Correlation Between Bone Marrow Dysplasia and Genomic Profile in De Novo Acute Myeloid Leukemia (AML): A Study By the ALFA Group

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2568-2568
Author(s):  
Thomas Cluzeau ◽  
Orianne Wagner-Ballon ◽  
Thomas Boyer ◽  
Estelle Guerin ◽  
Emmanuel Benayoun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Trials ◽  
Cell Lines ◽  
Research Funding ◽  
De Novo ◽  
Normal Karyotype ◽  
The Other ◽  
Molecular Abnormalities ◽  
Other Hand ◽  
De Novo Aml

Abstract Introduction: AML with multilineage dysplasia (MLD) are included in the WHO subset of "AML with myelodysplasia-related changes" (AML-MRC), together with AML arising from previous MDS or AML with MDS-related cytogenetic abnormalities. In the WHO classification, MLD is defined by dysplasia in at least 50% of the cells in at least two bone marrow (BM) myeloid cell lines. On the other hand, some genetically defined AML subgroups are specifically associated with morphologic changes, but close correlations do not exist for most of these entities. We searched for correlations between BM dysplasia and molecular aberrations in de novo AML patients included in 2 ALFA clinical trials Methods: BM cytomorphology was retrospectively reassessed in 192 patients with de novo AML (excluding CBF-AML), aged 18 to 70 enrolled in ALFA-0702 (n=123) and ALFA-0701 (n=69) clinical trials in 5 centers. 4 distinct morphologists performed the analysis from BM smears. Dysmegakaryopoiesis (DM), dyserythropoiesis (DE) and dysgranulopoiesis (DG) were quantified (respectively on 30, 200 and 200 cells) using 22 criteria designed by GFHC, which allow better evaluation of cytoplasmic and nuclear dysplasia in all BM lineages. Dysplasia was also evaluated using WHO criteria. NPM1, FLT3, MLL, CEBPA, IDH1, IDH2, WT1, DNMT3A, RUNX1, TET2 and ASXL1 gene mutations and EVI1 gene overexpression were detected by standard methods, as previously published (Renneville et al. Oncotarget 2014). Results: In the 192 patients analyzed, the incidence of molecular abnormalities was: MLL-PTD 5% (8/155), NPM1 31% (52/170), FLT3-TKD 9% (15/171), FLT3-ITD 19% (34/171), CEBPA double mutated (CEBPA-dm)11% (17/152), EVI1 overexpression 11% (17/152), IDH1 R132 9% (14/146), IDH2 R140 6% (10/159), IDH2 R172 2% (2/92), RUNX1 8% (6/67), DNMT3A 26% (11/43), TET2 12% (5/43) and ASXL1 7% (4/62). DG, DE and DM was evaluable in 59%, 83% and 85% of the patients, respectively. WHO-MLD was identified in 43/192 (22%) patients, and was not significantly associated with any genetic marker, even in AML with normal karyotype (Table 1). On the other hand, when using GFHC criteria, we observed in NPM1 mutated patients a higher % of bi-tri or multi nucleated megakaryocytes (25% vs 10%, p=0.03), of cytoplasmic DG (74% vs 58%, p=0.03); and more dysplasia in other cell lines including eosinophils, basophils, mastocytes, monocytes (p=0.008). In CEBPA-dm patients, lower % of global DG (21% vs 54%, p=0.04) was seen. In EVI1 overexpressing patients, we found a higher % of global DM, of micromegacaryocytes and of hypolobulated megacaryocytes (80% vs 31%, p=0.01; 18% vs 2%, p=0.01 and 19% vs 6%, p=0.001 respectively). In DNMT3A mutated patients, we observed a lower % of bi-tri or multi nucleated megakaryocytes (2% vs 28%, p=0.01) and a higher % of nuclear and cytoplasmic DG (21% vs 2%, p=0.005 and 1.2% vs 0%, p=0.03, respectively). In TET2 mutated patients, we observed less defects in nuclear segmentation and a higher % of abnormal chromatin condensation in granulocytes (1% vs 9%, p=0.02 and 6% vs 0%, p=0.008, respectively). Conclusion: Presence of WHO-MLD was not significantly correlated with any genetic subgroup. The 22 BM dysplasia parameters designed by the GFHC were evaluable in a majority of patients, and allowed us to find some specific cytomorphologic features in de novo AML with NPM1, CEBPA-DM, DNMT3A, TET2 mutation, or EVI1 overexpression. Those findings suggest that the definition of MLD may be refined by using more in depth quantification of dysplasia, especially with GFCH parameters. This study will be expanded with the inclusion of whole exome sequencing data (ongoing). Table 1. Correlation between MLD, normal karyotype and molecular abnormalities % AML-MLD % AML-MLD in AML with normal karyotype MLL-PTD 37,5% 29% NPM1 25% 28% FLT3-TKD 27% 33% FLT3-ITD 27% 25% CEBPA-dm 0% 0% IDH1 R132 21% 18% IDH2 R140 10% 0% IDH2 R172 0% 0% RUNX1 40% 40% DNMT3A 0% 0% TET2 0% 0% ASXL1 25% 50% EVI1 24% 0% Disclosures Fenaux: Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Bortezomib+Chemotherapy Based Salvage Combinations in Refractory AML, Preliminary Results

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4000-4000
Author(s):  
Miklos Udvardy ◽  
Attila Kiss ◽  
Bela Telek ◽  
Robert Szasz ◽  
Peter Batar ◽  
...  
Keyword(s):  
Cell Lines ◽  
De Novo ◽  
Case Reports ◽  
Fatal Outcome ◽  
Normal Karyotype ◽  
Secondary Aml ◽  
Poor Risk ◽  
Group 2 ◽  
De Novo Aml ◽  
Group 1

Abstract Bortezomib (Velcade) proved to be the standard element of refractory myeloma 2nd and 3rd line treatment, while many studies are suggesting excellent results in 1st line. Proteasome inhibition, the block of angiogenesis, modification of the NF-kappa-B system seems to be a challenging target in other malignant diseases, including refractory acute myeloid leukemia (AML), as well. In vitro data clearly support, that bortezomib exerts antiproliferative and pro-apoptotic effects in different AML cell-lines, along with human AML cell cultures, and moreover bortezomib was able to restore, or at least improve anthracyclin and possibly ARA-C sensitivity in different cell-lines (including AML). More recently, a Phase I trial showed bortezomib monotherapy efficient (only in few percents) in childhood refractory acute leukemia. Some case reports were shown at ASH 2007. We have tried bortezomib containing first or second line combinations in 27 (14 female, 13 male, mean age 57.6 years) patients with refractory or poor risk AML, in a small retrospective survey. The combinations were as follows: HAM or Flag-Ida, combined with bortezomib 1,3 mg pro sqm, day O and seven). The following groups were considered as refractory or poor risk AML: De novo AML, 2nd line: No response/remission to first line standard treatment (“3+7”), n=2 (Velcade- Flag-Ida treatment) De novo AML 1st line: bilineal or biphenotypic (flow-cytometry) n=2 (Velcade-Flag- Ida treatment) De novo AML with complex (numerical or more than 3 abnormalities) karyotype or normal karyotype with flt-3 TKD mutation, n=9, 1st line (Velcade-Flag-Ida n=6, Velcade- HAM protocol, n=3) Secondary AML or AML with evidence of previous more than 6 mo duration high grade MDS, n=14, 1st line: (Velcade-Flag-Ida n=9, Velcade-HAM n=5) RESULTS: Complete remission (CR) 12/27, partial remission (PR) 9/27, no remission 5/27, progression during treatment: 1/27.Best responses were seen in de novo cases. CR had been achieved in all patients of group 1 (two standard risk patients not responding to 3+7 protocol), and group 2 (biphenotypic, bilineal). The CR rate was quite appreciable in group 3, i.e. 6/9 (complex karyotype or normal karyotype with FLt-3 mutation – the response rate was excellent with flt-3 mutated cases). In group 4. (MDS, secondary AML) the results were less impressive. There were no major differences according to protocol (Flag-Ida or HAM) Allogeneous stem cell transplantation could have been performed in 1st CR in two patients (one from group 1. and another from group 2.). One of them died due to relapse, the other one is in CR since then. The combinations seem to be relatively safe. Induction related death rate was low (1 elderly patient acute thrombocytopenic bleeding with refractory MDS-AML). 5 other patients had severe neutropenic sepsis (2 with fatal outcome). Pulmonary syndrome, which may follow Velcade+ARA-C had not been documented. Other adverse events did not differ from the pattern observed with standard induction therapies.

IDH1/2, TET2 and DNMT3A Mutations Are Not Mutually Exclusive in Secondary Acute Myeloid Leukemias,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3556-3556
Author(s):  
Olivier Kosmider ◽  
Olivier LaRochelle ◽  
Marie-Magdelaine Coude ◽  
Veronique Mansat-De Mas ◽  
Eric Delabesse ◽  
...  
Keyword(s):  
Bone Marrow ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Remission Rate ◽  
Normal Karyotype ◽  
Prognostic Impact ◽  
Mutational Status ◽  
Myeloid Leukemias ◽  
Melting Analysis ◽  
De Novo Aml

Abstract Abstract 3556 IDH1/2, TET2 and DNMT3A mutations have been reported in myeloid malignancies including de novo AML. In this study, we have analyzed the frequency and prognostic impact of these mutations in a large retrospective cohort of patients (pts) with secondary AML (SA) which encompass myelodysplasia-related changes (MRC) AML and therapy-related (TR) AML according to the WHO classification. Bone marrow samples were collected from 247 pts at diagnosis with SA and the mutational status of IDH1/2, TET2 and DNMT3A genes together with other genes frequently mutated in AML (NPM1, FLT-3, N and K-RAS, WT1) was determined by Sanger sequencing or high resolution melting analysis. The cohort of 247 pts consisted in 201 MRC AML and in 46 TR AML, 39.5% of which with a normal karyotype (NK). The frequency of IDH1/2, TET2 and DNMT3A mutations was 12.6, 19.8 and 4.5%, respectively. Two pts had both TET2 and IDH1/2 mutations, 2 pts had TET2 and DNMT3A mutations and 5 pts had both IDH1/2 and DNMT3A mutations showing that these mutations were not mutually exclusive in SA. IDH1/2 and TET2 mutations were significantly more frequent in MRC AML (14.1 and 22.3%) than in TR AML (6.4 and 8.7%) (P =0.04 and P =0.03) while the frequency of DNMT3A mutations was identical in the two subgroups. The SA pts harbouring at least one IDH1/2 or TET2 or DNMT3A mutation were significantly older (P <0.0001) and presented higher leukocyte count and lower MCV (P <0.05) than unmutated pts. Percentage of blasts in the bone marrow was similar in the two groups. Karyotype was normal in 48% of the IDH1/2 or TET2 or DNMT3A mutated pts and 18% of the unmutated patients, indicating that these mutations were strongly associated with NK (P < 0.001). A statistically significant link was found between TET2 or IDH1/2 or DNMT3A mutations and NPM1 mutations, but not with FLT-3, N/K-RAS or WT1 mutations. Complete remission rate and overall survival were evaluated in a group of 158 pts which had received intensive chemotherapy at diagnosis, and were identical in the IDH1/2 or TET2 or DNMT3A mutated and unmutated groups. These mutations did significantly influence survival neither in the subgroup of pts with normal karyotype, nor in the subgroup of MRC-AML, or TR-AML which were of very poor prognosis. These data show that IDH1/2, TET2 or DNMT3A mutations could modify the clinical presentation without impact on prognosis. Disclosures: No relevant conflicts of interest to declare.

Clinicopathologic Analysis of Follicular Lymphoma (FL) Patients with Coexisting Diffuse Large B-Cell Lymphoma (DLBCL) Undergoing Rituximab-Containing Chemotherapy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3693-3693 ◽  
Author(s):  
Suguru Fukuhara ◽  
Dai Maruyama ◽  
Ken-ichi Miyamoto ◽  
Sung-Won Kim ◽  
Takashi Watanabe ◽  
...  
Keyword(s):  
Research Funding ◽  
De Novo ◽  
International Prognostic Index ◽  
Prognostic Index ◽  
B Cell Lymphoma ◽  
Initial Diagnosis ◽  
The Other ◽  
Low Grade ◽  
Other Hand ◽  
Significant Difference

Abstract Abstract 3693 Background: Histopathologically, some FLs have components of DLBCL at diagnosis. In the pre-rituximab era, there were two reports regarding the characteristics and prognosis of patients (pts) with these particular FLs1,2). Hans et al. reported that the overall survival (OS) of pts with FL grade 3 having a predominant (> 50%) DLBCL component is similar to that of pts with DLBCL1). Ghesquières et al. concluded that pts with DLBCL presenting with a low-grade component have a similar OS to those with de novo DLBCL2). However, the clinical implications and prognosis of FL pts with coexisting DLBCL at diagnosis undergoing rituximab-containing chemotherapy remain unclear. Patients and Methods: We retrospectively analyzed the clinicopathologic features of 59 FL pts with coexisting DLBCL (FL/DLBCL) initially undergoing rituximab-containing chemotherapy. Furthermore, the prognosis of FL/DLBCL pts was compared with that of 223 FL pts without DLBCL as well as 285 DLBCL pts without FL3). All pts received a rituximab-containing regimen as the initial chemotherapy. These pts with FL/DLBCL or FL were diagnosed and treated at our institution between 2001 and 2010, and DLBCL pts between 2003 and 2010. Results: The median age of the 59 pts with FL/DLBCL was 54 years (range: 22–83). Among them, 41 (69%) pts had FL grade 1–3a and the remaining 18 (31%) had FL grade 3b components. Thirty (51%) pts had a predominant (> 50%) DLBCL component. With regard to treatment, all pts except one received the R-CHOP regimen. Forty-nine (83%) pts achieved CR, but 22 of them (37%) relapsed. With a median follow-up of 5.4 years, the estimated 5-year OS and progression-free survival (PFS) for all 59 pts were 83 and 64%, respectively. The International Prognostic Index (IPI) and FLIPI were not correlated with OS and PFS, whereas the revised-IPI (R-IPI) and FLIPI2 were significantly correlated with PFS. A predominant DLBCL component was predictive of neither OS nor PFS. In the 41 pts with FL grade 1–3a with DLBCL, %CR, 5-year OS, and PFS rates were 80, 84, and 60%, respectively. On the other hand, in the 18 pts with FL grade 3b with DLBCL, they were 89, 82, and 72%, respectively. The %CR and PFS rates of the latter cohort showed superior tendencies to those of the former cohort, with no statistically significant difference. Among 22 relapses, 10 were confirmed histologically: 5 DLBCL, 4 FL, and 1 HL. In 16 relapsed pts with FL grade 1–3a at initial diagnosis, 4 relapsed as FL, one each as DLBCL and HL. Two pts who had relapsed as FL and HL subsequently developed DLBCL. On the other hand, 4 out of 5 relapsed pts with FL grade 3b at the initial diagnosis relapsed as DLBCL. Additionally, the prognosis of FL/DLBCL pts was compared with that of FL and DLBCL pts. The 5-year OS rate of FL/DLBCL pts (83%) was significantly worse than that of DLBCL pts (91%, p=0.039) as well as FL pts (97%, p=0.001) (Fig 1). The 5-year PFS rate of FL/DLBCL pts (64%) was not significantly different from that of DLBCL pts (72%, p=0.120). The PFS curves (Fig 2) suggested a slight increase in progression or mortality in FL/DLBCL pts during the first 2 years. However, the FL/DLBCL pts had a low incidence of events after 2 years. The PFS curve of FL/DLBCL pts was similar to that of DLBCL pts. By multivariate analysis of pts with FL/DLBCL and DLBCL, coexisting FL components was a significant predictor of inferior OS, but not PFS. Conclusions: The PFS curve of FL/DLBCL pts was similar to that of DLBCL pts, although pts with FL/DLBCL had poorer prognosis than DLBCL pts in the rituximab era. Furthermore, in our present analysis, predominant DLBCL component was predictive of neither OS nor PFS. Disclosures: Kobayashi: Nippon Shinyaku: Research Funding; Ariad: Research Funding; Ohtsuka: Research Funding; Celgene: Research Funding; Behringer : Research Funding. Tobinai:Grant Support: Zenyaku, Chugai/Roche, GSK, Biomedics Other.

scholarly journals BRCA1/2 Containing Complex 3 (BRCC36) Is Recurrently Mutated in AML with t(8;21) and Associated with Increased Sensitivity to Chemotherapy through Impairment of the DNA Damage Repair Pathway

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1487-1487
Author(s):  
Tatjana Meyer ◽  
Nikolaus Jahn ◽  
Anna Dolnik ◽  
Peter Paschka ◽  
Verena I. Gaidzik ◽  
...  
Keyword(s):  
Dna Damage ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
De Novo ◽  
Dna Damage Repair ◽  
Advisory Committees ◽  
Data Set ◽  
Damage Repair ◽  
De Novo Aml

Abstract Introduction BRCA1/BRCA2-containing complex 3 (BRCC36) is a Lys63-specific deubiquitinating enzyme (DUB) involved in DNA damage repair. Mutations in BRCC36 have been identified in 2-3% of patients with myelodysplastic syndromes (MDS) and secondary AML (sAML). The role of BRCC36 mutations in de novo AML and their impact on DNA damage-inducing cytotoxic chemotherapy sensitivity is not clear. Aim We aimed to determine the incidence of BRCC36 mutations in AML and their impact on outcome and drug sensitivity in vitro. Methods We analyzed the entire coding region of BRCC36 for mutations in 191 AML cases with t(8;21) (q22;q22.1) and 95 cases with inv(16) (p13.1q22) using a customized targeted sequencing panel. Data for de novo AML was derived from The Cancer Genome Atlas Research Network (TCGA) data set (NEJM 2013). Lentiviral CRISPR/Cas9 was used to inactivate BRCC36 in t(8;21)-positive AML cell lines - Kasumi-1 and SKNO-1 - and murine hematopoietic stem and progenitor cells (LSKs). Knockout was confirmed by a cleavage assay as well as Western blot. AML1-ETO-9a was expressed by a retroviral vector. Cell lines and LSK cells were treated with different concentrations of doxorubicin or cytarabine and their viability was assessed seven days post treatment. DNA damage was assessed through phospho-γH2AX staining using flow-cytometry. Results BRCC36 mutations were identified in 7 out of 191 patients (3.7%) with t(8;21) AML and none of 95 patients with inv(16). In the TCGA data set one out of 200 patients (0.5%) with de novo AML had a BRCC36 mutation. This patient had a complex karyotype and would be considered as secondary AML with myelodysplastic-associated changes according to the 2016 WHO classification. Six of the 7 mutations were missense or nonsense mutations that were predicted to be deleterious to BRCC36 function. One mutation affected a splice site at exon 6, resulting in an impaired splicing capability. With intensive standard chemotherapy all patients with BRCC36 mutations achieved a complete remission and had an estimated relapse-free and overall survival of 100% after a median follow up of 4.2 years. Given its role in DNA damage repair, we hypothesized that BRCC36 inactivation sensitizes AML cells to DNA-damage inducing drugs. In order to test this, we generated BRCC36 knockout Kasumi-1 and SKNO-1 cell lines using CRISPR-Cas9. BRCC36 inactivation had no impact on cell growth on either of the cell lines. However, we found that BRCC36 knockout cells were significantly more sensitive to doxorubicin as compared to the parental cells with normal BRCC36. This was accompanied by a significant increase in DNA damage as assessed by phospho-γH2AX in BRCC36 knockout vs control cells after doxorubicin treatment. In contrast, BRCC36 inactivation had no impact on cytarabine sensitivity. We next assessed drug sensitivity in primary murine leukemic cells expressing AML1-ETO-9a. Again, inactivation of BRCC36 resulted in a significant higher sensitivity to doxorubicin but not cytarabine. Conclusion We found BRCC36 to be recurrently mutated in t(8;21)-positive AML Inactivation of BRCC36 was associated with impairment of the DNA damage repair pathway and thus higher sensitivity to DNA damage-inducing chemotherapy. This might be also reflected by the favorable clinical outcome of patients with BRCC36 mutated t(8;21)-positive AML, a finding which has to be confirmed in a large patient cohort. Disclosures Paschka: Pfizer: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Travel support; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Otsuka: Membership on an entity's Board of Directors or advisory committees; Sunesis: Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Amgen: Other: Travel support; Janssen: Other: Travel support; Bristol-Meyers Squibb: Other: Travel support, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Travel support; Astex: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees. Bullinger:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Speakers Bureau; Bayer Oncology: Research Funding; Sanofi: Research Funding, Speakers Bureau; Janssen: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Döhner:Novartis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Janssen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Pfizer: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Pfizer: Research Funding; Agios: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Sunesis: Consultancy, Honoraria, Research Funding.

scholarly journals Frontline Selinexor and Chemotherapy Is Highly Active in Older Adults with Acute Myeloid Leukemia (AML)

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 24-25
Author(s):  
Timothy S. Pardee ◽  
Kristin M. Pladna ◽  
Susan Lyerly ◽  
Sarah Dralle ◽  
Megan Manuel ◽  
...  
Keyword(s):  
Cell Lines ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Topoisomerase Ii ◽  
De Novo ◽  
Preclinical Studies ◽  
Newly Diagnosed ◽  
Highly Active ◽  
De Novo Aml ◽  
Acute Myeloid

Background: Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow characterized by resistance to treatment and dismal outcomes, especially in the elderly. Novel approaches are desperately needed. Exportin 1 (XPO1) is involved in the selective nuclear export of certain proteins and RNA species. It is overexpressed in a subset of AML, conferring an adverse prognosis. Selinexor is a small-molecule inhibitor of XPO1 with activity in AML. Selinexor sensitizes AML cells to anthracyclines by retaining topoisomerase II in the nucleus resulting in increased DNA strand breaks. Furthermore, selinexor has shown encouraging results when combined with chemotherapy in AML. This abstract reports the ongoing results of a randomized phase II study of induction and consolidation with or without selinexor in newly diagnosed patients with AML, 60 years of age or older and preclinical studies to assess the mechanisms of chemo-sensitization. Methods: Patients 60 years of age or older with newly diagnosed de novo AML were randomized 3:1 between 7+3+selinexor or 7+3. Responding patients could go on to high dose cytarabine consolidation with or without selinexor as per initial randomization. Patients in the selinexor arm who completed all consolidation could then move to maintenance therapy with selinexor alone. Induction consisted of cytarabine 100 mg/m2/d by continuous infusion for 7 days and daunorubicin 60 mg/m2 on days 1-3. Consolidation consisted of cytarabine at 1.5 gm/m2 given Q12 hours days 1-3 with G-CSF given 24 hours following the last dose of cytarabine. Selinexor was dosed at 60 mg PO on days 1, 3, 8, 10, 15 and 17 during induction and consolidation and on days 1 and 8 every 21 days during maintenance. Preclinical studies were conducted with murine AML cell lines. Results: Twenty-seven of a planned twenty-eight patients were enrolled to date. Of the 27 evaluable patients, 20 were randomized to the selinexor arm and 7 to the control arm. Baseline demographics are listed in Table 1. In the standard arm, both 30- and 60-day mortality were 14% (1/7). In the selinexor arm, both 30- and 60-day mortality were 10% (2/20). In the standard arm, 43% (3/7) of patients achieved a complete remission (CR) or complete remission with incomplete count recovery (CRi). Of the 3 responders 1 has gone on to transplant. In the selinexor arm, 85% (17/20) of patients achieved a CR or CRi. Of the 17 responders, 4 have gone on to transplant. Progression free and overall survival both favor the selinexor arm with trends towards significance despite the small size of the trial (Figure 1 and Table 2). No difference in the AE profile was noted between arms and no unexpected side effects were observed. Selinexor induces retention of topoisomerase II in the nucleus increasing sensitivity to anthracyclines. To determine if selinexor sensitized to cytarabine viability assays using murine AML cell lines were done (Figure 2A). Selinexor significantly sensitized both cell lines to cytarabine. AML cells increase mitochondrial oxygen consumption in response to cytarabine leading to resistance. The ability of selinexor to interfere with this response was assessed using Seahorse flux analysis. AML cells treated with cytarabine for 16 hours showed a diminished mitochondrial oxygen response when co-treated with selinexor (Figure 2B). Conclusions: Selinexor in combination with standard induction and consolidation therapy appears highly active in older patients with de novo AML. Selinexor may increase response to cytarabine by interfering with nuclear-mitochondrial communication. Enrollment is ongoing. Disclosures Pardee: Rafael Pharmaceuticals: Consultancy; AbbVie: Consultancy; Genentech, Inc.: Consultancy; BMS: Consultancy, Honoraria, Speakers Bureau; Karyopharm: Research Funding; Rafael: Research Funding; Celgene: Consultancy, Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Pharmacyclics: Speakers Bureau. Ellis:Rafael Pharmaceuticals: Consultancy. Howard:Jazz Pharmaceuticals: Consultancy. Powell:Jazz Pharmaceuticals: Consultancy, Other: Advisor, Research Funding; Genentech: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Rafael Pharmaceuticals: Consultancy, Other: Advisor, Research Funding. OffLabel Disclosure: Selinexor for the treatment of AML

scholarly journals The Novel Small Molecule Inhibitor KS99 Targets AML and Inhibits Stemness Markers STAT3 and ALDH

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1440-1440
Author(s):  
Charyguly Annageldiyev ◽  
Krishne Gowda ◽  
Dhimant Desai ◽  
Viola Devine ◽  
Daniel Brosius ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Mononuclear Cells ◽  
De Novo ◽  
Annexin V ◽  
Hematopoietic Stem ◽  
Stat3 Phosphorylation ◽  
Preclinical Efficacy ◽  
De Novo Aml

Abstract Introduction: Signal transducer and activator of transcription 3 (STAT3) has been shown to be activated in leukemic stem cells (LSCs), especially in Pre-LSCs, and activation has been associated with poor prognosis in multiple cancers including AML. In AML, sorted CD34+/CD38- and aldehyde dehydrogenase-positive (ALDH+) cells have significantly phosphorylated STAT3 when compared to healthy controls. The Isatin analog, KS99 has shown selective anti-cancer activity against Multiple Myeloma (MM) which may, in part, be mediated by inhibition of BTK activation. Here we demonstrate that KS99 selectively targets AML CD34+/CD38- and ALDH+ cells and inhibits STAT3 phosphorylation while sparing normal hematopoietic stem and progenitor cells (HSPCs). Experimental Design: The pro-apoptotic activity of KS99 for primary human AML cells (n=21) and human AML cell lines (n=9) was evaluated using Annexin V and cell proliferation (MTS) assays. Colony forming assays were carried out to demonstrate the specific LSC-targeted effect of KS99. Flow cytometry was used to detect STAT3 phosphorylation and apoptosis in CD34+/CD38- LSCs and ALDH activity in a series of human AMLs. Next, to assess the preclinical efficacy, the human AML cell line U937 was injected intravenously (IV) into NSG mice. Mice with an established leukemic burden were randomized into treatment groups and treated through an intraperitoneal (IP) injection with either vehicle control or KS99 (2.5mg/kg) every other day x8. Two days after the final treatment, leukemic burden (hCD45+) was quantitated in the bone marrow. Results: AML cell lines exhibited dose-dependent sensitivity to KS99 in the nanomolar range (IC50: 150-400nM) as did most primary AML cells (Figure: A). Interestingly, poor prognosis AML subsets, AML with MDS related changes (MDS-RCs), were more sensitive than De Novo AML cases (p= 0.0077) with or without a NMP1 mutation (p= 0.012, p=0.045, respectively). Within De Novo AML, NMP1 wild-type cases were more sensitive than NMP1 mutant cases (p= 0.02) (Figure: B). Furthermore, KS99 treatment selectively reduced the clonogenicity of human AML patient cells vs. normal cord blood mononuclear cells (p<0.001) (Figure: C). KS99 induced apoptosis in the CD34+/CD38- gated cell population of primary AML, leading to reduced blast-like and stem-like cells, but not other cells. It also showed a reduction in ALDH+ cells in an Aldefluor assay. Likewise, KS99 treatment showed reduced pSTAT3 in viable CD34+/CD38- cells (Figure: D). Inhibition of pBTK, BCL-2, and pSTAT3 were seen in Western blot experiments. Animal studies showed 76 % reduction of hCD45+ U937 cells in bone marrow of KS99-treated mice when compared to bone marrow isolated from vehicle-treated animals (p<0.05) (Figure: E). Conclusion: KS99 selectively targets LSCs from poor prognosis AML patients while sparing normal HPSCs. This effect may be mediated via targeting of STAT3 signaling and ALDH, which are critical for proliferating LSCs, but not normal HPSCs. Further studies will aim to demonstrate mechanisms of activity and preclinical efficacy in patient-derived AML xenograft mouse models. Disclosures Dovat: Elf Zone, Inc.: Membership on an entity's Board of Directors or advisory committees.

Mutant NPM1 is a Reliable marker of Minimal Residual leukemia in patients with Acute Myeloid Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2484-2484
Author(s):  
Preetesh Jain ◽  
Hagop M. Kantarjian ◽  
Stefan Faderl ◽  
Keyur P. Patel ◽  
Guillermo Garcia Manero ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
De Novo ◽  
Response Rates ◽  
Analytical Sensitivity ◽  
Newly Diagnosed ◽  
Wild Type ◽  
Npm1 Mutation ◽  
De Novo Aml ◽  
Minimal Residual

Abstract Abstract 2484 Background: Mutations in NPM1 (Nucleophosmin-1) have been described in about 35% of adult patients with de novo AML and 45–60 % of AML patients with a diploid karyotype. NPM1 mutations predict for achieving higher complete remission (CR) rates and better outcomes in AML. Few studies have reported on the reliability of mutated NPM1 as a marker for minimal residual disease (MRD) detection in patients with AML. Methods: We conducted a retrospective analysis of patients (n=360) with newly diagnosed AML treated at our institution between 2008 and 2012. The study was approved by the Institutional Review Board. NPM1 mutation status was determined from DNA from unsorted bone marrow (BM) aspirate samples by a PCR-based method at baseline, remission, and relapse. Genomic DNA from bone marrow samples was isolated using the Autopure extractor (QIAGEN/Gentra, Valencia, CA). Mutations in exon 12 of NPM1 were assessed by a DNA-based semi-quantitative polymerase chain reaction capillary electrophoresis (PCR-CE) assay with analytical sensitivity of approximately 2.5%. Results: Data on remission and relapse samples from 360 newly diagnosed and previously untreated patients with AML, with available NPM1 analysis on their BM at the time of initial diagnosis, was collected (see flow chart below). Median age was 60 years (range 21 – 81 years). 262 patients (72%) had de novo AML, and 98 (27%) secondary AML. Cytogenetics was diploid in 137 (38%) patients, t(8;21) in 17 (5%), inversion 16 in 26 (7%), deletion 5 alone in 27 (7.5%), del 7 and 5 in 26 (7%), deletion 7 alone in 21 (5.8%), trisomy 8 in 17 (5%) and miscellaneous in 89 (24.7%) patients, respectively. Overall, 60 (16.6%) patients including 46 of the 137 (33.5%) diploid patients had NPM1 mutation at baseline. Secondary leukemia was more common in the NPM1 wild type (30%) than in the NPM1 mutated (13%) category. When analysed by age, in patients < 60 years (n=175), OS (overall survival), EFS (event free survival) and response rates were significantly superior in NPM1 mutated subgroup (p=0.001, 0.007, 0.02 respectively), while among patients ≥ 60 years (n=185) EFS and response rates were significantly higher in the NPM1 mutated subgroup (p=0.008, 0.03 respectively). Among the patients with diploid cytogenetics who were younger than 60 years (n=60) OS, EFS and CR duration was significantly better in the NPM1 mutated subset (p=0.007, 0.007 and 0.02 respectively), while in those ≥60 years (n=77) there was no statistically significant difference in the outcomes for the NPM1 mutated and wild-type subsets. Among the 60 NPM1 mutated patients 54 (90%) including 41/46 (89%) of those with diploid cytogenetics achieved complete response (CR) or CR without platelet recovery. Thirty nine patients (including 30 with diploid karyotype) had available NPM1 status at the time of CR and all (100%) were negative for NPM1 mutation. Among the patients with mutated NPM1 at baseline who have achieved a NPM1 negative status at CR, 10/39 overall (25%) and 7/30 diploid (23%) patients relapsed. NPM1 status was available for 6 patients overall including 4 with diploid karyotype at the time of relapse. Among them, 5/6 overall (83%) and 3/4 diploid (75%) patients had mutated NPM1, while 1/6 overall (16%) and 1/4 diploid (25%) patients remained NPM1 wild type. This patient relapsed with extramedullary disease (leukemia cutis) without any BM involvement. Among the 300 patients (including 91 with diploid karyotype) with wild type NPM1 at diagnosis, none acquired a mutated NPM1 clone, either at CR or at the time of relapse. Conclusions: These data suggest that mutated NPM1 is a reliable and stable marker for the detection of MRD at the time of CR. Hence, NPM1 mutations can be used to detect MRD and their recurrence may predict pending relapse. Disclosures: Cortes: Celgene: Research Funding. Ravandi:Johnson and Johnson: Honoraria; Celgene: Research Funding.

Genetic Profile and Novel Thrombospondin-1(THBS1) Mutation of Familial AML Syndrome: A Genomic Analysis By Whole Genome Sequencing and Whole Transcriptome Sequencing

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2595-2595
Author(s):  
Daeyoon Kim ◽  
Youngil Koh ◽  
Jeonghwan Youk ◽  
Kwang-Sung Ahn ◽  
Hwi-Joong Yoon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Genetic Factors ◽  
De Novo ◽  
The Other ◽  
Second Hit ◽  
Dna And Rna ◽  
Familial Form ◽  
De Novo Aml ◽  
Tumor Dna

Abstract Background Familial form of acute myeloid leukemia (AML) is not well known except for extreme cases. BRCA1/2 and p53 germline mutation are well known genetic changes that is related to familial AML¡¯s. Because of diverse penetrance potential of germline mutations and complex pathogenesis for the development of AML, it is not easy to discern familial form of AML. In this study, we performed next generation sequencing (NGS) study of a family who has been suspected to have familial form of AML. Material and method Two relatively young patients have been referred to our institution for AML. One was a 41 years old male (son) and the other was 58 years old female (mother). To discover genetic factors involved in hematological familial cancer syndrome, we performed WGS/WTS using DNA of mother and son who are AML patients and WGS using DNA of healthy father. Tumor DNA and RNA of mother and son were extracted from bone marrow samples which were collected at the time of diagnosis and control DNA and RNA was extracted from saliva and bone marrow samples, respectively, at the time of CR. WGS were performed using HiSeq X ten (illumina, San Diego, USA). For WTS, we used HiSeq2000 platform (illumina, San Diego, USA). For the analysis of WGS, we used GATK unifiedGenotyper for caller in this study. The first filter was set an average depth >10 and conf_cut > 50. And next filter step was using several databases (dbSNP, clinvar, cosmnic70, nci60) and filtering tools (Meta SVM and Meta LR) to filter out of called loci. Meerkat was used to analyze structure variations (SVs). To analyze DEGs using WTS, we used HTSeq-count. To analyze DEGs, we used WTS and HTSeq-count. Results and discussion A total of 3,695,266 loci of normal DNA and 3,977,321 loci of tumor DNA were found in mom samples and 3,622,083 of these were commonly found in both normal and tumor samples. Similarly, a total of 3,513,806 loci of normal and 3,935,873 loci of tumor DNA were found in son samples, of which 3,476,405 were commonly found. Of these common loci, we identified 2,799,429 that are universally present in both mum and son. To determine genes that are found only in patients (Mon and son), we filtered out the loci of father¡¯s SNPs and excluded 2,191,882 loci We subsequently identified a total of 607,547 candidate loci which have association with AML malignancies. Using Support vector machines (SVMs) and DNM filter, we found 37 significant genes that are considered to be related with de-novo AML Among these, 12 were (AGL, COL12A1, IMPDH1, LIPN, MET, MYH13, PBX3, ROBO3, SLC34A3, SMO, THBS1 and TP63) already reported to have an association with hematopoietic disorder, while 25 were a novel mutation genes. The most interesting gene is THBS1. It is reported to affect hematopoietic differentiation function via CD36 and CD47 and a greater than 3 fold change was found in both mum and son in DEG analysis. In DEG analysis, total 1317 gene in mother and 473 genes in son were shown differently expressed above 3FC. The number of recurrent DEGs between mother and son is 144 and these genes were estimated to involve in Systemic lupus erythematosus, Chemokine signaling and bladder cancer pathway. We used Mutect to detect somatic mutation that acts as second hit. 29 nonsynonymous-SNVs were detected in son sample and 43 nonsynonymous-SNVs were detected in mother sample. OR11H1 gene was recurrently shown in both mother and son. We performed Structure variants analysis to identify second hit SVs using Meerkat in mother and son separately SVs of mother were detected in several regions and 69 genes were detected in exonic regions, 39 genes were somatic SVs and 12 of these were filtered out because the genes were also detected in the normal sample of son. 27 SVs genes are considered as a candidate of AML second hit in mother. On the other hand, we found 5 genes somatic SVs in son using the same method. Of note, FAM231A gene overlapped with mom tumor sample and son tumor sample. In conclusion, we found 37 significant genes may be related with de-novo AML. In addition, several genetic factors affect tumorigenesis through second hit. Figure 1. Association between THBS1 and other genes by gene to gene networking Figure 1. Association between THBS1 and other genes by gene to gene networking Figure 2. Expression level and heatmap of gene expression of mother and son by DEGs analysis Figure 2. Expression level and heatmap of gene expression of mother and son by DEGs analysis Figure 3. Circosplot of Structure Variantions(SVs) of mother and son Figure 3. Circosplot of Structure Variantions(SVs) of mother and son Disclosures No relevant conflicts of interest to declare.

Phase 1 Results of FF-10501-01, a Novel Inosine 5'-Monophosphate Dehydrogenase Inhibitor, in Advanced Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS), Including Hypomethylating Agent (HMA) Failures

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1640-1640 ◽  
Author(s):  
Guillermo Garcia-Manero ◽  
Hui Yang ◽  
Zhihong Fang ◽  
Hagop M. Kantarjian ◽  
Courtney D. DiNardo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Disease Progression ◽  
Cell Lines ◽  
Dose Level ◽  
Research Funding ◽  
De Novo ◽  
Phase 1 ◽  
Clinical Activity ◽  
Guanine Nucleotide ◽  
Bone Marrow Blast

Abstract Inosine 5'- monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes de novo synthesis of the guanine nucleotide and is overexpressed in both hematologic and solid tumors. FF-10501-01 is a potent new competitive IMPDH inhibitor. We investigated the anti-leukemia effect of FF-10501-01 in a Phase 1 clinical study in advanced AML and MDS, including HMA failures. Previous preclinical studies demonstrated potent anti-proliferative and apoptotic effects of FF-10501-01 on AML cell lines, including HMA-resistant derivatives, through inhibition of de novo guanine nucleotide synthesis. Therefore, we performed a standard 3+3 dose-escalation Phase 1 trial to access the safety and clinical activity of FF-10501-01 in patients with advanced AML, MDS and chronic myelomonocytic leukemia (CMML). Eligibility criteria: age ≥ 18 years, high risk MDS/CMML, AML with documented PD following previous therapy, AML ≥ 60 years of age and not a candidate for other therapy, adequate renal and hepatic function, and no known history of significant cardiac disease. A total of 29 patients, 15M and 14F (23 AML, 6 MDS) have been treated in 7 dose cohorts (50 - 500 mg/m2 PO BID) for 14 days on and 14 days off, and 400 mg/m2 for 21 days on and 7 days off, each 28-day cycle. Median (range) values: age 75 yrs (59 - 88); baseline bone marrow blast counts for AML 34% (12 - 82), for MDS 10% (5 - 16), and overall 30% (5 - 82); and prior treatment regimens 2 (1 - 7). All patients relapsed from, or progressed on, prior HMAs. At baseline, mutations in FLT3, NPM1, GATA2, TET2, ASXL1, DNMT3A, NOTCH1, JAK2, IDH2, PTPN11, KRA, TP53, RUNX1, EZH2 and/or MDM2 were present in 13 of 29 (45%) of patients. Atrial fibrillation (Gr 2) was reported in 2 subjects at a dose of 500 mg/m2 BID. This met the definition of dose-limiting toxicity (DLT) and no further enrollment was made at this dose level. The maximally tolerated dose (MTD) was declared at 1 dose level lower, 400 mg/m2 BID, and this cohort was expanded to 6 subjects. No DLTs have been observed in N=7 total subjects treated at 400 mg/m2 BID x 14 days. FF-10501-01 has been very well tolerated through 24 cycles. The most frequent drug-related AEs have been Gr 1-2 nausea, diarrhea and fatigue. Drug-related thrombocytopenia, neutropenia and bone marrow aplasia (all Gr 4) were reported in 1 patient at 200 mg/m2 BID. The median number of FF-10501-01 cycles received to date is 2 (range 1 - 24). Partial remissions have occurred in 2 AML patients (50 and 100 mg/m2 BID) after 3 cycles, lasting for 5 and 24 cycles, respectively, with the higher dose patient still on study after 24 cycles. A total of 8/23 (34.8%) AML patients, including the 2 PRs, have attained stable disease (SD) control with no disease progression over 3 - 24 cycles. Three AML patients remain on study through 3, 23 and 24 cycles, respectively. A bone marrow complete response was achieved in 1 MDS patient treated at 400 mg/m2 BID after 1 cycle. Although the bone marrow blast counts have increased since, this patient remains stable and is still on therapy through 14 cycles. Three of 6 MDS patients (50%), including the marrow CR, attained SD control with no disease progression over 3, 14 and 14 cycles, and 2 remain on study through 3 and 14 cycles, respectively. FF-10501-01 was rapidly absorbed with mean Tmax of 2.74 hours and mean t1/2 of 4.05 hours. Drug exposure (AUC0-24 and AUCcourse) increased with dose in a near linear manner. Potent suppression of circulating xanthine monophosphate (XMP), a marker of IMPDH activity, has been observed following FF-10501-01 administration on Day 1 of Cycles 1 and 2 at dose levels of 50 mg/m2 BID and above. FF-10501-01 is a promising new agent for the treatment of advanced AML and MDS in patients who have failed or progressed on HMAs and with one or more baseline mutations in pathways known to be affected in AML and MDS. Preclinical activity was seen in multiple leukemia cell lines, including HMA-resistant derivatives. In a Phase 1 trial, clinical activity with a marrow CR, PRs, long-term disease stabilization (≥ 5 cycles) and a highly tolerable safety profile were observed. The Phase 2a expansion phase of the study is soon to begin. Disclosures DiNardo: Agios: Research Funding; Daiichi Sankyo: Research Funding; Celgene: Research Funding; Novartis: Research Funding; Abbvie: Research Funding. Jabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Daver:BMS: Research Funding; Kiromic: Research Funding; Pfizer: Consultancy, Research Funding; Otsuka: Consultancy, Honoraria; Ariad: Research Funding; Karyopharm: Honoraria, Research Funding; Sunesis: Consultancy, Research Funding. Denton:Westat Corporation: Employment. Smith:Westat Corporation: Employment. Tiefenwerth:Westat Corporation: Employment. Iwamura:FUJIFILM Corporation: Employment. Gipson:Strategia Therapeutics, Inc.: Employment. Rosner:Strategia Therapeutics, Inc.: Employment. Myers:Strategia Therapeutics, Inc.: Employment. Paradiso:Strategia Therapeutics, Inc.: Employment.

scholarly journals Integrated Genomic Analysis Identifies UBTF Tandem Duplications As a Subtype-Defining Lesion in Pediatric Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 2) ◽  
pp. LBA-4-LBA-4
Author(s):  
Masayuki Umeda ◽  
Jing Ma ◽  
Benjamin J. Huang ◽  
Kohei Hagiwara ◽  
Tamara Westover ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Research Funding ◽  
De Novo ◽  
Screening Method ◽  
Normal Karyotype ◽  
Rna Seq ◽  
Trisomy 8 ◽  
De Novo Aml ◽  
Pediatric Aml ◽  
Tandem Duplications

Abstract Children with acute myeloid leukemia (AML) have a dismal prognosis due to a high relapse rate; however, the molecular basis leading to relapsed pediatric AML has not yet been fully characterized. To define the spectrum of alterations common at relapse, we performed integrated profiling of 136 relapsed pediatric AML cases with RNA sequencing (RNA-seq), whole-genome sequencing, and target-capture sequencing. In addition to well-characterized fusion oncoproteins, such as those involving KMT2A (n=36, 26.5%) or NUP98 (n=18, 13.2%), we also identified somatic mutations in UBTF (upstream binding transcription factor) in 12 of 136 cases (8.8%) of this relapsed cohort. Somatic alterations of the UBTF gene, which encodes a nucleolar protein that is a component of the RNA Pol I pre-initiation complex to ribosomal DNA promoters, have rarely been observed in AML. In our cohort, all alterations can be described as heterozygous in-frame exon 13 tandem duplications (UBTF-TD), either at the 3' end of exon 13 of UBTF or of the entire exon 13 (Fig. A). As we noticed limited detection in our pipeline as a result of complex secondary indels alongside the duplications, we established a soft-clipped read-based screening method to detect UBTF-TD more efficiently. Applying the screening to RNA-seq data of 417 additional pediatric AMLs from previous studies and our clinical service, we identified 15 additional UBTF-TDs, many of which have not been previously reported. At the amino acid level, UBTF-TDs caused amino acid insertions of variable sizes (15-181 amino acids), duplicating a portion of high mobility group domain 4 (HMG4), which includes short leucine-rich sequences. UBTF-TD AMLs commonly occurred in early adolescence (median age: 12.6, range: 2.4-19.6), and 19 of the total 27 cases had either normal karyotype (n=12) or trisomy 8 (n=7). UBTF-TD is mutually exclusive from other recurrent fusion oncoproteins, such as NUP98 and KMT2A rearrangements (Fig. B), but frequently occurred with FLT3-ITD (44.4%) or WT1 mutations (40.7%). The median variant allele fraction (VAF) of the UBTF-TD was 48.0% (range: 9.7-66.7%). In four cases with data at multiple disease time points, the identical UBTF-TDs were present at high allele fractions at all time points, suggesting that UBTF-TD is a clonal alteration. tSNE analysis of the transcriptome dataset showed that UBTF-TD AMLs share a similar expression pattern with NPM1 mutant and NUP98-NSD1 AML subtypes, including NKX2-3 and HOXB cluster genes (Fig. C) . Altogether, these findings suggest that UBTF-TD is a unique subtype of pediatric AML. To address the impact of UBTF-TD expression in primary hematopoietic cells, we introduced UBTF-TD and UBTF wildtype expression vectors into cord blood CD34+ cells via lentiviral transduction. UBTF-TD expression promotes colony-forming activity and cell growth, yielding cells with a persistent blast-like morphology (Fig. D). Further, transcriptional profiling of these cells demonstrated expression of HOXB genes and NKX2-3, similar to UBTF-TD AMLs in patients, indicating that UBTF-TD is sufficient to induce the leukemic phenotype. To investigate the prevalence of UBTF-TDs in larger de novo AML cohorts, we applied the above UBTF-TD screening method to the available de novo AML cohorts of TCGA (n=151, adult), BeatAML (n=220, pediatric and adult), and AAML1031 (n=1035, pediatric). We identified UBTF-TDs in 4.3% (45/1035) of the pediatric AAML1031 cohort, while the alteration is less common (0.9%: 3/329, p=0.002) in the adult AML cohorts (Fig. E). In the AAML1031 cohort, UBTF-TDs remain mutually exclusive with known molecular subtypes of AML and commonly occur with FLT3-ITD (66.7%) and WT1 (40.0%) mutations and either normal karyotype or trisomy 8. The presence of UBTF-TDs in the AAML1031 cohort is associated with a poor outcome (Fig. F, median overall survival, 2.3 years) and MRD positivity; multivariate analysis revealed that UBTF-TD and WT1 are independent risk factors for overall survival within FLT3-ITD+ AMLs. In conclusion, we demonstrate UBTF-TD defines a unique subtype of AMLs that previously lacked a clear oncogenic driver. While independent of subtype-defining oncogenic fusions, UBTF-TD AMLs are associated with FLT3-ITD and WT1 mutations, adolescent age, and poor outcomes. These alterations have been under-recognized by standard bioinformatic approaches yet will be critical for future risk-stratification of pediatric AML. Figure 1 Figure 1. Disclosures Iacobucci: Amgen: Honoraria; Mission Bio: Honoraria. Miller: Johnson & Johnson's Janssen: Current Employment. Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company.

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