P=0.03). Furthermore, atypical CKs had a broader spectrum of recurrent mutations, with 6 mutations occurring in ≥10% of pts (IDH2, NPM1, PHF6, TET2 and ZRSR2 mutations and FLT3-TKD), compared with only 2 genes, TP53 and TET2, mutated in ≥10% of pts with typical CKs, suggesting major biological differences between typical and atypical CKs.CBF-AML pts with had few detectable mutations, with a median of 1 (range, 0-5) versus 3 mutations (range, 0-9) detected in the remaining AML pts (P<0.001).In AML with balanced rearrangements, AML pts with MLL-rearrangements often had mutations in RAS pathway genes, whereas they rarely harbored mutations in other functional groups compared with pts with non-MLL-rearranged balanced translocations [MLL-rearranged vs. non-MLL-rearranged: chromatin remodeling, 9% vs. 26%; methylation-related, 12% vs. 23%; spliceosome, 12% vs. 24%]. Some cytogenetic subgroups with particular recurrent chromosome abnormalities presented with specific molecular features, supporting claims that they represent separate AML entities. For instance, 51% of pts with t(6;11)(q27;q23) harbored KRAS mutations, compared with only 3% of all remaining pts. Among t(9;22)(q34;q11.2)-positive pts, 47% had mutations in RUNX1,and 27% mutations in ZRSR2. Pts with inv(3)(q21q26.2/t(3;3)(q21q26.2) had frequent mutations in SF3B1 and BCOR (both 38%).Pts with gains or losses of chromosome material in non-complex karyotypes (unbalanced chromosomal abnormalities) were characterized by high mutation frequencies in methylation-related (55%) and spliceosome genes (34%). Strikingly, spliceosome mutations were enriched in pts with chromosome gains, with almost half of these pts harboring one or more mutations. Except for pts with sole +11, who most often harbored U2AF1 mutations (43%), SRSF2 was the most frequently mutated spliceosome gene in pts with unbalanced chromosomal abnormalities.Our comprehensive depiction of frequencies of single gene mutations and functional groups in recurrent cytogenetic subsets may be useful in guiding mutation testing in AML pts, and result in more focused application of targeted therapy of AML. DisclosuresWang:Immunogen: Research Funding; Incyte: Speakers Bureau. Stone:Pfizer: Consultancy; Sunesis Pharmaceuticals: Consultancy; Novartis: Consultancy; Merck: Consultancy; Karyopharm: Consultancy; Agios: Consultancy; Juno Therapeutics: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy; ONO: Consultancy; Celator: Consultancy; Amgen: Consultancy; Jansen: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Xenetic Biosciences: Consultancy; Roche: Consultancy.