Integrated genetic, epigenetic, and immune landscape of TP53 mutant AML and higher risk MDS treated with azacitidine.

Background: TP53 mutations are associated with an adverse prognosis in acute myeloid leukemia (AML) and higher-risk myelodysplastic syndromes (HR-MDS). However, the integrated genetic, epigenetic, and immunologic landscape of TP53-mutated AML/HR-MDS is not well defined. Objectives: To define the genetic, epigenetic, and immunologic landscape of TP53-mutant and TP53 wild-type AML and HR-MDS patients. Design: Post hoc analysis of TP53-mutant and TP53 wild-type patients treated on the randomized FUSION trial with azacitidine ± the anti-PD-L1 antibody durvalumab. Methods: We performed extensive molecular, epigenetic, and immunologic assays on a well-annotated clinical trial dataset of 61 patients with TP53-mutated disease (37 AML, 24 MDS) and 144 TP53 wild-type (89 AML, 55 MDS) patients, all of whom received azacitidine-based therapy. A 38 gene-targeted myeloid mutation analysis from screening bone marrow (BM) was performed. DNA methylation arrays, immunophenotyping and immune checkpoint expression by flow cytometry, and gene expression profiles by bulk RNA sequencing were assessed at baseline and serially during the trial. Results: Global DNA methylation from peripheral blood was independent of TP53 mutation and allelic status. AZA therapy led to a statistically significant decrease in global DNA methylation scores independent of TP53 mutation status. In BM from TP53-mutant patients, we found both a higher T-cell population and upregulation of inhibitory immune checkpoint proteins such as PD-L1 compared to TP53 wild-type. RNA sequencing analyses revealed higher expression of the myeloid immune checkpoint gene LILRB3 in TP53-mutant samples suggesting a novel therapeutic target. Conclusion: This integrated analysis of the genetic, epigenetic, and immunophenotypic landscape of TP53 mutant AML/HR-MDS suggests that differences in the immune landscape resulting in an immunosuppressive microenvironment rather than epigenetic differences contribute to the poor prognosis of TP53-mutant AML/HR-MDS with mono- or multihit TP53 mutation status. Trial registration: FUSION trial (NCT02775903).

Survival outcomes in patients with acute myeloid leukaemia who received subsequent therapy for relapse in QUAZAR AML-001.

In the phase 3 QUAZAR AML-001 trial (NCT01757535) of patients with acute myeloid leukaemia (AML) in remission following intensive chemotherapy (IC) and ineligible for haematopoietic stem cell transplant (HSCT), oral azacitidine (Oral-AZA) maintenance significantly prolonged overall survival (OS) versus placebo. The impact of subsequent treatment following maintenance has not been evaluated. In this post hoc analysis, OS was estimated for patients who received subsequent AML therapy, and by regimen received (IC or lower-intensity therapy). First subsequent therapy (FST) was administered after treatment discontinuation in 134/238 Oral-AZA and 173/234 placebo patients. OS from randomization in patients who received FST after Oral-AZA versus placebo was 17.8 versus 12.9 months (HR: 0.82 [95% CI: 0.64-1.04], median follow-up: 56.7 months); OS from FST was similar between arms. Among patients who received injectable hypomethylating agents as FST, median OS was 8.2 versus 4.9 months in the Oral-AZA versus placebo groups (HR: 0.66 [95% CI: 0.41-1.06]). Forty-eight patients (16/238 Oral-AZA, 32/234 placebo) received HSCT following treatment discontinuation, including six Oral-AZA patients still in first remission; Oral-AZA OS benefit persisted when censoring these patients. Oral-AZA maintenance can prolong AML remission duration without negatively impacting survival outcomes after salvage therapies.

Quantification of azacitidine incorporation into human DNA/RNA by accelerator mass spectrometry as direct measure of target engagement.

We report an accelerator mass spectrometry (AMS) assay to quantify azacitidine (Aza) incorporation into DNA and RNA from human acute myeloid leukemia (AML) cells, mouse bone marrow (BM) and peripheral blood mononuclear cells (PBMCs). Aza, a cytidine nucleoside analogue, is a disease modifying pharmacological agent used for treatment of myelodysplastic syndromes (MDS) and AML. Our assay was able to directly quantify the complex of Aza incorporated into DNA/RNA, via isolation of DNA/RNA from matrix (i.e., cancer cells, BM and PBMC) and subsequent measurement of total radioactivity (i.e., 14C-Aza) by using AMS. The sensitivity of the method was able to quantify as little as a single Aza molecule incorporated into DNA with approximately 2 × 107 nucleotides from PBMCs. An in vivo mouse model was used for establishing the lower limits of quantification (LLOQs) for Aza incorporated into DNA/RNA in mouse PBMCs (∼ 3.7 × 105) and BM (∼27.8 mg) collected 24 h post-dose after total exposure of 18 nCi/mouse (Aza 1 mg/kg). The LLOQs for PBMC analysis were 2.5 picogram equivalents per microgram (pgEq/µg) DNA and 0.22 pgEq/µg RNA, and for BM analysis were 1.7 pgEq/µg DNA and 0.22 pgEq/µg RNA. A linear relationship (i.e., ∼10-fold) was established of radioactive dose from 14C-Aza 17 nCi/mouse to 188 nCi/mouse and AMS response (i.e., 14C/12C ratio ranging from 2.45 × 10-11 to 2.50 × 10-10), as Aza was incorporated into DNA in mouse BM. The current method enables the direct measurement of Aza incorporation into DNA and RNA from patient PBMCs and BM to provide dosing optimization, and to assess target engagement with as little as ∼5 mL whole blood and ∼3 mL of BM from patients.