Acute Myeloid Leukemia Risk Groups Research Articles

Venetoclax in Combination with Intermediate Doses of Cytarabine in Consolidation Phase for Acute Myeloid Leukemia Patients in First Complete Remission; Results of the Part 1 of the Phase 1/2 Multicentric Covenidac Study

For authors: Please: L Gastaud and P Peterlin: co-primary, H dombret et C Recher: co-last Background: The French ALFA-FILO AML intergroup launched the BIG-1 trial (EudraCT No.: 2014-000699-24) in 2015 to evaluate different intensive chemotherapy (ICT) strategies aiming at improving the survival of younger adults with acute myeloid leukemia (AML). All patients (pts) with previously untreated non-APL and non-CBF AML aged 18-60 years were eligible for this trial. The trial design included several randomization types. Results of the R1 and R2 randomizations, namely idarubicin vs daunorubicin for induction and IDAC vs HDAC for consolidation, will be reported at this meeting. In patients who achieved first complete remission (CR), randomizations R4 consisted of nested randomized Phase 2 or 1/2 studies evaluating the addition of new drugs to the IDAC/HDAC consolidation cycles. The protocol was designed to allow several sequential multicenter R4 evaluations of various new agents over the trial period. Venetoclax (VEN) has proved its efficacy when combined to azacitidine or low dose cytarabine in unfit AML pts (DiNardo et al. 2020; Wei et al. Blood 2020). Its role in fit patients treated with ICT is still under investigation. The COVENIDAC Phase 1/2 R4 sub-study is evaluating the safety and efficacy of VEN added to IDAC during the consolidation cycles of CR pts. We report herein the result of the Phase 1 part. Methods: The primary objective of this Phase 1 study was to determine the maximum tolerated dose (MTD) of VEN in combination with IDAC during the consolidation cycles, which will be the recommended dose for the Phase 2 (RP2D). Dose limiting toxicities (DLTs) were assessed during the first cycle of consolidation (C1) according to a 3+3 design. A maximum of 3 consolidation cycles was planned depending on the AML risk group. Hematological DLT was defined as no peripheral blood count recovery within 56 days from day 1 of the cycle. Non-hematological DLTs were defined as Grade ≥4 toxicities or Grade ≤3 toxicities which have not recovery by day 56 and deemed related to VEN per investigator opinion. VEN was started at dose level (DL) 1: 100 mg/d from day 1 to 8. In the absence of DLTs at DL1, 3 others DL were planned: 200 mg/d from day 1 to 8 (DL2); 400 mg/d from day 1 to 8 (DL3), and 400 mg/d from day 1 to 14 (DL4). IDAC was administrated at 1.5 gr/m² twice daily on day 1/3/5 for DL1, DL2 and DL3 pts, then on day 1/2/3 for DL4 pts after a protocol amendment. Main inclusion criteria were: pts in first CR following 1 or 2 courses of induction chemotherapy fit for further ICT; favorable and intermediate risk AML as defined by the protocol; ECOG PS ≤2; written informed consent. Measurable residual disease (MRD) was assessed by multiparametric flow cytometry (MFC) in all pts as well as NPM1-qPCR in NPM1-mutated pts. Results: Between 06/2021 and 04/2022, 15 pts from 9 centers were screened and enrolled. Pt characteristics were as follows: median age, 42 years (IQR, 30-49); 2 males/7 females; ECOG-PS, 0 in 10 pts and 1 in 5 pts; favorable/intermediate/adverse ELN-2017 risk, 9/2/4. Neutropenia and thrombocytopenia durations are shown in Table 1. Only 1 hematological DLT was observed at DL3 (platelet count of 96 giga/L at day 56 in a patient who was infected by Covid-19). Overall, there were very few non hematological AEs ( Table 2, no grade 4) and only 4 SAEs (1 related to IDAC/VEN during C1, 3 non-related). No non-hematological DLT was observed. No patient died during the DLT observation period, 1 experienced a septic shock (C2, DL2). One pt received only C1 (due to DLT), 7 received 2 cycles, and 7 received 3 cycles. Median time between initiation of subsequent consolidation cycles was 37.5 days (IQR, 35-41) for C1/C2 and 40 days (IQR, 35-43) for C2/C3. One pt had to stop VEN during C3. Six pts received an allogeneic HSCT (5 in CR1, 1 after salvage). With a median follow-up of 13.5 months, 3 pts relapsed and 2 died. MRD results will be presented. Conclusion: VEN/IDAC combination appears to be feasible and safe as consolidation therapy, with expected thrombocytopenia and neutropenia durations. These durations appeared even shorter at DL4, when IDAC was given on 3 consecutive days. VEN at 400 mg/d from day 1 to 14 combined with IDAC on day 1/2/3 was thus retained as RP2D for the randomized Phase 2 with RFS as primary endpoint. This phase 2 has already recruited 29 of the 200 pts planned.

Prognostic Role of Molecular MRD Variations during Treatment of Pediatric AML: A Retrospective AIEOP AML2013/01 Study

Background. Measurable residual disease (MRD) is a key parameter to evaluate response to cytotoxic treatment in leukemia patients and, thus, to guide therapeutic strategies. In fact, the identification of persisting leukemia cells after chemotherapy is associated with an increased risk of disease recurrence, and many patients with acute myeloid leukemia (AML) experiencing relapse have a fatal outcome. Many somatic genetic aberrations are under investigation for assessing molecular quantitative MRD (qMRD). However, while flow cytometry is already recognized as useful in measuring residual disease levels, the impact of qMRD monitoring is still debated. Methods and Results. We set up a qPCR assay to quantify mRNA fusion or mutated transcripts for 23 AML rearrangements identified at diagnosis in 247 patients (67% of the whole population) enrolled in our recently concluded AIEOP-AML2013/01 trial, with a sensitivity reaching 10 -5/-6. We measured qMRD after the induction therapy courses (n=202) and identified 66 cases with qMRD>1x10 -3 (cut-off determined by ROC curve method, qMRD with respect to disease levels found at disease onset) and 136 with qMRD<1x10 -3 after the II induction course. Patients with qMRD>1x10 -3 had a significantly worse OS and EFS with respect to patients with qMRD<1x10 -3 (77% vs 89%, p=0.016 and 61% vs 78%, p=0.016, respectively). qMRD assessment within each AML risk group, namely Standard (SR, n=64), Intermediate (IR, n=38) or High (HR, n=101), showed that the persistence of qMRD>1x10 -3 after the II induction course in SR patients did not correlated with worse EFS with respect to patients who reduced qMRD levels below the cut-off, mainly due to NPM1 mutated patients all relapsing (7/7) with qMRD<1x10 -3 after the second induction course. By contrast, qMRD>1x10 -3 was a predictive variable able to discriminate, among the patients assigned to the IR (EFS 46% vs 81% for IR, p=0.009) those children with poorer outcome. For the HR patients, qMRD 1x10 -3 predicted a worse, although not statistically significant, outcome (61% vs 80% for HR, p=0.08). When we interrogated qMRD role within the different genetic subtypes, we showed that qMRD significantly impacted on EFS of patients harboring t(8;21) RUNX1-RUNX1T1 (47% for qMRD>1x10 -3vs 88% for qMRD<1x10 -3, p=0.004), this finding corroborating results previously published by our group. A similar trend, although not significant, was also obtained for patients harboring isolated FLT3-ITD mutation, who had a dismal EFS when qMRD levels after the II induction course were above 1x10 -3 (40% (n=5) vs 71% (n=7) for qMRD<1x10 -3, p=ns). qMRD reduction for KMT2A-rearranged AML cases and for other genetic markers having a lower incidence in AML, needs to be evaluated in larger cohorts to obtain robust results. Additionally, we interrogated if qMRD maintains the same clinical relevance when measured after the III or IV chemotherapy blocks. We calculated proper cut-off for this analysis by ROC curve method, finding again 1x10 -3 for the third chemotherapy cycle; we observed that AML cases with qMRD>1x10 -3 had a worse EFS with respect to patients with lower levels of qMRD (39% n=30 vs 80% n=82, p<0.0001). As concerns the IV chemotherapy block, we calculated an appropriate cut-off corresponding to 1x10 -4, and once again we confirmed that patients with qMRD above the cut-off displayed a lower probability of experiencing adverse events (EFS 49% n=46 vs 85% n=65, p=0.0003). In conclusion, this is the first study that considers a serial and continuous qMRD monitoring all along an entire trial demonstrating a significant prognostic role of qMRD after II, III and IV therapy courses. These findings demonstrate that qMRD represents a useful tool in supporting therapeutic decisions and suggest that harmonization of currently used molecular MRD detection methods is the best solution to face MRD challenge in pediatric AML. Further comparisons between molecular and cytofluorimetric methods will increase the chances to identify those patients at high risk of relapse, despite negative MRD measured by flow cytometry.

AgNOR patterns and configurations in adult acute leukemia patients

The number of argyrophilic nucleolus organizer regions (AgNOR) is related to the proliferative activity of cells and the degree of neoplastic transformation. The surface area of AgNOR depending on nuclear structure may be a predictor of tumor recurrence, while research into acute leukemias is scarce. The aim of the study was to determine whether the assessment of AgNOR parameters is useful in the differentiation of acute leukemias and, together with cytogenetic changes, would allow for a quick evaluation of the risk group. The AgNOR structures were analyzed in terms of the shape, surface area and distribution in bone marrow blast cells in patients with acute leukemias. We observed significant differences in the AgNOR structures, simple, compound and complex patterns between acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Complex structures were more numerous in ALL than in AML patients. There were significant differences in the distribution of AgNOR configuration among various cytogenetic AML risk groups. We observed a significant difference in the mean number of AgNOR between ALL-T and ALL-B. We detected diversity in the AgNOR structures and pattern map in AML and ALL. Thus, presentation of a variety of AgNOR configurations is innovative and can be a useful method of differentiating patients with acute leukemia types and cytogenetic risk.

CHARACTERISTICS AND OUTCOME OF T(8;21)-POSITIVE CHILDHOOD ACUTE MYELOID LEUKEMIA: A SINGLE INSTITUTION'S EXPERIENCE

Compared with other cytogenetic acute myeloid leukemia (AML) groups, patients with core-binding factor AML (CBF-AML) are considered as a favorable AML risk group based on their high remission rate and survival probabilities. However, up to 30-40% of these patients can still relapse after standard intensive induction and consolidation chemotherapy. From 2004 to 2020, 147 AML patients reviewed. Ten of 147 patients were followed up with t(8;21) chromosomal anomaly. The t(8;21)(q22;q22) was detected by reverse transcription polymerase chain reaction (RT-PCR) and/or floresan in situ hibridizasyon (FISH). We analyzed patients’ demographic data: sex, white blood cell count at diagnosis, central nervous system status, additional cytogenetic anomaly and recurrence rates, stem cell transplant status and survival rates. Two of 10 patients were female. The median age was 10 years (3-17 years). Median followup was 36 months (2-114 months). The mean white blood cell count of 10 patients was 21.5 (× 109/l) at diagnosis. One out of 10 patients had granulocytic sarcoma and 2 had central nervous system involvement. Additional cytogenetic anomalies were detected in 90% of the patients, of which 2 relapsed and 3 died. One patient received hematopoietic stem cell transplantation and died because of HSCT complications. Recent studies show that CBF-AML includes different groups with different clinical outcomes. We found that 50% of our patients achieved complete remission and 50% experienced relapsed disease or death. After we were able to monitor the t(8;21) level with RT-PCR, we diagnosed relapsed disease in 1 patient with additional cytogenetic anomaly. RT-PCR is essential for optimal handling of these patients to predict patients’ relapse risk and to detect minimal residual disease.

MAPKAP1 (Sin1), a Key Component of the MTORC2 Complex, Confers Resistance to Sorafenib and Correlates with Adverse Clinical Outcomes in Acute Myeloid Leukemia

Introduction: The mammalian target of rapamycin (mTOR) functions as a critical catalytic core in two distinct intracellular complexes: the rapamycin-sensitive mTORC1 and the rapamycin-insensitive mTORC2. The latter is capable of activating Akt through phosphorylation at the Ser473 residue. Constitutive activation of Akt is identified in up to 70% of acute myeloid leukemia (AML) patients and has been associated with an adverse impact on survival. Akt activation also mediates, at least in part, the leukemogenic effects of activating FLT3 internal tandem duplication (ITD) mutations, which in turn lead to activation of mTORC1. Mitogen-activated protein kinase-associated protein 1 (MAPKAP1 or Sin1) is required for mTORC2 integrity and activation and is also required for phosphorylation and activation of Akt. In addition, the pleckstrin homology (PH) domain of MAPKAP1 directly binds the PI3K product PtdIns P3 to promote mTORC2 kinase activation and membrane localization, thereby revealing a mechanistic link between PI3K and mTORC2. However, the role of MAPKAP1 in AML pathogenesis and its impact on clinical outcomes are as of yet unknown.Methods: The in vitro system included 5 AML cell lines (2 FLT3 -mutated and 3 FLT3 -unmutated) and 6 BA/F3 (murine pro-B cells) stable clones bearing specific FLT3 mutations. Experiments were performed using transient transfections with siRNA or plasmids, pharmacologic studies, Western blot analysis, q-RT-PCR, flow cytometry, immunofluorescence, confocal microscopy and proteomics. The patient cohort included 224 adult AML patients diagnosed and treated at The University of Texas MD Anderson Cancer Center (Houston, TX) and the National University Hospital (Singapore) with available cytogenetics and mutation status data. Potential associations of MAPKAP1 with clinical outcomes were evaluated using mRNA expression data from the The Cancer Genome Atlas (TCGA) AML dataset and protein expression data assessed by immunohistochemistry on bone marrow biopsy specimens from patients in our study group using a monoclonal anti-MAPKAP1 antibody (Cell Signaling). Overall survival (OS) was defined as time from AML diagnosis to death or last follow-up. Event-free survival (EFS) was defined as time from AML diagnosis to relapse, death, or last follow-up. Survival analysis was based on the Kaplan-Meier method using the Log-rank test.Results: MAPKAP1 is frequently expressed in AML cells and regulated by STAT3, which is predominantly phosphorylated at Ser727 residue in AML cell lines and patient samples. Inhibition of STAT3 activity by pharmacologic agents, dominant negative constructs, or STAT3 gene silencing leads to decreased MAPKAP1 levels and MTORC2 activity associated with Akt dephosphorylation. In silico analysis reveals 4 potential STAT3 binding sites on the MAPKAP1 gene promoter suggesting transcriptional regulation. Knocking down the MAPKAP1 gene in AML cell lines results in Akt deactivation associated with decreased cell proliferation, and these effects are mediated by cell cycle regulators. MAPKAP1 also confers resistance to sorafenib in vitro, as MAPKAP1 is shown to be upregulated and more active in BA/F3 clones bearing sorafenib-resistant FLT3 mutations. Conversely, STAT3 or MAPKAP1 inhibition sensitizes resistant BA/F3 clones to sorafenib treatment. Using the TCGA dataset, high MAPKAP1 mRNA levels are significantly associated with poorer EFS and OS (Figure 1); these findings were independent of AML risk group or the presence of FLT3 or NPM1 mutations. Similarly, high MAPKAP1 protein levels assessed by immunohistochemistry on bone marrow specimens prior to treatment correlate with adverse clinical outcomes in a cohort of AML patients treated with equivalent regimens.Conclusions: MAPKAP1 is involved in AML pathogenesis, likely through a STAT3-MTORC2-Akt crosstalk mechanism, and may represent a novel target of investigational therapy. MAPKAP1 mRNA and protein levels are significantly associated with clinical outcomes in AML._ [Display omitted] DisclosuresKhoury:Pfizer: Research Funding; Angle: Research Funding; Stemline Therapeutics: Research Funding; Kiromics: Research Funding. Kantarjian:Delta-Fly Pharma: Research Funding; Amgen: Research Funding; ARIAD: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding; Novartis: Research Funding.

A 11-Year Clinical Summary of DC - CIK / NK Cell Immunotherapy for 152 Patients with Acute Myeloid Leukemia

Objective: To assess the efficacy of immunotherapy based on autologous dendritic cells-cytokine induced killer cells (DC-CIK) and allogeneic natural killer cells (NK) in treating low-risk and intermediate-risk acute myeloid leukemia (AML). The study was conducted over a period of 11 years (April 1, 2006 to April 1, 2017) across the Beijing Ludaopei hospital systems.Methods: DC-CIK cells were derived by culturing patients' peripheral blood mononuclear cells (PBMCs) in vitro with cytokines for 8-12 days. To generate NK cells, allogenic PBMCs were cultured for 3 to 6 days. After patients completed intensified chemotherapy consisting of 4 cycles of high-dose Ara-C within 6 months, DC-CIK or NK cells were infused and given once every 3 months for 2-4 cycles along with chemotherapy of Fludarabine, Cyclophosphamide and Ara-C given few days prior to each cycle of immunotherapy.Results: From April 1, 2006 to April 1, 2017, a total 152 patients in the low and intermediate risk groups of AML (except APL) underwent combined immunotherapy and chemotherapy in our center. Overall survival (OS) rate was 82% and disease-free survival (DFS) rate was 67% for the cohort. Beginning in June 2012, the low and intermediate risk groups were further stratified into very-good risk, good risk, and intermediate risk groups according to the specific AML related gene mutations. The OS rate of the 2012-2017 group was significantly better than that of the 2006-2011 group (91.7% vs. 71.6% p= 0.003). For patients treated prior to 2012, there were no significant differences between the low and intermediate risk group in OS (72.2% vs 71.4%) and DFS (72.2% vs 61.2%). From 2012 to 2017, the OS rates were 94.4%, 86.3%, and 93.3%, and the DFS rates were 83.3%, 81.8%, and 62.2%, (p=0.15) for the very-good risk, good-risk & intermediate risk groups, respectively, again suggesting no statistical differences among the groups. Side effects were mild with some fever, chills and fatigue.Patients who were treated with 2-4 cycles of immunotherapy were further divided into the DC-CIK alone group and the DC-CIK alternating with NK group. Importantly, the OS and DFS rates of 67 patients in the DC-CIK alternating with NK group were significantly better than those of 53 patients in the DC-CIK alone group (OS 95.5% vs 71.4%, p=0.0003), (DFS 85% vs 63.5%, p =0.0099). .Twenty-nine of 48 relapsed patients from both groups underwent allo-hematopoietic stem cell transplantation (allo-HSCT). The long-term OS after HSCT was 65.5%, closed to the OS of AML patients who transplanted in their first complete remission (CR) in our center. OS of 12 patients transplanted after 2012 was significantly better than that of 17 patients transplanted before 2012 (88.2% vs. 33.3%, p = 0.015). This because that after 2012, all patients have been checked minimal residual disease (MRD) by flow cytometry. Once patient was found to have relapse with MRD,immediately pursued HSCT while some patients transplanted prior to 2012 had morphological relapse.Conclusion: When combined with chemotherapy, DC-CIK/NK cells-based immunotherapy significantly improves the long-term OS and DFS rates of good and intermediate risk groups of AML. Auto DC-CIK alternating with allo NK is superior to auto DC-CIK alone. Relapsed patients could be safely and effectively treated with HSCT. If relapse patients with only positive MRD, then the outcome of HSCT will be comparable to those who received the HSCT during their first CR.Figure 1. OS of 152 patients treated with DC-CIK/NK immunotherapy from 2006 to 2017.Figure 2, OS of 67 patients treated from 2006 to 2012 vs 85 patients from 2012 to 2017 (71.6% vs 91.7%, p= 0.003).Figure 3, OS & DFS of DC-CIK+NK vs DC-CIK treated groups from 2012 to 2017 (p= 0.0003, p= 0.0099) [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genome Sequencing as an Alternative to Cytogenetic Analysis in Myeloid Cancers

BackgroundGenomic analysis is essential for risk stratification in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). Whole-genome sequencing is a potential replacement for conventional cytogenetic and sequencing approaches, but its accuracy, feasibility, and clinical utility have not been demonstrated.MethodsWe used a streamlined whole-genome sequencing approach to obtain genomic profiles for 263 patients with myeloid cancers, including 235 patients who had undergone successful cytogenetic analysis. We adapted sample preparation, sequencing, and analysis to detect mutations for risk stratification using existing European Leukemia Network (ELN) guidelines and to minimize turnaround time. We analyzed the performance of whole-genome sequencing by comparing our results with findings from cytogenetic analysis and targeted sequencing.ResultsWhole-genome sequencing detected all 40 recurrent translocations and 91 copy-number alterations that had been identified by cytogenetic analysis. In addition, we identified new clinically reportable genomic events in 40 of 235 patients (17.0%). Prospective sequencing of samples obtained from 117 consecutive patients was performed in a median of 5 days and provided new genetic information in 29 patients (24.8%), which changed the risk category for 19 patients (16.2%). Standard AML risk groups, as defined by sequencing results instead of cytogenetic analysis, correlated with clinical outcomes. Whole-genome sequencing was also used to stratify patients who had inconclusive results by cytogenetic analysis into risk groups in which clinical outcomes were measurably different.ConclusionsIn our study, we found that whole-genome sequencing provided rapid and accurate genomic profiling in patients with AML or MDS. Such sequencing also provided a greater diagnostic yield than conventional cytogenetic analysis and more efficient risk stratification on the basis of standard risk categories. (Funded by the Siteman Cancer Research Fund and others.)

Functional Impact of MacroH2A Histone Variants in Acute Myeloid Leukemia

Histone variants are emerging as key regulatory molecules in cancer. Recent data provided first evidence of how transcriptional deregulation and changes in the deposition of histone variants may affect malignant transformation. H2A variants are highly conserved and consist of 8 members. Among all histone variants, macroH2A variants hold a unique C-terminal macro domain, which is approximately twice the size of its H2A-like histone domain. In mammals, there are 3 macroH2A variants encoded by H2AFY (macroH2A1.1, macroH2A1.2) and H2AFY2 (macroH2A2). MacroH2A variants occupy large repressive domains throughout the genome, exerting a repressive role on transcription. So far, the function of macroH2A proteins in hematopoiesis and leukemic transformation is incompletely understood. Here, we report on a functional role of macroH2A histone variants that may influence cell competition in leukemia and normal hematopoietic stem and progenitor cell (HSPC) function. In published gene expression datasets, macroH2A variants H2AFY and H2AFY2 are highly expressed in hematopoietic stem cells (HSCs) compared to mononuclear cells (both p<0.0001****). Moreover, expression of H2AFY and H2AFY2 appeared even higher in acute myeloid leukemia (AML) (p=0.0750 and 0.0037**, respectively) when compared to the expression level in HSCs. This high expression is independent of different genetic subtypes and AML risk groups. To assess for the functional dependency of leukemic cells on the expression of macroH2A variants, we performed an in vitro CRISPR-Cas9 dropout screen focusing on macroH2A variants in MOLM-13 cells. Genetic inactivation of H2AFY but not H2AFY2 resulted in outcompetition of infected cells against non-infected competitors. This finding could be recapitulated in 3 additional AML cell lines (OCI-AML3, THP-1, HL-60), indicating a broader dependency on H2AFY irrespective of the oncogenic background or underlying driver mutation. To validate the functional impact of H2AFY on leukemia development in vivo, we generated a novel conditional knockout mouse model for H2afy and induced leukemia through retroviral infection of sorted Lineage-Sca1+cKit+ (LSK) cells with the oncogene MLL-AF9. Results of these functional studies will be presented. To understand the functional impact of macroH2A variants on normal HSPC function in vivo, we performed competitive repopulation studies. We used conventional knockout mouse models for the 2 H2afy isoforms: macroH2A1.1 and macroH2A1.2 In addition, for inactivation of both isoforms, we used the conditional macroH2A knockout mouse model. H2afy2 was either inactivated by RNAi or conditionally knockout through Mx1-Cre recombinase activation. In brief, whole bone marrow cells (WBMCs) were transplanted into primary recipient mice in a 1:1 competitive manner and compared to the respective wildtype littermate controls. Peripheral blood (PB) chimerism was monitored on a monthly basis 4 weeks post-transplantation for a total of 16 weeks. We observed no significant difference in the recipients of macroH2A1.1 (n=5/5) or macroH2A1.2 (n=10/9), suggesting both macroH2A isoforms are dispensable for intrinsic functions of HSCs. Remarkably, we found macroH2A2-deficient cells (n=6) competed significantly worse than macroH2A2+/+ (n=5) controls in PB (p=0.0043**) and in WBM (p=0.0043**), indicating a requirement for macroH2A2 in maintenance of HSPCs. These findings could be recapitulated by RNA-mediated inactivation of H2afy2 in sorted HSCs. We observed significant reduction of macroH2A2-depleted hematopoiesis after 16 weeks in WBM (p=0.0286*) and PB (p=0.0079**). Importantly, this functional impairment was less pronounced when investigating multipotent progenitor and committed progenitor cell function in spleen colony-forming unit (CFU) assay in vivo or CFU assay in vitro. Taken together, our data indicate distinct requirements for macrohistone variants in leukemic and normal hematopoietic cells. Dependency of leukemic cells but not normal HSPCs on macroH2A may indicate a potential therapeutic index. Disclosures No relevant conflicts of interest to declare.

Blast vacuolization in AML patients indicates adverse-risk AML and is associated with impaired survival after intensive induction chemotherapy.

IntroductionVacuolization is a frequently found morphological feature in acute myeloid leukemia (AML) blasts. Subcellular origin and biological function as well as prognostic impact are currently unknown. The aim of this study was to evaluate whether vacuolization correlates with clinically relevant AML features.Materials & methodsBone marrow smears of patients diagnosed with AML at the University Hospital Frankfurt between January 2011 and August 2013 were analyzed for blast vacuolization and correlated with clinically relevant AML features. Patients undergoing standard induction chemotherapy were further analyzed for molecular and cytogenetic features as well as treatment response and survival.Results14 of 100 patients diagnosed with AML receiving standard induction chemotherapy had evidence of blast vacuolization. Positivity for vacuolization correlated with a CD15 positive immunophenotype and with a higher incidence of high-risk AML according to the European LeukemiaNet risk stratification. AML patients with blast vacuolization had a poor blast clearance after standard induction chemotherapy and poor survival.DiscussionIn conclusion, our findings demonstrate that vacuolization can easily be determined in myeloid leukemia blasts and may be a useful biomarker to predict AML risk groups as well as early treatment response rates and survival.

Advances in Acute Myeloid Leukemia Genomics, Where Do We Stand in 2018?

The aim of this review is to summarize the data on commonly mutated genes and genomic pathways in acute myeloid leukemia (AML) with a focus on recently approved targeted therapies. AML is a heterogeneous disease with recurrent cytogenetic and genomic abnormalities that define the disease biology and pathogenesis. Classification of the disease categories and their prognostication was updated in the past 2 years to reflect the most recent advances in understanding the complex disease biology of AML. This review highlights major updates in the World Health Organization classification, including cytogenetic re-classifications, provisional entities, and updates to the European Leukemia Net (ELN) AML risk group stratification. An overview of pivotal studies that used novel sequencing techniques to define the mutational landscape of AML is also provided. In these studies, mutations are classified into subgroups based on functional pathways and are used to understand various interactions and mutual exclusivity of some mutations, suggesting important roles in disease evolution and AML pathogenesis. The complex interactions between mutations can dictate outcomes as well as possibly predict disease phenotypes after correcting for clinical variables. CONCLUSION: Genomic testing in AML using next generation sequencing has become widely available and a new standard of care for all patients. Therefore, it is vital to use novel methods to incorporate these data in clinical decision making.

Graft-Versus-Leukemia Effect of Allogeneic Stem-Cell Transplantation and Minimal Residual Disease in Patients With Acute Myeloid Leukemia in First Complete Remission.

The detection of minimal residual disease (MRD) in patients with acute myeloid leukemia (AML) may improve future risk-adapted treatment strategies. We assessed whether MRD-positive and MRD-negative patients with AML benefit differently from the graft-versus-leukemia effect of allogeneic hematopoietic stem-cell transplantation (alloHSCT). A total of 1,511 patients were treated in subsequent Dutch-Belgian Hemato-Oncology Cooperative Group and the Swiss Group for Clinical Cancer Research AML trials, of whom 547 obtained a first complete remission, received postremission treatment (PRT), and had available flow cytometric MRD before PRT. MRD positivity was defined as more than 0.1% cells with a leukemia-associated immunophenotype within the WBC compartment. PRT consisted of alloHSCT (n = 282), conventional PRT by a third cycle of chemotherapy (n = 160), or autologous hematopoietic stem-cell transplantation (n = 105). MRD was positive in 129 patients (24%) after induction chemotherapy before proceeding to PRT. Overall survival and relapse-free survival were significantly better in patients without MRD before PRT compared with MRD-positive patients (65% ± 2% v 50% ± 5% at 4 years; P = .002; and 58% ± 3% v 38% ± 4%; P < .001, respectively), which was mainly because of a lower cumulative incidence of relapse (32% ± 2% compared with 54% ± 4%; P < .001, respectively). Multivariable analysis with adjustment for covariables showed that the incidence of relapse was significantly reduced after alloHSCT compared with chemotherapy or autologous hematopoietic stem cell transplantation (hazard ratio [HR], 0.36; P < .001), which was similarly exerted in both MRD-negative and MRD-positive patients (HR, 0.38; P < .001; and HR, 0.35; P < .001, respectively). The graft-versus-leukemia effect of alloHSCT is equally present in MRD-positive and MRD-negative patients, which advocates a personalized application of alloHSCT, taking into account the risk of relapse determined by AML risk group and MRD status, as well as the counterbalancing risk of nonrelapse mortality.

The Novel BET Inhibitor PLX51107 Has In Vitro and In Vivo Activity Against Acute Myeloid Leukemia

Background:Acute myeloid leukemia (AML) has very poor long-term survival with traditional therapies. AML has a diverse pathogenesis and likely represents multiple different diseases. Various epigenetic effector proteins are altered in AML by mutation, over-expression, or compartmental displacement and these changes maintain transcriptional programs important for leukemogenesis. The bromodomain and extra-terminal domain (BET) proteins, including BRD2, BRD3 and BRD4, play roles in many cellular functions important to leukemogenesis, such as super-enhancer function, transcriptional elongation, histone acetylation and cell cycle progression. In particular, AML cells depend on BRD4 for expression of the pro-survival proteins MYC and BCL2. BRD4 has therefore become an attractive target for novel therapeutics. PLX51107 is a novel BET inhibitor with a unique binding mode in the acetylated lysine binding pocket of BRD4 that differentiates it from other compounds under investigation. Our group has previously shown this compound to have antineoplastic activity in models of aggressive B cell malignancies. We have now investigated the anti-leukemic properties of PLX51107 in both in vitro and in vivo models of AML.Results:PLX51107 treatment potently reduced viability and proliferation of the human AML cell lines MV4-11, MOLM-13, OCI-AML3, and Kasumi-1, with IC50 of 0.17, 1.8, 0.2 and 0.2 μM, respectively. We then evaluated the in vitro activity of PLX51007 in primary human AML samples. PLX51107 inhibited the proliferation of primary human AML cells co-cultured with HS5 stromal cells. For nearly all samples tested (n=9), the IC50 of PLX51007 was less than 1 μM (average = 0.41 μM, range 0.039 - 1.5 μM). Notably, PLX51107 showed efficacy across a broad range of AML risk groups, including samples with adverse risk features such as 11q23 abnormalities and FLT3-ITD mutations. In comparison, for the same AML samples, the average IC50 for JQ1 was 0.71 μM (range 0.02 - 3.3 μM) and for cytarabine was 3.5 μM (range 0.33 to >10 μM). Furthermore, PLX51107 treatment reduced the clonogenicity of primary AML cells. Following incubation of AML cells in 1 μM PLX51107, there was significantly decreased colony formation (p<0.05) in drug-free, cytokine-supplemented methylcellulose media.We next examined the efficacy of PLX51107 in vivo, utilizing luciferase labeled MV4-11 AML cells xenotransplanted into NOD / SCID / IL2rgnull (NSG) immunodeficient mice. Daily oral dosing with 20 mg/kg PLX51107 resulted in prolonged survival (median 47 days) compared to vehicle treated control animals (median 30 days, p< 0.001). Weekly measurement of bioluminescence showed decreased disease burden in PLX51107 treated mice. In addition, human peripheral blood CD45 / CD33 double positive cells were significantly decreased in treated animals. Histologic analysis conducted at day 16 showed decreased leukemic burden in the bone marrow of the PLX51107 treated animals. In addition, examination of tissues from moribund mice at time of euthanasia demonstrated fewer leukemia cells in the spleen, liver and bone marrow.Conclusions:Collectively, our results show pre-clinical activity of PLX51107 in AML, supporting further development of this compound in clinical trials for relapsed or refractory myeloid malignancies. We are currently working to define downstream targets of PLX51107 action and developing patient derived AML xenografts to further characterize the in vivo effects of PLX51107. DisclosuresWalker:Gilead Sciences: Research Funding. Bhatnagar:Karyopharm: Research Funding.

Copy-number analysis identified new prognostic marker in acute myeloid leukemia.

Recent advances in genomic technologies have revolutionized acute myeloid leukemia (AML) understanding by identifying potential novel actionable genomic alterations. Consequently, current risk stratification at diagnosis not only relies on cytogenetics, but also on the inclusion of several of these abnormalities. Despite this progress, AML remains a heterogeneous and complex malignancy with variable response to current therapy. Although copy-number alterations (CNAs) are accepted prognostic markers in cancers, large-scale genomic studies aiming at identifying specific prognostic CNA-based markers in AML are still lacking. Using 367 AML, we identified four recurrent CNA on chromosomes 11 and 21 that predicted outcome even after adjusting for standard prognostic risk factors and potentially delineated two new subclasses of AML with poor prognosis. ERG amplification, the most frequent CNA, was related to cytarabine resistance, a cornerstone drug of AML therapy. These findings were further validated in The Cancer Genome Atlas data. Our results demonstrate that specific CNA are of independent prognostic relevance, and provide new molecular information into the genomic basis of AML and cytarabine response. Finally, these CNA identified two potential novel risk groups of AML, which when confirmed prospectively, may improve the clinical risk stratification and potentially the AML outcome.

Valproic acid may exerts its cytotoxic effect through rassf1a expression induction in acute myeloid leukemia.

In acute myeloid leukemia (AML), despite the acceptance of standard intensive chemotherapy as an optimal induction regimen for all age groups, in the elderly patients, the best treatment should meet the challenge of multiple factors like age, comorbidities, and cytogenetics, making them ineligible for standard induction chemotherapy. Using the current low-intensity therapies like decitabine, azacitidine, and low-dose cytarabine as a single arm, outcomes for these patients remain poor. As a histone deacetylase inhibitor valproic acid (VPA) exhibit anticancer activity by triggering apoptosis, the mechanism of which is not yet completely clarified. To explore the possible connection between VPA treatment and the Hippo pathway as an apoptosis stimulating route, we also explore the expression of major components of this pathway and for the first time we postulate a relationship between VPA treatment and cell death induction through RASSF1A expression induction. Furthermore, we demonstrate that autophagy inhibition by chloroquine (CQ) significantly augmented the cytotoxic effect of VPA on AML cells, especially in those with unfavorable and normal karyotype. Regarding that VPA and CQ are well-tolerated drugs and our presumptive results of usefulness of VPA + CQ in three cytogenetic risk groups of AML, this combinatorial therapy could represent an attractive treatment option for older AML patients unfit for intensive therapy.

Association of acute myeloid leukemia's most immature phenotype with risk groups and outcomes.

The precise phenotype and biology of acute myeloid leukemia stem cells remain controversial, in part because the "gold standard" immunodeficient mouse engraftment assay fails in a significant fraction of patients and identifies multiple cell-types in others. We sought to analyze the clinical utility of a novel assay for putative leukemia stem cells in a large prospective cohort. The leukemic clone's most primitive hematopoietic cellular phenotype was prospectively identified in 109 newly-diagnosed acute myeloid leukemia patients, and analyzed against clinical risk groups and outcomes. Most (80/109) patients harbored CD34(+)CD38(-) leukemia cells. The CD34(+)CD38(-) leukemia cells in 47 of the 80 patients displayed intermediate aldehyde dehydrogenase expression, while normal CD34(+)CD38(-) hematopoietic stem cells expressed high levels of aldehyde dehydrogenase. In the other 33/80 patients, the CD34(+)CD38(-) leukemia cells exhibited high aldehyde dehydrogenase activity, and most (28/33, 85%) harbored poor-risk cytogenetics or FMS-like tyrosine kinase 3 internal tandem translocations. No CD34(+) leukemia cells could be detected in 28/109 patients, including 14/21 patients with nucleophosmin-1 mutations and 6/7 acute promyelocytic leukemia patients. The patients with CD34(+)CD38(-) leukemia cells with high aldehyde dehydrogenase activity manifested a significantly lower complete remission rate, as well as poorer event-free and overall survivals. The leukemic clone's most immature phenotype was heterogeneous with respect to CD34, CD38, and ALDH expression, but correlated with acute myeloid leukemia risk groups and outcomes. The strong clinical correlations suggest that the most immature phenotype detectable in the leukemia might serve as a biomarker for "clinically-relevant" leukemia stem cells. ClinicalTrials.gov: NCT01349972.

Identification of differentially methylated markers among cytogenetic risk groups of acute myeloid leukemia

Aberrant DNA methylation is known to occur in cancer, including hematological malignancies such as acute myeloid leukemia (AML). However, less is known about whether specific methylation profiles characterize specific subcategories of AML. We examined this issue by using comprehensive high-throughput array-based relative methylation analysis (CHARM) to compare methylation profiles among patients in different AML cytogenetic risk groups. We found distinct profiles in each group, with the high-risk group showing overall increased methylation compared with low- and mid-risk groups. The differentially methylated regions (DMRs) distinguishing cytogenetic risk groups of AML were enriched in the CpG island shores. Specific risk-group associated DMRs were located near genes previously known to play a role in AML or other malignancies, such as MN1, UHRF1, HOXB3, and HOXB4, as well as TRIM71, the function of which in cancer is not well characterized. These findings were verified by quantitative bisulfite pyrosequencing and by comparison with results available at the TCGA cancer genome browser. To explore the potential biological significance of the observed methylation changes, we correlated our findings with gene expression data available through the TCGA database. The results showed that decreased methylation at HOXB3 and HOXB4 was associated with increased gene expression of both HOXB genes specific to the mid-risk AML, while increased DNA methylation at DCC distinctive to the high-risk AML was associated with increased gene expression. Our results suggest that the differential impact of cytogenetic changes on AML prognosis may, in part, be mediated by changes in methylation.

Is it time to change conventional consolidation chemotherapy for acute myeloid leukemia in CR1?

Choosing the most appropriate postremission therapy (PRT) for a patient with acute myeloid leukemia (AML) in first complete remission remains a challenging task. Factors such as risk for disease relapse, nonrelapse mortality associated with different PRT approaches, donor availability, prospects for salvage should disease relapse, and patient preference all affect PRT choice. New genetic markers refine AML risk stratification and identify patients within the 'classical' risk groups who may benefit from transplant-based or chemotherapy-based PRT. The use of minimal residual disease in first remission to guide PRT choice and the application of novel, targeted therapies have the potential to alter PRT approaches across AML risk groups. The advent of alternative donor sources, use of reduced intensity regimens, and improved supportive care all affect the availability and safety of transplant-based PRT and challenge the relevance of the older legacy 'donor/no-donor' genetically randomized trials. Genetic risk assessment, monitoring of minimal residual disease in first remission, use of targeted agents, and the newer transplant strategies all have the potential to 'personalize' PRT choice in the AML patient. The clinical value of these novel interventions awaits validation in prospective, risk-adapted clinical trials.

An integrated approach of gene expression and DNA-methylation profiles of WNT signaling genes uncovers novel prognostic markers in acute myeloid leukemia.

BackgroundThe wingless-Int (WNT) pathway has an essential role in cell regulation of hematopoietic stem cells (HSC). For Acute Myeloid Leukemia (AML), the malignant counterpart of HSC, currently only a selective number of genes of the WNT pathway are analyzed by using either gene expression or DNA-methylation profiles for the identification of prognostic markers and potential candidate targets for drug therapy. It is known that mRNA expression is controlled by DNA-methylation and that specific patterns can infer the ability to differentiate biological differences, thus a combined analysis using all WNT annotated genes could provide more insight in the WNT signaling.ApproachWe created a computational approach that integrates gene expression and DNA promoter methylation profiles. The approach represents the continuous gene expression and promoter methylation profiles with nine discrete mutually exclusive scenarios. The scenario representation allows for a refinement of patient groups by a more powerful statistical analysis, and the construction of a co-expression network. We focused on 268 WNT annotated signaling genes that are derived from the molecular signature database.ResultsUsing the scenarios we identified seven prognostic markers for overall survival and event-free survival. Three genes are novel prognostic markers; two with favorable outcome (PSMD2, PPARD) and one with unfavorable outcome (XPNPEP). The remaining four genes (LEF1, SFRP2, RUNX1, and AXIN2) were previously identified but we could refine the patient groups. Three AML risk groups were further analyzed and the co-expression network showed that only the good risk group harbors frequent promoter hypermethylation and significantly correlated interactions with proteasome family members.ConclusionOur results provide novel insights in WNT signaling in AML, we discovered new and previously identified prognostic markers and a refinement of the patient groups.

Phase I Study of the Combination of Azacitidine (AZA) with MEC (Mitoxantrone, Etoposide and Cytarabine) Salvage Chemotherapy in Relapsed/Refractory Acute Myeloid Leukemia (AML): Early Results

AbstractAbstract 3616We recently reported that in addition to down-regulating DMNT1 mRNA and inducing DNA hypomethylation, AZA has the ability to reduce the mRNA and protein expression of M2, a subunit of Ribonucleotide Reductase (RNR) in AML cells [Blood 119(22): 5229–38, 2012]. RNR is an enzyme critical to DNA synthesis and repair that converts endogenous ribonucleotides to deoxynucleotides. Thus, we hypothesize that AZA-mediated downregulation of M2 results in decreased levels of endogenous triphosphate deoxynucleotides (dNTP). Thus, when AZA is combined with cytarabine (AraC), the backbone for several primary and salvage regimens in AML, the AraCTP/dNTP ratio would be expected to increase in favor of AraCTP DNA incorporation, thereby overcoming AraC resistance. Based on this rationale, we conducted a phase I trial of escalating doses of AZA (q24 h) on days 1–8 combined with unchanged mitoxantrone (6 mg/m2 q24h), etoposide (80 mg/m2 q24 h), and AraC (1 g/m2 q24 h) (MEC) salvage chemotherapy on days 3–8 in patients (pts) with relapsed or refractory AML. AZA doses were escalated (30, 50, or 75 mg/m2 q24h) with each dose level (DL) at which 3–6 pts were treated. Thirteen pts (39% male) with median age 52 (range 36–64) were enrolled. Five pts had primary refractory disease and 8 had relapsed AML (4 with 1st complete remission duration <12 months). Four pts had prior allogeneic stem cell transplant, and 4 pts had secondary AML (2 treatment related, 2 prior MDS). According to European LeukemiaNet AML Risk Groups, 2, 4, 4, and 3 pts had favorable, intermediate-I, intermediate-II, and adverse disease, respectively. One pt died on day 18 of sepsis, was not evaluable for response, and was replaced. Three pts are currently being treated on the last planned DL. In addition to anticipated myelosuppression, grade 3/4 non-hematologic adverse events at least possibly related to AZA included infections (n=10), diarrhea (n=5), increased bilirubin (n=1), increased AST/ALT (n=1), and vomiting (n=1). One pt (relapsed within 100 days of allogeneic transplant) did not recover platelets until day 58 after achieving morphologic complete remission (CR). Maximally tolerated dose has not been reached. CR and CR with incomplete count recovery (CRi) were achieved in 6/9 (67%; 5 CR and 1 CRi) pts who completed treatment. In responding pts, median time to ANC>500 was 35 days (range 23–45) and median time to platelets>50K was 35 days (range 22–45). Pharmacokinetics (PK) of AZA showed post-infusion biexponential decays with fast and slow plasma half-lives in the range of 10 minutes and 2 hours, respectively. Significant pharmacodynamic reduction in M2 mRNA and/or protein levels was observed in 3 pts analyzed, thus far. In conclusion, the combination of AZA with MEC salvage chemotherapy in advanced AML is well tolerated, not significantly different than MEC alone, and without prolongation in anticipated cytopenias. Early response rate and evidence of reduction in M2 as a proposed target of AZA are encouraging considering that most of the pts had high risk clinical and cytogenetic features. Updated clinical, PK and pharmacodynamic data will be presented. Supported by the National Cancer Institute of the National Institutes of Health Grant Number U01CA076576. Disclosures:Off Label Use: Azacitidine in AML.

Identification of Differential Methylation Markers Among Cytogenetic Risk Groups of Acute Myeloid Leukemia

Abstract 1464Aberrant DNA methylation has been shown as an important mechanism in the progression from myelodysplasia (MDS) to acute myeloid leukemia (AML), leading to use of the demethylating agents, 5-azacitidine and decitabine, for treatment of both disorders. While these drugs produce responses, the ability to distinguish potential responders from potential non-responders remains limited. The purpose of this work was to bring new technology to study this problem. Recent studies demonstrated that promoter DNA methylation is not randomly distributed in AML blasts but rather is highly organized and associated with biologically distinct AML subtypes. Because cytogenetics at presentation is the most important prognostic factors in predicting response to therapy, remission duration and overall survival in AML, we aimed to identify differentially methylated genomic regions (DMR) in cytogenetically defined risk groups of AML. Published literature suggested that the new comprehensive high-throughput array-based relative methylation analysis (CHARM) has the highest sensitivity and specificity among all the array-based genome profiling methods and should be the most accurate means to identify methylation markers. It is a customized NimbleGen HD2 array of tiled 50mer-probes typically separated by 30–40 bases covering approximately 4.6 million CpG sites across the genome. This assay is highly quantitative for approximately 100,000 independent CpG sites.We performed methylation profiling with CHARM on 15 age-matched patients divided into 3 groups (n=5 in each): (1) high-risk AML, defined as patients with a complex or monosomal karyotype, inv(3)/t(3;3), t(6;9), or FLT3-ITD; (2) intermediate-risk AML, defined as patients with normal karyotype, trisomy 8, t(9;11), or others; (3) low-risk AML, defined as patients with t(8;21) or inv(16). Five age-matched normal individuals served as control. Randomly fractionated DNA was divided into two equal portions with and without McrBC treatment, which cleaves methylated DNA, then size-fractionated, purified and subject to whole-genome amplification prior to hybridization with the CHARM array. Data analysis was performed with R and Bioconductor. AML patients showed a very strong hypermethylation signature as compared with the normal control blood. A unique set of DMRs was identified which distinguishes between any two risk groups. The number of DMRs and those with p -values < 0.01 are shown in Table 1. There were fewer methylation discriminators between low- and mid-risk groups than between high-risk and the other risk groups. Figure 1 shows the comparison between high-risk group and other risk groups highlighting the 12 top DMRs with lowest p -values. In silico validation of the DMRs identified by CHARM verified previously reported aberrant hypermethylation of tumor suppressor genes like p15CDKN2B, discriminating normal from AML patients, CDH1 promoter hypermethylation in high-risk AML compared with mid-risk AML, and HOXB3 hypomethylation in mid-risk AML compared with both low-risk and high-risk AML, etc. Technical validation using quantitative bisulfite pyrosequencing on 8 genes, including DCC, DUOX2, NEFL, and PITX1, demonstrated an 87.5% concordant rate with CHARM. Testing of additional AML samples with validated markers are underway to confirm the top DMRs identified, which may serve as useful biomarkers to predict response to azacitidine and decitabine.Table 1Number of DMRsNumber with p-value < 0.01Normal blood VS at risk3768311Low-risk VS mid-risk156530Low-risk VS high-risk247573Mid-risk VS high-risk2651107 [Display omitted] Disclosures:No relevant conflicts of interest to declare.