Akute myeloische Leukämie III
V444 Aktivität von Decitabine (DAC) in Kombination mit all-trans-Retinsäure (ATRA) bei oligoblastischer AML: Ergebnisse einer randomisisert 2x2 Phase II Studie (DECIDER)
Christoph Rummelt, Freiburg, D
Introduction: In elderly AML patients (pts) within the DECIDER trial, DAC+ATRA combination resulted in an improved response rate and survival compared to DAC alone (Lübbert et al., J. Clin. Oncol. 2020). We hypothesized that the outcome of pts with oligoblastic AML may also be improved by this combination. Therefore, pts with 20-30% bone marrow blasts were analyzed for clinical outcome in this subgroup analysis.
Patients and methods: Key inclusion criteria: newly diagnosed pts >60 years (yr), unfit for induction with non-M3 AML, ECOG performance status 0-2. Treatment: DAC 20 mg/m2 day 1-5 (treatment arms A/B/C/D), ATRA p.o. day 6-28 (arms C/D), VPA p.o. continuously from day 6 (arms B/D) of each 28-day course. Key endpoints: objective response rate (ORR): CR/CRi/PR, overall (OS) and event-free survival (EFS). 200 pts were randomized and treated. ATRA was investigated by comparing arms C+D vs arms A+B, ORR was analyzed with logistic regression estimating odds ratios (OR), OS/EFS with Cox regression estimating hazard ratios (HR), each with 95% confidence intervals (CI), and presented with descriptive two-sided p values of the tests of no treatment effect.
Results: Bone marrow blasts were 20-30% defining oligoblastic AML in 56/200 pts of the DECIDER cohort. The number of pts in the arms were: 13 in arm A, 21 in arm B, 9 in arm C, 13 in arm D. Baseline pt characteristics: male 77%, median age: 75 yr, median WBC: 3400/μl, adverse genetics (ELN 2010) present in 25%, ECOG 2 in 13%, HCT-CI ≥ 3 in 48%, AHD in 68%, tAML in 11%. A median of 5 DAC courses were administered (per arm: 2/5/11/4, respectively). Six pts attained a CR, 7 pts a CRi, and 1 pt a PR, resulting in an ORR of 25% (per arm: 7.7/28.6/33.3/30.8%, respectively). Effect on ORR of ATRA vs no ATRA (31.8 vs 20.6%): OR 1.85, CI [0.54,6.37], p=0.33. With 40 deaths, median OS was 9.5 mths (per arm: 7.6/8.9/37.2/11.2 mths, respectively). Effect on OS of ATRA vs. no ATRA (12.5 vs 7.6 mths median OS, Fig. 1): HR 0.47, CI [0.24,0.94], p=0.032. A comparable benefit on EFS of ATRA vs. no ATRA was observed.
Conclusion: In elderly pts with oligoblastic AML ineligible for induction chemotherapy, the addition of ATRA to DAC resulted in a clinically meaningful survival benefit. The combination of an HMA with a retinoid may therefore also be active in MDS pts with excess of blasts. A novel potent RARα receptor agonist is under clinical development combined with an HMA in AML/MDS (De Botton S. et al., ASH Abs. 112, 2020).
V481 Gesundheitsbezogene Lebensqualität (Hrqol) mit Oraler Azacitidin-Formulierung bei Patienten mit Akuter Myeloischer Leukämie (AML) in 1. Remission nach Intensiver Chemotherapie (IC): Ergebnisse der Phase 3 QUAZAR AML-001 Erhaltungstherapie-Studie
Uwe Martens, Heilbronn, D
Introduction: AML maintenance treatment (Tx) should decrease relapse risk and prolong survival without impairing health-related quality of life (HRQoL) or worsening fatigue. In the phase 3 randomized, placebo (PBO)-controlled QUAZAR AML-001 trial, Oral-AZA significantly improved overall (OS) and relapse-free survival (RFS) in patients (pts) with AML in first remission after IC.
Methods: Pts were aged ≥55 yrs with intermediate- or poor-risk cytogenetics and ECOG PS ≤3; achieved CR/CRi after IC (induction ± consolidation); and were not transplant eligible. Pts were randomized 1:1 to Oral-AZA 300mg or PBO QD ×14d/28d cycle. HRQoL was assessed by FACIT-Fatigue Scale and EQ-5D-3L health utility index (HUI), completed on d1 of each cycle and at end of Tx (EOT). HRQoL endpoints included mean score changes from baseline (BL) and clinically meaningful changes, defined as a ≥3-point change from BL on the FACIT-Fatigue, and +0.08 point (improvement) or -0.10 point (worsening) on the EQ-5D-3L HUI. Evaluable pts had BL and ≥1 post-BL assessments. Longitudinal mixed-effect model for repeated measures (MMRM) analysis assessed noninferiority of Oral-AZA to PBO.
Results: 225/238 pts (95%) in the Oral-AZA arm and 219/234 (94%) in the PBO arm were HRQoL- evaluable. Most pts (61%) were age 65-74 yrs. Median number of Oral-AZA cycles was 12 and of PBO was 7. At BL, pts in both arms had low levels of fatigue and generally good HRQoL, with FACIT-Fatigue and EQ-5D-3L HUI scores comparable to general populations. Compliance rates were >95% at BL and >85% at all post-BL visits except EOT.
No clinically meaningful differences occurred between Tx arms in mean changes from BL FACIT-Fatigue scores at any post-BL visit. Significant differences in EQ-5D-3L HUI scores between arms at cycles 22-23 were not clinically meaningful and likely due to chance (no adjustment was made for multiplicity). MMRM analysis confirmed noninferior HRQoL with Oral-AZA vs PBO. There was no significant difference between arms in meaningful deterioration in FACIT-Fatigue score except at cycle 29 (likely due to chance), or in EQ-5D-3L HUI score at any visit, and no differences between Oral-AZA and PBO in median time to deterioration in FACIT-Fatigue scores (41 vs 44 wks; P=0.70) or EQ-5D-3L HUI (200 vs 164 weeks; P =0.63).
Conclusion: Favorable HRQoL and low levels of fatigue at BL were preserved during Oral-AZA maintenance Tx. Oral-AZA significantly improved OS and RFS while maintaining HRQoL similar to PBO.
V539 Entschlüsselung molekularer Mechanismen, die Sensitivität und Resistenz gegen Menin-MLL1 Inhibition in NPM1 mutierter AML kontrollieren
Jonas Schönfeld, Mainz, D
Introduction: NPM1 mutant (NPM1mut) acute myeloid leukemias (AML) are dependent on the interaction between the oncogenic co-factor Menin and the histone methyltransferase MLL1. Pharmacologic inhibition of this interaction showed dramatic anti-leukemic activity in preclinical NPM1mut AML models and a novel small-molecule Menin-MLL1 inhibitor (Men-i) showed promising efficacy in an early clinical trial. However, no single drug alone is expected to induce long-term remission in AML. Developing synergistic drug combinations that target more than one vulnerability of AML cells will be key to overcome drug resistance, toxicity, and relapse from heterogenous AML subclones.
Methods: Here, we generated human NPM1mut OCI-AML3 cells with non-genetic resistance against Men- i and performed detailed phenotypic characterization of these cells. We integrated our findings with a high-resolution CRISPR/Cas9 domain scan of MEN1 (Menin) and MLL1 as well as mass spectrometry (MS).
Results: By exposing AML cells to increasing concentrations of the Men-i MI503, we developed three cell clones that demonstrated profound resistance against two novel Men-is compared to parental and vehicle-treated long-term cultured cells. Sanger sequencing confirmed that regions encoding the interaction domains of Menin and MLL1 were not mutated. Resistant cells exhibited upregulation of the monocytic differentiation marker CD11b. Gene expression changes associated with drug resistance were assessed using RNA sequencing paying particular attention to Menin-MLL1 target genes, including
MEIS1 and PBX3. These genes are important leukemic drivers and uniformly downregulated after Men-i in sensitive NPM1mut AML cells. While we found MEIS1 expression to be irreversibly silenced in resistant cells, the expression level of PBX3 was similar to the parental cells. Also, we employed a CRISPR/Cas9 domain scan with a library of 2,041 sgRNAs that targeted MEN1 and MLL1 on average every eight nucleotides and identified several protein domains that represent strong dependencies in NPM1mut OCI- AML3 cells. We mapped these domains to binding regions of 13 interaction partners identified by literature annotation and MS. Three co-factors of the Menin-MLL1 complex were found to be required for NPM1mut cell survival.
Conclusion: This study provides inside into the molecular mechanisms that control sensitivity and resistance to Men-i and will improve efforts to develop novel combinatorial treatment approaches against AML.
Estelle Erkner, Tübingen, D
Chromosomal translocation t(4;11) initiates malignant transformation of hematopoietic stem and progenitor cells (HSPCs) leading to the development of myeloid and lymphoblastic leukemia which are associated with a poor prognosis. Cholesterol metabolism is strongly up-regulated in human cancers to satisfy the demands of increased membrane biogenesis. By using a CRISPR/Cas9 generated MLL-AF4 translocation model, we could identify the sterol regulatory element-binding protein 2 (SREBP2) as key transcription factor being highly upregulated in our model but also in publicly available patient data. Recently, a new inhibitor blocking RAR-related orphan receptor gamma (RORɣ), the master regulator of sterol and fatty acid synthesis, has been synthesized allowing us to unravel the impact of dysregulated cholesterol metabolism in MLL leukemogenesis leading to new therapeutic targets.
CD34+ HSPCs were isolated from human cord blood and t(4;11) was induced using CRISPR/Cas9. Publicly available patient data sets were screened for SREBP2 expression in comparison to healthy donors (gent2 database) and the influence on overall survival (bloodspot.eu) was evaluated. We measured cholesterol target genes in MLL-AF4 cells via RNA sequencing (RNAseq), RT-qPCR, western blot and flow cytometry compared to non-translocated HSPCs. RORɣ-selective antagonist XY018
was used in our model and (non-) MLLr cell lines, respectively, to study the effect on cell proliferation, viability, cell cycle and expression of cholesterol target genes.
RNAseq of our MLL-AF4 model showed cholesterol target genes highly overexpressed in MLL-AF4 leukemia compared to healthy controls which could be confirmed by RT-qPCR and on protein levels. Moreover, SREBP2 was significantly upregulated in patients, which correlated with poor survival. Treatment with the selective inhibitor XY018 resulted in a dose-dependent reduced proliferation and viability, increased apoptosis, induction of cell cycle arrest and downregulation of metabolic activity in the cholesterol pathway. Furthermore, cells with t(4;11) showed an increased inhibitor response compared to non-translocated cells demonstrating the specificity towards MLL-rearranged (MLLr) leukemia.
In summary, our findings indicate the important role of cholesterol metabolism and related target genes in MLL-AF4 leukemia. We uncovered RORɣ as metabolic key regulator and promising new target to improve the poor outcome of MLL-rearranged leukemia.
V592 Risikostratifizierung der akuten myeloischen Leukämie auf Grundlage einer kalkulierten Karyotypisierung durch Next Generation Sequencing
Elisabeth Mack, Marburg, D
Introduction: Acute myeloid leukemia (AML) is heterogenous on the molecular level and prognosis varies substantially between subgroups. Rapid comprehensive genetic characterization of AML is essential for prompt initiation of risk-adapted therapy. We have recently developed an integrated Next Generation Sequencing (NGS) approach that identifies all currently known relevant translocations and sequence variants in AML along with copy number variations (CNVs) of large chromosomal regions within five days. Here, we validate our calculated karyotyping approach comprising fusion- and CNV-analysis in a larger cohort of patients in a blinded fashion.
Methods: Diagnostic bone marrow or peripheral blood samples of 48 AML patients from the Study Alliance Leukemia biobank were analysed without knowledge on cytogenetics or mutational status. Targeted sequencing of fusion genes was performed using the FusionPlex Myeloid Panel (Archer Dx). For the detection of CNVs, whole genome libraries were sequenced to a low depth of 1-3 M reads on an Illumina MiSeq instrument. Sequencing data were analysed using the Archer Analysis pipeline and a proprietary R-script to calculate numerical karyotypes. A researcher not involved in NGS karyotype construction compared the results to cytogenetic karyotypes.
Results: A complete calculated karyotype was obtained for all samples, including 10 cases for which cytogenetics had failed. In 31 of the remaining 38 patients (82%), the calculated karyotype corresponded exactly to reference results, which included both normal and aberrant karyotypes. NGS karyotyping missed CNVs that were present only in subclones comprising < 20% of analysed metaphases in four patients and a non-recurrent translocation in one patient. Three samples showed additional CNVs. All patients were classified in the same ELN risk group based on calculated karyotyping or cytogenetics.
Conclusions: Our results confirm that NGS karyotyping enables a higher diagnostic yield than conventional techniques. Calculated karyotypes correspond to cytogenetic karyotypes in 80% of cases with discrepancies resulting from limited sensitivity of CNV detection at shallow sequencing depth and a non-universal approach to fusion detection. Moreover, our method is highly compatible with the current ELN risk stratification system. Thus, calculated karyotyping by low coverage whole genome sequencing combined with panel-based fusion screening has the potential to replace classical cytogenetics.