Genetic and Epigenetic Changes at Secondary Resistance after Continued Treatment in the Randomized Phase II Study of All-Trans Retinoic Acid (ATRA) and/or Valproic Acid (VPA) Added to Decitabine (DAC) in Newly Diagnosed Elderly AML Patients (DECIDER Trial)

Introduction Hypomethylating agent (HMA)-based therapy is standard of care of patients (pts) with acute myeloid leukemia (AML) ineligible for intensive chemotherapy. However, hematologic remissions occur in only 10-30% (HMA monotherapy) or 40-70% (HMA + Venetoclax) of pts, and initially responsive disease will eventually develop secondary resistance. The mechanisms underlying secondary HMA resistance are not well understood (reviewed by Stomper et al. Leukemia 35:1873, 2021). A better knowledge could inform strategies for early detection and (potential) treatment adaptions. Here, we studied secondary resistance in pts who received decitabine (DAC) combination therapy in the randomized ph II DECIDER trial (NCT00867672, Lübbert et al. J Clin Oncol 3:257, 2020). Of pts on treatment for ≥6 months, AML blasts collected at treatment start and at resistance were analyzed by exome sequencing (WES) and DNA methylation profiling.

Patients and Methods Key inclusion criteria (DECIDER trial): newly diagnosed AML pts >60yr (non-M3) unfit for induction, ECOG performance status 0-2. Treatment (2x2 factorial design): DAC 20 mg/m² day (d) 1-5 (arms A/B/C/D), valproic acid (VPA) p.o. from d6 (arms B/D), all-trans retinoic acid (ATRA) p.o. d6-28 (arms C/D) of each 28-d cycle. Pts for the present study had to fulfill the following criteria: (1) achieved complete remission (CR), CR without regeneration of platelets and neutrophils (CRi), partial remission (PR), antileukemic effect (ALE) or stable disease (SD), (2) progressive disease (PD) or relapse after ≥6 months treatment, (3) available samples from both treatment start and resistance. WES was performed using the All Exon v7 Kit (Agilent) and a NovaSeq 6000 (Illumina; paired-end 50bp; DKFZ Heidelberg) and analyzed as previously described (Hoefflin et al. Cancers 13:1151, 2021). DNA methylation profiles were assessed by 450K arrays (Illumina).

Results In the DECIDER trial, 200 pts received DAC alone or in combination with VPA or ATRA or VPA and ATRA. Thirty-five (18%) pts achieved a CR, CRi, or PR; further 84 (42%) pts an ALE or SD. Of these 119 pts, we identified 14 pts with PD or relapse after ≥6 months of treatment and samples available from both treatment start and time of PD or relapse. Of these, 7 had an abnormal karyotype, including 3 with a complex karyotype. Two pts had an NPM1 mutation, none a FLT3 mutation. ELN 2010 genetic groups were as follows: favorable, n=2; intermediate-I/II, n=7; adverse, n=4. Per study protocol, all pts received DAC, 6 pts additionally received ATRA and 4 pts VPA. Six pts achieved a CR, CRi or PR as best response, the remaining an ALE or SD. Samples at resistance were collected a median of 10.1 months after treatment start. This corresponds to a median of 9 treatment cycles applied, including 6 pts who received ≥12 cycles. We compared the WES data at treatment start and resistance, and first focused on predefined cancer genes (OncoKB). All but 3 patients had gained or lost ≥1 mutation in a cancer gene (median: 3 mutations). Focusing on genes typically mutated in myeloid neoplasms, 2 pts acquired an IDH1 p.R132 mutation; 1 pt each gained an ETV6, FLT3 p.D835Y, JAK2 p.V617F or KRAS p.G12A mutation; 1 pt each lost a DNMT3A p.F732S, NRAS p.G12D or TP53 mutation. All pts additionally gained or lost mutations in genes not defined as cancer genes, including genes with mutations in ≥2 patients and of potential biological relevance, e.g. PPM1E (regulating AMPK-mTOR signaling) or SALL3 (decreasing CpG methylation via DNMT3A). However, no clear pattern in the changes of the WES profiles towards resistance were identified. With regard to the DNA methylation profiles, genome-wide beta values indicated a relative hypomethylation at secondary resistance.

Conclusions AML with secondary resistance after continued HMA treatment commonly exhibits changes in the mutation profile and hence clonal architecture, including gains and losses of mutations in genes typically altered in AML. However, no distinct patterns in the changes of the mutation profiles towards resistance were identified. Genetic changes may be inherent to the disease evolution under treatment rather than specific for HMA therapy. In an attempt to decipher non-genetically mediated resistance mechanisms, we studied DNA methylation profiles and observed relative hypomethylation at resistance. Further studies focusing on epigenetic alterations towards secondary HMA resistance are warranted.

Niemöller:Cytena: Current Employment. Thol:BMS: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Heuser:Abbvie: Consultancy, Honoraria, Research Funding; Eurocept: Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Agios: Consultancy, Research Funding; BMS: Consultancy; Daiichi Sankyo: Consultancy, Research Funding; Glycostem: Consultancy, Research Funding; Kura Oncology: Consultancy; Pfizer: Consultancy, Research Funding; PinotBio: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Tolremo: Consultancy; Astellas: Research Funding; Bayer Pharma AG: Research Funding; BergenBio: Research Funding; Loxo Oncology: Research Funding. Bug:Gilead: Consultancy, Honoraria; Novartis: Consultancy; Celgene /BMS: Consultancy, Honoraria; Jazz: Honoraria; Pfizer: Consultancy. Crysandt:Pfizer: Honoraria; Novartis: Honoraria; Astra Zeneca: Honoraria. Dohner:Abbvie: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Honoraria, Research Funding; AstraZeneca: Honoraria; Berlin-Chemie: Consultancy, Honoraria; Brystol Myers Squibb: Consultancy, Honoraria, Research Funding; Celgene: Honoraria; Daiichi Sankyo Co, LTD: Consultancy, Honoraria; Gilead Sciences Inc: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Jazz: Consultancy, Honoraria, Research Funding; Kronos Bio, Inc: Research Funding; Novartis AG: Consultancy, Honoraria, Research Funding; Pfizer Inc: Research Funding; Servier: Consultancy, Honoraria; Syndax Pharmaceuticals Inc: Consultancy, Honoraria. Becker:Pierre Fabre: Honoraria; Servier: Honoraria; BMS: Honoraria; Novartis: Honoraria; Roche: Honoraria. Luebbert:AbbVie: Honoraria; Astex: Honoraria; Janssen: Research Funding; Otsuka: Consultancy; Syros: Consultancy; Cheplapharm: Other: study drug.

decitabine, valproic acid, all-trans retinoic acid