Peripheral Blood Blasts Research Articles

Peripheral leukemia burden at time of apheresis negatively affects the clinical efficacy of CART19 in refractory or relapsed B-ALL

Our previous clinical study achieved complete remission (CR) rates of >90% following chimeric antigen receptor T cells targeting CD19 (CART19) treatment of refractory/relapsed B cell acute lymphoblastic leukemia (r/r B-ALL); however, the influence of the leukemia burden in peripheral blood (PB) blasts remains unclear. Here, we retrospectively analyzed 143 patients treated with CART19 (including 36 patients with PB blasts) to evaluate the effect of peripheral leukemia burden at the time of apheresis. One hundred seventeen patients with high disease burdens achieved 91.5% CR or incomplete count recovery CR and 86.3% minimal residual disease-negative CR, and 26 patients with low disease burdens obtained 96.2% MRD− CR. Collectively, 9 of 36 (25%) patients with PB blasts and 2 of 107 (1.87%) patients without PB blasts did not respond to CART19 therapy. The leukemia burden in PB negatively influenced ex vivo cell characteristics, including the transduction efficiency of CD3+ T cells and their fold expansion, and in vivo cell dynamics, including peak CART19 proportion and absolute count, fold expansion, and persistence duration. Further studies showed that these patients had higher programmed death-1 expression in CART19 products. Our data imply that PB blasts negatively affected CART19 production and the clinical efficacy of CART19 therapy in patients with r/r B-ALL.

Risk, Characteristics and Biomarkers of Cytokine Release Syndrome in Patients with Relapsed/Refractory AML or MDS Treated with CD3xCD123 Bispecific Antibody APVO436.

Simple SummaryCytokine release syndrome is a potentially life-threatening complication of therapy with T-cell engaging bispecific antibodies. Here we evaluated the risk, characteristics and biomarkers of treatment-emergent cytokine release syndrome in patients with relapsed/refractory acute myeloid leukemia or myelodysplastic syndrome who received weekly intravenous infusions of the CD3xCD123 bispecific antibody APVO436. Cytokine release syndrome was encountered in 10 of 46 patients (21.7%) treated with APVO436 with a cumulative Grade 3/4 cytokine release syndrome incidence of 8.7%. Cytokine profiling in patients who developed cytokine release syndrome after APVO436 infusion indicated that the predominant cytokine in this inflammatory cytokine response was IL-6. The findings from this research provide new insights regarding the biology and effective management of cytokine release syndrome in leukemia patients treated with T-cell redirecting bispecific antibodies. We evaluate the risk, characteristics and biomarkers of treatment-emergent cytokine release syndrome (CRS) in patients with relapsed/refractory acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) who received APVO436 during the dose-escalation phase of a Phase 1B study (ClinicalTrials.gov, identifier: NCT03647800). Of four patients who developed Grade ≥ 3 CRS, two received steroid prophylaxis. The dose level, gender, race, obesity, or baseline hematologic parameters in peripheral blood did not predict the risk of CRS. Patients with a higher leukemia burden as determined by a higher total WBC, higher percentage of blasts in bone marrow, or higher percentage of blasts in peripheral blood (by hematopathology or immunophenotyping) did not have a higher incidence of CRS. There was an age difference between patients who did versus patients who did not develop CRS (72.9 ± 1.6 years (Median 73.5 years) vs. 63.3 ± 2.3 years (Median: 65.0 years), which was borderline significant (p = 0.04). Premedication with steroids did not eliminate the risk of CRS. Cytokine profiling in patients who developed CRS after APVO436 infusion indicates that the predominant cytokine in this inflammatory cytokine response was IL-6. APVO436-associated CRS was generally manageable with tocilizumab with or without dexamethasone. Notably, the development of CRS after APVO436 therapy did not appear to be associated with a response. The prolonged stabilization of disease, partial remissions and complete remissions were achieved in both patients who experienced CRS, as well as patients who did not experience CRS after APVO436 infusions.

PATTERNS AND PROGNOSTIC IMPACT OF CNS INFILTRATION IN ADULTS WITH NEWLY DIAGNOSED ACUTE LYMPHOBLASTIC LEUKEMIA

Acute lymphoid leukemia (ALL) is a subtype of acute leukemia which portends a high risk of central nervous system (CNS) infiltration, which decreases overall survival (OS). Conventional cytology (CC) has been the standard for CNS disease assessment, although flow cytometry (FC) has gained space across several centers. In this study, we intend to: i) access the impact of CNS involvement (defined by CC or FC) on OS, event-free survival (EFS) and relapse of ALL patients treated in a public health center; ii) find variables associated with CNS infiltration. This retrospective study encompassed 105 cases of newly diagnosed ALL at Instituto do Cancer de São Paulo. Data were collected from Leukemia clinic database, approved by Local Ethics Committee. Variables related to cerebrospinal fluid (CSF) were collected from the first lumbar puncture (LP). Traumatic LP was defined as more than 10 red blood cells/μL. CNS status was assessed through the COG's classification and divided into positive/negative. Intensive intrathecal chemotherapy was given in CNS2 or 3 cases, while cranial irradiation was scheduled for CNS3 patients. A total of 105 cases were diagnosed from 2014 to 2020. Median age at diagnosis was 37 years (range, 15-79) with a male majority (58%). Most cases had a B-cell phenotype (76%), followed by T (19%) and ambiguous (5%). Philadelphia chromosome was found in 39%. Eighty-nine percent received corticosteroid at the time of LP with a median time of 7 days. Traumatic LP was reported in 50%. CSF was positive for disease in 15% (16/104) and 26% (19/72),by CC and FC, respectively. Among those with a positive CC, 56% (9/16) had a traumatic LP,but they were not statistically correlated (p = 0.8). Prior chemotherapy was associated to higher traumatic LP rates (56 vs 31%, p = 0.019). CNS1, CNS2 and CNS3 were found in 84%, 14% and 2% of cases, respectively. At the time of PL,44% of patients presented blasts in peripheral blood. By comparing CC and FC, we found one case with CC pos /FC neg whereas 9/19 with FC pos did not have CC pos . Sensitivity and specificity of CC for CNS disease were 53% and 98%. Higher white-blood cell (WBC) counts were found in subjects with positive CC or FC (Mann-Whitney test, p = 0.007 for CC). Also, the blast percentage was relevant for CNS positivity (p = 0.027). Genetic subgroup, age, CD34 expression and phenotype were not correlated with CNS invasion. Most patients received adapted versions of BFM (n = 43), GRAAPH (n = 24) and GRAALL-Elderly (n = 18) regimens. Median OS for the whole cohort was 16.4 months, with a 3y OS of 42.5% (95% CI 33.5-54). For BFM subset, OS and EFS were 53.5% and 37.3%. We did not find correlation between CNS disease with OS or EFS in univariate analysis. Three-year relapse rate was 38.5% (95% CI 29-49) whereas non-relapse mortality (NRM) was 33%. In multivariate analysis, a positive CC increased relapse rate (HR = 2.3 [95% CI 1.1-4.9, p 0.029]), aswell as initial WBC. Among relapses, only one was isolated in CNS and 13 had also concomitant bone marrow involvement. Several studies have demonstrated correlation between CNS infiltration and shorter OS in ALL, even though it relies on how these patients are managed and how CNS is assessed upfront. Our data showed that those with a positive CSF cytology had a higher risk of relapse, whereas FC was not able to discriminate this risk. Survival rates are increasing over the last years by the adoption of a stratified risk strategy. CSF contamination did not increase CNS involvement or relapse rates.

ABSENT CD33 IN AML PATIENT MIMICS MRD POSITIVE: A SINGLE CASE REPORT

Diagnosis and treatment of acute myeloid leukemia (AML) remains difficult to this day, cure rates remain low and measurable residual disease (MRD) assessment is still challenging. Early detection of residual leukemic cells is essential to predict therapeutic success and Multiparametric Flow Cytometry (MFC) has been an effective tool for it. Here, we focus on the importance in caution when interpreting MDR in AML. Bone marrow (BM) sample was assessed to FLT3 and NPM1 mutation, RUNX1/RUNX1T1 and CBFB-MYH11 rearrangements, karyotype (KT) and immunophenotyping. Using an 8-colour antibody panel (CD45, CD34, CD117, CD 33, HLA-DR, CD38, CD123, CD13, CD19, CD7, CD11b, CD4, CD56, CD64, CD11c, CD15, CD133, CD41a and NG2), we applied a gating strategy with fixed gates comparing normal to AML BM, settled to catch LAIPs (Leukemia Associated-Immunophenotypes), abnormal cell maturation pattern and Leukemic Stem Cell, that were detected on diagnosis and evaluated for MRD. Assays were performed at the Hematology Laboratory of the Medical School of Ribeirão Preto, University of São Paulo, in accordance to Local Ethical Boards. A 21-year-old male patient with AML without maturation, diagnosed after 60 days of symptoms, presented anemia (7.9 g/dL), thrombocytopenia (26 000/μL) and white blood cell count of 5710/μL with 73% blasts in peripheral blood and 84% in BM. MFC revealed CD45dim and low side scatter; positive staining for CD117, CD34, HLA-DR, CD38, CD123, CD11c, CD64, CD15 and CD133; negative staining for CD33, CD14, CD7, CD11b, CD4, CD56, CD19, CD41a and NG2. We found no metaphases (KT), no molecular abnormality and he failed risk stratification. Treatment consisted of 2 induction cycles (cycle 1: 3 days of Daunorubicin 60 mg/m2 and 7 days of cytarabine 200 mg/m2; cycle 2: 6 days of cytarabine 1 g/m2 twice a day) and 1 or 2 consolidation cycles (6 days of cytarabine 1 g/m2 twice a day). BM was obtained at diagnosis, 30 and 33 days after 1 st and 2 nd induction, and 3, 9, 12 and 15 months after consolidation of chemotherapy. The positivity of a specific phenotype (CD117+/CD34+/CD33wk/CD13+ ≥ 0.1%) was preserved at diagnosis and follow-ups: 0,506%, 0,681%, 0,402%, 0,101%, 0,158% and 0,122%. Complete remission was achieved by day 30 after 1st induction and no change in the outcome was reported. Absent CD33 was observed in diagnosis and follow-ups, during and after treatment. Post-analytical phase of MFC is the most vulnerable to wrong interpretations. CD33 is a common myeloid antigen expressed on malignant blasts in AML and had been reported as a potential target therapy. CD33 low blasts are associated to a more mature AML and its high expression is related to adverse landscapes, highlighting the importance of CD33 evaluation. Standard care for AML enrolls MRD monitoring and failure to achieve an MRD-negative, CR or detected MRD during or after therapy is associated with relapse or poor outcomes. Still, MRD analysis must embrace a set of standards in order to prevent post-analytic errors. Here we present a case report of AML with absent CD33 maintained from diagnosis to MRD follow-ups, mimicking MRD positivity. This data emphasizes the importance of caution in MFC analysis and correlation of diagnostic and follow-ups results interpretation, as well as constant training of the team.

Relationship between Effect of Induction Chemotherapy and Prognosis in AML Patients

To explore the relationship between effect of induction chemotherapy and prognosis in acute myeloid leukemia (AML) patients. The clinical data of 146 adult AML patients treated in Affiliated Hospital of Chifeng University from March 2015 to March 2018 were enrolled and retrospectively analyzed. Day 14 bone marrow biopsy (D14BM) cellularity and blast proportion, daily peripheral blood blast (PBB) clearance rate, time to PBB clearance and etc. were primarily observed after induction chemotherapy. All the patients were divided into Non-relapse survival group, Relapse survival group, Non-relapse death group and Relapse death group according to survival and recurrence situation during 2-year follow-up. The survival of the patients was analyzed by Kaplan-Meier. Univariate analysis of prognostic factors were performed by ordinal Logistic regression, and ROC curve was used to assess the prediction efficiency of those factors for the 2-year overall survival (OS) and relapse of the patients. A total of 138 patients were included since 8 cases failed to be assessed clinically. Their 2-year OS rate was 65.94%. Age of the patients in Non-relapse survival group was lower than that in Relapse death group. The D14BM cellularities in Non-relapse survival group and Relapse survival group were lower than those in Relapse death group (P<0.05). Daily PBB clearance rates in Non-relapse survival group and Relapse survival group were higher than those in Non-relapse death group and Relapse death group (P<0.05). There was a statistical difference among the four groups in the number of cycles of induction chemotherapy (P<0.05). The survival rate within 2 years in the patients with D14BM cellularity≤10% was higher than that in patients with cellularity >10%, while it was higher in patients with daily PBB clearance rate >20% than those with clearance rate≤20% (P<0.05). Age (HR=1.102, P=0.000), D14BM cellularity (HR=1.252, P=0.000) and the cycles of induction chemotherapy≥3 (HR=1.703, P=0.000) were the risk factors affecting the prognosis of AML patients, while daily PBB clearance rate was a protective factor (HR=0.799, P=0.000). The AUC of age, daily PBB clearance rate and D14BM cellularity in predicting 2-year OS of AML patients was 0.738, 0.817 and 0.807, respectively, whereas in predicting relapse within 2 years it was 0.691, 0.647 and 0.711, respectively. There was no statistical difference among the three factors in the sensitivity of 2-year OS (68.11%, 85.12%, 74.49%) and 2-year relapse (50.00%, 64.13%, 61.60%) (P>0.05). Bone marrow biopsy results and PBB clearance rate are related to prognosis in AML patients, which can offer certain predictive value in assessing 2-year OS of patients.

Expression of BECN1 gene in Acute Myeloid Leukemia: Association with Hematological and Clinical Parameters

Abstract Background In acute myeloid leukemia (AML), there is copy number loss in autophagic genes such as BECN1. Accordingly, decreased autophagy and the development of AML are related. BECN1 is a critical mediator that influences the onset and progress of autophagy. Objective To investigate the expression status of BECN1 gene in newly diagnosed adult AML patients and its association with various hematological parameters and clinical outcomes. Methods Case control study to study BECN1 gene expression variability between 50 newly diagnosed adult AML patients and 20 healthy age and sex matched controls, with follow up of the patients to detect its effect on induction therapy. All AML patients underwent full history taking, through clinical examination, laboratory investigations such as complete blood count (CBC) with examination of peripheral blood and bone marrow Leishman stained films, immunophenotyping, cytogenetic analysis (karyotyping/FISH analysis) and BECN1 gene expression analysis using real-time quantitative polymerase chain reaction (qRT-PCR). Results In our study, a highly significant difference was found as regards reduced expression of BECN1 gene in patients group compared to control group. We also found reduced BECN1 gene expression in both intermediate and adverse risk groups compared to favorable risk group. Reduced expression of BECN1 gene was associated with increasing age and total leukocytic count (TLC), peripheral blood (PB) and bone marrow (BM) blasts, the presence of FLT3-ITD mutation, CD34 and CD117 and in non-responders group. No statistically significant difference was found as regards haemoglobin (Hb) level, platelet (PLT) count and FAB subtypes. Conclusion Autophagy plays an important role in the pathogenesis of AML. Furthermore; the reductive regulation of the BECN1 gene may carry a poor prognosis and is associated with many well established bad prognostic factors especially FLT3-ITD mutation. Targeting autophagy pathways especially its major regulator (BECN1 gene) may become an effective and promising new line of therapy for AML patients.

CLINICAL, CYTOGENETIC AND MOLECULAR CHARACTERIZATION OF BRAZILIAN COHORT PATIENTS WITH PRIMERY MYELOFIBROSIS AND MYELOFIBROSIS POST-POLYCYTTHEMIA VERA AND POST-ESSENTIAL THROMBOCYTHEMIA

Myelofibrosis (MF) is the most severe myeloproliferative neoplasia (MPN), with a very variable survival, which can be beyond 20 years to less than two years. The only curative treatment is allogeneic bone marrow transplantation (ABMT), but this is restricted to patients of less advanced age and is associated with significant mortality and disease relapse. Adequate prognostic characterization is essential for therapeutic decision-making, and modern prognostic indexes using molecular and cytogenetic data have been replacing the traditional indexes. Clinical and laboratory data were evaluated, bone marrow and peripheral blood karyotype were studied, and BCR-ABL1, JAK2, CALR, MPL, ASXL1, SRSF2, EZH2, IDH1, IDH2, U2AF1 mutations were investigated. The classic IPSS, DIPSS, DIPSS plus, and the most recent MIPSS70, MIPSS70 plus, MIPSS 70 plus 2.0, GIPSS, and MYSEC-PM indexes were applied to a population of 230 patients from 8 Brazilian institutions with primary myelofibrosis (MFP) and secondary myelofibrosis polycythemia vera (PV-MF) and essential thrombocythemia (TE-MF) and compared among themselves. The median-age was 65 years-old, the overall survival was 8.1 years (CI 95%, 7,2 – 10,7), with a 5 years estimated survival of 61% (53 – 70). Comparing the prognostic indexes with each other identified a greater accuracy of the prognostic indexes that use mutational and cytogenetic data on the classic prognostic indexes. In the univariate analysis, advanced age, male gender, spleen size, hemoglobin, white blood cell count, peripheral blood blast percentage, SRSF2 mutation, triple-negative patients, IPSS, DIPSS and DIPSS plus stratification, MIPSS70 high risk and MIPSS70 plus high and very high risk showed impact in survival. In the multivariate analysis, triple-negative for JAK2 V617F, CARL , and MPL (p = 0,002) and stratification by IPSS (p < 0,0001) maintained significance as risk factors for overall survival. The present study is the most extensive Brazilian cohort study of patients with MF evaluating these variables.

LEUKEMIC PULMONARY INFILTRATION: A RARE COMPLICATION OF ACUTE LYMPHOBLASTIC LEUKEMIA

TOPIC: Pulmonary Manifestations of Systemic Disease TYPE: Fellow Case Reports INTRODUCTION: We report a rare case of pulmonary leukemic infiltration that posed a diagnostic dilemma. CASE PRESENTATION: A 61 year old female presented with weeks of weakness, nausea, vomiting, diarrhea. Initial laboratory evaluation revealed hyperleukocytosis of >100,000 cells/microliter with 86% blasts and thrombocytopenia. A bone marrow biopsy revealed B cell type ALL. Hydroxyurea was initiated for cytoreduction. On hospitalization day 3 she developed hypoxemia requiring 2-3 L supplemental oxygen. Chest radiograph revealed mild patchy airspace opacities. Extensive non-invasive infectious work up was negative. Over next week she developed worsening dyspnea and hypoxemia, with near tandem progression of radiographic airspace disease, despite antibiotics and stabilization of blood count. CT chest revealed diffuse patchy ground glass opacities, small pleural effusion and adjacent atelectasis. Bronchoscopy showed bloody secretions, with BAL differential showing abundant atypical cells with irregular nuclear contours, fine chromatin and prominent nucleoli, consistent with lymphoblastic leukemic blast cells. Concern was for pulmonary leukemic infiltration vs DAH with peripheral blood blast spillage. High dose corticosteroids were initiated. Her clinical condition deteriorated requiring endotracheal intubation and ICU admission. Due to her critically ill status, she was treated dose reduced, moderate intensity Cyclophosphamide and Vincristine chemotherapy. A repeat BAL revealed similar findings of ~30% blasts on differential. Subsequently her respiratory status improved, she was extubated and discharged on low supplemental oxygen. Bone marrow restaging 4 weeks after initial treatment showed persistent B acute lymphoblastic leukemia in the bone marrow. Her oncology team is planning for mini-HOP induction with Larson consolidation. DISCUSSION: Thoracic involvement of leukemia is varied, from leukemic pulmonary infiltration, hyperleukocytopathic reaction, leukostasis, DAH, lymphadenopathy, pleural effusion etc. CT findings of leukemic pulmonary infiltration are nonspecific, however leukemic cells have a tendency to involve the perilymphatic interstitium, producing smooth or nodular thickening of the bronchovascular bundles and interlobular septa. Nodules are also common and may be peribronchovascular, centrilobular, or random in distribution. Ground-glass and consolidative opacities are less common, predominately in peribronchial regions, by infiltration of the parenchyma adjacent to the perilymphatic interstitium. Accurate diagnosis usually requires bronchoalveolar lavage or lung biopsy to rule out other common etiologies. Response to primary chemotherapy usually depends on primary leukemia and patient's clinical status, but usually portends a poor prognosis. CONCLUSIONS: Clinicians need to thoroughly investigate pulmonary infiltrates in a leukemic patient in order to ensure appropriate therapy. REFERENCE #1: Shroff, G. S., et al (2019). Leukemic involvement in the thorax. Radiographics, 39(1), 44-61. DISCLOSURES: No relevant relationships by Jyotirmayee Lenka, source=Web Response No relevant relationships by Naman Sharma, source=Web Response

Mebendazole Mediates Proteasomal Degradation of GLI Transcription Factors in Acute Myeloid Leukemia.

The prognosis of elderly AML patients is still poor due to chemotherapy resistance. The Hedgehog (HH) pathway is important for leukemic transformation because of aberrant activation of GLI transcription factors. MBZ is a well-tolerated anthelmintic that exhibits strong antitumor effects. Herein, we show that MBZ induced strong, dose-dependent anti-leukemic effects on AML cells, including the sensitization of AML cells to chemotherapy with cytarabine. MBZ strongly reduced intracellular protein levels of GLI1/GLI2 transcription factors. Consequently, MBZ reduced the GLI promoter activity as observed in luciferase-based reporter assays in AML cell lines. Further analysis revealed that MBZ mediates its anti-leukemic effects by promoting the proteasomal degradation of GLI transcription factors via inhibition of HSP70/90 chaperone activity. Extensive molecular dynamics simulations were performed on the MBZ-HSP90 complex, showing a stable binding interaction at the ATP binding site. Importantly, two patients with refractory AML were treated with MBZ in an off-label setting and MBZ effectively reduced the GLI signaling activity in a modified plasma inhibitory assay, resulting in a decrease in peripheral blood blast counts in one patient. Our data prove that MBZ is an effective GLI inhibitor that should be evaluated in combination to conventional chemotherapy in the clinical setting.

INCIDENCE, RISKS AND OUTCOME OF EXTRA MEDULLARY RELAPSE IN ACUTE LYMPHOBLASTIC LEUKEMIA IN CHILDREN AND ADULTS: A RETROSPECTIVE COHORT STUDY

Extra medullary relapse (EMR) is the major concern of treatment failure in acute lymphoblastic leukemia (ALL). Hence, their early detection may improve prognosis. Aim of the study is to identify risk factors and outcome of EMR patients with and without concurrent medullary relapse. We retrospectively analyzed 113 patients of ALL who presented with EMR in a tertiary cancer care hospital. Patients were divided into two groups; Group 1, combined EMR (with medullary relapse) and Group 2, isolated EMR (without medullary relapse). Clinical characteristics including total leukocyte count (TLC) &amp; peripheral blood blast percentage during presentation, phenotype, initial central nervous system (CNS) involvement and BCR-ABL translocation were analyzed to identify risk factors for EMR. Incidence of EMR in our study was 15.7% (113/720 patients). Two year incidence of combined EMR and isolated EMR was 10.1% and 5.5% respectively. CNS, followed by testis was common sites of EMR. TLC &gt;50x109/L and peripheral blood blast percentage &gt;50% at presentation was common in Group 1. BCR-ABL translocation was frequent in Group 2 and in adults compared to children (p=0.027). While Group 1 patients presented with leukocytosis (TLC &gt;10X109/L), Group 2 patients presented with leukopenia (TLC &lt;4X109 p=0.001). p=0.001).&gt;50X109/L and BCR-ABL translocation were identified as risk factors for combined EMR (p=0.004) and for in adults with isolated EMR (p=0.001) respectively.

The Relationship between the Connexin 32 and Connexin 43 Genes and the Pretreatment Stage and Short-term Follow-up of Patients with Acute Myeloid Leukemia.

Background: Connexins (Cxs) are gap junction proteins involved in the communication between acute myeloid leukemia (AML) and stromal cells. They consist of intercellular channels termed “connexions”, which can cause uncontrolled cell proliferation if dysregulated. This study aimed to evaluate the expression levels of the Cx32 and Cx43 genes and their correlations with other prognostic markers in patients with AML.Methods: This cross sectional study was performed on peripheral blood samples from 60 newly diagnosed patients with AML and 40 healthy control subjects at Kasr Alainy School of Medicine, Cairo University, from June 2016 to December 2017. The quantitative real-time polymerase chain reaction (qRT-PCR) test was used to examine the relative expression level of Cx43 and Cx32 genes in the patients and the control subjects. The Chi square test or the Fisher exact test was employed to examine the relationship between qualitative variables, while the independent t test or the Mann–Whitney test was employed for quantitative data. All the tests were two-tailed, and a P value of less than 0.05 was considered significant.Results: Among the patients with AML, 65% had a high Cx32 expression level, whereas 63.3% had a low Cx43 expression level. There was a statistically significant difference in the fold change values of Cx32 and Cx43 expression between the patient group and the control group (P=0.009 vs P=0.013, respectively). There was a remarkable association between both Cxs and CD34 and HLA-DR cells.Conclusion: Cx expression in samples may add to the diagnostic workup of AML. Although we found a negative correlation between Cx43 expression and the peripheral blood blast percentage, the response after the first induction of chemotherapy showed no significant relationship with Cx43 and Cx32.

AML-320: A Prospective Evaluation of WT1 Gene Expression in Primary Acute Myeloid Leukemia: Bench to Bedside

Context: Acute myeloid leukemia (AML) is a heterogeneous disease with marked variation in response to therapy. New prognostic markers, such as gene mutations, including NPM1, FLT3, and WT1, have been identified. Objective: Evaluation of WT1 gene expression and mutations, including AML-ETO, CBFB-MYH11, NPM1, FLT3-ITD, and PML-RARA, as biomarkers for therapy response in primary AML. Design: Prospective clinical research Setting: Study performed at a tertiary cancer care center. Patients or Other Participants: A total of 114 cases of diagnosed AML, having blast percentage of ≥20% blasts in peripheral blood or bone marrow on day 0, and treated with induction therapy for 28 days, after which morphological remission occurred (blast percentage ≤5%), were enrolled. Materials and Methods: RNA was extracted from blood/bone marrow samples at diagnosis (day 0) and on completion of induction therapy (day 28). Hematological workup and flowcytometric immunophenotypes were done. WT1 expression was done using real-time qPCR and was normalized against endogenous control genes: HBG2, GAPDH, and β-2-microglobulin. Detection of mutations, including AML-ETO, CBFB-MYH11, NPM1, FLT3-ITD, and PML-RARA, was done in 47 cases. Cytogenetics was done in 40 cases, and FISH for PML-RARA in 9 cases. Results: WT1 expression was seen to be reduced at day 28 in 96/114 (84.2%) cases, whereas in 18/114 (15.8%) cases, WT1 expression was seen to have increased at day 28 compared to day 0 levels. Blast count percentage in these 18 cases varied from 28% to 80%. AML-ETO mutation was detected in 14/47 (29.7%) cases, CBFB-MYH11 in 3/47 (29.8%) cases, NPM1 mutations in 5/47 (10.6%) cases, and FLT3-ITD in 8/47 (17%) cases. PML-RARA mutation was detected in 9 cases of APML (confirmed by cytogenetics and FISH in 6/9 cases). Conclusions: WT1 gene expression shows significant reduction in more than 80% of cases showing morphological remission at the end of induction therapy. WT1 expression along with blast count percentage can act as a good biomarker for assessment of remission. Molecular analysis in AML cases has diagnostic as well as prognostic significance. More prospective studies with larger sample sizes are needed to further validate these results. Funding Acknowledgment: The study was supported by SERB, DST, Government of India.

MPN-161: A Phase I Study of TP-3654, an Oral PIM Kinase Inhibitor, in Patients with Intermediate-2 or High-Risk Primary or Secondary Myelofibrosis (MF)

Context: MF is characterized by bone marrow fibrosis (BMF), ineffective hematopoiesis, splenomegaly, and debilitating symptoms. Expression of PIM-1 is significantly upregulated in MF hematopoietic cells, indicating that PIM-1 is a potential therapeutic target in MF. TP-3654, an oral small-molecule investigational PIM-1 kinase inhibitor with favorable selectivity against other kinases, has been shown to inhibit proliferation and increase apoptosis in murine and human hematopoietic cells expressing the JAK2V617F mutation. TP-3654 alone and in combination with ruxolitinib normalizes leukocyte counts and reduces spleen size and BMF in JAK2V617F and MPLW515L murine MF models. Objective: To evaluate TP-3654 monotherapy in patients with MF. Design: Phase I, open-label, dose-escalation study (NCT04176198). Setting: Multicenter, US only. Patients or Other Participants: Key eligibility criteria include primary or secondary MF (post-polycythemia vera-MF/post-essential thrombocythemia-MF); intermediate-2/high-risk MF per Dynamic International Prognostic Scoring System (DIPSS); previously failed or are ineligible to receive Janus kinase inhibitors (e.g., ruxolitinib, fedratinib); grade ≥2 BMF; platelet count ≥50x109/L; absolute neutrophil count ≥1x109/L; peripheral blood blast counts Interventions: Patients receive oral TP-3654. Dose escalation, performed using a Bayesian Logistic Regression Model with Escalation with Overdose Control, will continue until identification of the maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D) is achieved. Dose expansion will follow. Main Outcomes Measures: Primary objectives: to determine MTD and/or identify the RP2D of TP-3654 monotherapy. Secondary objectives: to assess preliminary activity (spleen volume reduction, symptom improvement, and BMF reduction), determine QT interval changes, and establish the pharmacokinetic profile. Exploratory objectives: to study pharmacodynamic markers in peripheral blood and BM biopsies. This study is currently recruiting.

ALL-003: Value of Autophagy Related Gene 5 (ATG5) Expression as a Prognostic Marker in Adult Patients with Newly Diagnosed Acute Lymphoblastic Leukemia (ALL)

Context: Autophagy is considered a pivotal cellular mechanism to cope with stress and maintain homeostasis. It is frequently dysregulated in different types of cancers with some exhibiting excessive and others having reduced autophagy levels. Objectives: To determine autophagy status in newly diagnosed adult acute lymphoblastic leukaemia (ALL) patients compared to age and sex matched controls. Moreover, its prognostic impact on disease characteristics and response to treatment was evaluated. Design: Prospective study. The patients were followed up to 12 months. Setting: Clinical Hematology and Oncology unit, Internal Medicine department, Faculty of Medicine, Ain Shams University, Cairo, Egypt. Patients: Thirty-five newly diagnosed adult ALL patients were recruited and age and sex matched to 15 healthy control subjects. Intervention: ATG5 expression was measured on diagnosis using real-time quantitative polymerase chain reaction (qPCR). Cases have been followed up for one year after enrollment into the study. Main Outcome Measures:: To measure ATG5 in newly diagnosed ALL patients and correlate its level with clinical outcome and treatment response. Results: Mean ATG5 expression in ALL was twice that of controls which exhibits high statistically significant difference (2.15 + 2.2 vs 1.09 + 0.47, P value = 0.01). Its high expression has been significantly associated with B-ALL phenotype, lower haemoglobin and lower platelet counts. Of note, higher expressor group had higher bone marrow and peripheral blood blast percentages, more likelihood to develop chemoresistance to frontline agents; however, this did not show statistical significance. Further studies with the recruitment of larger sample of patients is needed to examine a relationship between autophagy activation and therapeutic outcome in ALL. Conclusion: Our study demonstrates that classic autophagic pathway may be activated in ALL patients and that it may be associated with more aggressive tumour behaviour and worse outcome.

Refractory Anemia With Excess Blasts In Transformation (Acute Myeloid Leukemia with 20-29% Blasts) in Older Adults is Less Aggressive Than Acute Myeloid Leukemia With ≥30% Blasts

BackgroundThe current WHO Classification defines acute myeloid leukemia (AML) in most cases as a myeloid neoplasm with ≥20% bone marrow (BM) or peripheral blood (PB) blasts. However, it is controversial whether AML cases with 20-29% BM blasts, referred to as oligoblastic AML or refractory anemia with excess blasts in transformation (RAEBT), should be considered as AML or myelodysplastic syndrome (MDS) in terms of treatment approach and prognosis. MethodsWe retrospectively studied 573 patients aged ≥50 years diagnosed after the year 2004 with de novo AML, including 142 with 20-29% BM blasts (RAEBT) and 431 with ≥30% BM blasts (AML30). Patients with t(15;17), t(8;21), inv(16)/t(16;16), or history of any prior myeloid neoplasm were excluded. Clinicopathologic and genetic features, treatments, and outcome of RAEBT were compared to AML30. The clinicopathologic and genetic features of RAEBT were also compared to 152 de novo MDS patients with 10-19% BM blasts (RAEB2) diagnosed over the same time period. Fisher's exact and Wilcoxon tests were used to compare categorical and continuous variables between groups, respectively. Overall survival (OS) was estimated using the method of Kaplan and Meier and the log-rank test was used to compare OS between groups. Univariate and multivariable Cox proportional hazards regression was used to assess the impact of the two groups along with other risk factors in OS. ResultsCompared to AML30 patients, RAEBT patients were older and had lower white blood count and BM cellularity, but had higher hemoglobin, platelet count, and UKMRC karyotype risk score and greater morphologic dysplasia (Table 1).RAEBT and RAEB2 patients were similar with respect to age, peripheral counts, BM cellularity, and karyotype risk scores. In the subsets of patients in whom they were assessed, FLT3 and NPM1 mutations and monocytic features occurred more commonly in AML30 than in RAEBT (Table 1). RAEBT patients were treated more often with hypomethylating agents and less often with intensive therapy (anthracycline/cytarabine) compared to AML30 patients, whereas allogeneic stem cell transplant frequency was similar. With 55.5 months median follow-up, OS of RAEBT patients was superior to that of AML30 patients (p=0.003)(Figure 1). Multivariable analysis showed that RAEBT (p<0.0001), hemoglobin ≥10.3 g/dL (p=0.045), intermediate UKMRC karyotype score (p<0.0001), normal bone marrow karyotype (p=0.0005), intensive therapy (p<0.0001), and stem cell transplant (p<0.0001) were associated with longer OS.Table 1Comparison of RAEBT and AML30 groupsRAEBT (n=142)AML30 (n=431)p valueMedian age in years6763<0.0001Median white blood count (x 109/L)2.389.20<0.0001Median hemoglobin (g/dL)9.809.200.0007Median platelets (x 109/L)76600.01Median BM cellularity (%)6090<0.0001Morphologic dysplasia(% with >10% erythroid / myeloid / megakaryocytic dysplasia)53 / 53 / 4631 / 25 / 27<0.0001 / <0.0001 / <0.0001Monocytic or myelomonocytic differentiation23/115 (20%)137/342 (40%)<0.0001UKMRC karyotype scores(% intermediate / adverse / missing)61 / 35 / 569 / 26 / 50.049FLT3-ITD mutation3/94 (3%)59/247 (23%)<0.0001NPM1 mutation1/55 (2%)46/151 (30%)<0.0001Supportive care only11/142 (8%)38/431 (9%)0.86Hypomethylating agent as initial therapy36/142 (25%)36/431 (8%)<0.0001Intensive therapy as initial therapy73/142 (51%)310/431 (72%)<0.0001Stem cell transplant41/142 (29%)118/431 (27%)0.7412-month OS [95% CI]60% [46-74%]51% [47-55%]0.00348-month OS [95% CI]29% [22-36%]20% [17-24%]▪ ConclusionsRAEBT is more similar to RAEB2 than to AML30 in terms of its clinicopathologic and cytogenetic features. In spite of less frequent treatment with intensive chemotherapy and higher karyotype risk, RAEBT patients had superior OS compared with AML30 patients. In a prior study (Estey E et al. Blood 1997;90:2969), RAEBT and AML30 patients treated with intensive therapy had similar outcomes; the advent of hypomethylating agent therapy may in part account for the differences found in our study. Our findings favor considering RAEBT in a disease category separate from AML with higher blast counts. Disclosures:No relevant conflicts of interest to declare.

Abstract CT236: A Two-Part Phase 2 Study of Itacitinib Immediate Release in Patients with Primary or Secondary Myelofibrosis Who Have Received Prior Ruxolitinib and/or Fedratinib Monotherapy

Abstract Background: Ruxolitinib (JAK1/JAK2 inhibitor) and fedratinib (JAK2/FLT3 inhibitor) are indicated in the US for myelofibrosis (MF); however, some patients fail to achieve adequate/sustained response to initial JAK-inhibitor therapy. Itacitinib is a potent JAK inhibitor selective for JAK1 over JAK2 when administered as a once-daily sustained-release (SR) formulation. In prior clinical trials, the SR formulation improved MF-related symptoms but had less effect with respect to spleen volume reduction. When dosed as a twice-daily (BID) immediate release (IR) formulation, itacitinib may offer increased JAK2 inhibition (in addition to JAK1 inhibition) to better address the JAK2-mediated myeloproliferative features of MF. This open-label phase 2 study is designed to determine a tolerable and safe dose of itacitinib IR that results in clinically significant reductions in symptoms and spleen volume in patients with MF who have previously received ruxolitinib and/or fedratinib monotherapy (INCB 39110-213; NCT04629508). Methods: Eligible patients are aged ≥18 years, diagnosed with at least INT-1 risk (Dynamic International Prognostic Scoring System [Passamonti. Blood. 2010;115:1703-1708]) primary or secondary (post-polycythemia vera or post-essential thrombocythemia) MF and have received ruxolitinib and/or fedratinib monotherapy. Patients must also have palpable splenomegaly and platelets ≥50×109/L at screening. Exclusion criteria include receipt of a JAK inhibitor other than ruxolitinib or fedratinib, ≥10% myeloid blasts in peripheral blood or bone marrow, or inability to taper ruxolitinib/fedratinib over 14 days without use of other agents. Two itacitinib IR dose levels (DLs) will be evaluated in part 1 following a Bayesian optimal interval design algorithm: 3-9 patients will be enrolled at DL1 (300 mg BID) and observed for 28 days for dose-limiting toxicity before enrollment at DL2 (600 mg BID). Part 2 will enroll ~55 patients at the recommended phase 2 dose (RP2D) determined in part 1. Patients may remain on treatment until week 48 if they are receiving clinical benefit and have not met study withdrawal criteria. A safety follow-up will occur 30 days after treatment completion. Primary objectives: part 1 - evaluate safety and tolerability of itacitinib IR and select the RP2D; part 2 - evaluate efficacy of itacitinib IR at the RP2D based on spleen volume reduction at week 24. Secondary objectives (part 2 only): evaluate safety and tolerability of itacitinib IR; evaluate efficacy of itacitinib IR with respect to MF symptom improvement at week 24 in patients with baseline total symptom score ≥10, quality-of-life improvement, and patient global impression of change. Sites are opening in the US and EU. Citation Format: Andrew T. Kuykendall, Lea Burke, Mani Lakshminarayanan, Philomena Colucci. A Two-Part Phase 2 Study of Itacitinib Immediate Release in Patients with Primary or Secondary Myelofibrosis Who Have Received Prior Ruxolitinib and/or Fedratinib Monotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT236.

Mature Analysis of the Prospective Oxford Down Syndrome (DS) Cohort Study: Timing and Clinical Impact of Preleukemic GATA1 Mutations and Lessons for Management of Newborns with DS

Children with Down syndrome (DS) have a high risk of developing a unique form of AML known as myeloid leukaemia of DS (ML-DS) defined by the presence of one or more acquired N-terminal truncating mutations in the hematopoietic transcription factor gene GATA1 . ML-DS is confined to children <4 y of age and preceded by a neonatal preleukemic syndrome, Transient Abnormal Myelopoiesis (TAM). Although the same GATA1 mutations are present in ML-DS and TAM, indicating they are clonally-linked disorders, chemotherapy is essential to cure ML-DS whereas most cases of TAM spontaneously resolve without treatment.To determine the natural history of GATA1 mutations in DS and the true risk of subsequent leukemia conferred by GATA1 mutations we established the Oxford DS Cohort Study that prospectively analyses serial clinical, hematological and GATA1 mutational data from children with DS from birth to age 4y. All children had serial blood counts and smears and mutation analysis of exon 2/3 of GATA1 by next-generation-sequencing (NGS). A diagnosis of TAM was made if peripheral blood (PB) blasts were >10% and a GATA1 mutation was detected. DS neonates with PB blasts <10% and a GATA1 mutation were designated Silent TAM as our previous data on 18/80 DS neonates with blasts <10% showed that small mutant GATA1 clones were clinically and hematologically undetectable [Roberts et al, Blood 2013].We have now studied 471 neonates (gestational age [GA] at birth 29-42 wks) with karyotypically-confirmed DS. 130/471 (27.6%) had >1 GATA1 mutation confirming the high frequency of GATA1 mutations in DS neonates. Multiple GATA1 clones (2-7) were detected in 19% of TAM and 21% of Silent TAM (p=ns). A diagnosis of TAM was made in 55/471 (11.7%). 75/471 (15.9%) had Silent TAM and 341/471 had no GATA1 mutations. Typical clinical findings of TAM (hepatomegaly, splenomegaly, skin rash and effusions) were uncommon in Silent TAM, affecting 7%, 1.3%, 1.3% and 3% neonates respectively compared to 40%, 29%, 17% and 10% of neonates with TAM (p<0.0001). There were no specific hematological abnormalities in Silent TAM: median WBC and blasts were not increased: 13.1x109/L v 14.5x109/L and 4% v 4% (Silent TAM v no GATA1 mutation) and no neonates with Silent TAM had leucocytosis (WBC >35x109/L) or anemia. There was a strong correlation (r=0.753; p<0.0001) between PB blast% and mutant GATA1 clone size (assessed by VAF) with a greater mutant clone size in TAM v Silent TAM (median VAF 17.1% v 1.2%; p<0.0001).Unexpectedly, clone size was inversely proportional to GA (r=-0.2569; p=0.004). Despite similar median GA at delivery for all 3 groups, no GATA1 mutations were detected before 31 wks GA and no cases of TAM before 33 wks; the highest frequency of GATA1 mutations was seen at 34-35 wks GA (40%) reducing to 7% at >40 wks. This suggests GATA1 mutations are likely to be acquired and expand during a narrow time window early in the 3rd trimester of fetal life. Consistent with this, GATA1 mutations were not detected in 16 DS fetal liver samples (GA 8-19 wks) analysed over the same period. Similarly, in a case of TAM presenting in utero with fetal pericardial effusion and 96% PB blasts at 27 wks GA, the blast% and mutant GATA1 VAF spontaneously fell progressively (blasts 19%; VAF 23% at birth at 34 wks GA; undetectable at 4 wks postnatal age).Outcome: 7 neonates developed ML-DS: 6/52 survivors with TAM; 1/71 survivors with Silent TAM and 0/328 survivors without neonatal GATA1 mutations; 2/451 survivors developed ALL (1 Silent TAM, 1 no GATA1 mutation). 5 yr overall survival was the same in all 3 groups (95.1%, 95.8%, 95.1% for TAM, Silent TAM and no GATA1 mutations; Plog-rank 0.8994) while 5 yr event-free survival was lower in TAM (89.2%, 92.8%, 95.1% in TAM, Silent TAM and neonates with no GATA1 mutations; Plog-rank=0.0262). ML-DS was diagnosed at a median age of 14 m (range 3-18 m) preceded in all cases by a fall in platelet count. No single risk factor at birth specifically predicted for ML-DS although all 6 neonates with TAM who developed ML-DS had a mutant GATA1 VAF of >15% and blasts >20% at birth. The DS neonate with Silent TAM had a VAF of 2.5% and 4% blasts; this neonate was the only one of the cohort with a partial trisomy 21. Conclusion: Acquired N-terminal mutations in GATA1 are very frequent in neonates with DS and most probably occur or expand in the 3rd trimester; in the majority of cases mutant GATA1 clones are very small, clinically silent, resolve spontaneously and confer an extremely low risk of ML-DS. DisclosuresVyas:Jazz Pharmaceuticals: Speakers Bureau; Celgene Corporation: Speakers Bureau.

Ruxolitinib or Dasatinib in Combination with Chemotherapy for Patients with Relapsed/Refractory Philadelphia (Ph)-like Acute Lymphoblastic Leukemia: A Phase I-II Trial

Ruxolitinib or Dasatinib in Combination with Chemotherapy for Patients with Relapsed/Refractory Philadelphia (Ph)-like Acute Lymphoblastic Leukemia: A Phase I-II Trial

Day 5 Peripheral Blood Blasts and Day 14 Bone Marrow Studies As Predictors for Response to Therapy in Acute Myeloid Leukemia (AML): An Experience from Indian Subcontinent

Day 5 Peripheral Blood Blasts and Day 14 Bone Marrow Studies as Predictors for Response to Therapy in Acute Myeloid Leukemia (AML): An Experience from Indian SubcontinentAuthorsShilpa Prabhu1, Swasti Sinha1, Vellaichamy M. Annapandian2, Sharat Damodar1, Shoba Badiger1, Sunil Bhat1, K.S. Nataraj1Author affiliations1 Department of Hematology and Bone Marrow Transplant, Narayana Health City, Bangalore, India.2 Department of Academic Research, Narayana Hrudayalaya Foundations, Bangalore, India.AbstractBackgroundThe major limitations of treating Acute Myeloid Leukemia (AML) in India are limited resources, patient affordability and utilization of health care services. It is vital to identify easily evaluable prognostic markers to plan for appropriate treatment that enables healthcare providers to deliver treatment at an affordable cost. AML is a most common leukemia in adults and has poor survival and response to treatment. However, with advances in treatment strategies, overall survival has improved in adults and there has been marginal increase in overall survival in older individuals. Studies have showed that immunophenotying to detect blasts by multiparameter flow cytometry on day 5 of induction chemotherapy as a predictor to remission. Financial constraint is the major concern in developing countries, hence peripheral smear examination can be used as an cheaper alternative to look for morphological evidence of blast. Morphological assessment at day 5 peripheral blood blasts can be used as a predictor for remission status, in addition to day 14 bone marrow studies. This strategy will enable us to identify a subset of patients who will benefit from early haematopoeitic stem cell transplant (HSCT) and prognosticate the patients. In this present study we assessed peripheral blast at day5 and day 14 bone marrow studies to predict remission on day 28.MethodsWe analyzed a cohort of 80 AML patients who presented to our centre (Mazumdar Shaw Medical Center, Narayana Health City, Bangalore, India) between April 2013 and April 2017. Diagnosis was confirmed by bone marrow studies based on the morphology, cytochemistry, immunophenotyping and cytogenetics. Clinico-pathological features, immunophenotyping, cytogenetics, mutation profile were recorded at the time of presentation. Day 5 peripheral blood blast percentage, day 14 bone marrow studies (aspiration & biopsy) and day 28 remission bone marrow (or bone marrow done at later dates) studies were recorded.ResultsAmong 80 patients, 48(60%) were male. The median age was 35 years (range; 10, 59 years). Seventeen (20%) were pediatric patients. Fifty-six (70%) patients were denovo and 24 (30%) were relapse at presentation. The most common presenting symptom was fever 68.75%(n=55). The median WBC count at presentation was 11.95x103/µl (1.5-280 x103). The median blast percentage on the day of diagnosis, day 5 peripheral blood , day 14 bone marrow were 68%, 3% and 4% respectively. According to ELN risk stratification, patients were grouped as favorable risk (3.75%, n=3), intermediate risk (51.25%, n=41) and adverse risk (37.5%, n=30). Thirty eight (47.5%) patients had no blasts in peripheral blood on 5th day of chemotherapy. Of, whom 34(89.4%) had attained remission on day 28 (p<0.01). Day 14 bone marrow blast was absent in 27 cases out of whom 26(96.3%) attained remission on day 28 (p<0.01). The clinicopathological details of the study patients are summarized in table 1.The overall survival rate was 62% in patients with absence of day 14 bone marrow blast and 32% in patients with persistent disease at the end of 48 months (Figure 1). Patients who attained remission on day 28 had 55% survival at the end of 48 months compared to those who had persistent disease on day 28 (16% survival). Analysis of risk stratification of patients showed that those in favorable risk, intermediate risk and adverse risk had overall survival of 80%, 39% and 21% respectively at the end of 48 months.ConclusionsDay 5 peripheral blood blasts and day 14 bone marrow status are most important factors that help predicting remission on day 28 in patients with AML, surpassing the prognostic information obtained from analysis of pretreatment characteristics alone. Day 14 bone marrow blasts can be used as a surrogate marker in predicting overall survival and disease free survival. Also, day 14 bone marrow studies can help in identifying subset of patients who can be benefit from HSCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Predictors of Response and Survival in 206 AML Patients Treated with Guadecitabine in a Phase 2 Study

Background: Guadecitabine is a next generation hypomethylating agent (HMA) resistant to degradation by cytidine deaminase which results in prolonged in vivo exposure to the active metabolite decitabine. We conducted a prospective phase 2 study testing different schedules of guadecitabine in 206 AML patients. We present here the results of multiple logistic regression, and Cox regression analyses of predictors of composite Complete Response or CRc (CR+CRp+CRi) and overall survival (OS).Methods: Multiple logistic regression analysis of response (CRc), and Cox regression analysis of OS were conducted with inclusion of the following baseline variables: disease state (relapsed/refractory (r/r) vs. treatment naïve (TN) AML); guadecitabine schedule (10-day vs. 5-day); age (<75 vs. ≥75); ECOG PS (0-1 vs. ≥2); Cytogenetics (others vs. poor risk); baseline BM blasts (≤40% vs. >40%); baseline Peripheral blood (PB) blasts (≤30% vs. >30%); baseline WBCs count (<20,000/µL vs. ≥20,000/µL); Flt-3 ITD, NPM, and TP53 mutations (each present vs. not detected). Cutoff values for blasts % for BM and PB were chosen based on the median or mean values respectively, and cutoff value for WBCs count was chosen as a common cutoff used for proliferative AML. Backward elimination method with alpha =0.05 was used to reach the final models.Results: We treated 206 AML patients (103 patients each for TN or r/r AML); 101 with 5-day schedule and 105 with 10-day schedule. There were 91 patients (44%) ≥75 y; 53 (26%) with ECOG PS≥2; 85 (41%) with poor risk cytogenetics; 99 (48%) with baseline BM blasts >40%; 65 (32%) with baseline PB blasts >30%; 20 (9.7%) with WBCs ≥20,000/µL. Flt-3, NPM, and TP53 mutations were present in 8%, 9%, and 4% of patients respectively. The final logistic regression model indicated that patients with ECOG PS 0-1 and those with baseline PB blasts ≤ 30% have two-fold higher odds of response than those with ECOG PS ≥2 and PB blasts >30% (Odds ratio 2.18; and 2.03 respectively; p<0.05 for both). Patients with TN AML had five-fold higher odds of response to guadecitabine than r/r AML (Odds ratio of response for r/r AML 0.22; p <0.0001). The final Cox regression model showed that both ECOG PS 0-1 and PB blasts ≤30% retained their significance for OS (HR 0.69 with p=0.03; and 0.61 with p= 0.004 respectively). However disease state (r/r AML vs TN AML) lost significance for OS while cytogenetics risk level (others vs poor risk) became significant with a HR for OS of 0.68, p=0.016.Summary/Conclusions: In a prospective series of AML patients treated with guadecitabine in a phase 2 study, better ECOG PS 0-1 and lower baseline PB blasts ≤30% were associated with a significantly higher likelihood of response and a longer OS. Patients with TN AML had significantly higher likelihood of response than those with r/r AML but this was not a significant factor for OS when other factors are present in the model such as ECOG PS, cytogenetics risk and PB blasts. On the other hand, the presence of poor risk cytogenetics did not alter the likelihood of response to guadecitabine but still had shorter survival compared to patients with other cytogenetics risk levels. Other variables such as age, baseline BM blasts %, and baseline WBCs count did not significantly impact response or OS in AML patients treated with guadecitabine when all other factors are present in the models. The analysis of genetic mutations was limited by the small number of patients where these mutations were present. The analysis of the treatment schedule is limited by the different effect of the schedule on r/r AML compared to TN AML where the 10-day schedule did better than the 5-day schedule in r/r AML but not in TN AML patients. DisclosuresKantarjian:Novartis: Research Funding; ARIAD: Research Funding; Delta-Fly Pharma: Research Funding; Amgen: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding. Roboz:Cellectis: Research Funding; AbbVie, Agios, Amgen, Amphivena, Array Biopharma Inc., Astex, AstraZeneca, Celator, Celgene, Clovis Oncology, CTI BioPharma, Genoptix, Immune Pharmaceuticals, Janssen Pharmaceuticals, Juno, MedImmune, MEI Pharma, Novartis, Onconova, Pfizer, Roche Pharmace: Consultancy. Kropf:Takeda: Honoraria; Celgene: Honoraria; Astex: Consultancy. Jabbour:Bristol-Myers Squibb: Consultancy. Yee:Oncoethix: Research Funding; Celgene Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Canada: Honoraria; Astex: Research Funding; Karyopharm: Research Funding. O'Connell:Astex Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Otsuka: Membership on an entity's Board of Directors or advisory committees. Stock:Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees. Podoltsev:Ariad: Consultancy; Incyte: Consultancy; CTI biopharma/Baxalta: Consultancy; Alexion: Consultancy. Hao:Astex Pharmaceuticals, Inc.: Employment. Ahanonu:Astex Pharmaceuticals, Inc: Employment. Azab:Astex Pharmaceuticals, Inc.: Employment. Issa:Astex Pharmaceuticals, Inc.: Consultancy, Research Funding; Teva: Consultancy, Research Funding; GSK: Consultancy.