Molecular Measurable Residual Disease Research Articles

Clonal relapse dynamics in acute myeloid leukemia following allogeneic hematopoietic cell transplantation

AbstractPatients with acute myeloid leukemia (AML) who experience relapse following allogeneic hematopoietic cell transplantation (alloHCT) face unfavorable outcomes regardless of the chosen relapse treatment. Early detection of relapse at the molecular level by measurable residual disease (MRD) assessment enables timely intervention, which may prevent hematological recurrence of the disease. It remains unclear whether molecular MRD assessment can detect MRD before impending relapse and, if so, how long in advance. This study elucidates the molecular architecture and kinetics preceding AML relapse by using error-corrected next-generation sequencing (NGS) in 74 patients with AML relapsing after alloHCT, evaluating 140 samples from peripheral blood collected 0.6 to 14 months before relapse. At least 1 MRD marker became detectable in 10%, 38%, and 64% of patients at 6, 3, and 1 month before relapse, respectively. By translating these proportions into monitoring intervals, 38% of relapses would have been detected through MRD monitoring every 3 months, whereas 64% of relapses would have been detected with monthly intervals. The relapse kinetics after alloHCT are influenced by the functional class of mutations and their stability during molecular progression. Notably, mutations in epigenetic modifier genes exhibited a higher prevalence of MRD positivity and greater stability before relapse, whereas mutations in signaling genes demonstrated a shorter lead time to relapse. Both DTA (DNMT3A, TET2, and ASXL1) and non-DTA mutations displayed similar relapse kinetics during the follow-up period after alloHCT. Our study sets a framework for MRD monitoring after alloHCT by NGS, supporting monthly monitoring from peripheral blood using all variants that are known from diagnosis.

Targeting Molecular Measurable Residual Disease and Low-Blast Relapse in AML With Venetoclax and Low-Dose Cytarabine: A Prospective Phase II Study (VALDAC).

A prospective phase II study examined the safety and efficacy of venetoclax combined with low-dose cytarabine (LDAC) in AML at first measurable residual disease (MRD) or oligoblastic relapse. Patients with either MRD (≥1 log10 rise) or oligoblastic relapse (blasts 5%-15%) received venetoclax 600 mg once daily D1-28 plus LDAC once daily D1-10 in 28-day cycles. The primary objective was MRD response in the MRD relapse cohort or complete remission (CR/CRh/CRi) in the oligoblastic relapse cohort. Forty-eight adults with either MRD (n = 26) or oligoblastic (n = 22) relapse were enrolled. Median age was 67 years (range, 18-80) and 94% had received previous intensive chemotherapy. Patients received a median of four cycles of therapy; 17% completed ≥12 cycles. Patients with oligoblastic relapse had more grade ≥3 anemia (32% v 4%; P = .02) and infections (36% v 8%; P = .03), whereas grade 4 neutropenia (32 v 23%) or thrombocytopenia (27 v 15%) were comparable with the MRD relapse cohort. Markers of molecular MRD relapse included mutant NPM1 (77%), CBFB::MYH11 (4%), RUNX1::RUNX1T1 (4%), or KMT2A::MLLT3 (4%). Three patients with a log10 rise in IDH1/2 (12%) were included. By cycle 2 in the MRD relapse cohort, a log10 reduction in MRD was observed in 69%; 46% achieved MRD negative remission. In the oligoblastic relapse cohort, 73% achieved CR/CRh/CRi. Overall, 21 (44%) underwent hematopoietic cell transplantation. Median overall survival (OS) was not reached in either cohort. Estimated 2-year OS rate was 67% (95% CI, 50 to 89) in the MRD and 53% (95% CI, 34 to 84) in the oligoblastic relapse cohorts. For AML in first remission and either MRD or oligoblastic relapse, venetoclax plus LDAC is well tolerated and highly effective.

Postinduction molecular MRD identifies patients with NPM1 AML who benefit from allogeneic transplant in first remission

AbstractSelection of patients with NPM1-mutated acute myeloid leukemia (AML) for allogeneic transplant in first complete remission (CR1-allo) remains controversial because of a lack of robust data. Consequently, some centers consider baseline FLT3–internal tandem duplication (ITD) an indication for transplant, and others rely on measurable residual disease (MRD) status. Using prospective data from the United Kingdom National Cancer Research Institute AML17 and AML19 studies, we examined the impact of CR1-allo according to peripheral blood NPM1 MRD status measured by quantitative reverse transcription polymerase chain reaction after 2 courses of induction chemotherapy. Of 737 patients achieving remission, MRD was positive in 19%. CR1-allo was performed in 46% of MRD+ and 17% of MRD− patients. We observed significant heterogeneity of overall survival (OS) benefit from CR1-allo according to MRD status, with substantial OS advantage for MRD+ patients (3-year OS with CR1-allo vs without: 61% vs 24%; hazard ratio [HR], 0.39; 95% confidence interval [CI], 0.24-0.64; P < .001) but no benefit for MRD− patients (3-year OS with CR1-allo vs without: 79% vs 82%; HR, 0.82; 95% CI, 0.50-1.33; P = .4). Restricting analysis to patients with coexisting FLT3-ITD, again CR1-allo only improved OS for MRD+ patients (3-year OS, 45% vs 18%; compared with 83% vs 76% if MRD-); no interaction with FLT3 allelic ratio was observed. Postinduction molecular MRD reliably identifies those patients who benefit from allogeneic transplant in first remission. The AML17 and AML19 trials were registered at www.isrctn.com as #ISRCTN55675535 and #ISRCTN78449203, respectively.

An audit of molecular measurable residual disease testing for cases of acute myeloid leukaemia diagnosed in the Western Australian public health system between 2019 to 2022

An audit of molecular measurable residual disease testing for cases of acute myeloid leukaemia diagnosed in the Western Australian public health system between 2019 to 2022

Prophylactic maintenance with venetoclax/azacitidine after reduced-intensity conditioning allogeneic transplant for high-risk MDS and AML

AbstractWe conducted a phase 1 trial assessing safety and efficacy of prophylactic maintenance therapy with venetoclax and azacitidine (Ven/Aza) for patients with high-risk myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML) undergoing reduced intensity allogeneic stem cell transplantation (allo-SCT) after Ven and fludarabine/busulfan conditioning (Ven/FluBu2 allo-SCT) with tacrolimus and methotrexate as graft-versus-host disease (GVHD) prophylaxis. Among 27 patients who underwent Ven/FluBu2 allo-SCT (55.6% with prior Ven exposure, and 96% with positive molecular measurable residual disease), 22 received maintenance therapy with Aza 36 mg/m2 intravenously on days 1 to 5, and Ven 400 mg by mouth on days 1 to 14 per assigned dose schedule/level (42-day cycles × 8, or 28-day cycles × 12). During maintenance, the most common grade 3-4 adverse events were leukopenia, neutropenia, and thrombocytopenia, which were transient and manageable. Infections were uncommon (n = 4, all grade 1-2). The 1-year and 2-year moderate/severe chronic GVHD rates were 4% (95% confidence interval [CI], 0.3%-18%) and 22% (95% CI, 9%-40%), respectively. After a median follow-up of 25 months among survivors, the median overall survival (OS) was not reached. Among the 22 patients who received Ven/Aza maintenance, the 2-year OS, progression-free survival, nonrelapse mortality, and cumulative incidence of relapse rates were 67% (95% CI, 43%-83%), 59% (95% CI, 36%-76%), 0%, and 41% (95% CI, 20%-61%), respectively. Immune monitoring demonstrated no significant impact on T-cell expansion but identified reduced B-cell expansion compared with controls. This study demonstrates prophylactic Ven/Aza maintenance can be safely administered for patients with high-risk MDS/AML, but a randomized study is required to properly assess any potential benefit. This trial was registered at www.clinicaltrials.gov as #NCT03613532.

Multi-gene measurable residual disease assessed by digital polymerase chain reaction has clinical and biological utility in acute myeloid leukemia patients receiving venetoclax/azacitidine

Venetoclax with azacitidine (ven/aza) is a lower-intensity therapeutic regimen that has been shown to improve outcomes in elderly patients with acute myeloid leukemia (AML). Measurable residual disease (MRD) using flow cytometry is a valuable tool for the prediction of relapse in AML using conventional therapies and ven/aza; however, the prognostic value for broad-scale molecular MRD after ven/aza treatment is less clear. We aimed to determine the utility of retrospective assessment using multi-gene molecular MRD by droplet digital PCR (ddPCR). We found this approach correlates with outcomes in a cohort of patients receiving frontline ven/aza for AML. The predictive value of ddPCR MRD persisted when NPM1 mutations were removed from analysis, as well as after adjustment for the impact of stem cell transplant (SCT) on outcomes. Late achievement of MRD negativity, including after SCT, was still associated with superior outcomes compared to persistently detectable MRD. We further explored the impact of ven/aza on the burden of different classes of mutations, and identified the persistence of splicing factor mutations, commonly associated with MDS, as a consistent finding after ven/aza treatment. These data add to our understanding of the effects of ven/aza on AML disease biology and provide details on molecular depth of remission that can guide prospective trials in the future.

Development of Circulating Tumor DNA (ctDNA) for Molecular Measurable Residual Disease (MRD) in Acute Myeloid Leukemia (AML)

Introduction: Despite advances in therapeutic options, the majority of AML patients experience relapsed or refractory disease. Standard-of-care (SOC) methods for MRD detection (multiparametric flow cytometry and single gene or structural variant molecular assays) are hampered by (1) inadequate lower limits of detection (LOD), (2) paucity of recurrent mutations across AML genomes, (3) insufficient numbers of identified mutations to characterize clonal evolution, and/or (4) need for invasive bone marrow biopsy. Herein we develop and apply non-invasive circulating tumor DNA (ctDNA) liquid biopsies using a customized Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) approach to simultaneously quantify MRD and characterize clonal dynamics in AML. Methods: To increase the number of tumor-specific mutations per case for MRD detection and improve the LOD for ctDNA-MRD, we performed whole exome sequencing (WES) on purified blasts and paired T cells (latter as germline control) from 24 patients with AML (n = 20 previously untreated, n = 4 with relapsed/refractory disease) and designed personalized hybrid capture panels. We combined the personalized panels with a shared hybrid capture panel of 58 recurrently mutated (‘canonical‘) AML genes covering 225kB as a composite approach to generate patient-specific AML-CAPP-Seq assays. We applied AML-CAPP-Seq to track single nucleotide variants (SNVs) in 468 biological samples from the 24 AML patients and 21 healthy control subjects. We compared the performance of MRD detection in cellular (bone marrow mononuclear cell/BMMC, peripheral blood mononuclear cell/PBMC) and cell-free (BM-ctDNA, PB-ctDNA) compartments against SOC clinical MRD assays as well as patient specific clinical outcomes. Results: We observed a median of 30 SNVs (IQR 16.5-40.25) per patient from WES, compared to only 2 (IQR 1-3.25) if using the AML-focused canonical panel alone (p &amp;lt; 0.0001) ( Fig. 1A). This number of variants enabled sensitive molecular disease monitoring in 96% (23/24) of patients with a LOD of &amp;lt; 0.01% tumor fraction from typical quantities of cell-free DNA. In contrast, when using the canonical panel alone, only 83% (20/24) of patients had 1 or more SNV to allow MRD detection. Notably, only 12.5% (3/24) of patients in this cohort had a feature amenable to SOC molecular MRD methods. Upon comparing quantitative levels of MRD in each biological compartment, we found that tumor burden in PB-ctDNA, BM-ctDNA and BMMC samples were highly correlated (r &amp;gt; 0.92, p &amp;lt; 0.0001) with similar amounts of disease. Interestingly, PB-ctDNA contained quantitatively more tumor DNA than corresponding PBMC samples (p = 0.0002), most pronounced at low tumor burdens (median 8.2-fold enrichment when mean tumor allele fraction in PBMCs was &amp;lt; 1%). These results suggest that PB-ctDNA effectively captures total body BM disease and mutational burden in AML and serves as a superior analyte for MRD monitoring compared to PBMCs. When evaluating the performance of AML-CAPP-Seq against clinical SOC methods, our assay correctly detected molecular disease in 99.6% (237/238) of samples known to contain measurable disease, including 98.2% (54/55) of samples at timepoints of clinical MRD positivity during clinical remission (CR). However, restricting trackable SNVs to only ‘canonical’ genes led to significant degradation of performance. Specifically, only 69.1% (38/55) of samples with positive MRD during CR by clinical SOC assay were detected by ctDNA if canonical genes alone were employed (p = 0.0001). Intriguingly, when using the complete gene panel, AML-CAPP-Seq also identified molecular MRD in 37.5% (30/80) of samples with undetectable MRD by SOC assays. Finally, stratifying patients who achieved CR after 1st treatment line based on PB-ctDNA MRD significantly predicted relapse free survival (p = 0.046, hazard ratio (HR) = 4.5). Moreover, attaining an MRD negative CR in PB-ctDNA at any timepoint was strongly prognostic of overall survival (p = 0.0004, HR = 8.5) ( Fig. 1B). Conclusions: ctDNA is a promising biomarker for ultrasensitive detection and tracking of MRD in AML without need for invasive bone marrow assessment. Tracking mutations beyond canonical AML genes improves performance of such assays. ctDNA analysis has numerous areas of clinical utility, including response and relapse prediction, evaluation of clonal dynamics, and exploration of mechanisms of therapy resistance.

Molecular Measurable Residual Disease (MRD) Clearance (≤1%) Is Associated with Improved Clinical Outcomes in Patients with Higher-Risk Myelodysplastic Neoplasms (HR-MDS): An Exploratory Analysis of Stimulus-MDS1 in Patients Receiving Sabatolimab or Placebo + Hypomethylating Agent (HMA)

Background: Sabatolimab (MBG453) is a novel immunotherapy targeting TIM-3, an immuno-myeloid regulator expressed on both immune and leukemic stem cells. In the randomized, double-blind, placebo-controlled, Phase (Ph) II STIMULUS-MDS1 study (NCT03946670) in patients (pts) with HR-MDS, although improvements in complete remission (CR) and progression-free survival (PFS) were not statistically significant, sabatolimab + HMA was associated with longer duration of response (DoR), and the data suggest a delayed-onset benefit in the sabatolimab arm, as well as a potential treatment effect in pts with lower disease burden (Zeidan AM, et al. ASH 2022). The clinical significance of MRD in HR-MDS is not well established. We report an exploratory analysis of mutational clearance and MRD by next-generation sequencing (NGS) to evaluate the association with clinical outcomes. Methods: Treatment-naive pts aged ≥18 years with intermediate (+ ≥5% bone marrow [BM] blasts), high or very high risk MDS by Revised International Prognostic Scoring System were randomized to sabatolimab or placebo added to azacitidine/decitabine (Zeidan AM, et al. ASH 2022). Peripheral blood (PBMC) and/or BM mononuclear cell (BMMC) samples were collected and NGS performed on genomic DNA extracted from baseline (BL) and on-treatment samples using a 38-gene NGS-error-corrected panel with sensitivity of 2% and 0.2% variant allelic frequency (VAF), respectively. Concordance and correlation between PBMC and BMMC were assessed. The prognostic value of clonal clearance at different VAF cut-offs (0.2%, 0.5% or 1%) was assessed. MRD-x status was defined as any mutation call above VAF cut-off x, excluding mutations in DNMT3A, TET2 and ASXL1 ( DTA). Association between MRD status and clinical outcomes was analyzed regardless of treatment to increase sample and used best overall response, 6-month landmark analysis, and a time-dependent Cox model to rule out immortal bias. Results: NGS was run on 332 BMMC samples from 112 pts and 439 PBMC samples from 123 pts (127 pts randomized). 112 pts had ≥1 qualifying BL mutation (NGS cohort) and 106 pts (sabatolimab + HMA, N=56; placebo + HMA, N=50) had ≥1 at follow-up (MRD cohort). BL characteristics were balanced between NGS/MRD cohorts and between treatments in the MRD cohort. Overall agreement in mutation calling at variant level between BMMC and PBMC was &amp;gt;97% (8312/8566 total mutation calls), with &amp;gt;88% positive (817/926) and &amp;gt;98% negative agreement (7495/7640). Across reported mutated variants (N=817), there was good correlation (pearson cor=0.88) in VAF between BMMC and PBMC. When considering all paired samples, concordance of best MRD status using PBMC or BMMC results at the pt level (N=95) was: MRD-0.2, 97%; MRD-0.5, 89%; MRD-1, 90%. Given good PBMC and BMMC agreement, results were combined (higher VAF used if both) for outcomes analysis. In the MRD cohort, 11% (N=12), 18% (N=19) and 27% (N=29) of pts achieved MRD-0.2, MRD-0.5 and MRD-1 negativity, respectively. Most pts with MRD-0.2, -0.5 and -1 clearance had documented response of CR/marrow CR [mCR] (&amp;gt;80%) or CR/partial remission [PR]/hematologic improvement [HI] (&amp;gt;75%). Best overall response of MRD-1 negativity achieved anytime on treatment was associated with improved PFS and overall survival (OS) in the overall population and for responding pts (CR/mCR or CR/PR/HI). Similar results were obtained in a landmark analysis at 6 months ( Fig 1). This was supported by a time-dependent Cox-model indicating that MRD-x negativity was associated with a lower hazard ratio for PFS and OS vs MRD-x positivity ( Fig 2). A consistently higher proportion of pts in the sabatolimab (N=56) vs placebo (N=50) arms had on-treatment mutation clearance at different cut-offs: MRD-0.2, 16.1% vs 6.0%; MRD-0.5, 25.0% vs 10.0%; MRD-1, 35.7% vs 18.0%, respectively. Conclusions: We present the first results from a prospective, controlled, randomized study demonstrating the potential prognostic value of MRD for PFS and OS in HR-MDS. Our results demonstrate high NGS concordance between PBMC and BMMC on-treatment samples, indicating that PBMC could represent an alternative for clonal monitoring. Interestingly, more pts treated with sabatolimab + HMA reached MRD negativity while in remission, potentially explaining the longer DoR in pts receiving sabatolimab + HMA vs placebo + HMA. These findings will be further explored in the ongoing Ph III STIMULUS-MDS2 study (NCT04266301).

Molecular Measurable Residual Disease in Patients with Newly Diagnosed m IDH1 Acute Myeloid Leukemia Treated with Ivosidenib + Azacitidine

Background: Ivosidenib (IVO) + azacitidine (AZA) improved complete remission (CR) rates and overall survival (OS) relative to placebo (PBO) + AZA in patients (pts) with newly diagnosed mutant isocitrate dehydrogenase 1 (m IDH1) acute myeloid leukemia (AML) in the pivotal AGILE study (Montesinos et al. NEJM 2022). Prior analyses showed clinical responses to IVO+AZA were associated with deep clearance of m IDH1 (limit of detection [LOD]: 0.02-0.04% variant allele frequency [VAF]), as well as clearance of baseline co-mutations below the threshold of conventional next-generation sequencing (NGS) (LOD: 2.0% VAF) (Döhner et al. Blood 2022; Daigle et al. Blood 2021). Measurable residual disease (MRD), a negative prognostic marker, following chemotherapy is often detected via multiparametric flow cytometry or quantitative polymerase chain reaction although both techniques have their limitations. (Heuser et al. Blood 2021). NGS can detect a wide range of AML mutations and studies show that NGS-assessed MRD is highly predictive of both relapse and survival (Thol et al. Blood 2018; Tsai et al. Blood Adv 2021). However, a limitation of NGS is difficulty in distinguishing between somatic leukemia-associated mutations and clonal hematopoiesis of indeterminate potential (CHIP) (Bacher et al. Blood Cancer J 2018). We aimed to perform an NGS analysis of MRD responses in the AGILE study. Methods: All AGILE pts who had a best overall response (BOR) of CR, CR with incomplete count recovery, or CR with incomplete platelet recovery, and had ≥1 on-treatment bone marrow mononuclear cell (BMMC) sample available were included. Suggested timepoints for MRD testing included day 1 of treatment cycles 3, 5, 7, 9, 11, 14, 20, 26, and 32. Baseline and on-treatment BMMC DNA samples were analyzed using a diagnostic 51-gene myeloid NGS panel (LOD: 3% VAF) and a 26-gene AML MRD panel, respectively. For variants detected at baseline, the LOD was 0.1% VAF; for variants not detected at baseline (either emerging mutations or if baseline samples were unavailable), the LOD was 0.5% VAF. All variants above the applicable LOD with known or potential clinical significance were considered evidence of positive MRD, aside from DNMT3A, TET2 and ASXL1 (“DTA”) mutations to reduce the risk of false-positive MRD due to CHIP. Results: The analysis set comprised 173 samples from 33 IVO+AZA-treated pts and 10 PBO+AZA-treated pts (Fig. 1). Clinical response data were based on a March 18, 2021 primary analysis data cut; OS data were based on an updated June 30, 2022 data cut. The median number of baseline mutations was 4 (range: 1-10) per patient, median number of MRD assessments was 3 (range: 1-9) and median follow-up period was 189 days (range: 49-875). Ten (30.3%) IVO+AZA-treated pts became MRD-negative (MRD neg), compared with 2 (20.0%) PBO+AZA-treated pts. All pts attaining an MRD neg response had a BOR of CR (10 [34.5%] of 29 IVO+AZA-treated pts in CR; 2 [22.2%] of 9 PBO+AZA-treated pts in CR). Seven (70.0%) of the 10 MRD neg IVO+AZA-treated responding pts converted to an MRD neg response by day 1 of cycle 7 (C7D1). Fewer pts with an MRD neg response had an Adverse risk at baseline according to European LeukemiaNet 2022 genetic risk classification versus those pts with an MRD-positive (MRD pos) response (26.3% vs 73.7%) (Table 1). In the IVO+AZA arm, rates of event-free survival at 12 months and OS at 24 months were numerically higher in MRD neg vs MRD pos responders (80.0% vs 60.6% and 88.9% vs 72.3%, respectively) but did not reach statistical significance. All 5 (15.2%) IVO+AZA-treated pts with confirmed relapses had detectable MRD at the last completed assessment prior to relapse. One pt converted from an MRD neg to an MRD pos response (“MRD relapse”) 169 days prior to overt clinical relapse. Among the 23 IVO+AZA-treated pts without an MRD neg response, 16 were alive as of the updated data cut; OS duration ranged from 20.3-48.9 months. Conclusions: IVO+AZA induced molecular MRD negativity in approximately one third of responding pts with newly diagnosed m IDH1 AML; MRD neg responses were most often observed on or before C7D1. Interestingly, several pts had durable clinical responses and OS despite ongoing MRD positivity, potentially due to persistent CHIP rather than true MRD, illustrating the complexity of interpreting NGS MRD data. Additional analyses, including associations between specific molecular alterations, MRD response, and OS are ongoing and will be presented.

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&amp;gt;1x10 -3 (cut-off determined by ROC curve method, qMRD with respect to disease levels found at disease onset) and 136 with qMRD&amp;lt;1x10 -3 after the II induction course. Patients with qMRD&amp;gt;1x10 -3 had a significantly worse OS and EFS with respect to patients with qMRD&amp;lt;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&amp;gt;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&amp;lt;1x10 -3 after the second induction course. By contrast, qMRD&amp;gt;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&amp;gt;1x10 -3 vs 88% for qMRD&amp;lt;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&amp;lt;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&amp;gt;1x10 -3 had a worse EFS with respect to patients with lower levels of qMRD (39% n=30 vs 80% n=82, p&amp;lt;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.

Long-Term Follow-up of AML Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation after Primary Induction Failure

Introduction Allogeneic hematopoietic stem cell transplantation (HCT) is the standard consolidation treatment for most patients with adverse-risk acute myeloid leukemia (AML). Achieving complete remission (CR) before HCT has been associated with improved clinical outcomes. Thus, AML patients with primary induction failure (PIF) are often not considered for immediate allogeneic HCT, despite being the only curative option. Most recently, Stelljes et al. reported similar survival rates after a median follow-up of 37 months for relapsed/refractory AML patients who either proceeded directly to allogeneic HCT or underwent intensive remission induction prior to HCT within the ASAP trial (Stelljes et al., ASHAnnual Meeting 2022). Yet, the effect of immediate allogeneic HCT in this clinical setting for long-term prognosis is still largely unclear. Thus, we explored long-term clinical outcomes of AML patients with PIF undergoing allogeneic HCT with active disease at our institution over a period of 30 years between 1989 and 2019, and investigated associations with molecular and clinical features. Methods We retrospectively analyzed AML patients undergoing allogeneic HCT after PIF at the University Medical Center Freiburg (Germany) between 1989 and 2019. PIF was defined as never achieving CR during induction or re-induction, assessed either by cytomorphology or molecular measurable residual disease as defined by the NCI (Version 23.06d, Code C70622). All clinical examinations and laboratory analyses were performed as part of standard clinical care. Statistical analyses were performed considering the entire cohort as well as distinct transplant periods: i) 1989-2000, ii) 2001-2010, and iii) 2011-2019. Primary endpoints were disease-free survival (DFS) and overall survival (OS). Other endpoints were time to relapse and time to non-relapse mortality (NRM). Results We included 220 patients in this retrospective single-center study, with a median follow-up of 8.5 years (range: 0.06-25.4). Median age of patients was 55 years (range: 20-75), increasing over time as reduced toxicity conditioning became more established (1989-2000: 13.9%; 2011-2019: 61.8%). Patients received in median two lines of treatment before HCT (range: 1-6). Blast count in bone marrow before initiation of allogeneic HCT was in median 36% (range: 0-95%). Molecular risk was assessed in 63.2% of patients, while genetic information was missing in 36.8% of AML cases. 39.8% of AML patients were disease-free after 1 year, 25.2% after 5 and 18.7% after 10 years from allogeneic HCT (Fig. 1A). 206 patients (93.6%) were alive at day 30 after HCT and 59 patients (26.8%) were alive at the time of the analysis. Median OS was 1.05 years, with 1-year, 5-year, and 10-year OS rates being 50.4%, 29.8%, and 21.6%, respectively (Fig. 1A). Survival rates were largely stable over time. 5-year OS in patients treated between 1989-2000 was 28.7%, in those receiving HCT between 2001 and 2010 28.7%, and 30.3% in patients transplanted between 2011 and 2019 (Fig. 1A). NRM was 29.3% after 5 years and 32.5% after 10 years (Fig. 1A). Over time, relapse rates decreased, with 5-year relapse rates of 45.7% between 1989 and 2000 and 33.3% between 2011 and 2019 (Fig. 1A). The main cause of death after HCT was AML progression/relapse (57.8%), while infections (19.3%), GvHD (11.2%), and treatment-related toxicity (8.4%) were leading causes of NRM. In Cox regression analyses incorporating known molecular and clinical risk factors, poor performance status (ECOG &amp;gt; 1 vs. 0-1; DFS: p=0.001, HR 2.26, 95% CI 1.37-3.73; OS: p=8*10 -5, HR 2.76, 95% CI 1.66-4.58,), adverse genetic risk at AML diagnosis (vs. intermediate/favorable risk; DFS: p=0.03, HR 1.6, 95% CI 1.1-2.4; OS: p=0.01, HR 1.7, 95% CI 1.1-2.6), and long diagnosis-to-HCT interval (DFS: p=0.01, HR 2.7, 95% CI 1.2-6.1; OS: p=0.005, HR 3.1, 95% CI 1.4-6.8) were strongly and independently associated with unfavorable DFS and OS (Fig. 1B). Conclusion We here demonstrated that immediate allogeneic HCT in AML patients with active disease represents a valid alternative to intensive remission induction prior to HCT and provides long-term survival and cure in a significant proportion of patients, suggesting to proceed with allogeneic HCT in patients with refractory disease as soon as a donor is available. The benefit of hypomethylating agents and venetoclax therapy prior to allogeneic HCT remains to be elucidated.

The Benefit of Allogeneic Transplant in 1 st Complete Remission in NPM1 Mutated AML with or without FLT3 ITD Is Restricted to Those Testing MRD Positive after Induction - an Analysis of the UK NCRI AML17 and AML19 Studies

Background While there is accumulating evidence that measurable residual disease (MRD) negative patients with intermediate risk AML may not benefit from allogeneic transplantation in 1 st complete remission (CR1-allo), many consider the presence of a FLT3 Internal Tandem Duplication (ITD) an indication for this procedure. However, the studies supporting this practice were mainly performed before the development of sensitive molecular MRD techniques (Schlenk NEJM 2008).The UK NCRI AML17 and AML19 trials studied, then incorporated, MRD by RT-qPCR into the management of patients with NPM1 mutated AML. Methods NCRI AML17 (April 2009 to December 2014) and AML19 (November 2015 to November 2020) were sequential prospective randomised trials for younger adults (18 - 60 years) with newly diagnosed AML. In AML17, decisions regarding CR1-allo were made using a validated risk score incorporating baseline factors and response to induction. In AML19, for patients with NPM1 mutated AML ( NPM1mut) the protocol recommended CR1-allo only in patients testing MRD positive in the peripheral blood (PB) post cycle 2 (PC2), regardless of other baseline risk factors such as FLT3 ITD, based on findings from AML17 (Ivey et al, NEJM 2016). Both trials were performed prior to the availability of midostaurin and FLT3 ITD MRD assays. Here we compare the outcomes of patients with NPM1mut in these trials and assess the benefit of CR1-allo according to FLT3 ITD and molecular MRD status. Time-to-event variables are analysed using the Kaplan-Meier method, and the impact of alloSCT assessed using time-dependent Cox regression and visualised with Simon-Makuch plots. Molecular relapses were included as relapse events, and alloSCT performed after molecular relapse was not considered as CR1. Results 737 patients with NPM1mutAML who achieved CR/CRi and had a valid PC2 PB MRD result were included in the analysis. 60/348 (17%) patients from AML17 and 83/389 (21%) from AML19 tested PB PC2 MRD positive. Overall, CR1-allo was performed in 20% of patients in AML17 and 25% in AML19. Of the MRD positive patients CR1-allo was performed in 16/60 (27%) in AML17 compared to 50/83 (60%) in AML19 where these patients were recommended for transplant. CR1-allo was performed in 18% and 16% of MRD negative patients in AML17 and AML19 respectively. In AML17, as previously reported (Ivey et al, NEJM 2016), patients who were MRD+ had poor outcomes, with 3-year cumulative incidence of relapse (CIR) of 84% and overall survival (OS) of 25%. These outcomes were improved in AML19, with 3y CIR 50% and 3y OS 51%. Across both trials, CR1-allo was associated with a significant survival benefit in MRD+ patients (3y OS 61% vs 24%, HR 0.42, 95%CI 0.26-0.69, p&amp;lt;0.001, Figure A). Patients who were MRD negative had excellent outcomes in both trials (3y CIR 33% and 24% and 3y OS 75% and 83% for AML17 and AML19 respectively). AlloSCT in CR1 provided no survival advantage for MRD- patients (HR 0.83, 95%CI 0.51-1.34, p=0.4). There were a total of 286 patients with NPM1mutFLT3 ITD mut AML with a PC2 PB MRD result across both trials. 81/286 (28%) were PB PC2 MRD+ of whom 32/81 (40%) received CR1-allo (28% in AML17 and 49% in AML19). Outcomes in these high-risk patients were improved in AML19, with 3y CIR 91% vs 68% and 3y OS 22% and 31% for AML17 and AML19 respectively. Those who received CR1-allo had improved outcomes (3y OS 45% vs 18%, HR 0.52, 95% CI 0.29-0.93, p=0.03) consistent with the overall MRD+ population. In the 70% of NPM1mutFLT3 ITD mut AML patients who achieved PB PC2 MRD-, outcomes were encouraging in both AML17 (3y CIR 37%, 3y OS 75%) and AML19 (3y CIR 27%, 3y OS 80%). CR1-allo was performed in 20% (19% in AML17, 21% in AML19), with no survival benefit for transplant (HR 0.80, 95% CI 0.37-1.72, p=0.6, Figure B). There remained no benefit when restricted to those with high (&amp;gt;0.5) FLT3 ITD allelic ratio (HR 0.63, 95% CI 0.2 - 2.23, p = 0.50) and there was also no evidence of an interaction between allelic ratio and benefit from transplant for MRD negative patients (p=0.86). Conclusion Molecular MRD can be used to stratify decision making concerning post remission therapy in patients with NPM1mut AML. Patients who achieve MRD negativity in the peripheral blood by the end of course 2 are at low risk of relapse and we found no evidence of benefit from allogeneic transplant in CR1, including in the subset with FLT3 ITD. This finding contrasts with MRD+ patients where there was a significant survival benefit for CR1 transplant.

Deep Sequencing Is a Widely Applicable Tool for Relapse Prediction in Acute Myeloid Leukemia with Mutated NPM1

Analysis of measurable residual disease (MRD) is a powerful tool for assessment of treatment response in acute myeloid leukemia (AML). In patients with mutation in NPM1, reverse transcription quantitative polymerase chain reaction (RT-qPCR) is recommended by European Leukemia Net for refinement of risk stratification and monitoring after treatment. However, since RT-qPCR is mutation-specific and requires stringent quality control, many clinical laboratories restrict NPM1 monitoring to the type A mutation (c.860_863dupTCTG) although &amp;gt;50 mutations have been reported in exon 12. Deep sequencing is an alternative method, performed on DNA, that covers all NPM1 exon 12variants in the same assay. Guidelines for its clinical use are however lacking. We here performed a retrospective analysis in a population-based cohort of AML patients to evaluate if deep sequencing MRD analysis of NPM1 during chemotherapy provides prognostic information. Adult patients with AML with mutated NPM1 treated with curative intent in our region during 2006-2016 who achieved complete remission (CR) or CR with incomplete count recovery (CRi) were eligible for the study. This time period was chosen to avoid inclusion of patients treated based on molecular MRD status. Through review of the national AML registry, clinical charts, and screening of diagnostic samples, 99 patients with mutated NPM1 were identified. Of these, 97 patients (60 women and 37 men, median age 64 years, range 19-82) had bone marrow slides for isolation of DNA from at least one time point during treatment. DNA from 257 bone marrow aspirate slides were assessed for MRD using deep sequencing of NPM1. Based on previous comparison with RT-qPCR (Pettersson et al, Int J Lab Hematol 2021;43:664-674), variant allele frequency (VAF) of ≥0.05% was chosen as cut-off to define deep sequencing MRD positivity (MRD pos). For subgroup analyses, patients were divided into groups of treatment intensity; high-dose (cytarabine- and daunorubicin-based, n=67) or low-dose (cytarabine- and daunorubicin-, or azacytidine-based, n=30) treatment regimens. Thirty-one patients underwent allogeneic stem cell transplantation (alloSCT). We first evaluated if deep sequencing MRD status during consolidation (n=70, analyzed after 2 or 3 cycles) had an impact on prognosis. Both 3-year relapse-free survival (RFS) (26.7±11.4% vs 71.6±6.2%, p=&amp;lt;0.001, Figure) and overall survival (OS) (33.3±12.2% vs 72.7±6%, p=0.004) were lower for patients that were MRD pos at any time point during consolidation than for MRD neg patients. Among MRD pos patients, 12/15 (80%) relapsed and 13/15 (87%) died, compared to 19/55 (35%) and 23/55 (42%) MRD neg patients. In univariate analyses, also age and treatment intensity, but not alloSCT or DNMT3A mutation status, predicted RFS and OS. FLT3-ITD predicted OS but not RFS, and there was no effect of FLT3-ITD allelic ratio. In Cox regression multivariate analysis including MRD, age, FLT3-ITD and treatment intensity, MRD pos was the only significant predictor of RFS (Hazard ratio (HR): 2.54, p=0.019). In the multivariate analysis, FLT3-ITD (HR: 2.43, p=0.014) and treatment intensity (HR: 0.43, p=0.035) were significant for OS (MRD pos HR: 1.75, p=0.13). When dividing the cohort based on treatment intensity, the effect of MRD status was most obvious in patients treated with low-dose regimens. When deep sequencing MRD analysis was performed post first cycle (n=96), it had no statistically significant effect on either RFS or OS. At the end of treatment (n=51), deep sequencing MRD status had a strong prognostic value with 3-year RFS and OS for MRD pos patients 30±14.5% and 40±15.5%, compared to 73.2±6.9% and 78±6.5% for MRD neg patients ( p=0.003 and p=0.006, respectively). In summary, this population-based study shows that MRD status by deep sequencing of NPM1 is predictive of both RFS and OS when assessed during or after consolidation. The study confirms the threshold of deep sequencing MRD pos of VAF 0.05% previously obtained by comparison with RT-qPCR. Since deep sequencing can be used for all NPM1 mutations, it is widely applicable and extends the use of molecular MRD analysis and risk refinement to patients with rare mutations in NPM1.

The Use of Targeted RNA- Sequencing Assay in the Diagnostic Evaluation of Acute Myeloid Leukaemia(AML)

Background: Acute Myeloid Leukaemia (AML) is a clinically heterogenous disease characterised by chromosomal and genetic abnormalities which guide risk stratification and management. Hence timely cytogenetic and molecular profiling is essential to initiate appropriate treatment. Approximately 30% of AML cases have fusion genes. We use a combination of fluorescence in situ hybridization (FISH) and the Acute Leukaemia Diagnostic Panel Real-Time PCR assay and targeted RNA-sequencing (Archer Fusion Plex Pan-Heme panel) to rapidly screen for common chromosomal abnormalities and fusion genes. The Fusion Plex Pan-Heme panel is an Anchored Multiplex PCR (AMP™)-based next-generation sequencing (NGS) product to detect and identify fusions, point mutations and expression levels from ribonucleic acid (RNA) input. Both known and novel fusions can be identified. The Acute Leukaemia Diagnostic Panel (TruPCR Acute Leukaemia Diagnostic Panel) is a real time PCR assay that detects 14 fusion genes with 28+ breakpoints commonly associated with AML and ALL, and variants in 5 genes associated with AML. Aims: To evaluate the RNA-sequencing Kit to detect rearrangements, gene fusions on bone marrow specimens from newly diagnosed AML patients; and compare concordance of targeted RNA-sequencing, FISH and qPCR in identifying leukaemia fusion genes. Methods: We retrospectively reviewed FISH, qPCR and targeted RNA-sequencing data for new AML cases diagnosed in the specialist integrated haematological malignancy diagnostic service (SIHMDS) at University College London Hospital over a one year period: 01 January 2022 to 31 December 2022. Results: 87 cases were included. They are classified based on ELN Cytogenetic Risk Group and types of AML as per Table 1. 28 (31%) of 87 patients had a detectable fusion gene by targeted RNA-sequencing (Figure 1). 22/28 were detected by targeted RNA-sequencing, FISH and qPCR. The 6/28 fusion genes detected by targeted RNA-sequencing, but not by FISH /qPCR were: - TFG-ADGRG7 (n=1) - NUP98-NSD1 (n=1) - JAK2-ETV6 (n=1) - LMO1-RIC3(n=1) - SSBP2-CHD1 (n=1)-clinical significance not known - CBFB-MYH11 (n=1) rare breakpoint - detected by FISH, not by PCR The discordant results were due to unavailability of probes/genes in FISH and qPCR panels. Summary/Conclusion: The ability to risk-stratify newly diagnosed AML patients allows earlier initiation of targeted therapies. We found that targeted RNA-sequencing (Archer Fusion Plex) is sensitive and showed 100% concordance in identifying fusions associated with good cytogenetic risk AML. Targeted RNA-sequencing can also identify high risk fusion genes such as NUP98-NSD1 for which FISH fusion probes are not routinely used. These may have been otherwise undetected, but can subsequently be confirmed by RT-PCR and importantly allow the generation of molecular measurable residual disease (MRD) assays to assess treatment response. We conclude that targeted RNA-sequencing can be implemented in a diagnostic laboratory to complement FISH-analysis for the detection of recurrent and rare gene fusions in AML diagnosis.

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.

Real-World Experience with Selinexor -Containing Regimens in Post-Transplant AML/MDS Patients Who MRD Still Positive after First-Line Preemptive Treatment

Background: Hematopoietic stem cell transplantation (HSCT) is the only cure for acute myeloid leukemia (AML), but relapse is the most important cause of transplant failure. Patients with remission but measurable residual disease (MRD) positive have a higher probability of hematologic relapse. The MRD-directed preemptive treatments can spare some patients in remission from further therapy. The conventional first-line preemptive treatment has been ineffective in some patients, so it's necessary to find a new preemptive treatment to prevent relapse and improve the prognosis. Our study aimed to evaluate the efficacy and safety of selinexor-containing regimens in the preemptive treatment of MRD positive in post-transplant patients with AML or myelodysplastic syndrome (MDS). Methods: The study was conducted in Tongji Hospital from March 2022 to May 2023. The inclusion criteria of post-transplant AML/MDS patients included the followings: (a) Persistent MRD positive. (b) MRD turned negative after first-line preemptive treatment but was positive again. (c) Patients at high risk for hematologic relapse after transplantation; (d) AML-M4/5. The end of the follow-up was in June 2023. Bone marrow aspiration was assessed for MRD using multiparameter flow cytometry (MFC) combined with real-time quantitative polymerase chain reaction (RT-qPCR) as follows: 1) MRD assessed by MFC: MRD negativity was defined as &amp;lt;0.1%. 2) MRD assessed by RT-qPCR: Abnormal genes associated with prognosis were used for molecular MRD detection. 3) WT1 was used for molecular MRD assessment if no molecular marker was available at diagnosis. MRD negativity was defined as &amp;lt;0.6%. Results: A total of 11 patients were included in the study. There were 10 patients with AML and one patient with MDS, with a median age of 43 years old. Two patients had received autologous HSCT, nine patients had received allogeneic HSCT, of which two matched sibling donor, one matched unrelated donor, and six were haploidentical donor. All patients were WT1 positive and two of the cases were both WT1 and MFC positive. Seven patients with post-transplant MRD positive were treated with selinexor-containing regimens after first-line preemptive therapy (azacytidine, azacytidine combined with venetoclax, decitabine combined with venetoclax, etc.) failed or MRD positive again. The remaining four patients were treated directly with selinexor-containing regimens for personal reasons or with a diagnosis of AML-M4/5 may be resistant to venetoclax. The selinexor-containing regimens used in this study were either selinexor (35mg/m 2 biw) alone or in combination with other drugs (decitabine, azacytidine, venetoclax, low-dose cytarabine, and CAG (cytarabine, aclarubicin, G-CSF) regimen). MRD was evaluated after two weeks of the selinexor-containing regimens. And selinexor 20 to 40 mg per week was used as maintenance therapy after MRD converted to negative. Eight patients (72.7%) successfully cleared the MRD. Of the three patients who didn't clear the MRD, two patients successfully converted to negative after continuing the selinexor-containing regimens. It is worth mentioning that 7 of the 11 patients were treated with the selinexor-containing regimens after first-linepreemptive treatment failure, and six of them had successfully cleared the MRD (71.4%). At the end of follow-up, 10 (90.9%) patients survived and one (9.1%) died due to disease progression. In terms of hematological adverse events (AEs), grade 3-4 neutropenia occurred in nine (81.8%) patients, the median duration was 2 days (IQR 1,4). Grade 3-4 thrombocytopenia occurred in three (27.3%) patients, and the median duration was 5 days (range 4-7). Although the number of people with grade 3-4 neutropenia was large, the duration was short after using granulocyte colony-stimulating factor, and no patient developed a severe infection. Most of the non-hematological AEs were mild, including nausea (100.0%), fatigue (63.6%), hyponatremia (58.3%), and vomiting (45.5%). The AEs can be alleviated after symptomatic treatment and no patient discontinued treatment. Conclusion: Selinexor-containing regimens have been shown to be effective and well tolerated in post-transplant AML/MDS patients with MRD-positive, including those who have failed first-line preemptive treatment.

Multiparametric Flow Cytometry-MRD Assay: Lesson from Phase II Trail REL AML 001

Background: Core-binding factor (CBF) AML with RUNX1/RUNX1T1 (AML1/ETO) and CBFB/MYH11 fusion genes is known as a heterogenous group, accounting for about 10% to 15% of AML. In the Phase II Trial REL AML 001 (EudraCT Number 2017-002094-18; ClinicalTrials ID: NCT 03686345) adult CBF leukemia patients treated with a continuation therapy with Midostaurin were included, and the measurable residual disease (MRD) was performed by molecular MRD assessment (qPCR) and multiparametric flow cytometric-MRD (MCF-MRD) assay. The findings of MCF-MRD assay were compared to qPCR. Methods: 31 patients with AML1/ETO (n=15) and CBFB/MYH11 (n=16) were evaluated between December 2018 to July 2022 with both techniques. A total of 145 determinations (n. AML1/ETO = 56 and n. CBFB/MYH11= 89) were performed. MCF panel included 2 10-color tubes: CD15 FITC/ CD56 PE/ CD34 PerCP-cy5.5/ CD117 PE-cy7/ CD7 APC/ CD13 APC-R700/ CD19 APC H7/ HLADR V450/ CD45 V500C/ CD33 BV630, specific for RUNX1/RUNX1T1 cases and CD64 FITC/ CD2 PE/ CD34 PerCP-cy5.5/ CD117 PE-cy7/ CD200 APC/ CD13 APC-R700/ CD14 APC H7/ HLADR V450/ CD45 V500C/ CD33 BV630, specific for CBFB/MYH11 cases. A mean of 1,733,240 CD45 positive events were acquired, with a mean lower limit of detection (LLOD) of 0.001% (ranging 0.01-0.0003%). In 10 cases (6 RUNX1 and 4 CBFB/MYH11) the molecular and phenotypical features at onset were not available. MCF analyses were performed by InfinicytTM software and reference image tool was used wherever possible. Results: We found a concordance of 76% between MCF cases and RUNX1/RUNX1T1 qPCR, and a concordance of 83% between MCF cases and CBFB/MYH11 qPCR (Tables 1a and 1b). In relapsed cases we observed a dynamic and continuous increase of abnormal MCF events, that was not found in cases without clinical relapse although with positive qPCR (Table 2). The HLADR expression evaluated as the geo mean intensity at presentation and/or at relapse resulted higher in RUNX1/RUNX1T1 patients than in CBFB/MYH11 cases (29845 vs 8979, respectively, p=0.0039 Figure 1). Interestingly, we found in a single case with atypical CBFB/MYH11 fusion gene a high value of HLADR geo mean (44522). The down-regulated expression of HLADR in CBFB/MYH11 blasts may be a useful clue to distinguish AML blasts from the scarcely represented normal CD2 positive myeloid precursors, and from the strongly HLADR positive elements that are found in a regenerating bone marrow background. Conclusion: Although MCF-MRD assay showed lower sensitivity than CBF qPCR, MCF offered interesting informations about the dynamics of the abnormal clone size with time, evaluated as the increasing number of MRD clustering events, which may predict an impending relapse.

Myelodysplasia-Related Gene Mutations for NGS-Based MRD Assessment in Acute Myeloid Leukemia

Introduction: Measurable residual disease (MRD) plays an increasing role for treatment management of AML patients. While next-generation-sequencing (NGS) based MRD assessment can be well standardized, there is uncertainty about the type of gene mutations that associate with prognosis and are useful for MRD assessment. The 2022 ELN recommendations assign patients with at least one mutation in the genes ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1 or ZRSR2 to the subgroup of myelodysplasia-related gene mutations (MRGM), which is associated with poor prognosis. We aimed to define the prognostic effect of MRD after induction chemotherapy in AML patients with MRGM using MRG mutations for MRD assessment by NGS. Methods: We retrospectively collected bone marrow and peripheral blood samples from AML patients in complete remission (CR) or CR with incomplete hematologic recovery (CRi) after one to two cycles of 7+3-based standard induction chemotherapy (IC) with at least one MRGM detected at diagnosis by myeloid panel sequencing covering 48 AML associated genes. Patients treated with one course of IC were included if they proceeded to allogeneic hematopoietic cell transplantation (alloHCT) already after one cycle. Molecular MRD status was assessed using targeted NGS and bioinformatics error-correction with 0.01% sensitivity as described before using only MRGM as MRD markers. Results: MRD was assessed on 104 adult newly-diagnosed AML patients (median age 58.6) with a total of 166 MRGM, resulting in a median of one MRGM per patient (range 1-4). 43 (41.3%) patients had secondary or therapy-related AML and 20 (19.2%) had myelodysplasia-related cytogenetic abnormalities. 68 (65.4%) patients were MRD positive for at least one MRGM after one (18 patients (17.3%)) or two (86 patients (82.7%)) cycles of intensive chemotherapy, while 36 (34.6%) became MRD negative. Measured variant allele frequencies (VAF) of positive samples ranged from 0.02-43.1% (median VAF 1.34%).MRD positive patients were older and more likely to be male compared to MRD negative patients, while cytogenetic risk, WBC count, Hgb, platelet count and ECOG status at diagnosis were similar to MRD negative patients. 53 (77.9%) MRD positive and 23 (63.9%) MRD negative patients underwent alloHCT in first CR/CRi.Median follow-up was 3.35 years. OS (P = 0.92), RFS (P = 0.99) and CIR (P = 0.98) were similar in MRD positive (at least one MRGM detectable) and negative (no MRGM detectable) patients (Figure 1). OS, RFS and CIR were also similar between MRD positive and negative patients, when MRD negativity was defined by conversion of at least one MRGM to negativity (55 (52.9%) MRD positive, 49 (47.1%) MRD negative), and when ASXL1 was excluded from the analysis. As variants with VAF &amp;gt;5% in CR/CRi samples may indicate clonal hematopoiesis, we excluded all variants with VAF &amp;gt;5% and repeated the analysis with the 88 (84.6%) remaining patients. Again, OS, RFS and CIR were similar for MRD positive and negative patients. We next reasoned that reduction of VAF correlates with the sensitivity of leukemic cells and possibly with depth of remission and long-term outcome. The fold-reduction of VAFs from diagnosis to the time of MRD analysis was calculated for all variants. For patients with several MRGMs, the variant with the largest fold-reduction was considered including undetectable variants. Patients with more or less than a 2-log, 3-log or 4-log reduction (log 10) had similar OS, RFS and CIR, respectively. Evaluating each MRGM individually, only RUNX1-MRD associated with RFS and by trend CIR (comparing MRD positive to negative patients for OS, HR 3.41, 95% CI 0.43-26.82, P = 0.24; RFS, HR 4.37, 95% CI 1.07-17.79, P = 0.04; CIR, HR 3.30, 95% CI 0.79-13.82, P = 0.1). Conclusion: 65.4% of AML-MRGM patients in CR/CRi remained MRD positive when MRGMs were used for MRD assessment by NGS. MRD positivity was associated with age above 60 years and male sex.Using all MRG mutations for MRD assessment at a threshold of 0.01% or by various degrees of log-reduction was not predictive of outcome after intensive induction chemotherapy. This limits the number of potential MRD markers for NGS analysis before alloHCT, although individual markers like RUNX1 may be of prognostic value.

Interaction between Presenting Features, Co-Occurring Mutations, MRD and Induction Treatment Influences Outcome in Adults with NPM1 Mutated AML - an Analysis of 1357 Patients in the UK NCRI AML17 and AML19 Studies

Background NPM1 mutated (mut) AML is considered favourable risk unless associated with FLT3 internal tandem duplication (ITD) or adverse karyotype. However many other genetic and clinical risk factors have been identified, including high white cell count (WCC), secondary disease, co-mutations and presence of a “triple hit” genotype ( NPM1mut, FLT3 ITD and DNMT3Amut). Although alternative classification systems have been proposed, few studies have been large enough to robustly evaluate clinical and molecular subgroups within NPM1mut, and none have incorporated the effects of measurable residual disease (MRD) status or treatment intensity. Therefore, clinical decision making remains challenging and inconsistent. Here, we performed comprehensive genotyping and molecular MRD assessment in two consecutive prospective randomised studies to clarify these issues. Methods NCRI AML17 (2009-2014) and AML19 (2015-2020) were large randomised trials for younger adults with newly diagnosed AML. Induction regimens included DA +/- etoposide (ADE) and FLAG-IDA, with or without Gemtuzumab. Patients underwent sequential MRD assessment by RT-qPCR, and diagnostic DNA samples were analysed by targeted NGS. Overall survival (OS) was calculated with the Kaplan-Meier method, while cumulative incidence of relapse (CIR) considered death as a competing risk. Relapse events included MRD relapse. All reported hazard/odds ratios are from multivariable analyses including age, prior haematological malignancy, prior chemo/radiotherapy, WCC (log transformed), cytogenetic risk and mutation profile ( FLT3 ITD, DNMT3A, PTPN11, N/KRAS, TET2, IDH1, IDH2, WT1, TP53 MDS-related genes). Results 1357 patients with NPM1mutAML were identified, 888 in AML17 and 469 in AML19. 1100 patients had DNA available for panel sequencing, and 981 underwent MRD monitoring of whom 737 had an evaluable post course 2 (PC2) peripheral blood (PB) sample. Morphological remission (CR+CRi) was achieved in 1264/1357 patients (93%) and PC2 PB MRD negativity in 594/737 (81%). We confirmed the adverse impact of PB PC2 MRD positivity: 3y CIR was 65% vs 29% in those MRD- (p&amp;lt;0.001), and 3yr OS was 40% vs 79% (p&amp;lt;0.001). For PB PC2 MRD+ patients, CIR was &amp;gt;50% in all subgroups. There was a lower rate of PC2 PB MRD negativity in patients with high presenting WCC (OR 1.14, p=0.05), FLT3 ITD (OR 2.58, p&amp;lt;0.001), DNMT3Amut (OR 2.2, p&amp;lt;0.001) and WT1mut (HR 2.04, p=0.02). There was no effect of FLT3 allelic ratio, while patients with both FLT3 ITD and DNMT3A mutations had the lowest rate of MRD- (64%, Figure A). Of the 594 PC2 MRD- patients, we observed higher 3y CIR in patients with high WCC (HR 1.14, p=0.01), DNMT3Amut (HR 1.83, p=0.001), WT1mut(HR 1.96, p=0.01) and IDH1mut (HR 1.9, p=0.002), and lower CIR in patients with N/KRASmut(HR 0.53, p=0.01). Although presence of FLT3 ITD alone was not associated with CIR (HR 0.88, p=0.7), patients with both FLT3 ITD and DNMT3A mutations had 3y CIR from MRD- of 40% (HR 1.74, p=0.03). MDS-related gene mutations had no impact on achieving MRD-, nor relapse from MRD-. Considering all patients, OS was poorer with increasing age (HR 1.03, p&amp;lt;0.001), WCC (HR 1.07, p&amp;lt;0.001), adverse karyotype (HR 2.48, p=0.01), FLT3 ITD (HR 1.34, p=0.01), DNMT3Amut (HR 1.56, p&amp;lt;0.001) and WT1mut(HR 1.71, p&amp;lt;0.001). 3y OS was 52% in patients with both FLT3 ITD and DNMT3Amut, 63% with FLT3 ITD only, 67% with DNMT3A only and 75% with neither. Considering MRD- patients, only age (HR 1.02, p=0.04), DNMT3Amut(HR 1.75, p=0.007) and WT1mut (HR 1.8, p=0.048) remained associated with OS. There was no benefit to CR1 allogeneic transplant in MRD- patients (HR 0.83, 95%CI 0.51-1.34, p=0.4), including in any subgroups (heterogeneity p=0.9). Compared with other treatments, FLAG-Ida increased MRD- (87% vs 79%, p=0.009), reduced relapse from MRD- (16% vs 34%, p&amp;lt;0.001) and improved 3y OS (79% vs 63%, HR 0.52, p&amp;lt;0.001). The OS benefit of FLAG-Ida was greatest in patients with at least one of the features associated with failure to achieve MRD- and relapse from MRD- (high WCC, triple hit or WT1mut, HR 0.39 95%CI 0.26-0.58, heterogeneity p&amp;lt;0.01). Conclusion The “triple hit” ( NPM1, DNMT3A and FLT3 ITD) genotype, WT1mut and high WCC were associated with poor outcomes, due to a lower probability of achieving MRD-, and a higher relapse risk from MRD- remission. Survival was improved for patients receiving intensified induction with FLAG-Ida including patients in these high-risk groups.

Venetoclax Combined with “7+3” Induction Chemotherapy Induces High Rates of MRD-Negative Remission in Newly Diagnosed AML Patients Fit for Intensive Chemotherapy Irrespective of Age

Introduction: Approximately 70% of patients (pts) with acute myeloid leukemia (AML) treated with the “7+3” regimen achieve complete remission (CR), with 30-50% of those attaining measurable residual disease (MRD) negative state by multiparameter flow cytometry (MFC). However, despite initial responses, the majority of pts eventually relapse. Venetoclax (Ven) added to intensive chemotherapy (IC) facilitates apoptosis of leukemic stem cells (LSC), and may result in deeper, more durable remissions. Moreover, IC induces MCL-1 downregulation and may overcome Ven resistance. We present safety and efficacy results from a phase 1b study of escalating cumulative Ven doses combined with daunorubicin (Dauno)+cytarabine (AraC) followed by high-dose cytarabine (HiDAC) chemotherapy in pts with newly diagnosed AML (NCT05342584). Objectives: The primary objective of this study is to determine the optimal dose of Ven in combination with Dauno &amp; AraC and HiDAC chemotherapy. Secondary objectives are response per 2022 ELN, survival (OS), event-free survival (EFS) and duration of response (DoR). A key exploratory objective is to assess rates of LSC eradication by means of MFC and single cell DNA (Tapestri, Mission Bio) and RNA sequencing (“high-resolution MRD assay”). Methods: Eligible pts must have a new diagnosis of AML, be 18-75 yrs of age and deemed fit for intensive chemotherapy. In a 3+3 design, Ven in escalating duration (400 mg D1-8 or -11 or -14, including 2-day ramp-up) is combined with AraC 100mg/m2 (D2-8) and Dauno 60mg/m2 (D2-4) in 2 separate age groups (&amp;lt;60 and ≥60yrs); Dauno 90mg/m2 was initially studied in pts &amp;lt; 60yrs, but since 04/2023 the dose is no longer explored. During consolidation, fixed dose Ven (200 mg x7days) is used in combination with age-adjusted HiDAC (1.5g/m2 in &amp;lt;60yrs and 1g/m2 in ≥60yrs, every 12hrs on days 1,3,5). In the expansion phase, additional pts in each age group will undergo 1:1 randomization between the starting and highest tolerated dose determined during dose escalation, for a total of 20 pts per dose cohort in both phases. Results: Twenty-seven AML pts enrolled and 25 completed the induction phase to date, 2 are actively treated. Median age was 59yrs, 44% were females and 67% ethnic or racial minorities. Fourteen (52%) pts had poor-risk genotypes by ELN 2022. Fourteen pts were treated with Ven x 8d, 11 in Dauno60 and 3 in Dauno90 cohorts, 7 pts with Ven x11d and 6 pts with Ven x14d. No DLTs were observed, and the toxicity profile was similar to historical experience with the “7+3” regimen. There were no induction mortality deaths. Twenty pts (20/25, 80%) achieved a composite complete remission (CRc) with single induction (19 CRs and 1 CRi), 10/13 in &amp;lt;60 and 10/12 in ≥60yr groups. Median time to ANC≥0.5K/uL and PLT≥50K/uL was 26 days and time to CR was 32 days. Of those in CRc, all except three pts &amp;lt; 60yrs were MFC-MRD negative (17/20, 85%). Among 8 patients with available MRD PCR testing, 6 achieved molecular MRD-negative and 2 low-level molecular MRD CRs. With a median 5.6-month follow-up time, median EFS or OS have not been reached, 25/27 (93%) patients are alive (10/12 ≥60yrs), 12 pts have completed the protocol, 5 continue on therapy and 5 underwent allogeneic transplantation. From those in CRc, 7 younger and 8 older pts (75%) remain in remission without evidence of hematologic or molecular relapse. Of 5 pts with complex karyotype plus TP53 mutated AML, 3 did not respond and 2 had an early treatment failure. Table 1 summarizes patient characteristics, toxicity and outcomes. Conclusions: Ven 400mg in combination with “7+3” induction chemotherapy is safe and highly effective in achieving MRD negative remissions in the vast majority of patients. Responses were low in TP53 mutated pts, suggesting no benefit of the addition of venetoclax to intensive chemotherapy, similar to what has been observed when venetoclax is added to standard dose hypomethylating agents in those pts, for whom non-cytotoxic approaches are being explored. The study is completing enrollment in the Dauno60 + Ven x14d cohorts and is entering the expansion phase in order to better explore the optimal Ven dose to combine with the “7+3” regimen. Updated results, as well as rates of LSC elimination post-induction will be presented at the meeting.