Minimal Residual Disease Flow Research Articles

Clonotypic VDJ rearrangements in Mixed Phenotype Acute Leukemia can be Successfully Utilized to Track Minimal Residual Disease (MRD).

Multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) can both be used to identify a neoplastic clonotype by targeting CDR3 and assessing rearrangements in IgH, IgK, IgL, TCR-β, and TCR-gamma loci. The clonotypic sequence can be robustly used to track minimal residual disease (MRD). The ability to track MRD by NGS in mixed phenotype acute leukemia (MPAL) is unknown and warrants investigation. Institutional Review Board (IRB) approval was obtained. Central Moffitt Cancer Center (MCC) database was searched to locate any patients with MPAL from over 600,000 entries. Patient charts were manually curated to identify those with clonoSEQ data, and clinical data was procured from the electronic medical record (EMR). Twenty-nine patients with MPAL were identified. Only 2 patients with clonoSEQ testing were found. Both demonstrated a B/myeloid phenotype, and both were bilineal. NGS (clonoSEQ) identified 4 dominant (IGH) (patient A; 8/2019) and 2 dominant sequences (patient B; 10/2019), respectively. In both patients, clonoSEQ testing successfully tracked minimal residual disease and mirrored clinical disease burden. This report is the first to confirm the utility of NGS-based MRD tracking in patients with MPAL and shows increased sensitivity of NGS over MRD flow cytometry.

Ixazomib, Lenalidomide, and Dexamethasone (IRD) Treatment with Cytogenetic Risk-Based Maintenance in Transplant-Eligible Myeloma: A Phase 2 Multicenter Study by the Nordic Myeloma Study Group.

Scarce data exist on double maintenance in transplant-eligible high-risk (HR) newly diagnosed multiple myeloma (NDMM) patients. This prospective phase 2 study enrolled 120 transplant-eligible NDMM patients. The treatment consisted of four cycles of ixazomib-lenalidomide-dexamethasone (IRD) induction plus autologous stem cell transplantation followed by IRD consolidation and cytogenetic risk-based maintenance therapy with lenalidomide + ixazomib (IR) for HR patients and lenalidomide (R) alone for NHR patients. The main endpoint of the study was undetectable minimal residual disease (MRD) with sensitivity of <10-5 by flow cytometry at any time, and other endpoints were progression-free survival (PFS) and overall survival (OS). We present the preplanned analysis after the last patient has been two years on maintenance. At any time during protocol treatment, 28% (34/120) had MRD < 10-5 at least once. At two years on maintenance, 66% of the patients in the HR group and 76% in the NHR group were progression-free (p = 0.395) and 36% (43/120) were CR or better, of which 42% (18/43) had undetectable flow MRD <10-5. Altogether 95% of the patients with sustained MRD <10-5, 82% of the patients who turned MRD-positive, and 61% of those with positive MRD had no disease progression at two years on maintenance (p < 0.001). To conclude, prolonged maintenance with all-oral ixazomib plus lenalidomide might improve PFS in HR patients.

Abstract PR008: Cell state transitions drive the evolution of disease progression in B-cell acute lymphoblastic leukemia

Abstract A mathematical framework is constructed to predict the risk of B-lymphoblastic leukemia (B-ALL) relapse post-induction chemotherapy. The framework is a Markov chain model that quantifies spontaneous cell state transitions between distinct immunophenotypic subpopulations defined by CD34 and CD38 expression relative to neutrophils. Cell states are analyzed via flow cytometry pre- and post-treatment, providing insight into the evolution of cell state transition rates during disease progression. Cancer stem cells (CSCs) are hypothesized to promote tumor progression through innate chemoresistance and self-renewal. Ostensible CSCs were first identified in acute myeloid leukemia and were found to have a CD34+/CD38- immunophenotype similar to hematopoietic stem cells. However, the isolation of CSCs from B-ALL has proved more difficult. B-ALL cells with stem cell-like properties have been reported with variable immunophenotype, perhaps due to temporal variation of CD34 and CD38 expression in this setting. We hypothesized that transitions between stem cell-like, hematogone-like, and naive B-cell-like leukemia subpopulations play a significant role in B-ALL disease progression. To test this hypothesis, we trained a Markov chain mathematical model using flow cytometry characterization of four B-ALL cell states with their normal counterpart appearing in parentheses: CD34+/CD38- (hematopoietic stem cells), CD34+/CD38+ (stage 1 hematogones), CD34-/CD38+ (stage 2 and 3 hematogones), and CD34-/CD38- (naïve B-cells). An iterative numerical search procedure was used to derive patient-specific Markov matrices, describing the stochastic cell state transitions. This flow cytometry evaluation was performed on a cohort of patient samples of peripheral blood (N=46) and bone marrow (N=63) with matched clinical features such as BCR::ABL1 status, comprehensive genomic profiling, minimal residual disease (MRD) post-induction chemotherapy, and 3-year relapse. Critical to our goal of quantifying the evolution of state transition rates, we also obtained bone marrow measurements for a cohort of normal/healthy individuals. Patients were divided into post-induction flow MRD positive (N=16), MRD negative (N=30), healthy (N=6) cohorts, as well as relapsed and non-relapsed cohorts to compare features of the transition matrices. Importantly, pre-treatment flow cytometry derived cell state distribution alone is not predictive of relapse or MRD. In contrast, pre-treatment Markov chain transition parameters are found to be clinically predictive of relapse and MRD. MRD correlates to high reciprocity (the product of incoming and outgoing transitions) of the stem cell state. This approach provides supporting evidence that cell state transitions drive B-ALL disease progression. There is an additional strong correlation between Markov transition parameters and both BCR::ABL1 and BCR::ABL1-like B-ALL classification. Finally, comparison of Markov parameters pre- and post-treatment quantifies the evolutionary selection pressures acting on transition rates induced by chemotherapy treatment. Citation Format: Curtis Gravenmier, Ling Zhang, Lynn Moscinski, Jeffrey West. Cell state transitions drive the evolution of disease progression in B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr PR008.

Efficacy and Safety Analysis of Venetoclax Combined with Azacitidine in Real-World Treatment of Patients with AML

Objective: To evaluate overall survival (OS), response rate, time to first response, duration of response, depth of response, and safety in real-world treatment with Venetoclax (Ven) combined with Azacitidine (AZA) in patients with Acute Myeloid Leukemia (AML). Methods: From January 2019 to December 2021, 31 patients with AML who completed at least 2 courses of Ven (100-400mg, 28 days), the Ven dose was adjusted based on drug interactions particularly with azole antifungal prophylaxis, and AZA (75mg/m 2,7 days). Baseline characteristics: Median age 65 years (30-82 years), including 5 patients ≥75 years. ECOG score: 19 patients (61.3%) scored 0-1, 12 patients (38.7%) scored 2-3. Type of AML: 20 De novo cases (64.5%), 8 MDS or MPN conversion cases (25.8%), 3 primary AML-MRC cases (9.7%). Previous treatment: 22 patients (71%) were initially treated, 8 patients (25.8%) were relapsed/refractory, and 1 patient (3.2%) was maintained after remission with standard therapy. At baseline, grade 3 or greater cytopenia was associated: 3 cases were anemia (9.7%), 13 cases were neutropenia (41.9%), and 14 cases were thrombocytopenia (45.2%). Bone marrow original cell count: &amp;lt; 30% (10 cases), 30-50%(9 cases), ≥50% (12 cases). Cytogenetic risk: 1 case was low risk (3.2%), 19 cases were medium risk (61.3%), 10 cases were high risk (32.3%), and 1 case was no dividing phase (3.2%). Molecular mutations included: NRAS/KRAS mutation in 12 cases, DNMT3A mutation in 11 cases, NPM1 mutation in 9 cases, IDH1/2 mutation in 6 cases, FLT3-ITD mutation in 5 cases each, HOX11 positive, ASXL1, RUNX1 and NPM1+FLT3-ITD mutation in 3 cases each. Results: After a median of 3 courses of treatment (C2-C15), 3 of the patients underwent bridge hematopoietic stem cell transplantation after remission. The best efficacy was evaluated in CR+CRi: 22 patients (71%), PR: 5 patients (3 of whom were refractory to recurrence), SD: 3 patients (1 of whom was refractory to recurrence), and NR: 1 patient (refractory to recurrence). First-line efficacy was better than second-line or multi-line. Median time to start treatment to reach CR/CRi: 45 days, DOR: 314 days. The negative response rate of flow MRD in CR/CRi patients was 81.8%, including 11 cases of C1 conversion, 3 cases of C2 conversion, 3 cases of C3 conversion and 1 case of C4 conversion. Flow minimal residual disease (MRD) persisted in AML1-ETO subgroup. The early combined response rate of patients with important gene mutations: ASXL1 and RUNX1 mutation subgroup 100%, NPM1 mutation subgroup 77.78%, IDH1/2 mutation subgroup 83.33%, FLT3-ITD mutation subgroup 80%, HOX11 positive subgroup 80%, 66.67% in the NPM1+FLT3-ITD and NRAS/KRAS mutation subgroups, 53.55% in the DNMT3A mutation subgroup, and 1 patient with TP53 mutation was not in remission. The median OS of the first-line/ maintenance treatment group was 19 months, which was better than the median OS of the second-line or multi-line treatment group was 7 months. Safety: The incidence of tumolysis syndrome and early treatment-related mortality was 0; The incidence of early reduction or treatment interruption was higher (25.8%). Severe adverse reactions included sepsis in 2 cases, severe pneumonia in 2 cases and septic shock in 1 case. Conclusions: In the real world first-line treatment of high-risk AML patients with Ven combined with AZA, the disease control and survival were initially improved, and the early combined response rate (CR+CRi) of molecular subgroups IDH1/2, NPM1, RUNX1, ASXL1, FLT3-ITD and HOX11 were higher. The higher incidence of adverse events in early treatment and delayed treatment may be related to 41.9% of patients with grade 3 neutropenia at baseline, partly AML-MRC. So, based on Ven-28d/AZA, We still need to explore new therapeutic modalities which should balance the efficacy and safety.

Minimal residual disease assessment in B-cell precursor acute lymphoblastic leukemia by semi-automated identification of normal hematopoietic cells: A EuroFlow study.

Presence of minimal residual disease (MRD), detected by flow cytometry, is an important prognostic biomarker in the management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, data-analysis remains mainly expert-dependent. In this study, we designed and validated an Automated Gating & Identification (AGI) tool for MRD analysis in BCP-ALL patients using the two tubes of the EuroFlow 8-color MRD panel. The accuracy, repeatability, and reproducibility of the AGI tool was validated in a multicenter study using bone marrow follow-up samples from 174 BCP-ALL patients, stained with the EuroFlow BCP-ALL MRD panel. In these patients, MRD was assessed both by manual analysis and by AGI tool supported analysis. Comparison of MRD levels obtained between both approaches showed a concordance rate of 83%, with comparable concordances between MRD tubes (tube 1, 2 or both), treatment received (chemotherapy versus targeted therapy) and flow cytometers (FACSCanto versus FACSLyric). After review of discordant cases by additional experts, the concordance increased to 97%. Furthermore, the AGI tool showed excellent intra-expert concordance (100%) and good inter-expert concordance (90%). In addition to MRD levels, also percentages of normal cell populations showed excellent concordance between manual and AGI tool analysis. We conclude that the AGI tool may facilitate MRD analysis using the EuroFlow BCP-ALL MRD protocol and will contribute to a more standardized and objective MRD assessment. However, appropriate training is required for the correct analysis of MRD data.

Identification of Minimal Residual Disease in B-Acute Lymphoblastic Leukaemia Using Uniform Manifold Approximation Projection

Background and objective: Acute Lymphoblastic Leukaemia (ALL) is the most common childhood cancer, with B lineage ALL accounting for approximately 85% of cases. Minimal residual disease (MRD) levels during induction therapy are important prognostic biomarkers that allow patient stratification dependent on their risk of relapse. MRD is quantified by molecular analyses of antigen receptor gene rearrangements or by flow cytometric evaluation of aberrant immunophenotypes. The latter method is increasingly being used worldwide, due to its reduced cost, its provision of predictive biomarkers for antigen therapies and the inclusion of both MRD methodologies in contemporary clinical trials. However, accurate detection of MRD by flow cytometry is complex, time-consuming and relies heavily on user training and expertise. Machine learning has been evolving in the field, with the aim of superseding manual analysis of Flow MRD data. This study aimed to test the ability of the unsupervised machine learning dimensional reduction algorithm, Uniform Manifold Approximation Projection, (UMAP) to visualise and accurately quantify 'MRD islands' from normal cell populations with minimal human intervention. Methods: Flow cytometric data from bone marrow samples from children entered on the UKALL2011 clinical trial were used in the study. For B lineage ALL, the protocol included the identification of a leukaemia associated immunophenotype at diagnosis using 2 antibody combinations and 'on treatment' samples were then ran with the most appropriate antibody tube. In this study, we used data from Euro tube 1 which included the antibodies, CD20 FITC, CD38 PE, CD34 PerCP, CD10 PE-Cy7, CD19 APC, CD45 APC-Cy7 and the nuclear dye, Syto 41, tagged with Pacific Blue. FCS files were uploaded to Cytobank and pre-gated for CD19 positive cells prior to running the UMAP algorithm. Each data set was ran alongside data from 10 normal marrows stained with Euro tube 1. Results: Initially, a selection of samples with varying MRD levels (n=6) were ran using UMAP with a range of minimum distance and number of neighbours parameters. A minimum distance of 0.1 and number of neighbours of 13 was the optimal UMAP settings to produce a very clear separation of MRD islands from healthy cells populations across all samples. (Figure 1). These setting were then used for 341 B-ALL MRD samples at different time points day 8 (n=116), day 15 (n=32), days 29 (n=118), weeks 6-20 (n= 75). MRD levels from UMAP were compared to those generated by the gold standard sequential gating. Excellent concordance was seen, as demonstrated by an R2 value of 0.99 (linear correlation). For 'on treatment samples', UMAP showed excellent accuracy with sensitivity and specificity values of 93% and 100%, respectively. Positive predictive values were 100% while negative predictive values were 94% due to UMAP analysis failing to identify an MRD island in 11 'on treatment' samples, 9 at day 8 and 2 at day 28. The average UMAP run time was less than 2 minutes. Conclusion: Detection of MRD using the UMAP algorithm showed an excellent correlation with the gold standard sequential gating technique. False negatives were due to overlap of antigen expression with normal cells and/or low ALL cell numbers. Current Flow MRD methodologies use 8 or more antibodies and higher target cell numbers, which may reduce the false negative UMAP call rate. In summary, UMAP has promising ability to simplify the downstream data analyses of Flow MRD, overcoming the extensive training needed, human subjectivity and will be time and thus cost saving. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Updated Outcomes of the University of Southern California (USC) ALL Frontline Regimen (based on CCG-1882) after Protocol Modification in the Era of Novel Agents in Ph-Negative ALL Patients

INTRODUCTION: Pediatric inspired regimens in adolescent young adults and adults have improved outcomes in acute lymphoblastic leukemia (ALL). CALGB 10403 was prospectively tested in patients 17-39 years and achieved a 3-year event free survival of 59%. In 2007, we reported outcomes after incorporating peg-asparaginase (PEG) dosed at 2000mg/m in induction for patients aged 17- 55 years. This was well tolerated and achieved a complete remission (CR) rate of 96%. In 2014, we reported experience with the use of multiple doses of PEG (CCG-1882) for newly diagnosed ALL adult patients achieving a 7-year overall survival (OS) of 51%. Since then, the USC ALL regimen consisting of 2 induction phases, was further modified with a goal of maintaining good outcomes and improving compliance and toxicities. Fractionated doses of cytarabine were changed to a single dose, the methotrexate dose of 1g/ m2 (2.5 g/m2 if T cell) given D1,15 in intensification phases was changed to 3g/ m2 (B and T cell) given in single doses, and consolidation was increased to six cycles allowing for PEG holidays. Moreover, the approval and incorporation of novel agents such as blinatumomab and inotuzumab also changed outcomes in ALL. Therefore, this study reports an update of outcomes since further modification of the USC ALL pediatric-based regimen in the era of novel agents. METHODS: This is a retrospective review which included adults aged 18 years with newly diagnosed Philadelphia negative ALL (2016-2020) treated at the USC/Norris Comprehensive Cancer Center and the Los Angeles County Hospital (LAC). Primary objectives were over-all survival (OS), event-free survival (EFS), disease-free survival (DFS) at 3 years. Secondary outcomes were complete remission/complete remission with incomplete recovery (CR/CRi) and minimal residual disease (MRD) by flow cytometry rates. OS, DFS, EFS were reported through Kaplan Meier curves and Log-rank tests. Two-sided p value ≤0.05 was significant. RESULTS: 121 patients with Ph-negative ALL were reviewed (49.6% Ph-negative B-ALL, 23.9% Ph-like B-ALL, 0.8% B cell lymphoblastic leukemia (LBL), 9.1% T cell ALL, 4.1% T cell LBL, 4.1% early T-cell, 5.8% mixed phenotype acute leukemia (MPAL). Median age at diagnosis was 39.3 years. There were 71.9% Hispanic, 15.7% White, 9.9% Asian, 2.5% African Americans. 57.9% males, 42.1% females. In terms of risk stratification, the majority were unfavorable at 54.6% and 37.8% unknown. Median BMI was 29.6 kg/ m2. 52.5% had hepatic steatosis and 5% had CNS disease pre-induction. 68.7% received PEG during induction 1, and 67.9% during induction 2. Post-induction 1, 83.5% patients achieved CR/Cri with 49% MRD negativity by flow. Post-induction 2, 75% achieved CR/Cri with 57.1% MRD negativity by flow. 26.4% received blinatumomab but not allo-HSCT. 20% received blinatumomab and underwent allo-HSCT. 34.7% received blinatumomab for MRD flow positivity, median 2 cycles, only 5.8% received inotuzumab, 35.5% underwent allogenic stem cell transplant. 28.9% and 9.1% had any relapse and refractory disease, respectively. Mortality rate was at 14.9%. 3y OS was at 80.7%, 3y EFS at 54.3% and 3y DFS 61.3%. When stratified by MRD flow negativity after induction 1 and 2, 3y OS was 83.7% vs 91.1% (p=0.55), 3y DFS was 68% vs 64.1%(p=0.98) and 3y EFS was 68% vs 59.5% (p=0.84). When stratified by blinatumomab use, 3y OS 72.5% vs 85.1% (p=0.15), 3y EFS was 31.6% vs 67.5% (p=0.001), 3y DFS was 40.1% vs 73.1% (p=0.001) (Fig 1-2). Use of PEG resulted in grade 3/4 toxicities including hypersensitivity (4.1%), transaminitis (21.5%), pancreatitis (5.4%), hypertriglyceridemia (49.5%), hypofibrinogenemia (45.5%), hyperbilirubinemia (21.5%) and thrombosis (16.5%). CONCLUSIONS: The modified pediatric-based USC ALL induction regimen using PEG maintained a high 3y OS at 85.3% and appreciable 3y EFS at 54.3% and 3y DFS at 61.3%, with tolerable toxicities in an era of novel agents like blinatumomab. This compares favorably with the original USC ALL regimen, CALGB 10407 and GRAALL 2005. Of note, 3y EFS and 3y DFS were significantly lower in those who underwent blinatumomab since these patients were MRD flow positive and lacked deeper remissions, but 3y OS (72.5%) remained high regardless, even though 26.4% who received blinatumomab were not able to go for allo-HSCT. This suggests the beneficial effect of blinatumomab. Further follow-up is necessary to provide a more robust and mature survival data. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Pilot Study of Azacitidine As Epigenetic Priming for Chemotherapy in Infants Less Than 1 Year of Age with KMT2A-Rearranged Acute Lymphoblastic Leukemia (ALL); Results from the Children's Oncology Group (COG) Trial AALL15P1

Introduction Acute lymphoblastic leukemia (ALL) with KMT2A-rearrangement (KMT2A-r) in infants <1 year is a high-risk childhood ALL subtype, with consistently poor event-free survival (EFS) of approximately 35% when treated with intensive chemotherapy with or without hematopoietic stem cell transplant. Infant ALL blasts are characterized by DNA hypermethylation, which is hypothesized to contribute to chemoresistance by altering transcriptional regulation of gene expression. In preclinical studies of KMT2A-r blasts, epigenetic priming with DNA methyltransferase inhibitors improved the in vitro cytotoxicity of chemotherapy. Azacitidine, a pyrimidine nucleoside analog of cytidine and hypomethylating agent, has been used in combination with chemotherapy in children with leukemia. We previously reported that azacitidine was safe and well tolerated in AALL15P1 (ASPHO 2021) and herein we report survival outcomes. Methods The Children's Oncology Group (COG) trial AALL15P1 (NCT02828358) was a single arm, open label, groupwide pilot trial. The primary aim of the trial was to evaluate the tolerability of azacitidine in addition to Interfant-06 standard chemotherapy in infants with newly diagnosed KMT2A-r ALL. Estimation of 5-year EFS was an exploratory aim, given the small sample size. Eligibility criteria included B-ALL or acute leukemia of ambiguous lineage with ≥50% B-lymphoblasts, <366 days of age at diagnosis, and >36 weeks gestational age at enrollment. Exclusions included Down syndrome, secondary ALL, and prior cytotoxic therapy (except intrathecal chemotherapy or corticosteroids). Following an Interfant-based induction, infants with KMT2A-r received 4 courses of azacitidine, 2.5 mg/kg/dose intravenously over 10-40 minutes daily for 5 consecutive days in each course, immediately preceding the start of a chemotherapy course on day 6. Infants without KMT2A-r were removed from protocol following induction and did not receive azacitidine. Treatment failure was defined as failure to achieve M1 marrow status (<5% blasts by morphology) with resolution of extramedullary leukemia by the end of consolidation. EFS and overall survival (OS) were measured from the time of enrollment. Results The study accrued from March 2017 to December 2019 and all protocol-directed treatment concluded in December 2021. Of the 78 infants enrolled, 56 had KMT2A-r (72%), and 53 completed induction therapy and received at least 1 course of azacitidine. No patients were ineligible. Diagnostic clinical characteristics of infants with KMT2A-r included four infants age <7 days (7%), 13 age <90 days (23%), 18 with white blood cell count ≥300,000/µL (32%), 32 CNS2 (57%), and four CNS3 (7%). As of the data cutoff (06/30/2022), the median follow-up is 3.8 years, and the 3-year EFS (SE) and OS (SE) rates are 34.2% (+/- 0.08) and 63.8% (+/-0.08), respectively, for infants with KMT2A-r. Six infants experienced treatment failure. Minimal residual disease (MRD) levels of marrow blasts by flow cytometry in COG-approved laboratories were submitted for 49 KMT2A-r patients at the end of induction. Of those, 32 were MRD negative <0.01% (65%), eight MRD positive 0.01%-<1% (16%) and nine MRD positive ≥1% (18%). Event-free survival was significantly associated with MRD; the 3-year EFS of patients with any positive MRD was 20.6% (+/-0.13) vs. 40.1% (+/-0.09) (p=0.0185) for those without MRD. As previously reported, at no time did the trial meet or exceed the pre-determined dose limiting toxicity boundaries, and the rates and types of toxicities observed were within the expected range for infants receiving standard chemotherapy alone. Conclusions Epigenetic priming with azacitidine prior to standard chemotherapy was well tolerated in infants with KMT2A-r ALL, but the EFS was consistent with the poor survival of historical outcomes. Positive flow MRD at the end of induction predicted a higher risk of treatment failure, relapse, or death, in comparison to negative MRD, but EFS was still unacceptably low for MRD-negative patients. There remains an urgent need for improved therapies for infants with KMT2A-r ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P859: ANTI-CD38 NANOBODY JK36 ALLOWS RELIABLE MRD DETECTION IN DARATUMUMAB TREATED MULTIPLE MYELOMA PATIENTS

Background: The introduction of immunotherapies such as daratumumab, a CD38 monoclonal antibody has improved survival in multiple myeloma (MM), however a few considerations on minimal residual disease (MRD) assessment by flow cytometry have been raised and deserve our attention. In flow cytometry CD38 is frequently used as a gating marker of plasma cells (PCs), but daratumumab can hampering myeloma cell detection. A strategy to overcome this interference is to staining PCs with anti-CD38 nanobody JK36. Nanobodies are single variable domain antibody fragments (VHH) that often expose a long complementarity-determining region 3 (CDR3), feature that allows anti-CD38 nanobody JK36 recognizing a cryptic epitope not masked by anti-CD38 therapies. Aims: To compare the median fluorescence intensity (MFI) of CD38 multiepitope (ME) antibody and anti-CD38 nanobody in bone marrow samples from MM patients not treated with daratumumab. Secondly, to compare flow cytometric MRD data obtained using CD38-ME and anti-CD38 nanobody from daratumumab treated MM patients. Methods: Samples were obtained from 10 MM patients not treated with daratumumab to compare the MFI of CD38-ME vs CD38-nanobody at baseline and after incubation with daratumumab at a concentration of 10 nM for 60 min at room temperature. Marrow samples were stained using panel CD38/CD56/CD19/CD138/CD45 with variable CD38, CD38-ME vs anti-CD38 nanobody. Data were analyzed using Kaluza 2.1.1 software (Beckman Coulter). Moreover, 17 bone marrow samples were processed using bulk lysis and subsequently stained using the MM-MRD EuroFlow 8-color antibody combination panel and tubes 1 and 2 with variable CD38, CD38-ME vs anti-CD38 nanobody. Samples were acquired on CytoFlex flow cytometer (Beckman Coulter) and data were analyzed using Infinicyt 2.0 software (Cytognos). Statistical analysis was performed using SPSS version 24, verifying the assumption of normality of the variable, pairwise comparisons were carried out using the T-tests for related samples. Results: The MFI values of CD38 ME on samples from daratumumab not treated patients was higher than samples after incubation with daratumumab (p=0.003). For CD38 nanobody, no significant differences were observed for the MFI values of samples, independent of daratumumab incubation (p=0,139) (figure 1A). The percentage of loss of MFI were higher in the CD38 ME group (-36,32%) than in the CD38 nanobody group (-0,35%). Regarding 17 patients treated with anti-CD38 therapies, 14 were treated with daratumumab and 3 with isatuximab. Sixteen samples were MRD positive with a sensitivity of 10-5 or 10-6 by both approaches. As shown in figure 1B, CD38-ME and CD38 nanobody both showed high and comparable MFI on PCs from patients treated with anti-CD38 therapies. Image:Summary/Conclusion: The anti-CD38 nanobody JK36 construct has lower coefficient of variation than CD38 ME antibody. It is a viable alternative to CD38 ME antibody to gate PCs by flow cytometry allowing reliable MRD detection to identify PCs in the presence of anti CD38 biologics. Nanobody technology open new avenues in the detection capability of MRD flow cytometry studies in the era of immunotherapy.

Prognostic Value of Flowcytometric Minimal Residual Disease Combined with PET/CT at Multiple Time Points before and after Autologous Stem Cell Transplantation in Multiple Myeloma

Background: Multiple myeloma is a plasma cell malignancy. With the emerging novel therapy and deepening response to therapy, minimal residual disease (MRD) now has been widely accepted as the response assessment beyond complete response. New generation flow (NGF) and new generation sequencing (NGS) assess the MRD status of the bone marrow, which might result in false-negative MRD because of the well-known spatially heterogeneous and extramedullary involvement in MM. To overcome this limitations, whole body functional imaging technique such as positron emission tomography/computed tomography (PET/CT) is required. To our knowledge, there is limited data examining the prognostic value of flow MRD and FDG-PET/CT at multiple time points before and after ASCT in MM. In this study, we aimed to elucidate the prognostic valued of MRD by flow combined with PET/CT at different time points in the long-term outcomes for transplant-eligible NDMM patients.Methods: This observational cohort study was approved by the ethnic committee of Sun Yat-sen University. The inclusion criteria were 1) patients with transplant eligible NDMM; 2) received uniform induction regimen of PAD and proceeded to the following ASCT; 3) received PET/CT scan in our hospital before receiving any anti-myeloma therapy. Whole-body 18F-FDG PET/CT was carried out in all patients at baseline and was then repeated post-induction (median 38days after the completion of induction), and at 6th, 12th and 24th month post-transplant. Flow MRD status were analyzed by 8-color multiparameter flow cytometry with the sensitivity of 10 -5.Results:From July 2012 to Apr 2017, 94 consecutive patients with transplant eligible NDMM were included in the final analysis. To standardize the PET/CT interpretation, 5-points scale of Deauville score (DS) was applied to evaluate the FDG uptake of bone marrow and focal lesions together.“Flow MRD” and “PET Deauville score” were highly discordant. No matter choosing DS 1, 2 or 3 as the cutoff for PET/CT positivity, there was no significant agreement between PET/CT and flow MRD assessment after induction. At 6th and 12th month post-transplant, neither existed the significant agreement between these 2 assessments if DS 1 or 2 as the cutoff; even if DS 3 as the cutoff, the agreement degree was very poor at these early time points post-transplant. At the 24th month post-transplant, adopting DS3 as the cutoff for PET/CT positivity, the agreement degree between flow MRD and PET/CT was significantly improved to moderate level with the kappa score of 0.579.In the univariate analysis, no matter the flow MRD status was positive or negative, patients with PET DS1-2 at each time points had similar favorable TTP (table 2) whereas patients with PET DS4-5 at each time points had similar poor TTP. In the subgroup of PET DS3, as early as after the induction, patients with flow MRD-negative already show the trend of better TTP (p = .083); while at the 6th, 12th, 24th month post-transplant, patients with flow MRD - negative had significantly better TTP than those with flow MRD - positive.In the multivariate analysis (table3), the lesion DS≤4 at baseline and standard risk FISH were Independent predictive factors for TTP and the lesion DS ≤4 was the only independent predictive factor for OS. After induction and 6th month after transplant, the baseline DS were excluded from the Cox model. The post-therapy PET DS 3 might be a good cutoff value for PET/CT MRD positivity because it significantly predicted both the TTP and OS. However, since 12th month after transplant, compared to patients with DS3, those with DS 1-2 had favorably TTP. The cutoff value shifted to DS2.Conclusion: PET/CT and flow MRD are highly discordant until 2 years after transplant for patients with NDMM. Patients with low Deauville score (1-2) had good prognosis at different time points no matter the MRD status, reflecting the importance of PET/CT in evaluation the aggressive degree of MRD clone. Patients with high Deauville score (4-5) had poor prognosis at different time points no matter the MRD status, reflecting the importance of PET/CT in overcoming the uneven and extramedullary disease in MM. The Deauville score of PET/CT remained as the independent prognosis factor at post-induction, 6 th -, 12 th -, and 24 th month post-transplant. However, the cut-off value of PET/CT Deauville score shift from score 3 before 1-year post-transplant to score 2 at and beyond 1-year post-transplant. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Retrospective Analysis of Minimal Residual Disease Testing By High Throughput Immunosequencing Versus High Sensitivity Flow Cytometry in Multiple Myeloma

Introduction:Detection of minimal residual disease (MRD) is one of the strongest predictors of outcome in multiple myeloma (MM). Until recently, the most commonly available method to detect MRD in clinical practice has been high sensitivity flow cytometry (FC) which can detect MRD with at 10 -5 sensitivity. In recent years, next-generation sequencing (NGS) has become a viable method to assess the MRD in MM patients with a 10 -6 sensitivity. NGS appears to have some advantages over HC-FC by circumventing subjectivity of analysis. However, real-world comparison between these two methodologies in the literature is limited and is important to inform daily hematopathology and oncology ordering practices.Methods:We retrospectively identified all cases of MM with NGS MRD data from bone marrow specimens at the Moffitt Cancer Center and collated corresponding flow MRD data and clinical data (OS, patient demographics) electronically and via chart review.10-color flow cytometry was performed on a Gallios System and analyzed on Kaluza (Beckman Coulter, IN). Two million events were collected on all cells. Validated lower limit of detection was at least 0.01%. Antibodies included CD28, CD81, CD56, CD138, CD319, CD20, CD19, CD117, CD38, CD45, CD27, CD200 (BD, Biolegend, Beckman Coulter).clonoSEQ ® (Adaptive Biotechnologies, Seattle, WA) testing was performed which uses multiplex polymerase chain reaction (PCR) and NGS to identify, characterize, and monitor clonotypes of immunoglobulin (Ig) IgH (V-J), IgH (D-J), IgK, and IgL receptor gene sequences, and translocated BCL1/IgH (J) and BCL2/IgH (J) sequencesStatistical analysis was performed by Spearman correlation coefficient and Kaplan-Meier analysis.Results:192 samples from 122 unique patients were identified that had both NGS and FC data performed on the same sample. FC+ values ranged from 1x10 -7 to 0.39. NGS+ values ranged from 2.3 x 10 -7 to 0.15. Spearman correlation coefficient showed moderate concordance between NGS and FC at r=0.67 (p<0.001). Six samples were positive by FC (mean tumor burden (MTB)= 0.0007) but missed by NGS; whereas 59 samples were positive by NGS (MTB= 0.002) but missed by flow cytometry. Two cases by FC were equivocal and these were both definitively designated as MRD+ by NGS. Overall survival was worse for MRD+ (by NGS or FC) vs MRD(-) (Figure 1).Conclusion:Our study confirms the importance of MRD detection in MM and shows the robust utility of NGS for MRD detection in routine hematopathology practice. While both FC and NGS are complementary given that each can potentially detect MRD missed by another method, the data supports the increased sensitivity of NGS over FC. [Display omitted] DisclosuresNishihori: Novartis: Research Funding; Karyopharm: Research Funding. Hussaini: Stemeline Therapeutics: Honoraria.

An Update on Outcomes Using the USC ALL Frontline Regimen (based on CCG-1882) after Further Modification of Protocol in the Era of Novel Agents in Ph-Negative ALL Patients; A Retrospective Study

▪INTRODUCTION: The use of pediatric inspired regimens in adolescent young adults and adults has improved outcomes in acute lymphoblastic leukemia (ALL). CALGB 10403 was prospectively tested in patients 17-39 years, showing a 3-year event free survival of 59%.In 2007, our institution reported outcomes after incorporating peg-asparaginase (PEG) dosed at 2000mg/m 2 in induction for patients aged 17- 55 years. This regimen was well tolerated and resulted in a complete remission (CR) rate of 96%. In 2014, we reported experience with the use of multiple doses of PEG (CCG-1882) for newly diagnosed ALL adult patients, with a 7-year overall survival (OS) of 51%. Since our last report, the USC ALL regimen consisting of 2 induction phases, has been further modified with a goal of maintaining good outcomes, and improving compliance and toxicities. Fractionated doses of cytarabine were changed to a single dose, the methotrexate dose of 1g/m 2 (2.5 g/m 2 if T-cell) given D1,15 of intensification phases was changed to 3g/m 2 (B and T cell) given in single doses, and consolidation was increased to six cycles allowing for PEG holidays (Table 1).Moreover, the approval and incorporation of novel agents such as blinatumomab and inotuzumab also changed outcomes in ALL. Therefore, this study reports an update of outcomes since further modification of the USC ALL pediatric-based regimen in the era of novel agents. METHODSThis is a retrospective review which included adults aged >18 with newly diagnosed Philadelphia negative ALL between 2016 and 2020 treated at USC/Norris Cancer Center and Los Angeles County Hospital (LAC). Primary objectives were over-all survival (OS), event-free survival (EFS), disease-free survival (DFS) at 3 years, and secondary outcomes were complete remission/complete remission with incomplete recovery (CR/CRi) rates and minimal residual disease (MRD) by flow cytometry. OS, DFS, EFS were reported through Kaplan Meier curves and Log-rank tests. Two-sided p value ≤0.05 was significant.RESULTS: 121 patients with Ph-negative ALL were identified (49.6% Ph-negative B-ALL, 24% Ph-like B-ALL, 0.8% B cell lymphoblastic leukemia (LBL), 9.1% T cell ALL, 4.1% T cell LBL, 4.1% early T-cell, 5.8% mixed phenotype acute leukemia (MPAL). Median age at diagnosis was 38.5 years and maximum age of patient to receive pegasparagase during induction 1 was 63 years. 71.9% Hispanic, 15.7% white, 9.9% Asian, 2.5% African American. 57.9% males, 42.1% females. 3.4% were favorable, 4.2% intermediate, 54.6% unfavorable and 37.8% unknown by karyotypic risk stratification. Median BMI was 28.9 kg/m 2.54.6% had hepatic steatosis either on history or imaging and 5.1% had CNS disease pre-induction.Post-induction 1, 81.4% of patients achieved CR/CRi and 50% MRD negative. Post induction 2, 82.2% achieved CR/CRi, 67.7% MRD flow negative. Post-consolidation 1, 90.9% were MRD flow negative. 33.1% subsequently received blinatumomab for MRD positive disease, 5% given inotuzumab for relapsed disease, 32.2% underwent allogenic hematopoietic stem cell transplant (allo-HSCT). Median number of pegasparagase doses received during treatment was 2, rate of relapse and mortality was 27.3% and 13% respectively.Median OS and DFS were not reached but median EFS was 35 months. 3-year OS was 85.3%, when stratified according to MRD post induction 2; 3-year OS was 91.7% for MRD positive patients and 91.2% for MRD negative patients (p=0.55). 3-year DFS was 83.2%; 88.2% for MRD positive patients and 97.4% for MRD negative patients (p=0.22). 3-year EFS was 47.3%; 51.3% for MRD positive patients and 50.6% for MRD negative patients (p=0.49) (Tables 2-3).Use of pegasparagase resulted in grade 3/4 toxicities including hypersensitivity (4.1%), transaminitis (21.5%), pancreatitis (5.4%), hypertriglyceridemia (49.5%), hypofibrinogenemia (45.5%), hyperbilirubinemia (21.5%) and thrombosis (16.5%). CONCLUSIONSPediatric-based modified USC ALL induction regimen led to a high 3-year OS (85.3%), DFS (83.2%) and EFS (47.3%) with predictable toxicity and compares favorably with original USC ALL regimen, CALGB 10407, GRAALL 2005 and USC/MSKCC regimen.Interestingly, MRD positivity after induction 2 did not adversely affect OS, DFS or EFS, which is likely due to incorporation of blinatumomab and inotuzumab. These agents could have allowed for deeper remissions allowing Ph-negative patients with residual disease to receive allo-HSCT. [Display omitted] DisclosuresChaudhary: Oncotartis: Consultancy; Pancella: Consultancy; Moderna: Current equity holder in publicly-traded company; Celldex: Current equity holder in publicly-traded company; TCR2: Current equity holder in publicly-traded company; Allogene: Current equity holder in publicly-traded company; Athelas: Consultancy, Current holder of stock options in a privately-held company; Angeles Therapeutics: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties: Cell therapy . Douer: Servier: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Adaptive: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Speakers Bureau; Jazz: Consultancy. Yaghmour: Novartis: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Alexion: Speakers Bureau; Astellas: Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Jazz: Speakers Bureau; Agios: Consultancy, Speakers Bureau.

ALL-323: Preliminary, Updated Outcomes of the Modified, Pediatric-Based USC ALL Frontline Regimen in Hispanics and Non-Hispanics

<h3>Context:</h3> In 2014, USC reported use of multiple doses of pegaspargase (PEG) for newly diagnosed ALL patients (7-year overall survival [OS] 51%). This is currently part of the NCCN Guidelines for frontline ALL treatment. Since then, the regimen consisting of 2 induction phases (IP) was modified, with fractionated doses of cytarabine changed to a single dose and consolidations increased to six cycles (six doses of PEG total). <h3>Objective:</h3> Outcomes since modification of the USC ALL induction regimen, with immunotherapies given when appropriate, in Hispanics <i>versus</i> non-Hispanics. <h3>Design:</h3> Retrospective chart review. <h3>Setting:</h3> Single institution, academic center/county hospital. <h3>Patients or Other Participants:</h3> Male/female patients (18–99 years) with any type of ALL diagnosed by bone-marrow/tissue biopsies during 1/2016–12/2020, treated at our institution. Patients who did not receive the modified USC ALL induction regimen with/without PEG were excluded. <h3>Interventions:</h3> Ninety-four ALL patient chart reviews. <h3>Main Outcomes Measures:</h3> OS, event-free survival (EFS) at 5 years, CR/CRi, minimal residual disease (MRD) flow; 2-sided p-value ≤0.05 was considered significant. <h3>Results:</h3> Among 63 Hispanics and 31 non-Hispanics, 67 achieved CR/CRi, 39 had MRD flow negativity after IP1; 50 CR/CRi, and 29 had MRD flow negativity after IP2. PEG was given in 59% Hispanics and 60% non-Hispanics. Salvage BiTE was given to 30 patients (Hispanic, 63%). Median follow-up for Hispanics was 17 (0.5–60) months and 21 (0.1–53) months for non-Hispanics. Median OS not-reached (NR) in both groups. Estimated OS for Hispanics at 5 years was 80% and 51% for non-Hispanics (p=0.020). Median EFS for Hispanics was 46 months and 34 months for non-Hispanics (p=0.306). <h3>Conclusions:</h3> CR/CRi and MRD flow negativity were appreciable with the median OS NR. Interestingly, a significant improvement in Hispanic OS <i>versus</i> non-Hispanics was noted. An explanation could be on how more Hispanics received salvage BiTE (63.3% of 30 patients). Also, 40% of Hispanics were able to receive stem cell transplant <i>versus</i> 33% of non-Hispanics. No significant difference in median EFS between Hispanics and non-Hispanics was noted.

Minimal Residual Disease (MRD) Detection By Flow Cytometry during Aplasia and Post-Induction Is an Independent Prognostic Parameter for Relapse-Free Survival in AML

The identification of patients at high risk of relapse is crucial for treatment stratification in acute myeloid leukemia (AML). Persistence and reoccurrence of AML cells with aberrant phenotypes can be assessed by flow cytometry at high sensitivity in more than 95% of patients. Therefore, assessment by flow cytometry enables determination of MRD status in patients without suitable molecular markers (e.g. NPM1). Previously, we demonstrated that early detection of MRD during aplasia is associated with shorter relapse-free survival (RFS) after intensive induction chemotherapy (Köhnke T et al., Leukemia 2015). Here, we aimed to validate these findings in an independent patient cohort. In addition, we assessed the prognostic value of a combined MRD determination during aplasia and post induction for improved prediction of relapse-free survival (RFS).Flow cytometry MRD was analyzed in patients with AML (excluding APL) diagnosed between 2012 and 2016 receiving intensive induction chemotherapy (sHAM or 7+3). MRD flow analysis was performed using a NAVIOS flow cytometer. Presence ≥0.1% Leukemia-associated immunophenotype (LAIP)-positive cells (identified by comparison with bone marrow from healthy donors) during aplasia or post-induction defined MRD positivity. Kaplan-Meier estimators and log-rank tests were used to retrospectively analyze survival data. Cox's proportional hazards regression model was used to determine the influence of individual factors in multivariate analyses. Pearson's chi square test was used to compare categorical variables between MRD groups.A total of 105 patients were included. In 3 cases (2.9% of all cases), no LAIP could be identified, and these patients were excluded from further analyses. MRD assessment during aplasia was available in 87 cases. Regarding flow MRD assessment post induction, patients refractory to induction therapy were excluded. Therefore, 72 patients had flow MRD assessments available post induction therapy. 59 patients achieving CR or CRi after induction therapy had flow MRD assessments available at both time points.MRD positivity during aplasia was associated with refractory disease (36.2% vs. 10.0% for MRD pos. vs. MRD neg., p=0.017) and shorter median event-free survival (EFS, 5.8 vs. 15.7 months, p=0.011). Importantly, early blast clearance by morphology had no prognostic value if flow MRD assessment was available (HR for EFS 2.0, p=0.03 for flow MRD vs. HR 1.6 and p=0.17 for Blast clearance by morphology). However, differences in median RFS for MRDpos vs. MRDneg patients (10.3 vs. 20.5 months) for assessment during aplasia have not reached significance yet (p=0.135). MRDpos patients post induction had significantly shorter median EFS (10.8 vs. 15.7 months, p=0.032) and RFS (7.7 months vs. not reached, p=0.007) than MRDneg patients. Using a combination strategy for aplasia and post induction assessments, we grouped patients with MRD positivity at both timepoints (confirmed MRD positivity) vs. all others (MRDneg or MRDuncertain). Confirmed MRD positivity was associated with significantly shorter median EFS (6.7 vs. 15.7 months, p=0.003) as well as RFS (4.4 months vs. not reached, p=0.002). Confirmed MRD positivity was an independent predictor of EFS and RFS (HR 2.7 and 2.8, p=0.017 and p=0.018, respectively).Taken together, patients with confirmed two-time flow MRD positivity showed lower EFS and RFS compared to MRDneg or MRDuncertain patients, respectively. Identification of patients with poor treatment response was improved in comparison with cytomorphologic assessment or independent flow MRD assessments at both timepoints alone. Using this approach, early identification of patients with adverse prognosis is possible in >95% of AML patients. MRD flow will be prospectively evaluated as a therapy stratification tool in patients with AML receiving intensive induction chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Flow Cytometry Based MRD Monitoring Is an Accurate Risk Stratification Tool in Acute T-Lymphoblastic Leukemia

▪Minimal residual disease (MRD) measured by PCR of clonal TCR/IGH rearrangements during early phases of chemotherapy is a strong predictor of relapse risk in acute T-lymphoblastic leukemia (T-ALL), and has thus served as the most important tool for stratification to reduced therapy (to avoid toxicity) or intensified therapy. In 8-15 % of T-ALL patients no useful PCR marker is available and flow cytometry (FCM) has been used for monitoring by some collaborative groups. However, studies of the usefulness of FCM-based MRD measurements are scarce, limiting the widespread use of the method. Furthermore, it is unknown whether the generally applied end-of-induction PCR MRD cut-off level of 10-4 should be the same for FCM-based MRD. In addition, it is not known whether earlier identification (day15) of low risk patients by FCM is possible, in which case it would allow elimination of anthracyclines during the latter part of induction therapy thus lowering risk of cardiotoxicity.Aim: To evaluate flow and PCR based MRD measurements as risk stratification tools in T-ALL.Material and methods: The study included 274 patients with T-ALL (193 children (<18y) and 81 adults (18-45y)) diagnosed between July 2008 and February 2016 and treated according to NOPHO ALL2008 in the Nordic and Baltic countries. Median follow up was 32 (range 0-103) months. MRD was measured by 6-colour FCM and by RT-QPCR at days 15, 29 (end of induction) and 79 (only intermediate risk, IR) and prior to and post high risk (HR) blocks. End-of-induction MRD (cut-off 10-3) was used to stratify for IR or HR consolidation therapy. In analyses of day29 MRD data and prognosis, patients who were stratified for HR blocks at day 15 or directly to SCT at day29 were excluded from analyses.Results: Overall 5y EFS was 72.7% (CI 67-79%) with relapse risk (RR) of 15.5% (CI 8-16%). End of induction FCM MRD separated patient risk groups well (5y EFS 84.2% (CI 78-91%) and RR 8.3 % (CI 4-13%) for MRD<10-3 versus EFS 63.4% (CI 50.1-80.3%) and RR 21.8% (CI 9-35%) for MRD>=10-3 (p=0.002 for EFS, p=0.049 for RR) although PCR-MRD gave better discrimination (5y EFS 90.2 (CI 85-96%) and RR 5.5% (CI 1-10%) for MRD<10-3 versus EFS 62.5 (CI 51-78%) and RR 23.4% (CI 12-35%) for MRD>=10-3 (p=0.0002 for EFS, p=0.005 for RR).Adjusting for WBC and age at diagnosis, patients with end-of-induction FCM-MRD >=10-3 had a higher risk of relapse (hazard rate 3.04, p=0.025) compared with MRD <10-3. For PCR the hazard rate was 4.4 (p=0.007). Day29 MRD was measured by both methods in 159 patients. There was an overall good correlation between day29 FCM- and PCR-MRD: (r=0.72, p<0.0001 (Spearman)), though PCR values were generally higher. These differences would give discrepant day29 risk classification in 43/159 patients (36 with PCR>FCM, 7 with FCM>PCR) with the 10-3 cut-off.For both FCM- and PCR-MRD a cut-off level of 10-3 was superior to 10-4 in separating risk groups at end of induction. Patients with d29 PCR MRD between 10-3 and 10-4 were stratified to IR therapy in NOPHO ALL2008 without decreasing EFS or relapse risk (5y EFS 91.4% (CI 82.6-100%), 0 relapses, n=35) compared to patients with MRD<10-4 (EFS 89.6% (CI 83-97%), relapse risk 7.9%, n=81).A day 15 FCM-MRD cut-off of 10-2 provided good prediction of the final risk groups (5y EFS 87% (CI 81-93%) and RR 8% (CI 3-13%) for day 15 FCM-MRD<10-2 vs. EFS 65.4% (CI 53-80%) and RR 16% (CI 6-26%) for FCM-MRD between 10-2 and 2.5x10-1 vs. EFS 24% (CI 8-71) and RR 45.8% (CI 19-73%) for flow MRD>=2.5x10-1 (patients with MRD>=2.5x10-1 went to HR therapy day 15)), but only 35 of the 145 patients with day15 flow MRD<10-2 had no detectable PCR MRD at day29. Only 27 out of 62 patients with day15 FCM-MRD <10-4 had no detectable PCR MRD at day29. Three out of these 62 patients experienced relapse, but none of these had a fully informative FCM marker at diagnosis.Conclusion: End-of-induction FCM-MRD provides a reliable indicator of outcome in T-ALL and can serve as second line method when no suitable PCR marker is found. Regardless of method, 10-3 seems to be superior to 10-4 as end of induction stratification cut-point, allowing patients with PCR MRD between 10-3 and 10-4 to receive intermediate risk therapy without increased relapse risk. Stratification at day 15 does not allow early and unambiguous identification of low risk patients eligible for reduced intensity induction therapy, but randomized studies are needed to clarify this. DisclosuresNo relevant conflicts of interest to declare.

Clearance of leukemic blasts by flow cytometry versus minimal residual disease as predictors of clinical outcome in elderly patients with acute myeloid leukemia.

e19514 Background: Lower-intensity regimens, such as hypomethylating agents (HMAs) are better tolerated and more effective than standard chemotherapy in elderly patients( &gt; 65 years old) with acute myeloid leukemia (AML). Scoring systems to predict clinical outcome in these patients are mainly based on cytogenetic assays and morphological response to therapy. Minimal residual disease (MRD) represents a powerful risk- stratification in younger AML patients but it remains poorly investigated in older patients, particularly in those undergone HMAs. In this subset, the survival benefit is not limited to patients achieving morphologic complete remission (CR). The clearance of leukemic blasts was never be valued as a predictive parameter of outcome in older patients. Thus, in this study we evaluated the most effective and predictive factor of outcome at the optimal time-point among leukemic blasts clearance by flow cytometry (FC), MRD and morphological response in older patients treated with HMAs. Methods: From april 2015 to may 2019, 76 older patients ( &gt; 60 years old) with newly diagnosed AML were treated con azacitidine and decitabine. Among them, 56 completed at least six cycles but 31were properly monitored by FC. Log Clearance was defined as logarithmic ratio between the percentage of blast cells at specific time point and that at the diagnosis. Results: A total of 31 elderly patients with a median age 72 (60-85) were evaluated after 4(T4) and 6(T6) cycles of HMAs. A Median of 10 cycles were administered, equally distributed between azacitidine and decitabine. A higher frequency of CR (73.3%vs 20%) and MRD negativity (16.6% vs 0) was found at T6 compared to T4, thus T6 was chosen for the analysis. Performance status (PS &gt; 2),Hgb &lt; 10 g/dl, MRD positivity and a Log-reduction lower than 2 log predicted a significantly shorter DFS at univariate analysis. However, Log-reduction and PS only confirmed to predict a short DFS also at multivariate analysis, together with CRi (CR with incomplete recovery) and partial remission (PR). When the OS was investigated, only PS represented a strong indicator of prognosis at both univariate and multivariate analysis. Conclusions: Our results suggest that patients achieving 2 Log at least of blasts-reduction by FC had a longer DFS. Dynamic monitoring of blast –reduction stratified better and more widely than the absolute value of MRD in older AML patients. Having a high PS and achieving a CRi instead of a CR showed an unfavorable outcome in terms of both DFS and OS

Preliminary Results from CPX-351 Italian Compassionate Use Program Show High Response Rate and Good Tolerability in Poor Prognosis AML Patients

Background: Therapy-related acute myeloid leukemia (t-AML) or AML evolving from a myelodisplastic syndrome are characterized by a low response rate to conventional chemotherapy and high relapse rate with poor overall survival (OS) despite intensive treatment and allogeneic stem cell transplantation consolidation (HSCT). CPX-351 is a dual-drug liposomal encapsulation of cytarabine and daunorubicin, in a fixed synergistic 1:5 molar ratio. CPX-351 has been approved by FDA for the treatment of patients affected by t-AML or AML with myelodisplasia-related changes (MRC-AML) based on the results of a randomized phase III trial where the drug was compared with conventional 3+7 induction (Lancet et al, JCO 2018). Notably, CPX-351 proved to increase survival probabilities in comparison with standard chemotherapy even among patient achieving complete remission (CR) and proceeding to HSCT, suggesting that CPX-351 may allow deeper responses. However, few information is available on minimal residual disease (MRD) assessment after CPX-351 treatment, or on the impact of molecular alterations on response probability. Aims: The aim of this study was to evaluate the clinical activity of CPX-351 in a real life setting, with particular focus on molecular characterization at diagnosis and MRD evaluation in responding patients. Methods: Seventy five patients were enrolled in a compassionate use program (CUP) in 37 Italian Hematology Centers. CUP started on December 2018 and closed on June 2019. Data collection began on July 2019 and was completed, at the time of writing, for 25/75 patients, enrolled in 9 Centers. Median age was 69 years (56-73), 10 patients were female. Molecular and MRD analysis were performed in each Center as per internal standard. MRD was assessed in most Centers with multicolor flow cytometry. NPM1 mutation was found in 2/22 assessed patients, FLT3-ITD in 3/22, with low allelic burden in 2/3 patients. TP53 mutations have been found in 4/12 patients.Four patients had complex Karyotype, one had isolated del(7q) whereas the remaining 19 had normal karyotype. Six patients had t-AML; 15 patients were previously diagnosed with MDS and 5 of them had already received hypomethilating agents for a median of 5 cycles (2-49). European Leukemia net risk score was low in 2 (8%), intermediate in 12 (48%) and high in 11 (44%) patients. Most patients (20/25) had relevant comorbidities upon enrollment, mostly COPD, diabetes and/or hypertension. As per CUP inclusion criteria, all patients had normal left ventricular function at the time of enrollment (defined by a normal ejection fraction). Results: Induction-related mortality was 2/25 (8%). Fourteen patients experienced grade &amp;gt;1 extra hematological adverse event during induction (mainly infections). Alopecia was observed in 4/25 patients (16%).Response was assessed in 20 patients:2 patients died during induction and 3 patients were not yet evaluated for response at the time of analysis. CR or CRi was observed in 19/22 (86.3%). MRD was performed in 11 patients, with 4 of them achieving flow MRD negativity after first cycle (defined as &amp;lt;0.1%, Minetto et al. BJH 2019). Median time to neutrophil and platelets recovery in responding patients were 29 (18-60) and 24.5 (19-60) days, respectively. Among responding patients, 10 received further CPX-351 consolidation, which was very well tolerated. One patient proceeded directly to HSCT, one is currently waiting for HSCT to be performed and one patient received a conventional chemotherapy consolidation. The remaining patients are currently waiting for administration of consolidation therapy with CPX-351. Four patient had completed so far all planned consolidation therapy with CPX-351 and 2/4 of them were flow MRD negative at the end of therapy. Of note, 3/4 patients with mutated TP53 achieved MRD negative CR. After a median follow-up of 5 months, relapse was observed in 2/20 responding patients and 21 patients are alive at the time of analysis. Conclusions: Our preliminary data confirm the high clinical activity and good tolerability of CPX-351 in a challenging AML cohort. The high median age and the high incidence of severe comorbidities did not result in unacceptable risk of death during induction. With the limitation of very small numbers, CPX-351 showed good antileukemic activity among TP53 mutated patients. Disclosures No relevant conflicts of interest to declare.

Three Year Update of the Phase II ABT-199 (Venetoclax) and Ibrutinib in Mantle Cell Lymphoma (AIM) Study

Background: The phase II AIM trial (Tam et al, NEJM 2018; NCT02471391), using combination BTK inhibitor ibrutinib (IB) and BCL-2 inhibitor venetoclax (VEN) therapy achieved excellent 16 week complete remission rates (CRR) (primary endpoint; 62%) in patients (pts) with poor prognosis mantle cell lymphoma (MCL), higher than currently approved second line IB monotherapy. Additionally, overall response rate (ORR) by PET of 71%, with attainment of minimal residual disease (MRD) negativity was reported. At the time of primary endpoint analysis (median follow up 15.9 months), median progression free survival (PFS), duration of response (DOR), time to progression (TTP) and overall survival (OS) had not been reached. Herein, we update our results with an additional 21.6 months of follow-up and report the outcomes of pts who electively interrupted treatment. Methods: Pts with MCL who had relapsed or refractory disease (R/R; n=23) or were treatment naïve but inappropriate for chemotherapy (n=1) were enrolled. Pts commenced treatment with IB 560mg/d for 4 weeks, followed by weekly ramp-up of VEN to 400mg/d. Initially, pts were intended to continue IB and VEN until progressive disease or unacceptable toxicity; a later amendment allowed pts to electively interrupt both drugs if MRD-negative CR was reached. Pts who elected to interrupt therapy were closely monitored by peripheral blood flow MRD testing and regular CT scans, and were allowed to recommence both study drugs at MRD recrudescence or clinical progression. Results: Median age was 68 years (range, 47-81), median number of previous treatments was 2 (range, 0 to 6), and 50% had aberrations of TP53. As previously reported, the flow MRD-negative and ASO-PCR MRD-negative rates were 67% and 38%, respectively. For the current analysis, the median follow-up was 37.5 months (range, 1.4-45.3).The median DOR and TTP had not been reached and were estimated to be 74% and 60% at 30 months, respectively. The median PFS was 29 months (95% CI of 13-NE) (Figure 1), and the median OS was 32 months (95% CI of 27-NE) (Figure 2). For pts with TP53 aberrant MCL (n=12), the CRR was 50% (95% CI 21-79) with and without PET. In this group the ORR was 58% (95% CI 28-85) without PET and 50% (CI 21-79%) with PET. The DOR in 6 responders with TP53 aberrant MCL (excluding one pt with CRu) was 12+, 24+, 26+, 35+, 36+ and 38+ months from study commencement. In contrast, pts with ATM aberrant MCL (n=10) the CRR was 90% (95% CI 55-100) with and without PET, and the ORR was 90% (95% CI 55-100) with and without PET. Of the 9 responders with ATM aberrant MCL, the DOR was 9+, 9+, 12+, 24+, 31+, 35+, 36+, 37+ and 38+ months from study commencement. Of 13 deaths, 8 were due to PD. Of the other 5 deaths, 2 were due to infection, and 1 each to cardiac failure, glioblastoma, and graft versus host disease after an allograft that occurred after PD on trial. Five pts in MRD negative PET-confirmed CR electively interrupted treatment after a median 18.5 months (range 18-33) of therapy: one pt had radiological progression after 7 months, and the other 4 remained free of clinical or MRD progression after 6, 13, 17 and 18 months off therapy. Conclusions: The median PFS for the IB-VEN combination was 29 months. Treatment interruption was feasible for pts in MRD-negative complete remissions, raising the prospect of limited duration targeted-agent therapy in the management of R/R MCL. Disclosures Handunnetti: Gilead: Honoraria; Abbvie: Other: Travel Grant. Anderson:Walter and Eliza Hall Institute: Employment, Patents &amp; Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.. Hicks:Clarity: Research Funding; Ipsen: Research Funding; Telix Pharmaceuticals: Equity Ownership. Bressel:Regeneron Pharmaceuticals: Consultancy. Koldej:NanoString Technologies: Other: Travel grant. Ritchie:Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy; BMS: Research Funding; Takeda: Research Funding; Beigene: Research Funding; Imago: Research Funding; Novartis: Honoraria; Sanofi: Honoraria. Seymour:Roche: Consultancy, Research Funding, Speakers Bureau; Acerta: Consultancy; Takeda: Consultancy; Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau. Roberts:Walter and Eliza Hall Institute: Patents &amp; Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.; AbbVie: Other: Unremunerated speaker for AbbVie, Research Funding; Janssen: Research Funding; Australasian Leukaemia and Lymphoma Group: Membership on an entity's Board of Directors or advisory committees; BeiGene: Research Funding. Tam:AbbVie: Honoraria, Research Funding; Roche: Honoraria; BeiGene: Honoraria; Novartis: Honoraria; Pharmacyclics LLC, an AbbVie company: Honoraria; Janssen: Honoraria, Research Funding. OffLabel Disclosure: While ibrutinib is FDA approved for use in relapsed and refractory mantle cell lymphoma, venetoclax and combination venetoclax and ibrutinib are not licensed for use in mantle cell lymphoma. The use of venetoclax and ibrutinib for this indication is investigational.

Combination of flow cytometry and functional imaging for monitoring of residual disease in myeloma.

The iliac crest is the sampling site for minimal residual disease (MRD) monitoring in Multiple Myeloma (MM). However, the disease distribution is often heterogeneous, and imaging can be used to complement MRD detection at a single site. We have investigated patients in complete remission (CR) during first-line or salvage therapy, for whom MRD flow-cytometry and the two imaging modalities positron-emission-tomography (PET) and diffusion-weighted magnetic resonance imaging (DW-MRI) were performed at the onset of CR. Residual focal lesions (FLs), detectable in 24% of first-line patients, were associated with short progression-free survival (PFS), with DW-MRI detecting disease in more patients. In some patients, FLs were only PET-positive, indicating that the two approaches are complementary. Combining MRD and imaging improved prediction of outcome, with double-negative and double-positive features defining groups with excellent and dismal PFS, respectively. FLs were a rare event (12%) in first-line MRD-negative CR patients. In contrast, patients achieving an MRD-negative CR during salvage therapy frequently had FLs (50%). Multi-region sequencing and imaging in an MRD-negative patient showed persistence of spatially separated clones. In conclusion, we show that DW-MRI is a promising tool for monitoring residual disease that complements PET and should be combined with MRD.

Treatment approach for young, fit, newly diagnosed multiple myeloma patients

Treatment approach for young, fit, newly diagnosed multiple myeloma patients