Acute Myeloid Leukemia In Remission Research Articles

Peripheral blood stem cell versus bone marrow graft for patients ≥60 years undergoing reduced intensity conditioning haploidentical transplantation for acute myeloid leukemia in complete remission: An analysis of the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation.

In the context of T-cell replete haploidentical stem cell transplantation (Haplo-SCT) using post-transplantation cyclophosphamide (PT-Cy), it is still unknown whether peripheral blood (PB) or bone marrow (BM) is the best graft source. While PB is associated with a higher incidence of graft-versus-host disease (GVHD), it may induce a stronger graft-versus-leukemia effect compared to BM, notably in acute myeloid leukemia (AML). From the EBMT registry database, we compared T-cell replete PB (n = 595) versus BM (n = 209) grafts in a large cohort of 804 patients over the age of 60 years who underwent Haplo-SCT with PT-Cy for an AML in first or second complete remission. The risk of acute GVHD was significantly higher in the PB group (Grade II-IV: HR = 1.67, 95% CI [1.10-2.54], p = 0.01; Grade III-IV: HR = 2.29, 95% CI [1.16-4.54], p = 0.02). No significant difference was observed in chronic GVHD or non-relapse mortality. In the PB group, the risk of relapse was significantly lower in the PB group (HR = 0.65, 95% CI [0.45-0.94], p = 0.02) and leukemia-free survival was significantly better (HR = 0.76, 95% CI [0.59-0.99], p = 0.04), with a trend toward better overall survival (HR = 0.78, 95% CI [0.60-1.01], p = 0.06). We conclude that in the specific context of Haplo-SCT with PT-Cy, PB grafts represent a valid option to decrease the risk of relapse and improve outcome of older AML patients who usually do not benefit from conditioning intensification.

Acute myeloid leukemia with paraneoplastic pemphigus successfully treated with a personalized antileukemic and immunosuppressive strategy.

Bullous pemphigoid (BP) is a rare blistering disease often considered a primary sign of a paraneoplastic syndrome. Retrospective studies have established its link with hematological malignancies, particularly lymphoproliferative disorders. Here, we present what we believe to be the inaugural case of successful simultaneous management of BP and de novo acute myeloid leukemia (AML) in a 28-year-old male patient. Given the rarity and severity of both conditions, our treatment strategy aimed to maximize efficacy by combining immunosuppressive therapy (initially plasmapheresis with high-dose corticosteroids, followed by anti-CD20 monoclonal antibody and intravenous immunoglobulins 2 g/m2) with lymphodepleting antileukemic chemotherapy utilizing Fludarabine (FLAG-IDA induction regimen). Following diagnosis, considering the patient's youth and the concurrent presence of two rare and potentially life-threatening diseases, we opted for an aggressive treatment. Upon achieving complete morphological remission of AML with measurable residual disease (MRD) negativity, despite incomplete resolution of BP, we proceeded with high-dose cytarabine consolidation followed by peripheral stem cell harvest and autologous stem cell transplantation (ASCT). Our conditioning regimen for ASCT involved Bu-Cy with the addition of anti-thymocyte globulins. At day + 100 post-ASCT, bone marrow evaluation confirmed morphological remission and MRD negativity. Meanwhile, BP had completely resolved with normalization of BP180 antibody levels.

Higher TIGIT+ γδ TCM cells may predict poor prognosis in younger adult patients with non-acute promyelocytic AML.

γδ T cells recognize and exert cytotoxicity against tumor cells. They are also considered potential immune cells for immunotherapy. Our previous study revealed that the altered expression of immune checkpoint T-cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) on γδ T cells may result in immunosuppression and is possibly associated with a poor overall survival in acute myeloid leukemia (AML). However, whether γδ T-cell memory subsets are predominantly involved and whether they have a relationship with clinical outcomes in patients with AML under the age of 65 remain unclear. In this study, we developed a multicolor flow cytometry-based assay to monitor the frequency and distribution of γδ T-cell subsets, including central memory γδ T cells (TCM γδ), effector memory γδ T cells (TEM γδ), and TEM expressing CD45RA (TEMRA γδ), in peripheral blood from 30 young (≤65 years old) patients with newly diagnosed non-acute promyelocytic leukemia (also known as M3) AML (AMLy-DN), 14 young patients with AML in complete remission (AMLy-CR), and 30 healthy individuals (HIs). Compared with HIs, patients with AMLy-DN exhibited a significantly higher differentiation of γδ T cells, which was characterized by decreased TCM γδ cells and increased TEMRA γδ cells. A generally higher TIGIT expression was observed in γδ T cells and relative subsets in patients with AMLy-DN, which was partially recovered in patients with AMLy-CR. Furthermore, 17 paired bone marrow from patients with AMLy-DN contained higher percentages of γδ and TIGIT+ γδ T cells and a lower percentage of TCM γδ T cells. Multivariate logistic regression analyses revealed the association of high percentage of TIGIT+ TCM γδ T cells with an increased risk of poor induction chemotherapy response. In this study, we investigated the distribution of γδ T cells and their memory subsets in patients with non-M3 AML and suggested TIGIT+ TCM γδ T cells as potential predictive markers of induction chemotherapy response.

Abstract SY45-02: Engineering strategies based on receptors derived from gdT cells

Abstract SY45-02: Engineering strategies based on receptors derived from gdT cells

Transient Spontaneous Remission of Acute Myeloid Leukemia with Mutated RUNX1: A Rare Report

Transient Spontaneous Remission of Acute Myeloid Leukemia with Mutated RUNX1: A Rare Report

High Expression of ENO1 and Low Levels of Circulating Anti-ENO1 Autoantibodies in Patients with Myelodysplastic Neoplasms and Acute Myeloid Leukaemia.

In solid tumours, high expression of the glycolytic enzyme, α-enolase (ENO1), predicts for poor patient overall survival (OS), and circulating autoantibodies to ENO1 correlate positively with diagnosis and negatively with advanced disease. Although ENO1 is one of the most highly expressed genes in acute myeloid leukaemia (AML), its potential role as a biomarker in AML or its precursor, myelodysplastic neoplasms (MDS), has not been investigated. A meta-analysis of nine AML online datasets (n = 1419 patients) revealed that high ENO1 expression predicts for poor OS (HR = 1.22, 95% CI: 1.10-1.34, p < 0.001). Additionally, when compared to AML in remission (n = 5), ENO1 protein detected by immunohistochemistry was significantly higher at diagnosis in bone marrow from both AML (n = 5, p < 0.01) and MDS patients (n = 12, p < 0.05), and did not correlate with percentage of blasts (r = 0.28, p = 0.21). AML patients (n = 34) had lower circulating levels of ENO1 autoantibodies detected by ELISA compared to 26 MDS and 18 controls (p = 0.003). However, there was no difference in OS between AML patients with high vs. low levels of anti-ENO1 autoantibodies (p = 0.77). BM immunostaining for ENO1 and patient monitoring of anti-ENO1 autoantibody levels may be useful biomarkers for MDS and AML.

Matched unrelated donor transplantation versus haploidentical transplantation with post-transplant cyclophosphamide in children with acute myeloid leukemia: a PDWP-EBMT study.

In children with acute myeloid leukemia (AML) who lack an HLA identical sibling, the donor can be replaced with an HLA matched unrelated donor (MUD) or a haploidentical donor (haplo). We compared outcomes of patients <18 years with AML in first and second complete remission (CR1 and CR2) undergoing a hematopoietic stem cell transplantation (HCT) either with a MUD with anti-thymocyte globuline (ATG) (n=420) or a haplo HCT with PT-CY (n=96) after a myeloablative conditioning regimen (MAC) between 2011 and 2021, reported to EBMT. A matched pair analysis was performed to adjust for differences among groups. The final analysis was performed on 253 MUD and 95 haplo-HCTs. In the matched cohort, median age at HCT was 11.2 and 10 years and median year of HCT was 2017 and 2018, in MUD and haplo- HCT recipients, respectively. The risk of grade III-IV aGvHD was significantly higher in the haplo group (HR=2.33, 95%CI1.18-4.58, p=0.03). No significant differences were found in 2 years overall survival (OS; 78.4%vs71.5%; HR 1.39, 0.84-2.31, p=0.19), leukemia-free-survival (LFS; 72.7%vs69.5%; HR1.22, 0.76-1.95, p=0.41), CI of relapse (RI; 19.3%vs19.5%; HR=1.14, 0.62-2.08, p=0.68) non-relapse-mortality (NRM; 8%vs11%; HR=1.39, 0.66-2.93, p=0.39) and graft versus host free-relapse free survival (GRFS; 60.7%vs54.5%, HR=1.38, 0.95-2.02, p=0.09) after MUD and haplo-HCT respectively. Our study suggests that haplo-HCT with PT-CY is a suitable option to transplant children with AML lacking a matched related donor.

Comparison of fludarabine/melphalan (FluMel) with fludarabine/melphalan/BCNU or thiotepa (FBM/FTM) in patients with AML in first complete remission undergoing allogeneic hematopoietic stem cell transplantation - a registry study on behalf of the EBMT Acute Leukemia Working Party.

Conditioning protocols for patients undergoing allogeneic hematopoietic cell transplantation (allo-HCT) are being developed continuously to improve their anti-leukemic efficacy and reduce their toxicity. In this study, we compared the conditioning protocol of fludarabine with melphalan 140 mg/m2 (FluMel) with conditioning protocols based on this same backbone but with an additional alkylating agent i.e., either fludarabine/BCNU (also known as carmustine)/melphalan (FBM), or fludarabine/thiotepa/melphalan (FTM) 110 mg/m2. We included 1272 adult patients (FluMel, n = 1002; FBM/FTM, n = 270) with acute myeloid leukemia (AML) with intermediate/poor cytogenetic risk in first complete remission (CR) from the registry of the EBMT Acute Leukemia Working Party. Despite patients in the FBM/FTM group were older (64.1 years vs. 59.8 years, p < 0.001) and had a worse Karnofsky performance score (KPS < 90, 33% vs. 24%, p = 0.003), they showed a better overall survival (OS) (2 y OS: 68.3% vs. 58.1%, p = 0.02) and less non-relapse mortality (NRM) (2 y NRM: 15.8% vs. 22.2%, p = 0.009) compared to patients treated with FluMel. No significant differences were observed in relapse incidence (RI) (2 y RI: 24.9% vs. 23.7%, p = 0.62). In conclusion, the addition of a second alkylating agent (BCNU/carmustine or thiotepa) to FluMel as FBM/FTM conditioning, improves OS in AML patients in first CR with intermediate/poor risk cytogenetics after allo-HCT.

Multiple Hepatic Microabscesses Caused by Magnusiomyces Clavatus in a Patient with Acute Myeloid Leukemia: A Case Report

Abstract Introduction/Objective Invasive fungal infections can result in significant complications for acute myeloid leukemia (AML) patients undergoing induction chemotherapy. While Candida and Aspergillus are the most common fungal pathogens, Magnusiomyces clavatus (Geotrichum clavatum) is a rare fungal pathogen that has been reported to cause invasive infection in immunocompromised patients. Here, we report a case of fungemia and hepatic microabscess with Magnusiomyces clavatus in an 18-year-old female with AML. Methods/Case Report An 18-year-old female with AML in remission after induction chemotherapy presented with fevers. Further work-up revealed fungemia on her blood culture. While awaiting culture speciation, numerous hepatic lesions were identified on the abdominal CT scan, which were biopsied. The liver biopsy showed aggregates of fungi with septate hyphae on silver-stained sections. The initial morphologic differential diagnosis included Aspergillus and Mucormycosis. Her blood cultures ultimately revealed fungemia with Magnusiomyces clavatus (Geotrichum clavatum), a yeast that is rising in prevalence, particularly among neutropenic and leukemic patients. The morphology of fungi detected in the liver biopsy resembled Magnusiomyces clavatus and a subsequent PCR testing confirmed that the fungi present was Magnusiomyces clavatus. She was started on voriconazole and liposomal amphotericin B. She became afebrile with no growth on blood cultures for several days and was discharged to outpatient follow-up. Results (if a Case Study enter NA) N/A Conclusion This case highlights the clinical significance of Magnusiomyces clavatus in immunocompromised patients, particularly those with AML, and emphasizes the importance of considering rare fungal pathogens in such patients who present with fever. Treatment with voriconazole and liposomal amphotericin B can be helpful in relieving symptoms.

Comparing Older Matched Related to Younger Matched Unrelated Donors in Acute Myeloid Leukemia in Remission Using Post-Transplant Cyclophosphamide

Backgroud. Over the past 25 years, the landscape of allogeneic stem cell transplantation (allo-SCT) changed from being rarely performed in patients 50 years or older to accounting for a little less than half of the transplantations reported in USA and Europe. Older patients will on average have older matched related siblings (MRD) being considered as donors with issues such as comorbidities, risk of clonal haematopoiesis of indeterminate potential, and impaired immune and regenerative potential of stem cells. Although younger HLA-matched unrelated donors (MUD) are an important alternative in this context, it remains unclear whether the use of MUD is associated with better outcomes. This is especially true in the context of post-transplant cyclophosphamide (PTCy) as graft-versus-host disease (GvHD) prophylaxis. Methods. The aim of this study was to compare the graft-relapse-free survival (GRFS) for patients diagnosed with acute myeloid leukemia (AML) in first complete remission (CR1), aged 50y or more, and having received a first allo-SCT from a 10/10 MUD younger than 40y or a MRD older than 50y using PTCy for in-vivo T-cell depletion. All transplants were performed between 2015-2021 at the EBMT centres. Results. A total of 410 consecutive patients were included, 172pts receiving a MRD, 238pts transplanted from a MUD. All patients with detailed conditioning regimen dosages were classified according to the transplant conditioning intensity (TCI) score in low (1-2), intermediate (2.5-3.5), high (4-6). TCI-score was available for 345 patients and was as follows: low in 53pts (36.8%), intermediate in 79pts (54.9%) and high in 12pts (8.3%) for MRD, low in 91pts (45.3%), intermediate in 93pts (46.3%) and high in 17pts (8.5%) for MUD (p=0.26). Only patients with available TCI-score were analysed. In univariate analysis the 2-year GRFS was 58% (49.8-65.3%) for TCI intermediate-high and 43.1% [34.3-51.5] for TCI low (p=0.005). In TCI-score intermediate-high non-relapse mortality (NRM) wasn't higher compared to TCI-score low (12.7% [12.2-24] vs 13.7% [8.4-20.3], p=0.53) but relapse incidence (RI) was lower (17.7% [12.2-24] vs 33.2% [25.1-41.5], p=0.003). As a strong interaction between TCI-score and type of donor was found, we compared transplant outcomes between MRD and MUD separately for TCI-score low and TCI-score intermediate-high. In TCI-score low, 2-year GRFS was 44.4% [33.5-54.8] for MUD and 40.9% [26.4-54.9] for MRD (p=0.83) (Figure 1A). In multivariable analysis (MVA) type of donor was not a risk factors for neither GRFS (HR 0.98, CI: 0.57-1.69; p=0.95) nor for any other transplant outcome. In TCI-score intermediate-high, 2-year GRFS was 67.2% [55.8-76.2] for MUD and 46.1% [34.4-57.1] for MRD (p=0.001) (Figure 1B). In MVA MUD were associated to better GRFS (HR 0.41, CI: 0.24-0.7; p=0.001), lower NRM (HR 0.19, CI: 0.07-0.55; p=0.002) and lower RI (HR 0.32, CI: 0.14-0.73; p=0.007) without advantages in terms of neither aGvHD nor cGvHD. Conclusions. In patients with AML in CR1, aged 50y or more and receiving a conditioning regimen with TCI-score ≥ 2.5 in the PTCy setting, a MUD younger than 40y is associated to longer GRFS, lower NRM and lower RI compared to a MRD older than 50y. For patients receiving regimens with TCI-score low transplant outcomes are independent on the type of HLA-matched donor.

Clonal Dynamics of Gene Mutations during Oral Azacitidine Maintenance Therapy in Patients with Acute Myeloid Leukemia (AML): Outcomes from the QUAZAR AML-001 Trial

Background: Acute myeloid leukemia (AML) is a genetically heterogeneous disease and despite some patients (pts) achieving remission with frontline intensive chemotherapy (IC), most eventually relapse. In the QUAZAR trial (NCT01757535), oral azacitidine (Oral-AZA) prolonged overall survival and relapse-free survival (RFS) vs placebo (PBO) in older pts with AML in remission post IC (Wei et al, N Engl J Med 2020). Here, we studied the molecular landscape and clonal dynamics of pts treated with Oral-AZA/PBO in the QUAZAR study. The mutational landscape for older patients with AML in remission after IC may be confounded by persistence of pre-leukemic (PL) or age-related clonal hematopoietic (CH) variants. The implications of these residual mutations on disease relapse and treatment (Tx) outcome are largely unknown. Aims: 1) characterize the mutational landscape from remission bone marrow at baseline (BL) prior to Oral-AZA maintenance (i.e., in remission post-IC); 2) determine the fate of variants over time and at relapse in the Oral-AZA vs PBO arms; 3) examine associations between mutational landscape and relapse risk between Tx arms. Methods: In QUAZAR, 472 pts (≥55 years) with AML with intermediate- or poor-risk cytogenetics in remission after IC (BL) were randomized 1:1 to Oral-AZA or PBO. Among pts who consented to biomarker analyses (n=310), targeted NGS (37 myeloid genes) was performed on bone marrow DNA at BL (Oral-AZA/PBO: n=165/145), cycle 6 (n=107/79) and relapse (n=83/77). Mean NGS coverage was 13K reads and median minimal detectable variant allele frequency (VAF) was 0.12% (range: 0.02-2.79). Clonal variants were categorized by the longitudinal association between VAF and blast percentage (slope of leukemic variants &amp;gt;0.1; PL/CHIP &amp;lt;0.1). RFS was computed from time of randomization to relapse (≥5% BM blasts) or death, estimated by Kaplan-Meier methods. Hazard ratios (HR) and 95% confidence intervals (CI) were obtained from Cox regression models. Nominal P values were derived from log-rank tests. Results: In the NGS cohort (n=310), median RFS (mRFS) for Oral-AZA vs PBO was 10.2 vs 4.7 months (mo), respectively. At BL, prior to maintenance Tx, 221 (71.3%) had detectable mutations, the most frequently occurring mutations (&amp;gt;5% of pts) were in DNMT3A (28.4%), TP53 (15.5%), IDH2 (12.3%), TET2 (11.9%), SRSF2 (11.0%), IDH1 (6.1%) and ASXL1 (5.5%). At BL, 110/310 (35.5%) pts had VAF &amp;gt;5%, potentially representing persistence of PL/CH variants in remission. Comparative analysis of all gene variants at BL and relapse revealed that some VAFs increased with blast frequency, while other variants remained largely static. Applying a variant classification algorithm, 97/258 pts had potential leukemic variants detected at BL. These involved DNMT3A (16.1%), SRSF2 (7.1%), TP53 (7.1%) and IDH2 (5.7%). PL/CH variants included DNMT3A (17.5%), TP53 (8%), IDH2 (5.7%) or TET2 (5.7%). TP53 was detected in 19.4% PL/CH variants (mean VAF 1.7%; range 0.2-13.4). Notably, when analyses were limited to potential leukemic variants at BL (&amp;lt;5% VAF), their presence was correlated with worse RFS (PBO: mRFS for 0, 1 or 2+ mutations was 6.1, 4.7 or 1.9 mo, respectively). The presence of ≥2 leukemic mutations was associated with shorter RFS only in PBO arm (mRFS vs &amp;lt;2 mutations: 1.9 vs 5.7 mo, P=0.02; Oral-AZA: 10.2 vs 10.2 mo). RFS favored Oral-AZA in pts with low mutational burden at BL (&amp;lt;2 mutations: mRFS 10.2 vs 5.7 mo [ P=0.003]; n=145 vs 122 [PBO]), and in a small subset of pts with higher mutational burden (≥2 mutations: mRFS 10.2 vs 1.9 mo [ P=0.008]; n=14 vs 13 [PBO]). Oral-AZA prolonged RFS vs PBO in pts across most mutational subtypes, when assessed for mutations that were deemed potentially leukemic (Figure). At relapse, the frequency of mutations, hotspots variants and co-mutations were comparable between Tx arms. Mutation-based pathway analysis indicated Ras pathway genes were enriched at relapse in PBO (28.6%) vs Oral-AZA arm (14.5%, P=0.03). Summary: In pts with AML in remission post IC, post-hoc analyses showed that Oral-AZA improved RFS vs PBO regardless of the mutational landscape at baseline. The spectrum of mutations at relapse was similar between Tx arms, suggesting that Oral-AZA maintenance prolongs remission without altering mutational heterogeneity.

Persistence of FLT3-TKD in Blood Prior to Allogeneic Transplant Is Associated with Increased Relapse and Death in Adults with AML in First Remission

Introduction: Next-generation sequencing (NGS) represents a promising modality for measurable residual disease (MRD) testing in patients with acute myeloid leukemia (AML), but systematic efforts are required to define the appropriate targets, test characteristics, and clinical implications. We recently reported that detection of persistent NPM1 or FLT3 internal tandem duplication (ITD) mutations in adults with AML in first complete remission (CR1) prior to allogeneic hematopoietic cell transplantation (alloHCT) is associated with increased relapse and death compared with those testing negative (JAMA 2023, PMID: 36881031). FLT3 tyrosine kinase domain (TKD) variants are present in 7-10% of adult patients with AML but the utility of this target for AML MRD testing in CR1 prior to alloHCT was previously unknown. Methods: Patients aged 18 or older who underwent alloHCT for AML in CR1 between 2013 and 2019 reported to have a FLT3-TKD variant at diagnosis and a pre-conditioning remission blood sample available in the CIBMTR biorepository were eligible for this study. Single-amplicon NGS (SA-NGS) library generation utilizing UMI-tagged primers targeting the D835 and I836 codons of FLT3 was performed on 400ng genomic DNA sequenced with unique dual indices, with error-corrected variant calling. Primary outcomes were overall survival (OS) and cumulative incidence of relapse (CIR). Secondary outcomes were relapse-free survival (RFS) and non-relapse mortality (NRM), with NRM being considered as a competing risk. Results: A total of351 patients met inclusion criteria for this study, and 342 (97.4%) had sufficient DNA for SA-NGS and were analyzed for clinical outcomes. Of these patients, 81 (23.7%) experienced relapse at a median of 5 months post-alloHCT. Using the current ELN-recommended variant allele fraction (VAF) threshold of 0.1% for NGS-based AML MRD, 14 patients (4.1%) tested positive. Patients with FLT3-TKD MRD VAF≥0.1% experienced higher rates of relapse (73.8% vs. 20.5%, p &amp;lt; 0.0001) and decreased OS (11.4% vs. 66.8%, p &amp;lt; 0.001) compared to VAF&amp;lt;0.1%. The highly sensitive SA-NGS method allowed for exploration to determine if a deeper VAF threshold of 0.01% (previously validated for NPM1 and FLT3-ITD in the Pre-MEASURE study) was prognostic for FLT3-TKD as an MRD target. This led to an additional 20 patients being reclassified as positive (n = 34 with VAF above 0.01%, =9.9%). However, there was no significant difference in CIR (33.6% vs. 19.7%, p = 0.089) or OS (65.2% vs. 66.9%, p = 0.267) between patients with 0.01%≤VAF&amp;lt;0.1% and VAF&amp;lt;0.01% ( Figure). Out of 28 SA-NGS positive calls, 6 could not be validated by orthogonal digital droplet PCR testing, and interestingly, none of those 6 patients experienced relapse. Site-reported pre-transplant remission multiparametric flow cytometry (MFC) was available for 335 patients (98%). Results of MFC were not associated with differences in CIR (HR 1.2, 95% CI 0.52 - 2.76, p = 0.67) or OS (HR 0.78, 95% CI 0.34 - 1.77, p = 0.551).In multivariable analysis, FLT3-TKD NGS-MRD VAF≥0.1% remained prognostic for CIR (HR 6.1, 95% CI 3.1 - 12.0, p &amp;lt; 0.001) and OS (HR 3.2, 95% CI 1.7 - 5.9, p &amp;lt; 0.001). Conclusions: Detectable persistence of FLT3-TKD variants in blood from AML patients in first remission prior to first alloHCT is rare, but at a VAF level of 0.1% was strongly associated with increased relapse and death after transplant compared to those testing negative. In contrast to FLT3-ITD NGS-MRD, no evidence was found to support lowering the NGS-MRD VAF threshold from 0.1% to 0.01% for FLT3-TKD. Alternative methodology may further improve NGS-MRD test performance. Site-reported MFC prior to alloHCT did not stratify for post-transplant relapse or survival. Optimal NGS-MRD testing for patients with AML will likely require a multi-target approach and testing for persistence of FLT3-TKD MRD is shown here to represent an important component of this strategy.

PPARG and ASS1: Targets of Metabolic Alteration in Venetoclax / Decitabine Resistant Acute Myeloid Leukemia

The combination of venetoclax (VEN), an inhibitor of the anti-apoptotic factor BCL2, with the hypomethylating agent (HMA) decitabine (DEC) is the current frontline standard therapy for elderly patients with acute myeloid leukemia (AML). Despite the success of VEN/HMAs in inducing remission in AML, significant proportion of patients remain refractory to the combination therapy and others relapse after initial response. To elucidate underlying mechanisms of resistance, we performed transcriptomic and DNA methylation analyses on VEN/DEC pre- and post-treatment samples from AML patients participating in a prospective clinical trial of DEC10-VEN (NCT03404193). We have previously reported that AML cells from non-responders demonstrate significant upregulation of mRNA expression of the fatty acid metabolism activator PPARG ( Peroxisome Proliferator-Activated Receptor γ) and ASS1 ( Argininosuccinate Synthase coding rate-limiting enzyme in the arginine biosynthesis) after VEN/DEC treatment, both accompanied by their transcription start site demethylation ( Yamatani et al. ASH 2022). In this study, we performed single cell (sc) ATAC-seq to profile the accessible chromatin changes that are associated with VEN/DEC resistance. BM samples from one patient who achieved complete remission with incomplete count recovery and later relapsed (pre- and post- VEN/DEC treatment) and 2 healthy control bone marrow cells were utilized. A total of 23,803 cells were sequenced, read alignment and initial stages of data processing were performed using Cell Ranger (version 7.0.1; 10x Genomics), then cells were clustered by Uniform Manifold Approximation and Projection (UMAP) and annotated using the Azimuth application, which distinguished leukemia-associated clusters. In the leukemia-associated cell cluster, PPARG motifs in accessible chromatin were significantly enriched compared to other clusters. After VEN/DEC treatment, further increase in the relative abundance of PPARG motifs along with chromatin accessibility of CXCR4 and CDC42EP4 genes, which have RRARG accessible motifs, was observed. Next, the role and mechanism of PPARG in the resistance to VEN/DEC was investigated using four AML cell lines with different sensitivity to DEC. PPARG expression was increased after DEC treatment in the less sensitive cells (HL-60 and MV4-11) but was unchanged in the more sensitive cells (OCI-AML3 and THP-1). To confirm that PPARG upregulation is associated with VEN/DEC resistance, we generated PPARG overexpressing HL-60 and MV4-11 cells using a lentiviral system. In the PPARG-overexpressing (OE) cells, VEN/DEC-induced cell growth inhibition and apoptosis were significantly reduced compared to the mock-transfected cells (PPARG -OE vs mock; cell growth inhibition: HL-60, 30% vs. 54%; MV4-11, 33% vs. 56%; apoptosis induction: HL-60, 14% vs. 38%, MV4-11, 25% vs. 36%; p &amp;lt; 0.05). To determine whether the suppression of energy metabolism by VEN/DEC is inhibited by PPARG overexpression, we performed an extracellular flux assay using PPARG-OE cells and mock HL60 cells. The results showed that VEN/DEC treatment reduced reserve respiratory capacity by 32% in mock cells, while VEN/DEC therapy was associated with an increase of 37% in PPARG-OE cells (p &amp;lt; 0.05). ASS1 is another potential target associated with metabolic alterations in VEN/DEC-resistant AML. We next investigated the effects of arginine deprivation by combining the arginine-degrading enzyme pegylated arginine deiminase (ADI-PEG 20) with VEN/DEC. We used HL-60 cells with low baseline ASS1 expression, which was increased by DEC treatment, and MV4-11 cells with high ASS1 expression, which was not altered by DEC. HL-60 was more sensitive to ADI-PEG 20 than MV4-11 cells (IC 50: HL-60, 20ng/mL; MV4-11, 100ng/ml); however, no further increase of cell growth inhibition by ADI-PEG 20 was observed in the combination with VEN/DEC , which enhances ASS1 expression. In MV4-11, ADI-PEG 20 combination significantly facilitated cell growth inhibition by VEN/DEC (increased VEN/DEC growth inhibition by ADI: HL60, 7%; MV4-11, 57%, p &amp;lt; 0.05). Taken together, this study demonstrated that VEN/DEC treatment facilitates energy metabolism in AML cells through increased expression of PPARG and ASS1, which may in turn translate into therapeutic resistance, and that VEN/DEC /ADI-PEG 20 combination may represent a potential novel therapeutic strategy (Figure 1).

Post-Transplant T Cell Clonotype Diversity Is Associated with Survival in Patients with TP53-Mutated Acute Myeloid Leukemia

Background TP53 mutations are associated with unfavorable outcomes in various cancer types and present an obstacle to achieving sustained remission in acute myeloid leukemia (AML). Even with allogeneic hematopoietic stem cell transplantation (HSCT), the risk of relapse approximates 80%, with dismal long-term survival. This could imply methods of immune escape in TP53 mutated leukemias that allow them to evade the graft-versus-leukemia (GVL) effect, which is a critical mechanism of disease control with HSCT. This study investigates the dynamics of immune reconstitution at single-cell resolution in TP53-mutated AML at pre- and multiple post-HSCT timepoints in patients who either remain in long-term remission or relapse following HSCT. Methods We collected 26 longitudinal bone marrow aspirates and 1 peripheral blood sample from 12 patients with TP53-mutated AML who underwent HSCT at the Dana-Farber Cancer Institute between 2015-21. All patients received HLA-matched T-cell replete HSCT in morphologic remission, with standard tacrolimus/methotrexate-based prophylaxis and peripheral blood stem cells. We categorized the patients into two cohorts based on their treatment response: long-term remission (&amp;gt;3.5 years, cohort 1, n=4) or relapse (cohort 2, n=8). We performed paired 5' single-cell RNA and T cell receptor (TCR) sequencing on sorted viable mononuclear from each sample and CD3+ T cells from selected samples using 10x Genomics procedures. In total, we analyzed 44 samples (27 mononuclear, 17 CD3+), resulting in 250,640 high-quality single-cell transcriptomes, including 137,649 T cells and NK cells. Data analysis was performed using Cell Ranger Multi (10x Genomics) and R 4.3 with the tidyverse 2.0, Seurat 5.1, and scRepertoire 1.1 packages. Results We focused our initial analysis on the 2-6-month post-HSCT period that is critical for immune reconstitution. This included eight samples from seven patients in the two cohorts (1: remission, 2: relapse, pink box in Figure 1). Of these 7 patients, 4 were in the relapse cohort, with these relapses occurring &amp;gt;4 months post-HSCT. No significant difference in T-cell chimerism was seen between these 7 patients in this 2-6 month period. Based on canonical gene expression signatures, we annotated cell types and selected T cells for further evaluation (Figure 2). First, we found that the CD4/CD8 ratio differed between the cohorts, indicating a relative CD8+ T cell expansion in the relapse cohort (cohort 1: 1.69, cohort 2: 0.55, P&amp;lt;0.0001). Second, we found lower TCR diversity in the relapse cohort (inverse Simpson index, cohort 1: 234.6, cohort 2: 83.0, P&amp;lt;0.0001). This difference remained significant when subsampling the same number of T cells per patient and when restricting all samples to the 3 to 4-month timepoint. Considering the disparity in CD8 T cell numbers between the cohorts, we also analyzed repertoire diversity, specifically in CD8+ T cells, and found that the observed significance persisted. To further investigate TCR diversity, we overlaid T cell clonotypes (cells that share the same TCR sequence) in a UMAP visualization, which highlighted increased hyperexpanded CD8+ T cells in the relapse cohort (Figure 2). Finally, we found that greater clonotype expansion was correlated with the expression of key T cell genes, including CD8A, CD8B, PRF1, NKG7, and GZMA. These results suggest that cytotoxic CD8+ T cells link lower TCR diversity to the likelihood of disease relapse. Conclusions By performing deep analysis of single-cell gene expression linked to TCR clonotypes, we discover new insights into the biology of hematopoietic reconstitution after HSCT of patients with TP53-mutated AML. Despite having analyzed a limited number of samples, we find that patients who remain in long-term remission have significantly higher CD8+ TCR diversity between 2-6 months post-HSCT, while those who relapsed between 5-18 months post-HSCT showed lower TCR diversity and hyperexpanded CD8+ clones at the same timepoints. This work suggests that early post-HSCT TCR diversity could be a biomarker associated with GVL and warrants further study. In ongoing and future analyses, we will mine the current dataset for further insights into the factors influencing survival in TP53-mutated AML patients. We also plan to extend the measurement of TCR diversity as a predictor of survival for validation in a larger cohort.

Prognostic Value of Copy Number Aberrations and Copy Neutral Loss of Heterozygosity in Acute Myeloid Leukemia

Background: Chromosomal abnormalities are key to the diagnosis and prognosis of patients with acute myeloid leukemia (AML). Chromosomal genomic array testing (CGAT) can identify copy neutral loss of heterozygosity (cnLOH) and genomic copy number alterations (CNAs) that are undetected by conventional karyotype or fluorescence in situ hybridization (FISH). Previous studies have shown that cnLOH is correlated with a shorter duration of complete remission (CR) and worse overall survival (OS) in AML (Grosneth et. al, Cancer, 2015). The primary objective of this exploratory study is to examine the predictive power of CGAT in a cohort of AML patients with majority normal karyotype. Methods: CGAT analyses were collected from 187 subjects with AML or other high grade myeloid neoplasm (myeloid blasts &amp;gt;10% in peripheral blood or bone marrow) between 2012 and 2022 at Fred Hutchinson Cancer Center. CGAT utilized CytoScan™ HD Array (ThermoFisher, MA), targeting genome-wide region with 2.4 million markers for copy number and approximately 750,000 genotype-able SNPs. The data were analyzed using the Nexus Copy Number software with manual curation. Based on the assay validation in our CLIA-certified diagnostic lab, the cutoff is 100 Kb for copy number aberrations and 10 Mb for cnLOH. Survival predictive power analysis (SPP), a log-rank based test using Nexus, was applied to identify aberrations significant for event free survival (EFS) and overall survival (OS). SPP analysis tabulated genomic segments based on p-values with the alpha value cutoff at 0.05. Using frequency as the filtering criteria (present in &amp;gt;5 patients), 132 SPP for EFS, and 73 SPP for OS were identified. SPP were then manually curated to (1) remove those representing constitutional absence of heterozygosity (AOH) and those overlapping with benign copy number variance and (2) connect adjacent SPP into contiguous regions. The presence and absence of the identified SPP were then tabulated, cleaned up, and used to generate Kaplan-Meier curves for EFS and OS. This process identified 5 SPP that are significant for EFS and 2 SPP for OS. A multivariate analysis was performed to determine the hazard ratio (HR) of these 5 genetic aberrations for EFS and OS, adjusted for patient age, secondary/treatment related AML, ELN 2022 risk, and receipt of allogeneic hematopoietic cell transplantation (HCT) as a time-varying covariate. Results: In our 187 subject cohort, the median age was 63 (range 20-87). 76 (41%) had secondary or treatment related disease. 97 (52%) went on to receive a hematopoietic stem cell transplant. 3 had favorable risk disease, 14 had adverse risk, and 170 had intermediate risk by karyotype and FISH using ELN 2022 criteria. 120 (64%) had normal karyotype. 5 subjects had 4q cnLOH, 6 had 5q ( EGR1) loss, 7 had 9p cnLOH, 10 had mixed-lineage leukemia gene partial tandem duplication ( MLL-PTD), and 4 had 7q deletion. Univariate analysis identified five SPP that are significant for EFS: 4q cnLOH (p=0.002), 5q ( EGR1) loss (p&amp;lt;0.001), 9p cnLOH (p&amp;lt;0.001), MLL-PTD (p&amp;lt;0.001), and 7q deletion (p&amp;lt;0.001). 2 SPP were significant for OS: 9p cnLOH (p=0.001) and MLL-PTD (p=0.036). Multivariate analysis for EFS adjusted for age, secondary/treatment related disease, ELN 2022 risk, and receipt of HCT as a time-varying covariate demonstrated a hazard ratio (HR) of 3.5 for 4q cnLOH (p=0.008), 2.3 for 5q ( EGR1) loss (p=0.16), 4.5 for 7q deletion (p=0.007), 5.7 for 9p cnLOH (p&amp;lt;0.001), and 2.9 for MLL-PTD (p=0.002). Multivariate analysis for OS adjusted for age, secondary/treatment related disease, ELN 2022 risk, and receipt of HCT as a time-varying covariate demonstrated a hazard ratio (HR) of 2.0 for 4q cnLOH (p=0.23), 0.5 for 5q ( EGR1) loss (p=0.36), 1.3 for 7q deletion (p=0.66), 4.9 for 9p cnLOH (p=0.001), and 2.3 for MLL-PTD (p=0.04). Discussion: This study has found 5 key genetic changes identifiable via CGAT that are significant for shorter EFS and/or OS in AML patients. Our next step is to validate these findings in a large independent cohort of AML patients. CGAT is a powerful adjunct to conventional karyotype and FISH for the detection of cnLOH and can be used to identify CNAs below the level of detection of karyotype. For these reasons, CGAT may be most useful in intermediate risk patients. Further research is warranted to fully understand the prognostic power of CGAT in AML.

Relative Prognostic Value of Flow Cytometric Measurable Residual Disease before Allogeneic Hematopoietic Cell Transplantation for Adults with MDS/AML or AML

Background: Measurable residual disease (MRD), assessed by multiparameter flow cytometry (MFC) before allogeneic hematopoietic cell transplantation (HCT), is strongly and independently associated with poor post-HCT outcomes in adults with acute myeloid leukemia (AML). On the other hand, the relative prognostic value of MFC-based MRD testing is unknown in patients with myelodysplastic neoplasm (MDS)/AML, a disease entity newly defined by the 2022 International Consensus Classification (ICC) that encompasses cases of MDS with 10-19% blasts and recognizes the diagnostic continuum between MDS and AML. Methods: To assess the relationship between pre-HCT, disease type, and post-HCT outcomes, we retrospectively analyzed 1,262 adults ≥18 years with MDS/AML (n=150) or AML (n=1,112), based on the ICC 2022 criteria, who received a first allograft while in first or second morphologic remission at a single institution between 4/2006 and 3/2023. Results: The MDS/AML and AML cohorts differed significantly regarding patient and disease-specific characteristics, including age at HCT (60 vs. 56 years in MDS/AML and AML patients, respectively, P&amp;lt;0.001), HCT-CI score ( P&amp;lt;0.001), adverse cytogenetic risk according to ELN 2022 criteria (39% vs. 26%, P&amp;lt;0.001), intensive therapy prior to allogeneic HCT (69% vs. 97%, P&amp;lt;0.001), time from last remission (76 vs. 98 days, P&amp;lt;0.001), first remission status (97% vs. 77%, P&amp;lt;0.001), pre-HCT MRD by MFC (35% vs. 19%, P&amp;lt;0.001), and peripheral blood stem cells as graft source (89% vs. 80%, P=0.015). There were no differences in donor source or conditioning intensity. With a median follow-up of 5.11 years (interquartile range [IQR]: 2.38-9.66) in survivors, there were 384 relapses, 590 deaths, and 258 non-relapse mortality (NRM) events contributing to the estimates for relapse, NRM, relapse-free survival (RFS), and overall survival (OS). At three years, NRM (21% [14-28%] for MDS/AML vs. 16% [14-19%] for AML, P=0.19), relapse (29% [22-36%] vs. 29% [27-32%], P=0.94), RFS (50% [42-59%] vs. 54% [51-58%], P=0.25), and OS (52% [44-61%] vs. 60% [57-63%], P=0.09) were similar between the two groups. After multivariable adjustment, relapse rates for patients with MDS/AML vs. those with AML were similar (hazard ratio [HR]=0.77 [0.50-1.17], P=0.2), as were NRM (HR=1.41 [0.91-2.18), P=0.12), RFS (HR=0.99 [0.73-1.35], P&amp;gt;0.9) and OS (HR=1.17 [0.85-1.61], P=0.3). Importantly, a significant interaction was found between pre-HCT MRD status and disease type (MDS/AML vs. AML) for relapse ( P=0.009), RFS ( P=0.013), and OS ( P=0.048). The interaction models indicated that the HRs for the association between pre-HCT MRD and post-HCT outcomes were lower in patients with MDS/AML (for relapse: HR=1.73 [0.96-3.11] in MDS/AML vs. HR=3.56 [2.90-4.38], in AML; for RFS: HR=1.60 [1.03-2.48] vs. HR=2.67 [2.25-3.16]; for OS: HR=1.53 [0.97-2.39] vs. HR=2.34 [1.96-2.78]). Pre-HCT MRD was associated with a higher risk of relapse in both MDS/AML and AML patients but to a lesser extent in the MDS/AML patients. In these patients, relapse at three years was 37% [24-51%] for those with pre-HCT MRD vs. 24% [16-33%] for those without ( P=0.068), translating into lower RFS (39% [28-56%] vs. 56% [47-67%], P=0.038) and OS (41% [29-58%] vs. 58% [48-70%], P=0.066) in MDS/AML patients with pre-HCT MRD. For comparison, in patients with AML, relapse at three years was 22% [20-25%] for those without MRD vs. 56% [50-62%] for those with ( P&amp;lt;0.001), translating into lower RFS (27% [22-31%] vs. 61% [58-64%], P&amp;lt;0.001) and OS (35% [29-42%] vs. 65% [62-68%], P&amp;lt;0.001) in those with pre-HCT MRD. In line with these findings, C-statistics showed that pre-HCT MRD was not as predictive in MDS/AML patients in comparison to AML patients (for relapse: 0.57 in MDS/AML patients vs. 0.62 in AML patients; for RFS: 0.57 vs. 0.59; for OS: 0.56 vs. 0.58). Conclusion: Our data suggest that, overall, patients with MDS/AML or AML in first or second morphologic remission have similar outcomes following allogeneic HCT. However, MFC-based pre-HCT MRD testing is prognostically substantially less informative for MDS/AML than AML patients, possibly because of increased rates of misclassification. These findings support the rationale to examine the prognostic value of molecular MRD testing in patients with MDS/AML. Figure. Cumulative incidence of relapse stratified (A) by disease type at diagnosis and (B) by disease type at diagnosis and pre-HCT MRD.

A Phase 2 Study of Ponatinib for Prevention of Relapse after Allotransplantation in FLT3-ITD + AML Patients : The Ponallo Trial

Background : For acute myeloid leukemia (AML) patients relapse remains a major issue after allogeneic stem cell transplantation (allo-SCT). In those with a FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD+), survivals have been improved with the use of small molecule Flt3 receptor protein-tyrosine kinase inhibitors in maintenance due to a significant decrease of relapse after allo-SCT. Ponatinib (pona) is a multikinase inhibitor that is approved for the treatment of Philadelphia chromosome-positive acute lymphoblastic and chronic myelogenous leukemias. It has also activity against FLT3-mutants including ITD and ITD691L but not D835 within the Flt3 activation segment. Pona may then represent an alternative drug for maintenance after allo-SCT in FLT3-ITD+ AML patients. Methods : This was a multicenter phase 2 non-randomized prospective study testing the efficacy and tolerability of pona after allo-SCT in FLT3-ITD+ AML patients (ClinicalTrial.gov ID : NCT03690115). The main objective was the incidence of relapse at 2 years post-transplant with the hypothesis that this incidence at 2 years will be reduced at 15% instead of 30%. Seventy-seven patients had to be included. Inclusion criteria were all patients, aged between 18 and 70 yo, with a FLT-3 ITD+ AML in cytologic complete remission (&amp;lt;5% of bone marrow blasts) and a documented engraftment at the time of starting pona after an allo-SCT using any type of conditioning regimen and donor. The control of a GVHD (if present) was mandatory as well as a platelets count ≥70 Giga/l and a neutrophils count ≥ 1 Giga/l at time of inclusion. The study was supported by Incyte. Initially, pona administration had to start between day +60 and +90/100 at the dose of 30 mg/day for 1-year. However, because of excesssive hematopoietic toxicity, an amendment was made after the 14th patient to start pona between day 100 and day 120 post-allo. During the study, It was allowed to restart pona at 15 mg/day after resolution of 30 mg dose related toxicities. After 1 year of pona it was not allowed to start a new FLT3 inhibitor or pursue pona. Results : Twenty-three patients were included between December 2019 and April 2022. Among them, 22 were effectively treated. Indeed, the last patient included did not receive pona due to hepatic abnormalities incompatible with the start of pona. Also, because of a very low rate of enrollment, the trial was closed prematurely in accordance with the sponsor in December 2022. Results for the 22 treated patients are reported here. Patient characteristics are given in Table 1. With a median follow-up of 740 days (range : 374-930), 1-y and 2-y overall survival were 81.3% (66.3-99.6) and 63.9% (45.4-90.0), and 1-y and 2-y leukemia-free survival were 54.5% (37.2-79.8) and 49% (31.8-75.7), respectively. Ten relapses occured at a median of 186 days (range=69-481) after allo-SCT with a CI of relapse of 45.5% at 2 years. Pona was started at a median of 69 days post-allo-SCT (range : 60-130). The median duration of treatment was 75.5 days (range : 4-369). Only 3 patients received one year of treatment and all at 30 mg/day as required by the protocol. The reasons for stopping pona were toxicity in 11 patients, relapse in 6, toxicity then relapse in 1, GVHD then relapse in 1. Among the 13 patients who stopped pona for toxicity or GVHD, 6 restarted pona (15 mg n=5, 30 mg n=1) then stoppped again for toxicity (n=4) or progression (n=2). A total of 36 adverse events related to pona were documented in 17 patients. The majority was grade 1 or 2 (n=27, 75%) while 6 grade 3 (17%) and 3 grade 4 (8%) occured. ( Table 2) Conclusion : Ponatinib administration after allo-SCT is associated with toxicity and do not reduce relapse or improve survival in this small cohort of FLT3+ AML patients. Other FLT3 inhibitors have to be considered in FLT3+ AML patients for maintenance after allo-SCT.

Decitabine/Cedazuridine (ASTX727) Combined with a Molecularly-Targeted Agent (Venetoclax, Gilteritinib, Ivosidenib, or Enasidenib) As Personalized Maintenance Therapy in Acute Myeloid Leukemia: First Results from a Phase 1b Study

Background: Disease relapse is the major cause of treatment failure and death in acute myeloid leukemia (AML). Currently, oral azacitidine (CC-486) is the only drug approved in the maintenance setting in patients with AML who are unable to complete standard therapy. The ECOG ACRIN E2906 trial suggested an overall survival (OS) benefit with 3-day IV decitabine maintenance. Decitabine/cedazuridine (ASTX727) is an oral formulation of decitabine that achieves systemic exposures as seen with IV decitabine. Multiple small-molecule agents are now available targeting key leukemic drivers (BCL-2, FLT3, and IDH1/IDH2 inhibitors). We developed a multi-arm phase 1b study (NCT05010772) to evaluate an oral maintenance regimen composed of an ASTX727 backbone combined with a molecularly-targeted agent (venetoclax, gilteritinib, ivosidenib, or enasidenib). Methods: Patients ≥ 18 years with AML in first complete remission (CR) or CR with incomplete blood count recovery (CRi) not currently eligible for stem cell transplantation (SCT) and unable/unwilling to complete standard therapy were eligible. Patients having received either intensive induction (at least 2 cycles of therapy based on intermediate to high dose cytarabine) or low-intensity induction (at least 3 cycles based on low-dose cytarabine or a hypomethylating agent) were permitted. Other inclusion criteria were ECOG ≤ 3, adequate hepatic/renal function, absolute neutrophil count &amp;gt; 0.5 x 10 9/L, and platelets &amp;gt; 50 x 10 9/L. Patients were enrolled in one of five arms per physician choice based on molecular profile. Arm A consisted of ASTX727 35/100 mg PO on D1-3. All other arms combined this ASTX727 backbone with either venetoclax 400 mg (adjusted for azoles) PO on D1-5 (arm B), gilteritinib 120 mg PO on D1-28 (arm C), enasidenib 100 mg PO on D1-28 (arm D), or ivosidenib 500 mg PO on D1-28 (arm E). Treatment was administered in 4-week cycles, up to 24 cycles. Each arm initially consisted of a safety lead-in cohort with predetermined dose reductions for dose-limiting toxicity (DLT) per a 3+3 dose de-escalation design. Prophylactic antibiotics, antifungals, and antivirals were encouraged. The primary objective was safety and tolerability. Secondary objectives included relapse-free survival (RFS) and OS. Patients becoming eligible for SCT were taken off study and censored at the time of SCT for time-to-event endpoints. Results: 23 patients, with a median age of 68 years (range 33-83), have been enrolled (3 on arm A, 19 on arm B, and 1 on arm C). 12 (52%) were enrolled following intensive induction and 11 (48%) after low-intensity induction. 16 (70%) had prior venetoclax. Best response at enrollment was CR in 22 (96%) patients and CRi in 1 (4%). 6/22 (27%) patients with available testing were measurable residual disease (MRD)-positive. 2 (9%) patients had antecedent myelodysplastic/myeloproliferative neoplasms and 1 (4%) had therapy-related AML. ELN 2022 risk was favorable in 10 (43%), intermediate in 3 (13%), and adverse in 10 (43%) patients. The most common grade 3/4 adverse events were neutropenia (96%), thrombocytopenia (74%), anemia (39%), lung infection (22%), hypertension (17%), and neutropenic fever (17%). Cycle 2 dose reductions due to myelosuppression occurred in 5/23 (22%) patients. No DLTs were observed. No deaths occurred on protocol. Two patients died in remission from SCT-related complications after going off maintenance. The current median follow-up time is 5.3 months (m). The median number of cycles given is 3 (1-15). Three relapses have occurred to date (1 in arm A, 2 in arm B). The median RFS for the full cohort is not reached (NR, 1-year 71%). The median OS for the full cohort (figure 1A) is NR (1-year 100%). The median RFS for arms A, B, and C are NR (1-year 67%), NR (1-year 84%), and NR, respectively (figure 1B). The median RFS is 14.5 m (1-year 50%) for patients who received intensive induction and NR (1-year 75%) for those who received low-intensity induction. The median RFS for patients who were MRD-negative and MRD-positive at enrollment were NR (6-month 93%) and NR (6-month 75%), respectively (p=0.50). Of the 6 MRD-positive patients, 2 (33%) cleared their MRD while on maintenance (both patients in arm B). Conclusions: A fully oral, targeted maintenance regimen is feasible in AML. Early results show encouraging RFS and OS. Further enrollment and follow-up are required to better assess the safety and efficacy of these regimens.

Azacitidine Plus Venetoclax Maintenance in Acute Myeloid Leukemia Produces Sustained Remissions with Low Toxicity: Results of a Phase 2 Study

Background: Most patients diagnosed with acute myeloid leukemia (AML) achieve remission following induction therapy. However, disease relapse remains a major clinical concern. The QUAZAR AML-001 study demonstrated improved overall and relapse-free survival with oral azacitidine (CC-486) maintenance. We designed a phase 2 study (NCT04062266) to evaluate low doses of azacitidine plus venetoclax as maintenance therapy following both intensive and low-intensity induction in AML. We present updated results of this study with close to 2 years of median follow-up. Methods: Patients ≥ 18 years of age with non-APL AML in first complete remission (CR) or CR with incomplete count recovery (CRi) not immediately eligible for allogeneic stem cell transplantation (SCT) were eligible. Cohort 1 enrolled patients who had received at least 2 cycles of intensive therapy (defined as containing intermediate to high-dose cytarabine). Cohort 2 enrolled patients who had received at least 3 cycles of low-intensity therapy (defined as hypomethylating agent or low-dose cytarabine-based). Patients with positive measurable residual disease (MRD) by flow cytometry were eligible in CR1 or beyond. Other inclusion requirements were ECOG ≤ 3, adequate hepatic/renal function, and adequate bone marrow reserve (defined as absolute neutrophil count &amp;gt; 0.5 x 10 9/L and platelets &amp;gt; 30 x 10 9/L). Treatment consisted of azacitidine 50 mg/m 2 SQ/IV on D1-5 and venetoclax 400 mg PO (adjusted for concomitant CYP3A inhibitors) on D1-14 in 28-day cycles, up to 24 cycles. Venetoclax duration could be reduced to D1-7 per physician discretion to mitigate myelosuppression and dose modifications during subsequent cycles were allowed. Antibacterial, antifungal, and antiviral prophylaxis were recommended in all patients. The primary objective was relapse-free survival (RFS, defined as time from enrollment to progression or death). Secondary objectives included overall survival (OS), safety/toxicity, and MRD clearance. Patients subsequently becoming eligible for SCT were taken off study and censored for analyses at the time of SCT. Results: 35 patients have been enrolled (25 in cohort 1 and 10 in cohort 2). The median age was 47 years in cohort 1 and 71 years in cohort 2. The baseline characteristics are shown in table 1. The median number of cycles given was 9 (range 1-24). 22/35 (63%) patients had 7 days of venetoclax during cycle 1. Median follow-up is currently 20.9 months (m). For the full cohort, the median RFS was not reached (NR; 2-year 60%) and the median OS was NR (2-year 75%). In cohort 1, the median RFS and OS were NR (2-year 71%) and NR (2-year 77%), respectively. In cohort 2, the median RFS and OS were 23.5 m (2-year 34%) and NR (2-year 67%), respectively. When stratified by ELN 2017, patients who were favorable (n=16), intermediate (n=11), and adverse (n=8) had a median RFS of NR (2-year 85%), NR (2-year 64%), and 4.0 m (2-year 0%), respectively. Corresponding median OS were NR (2-year 91%), NR (2-year 76%), and 14.9 m (2-year 27%), respectively. 4/8 (50%) of the ELN adverse patients had complex cytogenetics and/or TP53 mutations. Patients who were MRD-positive at enrollment (n=7, 3 with prior venetoclax exposure) had significantly shorter RFS compared to those who were MRD-negative (median 4.0 m versus NR, p&amp;lt;0.01). Only 2/7 (29%) MRD-positive patients converted to MRD-negative while on maintenance. As mutations in NPM1, IDH1, or IDH2 may sensitize AML to venetoclax, we analyzed RFS based on the presence or absence of these mutations (figure 1). Patients with sensitizing mutations had a trend towards longer RFS (median RFS NR versus 23.5 m, 2-year RFS 79% vs 36%, p=0.08). The most common grade 3/4 adverse events were infections (26%), neutropenia (17%), thrombocytopenia (17%), hypotension (6%), and neutropenic fever (6%). 4 (11%) patients required cycle 2 venetoclax dose reductions. 8 (23%) of patients went off protocol to receive SCT. All observed deaths (n=8) occurred following AML relapse (n=7) or SCT-related complications (n=1). Conclusions: Lower dose azacitidine plus venetoclax is a feasible and safe maintenance strategy in AML. This approach yields encouraging results in MRD-negative patients with non-adverse ELN risk (favorable or intermediate risk). A genetic-guided strategy may help select patients who benefit the most from this combination (i.e. those with NPM1, IDH1, or IDH2 mutations).

Post-Hoc Analysis of Measurable Residual Disease from BMT-CTN 1506/Morpho: FLT3-ITD Variant Allele Frequency and Survival Are Highly Correlated

Background: BMT CTN 1506/MORPHO was a randomized study of the FLT3 inhibitor gilteritinib versus placebo as post-transplant (HCT) maintenance therapy for patients (pts) with FLT3-ITD acute myeloid leukemia (AML). Patients with FLT3-ITD AML in first remission underwent HCT and then were randomized, in double-blind fashion, to either gilteritinib or placebo for 24 months. The primary endpoint was relapse-free survival (RFS), and a pre-specified secondary endpoint was the effect of measurable residual disease (MRD) on survival as detected by a highly sensitive assay for FLT3-ITD mutations. In the primary analysis, while RFS was higher for pts randomized to gilteritinib, the difference did not reach the pre-determined threshold for significance (HR: 0.679; 95% CI: 0.459, 1.005; 2-sided p-value: 0.0518). However, in the secondary analysis, the 50.6% of pts who had MRD detectable pre- or post-HCT had significantly higher RFS with gilteritinib (HR of relapse or death=0.515, 95% CI: 0.316, 0.838, p = 0.0065). In this post-hoc analysis, we examined 1) the impact on RFS of different levels of FLT3-ITD variant allele frequency (VAF) pre-randomization (immediately before or after HCT but prior to randomization to gilteritinib or placebo); 2) the impact of the presence of multiple mutations detected as MRD pre-HCT; and 3) the eradication of FLT3-ITD clones detected post-HCT during follow up on gilteritinib or placebo. Methods: First-pull marrow aspirates were collected from pts at two time points pre-randomization (pre-HCT and between 30-90 days post-HCT), as well as at 3, 6, 12, 18, and 24 months post-randomization. For MRD detection (performed at Invivoscribe; San Diego, CA), 700 ng input DNA was amplified by polymerase chain-reaction (PCR) using 25 cycles and primers flanking exons 14 and 15 of FLT3, followed by next-generation sequencing (NGS) analysis of the amplicons. Variant allele frequency (VAF) was calculated as FLT3 mutant reads/total FLT3 reads. For pts with multiple FLT3-ITD mutations, the VAF used in analysis was the sum of the VAFs for each FLT3-ITD variant. The lower limit of blank (LOB) of the assay is estimated to be 1 x 10 -6 VAF. For terminology, VAF &amp;gt; 1 x 10 -6 and &amp;lt; 1 x 10 -5 is referred to as MRD6, VAF &amp;gt; 1 x 10 -5 and &amp;lt; 1 x 10 -4 is MRD5, VAF &amp;gt; 1 x 10 -4 or greater is MRD4, and MRD0 equals no detectable MRD. Results: MRD was evaluated in 350/356 (98.3%) pts pre-HCT and 347/356 (97.5%) pts post-HCT. MRD was detected in 46% of pts pre-HCT and 19.9% prior to randomization, including 4.5% who did not have detectable MRD pre-HCT. The variant allele frequency (VAF) of detectable MRD ranged from a low of 1.09 x 10 -6 to a high of 3.0 x 10 -1. More than a single mutation was detected in 51 pts pre-HCT. Most of these 51 pts had 2 clones, but the total ranged from 2-9. The presence of more than one mutation detected as MRD immediately pre-HCT was associated with worse RFS compared with all other pts (MRD-positive with only one clone and MRD-negative), irrespective of treatment arm (Figure 1A; HR=2.428, 95% CI: 1.555, 3.792, P &amp;lt; 0.001). There was no significant difference in age, mutation length, VAF, or karyotype between pts with more than one mutation and those with single mutations. However, if the VAFs of pts with multiple FLT3-ITD mutations were summed and treated as a single value, then the median VAF for pts with multiple mutations was higher than for those with single mutations (2.14 x 10 -4 vs. 3.12 x 10 -5). The VAF (including the summed VAFs of pts with multiple mutations) at multiple levels correlated very closely with RFS after HCT. To illustrate this quantitative effect of peri-HCT FLT3-ITD VAF on RFS, Kaplan-Meier analysis of pts on the placebo arm grouped by MRD level is displayed in Figure 1B. In the 71 pts with MRD detectable post-HCT, the MRD was eradicated in 69% of pts on gilteritinib versus 44% of pts on placebo. Conclusions Pts with multiple FLT3-ITD mutations have a worse prognosis, which may simply be a reflection of increased disease burden (VAF). The quantity of FLT3-ITD MRD appears to highly correlate with outcome. Gilteritinib appears to augment the effect of HCT, as evidenced by increased eradication of MRD post-HCT. These data illustrate the potential utility of the PCR-NGS FLT3-ITD MRD assay in the management of pts with FLT3-ITD AML.