Survival In AML Patients Research Articles

Clonal Relapse Dynamics in Acute Myeloid Leukemia Following Allogeneic Hematopoietic Cell Transplantation

IntroductionThe 2-year survival for AML patients relapsing after allogeneic hematopoietic cell transplantation (alloHCT) is <20%, independent of the choice of relapse-treatment. Relapse detection in its molecular state enables early interventions and possibly prevention of hematological recurrence of the disease. The role of measurable residual disease (MRD) monitoring for risk stratification has been described for pre and post-alloHCT MRD analyses. Yet, it remains unclear, if and by which lead-time NGS assessment can detect MRD before impending relapse. We hypothesize that the functional class of mutations determines the relapse kinetics in AML after alloHCT.MethodsWe identified mutations present at AML relapse after alloHCT by Illumina myeloid panel sequencing covering 48 AML associated genes. Peripheral whole blood samples were retrospectively collected before hematological relapse, with a minimum of one sample per patient at three months prior to relapse and if available, additional monthly samples. Amplicon-based NGS and bioinformatics error-correction were performed on those samples as described in Thol et al. 2018. Positive MRD was defined as MRD detectable above the limit of detection. In the last step, we performed polynomic curve interpolation to model relapse dynamics.ResultsMRD was assessed in 75 AML patients after alloHCT using 203 AML-related mutations present at the time of relapse, corresponding to a median of 2.7 trackable mutations per patient (range 1-7). In total, 305 MRD analyses were performed from peripheral blood (median 1.5 per mutation, range 1-5) prior to relapse. VAFs measured above the limit of detection (median LOD across all targets 0.0315) ranged from 0.0048-26% (median 1.3%).In 45 of 75 patients (60%), we detected MRD in at least one sample and one marker before relapse. Of those, 23 patients (51%) were MRD positive in all markers before relapse and 22 patients (49%) were MRD positive in some, but not all markers before relapse. The majority of MRD-positive patients (30 of 45) were first detected three or fewer months before relapse, whereas 15 (33%) of 45 patients were MRD positive more than 3 months before relapse. The median time to relapse from the first MRD-positive sample to relapse was 2.9 months (range 0.6-10.2).Among the 203 mutations found in relapse, 93 (46%) were detectable by MRD monitoring before relapse while the remaining 110 markers (54%) remained undetectable prior to relapse. Of note, 88 of those 110 markers (80%) were measured only once before relapse, indicating that frequent sampling increases the likelihood of MRD detection. Genes in which mutations were found mostly MRD-positive were TET2 (6 out of 6), ASXL2 (4 out of 5), SF3B1 (4 out of 5), and RUNX1 (7 out of 9). Mutations in WT1 (1 out of 13), NRAS (1 out of 8), FLT3-ITD (9 out of 29), and PTPN11 (1 out of 5) were among the most common MRD negative mutations before relapse.To assess clonal relapse dynamics, pre-relapse samples were assigned to the monthly interval that best matched the sampling time. If MRD was measured positive at one time point, all the following monthly intervals were considered MRD-positive, whether a sample was available for that interval or not. The fraction of positive samples from all samples per time point was plotted against time to relapse and the function was approximated by fifth-order polynomials. The percentage of patients being MRD positive increased markedly with shortened distance to relapse. Thus, 29% of patients were MRD positive at 3 months, 44% at 2 months and 66% 1 month prior to relapse. Summarized by functional gene classes, mutations in tumor suppressor genes and especially signaling genes showed a higher slope and thus a shorter lead-time to relapse than mutations in epigenetic modifier genes (Figure 2).ConclusionIn summary, hematologic relapse can be detected in peripheral blood in 29, 44, and 66% of patients at 3, 2, and 1 months before relapse by NGS-MRD analysis, respectively. Mutations in epigenetic modifier genes show a higher fraction of MRD positivity before relapse than other mutations. In contrast, mutations in signaling genes show a shorter lead-time to relapse. [Display omitted] DisclosuresGanser: Celgene: Honoraria; Novartis: Honoraria; Jazz Pharmaceuticals: Honoraria. Thol: Abbvie: Honoraria; Astellas: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Jazz: Honoraria; BMS/Celgene: Honoraria, Research Funding. Heuser: BergenBio: Research Funding; Bayer Pharma AG: Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding; Tolremo: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Development and validation of a novel survival model for acute myeloid leukemia based on autophagy-related genes.

BackgroundAcute myeloid leukemia (AML) is one of the most common blood cancers, and is characterized by impaired hematopoietic function and bone marrow (BM) failure. Under normal circumstances, autophagy may suppress tumorigenesis, however under the stressful conditions of late stage tumor growth autophagy actually protects tumor cells, so inhibiting autophagy in these cases also inhibits tumor growth and promotes tumor cell death.MethodsAML gene expression profile data and corresponding clinical data were obtained from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, from which prognostic-related genes were screened to construct a risk score model through LASSO and univariate and multivariate Cox analyses. Then the model was verified in the TCGA cohort and GEO cohorts. In addition, we also analyzed the relationship between autophagy genes and immune infiltrating cells and therapeutic drugs.ResultsWe built a model containing 10 autophagy-related genes to predict the survival of AML patients by dividing them into high- or low-risk subgroups. The high-risk subgroup was prone to a poorer prognosis in both the training TCGA-LAML cohort and the validation GSE37642 cohort. Univariate and multivariate Cox analysis revealed that the risk score of the autophagy model can be used as an independent prognostic factor. The high-risk subgroup had not only higher fractions of CD4 naïve T cell, NK cell activated, and resting mast cells but also higher expression of immune checkpoint genes CTLA4 and CD274. Last, we screened drug sensitivity between high- and low-risk subgroups.ConclusionThe risk score model based on 10 autophagy-related genes can serve as an effective prognostic predictor for AML patients and may guide for patient stratification for immunotherapies and drugs.

Establishment and validation of prognosis predictive model using m6A RNA methylation regulators in children acute myeloid leukemia

Objective: To figure out the association between the expression of m6A RNA methylation regulators and the prognosis of children AML, and provide genetic markers for monitoring the progression and recurrence of AML. Methods: Twenty two m6A RNA methylation regulators were firstly analyzed using the data from Therapeutically Applicable Research To Generate Effective Treatments(TARGET) database and The Genotype-Tissue Expression(GTEx) database, Wilcoxon rank test was performed to analyze the differentially expression of m6A RNA methylation regulators between the AML and normal tissue, 296 AML children were divided into training cohort and validation cohort by simple random sampling method, Lasso regression was used to screen out the risk factors and the multivariate Cox regression was applied for establishing prognosis predicting model in training cohort. Kaplan-Meier survival curve, time-dependent ROC curve and multivariate Cox regression were used to estimate the efficiency of risk score calculated by predictive model in validation cohort. Results: Twenty one m6A genes were up regulated in AML compared to Normal patients. Five m6A RNA methylation regulators(ZC3H13, YTHDC2, HNRNPA2B1, METTL3, METTL5) were included in final predicting model. Risk score could independently predict the survival of AML patients in training cohort(HR:2.72, 95%CI: 1.54-4.81, P=0.000 6) and validation cohort(HR:2.01, 95%CI:1.14-3.50, P=0.016). Low-risk patients had better prognoses than high-risk patients both in training cohort(P=0.001 9) and validation cohort(P=0.023). Conclusion: This prognosis predicting model constructed by m6A RNA methylation regulators could independently predict the survival prognosis in AML children, and should be helpful for clinical therapy.

A Prognostic Autophagy-Related Long Non-coding RNA (ARlncRNA) Signature in Acute Myeloid Leukemia (AML).

BackgroundSome studies have proven that autophagy and lncRNA play important roles in AML. Several autophagy related lncRNA signatures have been shown to affect the survival of patients in some other cancers. However, the role of autophagy related lncRNA in AML has not been explored yet. Hence, this study aims to find an autophagy related lncRNA signature that can affect survival for AML patients.MethodA Pearson correlation analysis, a Kaplan–Meier survival curve, a univariate cox regression, and a multivariate cox regression were performed to establish an autophagy related lncRNA signature. A univariate cox regression, a multivariate cox regression, a Kaplan–Meier survival curve, and a ROC curve were applied to confirm if the signature is an independent prognosis for AML patients. The relationship between the signature and the clinical features was explored by using a T test. Gene Set Enrichment Analysis (GSEA) was used to investigate the potential tumor related pathways.ResultsA four-autophagy related lncRNA (MIR133A1HG, AL359715.1, MIRLET7BHG, and AL356752.1) signature was established. The high risk score based on signature was related to the short survival time of AML patients. The signature was an independent factor for the prognosis for AML patients (HR = 1.684, 95% CI = 1.324–2.142, P < 0.001). The signature was correlated with age, leukocyte numbers, and FAB (M3 or non-M3). The P53, IL6/JAK/STAT3, TNF-α, INF-γ, and IL2/STAT5 pathways might contribute to the differences between the risk groups based on signature in AML.ConclusionThe four autophagy related lncRNAs and their signature might be novel biomarkers for predicting the survival of AML patients. Some biological pathways might be the potential mechanisms of the signature for the survival of AML patients.

Overexpression of BRD4 Is an Adverse Prognostic Factor in Acute Myeloid Leukemia

Background:Bromodomain containing protein 4 (BRD4), a member of the bromodomain and extra terminal domain (BET) protein family, has been implicated in the pathogenesis of a number of hematological and solid tumors. BRD4 is an epigenetic reader that plays a central role in transcriptional regulation, cellular growth control, and cell cycle progression. As a consequence, BET inhibitors elicit anticancer activity in numerous malignant contexts. While deregulation of BRD4 activity has been shown to be essential for the maintenance of AML, its expression profile and prognostic value in AML have not been investigated. Additionally, how BRD4 is regulated in cancer cells remains largely unknown.Methodology: We therefore evaluated the level of BRD4 expression using Reverse Phase Protein Array (RPPA) technology in marrow and blood samples from 511 newly diagnosed AML patients, and determined a correlation between BRD4 expression and clinical characteristics and overall survival in AML patients. We also correlated expression of BRD4 with 232 other proteins (encompassing signal transduction, metabolism, apoptosis and cellular proliferation pathways) that were included in RPPA. Potential role of molecular chaperone heat shock protein 90 (Hsp90) in regulating BRD4 protein stability was also investigated.Results: BRD4 expression was similar in peripheral blood and bone marrow and expression was found to be higher at diagnosis than at relapse (P = .035). Compared with normal bone marrow derived CD34+ cells, BRD4 expression in AML was higher in 28% of patients and that higher BRD4 levels significantly predicted shorter overall survival (P = .002). There was no significant difference in BRD4 expression on the basis of age, sex, or performance status, antecedent malignancies, and previous chemotherapy and/or radiotherapy. BRD4 expression levels were not strongly correlated with French-American-British classification (FAB), although levels were slightly higher in M5 subtype. BRD4 levels were found to be significantly higher in patients with NPM1 mutation (P = .003) or FLT3-ITD (P = .057) or between those with both FLT3-D835F and NPM1 mutation (P = .01). BRD4 levels were not associated with the attainment of complete remission (CR), although rates were somewhat lower for those with high. However, patients with high levels of BRD4 had significantly inferior remission duration (25.9 vs 50 weeks; P = 0.03) compared with those with normal levels of BRD4. The overall survival (OS) of patients with higher levels of BRD4 was significantly inferior compared with those with normal levels of BRD4 (28.3 vs 55.3 weeks; P = .002). Patients with unfavorable cytogenetics and above-normal levels of BRD4 had inferior survival compared with those with normal levels of BRD4 (22.4 vs 38.9 weeks; P = .0043; Figure 3C). BRD4 expression was found to be strongly correlated (R > 0.2 and P < 0.0001 or 0.00001) either positively (n = 48) or negatively (n = 14) with proteins known to be important in AML. BRD4 expression was positively correlated with various cell proliferation-related proteins (Hsp90, pSTAT5, nucleolin and mTOR), anti-apoptotic protein (Bcl-2), oncoproteins (FLI1, CREB and ELK1), proteins important for stem cell functions (Ash2L and LSD1), hematopoietic transcription factors (ERG and PU.1/Spi1), and Smad proteins, signal transducers for the members of the transforming growth factor-β (TGF-β) superfamily. The stability, activity and function of several mutated, chimeric, and overexpressed signaling proteins crucial for oncogenesis are often maintained by Hsp90. Our study also demonstrates that BRD4 stability is dependent upon Hsp90 activity and pharmacologic inhibition of Hsp90 promoted ubiquitination followed by proteasome-dependent degradation of BRD4 protein.Conclusions: These findings suggest BRD4 as a prognostic biomarker for unfavorable outcomes and supports the clinical rational for using BRD4 inhibitors in AML. Future studies could identify mechanistic insights into BRD4-associated proteins as biomarkers for response to BET inhibition. DisclosuresKarnad:ADC Therapeutics: Research Funding. Weitman:Tolero Pharmaceuticals, Inc.: Employment. Iyer:Takeda: Research Funding; Genentech: Research Funding.

Integrative study of EZH2 mutational status, copy number, protein expression and H3K27 trimethylation in AML/MDS patients

BackgroundMutations in the EZH2 gene are recurrently found in patients with myeloid neoplasms and are associated with a poor prognosis. We aimed to characterize genetic and epigenetic alterations of EZH2 in 58 patients (51 with acute myeloid leukemia and 7 with myelodysplastic or myeloproliferative neoplasms) by integrating data on EZH2 mutational status, co-occurring mutations, and EZH2 copy number status with EZH2 protein expression, histone H3K27 trimethylation, and EZH2 promoter methylation.ResultsEZH2 was mutated in 6/51 acute myeloid leukemia patients (12%) and 7/7 patients with other myeloid neoplasms. EZH2 mutations were not overrepresented in patients with chromosome 7q deletions or losses. In acute myeloid leukemia patients, EZH2 mutations frequently co-occurred with CEBPA (67%), ASXL1 (50%), TET2 and RAD21 mutations (33% each). In EZH2-mutated patients with myelodysplastic or myeloproliferative neoplasms, the most common co-mutations were in ASXL1 (100%), NRAS, RUNX1, and STAG2 (29% each). EZH2 mutations were associated with a significant decrease in EZH2 expression (p = 0.0002), which was similar in patients with chromosome 7 aberrations and patients with intact chromosome 7. An association between EZH2 protein expression and H3K27 trimethylation was observed in EZH2-unmutated patients (R2 = 0.2, p = 0.01). The monoallelic state of EZH2 was not associated with EZH2 promoter hypermethylation. In multivariable analyses, EZH2 mutations were associated with a trend towards an increased risk of death (hazard ratio 2.51 [95% confidence interval 0.87–7.25], p = 0.09); similarly, low EZH2 expression was associated with elevated risk (hazard ratio 2.54 [95% confidence interval 1.07–6.04], p = 0.04).ConclusionsPerturbations of EZH2 activity in AML/MDS occur on different, genetic and non-genetic levels. Both low EZH2 protein expression and, by trend, EZH2 gene mutations predicted inferior overall survival of AML patients receiving standard chemotherapy.

The factors influencing clinical outcomes after leukapheresis in acute leukaemia

Leukapheresis is used for the mechanical removal of leukaemic cells in hyperleukocytosis. However, the effectiveness of leukapheresis remains unclear due to selection and confounding factors in the cohorts. We compared the effectiveness of leukapheresis among the subgroups according to either the 2016 World Health Organization classification or the number of cytogenetic abnormalities with a retrospective, single-centre study from January 2009 to December 2018. Acute myeloid leukaemia (AML, n = 212) and acute lymphoblastic leukaemia (ALL, n = 97) were included. The 30-day survival rates (95% confidence interval, 95% CI) for AML and ALL were 86.3% (81.6–90.9%) and 94.8% (90.3–99.2%), respectively. For AML, ‘primary AML with myelodysplasia-related changes’ and ‘AML with biallelic mutation of CEBPA’ showed better 30-day survival outcomes (P = 0.026) than the other subgroups. A higher platelet count after leukapheresis was associated with better 30-day survival in AML patients (P = 0.029). A decrease in blast percentage count after leukapheresis was associated with better 30-day survival in ALL patients (P = 0.034). Our study suggested that prophylactic platelet transfusion to raise the platelet count to 50 × 109/L or greater might improve clinical outcome in AML patients undergoing leukapheresis.

TERT rs2853669 as a predictor for overall survival in patients with acute myeloid leukaemia.

Introductionthe aim of the study was to investigate the contribution of TERT rs2736100 and rs2853669 gene polymorphisms in defining the genetic predisposition to acute myeloid leukaemia (AML), their association with different prognostic markers, and their impact on survival, outcome, and the prognosis of affected patients. Also, we investigated the association of TERT SNPs in AML in the presence or absence of DNMT3A (R882), NPM1, and FLT3 mutations.Material and methodsA total of 509 participants were enrolled in our study, consisting of 146 AML patients and 363 healthy participants, with no history of malignancy. TERT rs2736100 and rs2853669 polymorphisms were genotyped by using TaqMan SNP genotyping assay FLT3 (ITD, D835), DNMT3A (R882), and NPM1 c.863_864insTCTG (type A) mutations were analised in each AML case.ResultsTERT rs2736100 and rs2853669 were not associated with AML risk in the codominant, dominant, recessive, or allelic models. Multivariate Cox regression showed that TERT rs2853669 was a significant predictor for overall survival in AML patients. After adjusting for age, gender, cytogenetic risk group, ECOG status, FLT3, DNMT3A, NPM1 mutation, AML subtype, and treatment, the estimated adjusted hazard ratio (HR adjusted = 1.54, 95% CI: 1.01–2.35) showed that the TERT rs2853669 variant genotype had a negative influence on survival time.ConclusionsTERT rs2853669 and rs2736100 polymorphisms were not risk factors for developing AML in the Romanian population, but the TERT rs2853669 variant genotype had a negative effect on AML patients’ overall survival in the presence of other known prognostic factors.

Novel prognostic genes and subclasses of acute myeloid leukemia revealed by survival analysis of gene expression data

BackgroundAcute myeloid leukemia (AML) is biologically heterogeneous diseases with adverse prognosis. This study was conducted to find prognostic biomarkers that could effectively classify AML patients and provide guidance for treatment decision making.MethodsWeighted gene co-expression network analysis was applied to detect co-expression modules and analyze their relationship with clinicopathologic characteristics using RNA sequencing data from The Cancer Genome Atlas database. The associations of gene expression with patients’ mortality were investigated by a variety of statistical methods and validated in an independent dataset of 405 AML patients. A risk score formula was created based on a linear combination of five gene expression levels.ResultsThe weighted gene co-expression network analysis detected 63 co-expression modules. The pink and darkred modules were negatively significantly correlated with overall survival of AML patients. High expression of FNDC3B, VSTM1 and CALR was associated with favourable overall survival, while high expression of PLA2G4A was associated with adverse overall survival. Hierarchical clustering analysis of FNDC3B, VSTM1, PLA2G4A, GOLGA3 and CALR uncovered four subgroups of AML patients. The cluster1 AML patients showed younger age, lower cytogenetics risk, higher frequency of NPM1 mutations and more favourable overall survival than cluster3 patients. The risk score was demonstrated to be an indicator of adverse prognosis in AML patientsConclusionsThe FNDC3B, VSTM1, PLA2G4A, GOLGA3, CALR and risk score may serve as key prognostic biomarkers for the stratification and ultimately guide rational treatment of AML patients.

Towards better combination regimens of cytarabine and FLT3 inhibitors in acute myeloid leukemia.

AML patients with FLT3/ITD mutations have poor response to cytarabine-based chemotherapy. FLT3 inhibitors (FLT3i) may resensitize cells to cytarabine (CYT). Improving treatment outcome of this combination may benefit from a mechanistic extrapolation approach from in vitro data. The effects of CYT and several FLT3i on cell proliferation and cell cycle kinetics were examined in AML cell lines. The effect of FLT3i (quizartinib, midostaurin, sorafenib) on cell proliferation and cell cycle kinetics was assessed in AML cell lines with differing FLT3 status; HEL (negligible expression of wild-type FLT3), EOL1 (wild-type FLT3), MV4-11 (FLT3-ITD resulting in constitutively active isoform). Semi-mechanistic cell cycle models for CYT and FLT3i were developed. Clinical CYT and quizartinib pharmacokinetic dosage regimens were modeled. Survival of AML patients was described via a hazard model. Simulations exploring different CYT/quizartinib regimens were conducted with the goal of improving treatment outcome. FLT3 status was associated with sensitivity to CYT (HEL cells most sensitive > EOL1 > MV4-11 cells). This order of sensitivity is reversed for FLT3i. Cytarabine induced apoptosis in the S-phase while all FLT3i induced apoptosis and cell cycle arrest at G1 phase. Simulations of candidate clinical regimens predict better cell kill upon adding quizartinib simultaneously with or immediately after CYT exposure. Overall survival was predicted to be significantly better with quizartinib 200mg administered every 48h vs every 24h in patients with FLT3 aberrations. Simultaneous administration of quizartinib and CYT every other day is a promising combination regimen for AML patients with FLT3 mutations.

Clinical Analysis for Patients with AML Treated after Allo-HSCT

To analyze risk factors that affect survival and relapse of AML patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT), and to investigate the therapy choices after AML relapse. Clinical data of 180 AML patients achieved complete remission (CR) before HSCT from January 2009 to December 2018 treated in our center were analyzed retrospectively. Risk factors for survival and relapse after allo-HSCT were analyzed by COX regression. Among 180 AML patients, 134 survived (74.4%), 46 patients died (25.6%), and 40 patients relapsed (22.2%). The rate of overall survival (OS), event-free survival (EFS) and cumulative rate of relapse in 5-years was 74.3%、42.5% and 25.0%, respectively. High-risk, adverse cytogenetics, CR2 at HSCT and no cGvHD were independent risk factors that affect OS. CR2 at HSCT, high-risk were independent risk factors that affect EFS. High-risk, MRD+ after one course of induction therapy, adverse cytogenetics and no cGVHD were independent risk factors that affect relapse. The OS rate of relapse patients could be improved by the usage of hypomethylation agents combined with G-CSF mobilized donor lymphocyte infusion (DLI), and 2-year OS rate was 62.5%. The survival rate of AML is greatly improved by allo-HSCT, but relapse is still one of the most important factors that influence survival of the AML patients. The maintenance therapy of hypomethylation agents combined with DLI may be a new effective treatment option for patients who relapse after HSCT.

Genome-wide CRISPR screen reveals cancer cell resistance to NK cells induced by NKderived IFN-γ

Abstract The anti-leukemia activity of NK cells is important for preventing relapse during HSCT in leukemia patients. However, the factors that determine susceptibility of leukemia cells in the context of NK-mediated killing are not well established. Here we performed a genome wide CRIPSR screen in the human CML cell line K562 to identify genes that regulate vulnerability of leukemia cells to killing by primary human NK cells. Distribution of gRNAs in K562 cells that survived co-incubation with NK cells showed that loss of NCR3LG1 (encodes B7H6), the ligand of the activating receptor NKp30, predominantly protected K562 cells from killing. It indicates that B7H6 is a major ligand on K562 cells. In contrast, loss of genes that regulate pathways for antigen-presentation (B2M, HLA-I, PSMB5, TAP1, TAPBP) and IFN-γ signaling (IFNGRs, JAKs, STATs) increased the vulnerability of K562 cells. MHC-I on K562 cells was upregulated after co-culture with NK. Addition of IFN-γ neutralizing antibody during the co-culture increased the susceptibility of K562 cells to NK-mediated lysis. The results suggest that cancer cells respond to NK-derived IFN-γ and lead to NK-resistance and immune evasion. Moreover, Analysis of RNA-seq data from The Cancer Genome Atlas (TCGA) showed that low IFNGR2 expression in cancer tissues associated with improved overall survival in AML patients, indicating that the IFNGR-JAK-STAT pathway can be a contributor to worse prognosis of cancer patients, potentially related to IFN-γ–induced NK-resistance, despite the fact that IFN-γ signaling promotes T cell mediated immune surveillance. Our results suggest that targeting IFN-γ responses might be a promising approach to enhance NK cell anti-cancer efficacy.

Comparison of Post-Transplant Cyclophosphamide and Tacrolimus and Methotrexate Graft Versus Host Disease Prophylaxis in AML Patients Receiving Myeloablative Fractionated Busulfan Conditioning Regimen

Background Myeloablative conditioning can be given safely to older patients by simply administering busulfan over a longer period (fractionated busulfan regimen) than the standard four day regimen. (Popat et al Lancet Haematology 2018). Subsequently, we added post-transplant cyclophosphamide (PTCy) GVHD prophylaxis to our fractionated regimen with a hypothesis that it will reduce GVHD and improve outcomes. Herein, we report the results of our retrospective analysis comparing outcomes of patients with AML who received fractionated busulfan myeloablative conditioning and PTCy versus standard tacrolimus (Tac) and methotrexate (Mtx) GVHD prophylaxis. Methods Patients with AML between 18-70 years of age with adequate organ function and 8/8-HLA matched related or unrelated donor, who were treated on 4 consecutive protocols using fractionated busulfan were included in the analysis. All patients received two fixed doses of busulfan 80mg/m2 as outpatient either on days -13 and -12, or on days -20 and-13 followed by busulfan on day -6 to -3 dosed to achieve target area under the curve (AUC) of 20,000 mol/min for the whole course based on pharmacokinetic studies. Fludarabine ± cladribine were given on day -6 to -3. GVHD prophylaxis was PTCy 50mg/kg on days 3 and 4 and Tac ± mycophenolate mofetil in PTCy cohort (n=53), or standard Tac+ Mtx (n=53). Patients in both the cohorts were matched 1:1 by propensity score. Results Baseline characteristics were similar between the PTCy and Tac/Mtx cohorts. The median age was 60 (range, 18-70) and 58 (range, 24-70) years, respectively, (P=0.3). Forty-nine percent were in CR, and 30% had comorbidity index >3 in each arm. Similar proportion of patients had ELN adverse risk in both arms (43% vs 40%, respectively P=0.7). The median follow up was 19 months (range, 3-36) in the PTCy and 46 months (range, 14-73) in Tac/Mtx cohort. Overall survival at 2-year was 78% vs 58% (P=0.03), non-relapse mortality was 6% vs 13% (P=0.2), incidence of grade 3-4 acute GVHD at 1-year was 6% vs 19% (P=0.07) and chronic GVHD at 2-year was 10% vs 29% (P=0.03) [Table 1 and figure 1]. The median time to neutrophil engraftment was prolonged by 3 days (15 vs 12 days; P Conclusion As compared to Tac/Mtx, PTCy-based GVHD prophylaxis not only reduced severe acute GVHD and chronic GVHD, but also improved overall survival in AML patients up to the age of 70 years who received myeloablative fractionated busulfan conditioning. Prospective randomized trial is warranted to validate these findings.

MN1, FOXP1 and hsa-miR-181a-5p as prognostic markers in acute myeloid leukemia patients treated with intensive induction chemotherapy and autologous stem cell transplantation

BackgroundThe meningioma-1 (MN1) gene is expressed in hematopoietic CD34+ cells and down-regulated during myeloid differentiation. MN1 overexpression has been linked to shorter overall and disease free survival in AML patients treated with intensive induction chemotherapy. MN1 overexpression may still be an adverse prognostic marker in AML patients treated with autologous stem cell transplant (auto-SCT) after intensive induction chemotherapy. MethodsWe retrospectively analysed 54 peripheral blood mononuclear cell (PBMC) samples of AML patients who received auto-SCT at remission (CR1) after intensive induction chemotherapy. MN1 and putative MN1-associated mRNAs, as well as MN1-associated micro-RNAs were assessed at diagnosis in peripheral blood mononuclear cells using Taqman gene expression assays. ResultsAML patients with elevated MN1 or FoxP1 gene expression at diagnosis had a significantly shorter progression-free and overall survival after intensive induction chemo-therapy and auto-SCT. The presence of the favourable risk NPM1 mutation associated with reduced MN1 gene expression. In contrast to MN1 and FOXP1, elevated expression of the putative tumor suppressive micro-RNA hsa-miR-181a-5p was predictive for positive outcome. Correlation analysis of MN1 with myeloid gene expression levels revealed association of MN1 and BMI-1, CD34, FOXP1 and MDM2 expression. Analysis of non-coding RNAs revealed an inverse correlation of MN1 with hsa-miR-20a-5p and hsa-miR-181b-5p expression. ConclusionsMN1, FOXP1 and hsa-miR-181a-5p are prognostic markers in AML patients treated with intensive induction chemotherapy and auto-SCT. While MDM2 is a validated therapeutic target, the transcription factors MN1 and FOXP1, and the chromatin modulator BMI-1 are potential therapeutic targets in the treatment of AML. The tumor suppressor hsa-miR-181a-5p may be a candidate miRNA mimic for therapeutic use.

Reduced Severe aGVHD and Improved Survival Using Post Transplant Cyclophosphamide/Tacrolimus/Methotrexate Graft Versus Host Disease Prophylaxis in AML Patients Receiving Myeloablative Fractionated Busulfan Conditioning Regimen

Background: Myeloablative conditioning can be given safely to older patients by simply administering busulfan over a longer period (fractionated busulfan regimen) than the standard four day regimen. (Popat et al Lancet Haematology 2018). Subsequently, we added post-transplant cyclophosphamide (PTCy) GVHD prophylaxis to our fractionated regimen with a hypothesis that it will reduce GVHD and improve outcomes. Herein, we report the results of our retrospective analysis comparing outcomes of patients with AML who received fractionated busulfan myeloablative conditioning and PTCy versus standard tacrolimus (Tac) and methotrexate (Mtx) GVHD prophylaxis. Methods: Patients with AML between 18-70 years of age with adequate organ function and 8/8-HLA matched related or unrelated donor, who were treated on 4 consecutive protocols using fractionated busulfan were included in the analysis. All patients received two fixed doses of busulfan 80mg/m2 as outpatient either on days -13 and -12, or on days -20 and-13 followed by busulfan on day -6 to -3 dosed to achieve target area under the curve (AUC) of 20,000 mol/min for the whole course based on pharmacokinetic studies. Fludarabine ± cladribine were given on day -6 to -3. GVHD prophylaxis was PTCy 50mg/kg on days 3 and 4 and Tac ± mycophenolate mofetil in PTCy cohort (n=53), or standard Tac+ Mtx (n=53). Patients in both the cohorts were matched 1:1 by propensity score. Results: Baseline characteristics were similar between the PTCy and Tac/Mtx cohorts. The median age was 60 (range, 18-70) and 58 (range, 24-70) years, respectively, (P=0.3). Forty-nine percent were in CR, and 30% had comorbidity index &gt;3 in each arm. Similar proportion of patients had ELN adverse risk in both arms (43% vs 40%, respectively P=0.7). The median follow up was 19 months (range, 3-36) in the PTCy and 46 months (range, 14-73) in Tac/Mtx cohort. Overall survival at 2-year was 78% vs 58% (P=0.03), non-relapse mortality was 6% vs 13% (P=0.2), incidence of grade 3-4 acute GVHD at 1-year was 6% vs 19% (P=0.07) and chronic GVHD at 2-year was 10% vs 29% (P=0.03) [Table 1 and figure 1]. The median time to neutrophil engraftment was prolonged by 3 days (15 vs 12 days; P&lt;0.001) and platelet engraftment by 9 days (22 vs 13 days; P&lt;0.001) in the PTCy cohort. Full donor chimerism at day 30 was noted in 79% vs 28% in the PTCy and Tac/Mtx cohorts respectively, (P&lt;0.001). Conclusion: As compared to Tac/Mtx, PTCy-based GVHD prophylaxis not only reduced severe acute GVHD and chronic GVHD, but also improved overall survival in AML patients up to the age of 70 years who received myeloablative fractionated busulfan conditioning. Prospective randomized trial is warranted to validate these findings. Disclosures Popat: Bayer: Research Funding; Incyte: Research Funding; Jazz: Consultancy. Bashir:Imbrium: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; StemLine: Research Funding; Acrotech: Research Funding; Celgene: Research Funding. Ciurea:Kiadis Pharma: Membership on an entity's Board of Directors or advisory committees, Other: stock holder; MolMed: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Research Funding. Kebriaei:Kite: Honoraria; Pfizer: Honoraria; Amgen: Research Funding; Jazz: Consultancy. Nieto:Astra-Zeneca: Research Funding; Affimed: Consultancy; Affimed: Research Funding; Novartis: Research Funding. Oran:Astex pharmaceuticals: Research Funding; AROG pharmaceuticals: Research Funding. Qazilbash:Genzyme: Other: Speaker; Amgen: Consultancy, Other: Advisory Board; Autolus: Consultancy; Bioclinical: Consultancy. Molldrem:M. D. Anderson &amp; Astellas Pharma: Other: Royalties. Champlin:Johnson and Johnson: Consultancy; Actinium: Consultancy; Sanofi-Genzyme: Research Funding.

Residual TP53 Mutation Contributes to Chemoresistance through Clonal Expansion in Acute Myeloid Leukemia

TP53 mutation (TP53mut) associates with poor survival in AML patients. However, the risk of residual TP53mut has not fully characterized. We found there are around 3% of patients have detectable residual (less than 25%) TP53mut reads from TCGA AML patient exon sequencing data under current sequencing depth. Importantly, these patients also showed worse outcome as compared to the TP53 wild type patients. We therefore studied the effect of residual p53 mutation using two AML cell lines, OCI-AML2 and MV4-11. Both cell lines have been characterized as p53 wild type cells. However, a distinct p53 single hotspot mutation transcripts were detected in low frequency with RNA-seq analysis in both cells. TP53mut cells isolated from the parental cells show higher drug tolerance and have higher population of stem cell maker positive cells, a characteristic of chemoresistant cells. When mixed with p53 wild type cells, the TP53mut cells also show survival advantage under chemo-drug pressure in vitro and vivo (Fig. A), suggesting its advantage to become chemoresistant cells. Interestingly, the chemo-drug resistant cell lines derived from both lines have homogeneous TP53mut at same spot as parental residual mutation cells. Further, we found two p53 target genes p21 and GADD45A contribute to cell survival and chemoresistance. Decrease of these genes leads to cell less sensitive upon chemo-drug treatment. We previously showed that histone deacetylase inhibitor Romidepsin can resensitize chemoresistant cells. Here we further show that Romidepsin can reactivate p53 targeted genes which are failed to be activated by mutant p53 (Fig. B), including p21 and GADD45A, through increasing gene promotor histone acetylation. Moreover, it can preferentially target p53 mutant cell subpopulation (Fig. C). Therefore, either single treatment or combination with chemotherapy drug, Romidepsin can potentially cure or prevent residual p53 mutation caused chemoresistance and relapse in patient. Taking together, our study raise importance on previous under looked residual p53 mutation in AML patient and shed lights on therapeutic strategies for treatments on chemoresistance. Figure Disclosures No relevant conflicts of interest to declare.

Improved Survival in Patients Who Undergo Relapse of Acute Myeloid Leukemia Following Allogeneic Hematopoietic Cell Transplantation

Relapse of leukemia (RL) is the most common cause of treatment failure following allogeneic hematopoietic cell transplantation (alloHCT) in patients with AML. Historically, patients who suffer RL following alloHCT have had a dismal prognosis. A number of therapeutic options have recently become available for relapsed/refractory AML, including some that are associated with limited toxicity and may be well tolerated in the post-alloHCT setting. These therapies, and advances in supportive care have the potential to prolong survival of recently transplanted patients who suffer RL post post-alloHCT compared to prior cohorts. We hypothesized that patients suffering RL post-alloHCT within the last 5 years will have improved post-relapse survival compared to patients experiencing RL before this period. In order to test this hypothesis, we analyzed 309 patients who underwent a first allo-HCT for AML between Jan 2002 and Dec 2016 at our center and identified 112 patients (36%) who suffered RL post-alloHCT. Data were extracted from our institutional BMT database where they had been prospectively entered. Patient characteristics of those who experienced RL were: median age 53 (19-74); male - 51%; race- white-86%, black 12%, Asian -2%; donor-MRD 33%, MUD 41%, Haplo 26%; graft source - PBSC 86%, BM 11%, CBU 2%, PBSC+BM 1%; regimen intensity - MAC 61%, NST/RIC 39%%; DRI - intermediate 46%, high 50%, v. high 5%; HCT-CI 0-2 (58%), &gt;3 (42%); KPS &gt; 90 (39%) &lt;90 (61%), CMV - pos 70%, neg 29%, year of relapse -2003-2013 (68%), 2014-2018 (32%). Median time from alloHCT to relapse was 170 days (25-1106). Median follow-up of surviving patients was 50 months (15-143). DLI: A first DLI was administered to 19 patients at a median of 41 days post relapse. The corresponding number of patients receiving 2nd, 3rd and 4th DLI were 12, 6 and 2 patients given at a median of 62, 48 and 38 days following the prior DLI. A second alloHCT post RL was performed in 33 patients (29%) at a median of 85 days (22-772d) post relapse using the same donor as the first allo-HCT in 45% and a different donor in 55%. The second transplant was from a MRD, MUD and Haplo donor in 10, 11 and 12 patients respectively. Estimates of post-relapse survival at 1, 2 and 3 years following relapse for all patients are 39%, 23% and 15% (Fig 1). For patients whose AML relapsed between 2003-2013, and 2014-2018 estimated 1, 2, and 3 year survival rates following relapse were 34%, 18% and 9% vs 50%, 32% and 28% respectively (p=0.017, Fig 2). A multivariable Cox model was developed for post-relapse survival considering the following variables: at relapse, gender, race, time from transplant to relapse, donor type, graft source, regimen intensity DRI, HCT-CI, KPS, CMV status, DLI post relapse , second alloHCT post relapse, year of relapse. Second alloHCT post-relapse was tested as a time-dependent covariate. Variables were selected if the p value was less than 0.05. Proportionality was tested for selected variables and all variables passed the test. On multivariable analysis, survival was significantly better for patients who relapsed in 2014-2018 vs patients relapsing in prior years (2003-2013) - HR 0.55. p=0.018. Other variables associated with post-relapse survival were time from BMT to relapse &gt;250 d vs &lt;100 d - HR 0.55, p=0.025 and haplo donor vs MRD for first alloHCT- HR 2.41, p=0.002. The use of DLI or the occurrence of a second alloHCT post-relapse tested as a time-dependent co-variate were not significantly associated with survival in this analysis. These data suggest that survival for AML patients who suffer relapse of malignancy following alloHCT has improved in recent cohorts when compared to historic controls. Approximately one-half and one-third of the patients are now estimated to live 1 and 2 years following relapse and prolonged survival is possible in some cases. The impact of specific interventions that help promote survival following relapse should be further studied Disclosures Solh: ADC Therapeutics: Research Funding; Celgene: Speakers Bureau; Amgen: Speakers Bureau.

The Biological and Clinical Implications of the Alternative Splicing Landscape of 1,258 Myeloid Neoplasm Cases

NGS has led to the discovery of somatic mutations in splicing factors (SF), a group of functionally related genes previously not implicated in leukemogenesis. At least one genetic aberration in the most commonly affected 7 splicing factor genes is present in the majority of patients with MDS and related diseases (MDS/MPN and AML). The most popular and plausible hypothesis is that individual splicing mutations are associated with mis-splicing of key pathogenic genes in leukemia. However, searches for the essential mis-spliced gene or pathway in engineered cell lines and murine models have not been successful despite identification of many downstream gene targets. We have designed a strategy that overcomes pitfalls and advances results of previous attempts to identify the most essential targets. First, we have collected an expansive dataset (RNA-Seq and WGS of 1258 patient samples and 63 healthy controls) which allowed us to overcome sample size limitations and exclude cases with low tumor burdens, decreasing the analytic noise. In addition to studying the common mutant SRSF2 (n=208), SF3B1 (n=282), and U2AF1 (n=69) cases, we have also studied LOH lesions (fs, ns, deletions) in the less frequently affected splicing factors LUC7L2, DDX41, PRPF8, and ZRSR2 (n=211) (Fig.1A). Unsupervised hierarchical clustering segregated patient splicing signatures by disease type, SF mutation, and SF expression. To detect significantly dysregulated alternative splicing (AS) events, samples from each disease subtype, with mutations in SF3B1 (various), SRSF2P95, U2AF1S34, or U2AF1Q157, were compared to patients without SF mutations and also healthy controls. The disease cohorts were also stratified by LUC7L2, DDX41, PRPF8, and ZRSR2 expression levels, and the lower expression groups were compared to both the higher expression groups and healthy controls. Meta-analysis revealed over 17,000 splicing variations that were significantly dysregulated in at least one of 64 comparisons (PSI≥5%, q≤.05). Statistically significant AS events in each group were overlaid to identify commonly dysregulated AS events when compared to both the disease control and the healthy bone marrow controls (Fig.1B). We characterized AS events that were unique to the myeloid neoplasm subtypes as well as specific to genetic aberrations in SFs. We also identified genes and transcripts mis-spliced in multiple groups, suggesting a convergence of splicing factor mutations on a common target gene. The vast majority of our analysis identified alterations in isoform balance, however some splice sites that were activated only in the MDS and AML cohorts but never utilized in healthy controls. Examples of these tumor-specific splicing events are found in CERS2, which was found in a majority of patient samples, and in FMNL1, which was overwhelmingly mis-spliced in SF3B1 mutant patient samples (data not shown). We have highlighted the 52 AS events that were changed most often in comparisons against disease controls and/or healthy controls. Examples of targeted exons and introns included those in ubiquitination factors, transcription factors, DNA repair factors, and oncogenes. We classified significantly changed exons by the functional domains of the translated protein. The cohorts were then stratified according to the inclusion level of the exon or intron. The inclusion groups were compared to distinguish differences both in gene expression and in dysregulation of downstream pathways. Furthermore, the exons and introns were examined for any correlation with survival in the myeloid neoplasm subtypes. Integration of these datasets provided insights into the functional impact of AS in myeloid neoplasms, e.g., TDP1 exon 12, or exon 10b of NCOR1 inclusion both is positively correlated with expression of MYC targets and negatively associated with survival in AML patients (Fig.1C-D) In sum, we have identified strong isoform candidates for the practical study of AS driven pathogenesis, utilizing both RNA-seq and the integration of publicly available exon annotation and pathway databases. Notably, our analyses have unveiled hundreds of splicing changes dysregulated at a statistically significant level, thus warranting further assessments. This assemblage of splicing patterns found in myeloid neoplasms patients' samples is the largest in existence and should greatly advance the study of pathogenic AS. Disclosures Walter: MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

The Actin Binding Protein Plastin-3 Is Involved in the Pathogenesis of Acute Myeloid Leukemia.

Leukemia-initiating cells reside within the bone marrow in specialized niches where they undergo complex interactions with their surrounding stromal cells. We have identified the actin-binding protein Plastin-3 (PLS3) as potential player within the leukemic bone marrow niche and investigated its functional role in acute myeloid leukemia. High expression of PLS3 was associated with a poor overall and event-free survival for AML patients. These findings were supported by functional in vitro and in vivo experiments. AML cells with a PLS3 knockdown showed significantly reduced colony numbers in vitro while the PLS3 overexpression variants resulted in significantly enhanced colony numbers compared to their respective controls. Furthermore, the survival of NSG mice transplanted with the PLS3 knockdown cells showed a significantly prolonged survival in comparison to mice transplanted with the control AML cells. Further studies should focus on the underlying leukemia-promoting mechanisms and investigate PLS3 as therapeutic target.

Abstract 4610: Prognostic impact of KRAS driver mutations and GSTT1 expression in colorectal cancer to FOLFOX treatment

Abstract Colorectal cancer (CRC) is one of the most common cancer and the leading causes of cancer-related death worldwide. CRCs are neoplasms which have high propensity for metastasis, especially spreading to liver and lung. Liver metastasis accounts for around 50% of CRC patients, usually associated with cancer relapse and worse prognosis of the patient. The combination chemotherapy FOLFOX including 5-flurouracil, oxaliplatin and leucovorin is one of the most common and standard first-line chemotherapy utilized for adjuvant and/or neoadjuvant treatment of patients with CRC, which can reduce the risk of cancer relapse. However, a number of studies reported that chemotherapy alone cannot completely eradicate all the cancer cells, especially metastatic cells. Moreover, the mutations of oncogenes such as KRAS, existed in more than 40% of CRC patients, are often associated with poor response of anti-EGFR therapy either as single agent or in combination with FOLFOX. There is still no direct evidence on the association of KRAS or other oncogenic drivers with the resistance to FOLFOX. To understand if any of the potential biomarkers have the predictive value of FOLFOX treatment for CRC patients, especially later stage diseases, we have selected 16 CRC PDX models derived from patients who are in stage II or later where most of patients’ tumors present invasion and metastatic features. We have evaluated the drug efficacy of FOLFOX treatment in vivo for these 16 CRC PDX models and found that 6 of them were sensitive to FOLFOX and 10 models have poor responses. To study the potential biomarkers which affect the efficacy of FOLFOX in CRC PDX models, we analyzed the correlation of tumor growth inhibition (TGI) with the genomic background of these PDX models and identified the genes with significant correlation. Through filtering the genes by their functional relevance to FOLFOX treatment, we found that low expression of GSTT1 was highly related to FOLFOX resistance in these CRC PDX models. GSTT1 deletions was found to enhance the resistance to chemotherapy and a shorter survival in AML patients, which is consistent with our observation. Furthermore, we have also run the driver mutation analysis for these 16 PDX models, and found that models without KRAS driver mutations are more sensitive to FOLFOX treatment. Interestingly, in 32 CRC patients treated with FOLFOX, we found the sensitivity of FOLFOX is correlated with wild type KRAS and high expression of GSTT1, similar to our findings in PDX studies. In conclusion, our studies demonstrated that KRAS driver mutations or low expression of GSTT1 gene may render higher chances of resistance to FOLFOX treatment, these biomarkers may be beneficial for clinicians to choose effective therapies for advance CRC patients. Citation Format: Jessie Jingjing Wang, Binchen Mao, Sheng Guo, Davy Xuesong Ouyang, Henry Qixiang Li. Prognostic impact of KRAS driver mutations and GSTT1 expression in colorectal cancer to FOLFOX treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4610.