Aberrations In Acute Myeloid Leukemia Research Articles

IKZF2 Specifically Participates in AML1-ETO Mediated Blockage of Myeloid Differentiation

Objectives:The chromosomal translocation (8;21) is one of the most recurrent cytogenetic aberrations in acute myeloid leukemia (AML). Although most patients achieve complete remission, one third still relapse due to the persistent leukemia stem cells (LSCs). The lack of significant improvements in outcomes, especially in elderly patients ineligible for standard chemotherapy, has spurred further investigation in t(8;21) leukemia. In this study, we investigate the mechanism of AML1-ETO (AE) fusion gene induced differentiation blockage from earlier stages of HSPCs, aiming to identify potential therapeutic targets that can effectively overcome treatment resistance and minimize long-term side effects. Methods:The AML1-ETO conditional knock-in mouse (Aml1 Eto/+; Mx1-Cre) was previously established, hereafter called AE KI. The Cas9-expressing AE KI model, generated by crossing AE KI and Cas9 mice strains, was employed to define the regulatory program of AE fusion gene. For knockout of AE and IKZF2, Kasumi-1 cells were transduced with the single-vector lentiviral system containing sgRNAs for the respective genes, or a nontargeting control. Colony formation and serial bone marrow transplantation assay were performed to evaluate the function of HSPCs. ATAC sequencing, transcriptional profiling (RNA-seq) and Cut&Tag for H3K4me3, H3K27me3, H3K27ac were performed and analyzed comprehensively to characterize the regulatory profile of AE fusion gene. Results:Our previous research demonstrated that induction expression of AE fusion gene in vivo resulted in excessive accumulation of Lin -Sca-1 +c-Kit + (LSK) cells, leading to differentiation blockage from HSCs to HPCs. IKZF2 showed significantly higher expression in LSK cells following PIPC induction of AE expression compared to that of the control group. IKZF2 was highly increased in AML patients harboring AE fusion gene. And the level of IKZF2 was significantly higher in human t(8;21) cell lines Kasumi-1 and SKNO-1 when compared to that of non-AE controls. Depletion of the AE fusion gene led to a dramatic decrease in IKZF2 expression, while constitutive expression of the AE fusion protein in U-937 cells resulted in elevated levels of IKZF2. Loss of IKZF2 resulted in apoptosis in AE-expressing leukemia cells, and led to an increase in the myeloid differentiation markers CD11b and CD14. These findings suggested a functional relevance of IKZF2 in AE-driven AML. In our Cas9-expressing AE KI model, a significant improvement in the peripheral chimerism rate was observed following IKZF2 knockout at various time points. The population of HSCs, mainly the ST-HSCs, was reduced, while the more differentiated MPP and Lin -Sca-1 -c-Kit + (LK) pools, especially the GMP and CMP groups, were expanded. These findings indicated that the differentiation block triggered by AE was partially relieved by IKZF2 knockout. Furthermore, the deletion of IKZF2 had minimal impact on normal myeloid differentiation, highlighting its specific role in AE settings. We further investigated the underlying mechanisms through multi-omics analysis. The induction of AE led to increased accessibility of the myeloid differentiation inhibitory elements, including IKZF2. CUT&Tag data confirmed a significant decrease in H3K27me3 signals at the promoter regions of IKZF2 compared to that of the control group. Regarding the role of IKZF2 in chromatin remodeling, it is likely to interact with AE to form an epigenetic-transcriptional control module. This may expand the signal for differentiation blockage and drive critical leukemogenic gene expression profiles. Conclusions: Our results demonstrate that IKZF2 is a target gene of AML1-ETO crucial for AML1-ETO-induced differentiation blockage from HSCs to HPCs. The epigenetic regulation mechanism may contribute to the AML1-ETO-IKZF2 regulatory axis. And targeting IKZF2 may become a potential therapeutic strategy in human t(8;21) AML especially for the eradication of AML1-ETO-associated LSCs.

ETV6-ABL1 Fusion Gene Was Associated with a Poor Prognosis in Patients with Acute Myeloid Leukemia

Background:Acute myeloid leukemia (AML) is commonly characterized by chromosomal abnormalities that result in the formation of chimeric genes involved in leukemogenesis. ETV6-ABL1-a fusion gene results from a complex rearrangement involving translocation, inversion, or insertion of ETV6 into chromosomal band 9q34 or ABL1 into 12p13. This rare but recurrent genetic aberration is found in AML patients and is associated with a poor prognosis. Methods:A retrospective review was conducted of three relapsed AML patients with ETV6-ABL1 fusion genes screened by multiplex RT-PCR who were treated at Beijing Lu Daopei Hospital and Hebei Yanda Lu Daopei Hospital between November 2019 and January 2022. Results:At diagnosis, the median age was 32 years (range 32-42 years), and two patients were male(Pt.2,3). AML subtypes included M2 (Pt.2) and M4 (Pt.1,3), and no patient had a prior history of myeloproliferative neoplasms. Clinical and laboratory characteristics at diagnosis included: hepatosplenomegaly (Pt.3), lymphadenopathy (Pt.1), leukocytosis (median 209.71×10 9 /L, range, 60.28-687.2×10 9/L),but no significant eosinophilia(≥1.5×10 9/L). Cytogenetic analyses showed a normal karyotype in all three patients. FLT3-ITD gene mutation was found in two patients(Pt.1,2). All there patients received the same IA regimen(idarubicincytarabine) as induction chemotherapy and achieved complete remission, followed by multi-agent chemotherapy as consolidation treatment.Pt.1 received additional targeted therapy with sorafenib. However, after a median duration of remission (DOR) of 4 months (range 3-8 months), all patients experienced relapse and sought for further therapy at our hospital. Upon re-evaluation by laboratory tests, ETV6-ABL1 fusion gene was detected by multiplex RT-PCR, and additional NUP98-NSD1 fusion was also found in Pt.2. FLT3-ITD gene mutation was confirmed in Pt. 1 and Pt. 2, but the characteristic t(9;12) (q34;p13) chromosomal abnormalities were not present in any of the patients. Pt. 2 presented with an abnormal karyotype of 46, XY, t(3;17)(q12;p11.2),add (10)(q22)[5] / 46,XY[15], while the rest 2 patients had a normal karyotype. Pt.1 died rapidly from serious infection after relapse. Pt. 2 and Pt. 3 received dasatinib-containing combination regimens as salvage therapy and achieved partial remission. Pt. 2 underwent haploidentical allogeneic hematopoietic stem cell transplantation (allo-HSCT) with an enhanced myeloablative conditioning regimen in March 2021, and has maintained molecular remission until now. Pt. 3 is currently undergoing HSCT preparation. Conclusion: ETV6-ABL1 fusion gene is a rare but recurrent genetic aberration in AML, and its rearrangement is not uniform across eachpatient, typically involving cryptic insertions. Routine chromosome G-banding analysis may not identify this fusion gene, and the combined use of fluorescence in situ hybridization and PCR is recommended for better detection for ETV6-ABL1. Patients with this fusion gene have a high relapse rate and a poor prognosis. Tyrosine kinase inhibitors (eg. Dasatinib)are a reasonable treatment option, and allo-HSCT may be considered for potential cure.

Somatic exonic deletions in RUNX1 constitutes a novel recurrent genomic abnormality in acute myeloid leukemia.

In acute myeloid leukemia (AML), somatic mutations (commonly missense, nonsense and frameshift indels) in RUNX1 are associated with a dismal clinical outcome. Inherited RUNX1 mutations cause familial platelet disorder. As approximately 5-10% of germline RUNX1 mutations are large exonic deletions, we hypothesized that such exonic RUNX1 aberrations may also be acquired during the development of AML. Sixty well-characterized AML patients were analyzed with Multiplex Ligation-dependent Probe Amplification (MLPA, n=60), micro-array (n=11) and/or whole genome sequencing (WGS, n=8). In total, 25 (42% of the cohort) RUNX1-aberrant patients (defined by the presence of classical mutations and/or exonic deletions) were identified. Sixteen patients (27%) carried only exonic deletions, 5 (8%) carried classical mutations and 4 (7%) carried both exonic deletions and mutations. No significant difference was observed between patients with classical RUNX1 mutations and RUNX1 exonic deletions in median overall survival (OS, 53.1 vs 38.8 months respectively, p=0.63). When applying the European Leukemia Net (ELN) classification including the RUNX1-aberrant group, 20% of the patients initially stratified as intermediate-risk (5% of the whole cohort) were re-assigned to the high-risk group, which improved the performance of ELN classification regarding OS between intermediate and high-risk group (18.9 vs 9.6 months, p=0.09). Somatic RUNX1 exonic deletions constitute a novel recurrent aberration in AML. Our findings have important clinical implications regarding AML classification, risk-stratification and treatment decision. Moreover, they argue in favor of further investigating such genomic aberrations not only in RUNX1 but also in other genes implicated in cancer biology and management.

Frequency and pattern of chromosomal abnormalities in acute myeloid leukemia from Western India: A retrospective study

Chromosomal abnormalities play an important role in diagnosis and prognosis of hematological diseases. The aim of the present study was to study the pattern and frequency of chromosomal aberrations in acute myeloid leukemia (AML) subgroups from western India. A retrospective study was conducted through evaluating laboratory proforma which were filled during 2005 to 2014 for diagnosis and treatment of AML subjects. We have studied chromosomal aberrations in 282 subjects with AML from western India. AML patients were sub-grouped according to FAB classification. Cytogenetic study using conventional cytogenetics (GTG-banding) and Fluorescence in situ hybridization (FISH) was carried out using FISH probes (AML1/ETO, PML/RARA, CBFB). Student's t test for continuous variables and Pearson's Chi-squared test for categorical variables were used to identify the relationship between variables. Cytomorphological study revealed AML- M3 as most frequent (32.3%) group followed by AML-M2 (25.2%) and AML-M4 (19.9%). Chromosomal abnormalities were identified in 145 (51.42%) of the total AML cases. A high frequency (38.6%) of chromosomal abnormalities was identified in AML-M3 subgroup as compared to AML-M2 (31%) and AML-M4 (20.6%). Cytogenetic study is important for the diagnosis and management of the AML patients. Our study identified chromosomal abnormalities in AML subgroups with varied frequencies. It is important in diagnosis and monitoring of the disease. As younger AML patients were more affected in our study, etiological factors such as environmental factors need to be studied. Combination of conventional cytogenetics and FISH has an advantage of identifying high frequency of chromosomal aberrations in AML patients.

Identification of novel PIEZO1::CBFA2T3 and INO80C::SETBP1 fusion genes in an acute myeloid leukemia patient by RNA-seq.

Fusion genes are recurrent molecular aberrations in acute myeloid leukemia, with significant diagnostic and therapeutic value. The identification of novel fusion genes provides advanced biomarkers for diagnosis and facilitates the discovery of drug targets. Bone marrow sample was extracted from an acute myeloid leukemia patient and RNA-sequencing was performed. Several bioinformatic methods, including differential analysis and Gene Set Enrichment Analysis (GSEA) pathway analyses were conducted based on the expression data. Two novel fusion genes, PIEZO1::CBFA2T3 and INO80C::SETBP1, were identified by RNA-seq.Differential analysis found that SETBP1 and CBFA2T3 were overexpressed, and GSEA analysis showed the activation of immune-related pathways. These findings indicate dysfunction of the fusion related- genes and possible pathogenic effect of the fusion genes. We reported a male AML patient with presence of PIEZO1::CBFA2T3 and INO80C::SETBP1 fusion genes.

Molecular Aberrations in Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome with Excess Blast II (MDS-EB II) in Kuwait

Introduction: AML is a heterogeneous disease. Different mutational profiles have been described and correlate with variable outcomes. Genetic abnormalities are not well-described in our region. Our aim is to describe the molecular aberrations observed in the first cohort of patients. We recently introduced DNA based Oncomine Myeloid Gene Panel testing 55 genes. Method: we collected clinical, cytogenetic and molecular data prospectively since we introduced the test in late 2020. We also reviewed medical charts to collect baseline clinical data. We included patients aged 16 yrs and above with AML or MDS-EB II treated at Kuwait Cancer Center. Results: fifty-seven patients were included in the study with median age of 51 yrs. Almost one third of the patients were Kuwaitis (36%). Baseline demographic described in (table 1). We have 53 patients with AML (92%), 3 patients with MDS-EB II (5%) and 1 pt with chronic myeloid leukemia in myeloid blast crisis (CML-MBC). Most of the patients had at least one mutation (91%) detected by the panel. The most common mutations (figure1) were FLT3 (ITD & D835) detected 26.3%, followed by NPM-1 detected in 21%. We have noticed an increased PTPN11 mutation, detected in 17.5% of the cases. Conclusion: This is an important study showing extensive molecular abnormalities in AML patients in the region. This will facilitate more understanding of the disease biology and potentially guide therapeutic options

Epigenetic Landscape Predicts Overall Survival and Remission Duration in Adult Acute Myeloid Leukemia

INTRODUCTION: Epigenetic dysregulation is a hallmark of Acute Myeloid Leukemia (AML). Although recurrent epigenetic-related mutations have been identified, the understanding of how they affect pathophysiology and leukemogenesis remains incomplete. We used proteomics to assess the epigenetic landscape in AML and identified new prognostic phenotypes and therapeutic targets. METHODS: Using Reverse Phase Protein Array (RPPA) 405 proteins (327 total; 78 post-translational modified) were measured in 818 samples from newly diagnosed adult AML patients and normalized to CD34+ cells. These included Histone H3 total and H3K4Me 1, 2, 3, K9Me2, K27Me3, K27Ac and K36Me3 Modification Marks (HMM) expression. Categorical clinical variables were compared with Fisher's Exact with simulated p-values (10000 replicates), and continuous ones with Wilcoxon or Kruskal-Wallis tests. Univariate (UV) and Multivariate (MV) models for Overall Survival (OS) and Remission Duration (RD) were built with Cox proportional hazards model (CoxPH). Differentially Expressed Proteins were determined using the Kruskal-Wallis test (p<0.05) and pairwise Dunn's tests with p-values adjusted with the False Discovery Rate method (p<0.05). RESULTS: Using unbiased hierarchical clustering, patients were clustered into 4 groups (Fig. 1, C1 to C4) based on the expression of 2 Repressive HMM (H3K27Me3 and H3K9Me2), and 5 Activating HMM (H3K27Ac, H3K36Me3, H3K4Me3, H3K4Me2, H3K4Me1). High repression and low activation HMM were prominent in C1, while C4 had the opposite pattern. Higher expression of all marks and Total H3 defined C2, and C3 had lower expression of all marks, and Total H3. Importantly, when normalized by Total H3, HMM expression of both C2 and C3 are close to average, representing a chromatin state between C1 and C4. Compared to the other groups, patients in C4 were older (66yo vs 62yo; p=0.006), and had more secondary AML (55% vs 43%; p=0.007) and unfavorable cytogenetic risk (55% vs 37%; p<0.001). Mutations weren't exclusively associated with any cluster: ASLX1 and NPM1 mutants were more frequent in C1 and C2 (27% vs 13%; p<0.001; 23% vs 15%; p=0.015), WT1 more in C1 and C3 (9% vs 3.7%, p=0.018), and EZH2, PTPN11, RAS and RUNX1 happened more in C3 and C4 (7.7% vs 1.7%, p<0.001; 13% vs 5.6%, p=0.002; 26% vs 14%, p<0.001; 22% vs 14%, p=0.015). The effect of HMM expression on OS (Fig. 2) showed that patients with high repression and low activation (C1) did significantly better (Median OS (MS) = 19mo, Median RD (MRD)>102mo), and those with the opposite pattern of low repression and high activation (C4) did significantly worse (MS=7mo, MRD=16mo). Patients with an intermediary chromatin state were close to the average OS (C2: MS=14mo, MRD=20mo; C3: MS=12mo, MRD=24mo). In the UV CoxPH model, C1, C3 and C4 predicted OS (HR=1, 1.3, 1.4, 2.3; p=ref., 0.007, 0.001), as did age, secondary AML, complex karyotype, and CEBPA, FLT3, KIT, JAK2, NPM1, PTPN11, SRSF2 and TP53 mutations. In the MV model C1, C3 and C4 were again predictive for OS (HR=1, 1.5, 2.4, 2.7; p=ref., 0.003, 0.006), together with age, secondary AML, and PTPN11, SRSF2 and TP53 mutations. For RD, in the UV model, all clusters predicted RD (HR=1, 1.9, 1.8, 2.7; p=ref., 0.002, 0.005, 0.001), as did age, secondary AML, complex karyotype, and FLT3, NPM1, RUNX1 and TP53 mutations. In the MV model of RD, all clusters were predictors (HR=1, 1.8, 1.8, 2.7; p=ref., 0.045, 0.038, 0.003), together with age, complex karyotype and RUNX1 mutation. Differentially Expressed Proteins, from the other 397 proteins, for C4 revealed high activation of VEGF, ErbB and FcεRI signaling pathways, unveiling potential new druggable targets. CONCLUSION: HMM expression found that opposing chromatin states, with a more closed (C1) or open (C4) chromatin, significantly affected prognosis. Moreover, the epigenetic landscape was useful to predict OS and RD: compact chromatin equals high OS and RD (C1), and a loose one means poor OS and RD (C4), whereas an intermediary state (C2/C3) leads to average OS and RD. The expression pattern of the worst OS group showed high activation of specific signaling pathways which could be targeted to improve OS and RD. It would be worthwhile to correlate the findings with ATAC-seq, ChIP-seq, RNA-seq and DNA methylation analysis, in order to determine the loci responsible for the observed phenotypes in each cluster, and also WGS to identify possible new drivers of chromatin aberrations in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cytogenetics analysis as the central point of genetic testing in acute myeloid leukemia (AML): a laboratory perspective for clinical applications.

Chromosomal abnormalities in acute myeloid leukemia (AML) have significantly contributed to scientific understanding of its molecular pathogenesis, which has aided in the development of therapeutic strategies and enhanced management of AML patients. The diagnosis, prognosis and treatment of AML have also rapidly transformed in recent years, improving initial response to treatment, remission rates, risk stratification and overall survival. Hundreds of rare chromosomal abnormalities in AML have been discovered thus far using chromosomal analysis and next-generation sequencing. As a result, the World Health Organization (WHO) has categorized AML into subgroups based on genetic, genomic and molecular characteristics, to complementthe existing French-American classificationwhich is solely based on morphology. In this review, we aim to highlight the most clinically relevant chromosomal aberrations in AML together with the technologies employed to detect these aberrations in laboratory settings.

AML-419 Molecular Aberrations in Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome With Excess Blast II (MDS-EB II) in Kuwait

AML is a heterogeneous disease. Different mutational profiles have been described and correlate with variable outcomes. Genetic abnormalities are not well-described in our region. Our aim is to describe the molecular aberrations observed in the first cohort of patients. We recently introduced DNA based Oncomine Myeloid Gene Panel, testing 55 genes. We collected clinical, cytogenetic and molecular data prospectively since we introduced the test in late 2020. We also reviewed medical charts to collect baseline clinical data. We included patients aged 16 yrs and above with AML or MDS-EB II treated at Kuwait Cancer Center. Fifty-seven patients were included in the study with median age of 51 yrs. Almost one third of the patients were Kuwaitis (36%). We have 53 patients with AML (92%), 3 patients with MDS-EB II (5%), and 1 patient with chronic myeloid leukemia in myeloid blast crisis (CML-MBC). Most of the patients had at least one mutation (91%) detected by the panel. The most common mutations were FLT3 (ITD & D835), detected 26.3%, followed by NPM-1, detected in 21%. We have noticed an increased PTPN11 mutation, detected in 17.5% of the cases. This is an important study showing extensive molecular abnormalities in AML patients in the region. This will facilitate more understanding of the disease biology.

21q22 amplification detection in three patients with acute myeloid leukemia: cytogenomic profiling and literature review

Background21q22 amplification is a rare cytogenetic aberration in acute myeloid leukemia (AML). So far, the cytogenomic and molecular features and clinical correlation of 21q22 amplification in AML have not been well-characterized.Case presentationHere, we describe a case series of three AML patients with amplified 21q22 identified by fluorescence in situ hybridization using a RUNX1 probe. Two of these patients presented with therapy-related AML (t-AML) secondary to chemotherapy, while the third had de novo AML. There was one case each of FAB M0, M1 and M4. Morphologic evidence of dysplasia was identified in both t-AML cases. Phenotypic abnormalities of the myeloblasts were frequently observed. Extra copies of 21q22 were present on chromosome 21 and at least one other chromosome in two cases. Two showed a highly complex karyotype. Microarray analysis of 21q22 amplification in one case demonstrated alternating levels of high copy number gain split within the RUNX1 locus at 21q22. The same patient also had mutated TP53. Two patients died at 1.5 and 11 months post-treatment, while the third elected palliative care and died within 2 weeks.ConclusionsOur results provide further evidence that 21q22 amplification in AML is associated with complex karyotypes, TP53 aberrations, and poor outcomes. Furthermore, we demonstrate that 21q22 amplification is not always intrachromosomally localized to chromosome 21 and could be a result of structural aberrations involving 21q22 and other chromosomes.

EP050: Cytogenomic profiling and clinical correlation of 21q22 amplification in acute myeloid leukemia reveal distinct cytogenomic features and poor outcomes

21q22 amplification is a rare cytogenetic aberration in acute myeloid leukemia (AML). So far, the cytogenomic and molecular features and clinical correlation of 21q22 amplification in AML have not been well-characterized.

A rare der(10)t(X;10)(p11.2;p11.2) in an elderly patient with therapy-related acute myelomonocytic leukemia.

An 80-year-old male was referred to our hospital with dyspnea on effort. He had a history of diffuse large B-cell lymphoma 2 years ago, for which he was treated with chemotherapy, including rituximab, cyclophosphamide, vincristine, doxorubicin, and prednisolone. His laboratory data included a white blood cell count of 1.9 × 109/L, a hemoglobin concentration of 10.5 g/dl, and a platelet count of 92 × 109/L. His serum lactate dehydrogenase level was 196 U/L. A bone marrow examination revealed a hypercellular bone marrow with a blast frequency of 23.4%. The blast cells were positive for both nonspecific esterase and naphthol AS-D chloroacetate esterase staining (Figure 1). A cytogenetic study of the patient's bone marrow cells demonstrated the following karyotype: 47,XY, +8, der(10)t(X;10)(p11.2;p11.2)[7]/46,XY[13] (Figure 2). A diagnosis of therapy-related acute myelomonocytic leukemia was made due to his history of chemotherapy-treated lymphoma. Although the patient received two courses of azacitidine monotherapy, it was not effective, and he died 3 months after being diagnosed with leukemia. To the best of our knowledge, only five cases of acute myeloid leukemia (AML) involving a translocation consisting of 10p11 and Xp11 breakpoints have been reported [1-5]. Of these, one case demonstrated the t(X;10)(p11;p11) translocation as a sole abnormality [3]. This translocation has never been reported in other solid tumors. Hence, t(X;10)(p11;p11) is considered to be a rare, but recurrent, cytogenetic aberration in AML. The reported cases have several things in common, that is, they all involved balanced translocations, most of them involved therapy-related leukemia, and all of the patients except one (a 50-year-old [5]) were children. Although our case also involved therapy-related leukemia, it was very rare, as it involved an 80-year-old patient who harbored an unbalanced der(10)t(X;10)(p11;p11) translocation. As for the association between t(X;10)(p11;p11) and the pathogenesis of AML, our case involved der(10)t(X;10)(p11;p11), but lacked der(X)t(X;10)(p11;p11) due to the unbalanced nature of the translocation. Therefore, der(10)t(X;10)(p11;p11), rather than der(X)t(X;10)(p11;p11), is proposed to play a crucial role in the leukemogenesis in t(X;10)(p11;p11)-harboring AML. The patient's consent was obtained for the publication of the case report. The authors declare no conflict of interest. Masahiro Manabe, Nao Tanizawa, Satoru Nannno, and Ki-Ryang Koh designed the study. Masahiro Manabe, Yuuji Hagiwara, and Reiko Asada analyzed the data. Masahiro Manabe and Ki-Ryang Koh collected the clinical data and specimens. Masahiro Manabe, Yuuji Hagiwara, and Reiko Asada wrote the manuscript. All of the authors have reviewed and approved the final manuscript. All data generated during this study are included in this manuscript.

Risk Stratification, Measurable Residual Disease, and Outcomes of AML Patients with a Trisomy 8 Undergoing Allogeneic Hematopoietic Stem Cell Transplantation.

Simple SummaryTrisomy 8 is the most common numerical chromosome aberration in acute myeloid leukemia (AML). Although this AML type is often consolidated applying allogeneic hematopoietic stem cell transplantations (HSCT), detailed analyses of outcomes after HSCT are lacking. The purpose of this manuscript is to analyze biological and clinical features of patients with this cytogenetic aberration in the context of significant risk factors, including the ELN2017 risk stratification and measurable residual disease markers at HSCT. Our data provides evidence on the clinical disease courses and may aid in informed decisions on treatment and outcome prediction of trisomy 8 AML patients undergoing allogeneic HSCT.Background: For most patients with acute myeloid leukemia (AML) harboring a trisomy 8 an allogeneic hematopoietic stem cell transplantation (HSCT) is a suitable and recommended consolidation therapy. However, comparative outcome analyses between patients with and without trisomy 8 undergoing allogeneic HSCT have not been performed so far. Methods: We retrospectively analyzed clinical features, outcomes, and measurable residual disease (MRD) of 659 AML (12%, n = 81, with a trisomy 8) patients subjected to allogeneic HSCT as a consolidation therapy. Results: The presence of a trisomy 8 associated with a trend for higher age at diagnosis, AML of secondary origin, lower white blood cell counts at diagnosis, worse ELN2017 genetic risk, wild-type NPM1, and mutated IDH1/2 and JAK2. Outcomes after allogeneic HSCT in the entire cohort did not differ between patients with a sole trisomy 8, trisomy 8 with additional cytogenetic aberrations or without a trisomy 8. A trisomy 8 did not affect outcomes within the three ELN2017 risk groups. In accordance with findings in unselected patient cohorts, persistent MRD at allogeneic HSCT in patients with a trisomy 8 identified individuals with a higher risk of relapse following allogeneic HSCT. Conclusions: Outcomes of trisomy 8 patients after allogeneic HSCT did not compare unfavorably to that of other AML patients following allogeneic HSCT. Rather than the presence or absence of a trisomy 8, additional genetic aberrations and MRD at HSCT define outcome differences and aid in informed treatment decisions.

Efficacy Analysis of Different FLT3 Inhibitors in Relapsed/Refractory Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndrome Patients - a Systematic Review and Meta-Analysis

Background: Mutations in FMS like tyrosine kinase gene 3 (FLT3) are the most common genetic aberrations in acute myeloid leukemia (AML). Internal tandem duplications (ITD) are the most frequent FLT3 mutations, occurring in 20-30% of patients (pts) with newly diagnosed AML, and a minority are tyrosine kinase domain mutations. FLT3 mutation confers adverse prognosis in pts treated with standard chemotherapy (chemo) alone, and relapse risk is also higher compared to FLT3-wild type AML (HR-1.75). Studies of FLT3 inhibitors as monotherapy or in combination with chemo in the treatment of pts with FLT3-ITD relapsed/refractory (R/R) AML are associated with mixed results. No evidence synthesis has yet focused on the effect of FLT3 inhibitor alone in the treatment of R/R AML and high-risk myelodysplastic syndrome (HR-MDS). Hence, we analyzed the efficacy of various FLT3 inhibitors from different clinical trials used as monotherapy to treat pts with R/R AML and HR-MDS.Methods: We conducted a systematic review and meta-analysis of clinical trials of FLT3 inhibitors for pts with R/R AML and HR-MDS. We searched EMBASE and PubMed for clinical trials published between 1/1/2000 and 5/26/2021 using a combination of keywords and subject headings related to FLT3 inhibitors and AML. Titles/abstracts and full texts were screened by two independent reviewers, with conflicts arbitrated by a third reviewer. Studies were included if they were (1) full-length published journal articles that (2) reported the results of single-arm or double-arm phase I/II/III clinical R/R AML or HR-MDS. Outcomes of interest were composite response rate (CRc=complete response + complete response with incomplete count recovery) and overall response rate (ORR).Results: Thirty studies were included after the application of inclusion criteria (Figure 1). Two were excluded due to inadequate representation of pts with R/R AML and outcomes reported for stem cell transplant following FLT3 inhibitor use, respectively. Hence, 28 studies were included in the meta-analysis, with a total of 1927 pts. Quizartinib and sorafenib were the most frequently studied inhibitors (6 and 5 studies, respectively, Table 1). Heterogeneity testing was performed using Cochran's Q test and I 2 values. The Cochran's Q test p-value was less than 0.10 (p<0.01), and the I 2 value was more than 50% (I 2=87%), indicating the presence of heterogeneity. Thus, random-effects models were used. Asymmetry test was performed using Egger's linear regression test, which suggested the presence of publication bias (p=0.001; Figure 2). Thus, the trim-and-fill method was used to adjust for publication bias. Pooled ORR was 53% (95% CI, 43-63%) (Figure 2). Similarly, pooled CRc was 34% (95% CI, 26-44%, Figure 3). Pooled CRc and ORR were higher in studies involving quizartinib (40% and 61%, respectively) and gilteritinib (40% and 52%, respectively, Table 1). The CRc rates seen with FLT3 inhibitors are higher than the historical comparison of pts with FLT3-mutated-R/R AML treated with chemo only (CRc ~15%). This meta-analysis shows that FLT3 inhibitors as monotherapy leads to meaningful clinical responses in pts with FLT3-mutated-R/R AML.Conclusion: Most FLT3 inhibitors are effective as monotherapy for the treatment of pts with FLT3-mutated-R/R AML. Pooled response rates are notably higher in studies involving second-generation FLT3 inhibitors, particularly quizartinib and gilteritinib. Though not conclusive, the efficacy spectrum of various FLT3 inhibitors in the R/R setting could help design future studies and guide appropriate treatment selection; however, further validation is needed. Prospective clinical trials are required to compare the effectiveness of newer generation FLT3 inhibitors in pts with FLT3-mutated-R/R AML. [Display omitted] DisclosuresMaciejewski: Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy; Regeneron: Consultancy; Alexion: Consultancy. Balasubramanian: Servier Pharmaceuticals: Research Funding.

Modulation of FLT3-ITD Localization and Targeting of Distinct Downstream Signaling Pathways as Potential Strategies to Overcome FLT3-Inhibitor Resistance.

OBJECTIVES: Internal tandem duplications (ITDs) of the Fms-like tyrosine kinase 3 (FLT3) represent the most frequent molecular aberrations in acute myeloid leukemia (AML) and are associated with an inferior prognosis. The pattern of downstream activation by this constitutively activated receptor tyrosine kinase is influenced by the localization of FLT3-ITD depending on its glycosylation status. Different pharmacological approaches can affect FLT3-ITD-driven oncogenic pathways by the modulation of FLT3-ITD localization. AIMS: The objective of this study was to investigate the effects of N-glycosylation inhibitors (tunicamycin or 2-deoxy-D-glucose) or the histone deacetylase inhibitor valproic acid (VPA) on FLT3-ITD localization and downstream activity. We sought to determine the potential differences between the distinct FLT3-ITD variants, particularly concerning their susceptibility towards combined treatment by addressing either N-glycosylation and the heat shock protein 90 (HSP90) by 17-AAG, or by targeting the PI3K/AKT/mTOR pathway by rapamycin after treatment with VPA. METHODS: Murine Ba/F3 leukemia cell lines were stably transfected with distinct FLT3-ITD variants resulting in IL3-independent growth. These Ba/F3 FLT3-ITD cell lines or FLT3-ITD-expressing human MOLM13 cells were exposed to tunicamycin, 2-deoxy-D-glucose or VPA, and 17-AAG or rapamycin, and characterized in terms of downstream signaling by immunoblotting. FLT3 surface expression, apoptosis, and metabolic activity were analyzed by flow cytometry or an MTS assay. Proteome analysis by liquid chromatography–tandem mass spectrometry was performed to assess differential protein expression. RESULTS: The susceptibility of FLT3-ITD-expressing cells to 17-AAG after pre-treatment with tunicamycin or 2-deoxy-D-glucose was demonstrated. Importantly, in Ba/F3 cells that were stably expressing distinct FLT3-ITD variants that were located either in the juxtamembrane domain (JMD) or in the tyrosine kinase 1 domain (TKD1), response to the sequential treatments with tunicamycin and 17-AAG varied between individual FLT3-ITD motifs without dependence on the localization of the ITD. In all of the FLT3-ITD cell lines that were investigated, incubation with tunicamycin was accompanied by intracellular retention of FLT3-ITD due to the inhibition of glycosylation. In contrast, treatment of Ba/F3-FLT3-ITD cells with VPA was associated with a significant increase of FLT3-ITD surface expression depending on FLT3 protein synthesis. The allocation of FLT3 to different cellular compartments that was induced by tunicamycin, 2-deoxy-D-glucose, or VPA resulted in the activation of distinct downstream signaling pathways. Whole proteome analyses of Ba/F3 FLT3-ITD cells revealed up-regulation of the relevant chaperone proteins (e.g., calreticulin, calnexin, HSP90beta1) that are directly involved in the stabilization of FLT3-ITD or in its retention in the ER compartment. CONCLUSION: The allocation of FLT3-ITD to different cellular compartments and targeting distinct downstream signaling pathways by combined treatment with N-glycosylation and HSP90 inhibitors or VPA and rapamycin might represent new therapeutic strategies to overcome resistance towards tyrosine kinase inhibitors in FLT3-ITD-positive AML. The treatment approaches addressing N-glycosylation of FLT3-ITD appear to depend on patient-specific FLT3-ITD sequences, potentially affecting the efficacy of such pharmacological strategies.

The Clinical Characteristics, Genetic Alterations and Prognostic Significance of Acute Myeloid Leukemia (AML) with Trisomy 8

Introduction: Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy with prognosis and management that is dependent on underlying clinical characteristics, comorbidities, genetic and molecular features. Trisomy 8 is the most frequent numerical chromosome aberration in AML; however, its prognostic significance is not clear. Previous studies reported intermediate prognosis of isolated trisomy 8 (Schoch et al. 1997; Wolman et al. 2002); others concluded that it predicts adverse prognosis (Byrd et al. 1998). We investigated the clinical and genetic characteristics as well as prognostic significance of AML with trisomy 8.Methods: Clinical and laboratory information of adults with newly diagnosed AML excluding Acute Promyelocytic Leukemia at Beaumont Hospital, an academic medical center from January 2010 to December 2015 were retrospectively reviewed. Statistical analysis was performed using the SAS System version 9.3. Overall Survival (OS) was assessed by Kaplan-Meier method and associations of variables with survival investigated using log rank test and Turkey-Kramer method.Results:One hundred and thirty-seven AML patients were reviewed. Trisomy 8 was present in the karyotype of 24(18%), 70(51%) had normal karyotype and 43(31%) had other cytogenetic abnormalities without trisomy 8. Median age of patients with trisomy 8 was 71(47-89) and 67(21-91) for others without trisomy 8.Among the 24 patients with trisomy 8 karyotype, 9(38%) had isolated trisomy 8, 2(8%) had additional single chromosomal abnormality along with trisomy 8, 13(54%) occurred as part of complex karyotype and none was associated with favorable risk cytogenetics, t (8:21) and inversion 16. Secondary AML was present in 12(50%) patients and was preceded by Myelodysplastic Syndrome (MDS) in 11(92%). Only 3(25%) of these secondary AML were observed in the isolated trisomy 8 group. At presentation, 11(46%) of the trisomy 8 karyotype patients presented with leucopenia, 4(17%) had normal white blood cell count (WBC) and 8(33%) with leukocytosis. Hyperleukocytosis, leucocyte count above 100bil/L was present in only 1(4%) patient. The median percentage circulating blast was 10%(0-95) while median percentage bone marrow blast was 40%(20-95). Of the 13(54%) patients with trisomy 8 that had molecular testing; Nucleophosmin (NPM1) was negative in 12(92%) while FMS-like tyrosine kinase 3 (FLT3) was negative in 11(84.6%) patients.Induction therapy was with 3 days of Anthracycline and 7 days of Cytarabine (3+7) in 12(50%) patients, 5(21%) received hypomethylating agent while 7(29%) had best supportive care. Complete Remission (CR) was achieved by only 5(42%) patients in the “3+7” group, no CR was observed in the other treatment groups. OS appeared better in isolated trisomy 8 relative to combination with other cytogenetic abnormalities though not statistically significant (p=0.140; table 1). Superior OS in patients with trisomy 8 was associated with choice of induction treatment (p<0.0001; table 2). There was no significant association between OS and age, comorbidity score, WBC, percentage circulating or bone marrow blast, platelet count, hemoglobin level, or type of AML (de novo or secondary); table 2.Conclusion: Adult AML with trisomy 8 is commoner in older age group, with the youngest of our patients being 47 years. It is often preceded by MDS and rarely associated with hyperleukocytosis or high circulating blast. NPM1 and FLT3 mutations were negative in the majority of our patients with trisomy 8, which raises a question about possible mutual exclusion. A trend towards better overall survival was observed with isolated trisomy 8 compared to combination with other cytogenetic aberration though not statistically significant. Our findings are consistent with some prior studies; however larger patient cohort is needed to validate our results. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

OP340 - Optical genome mapping for assessment of genomic aberrations in acute myeloid leukemia: a multicenter evaluation

OP340 - Optical genome mapping for assessment of genomic aberrations in acute myeloid leukemia: a multicenter evaluation

Fludarabine, High-Dose Cytarabine and Idarubicin-Based Induction May Overcome the Negative Prognostic Impact of FLT3-ITD in NPM1 Mutated AML, Irrespectively of FLT3-ITD Allelic Burden.

Simple SummaryThe prognostic relevance of molecular aberrations in acute myeloid leukemia (AML) has been prevalently tested in patients receiving conventional 3+7 induction. Recently, there has been a renewed interest in intensified inductions, but very few data are available on the impact of the most frequent genetic alterations with these alternative treatments. We analyzed a large multicentric cohort of younger AML patients harboring NPM1 and FLT3-ITD mutations receiving an intensified fludarabine-containing regimen (FLAI). Our data suggest that in NPM1 mut patients, FLAI may overcome the prognostic influence of co-mutated FLT3-ITD. The increased efficacy of this treatment seems to reduce the need for early consolidation with allogeneic transplant in double-mutated patients. Our data strongly support FLAI as an ideal backbone for combination with innovative targeted drugs, in order to further improve patients’ outcome.The mutations of NPM1 and FLT3-ITD represent the most frequent genetic aberration in acute myeloid leukemia. Indeed, the presence of an NPM1 mutation reduces the negative prognostic impact of FLT3-ITD in patients treated with conventional “3+7” induction. However, little information is available on their prognostic role with intensified regimens. Here, we investigated the efficacy of a fludarabine, high-dose cytarabine and idarubicin induction (FLAI) in 149 consecutive fit AML patients (median age 52) carrying the NPM1 and/or FLT3-ITD mutation, treated from 2008 to 2018. One-hundred-and-twenty-nine patients achieved CR (86.6%). After a median follow up of 68 months, 3-year overall survival was 58.6%. Multivariate analysis disclosed that both NPM1mut (p < 0.05) and ELN 2017 risk score (p < 0.05) were significant predictors of survival. NPM1-mutated patients had a favorable outcome, with no significant differences between patients with or without concomitant FLT3-ITD (p = 0.372), irrespective of FLT3-ITD allelic burden. Moreover, in landmark analysis, performing allogeneic transplantation (HSCT) in first CR proved to be beneficial only in ELN 2017 high-risk patients. Our data indicate that FLAI exerts a strong anti-leukemic effect in younger AML patients with NPM1mut and question the role of HSCT in 1st CR in NPM1mut patients with concomitant FLT3-ITD.

Differential microRNA expression in childhood B-ALL with trisomy 8: A case report

B-Cell Acute Lymphoblastic Leukemia account for approximately 80% of leukemia cases in children and adolescents. Numerical chromosomal changes are important prognostic factors. Trisomy 8 is one of the most frequent numerical aberrations in acute myeloid leukemia but is rarely found in lymphoid malignancies. Here, we share our findings in a childhood acute lymphoblastic leukemia patient with trisomy 8, referred for diagnostic assessment. Physical examination, myelogram, complete blood count, cytogenetic, and RT-PCR analysis for TEL-AML1 and E2A-PBX1 rearrangements were carried out. Microarray methodology was used to assess the miRNA expression profile. We highlight 36 miRNAs differentially expressed (p < 0.001, fold change >2.0) comparing with mononuclear cells from five subjects without leukemia. Among the miRNAs differentially expressed in our patient, only two have been described in B-ALL, namely miR-133b and miR-181b-5p. Some miRNAs which was downregulated in our patient as miR-495 and miR-127 have tumor suppressor function. None of the miRNAs located on chromosome 8 were significantly upregulated. The relationship between the altered miRNA expression profiles and leukemogenesis can be associated with global changes in gene expression and regulation patterns caused by acquired trisomy 8, influencing the patient's response to treatment and prognosis.

Mutational landscape and clinical outcome of patients with de novo acute myeloid leukemia and rearrangements involving 11q23/KMT2A

Balanced rearrangements involving the KMT2A gene, located at 11q23, are among the most frequent chromosome aberrations in acute myeloid leukemia (AML). Because of numerous fusion partners, the mutational landscape and prognostic impact of specific 11q23/KMT2A rearrangements are not fully understood. We analyzed clinical features of 172 adults with AML and recurrent 11q23/KMT2A rearrangements, 141 of whom had outcome data available. We compared outcomes of these patients with outcomes of 1,097 patients without an 11q23/KMT2A rearrangement categorized according to the 2017 European LeukemiaNet (ELN) classification. Using targeted next-generation sequencing, we investigated the mutational status of 81 leukemia/cancer-associated genes in 96 patients with 11q23/KMT2A rearrangements with material for molecular studies available. Patients with 11q23/KMT2A rearrangements had a low number of additional gene mutations (median, 1; range 0 to 6), which involved the RAS pathway (KRAS, NRAS, and PTPN11) in 32% of patients. KRAS mutations occurred more often in patients with t(6;11)(q27;q23)/KMT2A-AFDN compared with patients with the other 11q23/KMT2A subsets. Specific gene mutations were too infrequent in patients with specific 11q23/KMT2A rearrangements to assess their associations with outcomes. We demonstrate that younger (age <60 y) patients with t(9;11)(p22;q23)/KMT2A-MLLT3 had better outcomes than patients with other 11q23/KMT2A rearrangements and those without 11q23/KMT2A rearrangements classified in the 2017 ELN intermediate-risk group. Conversely, outcomes of older patients (age ≥60 y) with t(9;11)(p22;q23) were poor and comparable to those of the ELN adverse-risk group patients. Our study shows that patients with an 11q23/KMT2A rearrangement have distinct mutational patterns and outcomes depending on the fusion partner.