De-novo Acute Myeloid Leukemia Patients Research Articles

Frameshift Mutations in Leukemia Associated Genes Correlate with Superior Outcomes in Patients Undergoing Allogeneic Stem Cell Transplant for De Novo Acute Myeloid Leukemia Compared to Other Types of Mutations

Background Allogeneic stem cell transplant (allo-SCT) is a mainstay of treatment for acute myeloid leukemia (AML) and offers the only chance of cure. The success of allo-SCT hinges greatly on the immune response of donor T lymphocytes against leukemia cells, known as graft versus leukemia effect (GvL). One of the key potential drivers of GvL is immune response to mutation derived neoantigens. Previous studies in solid tumors have demonstrated enhanced immunogenicity of frameshift derived peptides vs those from other mutation types (e.g. substitutions) due to the propensity of frameshifts to precipitate long novel amino acid strings distal to the site of mutation. We therefore hypothesized that AML cases bearing somatic frameshift mutations (FS) in leukemia associated genes would be more immunogenic than those with only other types of mutations (non-FS), and thus benefit more from allo-SCT via more robust GvL. Methods We retrospectively searched our institutional electronic medical records for de Novo AML patients who had undergone allo-SCT between 2009 and 2022 and had next generation sequencing data available on diagnostic specimens using a 42 gene hot spot panel. Via the Kaplan-meier method, we analyzed and compared the impact of tumor mutations present at diagnosis on overall survival and relapse free survival based on frameshift versus no frameshift status. Frameshift mutations only in DNMT3A, TET2 or ASXL1 (DTA) were not included in the frameshift group as previous studies have demonstrated DTA mutations to reflect mere clonal hematopoiesis and not to be strongly blast associated. Results 89 de Novo AML patients were identified who had at least one mutation detected by NGS or PCR fragment analysis. Frameshifted genes identified included RUNX1, WT1, TP53, BCOR, CEBPA, STAG2, PHF6, ZRSR2, TET2, ASXL1 and NPM1. Our data shows superior overall survival (p=0.038; HR 2.1) post-transplant in patients who have at least one gene with frameshift mutations (n=22) other than DTA or NPM1versus those with only other types of mutations [FIGURE 1]. Relapse free survival was also significantly better (HR 3.8) in the frameshift group by Gehan-Breslow-Wilcox analysis (p=0.038) but not by the log rank test (p=0.058). Patients with non DTA frameshifts other than NPM1 further showed superior overall survival to patients whose only frameshift was NPM1 (HR 2.3, p=0.045). Three patients showed both non-frameshift and frameshift mutations in the same gene and showed significantly poorer overall survival than those with at least one exclusively frameshifted gene (HR=4.3, p=0.013). Additionally, among patients with RUNX1 mutations (n=15) specifically, those with exclusively frameshifted RUNX1 (n=9) demonstrated superior overall survival (HR 4.8, p=0.042) to those who harbored non-frameshifted RUNX1 (n=6) [Figure 2]. Other mutations occurred with insufficient frequency to individually compare FS vs non-FS survival effects at the level of single genes. Conclusion Though limited by small sample size, our data suggest that frameshift mutated AML cases may benefit more from allo-SCT than those with only non-FS mutations, possibly due to increased generation of immunogenic neoepitopes. If validated in larger studies, incorporation of somatic frameshift mutation status in AML risk stratification models could improve existing patient selection algorithms for bone marrow transplant and thereby lead to superior outcomes.

Correlation between Number of Molecular Mutations and Outcomes in De Novo Acute Myeloid Leukemia

Introduction Genomic sequencing of cancer has led to a deeper genetic understanding of the pathogenesis of acute myeloid leukemia (AML). Studies in myelodysplastic syndrome have shown that as the number of oncogenic mutations increases, patient outcomes progressively worsen. We hypothesized that patients with AML may also have a worse prognosis if they have a higher number of somatic mutations. To test this hypothesis, we examined the somatic mutational profile of 225 patients with previously untreated AML. Methods A single-center retrospective analysis was done utilizing the Kentucky Cancer Registry (KCR) and evaluated patients with de novo AML diagnosed between January 2015 to January 2021. Patient characteristics, cytogenetics, and molecular data were collected by chart review. Patients with available cytogenetic and somatic mutations profile from next-generation sequencing (NGS) were included. Clinically significant (CS) mutations for AML were detected by NGS performed on the diagnostic bone marrow specimen. The list of clinically significant mutations was obtained from My Cancer Genome and was compared to the mutations present in our patients. Statistical analysis was done using the Mann-Whitney U test for continuous variables and proportions were compared using Chi-square test. Overall survival (OS) was calculated by the Kaplan-Meier method and compared using a log-rank test. P value was considered significant if < 0.05. Results A total of 225 De Novo AML patients met inclusion criteria. The median age at diagnosis was 61 (range, 21-86). The population was fairly distributed among males (52%) and females (48%). Approximately 51% (n=115) of patients had 0-2 CS mutations, 43% (n=97) had 3-5 mutations, and 6 % (n=13) had > 5 CS mutations. The median number of CS mutations for the entire cohort was 2 (range: 0-9). Survival was worse in the group with >5 mutations when compared to those with 5 or less mutations (23 vs 7 months, p= 0.012, Figure 1). When analyzing the number of CS mutations per cytogenetic group, those with intermediate risk cytogenetics had a higher median number of CS mutations compared to the favorable and adverse risk group combined (3 versus 1, p < 0.001). In patients with > 5 CS mutations, 11/13 had a mutation associated with an adverse prognosis. To determine whether overall survival was impacted by the presence of an adverse mutation, a subgroup analysis was done using 43 patients with a normal karyotype and at least 1 adverse mutation. The cohort with > 5 CS mutations had a significantly shorter OS compared to patients with ≤ 5 CS mutations (43 vs 9 months, p= 0.001, Figure 2). Conclusion In summary, we found that patients with intermediate-risk cytogenetics have a high number of CS mutations. When patients were stratified by number of mutations, we found that a higher number of mutations (>5) led to statistically significant difference in survival compared to those with ≤5 mutations. To determine whether this difference in survival was a result of the presence of adverse risk mutations vs. number of mutations alone, we analyzed our patients who had normal karyotype with at least one adverse genetic mutation and stratified them based on number of mutations. The presence of more than 5 CS mutations was associated with a worse OS compared to ≤ 5 mutations. It appears that the number of clinically significant mutations may be an independent predictor of overall survival in AML. This can serve as another tool in stratifying AML risk with normal karyotype. This finding will need to be validated in a large prospective clinical trial.

Prevalence of Gene Rearrangement by Multiplex PCR in De Novo Acute Myeloid Leukemia in Adult Iraqi Patients.

Gene rearrangements of acute myeloid leukemia (AML) play a significant role in categorizing patients and provide valuable information about prognosis and treatment choices. However, in Iraq, the prevalence and prognostic significance of gene rearrangements in AML have not been previously examined. This study utilized a multiplex reverse transcription real-time PCR (RT-qPCR) system to identify gene rearrangements in a group of 115 adult patients from Iraq who had been diagnosed with De Novo AML. The diagnosis of AML was confirmed through blood film and flow cytometry. The ethical committee of the College of Medicine at the University of Baghdad provided approval for this research study. In this study, 66.1% of the patients diagnosed with acute myeloid leukemia (AML) exhibited distinct genetic abnormalities. Among these abnormalities, the most frequent was the rearrangement involving the KMT2A gene, observed in 19.9% of the patients. The risk stratification analysis revealed that 40% of the patients were classified as having a favorable risk, 4.3% as intermediate risk, and 25.2% as adverse risk. A subtype of AML known as core-binding factor (CBF) AML was identified in 21.7% of the cases, with 84% of these patients achieving complete remission. The NPM-RARA gene rearrangement, found in 43% of acute promyelocytic leukemia (APL) cases, was associated with a 71% complete remission rate. Among patients with KMT2A rearrangement, which accounted for 19.9% of all AML cases, the MLL-AF10 rearrangement was the most common, although only one patient with KMT2A rearrangement achieved complete remission. Furthermore, the analysis of demographic data revealed a significant association between increased risk and advanced age, presence of comorbidities, and FAB classification (M0 subtype). The prevalence of genetic rearrangements in Iraqi De Novo AML patients is higher than the global trend, highlighting the importance of genetic characterization in risk assessment and treatment decisions.

Exposure-Response Relationship of Posaconazole Suspension in Theprophylaxis of Invasive Fungal Infections in Patients with Acute Myeloid Leukemia.

Antifungal prophylaxis with posaconazole has demonstrated a reduction in the risk of death due to Invasive fungal infections (IFI)in patients with acute myeloid leukemia (AML) during induction therapy. However, various factors affect the plasma levels of posaconazole and can potentially limit its efficacy. Therapeutic drug monitoring (TDM) can help optimize the dose, but literature is scant from centers with a high IFI burden. This study aimed to evaluate the proportion of de-novo AML patients on induction who could achieve the target level of 700ng/mL with posaconazole prophylaxis,factors that can influence the plasma levels, and the impact of plasma posaconazole levels on incidence of IFI. Patients with AML on induction therapy with no baseline IFI were enrolled at our tertiary cancer center which has high prevalence of IFI. These patients received posaconazole suspension as prophylaxis. Daily plasma levels were measured from Day 4 till Day 12 of posaconazole prophylaxis. All patients were monitored for the development of IFI. The data on adverse events, concomitant drugs, mucositis, vomiting, and diarrhea were recorded. A total of 411 samples from fifty patients were collected. Only 177 out of 411 samples had levels > 700 ng/mL. The median trough level was 610 ng/mL (range30-3000 ng/mL). The median time to achieve target trough concentration was four days (range 4-12 days) from the start of induction.Thirty-eight (76%) patients achieved target plasma levels by day 12 of induction.The median plasma level on day 12 was 690 ng/mL (range,30-1270) in patients who achieved target levels as compared to 340 (50-560) ng/mL in those who did not. Twenty-six (52%) patients had IFI in our study, and the median time to develop breakthrough IFI was 14 days (range 4-24 days). Median and range of plasma levels were 690 ng/ml (30-2410; n = 22) in those who developed IFI, while 590 ng/mL (50-2300 n = 24) in those who did not. The odds of developing IFI in patients who did not achieve the threshold trough concentration of 700 ng/mL was 7.14 (95% CI; 1.35-37.75, p = 0.0206). Occurrence of vomiting (p = 0.02), diarrhea (p = 0.0008), mucositis (p = 0.003) had adverse impact on achievement of target plasma posaconazole levels. A significant proportion of patients receiving posaconazole prophylaxis fail to achieve target plasma levels which can result in high risk of development of IFI. Occurrence of diarrhea, vomiting and mucositis can adversely affect the achievement target plasma levels.

Overexpression of BAMBI and SMAD7 impacts prognosis of acute myeloid leukemia patients: A potential TERT non-canonical role.

Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) and mothers against decapentaplegic homolog 7 (SMAD7) are important transforming growth factor-β (TGF-β) signaling antagonists, however their roles in acute myeloid leukemia (AML) remains unclear. Telomerase reverse transcriptase (TERT) may be involved in regulating BAMBI and SMAD7 expressions; a role beyond telomeres that is not clinically validated yet. In this study, we examined the expression levels and prognostic values of BAMBI, SMAD7 and TERT and their association with AML patients' outcomes. Blood samples were collected from 74 de-novo AML patients and 16 controls. Real-time quantitative PCR (qRT-PCR) was performed to analyze BAMBI, SMAD7 and TERT expressions. BAMBI and SMAD7 expression in AML were significantly upregulated versus controls (p< 0.05). BAMBI, SMAD7 and TERT levels were significantly correlated together (p< 0.001). Kaplan-Meier analysis indicated that patients with high BAMBI, SMAD7 and TERT expression levels had markedly shorter event free survival (EFS) and overall survival (OS) time (p< 0.01). Furthermore, multivariate analysis revealed that only high BAMBI expression was an independent risk factor for OS (p= 0.001). BAMBI is a novel biomarker in predicting prognosis in AML patients. Moreover, a potential interplay is found between BAMBI, SMAD7 and TERT in AML pathogenies.

Association of SDF-1 Gene Polymorphism with Increased Risk of Acute Myeloid Leukemia Patients.

Background:Acute myeloid leukemia (AML) is a heterogenous group of disorders that emerge from the malignant transformation of hematopoietic stem cells. Chemokine stromal cell-derived factor 1(SDF-1) and its receptor CXC receptor 4 (CXCR4) has an essential role in dissemination of blast cells. Study aimed to detect CXCR4 expression and the SDF-1 (rs1801157) gene polymorphisms and correlate them with prognosis and outcome in AML patients. Subjects and Methods:The study was conducted on 60 de-novo AML patients, and 60 healthy controls. SDF-1 (rs1801157) gene polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and CXCR4 expression was done using flow cytometry analysis. Results:SDF-1 dominant model (AG+AA) had higher risk AML (p 0.002). CXCR4 positive cases were associated significantly with toxic manifestations (p 0.019), lower CR rates (p 0.004), and unfavorable cytogenetics (p 0.027). Multivariate analysis showed that combined CXCR4positive with dominant SDF-1 considered as independent prognostic factor for shorter overall survival (OS) in AML patients (p 0.031). Conclusion:SDF-1 dominant model had a higher risk to develop AML, and CXCR4 positive expression predicts poor prognosis in AML patients and it could represent a targeted therapy in AML. In addition, CXCR4 could be easily integrated into the initial routine diagnostic work up of AML.

NPM1 and FLT3-ITD/TKD Gene Mutations in Acute Myeloid Leukemia.

Background: A number of mutations have been reported to occur in patients with acute myeloid leukemia (AML), of which NPM1 and FLT3 genes mutations are the commonest and have important diagnostic and therapeutic implications. Material and Methods: Molecular testing for NPM1 and FLT3 genes was performed in 92 de-novo AML patients. The frequency and characteristics of NPM1 and FLT3 mutations were analyzed. Results: Nucleophosmin 1(NPM1) and fms-like tyrosine kinase 3 (FLT3) mutations were seen in 22.8% and 16.3% of patients, respectively. Amongst FLT3 mutations, FLT3-ITD mutation was seen in 8.7% cases, FLT3-TKD in 5.4%, and FLT3-ITD+TKD in 2.2% cases. Certain associations between the gene mutations and clinical characteristics were found, including in NPM1 mutated group- female preponderance, higher incidence in M4/M5 categories and decreased expression of CD34 and HLA-DR; and in FLT3-ITD mutated group- higher age of presentation, higher total leucocyte count and blast percentage. Conclusion- AML patients with NPM1 and FLT3 mutations have differences in clinical and hematological features, which might represent their different molecular mechanism in leukemogenesis. The frequency of NPM1 and FLT3 mutations in this study was comparable to reports from Asian countries but lower than that reported from western countries. However, as the number of patients in the study was less, a larger number of patients need to be studied to corroborate these findings.

AML Cells Alter Bone Homeostasis By Promoting the Formation of Immature Bone and Resorption of Mature Bone That Supports Leukemia Progression

Background: Genetic alterations in the osteoprogenitor cells has been shown to induce acute myeloid leukemia (AML) in several mouse models. Moreover, we have recently reported that AML cells induce osteogenic differentiation in mesenchymal stromal cells (MSC) to gain growth advantage in the bone marrow (BM; Battula et al., JCI Insight, 2017). However, the effect of AML cells on bone homeostasis/turnover and its impact on AML progression is unknown. Here, we hypothesize that AML cells expand osteoprogenitor cells and alter the balance between bone formation and resorption. Methods: To investigate the effect of AML cells on osteoprogenitor cells and mature osteoblasts, we used triple transgenic reporter mice with the genotype Osx-CreERt2;Ocn-GFP;ROSA-tdTomato. Murine AML cells with MLL-ENL translocation were implanted into these transgenic mice, and Osteoprogenitor (Osx+) cells and mature osteoblasts (Ocn+) in femurs were measured by confocal microscopy. To investigate the effect of human AML cells on bone composition, patient-derived xenograft (PDX) cells were implanted into non-obese diabetic scid interleukin-2Rγnull (NSG) mice and bone histomorphometry was performed using H&amp;E and Goldner's trichrome staining. Computed tomography (micro-CT) was used to measure bone volume (BV) and mineral density (BMD) in mice. Tartrate-resistant acidic phosphatase (TRAP) staining was performed to measure osteoclast number/activation. Finally, bone density on the vertebral bone (T12) was measured in 263 de-novo AML patients and 23 normal individuals by CT imaging. Results: In transgenic mice implanted with syngenic AML cells, we found a 3-4 fold increase in Osterix+, but not Osteocalcin+ cells, suggesting AML cells expand osteoprogenitor cells in the BM during short term exposure (2-3 weeks). To investigate the effects of AML on bone during a long term exposure, we implanted 10 different AML-PDX models in NSG mice (3 mice per model) and analyzed femurs by micro-CT and bone histomorphometry analysis. Interestingly, we observed a dramatic increase in new web-like medullary bone formation in 5/10 of the PDX models tested. Moreover, higher bone volume is associated with less aggressive PDX models (which takes 4-6 months to reach 90% circulating leukemia), but not aggressive PDX models (only 4-8 weeks to reach 90% circulating cells). These findings were also confirmed by micro-CT of mouse femurs. Interestingly, in some PDX models, CT images revealed large cavities in cortical bones close to epiphysis and metaphysis areas in the femur and tibia of mice with AML suggesting bone resorption. To validate bone resorption, we performed TRAP staining and found a significant increase in osteoclast activity on the endosteal surface and massive bone resorption in AML bone compared to normal bone. These data indicate high bone turnover in mice with AML compared to control mice. Next, we measured bone densities in AML patients and normal individuals by chest CT imaging. We found bone densities were gradually decrease with age in both healthy individuals and AML patients. However, compared to healthy individuals, bone densities are significantly higher in majority of AML patients (~70%) (p&amp;lt;0.01). Interestingly, survival analysis revealed that higher bone density is associated with good prognosis in AML patients (p&amp;lt;0.01), suggesting high bone turnover alters patient outcomes. Conclusion: Our data suggest that AML cells expand osteoprogenitor-rich niche and alter BM microenvironment by high bone turnover. New bone induction by less aggressive AML-PDX models coupled with AML patient CT data suggests that high bone density and volume are associated with favorable prognostic factors in AML. Mechanisms underlying this observation are under investigation. Disclosures Andreeff: Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research &amp; Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees. Konopleva:Ablynx: Research Funding; AstraZeneca: Research Funding; Kisoji: Consultancy; Ascentage: Research Funding; Stemline Therapeutics: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents &amp; Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Rafael Pharmaceutical: Research Funding; Amgen: Consultancy; Agios: Research Funding; Sanofi: Research Funding; Cellectis: Research Funding; Calithera: Research Funding; Forty-Seven: Consultancy, Research Funding; Eli Lilly: Research Funding; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding. Battula:Leukemia Lymphoma Society: Research Funding; Tolero Pharmaceuticals: Research Funding; Golfers Against Cancer: Research Funding.

Upregulation of HOTAIR Predicts Poor Outcome in Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is a clonal hematopoietic malignancy, in spite of the marked improvement in the treatment of AML; Molecular biomarkers open the door to improve disease outcome. Accumulating evidence suggested that the long non-coding RNA “HOTAIR” has an oncogenic role in hemopoietic malignancies. Recently, it has been evident that knockdown of HOTAIR inhibits cell proliferation and induces apoptosis by modulating c-Kit expression via acting as competing for endogenous RNAs (ceRNAs) to sponge miR-193a at the post-transcriptional level. Objectives: we aimed to evaluate the diagnostic and prognostic value of HOTAIR in AML, to investigate its association with and c-Kit and miR-193a. Subjects &amp; Methods: we examined the expression levels of HOTAIR, miR-193a, and c-Kit in 100 de-novo AML patients using quantitative, the association of genes expressions with risk factors and patient’s outcome were statistically analyzed. Results: the expression of HOTAIR was significantly upregulated by four folds in AML compared to healthy controls; higher expression levels were associated with high-risk factors, poorer overall survival (OS) and shorter leukemia-free survival (LFS). In addition; a negative correlation was detected between Lnc-HOTAIR and miR-193a, although significance didn’t reach. Conclusion: The obtained results suggested that HOTAIR expression was upregulated in peripheral blood samples of de-novo AML patients and was associated with leukemic burden and disease outcome. Therefore, it may represent an effective diagnostic and poor prognostic tool for AML.

CD34 expression and FLT3 mutation are independent poor prognostic factors in normal karyotype acute myeloid leukemia

Background The prognostic impact of CD34 expression on malignant myeloid leukemia blast cells is still under debate. FLT3 mutation is a well-known poor prognostic factor in acute myeloid leukemia (AML). The aim of the study was to assess CD34 expression in this subgroup of AML patients to detect the association of CD34 expression with FLT3-internal tandem duplication (ITD) mutation and its impact on the outcome. Patients and methods This prospective study, carried out at Zagazig University Hospitals, included 33 de-novo AML patients with a normal karyotype. CD 34 expression was detected by flow cytometry using FITC florescent monoclonal antibody and it was considered positive if a cutoff level of 10% expression was exceeded. FLT3-ITD mutation was detected by PCR. Results CD34 was positive in 20 cases while 13 cases were negative. Twenty-one cases were FLT3-ITD wild type while 12 cases were mutated. FLT3 mutation was significantly linked to CD34 reactivity (P=0.048). CD34-positive cases were associated with a significantly less complete remission (CR) achievement rate (33 and 80% in CD34-positive and CD34 negative, respectively, P=0.03). Those with positive CD34 had significantly lower overall survival compared with CD34-negative cases. Multivariate cox regression survival analysis showed that positive CD34 was a predictor of poor survival and higher risk of mortality (HR=1.3 and P=0.027, confidence interval, 1.1–2.9). Conclusion CD34 expression is a poor prognostic biomarker in normal karyotype AML. It is associated with and further worsens the poor prognosis in FLT3-mutated cases. Its role in different subgroups of AML layered by different genetic aberrations needs further study.

High Dose Mitoxantrone Based “5+1” Induction Chemotherapy Regimen in Newly Diagnosed Acute Myeloid Leukemia

IntroductionIn acute myeloid leukemia (AML), dose intensification of “7+3” has not produced improved outcomes. At our center, we evaluated an intensive “5+1” regimen of high dose mitoxantrone (HDM) with high dose cytarabine (HiDAC) in all newly diagnosed AML patients. We present here our experience of this regimen at our institution.Patients and MethodsThe treatment regimen included bolus HiDAC at 3000 mg/m2 over three hours on days 1 to 5 and HDM 80 mg/m2 once on the second day immediately after the completion of cytarabine infusion. A total of 101 consecutive patients were treated from January 2009 to March 2013 with this regimen. There were twelve exceptions to the regimen dosages, where patients received a lower dose of 60 mg/m2 of mitoxantrone due to their significant comorbidities. The mean doses of mitoxantrone and Cytarabine administered for the whole cohort was 76 mg/m2 and 2844 mg/m2/day respectively. Patients who achieved CR were further consolidated by HiDAC chemotherapy cycles (1-4) and/or hypomethylating agent q28 day cycles. Patients with intermediate/adverse risk group were evaluated and considered for allogeneic transplant. Survival rates were calculated by the Kaplan-Meier method.ResultsThe median age was 65 years (range 18-90). The total number of patients in the age group <60, 60-69 and ≥70 were 30(29.7%), 35(34.6%) and 36(35.6%) respectively. Patients in favorable, intermediate and adverse cytogenetics group as per European Leukemia Net(ELN)-AML 2017 criteria were 16(15.8%), 40(39.6%) and 41(40.5%) respectively. 33 patients had secondary AML (therapy-related AML, AML with antecedent MDS or CMML, or de novo AML with MDS related cytogenetics abnormalities). The overall complete response (CR) was 76.2% (77/101). 22% (23/101) were either primary refractory or had partial responses. The CR rates in patients <60, 60-69 and ≥70 age groups were similar at 80% (24/30), 74.2% (26/35) and 77.1% (27/35) respectively. The de-novo AML patients had higher CR 83.5%(56/67) compared to secondary AML 63.6%(21/33), p=0.04. The 4 and 8-week mortality in our cohort was 3/101 (2.9%) and 7/99 (7%) respectively. Median follow-up among survivors was 62 months (range 1-109 months). The median overall survival (OS) stratified by age group < 60, 60-69 and >=70 years were 56, 31 and 9 months respectively (log-rank, p=0.02). The median overall survival in patients with secondary AML was 9 months versus 43 months in de-novo AML patients. Sixty patients underwent transplant during consolidation, 47 (46.5%) were allogeneic and 13 (12.8%) autologous. 51.7% (45/84) of patients with intermediate/adverse cytogenetic risk groups as per ENL/AML criteria proceeded to allogeneic transplants. Among these 84 patients, the percentage of patients able to proceed to transplant in age groups <60, 60-69 and ≥70 years were 75% (18/24), 60.7 % (17/28) and 31.2% (10/32) respectively.ConclusionIn conclusion, HDM based chemotherapy regimen produces high CR rates, is well tolerated and more patients can undergo curative post-remission therapy including stem cell transplant. Together with the low number of toxic deaths this induction regimen warrants further investigation. [Display omitted] DisclosuresRaffel:Magenta Therapeutics: Employment, Equity Ownership.

High Neuropilin-1 Expression in Acute Myeloid Leukemia Is Mainly Associated with Monocytic Subtypes and Extramedullary Invasion

Introduction: Neuropilins are single-pass transmembrane glycoproteins acting as receptors for the vascular endothelial growth factor (VEGF) family that mediates angiogenesis and vascular development. In Acute Myeloid leukemia (AML), Neuropilin-1 high expression was found to contribute in disease evolution.Aim of Work: To evaluate Neuropilin-1 (NRP-1) expression in AML patients especially monocytic subtypes and assess its role in disease progression and extramedullary invasion (EMI).Patients and Methods: We measured NRP-1 expression level in 50 denovo AML patients who presented to the National Cancer institute, Cairo university. Expression was assessed by Real time quantitative PCR (RQ-PCR) using normalized copy number equation (2 -ΔΔCT) in relation to normal healthy controls. AML patients were characterized according to their Bone marrow and peripheral blood morphology, karyotype, immunophenotyping and FLT3 internal tandem duplication (FLT3-ITD) by polymerase chain reaction (PCR). Patients were classified into 2 groups according to median NRP-1 expression into low ≤60 and high >60-fold expression.Results: 33 males (66%) and 17 females (34%) with a median age of 34 years (18-73) were included. Cytogenetic risk categories were low in 12 (24%), intermediate in 34 (68%) and high in 4 (8%) patients. No correlation was found between high NRP-1 and cytogenetic risk classes or FLT3-ITD.Out of 26 patients showing high NRP-1 expression; 19 (73%) were <40 years vs. 7(27%) >40 years (p=0.049) and 24 (92%) showed BM blasts >50% vs. 2 (8%) with < 50% BM blasts (p<0.001). A positive correlation was found between NRP-1 Fold -change and BM blasts (r=0.653, p=0.004). Out of 19 monocytic FAB subtypes (M4, M5), high NRP-1 expression was expressed in 16 (84%) vs. 10/31 (32%) patients presenting with other FAB subtypes (p=0.042). Out of 26 patients showing high NRP-1 expression, extramedullary invasion (EMI) was found in 23/26 (88%) vs. 3/26 (12%) patients only with no EMI (p=0.001).Conclusion: Neuropilin-1 is useful as a prognostic marker of extramedullary invasion for AML patients especially in monocytic subtypes . Its role in disease progression and severity is still to be evaluated in our patients.Key Words: Neuropilin-1, AML, extramedullary invasion DisclosuresNo relevant conflicts of interest to declare.

FLT3 and NPM1 Are Powerful Determinants of Outcome in Acute Myeloid Leukemia Patients Treated with Autologous Stem Cell Transplantation: An Analysis By the Acute Leukemia Working Party of the EBMT

Background: While intensive consolidation therapy with autologous stem cell transplantation (ASCT) can secure a remission in selected Acute Myeloid Leukemia (AML) patients with intermediate-risk cytogenetics, a substantial proportion will ultimately relapse. Knowledge of the mutational status of FMS like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) and nucleophosmin (NPM) 1 and their possible combinations could further refine a subset of intermediate cytogenetic risk patients who may benefit from ASCT. However, data are limited. We, therefore, set out to evaluate the impact of FLT3-ITD and NPM1 in a large cohort of patients undergoing ASCT.Methods: This was a retrospective analysis of the Acute Leukemia Working Parity of the European Society for Blood and Marrow Transplantation (EBMT) registry. We included 405 de-novo AML patients, from 51 European centers, with intermediate-risk cytogenetics (Grimwade et al., Blood 2010) and complete data on FLT3-ITD and NPM1 status, receiving an ASCT at first complete remission (CR1), between 2000-2014. Leukemia free survival (LFS) was the primary outcome. Secondary outcomes were overall survival (OS), transplantation-related mortality (TRM), and relapse incidence (RI). The latter two were considered as competing events. Univariate and multivariable Cox regression models, adjusted for recipient sex, age, Karnofsky performance status, FLT3/NPM1 combinations, days from diagnosis to transplantation, stem cell source -bone marrow or peripheral blood (PB), and use of total-body irradiation-based conditioning.Results: Patients included had a median age of 52 years and received an autograft at median of 5 months from diagnosis. PB-based autograft and non-TBI conditioning were used in the majority of patients (93% and 90%, respectively). FLT3-/NPM1- was the leading molecular combination (50%), followed by FLT3-/NPM1+ (30%), FLT3+/NPM1+ (11%), and FLT3+/NPM1- (9%). Age, time from diagnosis to transplantation, graft source, and use of TBI were similar between the molecular subgroups. The median year of transplantation was earlier in NPM1- patients (FLT3+/NPM1- 2008, FLT3-/NPM1- 2009, FLT3-/NPM1+ 2010, FLT3+/NPM1+ 2011, p<0.001). In the univariate analysis, age, molecular makers, time from diagnosis to transplantation, and use of TBI were associated with the risk for experiencing an LFS event. Patients in the FLT3-/NPM1+ subgroup had the most favorable outcome (5-years LFS [62%] and OS [74%]). Nonetheless, 5 years following ASCT the RI was 35% (Figure). FLT3+/NPM1- were the least likely to survive due to a tendency to relapse: 5-years LFS (21%), OS (33%),and RI (79%). In a Cox multivariable model, the combination of molecular markers were the strongest predictors of LFS: FLT3-/NPM1+ (reference), FLT3-/NPM1- HR 2.39 (1.65-3.46), FLT3+/NPM1+ HR 2.24 (1.33-3.75), FLT3+/NPM1- HR 5.09 (2.94-8.82). We did not find interactions between molecular markers and other covariates in the model.Conclusions: In this pioneering analysis of CR1-AML patients, with intermediate risk cytogenetics, receiving an ASCT as post-remission therapy, we show that the molecular subgroups of FLT3-ITD and NPM1 are highly prognostic. In line with previous publications in the allogeneic setting, patients with FLT3-/NPM1+ have promising outcomes, while those with FLT3+/NPM1- do poorly. Testing for the role of additional markers, such as isocitrate dehydrogenase (IDH), could further identify populations who may derive the most benefit from ASCT. DisclosuresBaerlocher:Novartis: Research Funding. Foà:NOVARTIS: Speakers Bureau; ABBVIE: Other: ADVISORY BOARD, Speakers Bureau; CELGENE: Other: ADVISORY BOARD, Speakers Bureau; JANSSEN: Other: ADVISORY BOARD, Speakers Bureau; ROCHE: Other: ADVISORY BOARD, Speakers Bureau; GILEAD: Speakers Bureau; CELTRION: Other: ADVISORY BOARD; AMGEN: Other: ADVISORY BOARD; INCYTE: Other: ADVISORY BOARD. Haenel:Novartis: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Roche: Honoraria. Zuckerman:Cellect Biotherapeutics Ltd: Consultancy. Mohty:MaaT Pharma: Consultancy, Honoraria.

Haploidentical transplantation is associated with better overall survival when compared to single cord blood transplantation: an EBMT-Eurocord study of acute leukemia patients conditioned with thiotepa, busulfan, and fludarabine

BackgroundThiotepa-busulfan-fludarabine (TBF) is a widely used conditioning regimen in single umbilical cord blood transplantation (SUCBT). More recently, it was introduced in the setting of non-T cell depleted haploidentical stem cell transplantation (NTD-Haplo). Whether TBF based conditioning provides additional benefit in transplantation from a particular alternative donor type remains to be established.MethodsThis was a retrospective study based on an international European registry. We compared outcomes of de-novo acute myeloid leukemia patients in complete remission receiving NTD-Haplo (n = 186) vs. SUCBT (n = 147) following myeloablative conditioning (MAC) with TBF. Median follow-up was 23 months. Treatment groups resembled in baseline characteristics.ResultsSUCBT was associated with delayed engraftment and higher graft failure. In multivariate analysis no statistically significant differences were observed between the two groups in terms of acute or chronic graft-versus-host disease (GvHD) (HR = 1.03, p = 0.92 or HR = 1.86, p = 0.21) and relapse incidence (HR = 0.8, p = 0.65). Non-relapse mortality (NRM) was significantly higher in SUCBT as compared to NTD-Haplo (HR = 2.63, p = 0.001); moreover, SUCBT did worse in terms of overall survival (HR = 2.18, p = 0.002), leukemia-free survival (HR = 1.94, p = 0.007), and GvHD relapse-free survival (HR = 2.38, p = 0.0002).ConclusionsOur results suggest that TBF-MAC might allow for a potent graft-versus-leukemia, regardless of the alternative donor type. Furthermore, in patients receiving TBF-MAC, survival with NTD-Haplo may be better compared to SUCBT due to decreased NRM.

Gene and protein analysis reveals that p53 pathway is functionally inactivated in cytogenetically normal Acute Myeloid Leukemia and Acute Promyelocytic Leukemia

BackgroundMechanisms that inactivate the p53 pathway in Acute Myeloid Leukemia (AML), other than rare mutations, are still not well understood.MethodsWe performed a bioinformatics study of the p53 pathway function at the gene expression level on our collection of 1153 p53-pathway related genes. Publically available Affymetrix data of 607 de-novo AML patients at diagnosis were analyzed according to the patients cytogenetic, FAB and molecular mutations subtypes. We further investigated the functional status of the p53 pathway in cytogenetically normal AML (CN-AML) and Acute Promyelocytic Leukemia (APL) patients using bioinformatics, Real-Time PCR and immunohistochemistry.ResultsWe revealed significant and differential alterations of p53 pathway-related gene expression in most of the AML subtypes. We found that p53 pathway-related gene expression was not correlated with the accepted grouping of AML subtypes such as by cytogenetically-based prognosis, morphological stage or by the type of molecular mutation. Our bioinformatic analysis revealed that p53 is not functional in CN-AML and APL blasts at inducing its most important functional outcomes: cell cycle arrest, apoptosis, DNA repair and oxidative stress defense. We revealed transcriptional downregulation of important p53 acetyltransferases in both CN-AML and APL, accompanied by increased Mdmx protein expression and inadequate Chk2 protein activation.ConclusionsOur bioinformatic analysis demonstrated that p53 pathway is differentially inactivated in different AML subtypes. Focused gene and protein analysis of p53 pathway in CN-AML and APL patients imply that functional inactivation of p53 protein can be attributed to its impaired acetylation. Our analysis indicates the need in further accurate evaluation of p53 pathway functioning and regulation in distinct subtypes of AML.

The role of the tumor-suppressive microRNA-370 and microRNA-29b in acute myeloid leukemia

Background MicroRNAs (miRNAs) are small noncoding RNAs that play important roles as diagnostic and prognostic markers in malignancy. They have diverse effects in acute myeloid leukemia (AML), mainly as tumor suppressors and occasionally provoke the disease. Aim The aim of this research was to study the expressions of both miRNA-370 and miRNA-29b in de-novo AML Egyptian patients and correlate their expression with clinical and laboratory parameters. Patients and methods This is a case–control study that was conducted in Alexandria Main University Hospital, Alexandria University. MiRNA quantification by real-time PCR was conducted on 30 de-novo AML patients admitted to the hematology unit of internal medicine department. Their age ranged between 13 and 63 years. Their results were compared with 10 age-matched and sex-matched controls. Descriptive statistics was performed using SPSS statistics software. Results Both miRNA-370 and miRNA-29b were significantly downregulated in AML patients (P=0.011 and 0.015, respectively). There was significant positive correlation between the expressions of miRNA-370 and miRNA-29b in AML patients (P=0.003). Neither miRNAs showed significant correlation with FAB subtypes. Moreover, miRNA-370 expression was significantly negatively correlated with the age of the patients (P=0.034) − that is, the older the patient the lower the expression level of miRNA-370. Conclusion There was a significant down-regulation of both miRNA-370 and miRNA-29b in de-novo AML patients. The positive correlation between both miRNA expressions might point to their potential roles as tumor suppressors.

FLT3 Gene Mutations in Acute Myeloid Leukemia

This study was aimed to detect the FLT3 gene mutation in patients with de-novo acute myeloid leukemia (AML), and to investigate its prognostic value and clinical significance. Polymerase chain reaction (PCR) was used to detect FLT3 gene mutation, in bone marrow samples of 54 patients with de novo AML. The incidence of FLT3-ITD mutation in 54 de-novo AML patients was 22.22%, 10 out of 12(83.3%) AML patients were identified with normal karyotype, while 16.7% patients were identified as with abnormal karyotype. The peripheral blood white cell count and bone marrow blast cells were significantly higher in the patients with FLT3-ITD mutation than those in patients without FLT3-ITD mutation (P<0.05), but there was no statistically significant difference in sex, age, CR rate of the first course induction chemotherapy, survival rate and so on between the two groups. Two cases had FLT3-TKD gene mutation; as compared with FLT3-TKD negative AML patients there was no statistical difference in sex, age, white blood cell count, the percentage of marrow blasts and CR rate of the first course of treatment at the initial diagnosis. FLT3-ITD mutation positive likely occurs in AML patients with normal karyotype, the FLT3-ITD mutation is associated with higher peripheral white cell count and higher percentage of bone marrow blast cells.

Population Pharmacokinetics of Daunorubicin in AML: Influence on Clinical Outcome

Daunorubicin (Dnr) has been the choice of anthracycline in the induction chemotherapy of Acute Myeloid Leukemia (AML) along with Cytarabine for the past 4 decades. Drug resistance and relapse of the disease has been the major concern in the success of AML therapy. Several factors, including pharmacokinetics (PK) of Dnr could have impact on treatment outcome as pharmacokinetic effects might limit the amount of drug that reaches the tumor. In this study, we analyzed the factors influencing plasma PK of Daunorubicin and its major metabolite Daunorubicinol (DOL) in AML patients receiving induction chemotherapy and compared PK parameters with clinical outcome (relapse).Sixty four adult denovo AML patients were enrolled in the study after obtaining informed consent. Dnr was given as 1 hour bolus infusion at a dose of 60mg/m2/day for the first 3 days of induction chemotherapy. Peripheral blood were collected at 0, 15min, 1hr, 2hr, 4hr, 6hr and 24hr on the first day of induction chemotherapy. Plasma levels of Dnr and DOL were measured using high performance liquid chromatography coupled with fluorescence detector. Population PK was analyzed by nonlinear mixed effects modeling using Monolix version 4.3.2. Common single nucleotide polymorphisms in ABCB1, ABCG2, CBR1 and CBR3 genes were screened by PCR followed by either sequencing or restriction digestion. In vitro cytotoxicity of the primary AML cells to Dnr was assessed by MTT cell viability assay and IC50 was calculated using ADAPT5 software. The role of plasma Dnr PK on relapse of disease was estimated using nonparametric Mann-Whitney U test.Demographics of the patients enrolled in the study are listed in table 1. PK parameters of Dnr and DOL showed wide inter-individual variation (table2). Older patients had a trend towards lower Dnr clearance (CL) (p=0.09). Patients harboring FLT3-ITD mutations had higher CL than the patients without mutation though not reaching statistical significance. Patients with variants of SNPs rs20572 (exon3) and rs9024 (3’UTR) in CBR1 (which were in complete linkage disequilibrium) had a trend towards significantly higher Dnr AUC [median-0.2711; (0.04903-2.165) vs. median 0.2117; (0.0546-0.9036); p=0.06] than patients with wild-type.Among the 64 patients, complete remission (CR) was documented in 42 (65%) of which, 31% relapsed eventually [median time to relapse 328 (104-864) days]. AML patients who relapsed had significantly lower Dnr AUC [median 0.17 (0.049-2.165) vs. median 0.31 (0.1-1.74); p=0.009] and higher DOL AUC [median 8.67 (1.6-20.1) vs.4.99 (1.3-16.5); p=0.015].Also, relapsed patients had higher Dnr clearance (CL)[median -546.54 (41.5-1632)vs. 329.3 (42.9-944.5); p=0.011] and lower DOL CL[median-9.8 (4.9-56)vs. median- 19.7 (6.1-57); p=0.015]than patients who did not relapse. IC50 of Dnr was higher in patients who relapsed (median-0.7 (0.02-1.46) vs. median- 0.23 (0.007-1.11); p=0.056) than in patients who did not relapse.To the best of our knowledge, this is the first population PK report in AML which is designed to evaluate various factors which might influence Dnr and DOL PK in AML. Though the number is small, our results suggest that plasma PK of Dnr influences clinical outcome in AML. Lower systemic exposure of Dnr in relapsed patients suggests that amount of drug required to completely eradicate the leukemic cells is not achieved and escalating dose might be an option to decrease relapse rates. Cellular pharmacokinetics of Dnr in these patients is ongoing in the lab, which will be more informative and help in prognosticating and dose adjustment for better treatment outcome.This data provides a probable biological basis for the previously reported clinical benefit of decreased relapses following the increase of the dose of DNR in induction to 90mg/m2/day (Tallman et al. NEJM 2009).Wide inter-individual variation in Dnr PK also suggests that this increased dose may not be necessary for all patients. Further studies are required to confirm this finding in a larger cohort of patients and to work towards strategies for TDM of Dnr in AML [Display omitted] [Display omitted] DisclosuresSrivastava:Octapharma: Consultancy, Other.

DNA nucleosomal fragments have a therapeutic significance in adult acute myeloid leukemia

Aim Nucleosomal DNA levels in plasma and serum can be correlated with the extent of cell death at a specific time point. The aim of this study was to evaluate the serum level of nucleosomal DNA fragments as an early predictive marker for acute myeloid leukemia (AML) therapy. Materials and methods This study included 77 participants: 14 healthy volunteers, included in the control group; they ranged in age between 20 and 55 years, median age 36.5 years. Routine investigations such as liver functions, blood urea, serum creatinine, and complete blood count were performed to confirm their healthy state. The patient group included 63 de-novo AML patients. Results In the patient group, serum nucleosome levels ranged between 25 and 760 AU, with a mean±SD of 203.7±197 and a median of 220. In the control group, serum nucleosome levels ranged between 38 and 99 AU, with a mean±SD of 72.8±20.8 and a median of 80; a statistically significant difference was found between nucleosome levels in patients and its level in the control group ( P P Conclusion It would be valuable to include these markers together with the kinetics of circulating nucleosomal DNA fragments in prospective trials to elucidate their potential additive role in the early prediction of response to therapy. In AML patients, the changes in nucleosomal DNA during the initial phase of induction chemotherapy are valuable markers for the early estimation of therapy response and should be validated in further prospective trials.

Prognostic Impact of the Levels of Expression of Cell Surface Proteins Commonly Expressed by Blasts and Hematopoietic Precursor Cells in De Novo AML Patients: A Report From the Spanish Cetlam Study Group

AbstractAbstract 1452▪▪This icon denotes a clinically relevant abstract Introduction:Acute myeloid leukemia (AML) is a heterogeneous group of neoplastic disorders characterized by the presence of acquired genetic alterations in hematopoietic progenitor and stem cells. The prognostic impact of immunophenotypic markers has long been controversial. Despite this, only a very limited number of studies have investigated the prognostic impact of the levels of cell surface membrane (Sm) proteins commonly expressed on AML blasts. In this study we analyzed the prognostic impact of the levels of expression of several markers associated with AML blasts and normal hematopoietic precursors, as asessed by Multiparameter Flow Cytometry (MFC) in de novo AML patients (pts). Methods:Overall, 122 adult de novo AML pts diagnosed between 12/2003 and 08/2011 were studied, after excluding acute promyelocytic leukemia. All cases were diagnosed and classified according to the WHO criteria and they were immunophenotyped using an extensive 4-color panel of monoclonal antibodies by MFC, as previously described. Patients were treated according to the multicenter CETLAM AML-03 trial. Blast cells were gated after excluding all other cell populations, as well-delineated clusters of SSC low/int/CD45dimevents (“blast gate”) using the merge function of the Infinicyt software (Cytognos SL, Salamanca, Spain). For each case, the reactivity for the following markers was evaluated in terms of mean fluorescence intensity (MFI; arbitrary units) in bone marrow samples: CD123, CD117, CD34 and CD7. Normal residual bone marrow lymphocytes were used as reference for internal quality control purposes. Cytogenetic and molecular risk stratifications were based on the European LeukemiaNet (ELN) recommendations and the molecular lesions were investigated using well established protocols. Overall survival (OS), disease-free survival (DFS) and relapse rate (RR) were measured by the Kaplan–Meier method and curves were compared with the log-rank test. Multivariate analysis was performed using the Cox regression model. P-values ≤0.05 were considered to be statistically significant. Results:Median AML patient age was of 54 years (range: 20–70 y) with a M/F ratio of 63/59. Twelve pts (10%) had good cytogenetic/molecular risk, 83 (68%) intermediate, 17 (14%) high risk and 10 (8%) pts were unknown. Fifteen cases (12%) had NPM1mut, 33 (27%) showed FLT3-ITDmut, 5 (4%) pts carried CEBPAmut, 41 (33.6%) pts with no mutations and 28 (23%) pts were unknown. The median follow-up of pts alive was 19 months (range 0–97 months) and the 5-year OS of all pts was 42±5%. Univariate analysis of prognostic factors showed an association between higher CD45 (MFI >232; p=.01), CD117 (MFI>259; p=.008), CD34 (MFI >242; p=.007) and CD7 (MFI> 19; p=.03) expression and both a shorter OS and DFS. In addition, high CD123 expression (MFI >248; p=.04) was associated with a shorter DFS. In multivariate analysis only a high CD45 (p=.03) and a high CD34 (p=.03) expression were identified as independent predictors for a shorter OS. In turn, higher levels of CD123 (p=.03) and of CD34 (p=.02) were independent predictors for DFS and relapse, even when age, gender and WBC were taken in account. Conclusion:In this study, we show that higher levels of expression of immunophenotypic markers associate with immature myeloid precursors (CD45, CD117, CD34 and CD7) as assessed by MFC, have a significantly adverse prognostic impact in AML, both as regards OS and DFS. Accordingly, higher levels of CD45 and CD34 were independent predictors for both a shorter OS whereas, greater CD123 and CD34 values were associated with an increased risk of relapse and a shorter DFS, supporting the adverse prognostic impact of a more immature blast cell phenotype in de novo AML. Disclosures:No relevant conflicts of interest to declare.