Acute Myeloid Leukemia Subgroups Research Articles

Single-Cell Transcriptional Profiling of Acute Myeloid Leukemia Identifies Prognostic Transcriptional Profiles at Diagnosis

Acute myeloid leukemia (AML) is a lethal cancer with a survival of less than 50%. Standard cytotoxic therapies frequently induce complete remission, but patients frequently relapse and die of their disease. Leukemia stem cells (LSCs) are the leukemia cells with self-renewal potential and ability to recapitulate the disease. In hematopoietic stem cells, the mechanisms of proliferation are distinct from self-renewal (Li et al. Nature 2013). Consequently, targeting proliferation may explain the failure of traditional chemotherapy to target LSCs and eradicate AML. Our goal is define the molecular mechanisms that allow AML to relapse. We previously showed that activated NRAS (NRASG12V) facilitates self-renewal in the LSC-enriched subpopulation of a mouse model of AML (Mll-AF9 / NRASG12V, Sachs et al. Blood 2014, Kim et al. Blood 2009). We hypothesize that understanding the NRAS-activated pathways required for self-renewal at the single-cell level can be used to identify clinically relevant features of human AML.We used single-cell RNA sequencing to identify the self-renewing cells among the LSC-enriched subgroup in this model (Mac1LowKit+Sca1+, “MKS”). We identified three discrete transcriptional profiles among the LSC-enriched subpopulation and found that that two of these profiles (Profile 1 and Profile 2) are NRASG12V-dependent. These two profiles can be differentiated by CD36 and CD69 expression. We sorted the MKS LSCs based on CD36 and CD69 expression. Sorted LSC subsets were transplanted into recipient mice to compare their ability to transfer leukemia as a measure of their self-renewal capacity. We found that MKS-CD36-CD69+ cells (consistent with Profile 1) rapidly transferred leukemia with high penetrance in 20 of 22 mice. In contrast, MKS-CD36+CD69- cells (Profile 2) failed transfer leukemia in most mice; only 2 of 25 of these mice developed AML (p < 0.004).In our previous work, we demonstrated that the NRASG12V-activated self-renewal gene expression profile that we identified in our murine model was expressed in human AML, suggesting that the gene expression behavior of LSCs from this model may recapitulate the gene expression behavior of human LSCs (Sachs et al. Blood 2014). We used our murine single-cell self-renewal transcriptional profile to define a 96-gene panel consisting of 88 genes from this profile and 8 housekeeping genes. We sorted primary, diagnostic human AML cells for leukemia stem and progenitor cells (CD34+CD38-) and performed single-cell qPCR on these cells using our 96-gene panel. We found that a subset of these human LSCs preferentially expresses Profile 1, the self-renewal gene expression profile that we identified in our murine model, and another subset preferentially expresses Profile 2 (the profile associated with no leukemia-reconstituting capacity).Next, we investigated whether CD36 and CD69 delineate clinically relevant transcriptional subgroups in human AML. We used previously published, clinically annotated gene expression datasets of human AML that include associated survival outcomes data (TCGA, GSE12417 (Metzeler et al. Blood 2008), and GSE6891 (Verhaak et al. Haematologica 2009)). Gene Cluster Expression Summary Score (GCESS, Scott et al. under review) analysis identified gene clusters associated with CD69 and CD36 in our murine single-cell dataset. We found that the CD36 and CD69-associated clusters are conserved in the human transcriptional datasets. More importantly, patients that express either the CD69 or the CD36-associated gene expression profile suffer from shorter survival times in these three, independent human transcriptional datasets.In these experiments, we use a murine model of AML to define the LSC self-renewal gene expression profile at the single-cell level and functionally validate this profile in vivo . Analogous to the murine model, a subset of human AML stem and progenitor cells expresses this LSC self-renewal gene expression profile at the single-cell level. Finally, we found that the gene expression profiles we identified in our murine single-cell data are associated with poor outcomes in human AML indicating that our single-cell gene expression analyses can be used to identify prognostic patient subgroups. These findings suggest that these gene expression profiles can be used to identify functional transcriptional modules associated with poor outcome in human AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Biology and Prognostic Markers in Young Children with Acute Myeloid Leukemia -the Jccg Study, JPLSG AML-05-

[Introduction] Acute myeloid leukemia (AML) in children aged < 12 months of age was conventionally defined as infant AML. On cytogenetic and molecular analysis, 11q23/ KMT2A rearrangement (KMT2A -R) was found to be the most frequent (approximately 50%) cause of infant AML. On prognostic analysis, infant AML was reported to have favorable outcome compared with AML in older children, and only KMT2A -R and RBM15-MKL1 were known to be intermediate-risk prognostic factors in infants. Although infants have been treated in the same regimens as older children, in some clinical trials, drug dosage for infants has been reduced to avoid severe toxicities. However, new findings in young children with AML have been recently accumulated. A recent report by an international expert panel of pediatric AML has proposed AML in children aged < 24 months of age as a distinct age-subgroup because their morphologic and cytogenetic/molecular characteristics were considered to be similar to those of infants (Creutzig et al, 2012). Moreover, recent studies have identified novel inv(16)(p13.3q24.3)/ CBFA2T3-GLIS2 and t(11;12)(p15;p13)/ NUP98-KDM5A fusion genes in AMKL. In this study, we investigated biology and prognosis of children aged < 5 years, to identify unique characteristics of young children with AML and propose a novel risk-stratification for them.[Patients and Methods] A total of 369 children with AML were enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial. In this study, we analyzed 133 children aged < 5 years (36%). We screened fusion genes of CBFA2T3-GLIS2, NUP98-KDM5A, RBM15-MKL1, KMT2A -Rs, NUP98-NSD1, FUS-ERG, DEK-NUP214, RUNX1-RUNX1T1, and CBFB-MYH11, and gene mutations of KIT, NRAS, KRAS, WT1, NPM1, KMT2A -PTD, FLT3 -ITD, CEBPA, ASXL1, ASXL2, and CSF3R .[Results] In clinical characteristics, no change was observed in the sex ratio with age. The WBC count at diagnosis was the highest in children aged 2 years (24-35months), and a trend of lower WBC in children aged > 2 years (> 35 months). On FAB classification, the proportion of M5 and M7 was observed to be decline with age. Among fusion genes, KMT2A -R was the most frequently identified fusion gene (23%), followed by RUNX1-RUNX1T1, CBFB-MYH11, and CBFA2T3-GLIS2 (13.5%, 9.8%, and 8.3%, respectively). Conversely, neither NUP98-NSD1 nor DEK-NUP214 was found. Although the frequencies of KMT2A -R and CBFA2T3-GLIS2 tended to decrease with age, the frequency of RUNX1-RUNX1T1 significantly increased with age. Among gene mutations, KIT and NRAS were the most frequent, and gene mutations other than ASXL1 and KMT2A -PTD were recurrently identified. When correlation of fusion genes with gene mutation was analyzed, 21 (68%) of 31 children with CBF-AML had gene mutations, whereas only 8 (16%) of 51 children with non-CBF-AML had gene mutations (p < 0.001). For fusion genes, 28 (90%) of 31 KMT2A -R-positive children were aged < 3 years (< 36 months) (90%), and 17 (94%) of 18 RUNX1-RUNX1T1 -positive children were aged > 2 years. Thus, according to these molecular characteristics of young children with AML, we defined children aged < 3 years as the youngest subgroup with distinct biology (n = 99). We further performed prognostic analyses of the youngest subgroup. According to recent previous studies that reported poor prognosis of CBFA2T3-GLIS2 and NUP98-KDM5A in AMKL, these fusions were combined to the same group. The Kaplan-Meier analyses revealed that CBFA2T3-GLIS2 / NUP98-KDM5A -positive children had the poorest outcome in OS, EFS, and CIR (38%, 13%, and 88%, respectively). Surprisingly, children with CBF-AML had the second poorest outcome in EFS and CIR (45% and 55%, respectively), although this subgroup had an excellent OS (100%), indicating that children with CBF-AML in the youngest subgroup have a high-risk of relapse and might need more intensified therapy as an initial treatment.[Conclusions] The youngest subgroup of pediatric AML had a unique biology. Children with CBFA2T3-GLIS2 and NUP98-KDM5A had the poorest outcome, and children with CBF-AML also had a high relapse rate. Because this subgroup is known to have a high incidence of severe side effects and late effects, a novel risk-stratified therapy restricted to young children is required. DisclosuresNo relevant conflicts of interest to declare.

Molecular and Pharmacological Inhibition of ATP Binding Cassette Transporter ABCB6 Decreases Chemoresistance in Acute Myeloid Leukemia

Cytarabine (Ara-C) and Daunorubicin (Dnr) remain the main stay treatment for patients with acute myeloid leukemia (AML). Drug resistance and relapse are the major hurdles in achieving long term event-free survival. ATP Binding Cassette transporter proteins (ABC) plays a prominent role in drug resistance by extruding the anticancer drugs from the cells. Among the mitochondrial ABC transporters, the heme protoporphyrin transporter ABCB6 was found to be highly expressed in cell lines- HT-29, A549, SK3, St-4, A2780, SKOV3, KK47, T24, K562, and U937 made resistant to camptothecin, cisplatin, etoposide (VP-16), adriamycin and also in HepG2 and Hep3B cell lines resistant to arsenite. We have previously reported that ABCB6 expression was significantly higher in primary AML samples resistant to Dnr (Varatharajan et al., 2017). AML patients with FLT3 -ITD showed low ABCB6 expression levels and were more sensitive to arsenic trioxide (ATO) compared to FLT3 wild type (Abraham et al., 2014). In the present study, using a molecular and inhibitor based approach we explored the functional role of ABCB6 in mediating resistance to Dnr and ATO in AML.Cell lines K562 and THP1 with high ABCB6 expression and high IC50 to both Dnr and ATO were transduced with an inducible lentiviral shRNA targeting ABCB6 . The efficiency of knock down was confirmed by real time PCR, immunoblot and immunofluorescence. As ABCB6 is known to localize in the mitochondrial membrane, its role in mediating reactive oxygen species (ROS) levels were assessed by flowcytometry using DCFDA. The effect of ABCB6 knock down in causing apoptosis to Dnr and ATO mediated apoptosis was evaluated using 7AAD and Annexin V. The effect of ABCB6 pharmacological inhibitor, tomatidine in improving chemosensitivity was also evaluated in combination with Dnr and ATO by MTT assay. The efficiency of knock down was >80% in K562 (p<0.001) and >60% in THP1 (p<0.001) cell lines. Concordant to the mRNA levels, there was a significant decrease in ABCB6 protein levels by western blot and immunofluorescence. Interestingly, ABCB6 knock down increased basal ROS levels in both K562 (18 fold) and THP1 (3.1 fold) compared to their respective doxycycline negative (Dox -ve) control. The combination of ABCB6 knockdown and Dnr treatment increased ROS by 2.1-fold in THP1 and by 21-fold in K562 cells and along with ATO by 1.7-fold in THP1 and 5.7-fold in K562 compared to the Dox -ve cells. Knockdown of ABCB6 also improved sensitivity to 0.125µM of Dnr (17.45% to 36.94%, p= ns) and 6µM ATO (10% to 39%, p=0.007). ABCB6 inhibitor tomatidine reduced the IC50 values of THP1 to Dnr (0.16µM to 0.05µM) and ATO (>6µM to 2.91µM) and K562 cells to ATO (12.08µM to 7.26µM). Our results suggest that targeting ABCB6 could sensitize different subgroups of AML to treatment with ROS inducing agents like Dnr and ATO. Further mechanistic studies are required to investigate if the effect of ABCB6 inhibition is selective to leukemic cells. DisclosuresNo relevant conflicts of interest to declare.

Obesity Is Associated with Favorable Cytogenetic Abnormalities in Pediatric Acute Myeloid Leukemia

Background: In adults, obesity has been associated with increased risk of developing acute myeloid leukemia (AML) with recurrent cytogenetic abnormalities and preclinical studies indicate that certain proteins associated with obesity are also involved in the development of subtypes of AML. Studies on the effect of body-mass index (BMI) in childhood AML have suggested differences in cytogenetic abnormalities across weight-groups, but the association has not been well-studied. Further, the effect of BMI on outcome in children with AML is controversial. While previous studies have found that overweight is associated with worse survival, mainly due to increased treatment-related mortality, others have not confirmed this association. We investigated differences in cytogenetic abnormalities, treatment-related mortality, relapse and overall survival in four weight groups in a large multi-institutional cohort of children diagnosed with AML.Methods: We included patients, age 2-17, diagnosed with de novo AML from the five Nordic countries (2004-2016), Hong Kong (2007-2016), the Netherlands and Belgium (2010-2016) and Canada and USA (1995-2012). The cohort was population-based for the European countries, Hong Kong and Canada from 1995-2004 for all provinces except for Saskatchewan. In USA and Canada from 2005-2012 the cohort was based on cases included for a prospective study at 15 Canadian and 2 American centers. Cases with Down syndrome, acute promyelocytic leukemia, isolated granulocytic sarcoma or missing data on BMI at diagnosis (n=7) were excluded. BMI standard deviation (SD) for age and sex was calculated and categorized as following: underweight: <-2 SD, healthy weight: -2-1 SD, overweight: >1-2 SD and obesity: >2 SD according to the World Health Organization. Treatment-related mortality was classified according to an international consensus-based definition of treatment-related mortality in children with cancer. Logistic regression was used to investigate associations between weight groups and cytogenetic abnormalities. Thirty-six cases had missing data on one or more cytogenetic abnormality and were excluded from the cytogenetic abnormality analyses. Time-to-event analyses and Cox regression was used to investigate associations with treatment-related mortality, relapse and overall survival. Reason for death was unknown in two cases (excluded from treatment-related mortality analyses). For all analyses, the healthy weight group was used as reference. For relapse analyses death was a competing risk and for treatment-related mortality analyses, death from progressive disease was a competing risk. Treatment-related mortality analyses were adjusted for sex, age, and year of diagnosis; relapse and overall survival analyses also for country.Results: In total 867 patients were included in the study. The median age was 10 (range 2-17) and 53% were male. Patients were treated on 17 different protocols with AAML0531, COG9421, NOPHO-AML 2004, DB AML 01 and NOPHO-DBH AML 2012 accounting for 79%. None of the protocols recommended chemotherapy dose capping for overweight patients. At diagnosis 32 (4%) were underweight, 593 (68%) were healthy weight, 160 (18%) were overweight and 82 (9%) were obese. Boys and older patients were more likely to be underweight or obese. North Americans were less likely to be underweight and more likely to be obese. There were 326 first relapses, 104 treatment-related mortalities and 251 deaths. The frequency of t(8;21) and inv(16) were higher in the obese group and the frequency of MLL rearrangements were higher in the overweight group. The frequency of t(8;21) and inv(16) increased with increasing weight-group (table 1). There was no evidence of difference in the risk of relapse, treatment-related mortality or overall survival based on weight groups (table 2).Conclusion: This multi-institutional study of 867 pediatric patients with de novo AML found an association between favorable cytogenetic abnormalities and obesity. The study did not confirm previous reports of overweight/obesity being associated with decreased overall survival, risk of relapse or treatment-related mortality. Our results could suggest that overweight is a risk factor for developing specific cytogenetic subgroups of AML in children. [Display omitted] DisclosuresKaspers:Janssen-Cilag: Research Funding.

Differential Ex Vivo Sensitivity Patterns to Small-Molecule Inhibitors and Combination Therapies Among Acute Myeloid Leukemia Patients Harboring Both FLT3 and NPM1 Mutations

Differential Ex Vivo Sensitivity Patterns to Small-Molecule Inhibitors and Combination Therapies Among Acute Myeloid Leukemia Patients Harboring Both FLT3 and NPM1 Mutations

Comparison of Survival Outcomes in Elderly Acute Myeloid Leukemia Patients (Age ≥ 70) When Treated with Azacitidine or Decitabine As an Initial Treatment Modality

BackgroundTherapeutic options for patients age ≥70 years with acute myeloid leukemia (AML) are limited and comprised mainly of hypomethylating agents (HMAs) including azacitidine (AZA) and decitabine (DEC) or clinical trials. Given a lack of direct comparisons between these agents, we aimed to explore clinical outcomes in older AML patients treated with AZA vs DEC.MethodsWe analyzed 983 patients with AML, age ≥ 70 years, treated at Moffitt Cancer Center between 1995 and 2016. Response rates and overall survival (OS) were compared among patients treated with azacitidine (AZA) or decitabine (DEC). The Kaplan Meier method was used for survival analysis and the log-rank test was used to determine significance (p-value ≤0.05). The World Health Organization (WHO) criteria were used to define complete response (CR) and complete response with incomplete hematologic recovery (CRi).ResultsOut of 983 patients analyzed, we identified 256 patients who were treated with HMA. Twenty four patients were excluded from the analysis due to prior treatment with HMA for non-AML diagnoses, yielding a cohort of 232 patients who were HMA naïve. The median age of all patients was 76 years (range 70-91) and 64% were male. The cohort included 108 patients (46.6%) with secondary AML and 124 patients (53.4%) with de novo AML. DEC was used in 72 patients (31%) and the remaining 156 patients (67%) received AZA as a frontline treatment. Response rates (CR + CRi) were similar in both AZA and DEC treated patients (24% vs. 30%, respectively, p=0.456). Median OS was 16.2 months (mo) in the AZA cohort vs 11.5 mo in the DEC cohort (p=0.331). Per European LeukemiaNet (ELN) cytogenetic risk classification for AML, patients with favorable-risk cytogenetics (FRC) comprised 1.92% and 0% of all patients treated with AZA or DEC, respectively. Patients with intermediate-risk cytogenetics (IRC) comprised 56.41% and 54.17%, respectively; and patients with poor-risk cytogenetics (PRC) comprised 34.6% and 34.72%. respectively. Median OS in the IRC patients was 20.5 mo in the AZA cohort vs 16.5 mo in the DEC cohort (p=0.638). The median OS in PRC patients treated with AZA was 11.5 mo compared to 7 mo with DEC (p=0.019) (Figure1A). Upon further refinement of PRC cohort into those harboring a monosomal karyotype (MK) vs a non-monosomal karyotype (NMK), the NMK patients treated with AZA (n=32) had a superior median OS of 13.6 mo compared to 6.8 mo with DEC (n=16) (p=0.0459) (Figure 1B). Such superiority was not observed in MK patients (8.3mo with AZA vs. 10.6 mo with DEC (p=0.490)).ConclusionsThese data suggest that overall survival in elderly AML patients (age ≥70) is not impacted by the choice of HMAs. However, patients with poor risk cytogenetics had a significantly longer OS when treated with AZA vs DEC, most apparent in poor-risk patients harboring a non-monosomal karyotype. Studies to molecularly characterize this poor-risk AML subgroup in relation to azanucleoside efficacy are warranted. [Display omitted] DisclosuresSallman:Celgene: Research Funding. Padron:Incyte: Honoraria, Research Funding. Komrokji:Celgene: Honoraria; Novartis: Honoraria, Speakers Bureau. Sweet:Pfizer: Consultancy; Otsuka: Consultancy; Ariad: Consultancy, Speakers Bureau; Incyte: Research Funding; Karyopharm: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Speakers Bureau. Lancet:BioSight: Consultancy; Bio-Path Holdings: Consultancy; Janssen: Consultancy; Erytech: Consultancy; Novartis: Consultancy; Pfizer: Other: Institutional research funding, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Boehringer Ingelheim: Consultancy.

Clofarabine-Based Consolidation Improves Relapse-Free Survival of Patients with Acute Myeloid Leukemia with Complex or Micro-Complex Karyotype: Results from the Randomized ALFA-0702 Study

Introduction. Acute myeloid leukemia (AML) encompasses heterogeneous entities, but global outcome remains poor. Among genetic AML subgroups, complex karyotypes represent one of the most difficult-to-treat. Genomic alterations arising from recent approaches in molecular genetics have allowed describing new subsets with potential therapeutic implications. Besides, the latter AML classification (“2017 European LeukemiaNet (ELN) risk stratification”) integrates both molecular alterations and conventional cytogenetics. High-resolution single-nucleotide polymorphism array (SNP-Array) identifies copy number alterations (CNA) and copy-neutral losses of heterozygosity (CN-LOH). In the randomized ALFA-0702 study (NCT 00932412), we have recently reported the effects of a CLARA combination (clofarabine + intermediate-dose cytarabine) as compared to standard high-dose cytarabine (HDAC) regimen as consolidation chemotherapy in younger adults with AML. Here, we aimed at evaluating the effects of the CLARA regimen according to patient subsets defined by SNP-array abnormalities.Methods. A total of 221 patients aged 18-59 years old with intermediate- or adverse-risk de novo AML in first complete remission (CR/CRp) after a common intensive induction course were randomized in the ALFA-0702 trial (Thomas et al. JCO 2017). The primary endpoint was relapse-free survival (RFS). Bone marrow or peripheral blood samples at diagnosis were analyzed by Cytoscan HD array (Affymetrix) according to the manufacturer's instructions. Data were screened according to the ChAS 3.1 algorithm, with stringent filter retaining CNA larger than 20 kb - 20 markers, telomeric CN-LOH > 3Mb and interstitial CN-LOH > 10Mb.Results. SNP-array was successfully performed in 187 out of the 221 randomized patients (92 CLARA arm, 95 HDAC arm). Twenty-three patients could not be analyzed in SNP-array for the following reasons: DNA sample was not available (15 patients) or in insufficient quantity (8 patients). SNP-array analysis failed in 11 patients. Median number of genomic SNP-Array abnormalities reached 2 per patient (range, 0-46) and did not significantly differ between both randomization arms (p=0.41). CNA accounted for the most common SNP-Array abnormalities (Loss=54%, Gain=38%, vs. CN-LOH=8%). Conventional cytogenetic analysis showed a complex karyotype (defined by at least 3 chromosomal abnormalities) in 37 patients (11 CLARA, 16 HDAC). In such patients, CLARA consolidation improved RFS (median RFS, 22 vs. 8 months; p=0.027). Interestingly, a similar impact was observed in the 56 patients (27 CLARA, 29 HDAC) with micro-complex karyotypes defined by 4 or more SNP-Array abnormalities. Notably, 7 patients with complex karyotype did not display micro-complex karyotype because of low bone marrow/blood blast infiltration and/or balanced alterations. In patients with micro-complex karyotypes, median RFS was 22 months in the CLARA arm compared to 10 months in the HDAC arm (p<.0001). The rate of patients who received allogeneic stem cell transplantation in first CR did not differ between the two arms (p=0.99). Importantly, CLARA consolidation tended to overcome the poor prognostic impact of patients with micro-complex karyotype. Indeed, patients with such lesions had similar 4-years RFS than other patients in the CLARA arm (44% vs. 47%) while it remained a poor prognosis factor in the HDAC arm (14% vs. 47%; Figure 1).Conclusion. As compared to conventional cytogenetics, SNP-array represents a high-resolution approach to better characterize molecular profile of AML patients by pointing out cryptic molecular abnormalities. Our results suggest that clofarabine-based consolidation benefits both patients with complex karyotypes and micro-complex karyotypes defined by 4 and more SNP-Array abnormalities. Therefore by delineating micro-complex karyotypes in SNP-array, we have defined a new subset of AML patients that could potentially benefit from a clofarabine-based consolidation regimen (21 additional patients in comparison with complex karyotypes). SNP-array could thus help to improve AML management by refining adverse patient subgroups that could potentially benefit from new alternative consolidation regimen. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Long-Term Follow-up on Adult Patients with Philadelphia-Chromosome Positive Acute Myeloid Leukemia Who Were Treated with Interim Imatinib Followed By Allogeneic Hematopoietic Cell Transplantation

▪Background: The incidence of Philadelphia chromosome (Ph)-positivity in adult patients with acute myeloid leukemia (AML) is very low ranging from only 1 to 2%. Ph-positive AML has been included in the high-risk group presenting lower complete remission (CR) rate and early relapse after standard chemotherapy. However, the treatment outcome in a large cohort is not well elucidated due to its low incidence and variable treatment strategies including the use of tyrosine kinase inhibitors. We attempted to unveil clinical outcomes of Ph-positive AML in association with concomitant chromosomal abnormalities.Methods: We retrospectively analyzed 29 patients with Ph-positive AML (median age: 43 years old [range; 18-80]) from 2002 to 2016. Except for the 2 patients who did not get treated at all, 3 were treated repeatedly with low-dose cytarabine and 24 were treated with 3+7 standard induction chemotherapy, followed with the application of interim imatinib 400mg until the starting day of next chemotherapy or conditioning for hematopoietic cell transplantation (HCT). Among them, 17 underwent allogeneic HCT from sibling donors (n=6), unrelated donors (n=7), and haploidentical donors (n=4).Results: In total of 29 patients, 7 (24.1%) had additional inv(16). There were 3 therapy-related AML in Ph-positive AML subgroup with inv(16), while none were observed in the subgroup without inv(16). In the Ph-positive AML subgroup with inv(16), median age was younger (31 vs. 44 years old, p= 0.083) and CR rate was higher (85.7% vs. 54.5%, p= 0.214) than the opposite. Among 27 treated patients, 20 (74.1%) patients achieved CR after standard chemotherapy with interim imatinib, and 2 achieved CR after low-dose cytarabine with interim imatinib. In the entire patients group, after a median follow-up duration of 41.5 months (range: 0.2 to 156.6), 5-year overall survival (OS) was 40.1%. However, 5-year OS of 17 patients (11 in subgroup without inv(16) and 6 in subgroup with inv(16)) treated with allogeneic HCT was 69.3%, and 6 patients among them with Ph-positive AML with inv(16) were all alive after allogeneic HCT. In contrast, all 12 patients who were not treated with allogeneic HCT expired within a median of 6.3 months [range: 0.2-18].Conclusion: This study revealed that interim imatinib combined chemotherapy produced an acceptable CR rate, and allogeneic HCT as a post-remission therapy was a feasible choice for providing a long-term disease control in adult patients with Ph-positive AML. We also identified that inv(16) was related to favorable outcomes regardless of the coexisting t(9;22) and to therapy-related AML. DisclosuresKim:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Il-Yang: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Genetically-Defined AML Subgroups Represent Diverse Epigenetic Heterogeneity Landscape

Epigenetic allele (epiallele) complexity and clonal evolution are linked to inferior clinical outcome in acute myeloid leukemia (AML). However, the source of epiallelic heterogeneity in AML is not known. Here we investigated whether the abundance and genomic distribution of epialleles might be linked to canonical recurrent somatic mutations. We performed mutational and methylome sequencing in a cohort of 119 clinically annotated AML patients curated to reflect some of the most common genetic and epigenetic lesions. Specifically, patients were selected based on presence of twelve highly recurrent genetic lesions: t(8;21), t(15;17), INV(16), t(8;21), Del(5/7q), EVI1, t(v;11q23), CEBPA double mutation, mutations in DNMT3A, IDH1/2, NPM1, and FLT3. Also included were biphenotypic patients with CEBPA silencing. We used 14 normal bone marrow CD34+ cells samples (NBM) as controls. Epiallele complexity was measured using orthogonal approaches, including “proportion of discordant reads (PDR)”, epipolymorphism, and Shannon entropy. We also measured the degree of global epiallele shift compared to NBM using Methclone.Strikingly, we found that AMLs with certain specific genetic lesions manifested characteristic and distinct genomic distribution and abundance of epialleles (p = 2.51-10, one-way ANOVA test). Overall, epiallele diversity was linked to inferior clinical outcome (p = 0.048, log-rank test of event-free survival). Moreover, epiallele complexity was significantly associated with high-risk cytogenetic AMLs (p = 0.002, Wilcoxon rank sum test). However, certain favorable risk genetic lesions - t(15;17) and CEBPA double mutation - were linked to high levels of epiallele diversity, while others manifested low burdens, including t(8;21), INV(16), and NPM1 mutations. Similarly, certain high-risk genetic lesions - Del(5/7q), CEBPA silencing, EVI1 - manifested high levels of epigenetic diversity whereas other high-risk alleles including t(v;11q23) and DNMT3A mutations had relatively low burdens. We used tSNE and various clustering metrics to determine the relative similarity in epiallele variance and location among genetically defined AMLs. These data indicated striking differences. For example, patients with t(15;17), INV(16), t(8;21), CEBPA silencing, CEPBA double mutation and t(v;11q23) formed unique and distinct clusters, as well as patients with IDH1/2 mutations but not DNMT3A mutations. Patients with Del(5/7q) and mutations in NPM1 did not form discrete clusters suggesting that these lesions do not drive formation or diversity of specific epiallele sets.We next wondered if cooperation between somatic mutations might increase epiallele diversity. We address this question using genetically engineered mice with uniform genetic backgrounds. We tested: i) Tet2-/-, Flt3ITD and Tet2-/-+ Flt3ITD (T2F3) and ii) Idh2R140Q, Flt3ITD and Idh2R140Q+ Flt3ITD (I2F3). In all cases, we obtained purified LSK cells at similar timepoints before development of leukemia, and normal controls. In all cases, the single mutants (Tet2-/-, Idh2R140Q or Flt3ITD) manifested modest if any increase in epiallele diversity. In contrast, both the T2F3 and I2F3 mice had significantly higher levels of epiallele complexity (p=0.01 and p = 0.006, t-test, respectively compared to the single mutants). Therefore, epiallele diversity can precede transformation to acute leukemia and is enhanced by cooperation between somatic mutations.Given that epiallele diversity is linked to inferior outcome, we wondered if it could be potentially reversed using epigenetic targeted therapy. Therefore, we treated T2F3 leukemic mice with 5'Aza, and I2F3 mice with the IDH2 inhibitor AG221. In T2F3, 5'Aza profoundly reduced epiallelic diversity (p = 0.002, t-test), whereas AG-221 was effective in I2F3 but not as strong as 5'Aza in the Tet2 leukemias (p = 0.03, t-test). Both mouse models were also treated with AC220 (FLT3 inhibitor). However, AC220 - while having some anti-leukemia activity - did not reduce epiallele diversity in either model. Our data show that epiallele complexity is potentially reversible by epigenetic therapy. Perhaps part of the benefit of epigenetic therapy is due to this effect, by reducing population fitness of AMLs. Moreover, we show that genetic lesions are a critical source of epigenetic heterogeneity driving the genomic location and abundance of epialleles. DisclosuresLi:The Jackson Laboratory for Genomic Medicine: Employment. Levine:Qiagen: Equity Ownership; Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership; Roche: Research Funding; Celgene: Research Funding.

High Frequency of P53 Mutations Detected by a Microarray Resequencing Assay, the AmpliChip P53 Test, in AML with a Complex Aberrant Karyotype.

Acute myeloid leukemia (AML) is heterogeneous in morphology, cytogenetics, and prognosis. On the basis of cytogenetics it can be subdivided into three groups with favorable, intermediate, or unfavorable prognosis. Accounting for 10%–20% of all AML cases, AML with complex aberrant karyotypes is more frequent at higher ages and is the subgroup with the worst prognosis. AML with complex aberrant karyotype is generally defined by the presence of three or more clonal chromosome abnormalities. Genetically, it has a characteristic pattern of chromosomal gains and losses, resulting also in a unique gene expression pattern including upregulation of genes involved in DNA repair. One of the important characteristics of AML with complex aberrant karyotype is the high incidence of deletions and/or mutations in the tumor suppressor p53 gene (60%–80%) which occur very unfrequently in other AML subgroups. Here we present data on 119 AML cases with complex aberrant karyotypes that were analyzed with the new AmpliChip p53 Test, a microarray-based resequencing test that is designed to detect single nucleotide changes in the entire coding region of the p53 gene. The AmpliChip p53 Test queries for the presence of sequence alterations through comparative analysis of the microarray hybridization pattern of a series of probes from sample DNA to wild type (wt) reference DNA. Starting from genomic DNA, the 10-hour assay includes PCR amplification, fragmentation and labeling, hybridization to the microarray, washing and staining, and mutation data analysis. By use of this technique we have detected p53 mutations located in exons 4–10 in 89/119 (74.8%) samples. These mutations included missense mutations, frameshift mutations, splice site mutations, silent mutations, and nonsense mutations. All 119 samples had also been analyzed in parallel for p53 mutations by a combination of several alternative methods including the predecessor Affymetrix p53 GeneChip microarray, denaturing high performance liquid chromatography (DHPLC), as well as conventional sequencing. The specificity and sensitivity of the AmpliChip p53 Test were compared to the results obtained with these alternative assays. Discordant results were observed in 27 samples when comparing the AmpliChip p53 Test to the alternative assays. Among these 27 samples the AmpliChip p53 Test detected mutations in Exon 10 in 2 samples which had not been tested by DHPLC and further exclusively detected the R72P polymorphism in 9 samples. The remaining group of discordant samples contained:5 samples with a I03 dup 16bp +40 (acctggagggctgggg);3 samples containing a > 2 bp deletion;2 samples with insertion mutations; andone with a mutation in intron 3.Due to technical reasons, all of these aberrations noted above are out of the specifications of the AmpliChip p53 Test. In 5 samples the AmpliChip p53 Test detected additional mutations that were not identified by conventional methods. In conclusion, the AmpliChip p53 Test is a robust and rapid diagnostic tool that allows to identify a poor prognostic subset of AML. Unraveling the biology of this disease may provide new pathophysiologic insights applicable to therapeutic intervention, especially for the targeted therapy of AML with complex aberrant karyotype.

Expression and prognostic relevance of long noncoding RNAs CRNDE and AOX2P in adult acute myeloid leukemia.

Several long noncoding RNAs (lncRNAs) have been demonstrated to play a critical role in the tumorigenesis of acute myeloid leukemia (AML), and altered expression of certain lncRNAs has been recognized as a potential prognostic marker in AML patients. Here, we sought to determine whether the expression of the lncRNA colorectal neoplasia differentially expressed (CRNDE) and aldehyde oxidase 2 pseudogene (AOX2P) is associated with clinicopathological features and clinical outcome of patients with AML. CRNDE and AOX2P expression levels were measured in diagnostic blood samples from 200 adult patients with de novo AML, along with 50 healthy control blood samples, using quantitative real-time polymerase chain reaction (qRT-PCR). The association of CRNDE and AOX2P expression with the clinicopathological characteristics and outcome of AML patients was analyzed. Upregulated CRNDE expression was independently associated with lower complete remission (CR) rates in the whole cohort of AML (P<.001). AOX2P overexpression was identified as an independent adverse prognostic marker for CR in the CN-AML (P=.009) and non-t (15;17) AML (P<.001) subgroups. Patients with high CRNDE expression had a significantly shorter event-free survival (EFS, whole cohort of AML: P=.017; CN-AML: P=.001; non-t (15;17) AML: P=.006) and inferior overall survival (OS, whole cohort of AML: P=.002; t(15;17) AML: P=.001) than those with low CRNDE expression. EFS and OS did not differ significantly between patients with high AOX2P expression and those with low expression. Aberrantly upregulated CRNDE expression and, to a lesser extent, AOX2P overexpression, are associated with poor prognosis in AML patients, suggesting that the determination of CRNDE and, perhaps, AOX2P, expression level at diagnosis provides valuable prognostic information, allows refinement of risk stratification, and helps clinical decision-making in AML.

Infant Acute Myeloid Leukemia: A Unique Clinical and Biological Entity.

Infant acute myeloid leukemia (AML) is a rare subgroup of AML of children <2 years of age. It is as frequent as infant acute lymphoblastic leukemia (ALL) but not clearly distinguished by study groups. However, infant AML demonstrates peculiar clinical and biological characteristics, and its prognosis differs from AML in older children. Acute megakaryoblastic leukemia (AMKL) is very frequent in this age group and has raised growing interest. Thus, AMKL is a dominant topic in this review. Recent genomic sequencing has contributed to our understanding of infant AML. These data demonstrated striking features of infant AML: fusion genes are able to induce AML transformation without additional cooperation, and unlike AML in older age groups there is a paucity of associated mutations. Mice modeling of these fusions showed the essential role of ontogeny in the infant leukemia phenotype compared to older children and adults. Understanding leukemogenesis may help in developing new targeted treatments to improve outcomes that are often very poor in this age group. A specific diagnostic and therapeutic approach for this age group should be investigated.

Acute Myeloid Leukemia with Platelet-Derived Growth Factor Receptor β (PDGFRβ) Rearrangement Successfully Treated with Intensive Chemotherapy in Combination Withimatinib- A Case Report

Acute myeloid leukemia (AML) is a heterogeneous clonal stem cell malignancy. Several subgroups of AML have been identified based on multiple genomic and proteomic aberrations; these demonstrate different clinical features, response to treatment and prognosis. Rearrangements in the platelet-derived growth factor receptor β (PDGFRβ) gene result in greater constitutive enzymatic activity (tyrosine kinase) and deregulation of haematopoiesis, similarly to BCR-ABL1 in chronic myeloidleukemia (CML). PDGFRβ gene rearrangements are infrequent entities, which are mostly diagnosed in patients presenting with atypical CML, chronic myelomonocytic leukemia (CMML), myelodysplastic/myeloproliferative disorders (MDS/MPN) or juvenile myelomonocytic leukemia (JMML). PDGFRβ-positive AML is extremely rare, accounting for well below 1% of all cases. In this article, we present the case of a 31-year-old woman with AML and abnormal karyotype with PDGFRβ rearrangement, who was treated with intensive chemotherapy combined with low-dose imatinib. Complete remission (CR) was achieved and imatinib was used as maintenance treatment. The patient remains in CR with disease-free survival (DFS) of 41 months.

Determination of drug sensitivity subgroups in acute myeloid leukemia to increase treatment efficacy by imatinib and its analog imatinib B

Determination of drug sensitivity subgroups in acute myeloid leukemia to increase treatment efficacy by imatinib and its analog imatinib B

A comprehensive assessment of the prognostic role of cell adhesion molecules in acute myeloid leukemia.

BackgroundThe outcomes for patients with acute myeloid leukemia (AML) have been shown to vastly differ, predominantly due to genetic heterogeneity. Cell adhesion molecules (CAMs) concluding numerous genes play an important role in AML. We aimed to systematically assess the expression characteristics of adhesion molecules and their correlation to the outcomes of AML.MethodA total of 173 patients with AML were enrolled in this study. The genetic expressional information and clinical data sourced in previous studies were collected from the Cancer Genome Atlas (TCGA) database. The expression profiles of 141 CAMs were assessed, and the AML subgroups with specific patterns of expression were identified. The outcomes and clinical features of each AML subgroup were compared to detect the factors associated with prognosis. The differentially expressed genes (DEGs) between each subgroup were identified and the prognostic roles of those molecules were evaluated.ResultsAccording to subgroup clustering, both the primary cluster_1 and subcluster_1 showed a favorable prognosis compared to that of the other patients (26.3 vs. 17.0 months of overall survival (OS) and 46.5 vs. 15.8 months of OS, respectively). Both of the two subgroups were characterized by depressed human leukocyte antigen (HLA) genes. Assessment of the expression of prognosis-associated CAMs revealed that the expressions of SELE, NRCAM, ITGA4, and SDC1 were positively correlated with AML prognosis, while the expression of L1CAM, PDCD1, CD276, SELPLG, and CLDN14 were negatively correlated with AML. Among the abovementioned genes, we detected that the individual gene expressions of NRCAM and VCAM1 were capable of independently predicting OS, and the OS was correlated with CAMs closely enough to enable the construction of models for prognosis prediction [area under the curve (AUC) =0.78 and AUC=0.77, respectively].ConclusionsThis study showed a landscape of the expression of CAMs in AML and identified a distinct subgroup with a significantly favorable prognosis. We detected that CAMs can assist in distinguishing the cohort with long term survival and constructed two models to predict the prognosis. Those CAMs have the potential to be developed as therapy targets in the treatment of AML.

Therapy-related myeloid neoplasm after peptide receptor radionuclide therapy (PRRT) in 1631 patients from our 20years of experiences: prognostic parameters and overall survival.

To determine prognostic factors and overall survival (OS) in therapy-related myeloid neoplasm (t-MN) of patients after receiving peptide receptor radionuclide therapy (PRRT). All patients treated from February 1999 until September 2019 at our center who had bone marrow biopsy-proven t-MN after PRRT were included. Patient characteristics, laboratory results, and all tumor-directed therapies before t-MN diagnosis were collected. Cox regression analysis was performed to identify parameters associated with OS. Receiver operating characteristic (ROC) curve analysis was used to define cutoff values as well as sensitivity and specificity of the parameters. Out of 1631 patients treated with PRRT, 30 patients developed t-MN comprising myelodysplastic syndrome (MDS) in 23 patients (77%) and acute myeloid leukemia (AML) in 7 patients (23%). The median OS of t-MN patients was 13months (range 9.1-16.9months): 6months for AML and 15months for the MDS subgroup, respectively. Higher platelet level was a significant prognostic parameter for longer OS (hazard ratio (HR): 0.99, P < 0.05). Using ROC analysis, the best cutoff value for thrombocyte count was 183.5Gpt/L, resulting in a sensitivity of 92.3% and a specificity of 50%. Other factors, such as hemoglobin level, did not show a significant correlation with OS. Even rarely occurred, the OS is gravely compromised in t-MN patients after PRRT, and even less in the AML subgroup (6months). Higher platelet value was a significant prognostic parameter for longer OS in t-MN patients.

Fanconi Anemia Gene Associated Germline Predisposition in Aplastic Anemia and Hematologic Malignancies

Introduction Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome with a surged cancer incidence, especially in hematologic malignancies, which has been listed in the entity of myeloid neoplasms with germline predisposition in the 2016 revision to the World Health Organization classification of tumors of hematopoietic and lymphoid tissues.Whether FA heterozygotes are predisposed to bone marrow failure and hematologic neoplasm is crucial but unsettled. We therefore retrospectively analyzed rare possibly significant variations (PSVs) in the five most obligated FA genes, BRCA2, FANCA, FANCC, FANCD2, and FANCG, in 788 aplastic anemia (AA) and hematologic malignancies patients to address this issue. Methods Patients diagnosed as AA, myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and acute lymphocytic leukemia (ALL) from April 2015 to December 2018 in Hebei Yanda Lu Daopei Hospital were enrolled with the ones diagnosed as FA via chromosome breakage test and/or genetic test excluded. Detailed disease histories and workup files were retrieved from the electronic medical record. Amplicon-based high throughput sequencing of aforementioned five genes were performed. The significance of the germline missense variants was assessed by in silico prediction algorithms, including SIFT, Polyphen2, PROVEAN, FATHMM, MutationTaster, and MutationAssessor. Variant predicted as deleterious/possibly deleterious by ≥ 3/6 scoring tools was defined as rare PSV and further included in statistical analysis. For splice site mutations, GeneSplicer, Human Splicing Finder, NetGene2, and FSPLICE were employed and only when ≥2/4 algorithms predict to affect/possibly affect splicing, would the variant be regarded as possibly significant. Same criteria were adopted when stratifying variants recorded in the ExAC. All reported variants in this study were confirmed germline variants by Sanger sequencing with fingernail specimens and/or pedigree analysis. WFisher exact two-tailed test was adopted for variant frequency comparison. Development of disease was analyzed by cumulative incidence method and Kaplan-Meier method. p &amp;lt; 0.05 was considered as statistically significant. Results A total of 788 patients, who were all of the East Asian ancestry, were enrolled, including 341 females and 447 males (Figure 1A). The median age of onset was 10 (1-63), 27 (1-65), 14.5 (1-65), and 6 (1-53) years in AA, MDS, AML, and ALL subgroup, respectively. Sixty-eight variants were identified in 66 patients (8.38%). FANCA is the most frequently mutated gene (n = 29), followed by BRCA2 (n = 20) (Figure 1B). When compared to the ExAC East Asian dataset, there was an overall higher rare PSVs incidence in our cohort (p = 0.016). BRCA2 PSVs showed a higher frequency in ALL (p = 0.038), and FANCA PSVs were significantly enriched in AA and AML subgroups (p = 0.020; p = 0.008). The patients with FANCA heterozygotes also tended to show an increased risk for developing MDS (p = 0.075) (Table 1). No impact of FA-PSV status was found neither on cumulative disease incidence (Figure 1C). FA-PSV + MDS/AML patients have a heavier tumor mutation burden, higher rate of cytogenetic abnormalities, and less epigenetic regulation and spliceosome gene mutations than those of FA-PSV - MDS/AML patients (p = 0.024, p = 0.029, p = 0.024, and p = 0.013) (Figure 1D). Discussion The overall PSVs enrichment in our cohort buttresses that heterozygous mutations of FA genes abate the capacity of DNA homologous recombination repair pathway and contribute to hematopoietic failure. Furthermore, we present the first evidence that BRCA2 heterozygotes have a significantly higher risk of developing into ALL. Instead of all or nothing, the impact of different variant imposed on protein might be seen as a continuous variation. Therefore, contributions of the FA pathway defect could be latent and subtle but be profound as time goes by. We further firstly observed a higher incidence of cytogenetic abnormalities and somatic mutations with statistical significance, and lower frequency of epigenetic regulation and spliceosome gene mutations in FA-PSV + myeloid malignancies. This provides evidence that these FA-PSV carriers are prone to accumulate chromosomal structural abnormalities, and confer the congenital susceptibility of myeloid malignancies. Disclosures No relevant conflicts of interest to declare.

Integration of Deep Multi-Omics Profiling Veals New Insights into the Biology of Poor-Risk Acute Myeloid Leukemia

Background: The poor-risk cytogenetic subgroup of acute myeloid leukaemia (AML) includes various chromosomal aberrations and represents a heterogeneous population of patients with a dismal 10-year overall survival. While the success of genetic landscaping studies is encouraging, it is debatable whether genomics, or indeed any single-omics platform alone, is sufficient to capture the biology of a disease that continues to evade our existing treatments so effectively. Instead, we need to develop a much better understanding of the complexity of this subgroup of AMLs: the relationship and interdependencies across biochemical pathways, how these may differ between patients and their impact on the leukemia and normal stem cell compartments. To launch this process, we have completed a multi-omics profiling programme to shed new light on the genetic and biochemical features of poor-risk AML (https://poor-risk-aml.bham.ac.uk/). Aims: Application of multi-omics and integrative approaches to decipher the complexities of cytogenetically poor-risk AML Methods: Sample inclusion criteria were based on cytogenetics and availability of sufficient diagnostic bone marrow or peripheral blood material for analysis. The 50 primary AMLs included 17 cases with complex karyotype, 13 -7/del(7), 11 KMT2A rearrangements (with the exception of t(9;11)), 4 t(6;9), 3 -5/de(5), 1 del(17) and 1 inv(3). Profiles consisted of a combination of genomics (whole genome sequencing (WGS, 60X for tumour and 30X for germ-line controls), targeted sequencing of 54 myeloid loci, and total RNA-seq (100 million reads per bulk sample), mass spectrometry proteomics and phosphoproteomics (with &amp;gt;6,000 proteins and &amp;gt; 25,000 phosphorylation sites detected and quantified), mass cytometry (CyTOF, 39 markers), drug screening (ranging from 200-500 approved or investigational compounds) and the selective generation of patient-derived xenograft (PDX) models. Results: Near complete datasets have been compiled on all 50 primary AMLs, with the exception of WGS analysis where profiling was restricted to cases where corresponding germline DNA was available. Integration of WGS and RNA-seq data identified 122 genes having notable allele-specific expression (ASE) in ≥ 5 samples supported by ≥ 3 SNPs and these included the transcription factor GATA2 and the DNA topoisomerase TOP1MT. Use of RNA fusion capture tools resolved novel inter- and intra- chromosomal gene rearrangements that were confirmed by WGS. The four t(6;9)(p23;q34)/DEK-NUP214 cases, with a mean age of diagnosis of 43.5 years and all harboring FLT3-ITD mutations, arose from the most immature hematopoietic compartment (CD34+CD117+ enrichment) and demonstrated a unique transcriptomic signature, which included upregulation of FOXO3 and GRP56. Collectively, t(6;9) primary samples also showed a selective drug sensitivity to XPO1 (selinexor and eltanexor) and JAK inhibitors (ruxolitinib, tofacitinib and momelotinib) compared to other cytogenetic risk groups. On the other hand, a comparison of in vitro drug sensitivity data with genomic data of our entire cohort of patients demonstrated that TP53 wt AMLs (n=37) were more sensitive to all four MDM2 inhibitors (AMG-232, idasanutlin, SAR405838 and NVP-CGM097) compared to TP53 mutated cases (n=13). Comparisons of transcriptomics with the in vitro sensitivity to drugs included in early/late phase AML clinical trials, identified signatures of response associated with MDM2 and Aurora B kinase (AZD1152-HQPA) inhibitors. Phosphoproteomics analysis and machine learning modeling separated KMT2A rearranged leukemias into 2 discrete groups (group A: MLLT4, MLLT10 and TET1; group B with MLLT6, ELL and SEP9 fusion partners). Functionally, group A presented with elevated HOXA10 protein expression and enhanced in vitro response to genotoxic drugs and cell cycle inhibitors when compared to group B leukemia. Conclusions: Our study demonstrates the feasibility of simultaneously generating omics data from several different platforms and highlights that a combination of genetic and proteomic profiles may help to inform the choice of therapies based on the underlying biology of a patient's AML. Disclosures Wennerberg: Novartis: Research Funding; Pfizer: Honoraria. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding; Innovative Mediicines Initiative project Harmony: Research Funding.

Prognostic Impact of Measurable Residual Disease on Survival in Acute Myeloid Leukemia: A Meta-Analysis of 81 Studies

Prognostic Impact of Measurable Residual Disease on Survival in Acute Myeloid Leukemia: A Meta-Analysis of 81 Studies

JAK/STAT Inhibition Targets TP53 altered Primary Human Acute Myeloid Leukemia Stem Cells

In acute myeloid leukemia (AML), the impact of genetic drivers on response to therapy and long-term survival has been well characterized. AML with complex cytogenetics and TP53 alterations (TP53Alt) is a poor-risk AML subtype that is largely insensitive to chemotherapy, modern targeted agents, and hematopoietic stem cell transplant leading to survival rates 0-10% at 1 year. In contrast, AML with favorable risk molecular features is highly sensitive to chemotherapy and confers survival rates of 50-70%. AML with intermediate risk molecular features can be responsive to chemotherapy and can be cured with hematopoietic stem cell transplant leading to overall survival rates of 30-60%. Leukemia stem cells (LSCs), the cells that recapitulate and propagate leukemia, are central to leukemia progression and relapse. Given the differences in chemo-sensitivity and clinical behavior of genetic subgroups of AML, we asked whether LSCs from poor risk AMLs exhibit distinct signaling activation profiles. We assembled a panel of 23 primary human AML samples with intermediate- and poor- risk genetics and used CyTOF (mass cytometry) to quantitatively measure the levels of immunophenotypic proteins and intracellular signaling molecules in each sample, at the single-cell level. We gated on CD34+CD123+CD3-CD19- cells (LSCs) and measured the level of intracellular signaling molecules within the LSCs of each sample. Notably, the intracellular signaling activation state of LSCs from each AML subtype was distinct; NFkB, pERK, p4EBP1, and pSTAT3 were uniquely upregulated in complex cytogenetics and TP53Alt LSCs, relative to LSCs from intermediate risk AML, suggesting that these signaling pathways may be important for LSC function in this AML subtype. Given that TP53Alt independently confer treatment resistance in AML, we focused on this genetic subgroup. We compared the gene expression profiles of TP53Alt and TP53-wild-type AML samples from the BEAT AML dataset (Tyner et al. Nature 2018) and found that the gene expression profiles of TP53Alt samples are enriched for gene sets representing JAK/STAT signaling, consistent with our CyTOF data, which identified activation of STAT3 in TP53Alt LSCs. A recent drug screen in AML demonstrated that a JAK1/2 kinase inhibitor, AZD1480, can reduce the in vitro viability of TP53-deleted AML cell lines (Nechiporuk et al. Ca Discovery 2019), but these effects were not tested in primary AML samples or on LSCs. Since LSCs confer treatment resistance, we investigated the effect of the AZD1480 on the LSC population in TP53Alt primary human AML samples. AZD1480 treatment abolished all colony formation in primary human TP53Alt AML samples (n=7, 6 replicates per sample, p&amp;lt;0.01). Treatment of these samples in liquid cultures led to a 50% reduction in LSC frequency. We used CyTOF to profile the intracellular signaling states of in vitro treated samples and found that AZD1480 attenuated pSTAT3, pSTAT5, p4EBP1, and NFkB in the LSCs of these samples. The mTOR/4EBp1 and NF༆B pathways have been implicated as drivers of self-renewal and LSC function in AML. Our data suggest that JAK/STAT inhibition may target these pathways in TP53Alt LSCs. These data demonstrate the unique signaling states of TP53Alt LSCs, relative to other LSCs, and show that inhibition of the JAK/STAT pathway specifically targets LSCs within human TP53Alt AML. Figure Disclosures No relevant conflicts of interest to declare.