Measurable Residual Disease Negative Patients Research Articles

Assessment of Measurable Residual Disease on Day 28 of First Induction Chemotherapy in Acute Myeloid Leukaemia Patients

Background: Measurable residual disease (MRD) is an independent, strong, prognostic marker in acute myeloid leukaemia (AML) that is important for risk stratification and treatment planning. This study assessed the MRD on day-28 after first induction chemotherapy in AML patients. Objective: To assess measurable residual disease status on day 28 after the first induction chemotherapy in newly diagnosed acute myeloid leukaemia patients. Methodology: This cross-sectional study was conducted at the Department of Haematology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka from April 2021 to March 2022 among a total of 25 newly diagnosed AML patients who were admitted and received induction chemotherapy with DA (7+3). On Day-28, bone marrow aspiration was done with standard procedure and first pull of 2ml bone marrow sample was sent to a laboratory for MRD evaluation by multiparameter flow cytometry. MRD was considered positive when value was ?0.1%. Results: On day 28 of post induction chemotherapy most of the patients (92%) achieved morphological remission and among them more than half (52.2%) were MRD positive. The presence of CD14 myeloid marker and baseline higher serum uric acid level was significant in MRD positive patients and cytarabine dose 150 mg/m2 was found significant in MRD negative patients. Conclusion: More than half of the Acute Myeloid Leukaemia patients, treated with standard induction chemotherapy and achieved morphological remission on day 28 of chemotherapy, had positive measurable residual disease assessed by multiparameter flow cytometry. Positive MRD indicates relatively poorer prognosis and may necessitate more cycles of induction followed by more intensive consolidation which may include haematopoietic stem cell transplantation.

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 >5% in CR/CRi samples may indicate clonal hematopoiesis, we excluded all variants with VAF >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.

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<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 (>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.

Measurable Residual Disease Status By Clonotypic Mass Spectrometry with Easym Assay Predicts Outcomes Following Autologous Hematopoietic Cell Transplant in Multiple Myeloma

Bone marrow (BM)-based measurable residual disease (MRD) has been established as one of the most important dynamic prognostic markers in multiple myeloma (MM). However, the necessity of bone marrow biopsies limits the feasibility of frequent MRD monitoring. Consequently, there is an urgent unmet need for peripheral blood-based MRD testing for serial monitoring of disease response to allow tailored therapy for patients achieving deep remissions. Quantification of myeloma (M)-protein using mass spectrometry (MS) is a promising approach for peripheral blood-based MRD monitoring due to its exceptional analytical sensitivity. Prior studies have examined the use of MALDI-TOF-based methods, which relies on the mass distribution of intact immunoglobulins light chains and is approximately 1-log more sensitive than traditional testing. However, recent advances in MS technology have allowed determination and tracking of a “clonotypic” MS signature in the peripheral blood which is highly sensitive and patient-specific (Liyasova et al, CCR 2021). Prior reports have demonstrated the feasibility of this approach, but data supporting the association of blood-based clonotypic MS MRD with clinical outcomes is currently lacking. Consequently, we performed a large retrospective analysis of patients enrolled in our institutional MM tissue bank to determine the association of clinical outcomes with clonotypic MS-based MRD testing (EasyM, Rapid Novor, Kitchener, ON). We identified patients in our institutional tissue bank who met the following criteria: 1) diagnosis of MM, 2) underwent autologous hematopoietic cell transplant (AHCT), 3) M-protein ≥0.2 g/dL by serum protein electrophoresis (SPEP) or >20 mg/dL (light chain) at diagnosis, 4) had no detectable M-protein by SPEP at day +100 and 5) had adequate samples for analysis collected at day +100 after AHCT. Patients were included regardless of M-protein isotype. Baseline and follow up samples were analyzed by Rapid Novor without access to the clinical outcomes. Percent residual EasyM at day +100, in respect to baseline EasyM, was used as the independent variable to predict Progression-free survival (PFS). 95 patients were included in the analysis. Median age was 63, 88% were White and 61% were male. 64% were IgG, 20% IgA and 16% were with light chain (LC) MM. 26%, 62% and 12% were R-ISS stage I, II and III, respectively. High-risk cytogenetics were present in 32%. 97% had received proteasome inhibitors, 89% received immunodulatory agents and 13% anti-CD38 antibodies prior to AHCT. The median year of transplant was 2018 (range: 2008 - 2022). 93% patients received AHCT as part of frontline therapy. 66% were in CR or better following AHCT. 95% received maintenance after AHCT. 9 patients had non-quantifiable EasyM at follow up and were excluded from derivation of the MRD definition. Of the remaining 86, patients with IgA/LC and IgG disease had significant different percent residual EasyM following AHCT (median: 0.19% vs. 1.00%, p < 0.001). A cutoff of 1.036% residual EasyM was derived for IgG and 0.048% residual EasyM for IgA/LC patients was determined to fit the PFS data best, and based on these cutoffs, 41 patients (47%) were MRD negative by EasyM. The incidence of MRD negativity between IgG and IgA/LC was similar (55% vs. 32%). MRD positivity by EasyM was a strong predictor of PFS following AHCT (Figure 1) (HR: 2.249, p = 0.017). The median PFS in MRD positive patients was 3.4 years versus not reached in MRD negative patients (log rank: p = 0.014) (Figure 1). Our study is the first, to our knowledge, to demonstrate the clinical utility of peripheral blood-based clonotypic mass spectrometry to predict outcomes for patients with MM. Notably, our results suggest that different cutoffs should be employed for IgG and IgA/LC myeloma if drawn at day +100 after transplant. This is possibly due to the differing half-lives of these molecules. Significant additional questions remain, including the utility of ongoing monitoring at multiple time points and the optimal time post-AHCT to assess for MRD, especially in patients with IgG myeloma. Additionally, these findings require prospective validation in an independent cohort of patients to demonstrate wider applicability.

Assessment of Outcomes of Consolidation Therapy By Number of Cycles of Blinatumomab Received in Newly Diagnosed Measurable Residual Disease Negative Patients with B-Lineage Acute Lymphoblastic Leukemia: In the ECOG-ACRIN E1910 Randomized Phase III National Clinical Trials Network Trial

Introduction: ECOG ACRIN E1910 is a randomized phase III trial that showed that adults with newly diagnosed BCR::ABL1 negative acute lymphoblastic leukemia (ALL) who become MRD negative (<0.01%) after induction chemo who receive blinatumomab with conventional chemotherapy (chemo) have improved survival compared with those who received chemo only (Litzow et al, Blood (2022) 140: Supplement 2, LBA-1). However, not all pts were able to receive all four planned cycles of blinatumomab in consolidation. In this report we assessed outcomes of pts in the blinatumomab arm of the trial who received all 4 cycles of blinatumomab compared to those who received 1-2 cycles. Methods: Patient between the ages of 30 and 70 with newly diagnosed BCR::ABL1 negative B-lineage ALL were enrolled and initially received 2.5 months of combination induction chemo utilizing a BFM-like regimen adapted from the E2993/UKALLXII clinical trial, with pegaspargase (after induction only for patients >=55 years of age) and addition of rituximab for CD20 positive patients. After remission induction (step 1) pts in morphologic complete remission (CR/CRi) received an intensification course of high dose methotrexate with pegaspargase for CNS prophylaxis (step 2). At the conclusion of step 2, remission and MRD status were determined centrally by 6-color flow cytometry with MRD negativity defined as <0.01%. In the primary analysis subset, MRD negative patients were randomized to receive an additional 4cycles of consolidation chemo or 2 cycles of blinatumomab at 28 mcg/day (flat dose) for 28 days each cycle followed by 3 cycles of consolidation chemo, another 4-week cycle of blinatumomab (3rd cycle of blinatumomab) followed by an additional cycle of chemo and then a 4th cycle of blinatumomab (step 3). Following completion of consolidation chemo +/- blinatumomab, pts were given 2.5 years of POMP maintenance therapy timed from the start of the intensification cycle (step 4). Estimates of OS were calculated using the Kaplan-Meier method. Comparison of OS between treatment arms were conducted using the stratified log-rank test and Cox proportional-hazards model with age, CD20 status, rituximab use, and whether pts intend to receive HSCT or not as stratification factors. As the number of cycles needed to optimize outcome has not been studied, an unplanned landmark analysis was used to compare OS of pts in the blinatumomab arm who received all 4 cycles o those who received 1-2 cycles. Time 0 was chosen as 9 months post step 3 randomization (the time that patients were supposed to complete 4 cycles of blina). Four pts who received blinatumomab but died within 9 months post step 3 randomization were therefore excluded from this analysis. Results: The study was activated on December 17, 2013 and closed to enrollment on October 15, 2019. 772 pts were screened for the trial and 488 were enrolled on step 1 induction therapy. The median age of the pts was 51 years (range 30-70). 224 MRD negative pts were randomized, 112 pts to each arm. In the blinatumomab arm 12 pts received 1 cycle (11%), 32 pts received 2 cycles (29%), 4 pts received 3 cycles (4%) and 63 pts received 4 cycles (57%). The OS of pts who received 1-2 cycles of blinatumomab was compared to control (Figure 1) The difference was not significant (hazard ratio 0.62, 95% CI 0.28 to 1.34, p=0.22). Fig 2 compares the survival of those who received 1-2 cycles to those who received 4 cycles ( (HR: 0.39, 95% CI 0.12 to 1.16, p=0.076). Conclusion: The addition of blinatumomab to consolidation chemotherapy resulted in a significantly better overall survival compared to chemotherapy alone in pts with newly diagnosed B-lineage ALL who were MRD negative after intensification. This represents a new standard of care for pts aged 30-70 years with BCR::ABL1 negative ALL. The optimal dose and number of cycles is however unknown. In this unplanned subgroup analysis we demonstrate that a survival benefit can only be confirmed in patients who receive the intended 4 cycles of blinatumomab during consolidation. Further studies will be needed to determine if fewer cycles can provide a similar benefit.

Measurable Residual IDH2 before Allogeneic Transplant for Acute Myeloid Leukemia

Introduction: Detection of measurable residual disease (MRD) in AML patients during remission is associated with adverse clinical outcomes after allogeneic hematopoietic cell transplant (alloHCT), including recent strong evidence from our group regarding residual NPM1 and/or FLT3-ITD variants (PMID: 36881031). Large studies examining the significance of other potential MRD targets, including isocitrate dehydrogenase isoform 2 ( IDH2), are needed. IDH2, which is mutated in approximately 12% of AML patients, is of particular interest due to the possibility of targeted clinical intervention. Methods: Patients over the age of 18 in first complete remission (CR1) with IDH2 mutated AML undergoing alloHCT transplant at a CIBMTR site between 2013-2019 with remission blood samples within 100 days of alloHCT and available clinical data were included in this study. Residual variants in IDH2, NPM1, and FLT3genes were detected by ultrasensitive error-corrected next-generation sequencing (NGS-MRD) on genomic DNA from pre-conditioning blood and validated by digital droplet PCR (ddPCR). The primary outcomes were overall survival (OS) and cumulative incidence of relapse with non-relapse mortality as a competing risk. The day of transplant was considered as time 0, and the median follow-up time was calculated for censored patients. Kaplan-Meier estimation (log-rank tests), Cox proportional hazards models, and Fine-Gray regression models were fitted to examine the clinical outcomes. Results: Among the 257 IDH2-mutated AML patients in this study, 57 (23%) experienced relapse, with 41 events occurring within 12 months after alloHCT. The median follow-up time was 25 months. Among 247 patients with pre-transplant remission flow cytometry results reported, 25% were positive, but no difference in rates of relapse or OS was observed between positive and negative patients. NGS MRD analysis detected residual IDH2 variants in remission samples of 130 patients (51%) with variant allele fraction (VAF) ranging from 0.05-56% (median: 3%), and 100% validation by ddPCR. NGS MRD IDH2 positive patients had increased rates of relapse (p = 0.03; 3yr: +11%, 95% CI: 0.2% to 22%) and decreased OS (p < 0.001; 3yr: -25%, 95% CI: -13% to -38%) compared to those who tested negative (Figure 1A). These OS differences were still observed between NGS MRD IDH2 positive and negative patients, regardless of age stratification (under 60yr p < 0.001, 3yr: -28%, 95% CI: -12% to -43%; over 60yr p = 0.04, 3yr: -19%, 95% CI: -1% to -40%); however, only patients over 60yrs had increased rates of relapse (p = 0.007; 3yr: +21, 95% CI: 6% to 36%). The impact of co-mutation status was examined by subdividing the cohort based on the presence of NPM1 or FLT3-ITD mutations at baseline. Residual IDH2 variants in patients without baseline NPM1 or FLT3-ITD co-mutations (n = 151) were associated with decreased OS and increased relapse ( IDH2+ vs IDH2-; OS p = 0.005, 3yr: -23%, 95% CI: -6% to -40%; Relapse p = 0.01, 3yr: +19, 95% CI: 6% to 32%; Figure 1B). In contrast, residual IDH2 variants were not predictive of relapse in co-mutated patients (n=106, p = 0.5). In these patients, the only prognostic marker for relapse was NPM1 and/or FLT3-ITD MRD, with similar relapse risk between IDH2 only positive and MRD negative groups (p < 0.001; 3yr: 83% vs. 11% vs. 15%; Figure 1B). In multivariable analysis, IDH2 MRD positivity was associated with decreased OS (HR 2.5, 95% CI: 1.5-4.2, p < 0.001) and increased rates of relapse (HR 2.1, 95% CI: 1.4-5.7, p = 0.003) after adjusting for other characteristics. Conclusion: In this study, we demonstrate that adult patients with IDH2-mutated AML who have IDH2 variants detected in CR1 prior to first alloHCT experienced increased relapse rates and decreased overall survival. However, this association is significant only in patients without baseline NPM1 and FLT3-ITD mutations. In co-mutated patients, NPM1 and/or FLT3-ITD MRD serve as superior prognostic markers. Subgroup analyses show the potential influence of factors such as conditioning intensity and age on clinical outcomes. Nevertheless, larger studies with balanced subgroups and more comprehensive baseline mutational information, including IDH2 mutation subtype, are needed to better define the clinical relevance of IDH2 MRD in this context.

Clinical Significance of Measurable Residual Disease (MRD) in Relapsed/Refractory Multiple Myeloma (RRMM) Patients (Pts) Treated with Chimeric Antigen Receptor (CAR) T Cells and T-Cell Engagers (TCE)

Clinical Significance of Measurable Residual Disease (MRD) in Relapsed/Refractory Multiple Myeloma (RRMM) Patients (Pts) Treated with Chimeric Antigen Receptor (CAR) T Cells and T-Cell Engagers (TCE)

Prognostic Significance of Measurable Residual Disease for Patients with Acute Lymphoblastic Leukemia Undergoing Allogeneic Stem Cell Transplant in Second Complete Remission and Beyond

Introduction: For patients with acute lymphoblastic leukemia (ALL) undergoing allogeneic hematopoietic stem cell transplantation (HCT) in first complete remission (CR1), measurable residual disease (MRD) prior to HCT is strongly associated with post-HCT relapse rates and overall survival (OS). However, for a growing number of patients with ALL, HCT is deferred until the second remission (CR2) or beyond. It is unclear whether pre-transplant MRD assessment is also prognostic in the setting of HCT beyond CR1. Methods: We performed a single center retrospective study to test the hypothesis that pre-transplant MRD positivity is associated with outcomes in patients receiving HCT in CR2 or beyond for ALL. MRD was measured by 6-color flow cytometry with a sensitivity of 0.01%. Consecutive pediatric and adult patients who underwent first allogeneic HCT between the years 2004-2021 for ALL in CR2 or beyond were included. Results: We identified 162 patients (Table 1), 123 (76%) with B-ALL and the remainder with T-ALL. The majority (80%, n=129) of patients were in CR2 at the time of HCT, the remainder in third CR or beyond (CR3+). One hundred and five (65%) patients were male and median age at transplant was 31 (range 4-69) years. Graft source was either matched unrelated donor (MUD) (43%, n=69), matched sibling donor (MSD) (24%, n=39), haploidentical (18%, n=29) or cord blood transplantation (CBT) (15%, n=25). Pre-HCT MRD status was determined for 150 (93%) of patients and of those was positive in 43 (26%). Subsequent analyses were limited to the subset of patients with known MRD status. There was no statistical difference in characteristics according to MRD status except for higher proportion of CR3+ patients (33% vs. 16%, p=0.02) and comorbidity index >2 (70% vs. 40%, p=0.001) in MRD positive vs. negative patients. The median follow-up in surviving patients was 62 (range 20-104) and 47 (range: 2-171) months in MRD positive and negative patients, respectively. In multivariate analysis (MVA) evaluating transplantation outcomes at 3 years, pre-HCT MRD positivity was associated with a higher rate of disease relapse (HR 1.9, 95% CI 1.1-3.6; p=0.02); but it had no impact on non-relapse mortality (NRM) (HR 0.8, 95% CI 0.4-1.8; p=0.6). Subset analyses showed that these trends were consistent for patients receiving HCT in CR2 or CR3+; however the impact on disease relapse was more pronounced for patients in CR2 at HCT. MRD positivity was also associated with less favorable overall (HR 1.96, 95% CI 1.2-3.3; p=0.01) and progression-free survival (PFS) (HR 2.5, 95% CI 1.5-4.2; p<0.001) for patients receiving HCT in CR2. MRD was not associated with OS (HR 0.9, 95% CI 0.4-2, p=0.8) or PFS (HR 0.8, 95% CI 0.3-1.7, p=0.5) for patients receiving HCT in CR3+. As shown in Figure 1 A-B, OS and PFS were significantly superior for MRD negative patients receiving HCT in CR2. OS and PFS for MRD positive patients in CR2 were comparable to patients receiving HCT in CR3+. Conclusions: Pre-transplant MRD positivity is predictive of disease relapse in patients undergoing HCT for ALL in CR2 or beyond; however, the survival advantage associated with MRD negativity maybe limited to patients undergoing HCT in CR2. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Single-Cell Immune Landscape of Measurable Residual Disease in AML after Allogeneic Hematopoietic Stem Cell Transplantation

Measurable residual disease (MRD) is the most important prognostic marker for relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in acute myeloid leukemia (AML). Unfortunately, our understanding of critical immune cell clusters in bone marrow (BM) which contribute to MRD occurrence is limited. Thus, we generated 184,231 single-cell transcriptomes to compare the BM immune cell landscape between MRD positive and negative patients. KLRG1-CD8+ T cell and KLRG1+CD8+ T cell clusters were prevalent in MRD positive and negative patients, respectively, demonstrating significantly different gene expression profiles (particularly cytotoxicity genes). KLRG1+CD8+ T cells showed a stronger cytotoxicity to leukemia cells compared with KLRG1-CD8+ T cells in vitro. Exhausted CD8+ T cell clusters were similar between MRD positive and negative patients. In addition, IGHG1+ B cells, IFITM3+ monocytes, and HSP90AB1+ erythrocytes were associated with MRD occurrence. MRD clearance by immunotherapies was accompanied with the increase of KLRG1+CD8+ T cell clusters and decrease of KLRG1-CD8+ T cell, IGHG1+ B cell, IFITM3+ monocyte, and HSP90AB1+ erythrocyte clusters, and vice versa. An independent cohort showed that the decrease of immune cell clusters which were prevalent in MRD positive patients was associated with MRD achieving negative after allo-HSCT. Macrophage migration inhibitory factor pathway might be the critical pathway among these immune cell populations, and it was weakened in MRD patients who showed response to immunotherapy. We firstly provided a complete landscape on BM immune cells in MRD stage which will facilitate a better understanding of the immune mechanisms for MRD occurrence in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Chemotherapy or Hematopoietic Stem Cell Transplantation in B-Lineage Acute Lymphoblastic Leukemia Patients Based on IKZF1 and MRD Status

Purpose： We aimed to investigate the outcomes of different post-remission treatment choices based on IKZF1 detection by RT-PCR and dynamic measurable residual disease (MRD) by multiparameter flow cytometry in B-ALL. Prospectively confirm the role of MRD and IKZF1 deletion as a significant predictor of post-remission treatment, replacing the definition of clinical risk categories for patients after complete remission (CR). Patients and Methods： There were 202 patients with B-ALL enrolled in this registry-based cohort study including 99 who received chemotherapy (CMT), and 103 allogeneic stem cell transplantation (allo-SCT). The primary endpoint was the 5-year overall survival (OS), event-free survival (EFS), and cumulative incidence of relapse (CIR). Subgroup analyses were performed based on IKZF1deletion and dynamic MRD 14,24,45 days after induction therapy. Results： In subgroups of patients with IKZF1wt and MRD negative patients， the 5-year overall survival (OS), event-free survival (EFS), and cumulative incidence of relapse(CIR) probabilities for patients assigned to chemotherapy were 67.6%, 55.2%,36.5% and 71.4%,60.7%,28.6%, for those assigned to HSCT respectively. In subgroups of patients with IZKF1 del or MRD positive patients after CR, with OS, EFS, and CIR rates for patients assigned to chemotherapy were 30.1%, 10.3% and76.2%, and 71.8%, 39.8%, and 43.7%for those assigned to HSCT, respectively. Conclusion： The results thus suggest a clear distinction between HSCT and CMT inIKZF1del or MRD positive Patients. While IKZF1wt patients and MRD negative had significant differences. Affected patients should thus be carefully monitored for IKZF1 and minimal residual disease to decide on the post-remission treatment after CR.

The HOVON 100 Study in Adult Acute Lymphoblastic Leukemia Re-Analysed By Multi-State Modeling: Benefits and Risks of Clofarabine

Background: Clofarabine (CLO) is an effective anti-leukemic drug that was recently evaluated in first line treatment of adult patients with acute lymphoblastic leukemia (ALL) in a prospective randomized study (HOVON 100). While more patients achieved eradication of measurable residual disease (MRD), no beneficial effect on the primary endpoint event-free survival (EFS) was noted (Rijneveld et al, 2022). To study these observations in more detail, we applied multi-state modeling to evaluate whether CLO affected intermediate events, including going to off protocol treatment (not due to relapse, but because of treatment-related toxicity) and achieving MRD negativity (defined as MRD<10-4), and the subsequent impact of these events on death or relapse. Methods: The HOVON 100 study randomized 334 adult ALL patients to the control (n=166) and to the CLO arm (n=168). Parameters determining disease risk were evenly distributed between the two study arms. Patients received induction and consolidation chemotherapy (Ind & Cons) followed by either allogeneic hematopoietic cell transplantation (alloHCT) or maintenance treatment, depending on risk status (irrespective of MRD), donor availability, and transplant eligibility. As recently reported, 5-year EFS was 50% (95%CI 42-57) in control patients compared with 53% (95%CI 45-60) in CLO treated patients. We developed a Markov time-inhomogeneous multi-state model describing the events between Ind & Cons (start state) until the date of last contact, incorporating maintenance treatment, off-protocol treatment, alloHCT, and relapse as intermediate events. Relapse mortality and non-relapse mortality were considered end states. We developed a second Markov time-inhomogeneous multi-state model that incorporated MRD negativity after consolidation as intermediate event, without considering off-protocol treatment. Patients with missing MRD data after consolidation (40%) were not considered MRD negative. All other states were evaluated as mentioned above. Effect estimation was done using a transition-specific Cox model including treatment arm (CLO vs control). Results: Overall, CLO treated patients went to off-protocol treatment almost as frequent as control patients (45/168 [27%] vs 36/166 [22%], P=0.31), but went to off-protocol treatment more frequently during maintenance treatment (13/44 (30%) vs. 6/56 (11%); HR 2.85 [1.08-7.50], P=0.035). Considering the treatment effect on the transitions to relapse, CLO treated patients had non-significantly different relapse during maintenance (HR 0.58 [0.24-1.39], P=0.22) than control patients. Such an effect was also not observed in alloHCT recipients (HR alloHCT to relapse: 0.86 [0.38-1.92], P=0.71) and was also not observed in patients who went to off-protocol treatment (HR 0.78 [0.31-1.98], P=0.60). With respect to MRD, patients in the CLO arm had a trend towards increased MRD negativity compared with control patients (HR Ind & Cons to MRD negativity: 1.36 [0.96-1.93], P=0.083). Additionally, MRD negative patients in the CLO arm were less likely to proceed to maintenance treatment (HR 0.45 [0.26-0.80], P=0.0065), did not proceed more often to alloHCT (HR 0.83 [0.50-1.38], P=0.46), and did not show less relapse than patients in the control arm (HR 1.08 [0.27-4.44], P=0.90). Transition probabilities of the multi-state "off-protocol” model per study arm are summarized in Figure 1. Conclusion: Multi-state modeling, taking going to off-protocol treatment and MRD negativity as intermediate events into account, identified that patients receiving CLO more often went to off-protocol treatment during maintenance, and that CLO treated patients less frequently proceeded to consolidation by alloHCT or maintenance treatment according to protocol. Despite a higher proportion of MRD negativity in patients receiving CLO, not completing protocol treatment might also explain similar relapse rates and EFS in the two arms. The addition of CLO to an already intensified chemotherapeutic schedule resulted in going to off-protocol treatment due to cumulative toxicities and not due to relapse for a higher proportion of patients. Future studies may evaluate CLO in a less intensified induction/consolidation/maintenance chemotherapeutic scheme, which differs from our pediatric inspired, intensified, backbone of the HOVON 100 ALL study. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Fla-IDA Chemotherapy with or without Venetoclax in Patients with Relapsed/Refractory Acute Myeloid Leukemia

Introduction: Refractory and relapsed AML (R/R AML) is difficult to treat with median overall survival of 6.5-39 months in patients undergoing allogeneic hematopoietic cell transplantation (alloHCT), and 1.5-11.9 months in patients not eligible for alloHCT. Venetoclax combined with fludarabine, cytarabine, and idarubicin (FLAVIDA) induces a high response rate in R/R AML patients. However, long-term outcome of this regimen has not been compared to FLA-IDA chemotherapy only. Objectives: To compare FLAVIDA with FLA-IDA as salvage chemotherapy in R/R AML and assess MRD response and overall survival (OS). Methods: This retrospective single center cohort study included patients with R/R AML treated with FLAVIDA or FLA-IDA. Venetoclax was dosed 100mg/day orally (days 1-7) due to co-medication with posaconazole. Eighty-one patients that were treated with FLA-IDA between 2000 and 2018 and fulfilled the selection criteria of the FLAVIDA patients served as control. Measurable residual disease (MRD) was assessed by NGS with a cutoff of 0.01% or multicolor flow cytometry with a cutoff of 0.1%. The overall response rate was defined by complete remission (CR) + CR with incomplete hematologic recovery (CRi) + morphologic leukemia-free state (MLFS). This study was approved by the local Ethics Review Committee in accordance with the Declaration of Helsinki. and is registered with ClinicalTrials.gov, NCT03662724. The data cut-off for this analysis was December 31, 2021. Results: Thirty-seven and 81 patients, who received one course of FLAVIDA and FLA-IDA, respectively, were included in the analysis. Baseline characteristics were balanced between FLAVIDA and FLA-IDA treated patients: median age 54 vs. 52 years; de novo AML 70% vs. 77%; 2017 ELN adverse risk 33% vs. 25%; relapsed AML 51% vs. 42%; prior alloHCT 22% vs. 28%. FLAVIDA and FLA-IDA treated patients received a median of one (range 1-5) prior lines of therapy. The ORR was 78% (n=29/37) in the FLAVIDA compared to 47% (n=38/81) in the FLA-IDA group (P=0.001) (Figure 1A). The CR/CRi rate was 59% and 30% in FLAVIDA and FLA-IDA patients, respectively (P=0.003). MRD negative response was achieved in a similar proportion of patients treated with the two regimens (50% and 57%; P=0.65). No differences in neutrophil and platelet recovery times were observed in the FLAVIDA compared to the FLA-IDA group. Most commonly observed treatment related toxicities of any grade were hematological adverse events (AEs) being reported in all FLAVIDA treated patients. Non-hematological AEs of all grades included bacteremia, sepsis, and fungal pneumonia occurring in 27%, 11%, and 11%, respectively. Eighty-one and 79% of patients proceeded to alloHCT or donor lymphocyte infusion (DLI) after FLAVIDA and FLA-IDA, respectively. Median follow up was 22.4 and 62.9 months in FLAVIDA and FLA-IDA cohorts. Despite a higher ORR in FLAVIDA patients, OS was similar between FLAVIDA and FLA-IDA-treated patients (HR 1.18; P=0.57) (Figure 1B). In the 26 responding FLAVIDA patients with available MRD data, survival was significantly longer in MRD negative patients (HR 0.1, 95%CI 0.01-0.82; P=0.009) while MRD was not prognostic for OS in responding FLA-IDA patients with available samples (n=23; HR 0.58, 95%CI 0.17-1.9; P=0.36). Subgroup analyses revealed that the only group with different EFS and OS after FLAVIDA or FLA-IDA was the non-responding patient group, in which FLA-IDA treated patients had improved EFS and OS after additional salvage treatment. Median OS from the time of non-response was significantly longer in non-responding FLA-IDA compared to non-responding FLAVIDA patients (HR 0.31, 95%CI 0.12-0.8; P=0.01), suggesting that non-responding FLA-IDA patients could be salvaged more successfully. Conclusions: Short-term venetoclax in combination with FLA-IDA represents an effective intensive treatment regimen in R/R AML. ORR was significantly higher in FLAVIDA compared to FLA-IDA treated patients. However, OS was comparable among patients treated with FLAVIDA or FLA-IDA. MRD negative response was associated with excellent OS in FLAVIDA treated patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Autologous stem cell transplantation in favorable-risk acute myeloid leukemia: single-center experience and current challenges.

Autologous stem cell transplantation (ASCT) has gained growing consideration as a treatment option for favorable-risk acute myeloid leukemia (FR-AML) in first complete remission (CR1), compared with chemotherapy. We report the long-term outcomes of 117 consecutive patients with FR-AML fit for intensive chemotherapy diagnosed in our center between 1999 and 2020, who underwent ASCT. Sixty-five of the 117 were eligible for intensive post-remission treatment, and 42 of those 65 received ASCT. Median follow up was 132 months. Overall survival (OS) and disease-free survival (DFS) were 75% and 76%. Higher doses of CD34 + stem cell infusions negatively impacted DFS in multivariate analysis. Core-binding factor (CBF) leukemia was an independent prognostic factor for improved DFS. No differences based on pre-transplant measurable residual disease (MRD) were observed. In CBF leukemia, 10-year DFS is 72% for MRD-positive patients versus 100% for MRD negative patients. ASCT is effective and safe in FR-AML patients. In CBF leukemia, ASCT provides excellent results regardless of achievement of bone marrow MRD negativity. In NPM1-mutated/FLT3-wild type (mNPM1) AML, early molecular response seems to have more impact on prognosis. Prospective investigation of the role of gemtuzumab ozogamicin in this setting is ongoing.

Real-World Experience of CPX-351 As First-Line Treatment in 188 Patients with Acute Myeloid Leukemia

Real-World Experience of CPX-351 As First-Line Treatment in 188 Patients with Acute Myeloid Leukemia

Targeting MDS Stem Cells with Omacetaxine and Azacitidine for Newly Diagnosed High Grade Patients: Phase 1 Trial Results and Preliminary Mechanistic Studies

BackgroundMyelodysplastic syndrome (MDS) arises from and is perpetuated by malignant stem cells. Curative therapy requires eradication of this population, but this is not achieved with currently available medical therapies. MDS stem cells possess unique molecular properties that can be therapeutically exploited, but in general these strategies have not been explored clinically. Our recent in vitro studies showed that combination treatment with the protein synthesis inhibitor omacetaxine (oma) and the hypomethylating agent (HMA) azacitidine (aza) efficiently targeted MDS stem cells with minimal impact on normal stem cells. Based on these findings we conducted a Phase 1 clinical trial for MDS patients with concomitant oma + aza (NCT03564873).MethodsPatients were eligible if they had newly diagnosed MDS with ≥5% blasts. Aza was given at 75 mg/m 2 IV days 1-7 of a 28-day cycle; oma was given subcutaneously twice daily on days 1-7 of a 28-day cycle with 4 dose cohorts (0.75, 1.0 and 1.25 mg/m 2, and a de-escalation cohort of 0.5 mg/m 2). Dose escalation was based on a 3+3 design. Bone marrow biopsies occurred at baseline and day 8 and 28 of cycle 1, and at the conclusion of odd-numbered cycles. The primary endpoint was the maximum tolerated dose (MTD) of oma with aza. Responses were assessed according to 2006 IWG criteria with hematologic improvement (HI). Using pre- and post-treatment patient samples, advanced single cell techniques including mass cytometry and antibody-based single-cell RNA sequencing (CITE-Seq) were used to elucidate mechanisms of sensitivity and resistance. High sensitivity DNA sequencing techniques were used to understand clonal evolution and detect measurable residual disease (MRD).ResultsNine patients were required to complete phase 1. See Table 1 for baseline characteristics. Two patients in cohort 1 experienced dose limiting toxicity (DLT) (grade 3 hypoxia and grade 3 respiratory failure). De-escalation to cohort 0 resulted in one DLT event (grade 4 GI bleed) among the six patients treated in this cohort. The MTD was established as 0.5 mg/m 2 oma days 1-7. Adverse events are detailed in Table 2. Median number of cycles was 2 (1-3). 8/9 patients achieved a marrow CR; 5 had HI. 6 patients proceeded to transplantation and none have relapsed with median time from transplant of 772 (183-1158) days. Median response duration is 587 days (29-938). 5 remain alive.MRD negativity, as measured with droplet digital PCR (sensitivity up to 0.1%), was achieved in 2 out of 4 patients measured. Both MRD negative patients had complete clearance of mutated splice factor genes; we previously showed no ability to clear these mutations (0/13 patients) using venetoclax+aza in AML (Nature Medicine (2018) 24:1859-1866).Baseline analysis of patient specimens by single cell transcriptomics demonstrates a distinct population of MDS stem cells (Figure 1A). This subset has overlapping transcriptional properties with known AML stem cell profiles as highlighted by increased geneset expression. Within the stem cell compartment, we observed up-regulation of protein synthesis pathways (Figure 1B) (e.g. KEGG ribosome pathway in MDS stem cells), indicating that oma/aza treatment may specifically target the malignant stem cell population. Subsequent studies are investigating the use of CITEseq as a means to evaluate drug response, which will provide very high resolution analyses of transcriptional signatures in MDS patients. By specifically monitoring eradication of MDS stem cells during the course of treatment, it should be possible to predict therapeutic efficacy and response duration while elucidating genes that correspond to oma/aza sensitivity and resistance.ConclusionsPhase 2 of this study is ongoing; 23 newly-diagnosed high risk MDS patients will be enrolled with a primary endpoint to determine the overall response rate. In addition a ten-patient HMA failure expansion cohort will be enrolled to determine preliminary toxicity and efficacy assessments. Correlative studies will seek to determine the impact of this regimen on the MDS stem cell population and the mechanism of this impact. [Display omitted] DisclosuresMcMahon: Takeda: Membership on an entity's Board of Directors or advisory committees. Smith: Syros: Research Funding; Kura: Research Funding; Argenx: Research Funding. Pollyea: Foghorn: Honoraria; Gilead: Consultancy, Honoraria; Celgene: Honoraria; Bristol Myers Squibb: Honoraria; Genentech: Consultancy, Honoraria; Jazz: Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Amgen: Honoraria; Astellas: Honoraria; Aprea: Honoraria; Karyopharm: Consultancy, Honoraria; Kiadis: Honoraria; Novartis: Consultancy, Honoraria; Syndax: Honoraria; Syros: Consultancy, Honoraria; Takeda: Honoraria; Teva: Research Funding. OffLabel Disclosure:Omacetaxine for MDS

Post-Transplant MRD Status and T Cell Chimerism Predict Outcomes in Patients Allografted for AML/MDS-a Prospective Analysis from the UK NCRI Figaro Trial

▪Allogeneic stem-cell transplantation is an important curative strategy in acute myeloid leukaemia (AML) consequent upon the development of a graft-versus-leukaemia (GVL) effect. Disease relapse is the major cause of transplant failure and novel therapeutic strategies are required in patients with measurable residual disease (MRD) post-transplant. Recognizing that full donor T cell chimerism serves as a biomarker of GVL and that mixed donor T cell chimerism reflects the presence of bi-directional tolerance, we report the first prospective correlation of the impact of post-transplant T cell chimerism and MRD in patients allografted for AML/MDS.Methods: 186 patients from FIGARO, a prospective randomized trial of reduced intensity conditioning regimens in AML/MDS, were alive and relapse free, at the first MRD timepoint and provided post-transplant bone marrow samples for flow cytometric MRD monitoring (day+42, month+3/+6/+9/+12) and peripheral blood samples for T cell chimerism analysis (3-monthly during the first year). Results were not available to clinicians. Flow cytometric MRD incorporating an unsupervised immunophenotypic analysis approach was used on 778 sequential samples. The prognostic significance of post-transplant MRD from each sampling timepoint was assessed by landmark analysis.Results: 29 (15.6%) patients were MRD+ at one or more timepoints in the first year after transplant. The presence of post-transplant MRD was associated with reduced OS (HR:2.19, p=0.0026) and RFS (HR:5.36, p<0.0001), when analyzed as a time varying Cox variable. We, and others, have shown that pre-transplant MRD status is an important determinant of post-transplant survival. We therefore performed a multivariate analysis of factors that may determine the prognoses of patients with pre-transplant MRD+ disease. This demonstrated that the presence of detectable post-transplant MRD was the only independent determinant of RFS in this group of patients (HR 9.05 (95%CI:1.70-48.2), p=0.0099), even when adjusted for cytogenetics, FLT3-ITD and GVHD. The highest frequency of post-transplant MRD positivity occurred at day+42/month+3. The 2yr OS of day+42 MRD+ patients (n=13/158) was 30.8% (95%CI 9.5-55.4) compared to 67.1% (95%CI 58.5-74.2) in MRD negative patients (p<0.001); and 2yr CIR in day+42 MRD+ patients was 92.3% (95% CI:35.8-99.4) versus 22.8%% (95%CI:16.3-29.9) when MRD negative (p<0.001). Day+42 MRD was associated with more rapid relapse kinetics (median RFS of 2 months) compared to when MRD was first detected at later timepoints.Next, we examined the impact of T-cell chimerism, and post-transplant MRD on outcomes. In patients with mixed-donor T-cell chimerism (MDTC) at month+3 or +6, the presence of post-transplant MRD (preceding or at time of first MDTC) was associated with decreased OS (p=0.001) and RFS. Specifically, 2yr RFS in MRD+ patients was 23.1% (95%CI:5.6-47.5), compared to 63.6% (95%CI:44.9-77.5) in patients who were MRD negative (p=0.004) (Figure). In contrast, in 48 patients with full donor T-cell chimerism (FDTC) at months 3 and 6, only 5 patients were MRD+ post-transplant and their outcomes were equivalent to patients who were MRD negative (FDTC/MRD negative 2yr RFS: 80.9% (95% CI:65.3-90.0)).Downregulation of HLA class II molecules on AML blasts after transplant may result in immune evasion from GVL: we examined whether this had an additional impact on outcome in post-transplant MRD+ patients. Both RFS and OS were significantly lower in MRD+ patients when MRD+ blasts had down-regulated HLA-DR expression. In MRD+ patients, 2yr OS was 20.0% (95%CI:3.1-47.5) in those with HLA-DR down-regulation, versus 57.9% (95%CI 33.2-76.3) in those without (p=0.001).Conclusion We demonstrate that dynamic assessment of post-transplant MRD is an important predictor of outcome in patients allografted for AML/MDS, with additional prognostic value to pre-transplant MRD assessment. Post-transplant MRD and T-cell chimerism results are most informative when combined, underlining the importance of a GVL effect in AML/MDS. This suggests post-transplant strategies which result in FDTC may improve outcomes in patients allografted for AML/MDS. Our data also supports the clinical relevance of monitoring for leukemic immune escape as HLA-DR down-regulation identifies a post-transplant MRD positive subgroup at risk of almost inevitable relapse. [Display omitted] DisclosuresLoke: Novartis: Other: Travel; Janssen: Honoraria; Amgen: Honoraria; Pfizer: Honoraria; Daichi Sankyo: Other: Travel. Protheroe: Jazz Pharmaceuticals: Honoraria; Astellas: Honoraria; Kite Gilead: Honoraria. Dillon: Amgen: Other: Research support (paid to institution); Astellas: Consultancy, Other: Educational Events , Speakers Bureau; Jazz: Other: Education events; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: educational events; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Session chair (paid to institution), Speakers Bureau; Menarini: Membership on an entity's Board of Directors or advisory committees; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research Support, Educational Events. Craddock: Novartis Pharmaceuticals: Other: Advisory Board ; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Next-Generation Sequencing-Based Measurable Residual Disease (MRD) Monitoring Is Highly Predictive for Outcome after Allogeneic Stem Cell Transplantation

Background: Molecular predictors for relapse following allogeneic haematopoietic stem cell transplantation (alloHSCT) are urgently needed as 30-40% of AML patients experience recurrence of disease after HSCT. Measurable residual disease monitoring (MRD) for nucleophosmin 1 (NPM1 ) mutations has proven to be highly predictive for relapse in AML patients treated with or without alloHSCT. However, the majority of AML patients undergoing alloHSCT do not have the favorable NPM1 marker so that alternative markers and techniques are required. We studied a next-generation sequencing (NGS)-based error-corrected sequencing approach that is applicable to all mutations for its ability to prognosticate relapse and survival following alloHSCT.Aim: To evaluate the prognostic impact of MRD before alloHSCT in AML patients in morphologic complete remission (CR) using a novel approach of error-corrected sequencing applicable to the majority of AML patients.Patients and Methods: We studied 75 patients who underwent myeloablative (MA) (n=36) or reduced-intensity conditioned (RIC) (n=39) alloHSCT for AML in morphologic CR for the presence of MRD shortly before alloHSCT. All patients had at least one mutation in genes other than NPM1 or DNMT3A at the time of diagnosis that was identified by NGS with a myeloid panel on the Illumina platform. Amplicon-based error-corrected sequencing and bioinformatics was developed and applied to CR samples before alloHSCT analyzing 1-3 diagnostic mutations. Genomic DNA from peripheral blood (n=46) or bone marrow (n=29) was used for MRD assessment. Mutations in the following genes were used for MRD analysis in the indicated number of patients: IDH2 (n=12), FLT3 (n=10), RUNX1 (n=7), SF3B1 (n=6), NRAS (n=5), IDH1 and TP53 (n=4 each), KRAS and WT1 (n=3 each), CBL, ETV6, EZH2, STAG2 (n=2 each), BCOR, BCORL1, DDX41, PHF6, PTPN11, RAD21 , and SMC3 (n=1 each). 55 mutations were single nucleotide variants and 14 mutations were indels. The sensitivity of the assay was determined for each analysis and ranged from 10-4 to 10-5. Six patients (8%) had molecular persistence at high copy number in CR (variant allele frequency, VAF 31-51%) and were excluded from further analysis.Results: 31 of 69 patients (45%) were found MRD positive before alloHSCT with a median VAF of 0.19% (range 0.012% to 14.5%). Clinical and transplantation-associated characteristics were similarly distributed between MRD positive and negative patients. Cytogenetic risk according to MRC, ELN risk groups and molecular aberrations showed no significant differences between MRD positive and negative patients apart from more MRD positive patients being SF3B1 mutated. The median follow up was 6.1 years. 19 of 31 MRD positive patients (61%) and 7 of 38 MRD negative patients (18%) relapsed after alloHSCT. In MRD positive patients the MRD marker was also found in the majority of available relapse samples (9 of 11 patients, 82%). MRD negative patients who relapsed had lost the MRD marker in 4 of 5 available relapse samples (80%), suggesting high accuracy of our MRD assay. By competing risk analysis for cumulative incidence of relapse (CIR) and non-relapse mortality (NRM) patients with positive MRD had a significantly higher CIR than MRD negative patients (P<.001; HR 4.85, 95% CI 1.95-12.1; 5 year CIR 67.7 vs 19%, Figure 1A), while NRM was not significantly different (P=.31; HR 0.44, 95% CI 0.09-2.14). Overall survival (OS) was significantly shorter in MRD positive compared to MRD negative patients (P=.029; HR 2.26, 95% CI 1.07-4.79; 5 year OS 38 vs 70%, Figure 1B). In multivariate analysis MRD status was an independent predictor of CIR (P<.001, HR 5.80, 95% CI 2.18-15.48) besides cytogenetic risk, and of OS (P=.013, HR 2.57, 95%CI 1.22-5.38) besides cytogenetic risk and WBC count.Conclusion: We provide evidence that NGS-based MRD monitoring can be applied to a large proportion of AML patients using almost any available molecular aberration. This approach requires morphologic confirmation of CR status in order to exclude patients with molecular persistence at high copy number, likely originating from preleukemic clones or germline mutations. NGS-based MRD is highly predictive of relapse and survival and may help refining transplant and posttransplant management in AML patients. [Display omitted] DisclosuresChaturvedi:Bayer Pharma AG, Berlin, Germany: Research Funding. Fiedler:Amgen, ARIAD/Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Patents & Royalties; Amgen, Pfizer: Research Funding; Amgen, Gilead, GSO, Teva, Jazz Pharmaceuticals: Other: Support for meeting attendance.

Clinical Outcomes Based on Measurable Residual Disease Status in Patients with Core-Binding Factor Acute Myeloid Leukemia: A Systematic Review and Meta-Analysis.

Measurable residual disease (MRD) response during acute myeloid leukemia (AML) treatment is a gold standard for determining treatment strategy, especially in core-binding factor (CBL) AML. The aim of this study was to critically review the literature on MRD status in the CBF-AML to determine the overall impact of MRD status on clinical outcomes. Published studies in the MEDLINE and EMBASE databases from their inception up to 1 June 2019 were searched. The primary end-point was either overall survival (OS) or recurrence-free survival (RFS) between MRD negative and MRD positive CBF-AML patients. The secondary variable was cumulative incidence of relapse (CIR) between groups. Of the 736 articles, 13 relevant studies were included in this meta-analysis. The MRD negative group displayed more favorable recurrence-free survival (RFS) than those with MRD positivity, with a pooled odds ratio (OR) of 4.5. Moreover, OS was also superior in the MRD negative group, with a pooled OR of 7.88. Corroborating this, the CIR was statistically significantly lower in the MRD negative group, with a pooled OR of 0.06. The most common cutoff MRD level was 1 × 10−3. These results suggest that MRD assessment should be a routine investigation in clinical practice in this AML subset.

Continued Long Term Responses to Ibrutinib + Venetoclax Treatment for Relapsed/Refractory CLL in the Blood Cancer UK TAP Clarity Trial

Continued Long Term Responses to Ibrutinib + Venetoclax Treatment for Relapsed/Refractory CLL in the Blood Cancer UK TAP Clarity Trial

Sensitive and broadly applicable residual disease detection in acute myeloid leukemia using flow cytometry-based leukemic cell enrichment followed by mutational profiling.

Persistent measurable residual disease (MRD) is an increasingly important prognostic marker in acute myeloid leukemia (AML). Currently, MRD is determined by multi‐parameter flow cytometry (MFC) or PCR‐based methods detecting leukemia‐specific fusion transcripts and mutations. However, while MFC is highly operator‐dependent and difficult to standardize, PCR‐based methods are only available for a minority of AML patients. Here we describe a novel, highly sensitive and broadly applicable method for MRD detection by combining MFC‐based leukemic cell enrichment using an optimized combinatorial antibody panel targeting CLL‐1, TIM‐3, CD123 and CD117, followed by mutational analysis of recurrently mutated genes in AML. In dilution experiments this method showed a sensitivity of 10−4 to 10−5 for residual disease detection. In prospectively collected remission samples this marker combination allowed for a median 67‐fold cell enrichment with sufficient DNA quality for mutational analysis using next generation sequencing (NGS) or digital PCR in 39 out of 41 patients. Twenty‐one samples (53.8%) tested MRD positive, whereas 18 (46.2%) were negative. With a median follow‐up of 559 days, 71.4% of MRD positive (15/21) and 27.8% (5/18) of MRD negative patients relapsed (P = .007). The cumulative incidence of relapse (CIR) was higher for MRD positive patients (5‐year CIR: 90.5% vs 28%, P < .001). In multivariate analysis, MRD positivity was a prominent factor for CIR. Thus, MFC‐based leukemic cell enrichment using antibodies against CLL‐1, TIM‐3, CD123 and CD117 followed by mutational analysis allows high sensitive MRD detection and is informative on relapse risk in the majority of AML patients.