Flow Cytometric Minimal Residual Disease Research Articles

The Evolving Landscape of Flowcytometric Minimal Residual Disease Monitoring in B-Cell Precursor Acute Lymphoblastic Leukemia.

Detection of minimal residual disease (MRD) is a major independent prognostic marker in the clinical management of pediatric and adult B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL), and risk stratification nowadays heavily relies on MRD diagnostics. MRD can be detected using flow cytometry based on aberrant expression of markers (antigens) during malignant B-cell maturation. Recent advances highlight the significance of novel markers (e.g., CD58, CD81, CD304, CD73, CD66c, and CD123), improving MRD identification. Second and next-generation flow cytometry, such as the EuroFlow consortium's eight-color protocol, can achieve sensitivities down to 10-5 (comparable with the PCR-based method) if sufficient cells are acquired. The introduction of targeted therapies (especially those targeting CD19, such as blinatumomab or CAR-T19) introduces several challenges for flow cytometric MRD analysis, such as the occurrence of CD19-negative relapses. Therefore, innovative flow cytometry panels, including alternative B-cell markers (e.g., CD22 and CD24), have been designed. (Semi-)automated MRD assessment, employing machine learning algorithms and clustering tools, shows promise but does not yet allow robust and sensitive automated analysis of MRD. Future directions involve integrating artificial intelligence, further automation, and exploring multicolor spectral flow cytometry to standardize MRD assessment and enhance diagnostic and prognostic robustness of MRD diagnostics in BCP-ALL.

Flow cytometric minimal residual disease measurement accounting for cytogenetics in children with non-high-risk acute lymphoblastic leukemia treated according to the ALL-MB 2008 protocol.

Quantitative measurement of minimal residual disease (MRD) is the "gold standard" for estimating the response to therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Nevertheless, the speed of the MRD response differs for different cytogenetic subgroups. Here we present results of MRD measurement in children with BCP-ALL, in terms of genetic subgroups with relation to clinically defined risk groups. A total of 485 children with non-high-risk BCP-ALL with available cytogenetic data and MRD studied at the end-of-induction (EOI) by multicolor flow cytometry (MFC) were included. All patients were treated with standard-risk (SR) of intermediate-risk (ImR) regimens of "ALL-MB 2008" reduced-intensity protocol. Among all study group patients, 203 were found to have low-risk cytogenetics (ETV6::RUNX1 or high hyperdiploidy), while remaining 282 children were classified in intermediate cytogenetic risk group. For the patients with favorable and intermediate risk cytogenetics, the most significant thresholds for MFC-MRD values were different: 0.03% and 0.04% respectively. Nevertheless, the most meaningful thresholds were different for clinically defined SR and ImR groups. For the SR group, irrespective to presence/absence of favorable genetic lesions, MFC-MRD threshold of 0.1% was the most clinically valuable, although for ImR group the most informative thresholds were different in patients from low-(0.03%) and intermediate (0.01%) cytogenetic risk groups. Our data show that combining clinical risk factors with MFC-MRD measurement is the most useful tool for risk group stratification of children with BCP-ALL in the reduced-intensity protocols. However, this algorithm can be supplemented with cytogenetic data for part of the ImR group.

The prognosis of haploidentical hematopoietic stem cell transplantation in infants and patients under 3 years old with acute leukemia.

The role of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients<3 years of age remains controversial. Data on haploidentical donor (HID) transplants in this age group is limited. We retrospectively analyzed the prognosis of 97 patients with acute leukemia aged<3 years who underwent HID transplantation at our institute. With a median follow-up of 45 months, the 3-year disease-free survival (DFS), overall survival (OS), and 3-year cumulative incidence rate of treatment-related mortality were 69.3% (95% confidence interval (CI): 59.9%-78.7%), 74.2% (95% CI: 65.2%-83.2%), and 3.6% (95% CI: 0.9%-9.7%) in all 97 patients, respectively. The 3-year DFS and OS rate in patients diagnosed<1 year and patients diagnosed ≥1 year were comparable: 77.8% (95% CI: 62.2%-93.4%) versus 66.3% (95% CI: 55.0%-77.6%, p=.253) and 82.5% (95% CI: 66.3-98.7%) versus 72.8% (95% CI: 61.9%-83.7%, p=.153), respectively. At the last follow-up, 23 patients had died, and 20 had died of relapse. Multivariate analysis revealed that positive pre-HSCT flow cytometric minimal residual disease (hazard ratio 5.605, p=.000) and AML-M7 expression (hazard ratio 2.906, p=.014) were independent adverse prognostic variables for relapse. HID transplantation is potent and safe for infants and young patients with acute leukemia. Relapse is the primary cause of treatment failure.

CD33 CAR T-Cells (CD33CART) for Children and Young Adults with Relapsed/Refractory AML: Dose-Escalation Results from a Phase I/II Multicenter Trial

Introduction: Current therapies for patients with acute myeloid leukemia (AML) push the limits of chemotherapy intensity but cure only 30% of adults and 70% of children. Recently developed antibody-based therapies and molecularly-targeted agents only modestly improve outcomes for select patient subsets. Thus, new approaches are needed. Given the success of chimeric antigen receptor (CAR) T-cell therapies for acute lymphoblastic leukemia (ALL), we developed and optimized a novel CD33 CAR T-cell (CD33CART) construct for clinical testing in a multicenter Phase I/II clinical trial in children, adolescents, and young adults (AYA) with relapsed/refractory (r/r) AML. ( Qin H, et al. JITC 2021) We report interim results following completion of the dose-escalation phase. Methods: This multicenter phase I/II clinical trial (NCT03971799) was conducted as a 3+3 dose escalation study through the Pediatric Transplantation and Cell Therapy Consortium with National Marrow Donor Program sponsorship and managed by CIBMTR CRO. Autologous CD33CART product were centrally manufactured on a ClinicMACS Prodigy® by the Biopharmaceutical Development Program at the Frederick National Laboratory for Cancer Research (NCI). Eligibility criteria were r/r AML in subjects &amp;lt; 35 years old with adequate organ/performance status and an identified allogeneic stem cell transplant (SCT) donor. Bone marrow assessment was used for standard morphologic evaluation and central flow cytometric minimal residual disease (MRD) quantification. All subjects received pre-CD33CART lymphodepleting chemotherapy (LD) with fludarabine (75-120 mg/m 2) and cyclophosphamide (900-1000 mg/m 2). CD33CART doses levels (DLs) were: DL1: 3 x 10 5 CD33 CAR+ T-cells/kg; DL2: 1 x 10 6/kg; DL3: 3 x 10 6/kg and DL4: 1 x 10 7/kg. Adverse event grading used CTCAE v5 with incorporation of ASTCT consensus definitions for cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS). Dose-limiting toxicities (DLTs) included &amp;gt; grade 3 CRS, &amp;gt; grade 3 ICANS, and persistent &amp;gt; grade 3 non-hematologic toxicity that did not resolve &amp;lt; grade 2 in 72 hours. CD33CART persistence was assessed via flow cytometric evaluation of the blood and bone marrow. Data cut-off was June 1, 2023. Results: A total of 24 subjects (median age of 16 years, range 1-34 years), were enrolled, 12 (50%) of whom underwent a prior SCT. CD33CART products were successfully manufactured for 23 subjects and infused into 19. In 4 non-infused subjects with manufactured products, 1 died from progressive disease (PD) prior to LD, 2 transitioned to palliative care and 1 withdrew consent to seek alternative therapy. Manufacturing was not performed in one subject due to death from PD after enrollment. One morbidly obese subject (BMI=49.3) enrolled at DL4 but received treatment at DL3 due to weight-based manufacturing limitations. The median time from enrollment to infusion was 47 days (range, 24-242). (Table) Three subjects each were infused at DL1 and DL2 without DLTs. At DL3, 1 subject experienced DLT (grade 4 CRS) prompting expansion at this dose level, and no subsequent DLTs were observed. At DL4, 1 subject experienced a prolonged grade 3 CRS (&amp;gt; 72 hours) and grade 3 ICANS, both constituting DLT and necessitating expansion of the cohort without additional DLTs seen. Across all dose-levels, CRS was seen in 13 (68%) patients and was &amp;gt; grade 3 in 4 (21%) with onset typically within the first 24 hours post-infusion. Complete remission (CR) was only seen at DL4 and achieved in 2 subjects, both achieving an MRD negative CR alongside myeloid aplasia. One SCT naïve subject developed candidemia during aplasia but was able to proceed to an allogenic SCT, achieved engraftment, and was in remission at Day +100 post SCT. The second patient (post-two prior SCTs) declined third SCT, had spontaneous count recovery and remained in remission until day +119 (MRD+ relapse). Transient CD33CART expansion was detected in 9 (47.4%) subjects overall and in all 6 (100%) subjects at DL4. Conclusions: CD33CART manufacturing is feasible in children and AYAs with r/r AML with acceptable toxicity experienced in treated subjects. CD33CART expansion was best in subjects treated at DL4 (1 x 10 7/kg) with MRD negative CRs and transient myeloid aplasia occurring in 2 of 5 (40%) subjects evaluable for response (Table). Based on early clinical efficacy at DL4, enrollment continues in the phase 2 portion.

Guidelines for the flow cytometric minimal residual disease monitoring in B-lineage acute lymphoblastic leukemia after CD19-directed immunotherapy

Multicolor flow cytometry is now routinely used in laboratory practice for the minimal residual disease (MRD) monitoring in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Wide application of CD19-directed immunotherapy leads to frequent loss of CD19 expression, that hampers significantly the flow cytometric MRD detection methodology. We developed an antibody panel and data analysis algorithm for multicolor flow cytometry, which is a reliable method for MRD detection in patients with BCP-ALL treated with CD19-directed therapy. We recommend a single-tube 11-color panel for MRD detection, which is adapted for the case of possible CD19 loss. Based on patterns of antigen expression changes and the relative expansion of normal CD19-negative BCPs, guidelines for multicolored flow cytometry data analysis and interpretation are established. The recommended approach is reliable tool for therapy response monitoring displaying the same effectiveness with the more laborious and costly molecular techniques.

Flow Cytometric Analysis of Bone Marrow Particle Cells for Measuring Minimal Residual Disease in Multiple Myeloma.

Simple SummaryThe hemodilution of bone marrow aspirates is the main factor affecting the reliability of the determination of the minimal residual disease (MRD) resulting in multiple myeloma (MM). A bone marrow particle cell (BMPL) enrichment assay may be applied to overcome the problems caused by the hemodilution of bone marrow aspirates and to improve the rate of myeloma cell detection. Assessment of BMPL samples could decrease the false-negative rate of the MRD assessed with routine bone marrow aspirates. This study validated that the BMPL was superior to the routine bone marrow aspirate samples in the MM and MRD analysis.Minimal residual disease (MRD) is one of the most relevant prognostic factors in patients with multiple myeloma (MM). However, the hemodilution of bone marrow (BM) aspirates, the most common preanalytical problem, is known to affect MRD detection. In the present study, we analyzed a preanalytical method for routine BM aspirates and a bone marrow particle cell (BMPL) enrichment assay and validated it as a reliable preanalytical method for flow cytometric MRD determination. A total of 120 BM samples were taken from 103 MM patients consecutively recruited; 77 BM samples had BMPL enrichment analysis and 99 BM samples were routinely analyzed. Then, the two different samples from patients with MM were sent for MRD detection using an eight-color flow cytometry. Our data showed that assessment of the BMPL enrichment samples attenuated the overestimation of MRD-negative assessed in the routine BM samples, which was mainly caused by hemodilution. In conclusion, the BMPL enrichment assay is a functional and practical preanalytical method for flow cytometric MRD analysis.

One-point flow cytometric MRD measurement to identify children with excellent outcome after intermediate-risk BCP-ALL: results of the ALL-MB 2008 study.

Measurement of minimal residual disease (MRD) with multicolor flow cytometry (MFC) has become an important tool in childhood acute lymphoblastic leukemia (ALL), mainly to identify rapid responders and reduce their therapy intensity. Protocols of the Moscow-Berlin (MB) group use a comparatively low (for standard risk; SR) or moderate (for intermediate risk; ImR) treatment intensity from the onset, based on initial patient characteristics. Recently, we reported that 90% of SR patients-50% B cell precursor (BCP-ALL)-MFC-MRD negative at end of induction (EOI)-had 95% event-free survival (EFS). METHODS: In the present study, we applied this method to children with initial ImR features. In study MB 2008, 1105 children-32% of BCP-ALL patients-were assigned to the ImR group. Of these, 227 were treated in clinics affiliated with MFC laboratories of the MB group network, and included in this MFC-MRD pilot study. A single-point MFC-MRD measurement at the EOI with the threshold of 0.01% identified 65% of patients-20% of all BCP-ALL patients-with EFS of 93.5%. Taking both studies together, the combination of clinical parameters and a one-point MRD measurement identifies 70% of BCP-ALL patients with an excellent outcome after low- or moderate-intensity therapy and avoids overtreatment of a significant proportion of patients.

B-lineage antigens that are useful to substitute CD19 for minimal residual disease monitoring in B cell precursor acute lymphoblastic leukemia after CD19 targeting.

The potential loss of CD19 during targeted treatment of B cell precursor acute lymphoblastic leukemia (BCP-ALL) can hamper flow cytometric minimal residual disease (MRD) monitoring. In the current study, we present expression data for antigens that are candidates for CD19 substitution: surface CD22, CD24, CD10, and intracellular (i) CD79a. Bone marrow samples from 519 consecutive children (below 18 y.o.) with primary BCP-ALL were studied with a focus on expression of CD19, CD10, CD22, CD24, and iCD79a. As these antigens are planned to be used as substitutions for CD19 for primary B cell gating, only total expression on the leukemic population (≥95% cells) was considered appropriate. It was found that each of these antigens is totally expressed in nearly 90% of patients. For each single marker, a subgroup of patients without complete positivity presented with BCP-ALL harboring diverse cytogenetic and molecular genetic aberrations. Based on expression data, we have developed algorithm of simultaneous application of these antigens for initial B-lineage compartment gating, that is applicable for nearly all patients after CD19 targeting. We conclude that the addition of CD22, CD24, and iCD79a to the conventional antibody panel and their application together with CD10 allow for the identification of B-lineage compartment including residual tumor blasts, for MFC-MRD searching in virtually all patients with BCP-ALL after CD19-directed treatment.

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.

Abstract 3633: Disparities in relapse among a large multi-ethnic population of children diagnosed with acute lymphoblastic leukemia (ALL): A report from the Reducing Ethnic Disparities in Acute Leukemia (REDIAL) Consortium

Abstract Introduction: While end-induction minimal residual disease (MRD) is the strongest prognostic factor for relapsed ALL, approximately half of all relapses occur in children who are MRD negative. Latino ethnicity is also a risk factor for relapse. To further explore these associations, we conducted an interim analysis of risk factors for relapse in a large multi-ethnic population of children diagnosed with ALL. Methods: The REDIAL Consortium includes patients diagnosed with ALL at six major pediatric cancer centers in the Southwestern U.S. The study period was 2004 to 2018, and we included individuals who were 1-23 years of age when diagnosed with ALL. Time to relapse was defined as time from ALL diagnosis to the initial relapse event, with individuals censored at date of death, last follow-up, or bone marrow transplant. Demographic and clinical factors evaluated included race/ethnicity (Latino, non-Latino Black, non-Latino White, non-Latino other), sex, age at diagnosis (1-5, 6-10, 11-15, &amp;gt;15 years), ALL immunophenotype (B-cell, T-cell), National Cancer Institute (NCI) risk group, central nervous system involvement, enrollment on a Children’s Oncology Group clinical trial, end-induction disease failure, and end-induction bone marrow flow cytometric MRD. Cox proportional hazards models were used to calculate adjusted hazards ratios (HRs) and 95% confidence intervals (CIs). Analyses were further stratified based on end-induction MRD status (positive ≥0.01%, negative &amp;lt;0.01%). Results: Overall, there were 1,710 ALL patients with a median age at diagnosis of 5 years (interquartile range: 3-11 years). The majority of patients were Latino (60.1%) and male (56.9%). Of the 379 MRD-positive patients, 74 (19.5%) relapsed, compared to 138 of 1,233 (11.2%) MRD-negative patients (p&amp;lt;0.0001). In adjusted models, factors associated with a greater risk of relapse included MRD positivity (HR=1.72, 95% CI: 1.26-2.36), older age at diagnosis (&amp;gt;15 vs. 1-5 years, HR=1.98, 95% CI: 1.19-3.29), and NCI high-risk group (HR=1.74, 95% CI: 1.20-2.52), while patients enrolled on a clinical trial were less likely to relapse (HR=0.76, 95% CI: 0.57-0.99). Among MRD-positive patients, Latinos were less likely to relapse (HR=0.60, 95% CI: 0.33-0.99) compared to non-Latino Whites, whereas Latinos who were MRD negative were more likely to relapse (HR=1.68, 95% CI: 1.09-2.59). Conclusion: In a large contemporary multi-ethnic cohort of &amp;gt;1,700 children with ALL, we observed significant disparities in relapse by MRD status, age at diagnosis, NCI risk group, clinical trial enrollment, and race/ethnicity. Notably, nearly 65% of relapse events occurred in MRD-negative patients. Further analyses are ongoing in REDIAL to evaluate the impact of other factors including cytogenetics and novel biomarkers of relapse. Citation Format: Pagna Sok, Austin L. Brown, Olga A. Taylor, M. Brooke Bernhardt, Juan C. Bernini, Rodrigo A. Erana, Timothy Griffin, Kenneth Heym, Van T. Huynh, Laura Klesse, Kathleen Ludwig, Sandi L. Pruitt, Karen R. Rabin, Michael E. Scheurer, Philip J. Lupo. Disparities in relapse among a large multi-ethnic population of children diagnosed with acute lymphoblastic leukemia (ALL): A report from the Reducing Ethnic Disparities in Acute Leukemia (REDIAL) Consortium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3633.

Flow cytometric minimal residual disease assessment in B-cell precursor acute lymphoblastic leukaemia patients treated with CD19-targeted therapies - a EuroFlow study.

SummaryThe standardized EuroFlow protocol, including CD19 as primary B‐cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B‐cell precursor acute lymphoblastic leukaemia (BCP‐ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP‐ALL patients treated with CD19‐targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19‐positive, whereas this was 81% in patients that became (partially) CD19‐negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct.

An Extensive Quality Control and Quality Assurance (QC/QA) Program Significantly Improves Inter-Laboratory Concordance Rates of Flow-Cytometric Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia: An I-BFM-FLOW-Network Report.

Simple SummaryStandardization of flow-cytometric assessment of minimal residual disease in acute lymphoid leukemia (ALL) is necessary to allow concordant multicentric application of the methodology. This is a prerequisite for internationally collaborative trials, such as the AIEOP-BFM-ALL and the ALL IC-BFM trial. We developed and applied a comprehensive training and quality control program involving a large number of international laboratories within the I-BFM consortium to complement standardization of the methodology with an educational component as well as with persistent quality control measures to allow large ALL treatment trials which use multi-laboratory FCM-MRD assessments for risk stratification of pediatric patients with ALL.Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.

Allogeneic-Hematopoietic Stem Cell Transplantation Is Effective and Safe for Relapsed/ Refractory B-ALL Patients Who Cannot Get Remission or MRD Negative after CART Treatment

▪Background: Chimeric antigen receptor T-cell (CART) Therapy has induced high rates of complete remission (CR) in the patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL), but a small portion of them still cannot obtain CR or minimal residual disease (MRD) negative. Salvaged allogeneic hematopoietic stem cell transplantation (allo-HSCT) could be the only curative treatment for this setting. According to previous reports, around 20% to 30% overall survival (OS) has been seen in the patients with r/r B-ALL treated with salvaged allo-HSCT.Aim: In current study, the efficacy and safety of allo-HSCT for the patients with r/r B-ALL who cannot achieve CR or MRD negative after CART therapy were evaluated.Methods: Between January 2018 and June 2021, 23 consecutive patients with r/r B-ALL who did not achieve CR or MRD negative after CART treatment and received allo-HSCT in our hospital were included. The median age was 10 (1-47) years old. The median disease course was 24 (8-89) months. Eight patients were in non-remission (NR) and 15 cases were MRD positive (10 detected by flow cytometry, and 5 detected by RT-PCR). The median blast in bone marrow (BM) in NR series was 39 (0.5-94)% in which two patients were only extramedullary leukemia, and the median flow cytometric MRD level in BM in MRD positive series was 0.11 (0-3)%. One patient had TP53 mutation, one had MLL-AF4 fusion gene and one had both TP53 mutation and MLL-AF4 fusion gene. Nineteen patients (82.6%) received at least two kinds of CART therapies (murinized CD19, humanized CD22, humanized CD19) and 4 patients (17.4%) received one kind of CART therapy (murinized CD19, humanized CD22 or humanized CD19). Three patents underwent second transplantation. Eighteen patients (78.3%) received allo-HSCT from haploidentical donors and 5 patients (21.7%) from unrelated donors (HLA 10/10 matched in 3 and 9/10 matched in 2). Myeloablative conditioning regimens with total body irradiation (fractionated, total 10 Gy) /etoposide (200mg/m 2 x 3) /fludarabine (30mg/m 2 x 5) or cyclophosphamide (1.8g/m 2 x 2) /rabbit anti-T-cell globulin were used. Cyclosporine, mycophenolate mofetil and short-term methotrexate were employed for graft-versus-host disease (GVHD) prophylaxis. Some patients received maintenance regimens with targeted medicine based on their fusion genes or gene mutations up to 2 years post-transplant.Results:All patients achieved durable engraftment. For the patients in NR (n=8) before transplantation, the median follow-up time was 336 (22-803) days. Four of them had been alive free of leukemia and MRD negative, and 4 patients died of relapse. One-year OS and leukemia-free survival (LFS) were 85.7% and 35.7%, respectively. No patient died of transplant-related events. Two patients developed grade III acute GVHD (aGVHD) and 1 patient had extensive chronic GVHD (cGVHD). All of them were resolved with immune-suppressants. CMV and EBV reactivation was detected in 6 and 1 patients, respectively. One patient had severe lung fungal infection and 1 case developed mild hemorrhagic cystitis. For the patients with MRD positive (n=15) before transplantation, the median follow-up time was 359 (42-872) days and one-year OS and LFS were 68.1% and 40.9%, respectively. Seven patients relapsed, three of them achieved MRD negative CR with donor-derived CART therapies, and 3 patients died of relapse. Six patients developed grade III~IV aGVHD and one patient had extensive cGVHD. All of them were resolved except one patient died from grade IV aGVHD. CMV and EBV reactivation was found in 6 and 1 patients, respectively. Three patients had mild hemorrhagic cystitis. No other severe infection occurred.Conclusion: Our results indicate that salvaged allo-HSCT has improved survival remarkably in r/r B-ALL patients who failed not only chemotherapy but also CART therapy.One-year OS and LFS can reach 85.7% and 35.7%, 68.1% and 40.9% in NR and MRD positive cohorts, respectively. Transplant-related mortality is quite low (1/23). The leading cause of death is disease recurrence (7/23). Under our protocol, allo-HSCT is a safe and effective salvaged modality for r/r B-ALL who cannot obtain CR or MRD negative with CART therapy. DisclosuresNo relevant conflicts of interest to declare.

Conventional and Flowcytometric Minimal Residual Disease Based Risk Stratified Post Remission Therapy Improves Survival in Ph-Negative Pediatric Acute Lymphoblastic Leukemia- Single Centre Experience from South India

Background: Children with Acute Lymphoblastic Leukemia (ALL) in Low Middle income countries(LMIC) face major challenges including treatment abandonment and poor overall survival (OS) and event-free survival (EFS).The preliminary data and results of outcome of Pediatric ALL from our pediatric hematology-oncology center which follows BFM based ALLtreatment protocol has been published in 2015 in ASH forum.Due to non availability of Polymerase chain reaction (PCR)based Minimal residual disease(MRD)analysis, we use multiparametric flowcytometry (FCM) based MRD analysis for remission assessment and risk stratification in our patients. Within resource constraints, we present evidence that outcomes comparable with that seen in high income (HIC)and upper middle income countries(UMIC) can be achieved with a post remission therapy guided by a risk stratification incorporating FCM MRD.Methods: Following IRB approval,an ambidirectional cohort study was performed using clinical information and outcomes of all patients aged 1to 14 years treated for newly diagnosed B- or T-ALL between January 1,2015 and December 31, 2020. AIEOP BFM ALL 2009 protocol with modifications was followed as the institutional protocol in all patients.The treatment algorithm is mentioned in Figure 1. Patients who underwent multiparametric FCM MRD analysis at the end of Induction IA and whose 6 months follow up details were available were included in the analysis. Patients with Ph positive ALL and those who died during Induction IA were excluded. FCM MRD analysis was performed after Induction IA on Day 33.MRD level above 0.01% was considered positive.MRD assessment was repeated following Induction IB on Day 74 in patients who had MRD positivity on Day 33. At the end of Induction IB patients were risk stratified into High risk(HR) and non High risk(Non HR).HR features included Hypodiploidy(<45chromosomes), Positivity for MLL/AF4 or t(4:11),poor prednisolone response(absolute blast count in peripheral blood ≥1 x 10^9/l on Day 8 of initiation of prednisolone) and non remission on Day33. Patients who had persistent MRD positive on Day74 were reallocated to high risk. Treatment with HR protocol was initiated for high risk patients whenever financially manageable.Statistical analysis was done using SPSS version 21 OS and EFS were assessed by Kaplan-Meier method Patients were censored at last follow-up.Results: Median follow-up time was 33.5(4-102) months . Study had 102(n=102) consecutive Ph negative patients who underwent induction therapy. Six patients (5.88%) died during induction IA;96(n=96) children who continued treatment were included in further analysis. The mean age at diagnosis was 6(1-14) years .Forty seven (48.95%)patients were male..B-ALL n=84(87.5%) and T ALL n=12.(12.5%). Four patients(4.16%) had CNS disease at diagnosis. Three children (3.13%) had high risk cytogenetics.Ten children (10.42%)in the cohort had poor prednisolone response. Five patients(5.20%) didnot achieve morphological remission on Day33. Fifteen (15.63%) patients were risk stratified as HR during IA. Four more (4.16%) were reallocated to HR in view of persistent MRD positivity after Induction IB. At median follow-up, the OS was 95.35%±2.65(95% CI 90.16-100.54) and the EFS 94.48%±2.74 (95% CI, 89.11%-99.84%).Female gender predicted better EFS (p=0.042).The EFS of patients without CNS disease at presentation was significantly better(93.5% Vs 23.2% p=0.000). The EFS at median follow up of patients in re HR cohort was 64.19% Vs 97.81% in non HR (p=0.010). EFS by risk stratification is shown in Figure II.Conclusion:Our study suggests that FCM MRD can be successfully incorporated into the treatment algorithm in a resource limited setting. With FCM MRD were able to identify an additional subset of HR patients who otherwise would have been stratified into non HR group. In a prospective cohort , FCM MRD could be tested at an earlier time point in IA induction to facilitate identification of early drug responsive patients with lowest risk of relapse. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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.

VS38c and CD38-Multiepitope Antibodies Provide Highly Comparable Minimal Residual Disease Data in Patients With Multiple Myeloma.

ObjectivesTo compare flow cytometric minimal residual disease (MRD) data obtained using the EuroFlow approach, including the CD38-multiepitope (ME) antibody or the VS38c antibody.MethodsWe evaluated 29 bone marrow samples from patients with multiple myeloma (MM), of whom 15 had received daratumumab within the past 6 months. We evaluated MRD data and fluorescence intensities.ResultsQualitative MRD data were 100% concordant between the 2 approaches. In MRD-positive samples (n = 14), MRD levels showed an excellent correlation (R2 = 0.999). Whereas VS38c staining was strong in both normal plasma cells and MM cells, independent of daratumumab treatment, staining intensities for CD38 were lower in MM cells compared with normal plasma cells, and on both cell types CD38 expression was significantly reduced in daratumumab-treated patients.ConclusionsBoth CD38-ME and VS38c allow reliable MRD detection in MM patients, but the high expression of VS38c allows easier identification of MM cells, especially in daratumumab-treated patients.

Harmonization of Flow Cytometric Minimal Residual Disease Assessment in Multiple Myeloma in Centers of Polish Myeloma Consortium.

Minimal residual disease (MRD) status is now considered as one of the most relevant prognostic factors in multiple myeloma (MM) while MRD negativity became an important endpoint in clinical trials. Here, we report the results of the first study evaluating the reproducibility of high-sensitivity flow cytometry MM MRD assessment in four laboratories in Poland. EuroFlow protocols for instrument setting standardization and sample preparation in MM MRD assessment were implemented in each laboratory. In the inter-laboratory reproducibility study, 12 bone marrow samples from MM patients were distributed and processed in participant laboratories. In the inter-operator concordance study, 13 raw data files from MM MRD measurements were analyzed by five independent operators. The inter-laboratory study showed high 95% overall concordance of results among laboratories. In the inter-operator study, 89% of MRD results reported were concordant, and the highest immunophenotype interpretation differences with regard to expression of CD27, CD45, CD81 were noticed. We confirmed the applicability and feasibility of the EuroFlow protocol as a highly sensitive method of MRD evaluation in MM. Results of our inter-center comparison study demonstrate that the standardization of MM MRD assessment protocols is highly desirable to improve quality and comparability of results within and between different clinical trials.

Hematopoietic stem cell transplantation for infants with high-risk KMT2A gene–rearranged acute lymphoblastic leukemia

AbstractThe role of allogeneic hematopoietic stem cell transplantation (HSCT) for infants with acute lymphoblastic leukemia (ALL) and KMT2A gene rearrangement (KMT2A-r) is controversial in terms of both its efficacy and potential for acute and late toxicities. In Japanese Pediatric Leukemia/Lymphoma Study Group trial MLL-10, by introducing intensive chemotherapy, indication of HSCT was restricted to patients with high-risk (HR) features only (KMT2A-r and either age <180 days or presence of central nervous system leukemia). Of the 56 HR patients, 49 achieved complete remission. Forty-three patients received HSCT in first remission including 38 patients receiving protocol-specified HSCT with conditioning consisting of individualized targeted doses of busulfan, etoposide, and cyclophosphamide. Three-year event-free survival (EFS) of 56.8% (95% confidence interval [CI], 42.4% to 68.8%) and overall survival of 80.2% (95% CI, 67.1% to 88.5%) were accomplished. Univariable analysis showed that Interfant-HR criteria and flow cytometric minimal residual disease (MRD; ≥0.01%), both at the end of induction and at the end of consolidation (EOC), were significantly associated with poorer EFS. In the multivariable analysis, positive MRD at EOC was solely associated with poor EFS (P < .001). Rapid pretransplant MRD clearance and tailored HSCT strategy in the MLL-10 trial resulted in a favorable outcome for infants with HR KMT2A-r ALL. However, considering the high rate of potentially life-threatening toxicities and the risk of late effects, its indication should be further restricted or even eliminated in the future by introducing more effective therapeutic modalities with minimal toxicities. This trial was registered at the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR) as #UMIN000004801.

Changes in Leukemic Blasts CD19 Expression in Children with Relapsed/Refractory B-Cell Precursor ALL Treated with Blinatumomab

Blinatumomab is a bispecific T-cell engager (BiTE) antibody construct targeting CD3 and CD19, resulting in T-cell-mediated lysis of malignant B cells. Currently the main application of blinatumomab is the treatment of relapsed or refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Although promising results were obtained therapy with blinatumomab fails in 30-60% of patients. CD19 downexpression is shown to be one of the possible mechanisms of tumor cells adaptation and subsequent escape from treatment. Moreover, loss of CD19 is a serious obstacle for flow cytometric minimal residual disease (MRD) assessment, necessary for control the leukemia response to therapy. The aim of the present study was to evaluate changes of CD19 expression in children with BCP-ALL treated with blinatumomab and possible monitoring of MRD using additional markers.Patients and methods. From December 2015 to July 201739 (18 F/21M) patients with median age of 8 years (8 mo-20 y) with relapsed/refractory (R/R) BCP-ALL completed at least one course of blinatumomab. 5 patients (13%) carried various types of KMT2A-gene rearrangements and in 3 (7,5%) children ETV6-RUNX1 fusion transcript was detected. MRD monitoring was performed by 8-color flow cytometry. After CD19-directed treatment CD22 and CD24 were added to conventional antibodies' combinations for MRD detection (as suggested by Cherian S. et al, Cytometry B. 2016) to avoid negative influence of potential CD19 loss on cytometric data analysis and interpretation. MRD-negativity was defined as undetectable at the sensitivity level of 0.01%.Results. 20 out of 39(51%) patients continue in complete continuous remission(CCR) after one or two blinatumomab courses, followed by alloHSCT.In 8 (40%) patients in CCR, MRD was never detected since completion of first blinatumomab course, and they were excluded from further analysis. In other 12 pts were found to be MRD-positive at least once after blinatumomab administration. Among them in 5 patients leukemic blasts were still CD19-positive and in 7 patients residual leukemic cells were CD19-negative.Among 19 out of 39 patients (49%) who were resistant to(n=7) or developed bone marrow relapse after blinatumomab (n=12), in 12 (63%) cases tumor cells at relapse were CD19-positive, whereas 5 children experienced CD19-negative relapse. Among them only CD19-positive MRD was detected in one case, only CD19-negative residual blasts - in two cases and in other two children both types of CD19 expression were found atdifferent time-points.Two additional patients, who had CD19-negative MRD, then developed relapse as a lineage “switch” from BCP-ALL to acute myeloid leukemia (AML). In these patients myeloid switch developed in a different ways: one patient carried KMT2A-AF4 fusion gene which is more or less typical for switching AL, the second patient with germline KMT2A hadvery small AML population at the time of relapse diagnosis. Thus in this case positive selection of myeloid subclone during the blinatumomab treatment could be the main pathway of AML development.Conclusions. In our group the remission rate after blinatumomab treatment was about 50%. In those children, who didn't respond and/or developed subsequent relapse, CD19 downexpression seems to be one of the significant pathways for tumor escape from blinatumomab - 26% of relapses were CD19-negative, and in 42% of all relapsed/resistant casesCD19-negative blasts were detected at least on MRD level. Rather frequent loss of CD19 could be a serious obstacle for flow cytometric MRD monitoring, but application of expanded antibodies panel and use of 10-12-color investigation allows residual blasts detection in nearly all cases. Other mechanisms of leukemia resistance including myeloid switch (even in KMT2A-germline patients) can cause blinatumomab failure. DisclosuresNo relevant conflicts of interest to declare.

Flow Cytometric Minimal Residual Disease As Risk Stratification Tool in Younger Adults with NPM1 Wild Type Standard Risk Acute Myeloid Leukemia

Response to induction therapy is a powerful prognostic indicator in acute myeloid leukemia (AML). Measurable/minimal residual disease (MRD) assays are being increasingly incorporated into response assessment; however, their value as a surrogate for long-term outcome remains unclear because of assay limitations and the effect of post remission therapies. This is particularly the case for the molecularly heterogeneous NPM1 wild type standard risk patients for whom treatment decisions, including predicting the benefit of allogeneic stem cell transplantation (SCT), are particularly challenging. We investigated the effect on outcome of MRD status compared to other response criteria after each cycle of induction for NPM1 wild type (wt) standard risk patients and other risk groups in a cohort of younger AML patients.Patients and MethodsAs part of the UK NCRI AML17 trial (ISRCTN55675535) for patients with AML or high risk MDS up to the age of 60, prospective flow cytometric MRD (MFC-MRD) monitoring was performed after each course of induction. Any level of MRD detected was considered MRD+ (sensitivity thresholds: ~0.02% by tracking diagnostic leukemic aberrant phenotypes/LAIP, ~0.05-0.1% by “different-from normal” blast LAIP). Clinicians were not informed of MFC-MRD results. Following cycle 1 of induction with daunorubicin/ara-C based therapy, patients were allocated a risk group by a validated score (comprising cytogenetics, WBC, age, secondary disease, blast response to cycle 1 and mutation status). Standard risk patients including ITD wt/ NPM1 wt received a second daunorubicin/ara-C induction with some proceeding to SCT after 1 or 2 consolidation cycles (high dose ara-C).Results2450 non APML patients, median age 51, randomised 4/09 to 12/14, contributed data. For the total cohort, 5 year overall survival (OS) excluding early deaths was 52% for those achieving CR/CRi vs 31% for those not in CR (p<.001).Post cycle 1 (C1) responses were categorised as resistant disease (RD), partial remission (PR) and CR/CRi by clinicians, with CR/CRi subdivided by MFC-MRD assay into MRD+ and MRD-. RD vs PR vs MRD+ vs MRD- frequencies were 15% vs 14% vs 40% vs 31% for all patients with response data (n=1443), 5% vs 7% vs 45% vs 43% for good / standard risk (n=785), 10% vs 8% vs 47% vs 35% NPM1 wt standard risk (n=320).Post C1 response was prognostic for survival: OS for RD vs PR vs MRD+ vs MRD- were 25% vs 36% vs 43% vs 63% all patients; 33% vs 49% vs 51% vs 70% good/standard risk and 28% vs 38% vs 47% vs 59% NPM1 wt standard risk. (p<.0001 throughout). For NPM1 wt standard risk as well as good/standard risk patients adjusted analyses confirmed significant OS differences between C1 RD vs PR or MRD+ but not PR vs MRD+. Results were similar when censored at SCT.Post C1 best response was CRi for 39/446 MRD- vs 49/575 MRD+ overall (11/111 vs 8/149 for NPM1 wt standard risk). C1 CRi was associated with reduced OS overall (39% vs 53% CR, p=.002) and in MRD+ (19% vs 45% CR, p=.001) but not for MRD- patients. OS for NPM1 wt standard risk MRD+ was 25% CRi vs 43% CR (p=0.4). No significant difference was detected for 5 year cumulative incidence of relapse (CIR).The effect on outcomes of MRD status post cycle 2 (C2) for the 806 patients in CR/CRi with MFC-MRD data appeared greater (relapse HR 1.88(1.50- 2.36), p<0.001; OS HR 1.77(1.41-2.22) p<0.001)than C1 (relapse HR 1.70(1.40- 2.06), p<0.001; OS HR 1.50(1.23-1.84) p<0.001). MRD+ levels of ≥0.1% predicted highest CIR in NPM1 wt standard risk patients at C1 (CIR 71%) and C2 (CIR 89%) but any MRD+ at C2 reduced OS (32% vs 63% MRD-, p=0.003) and OS censored at any SCT (35% vs 88%). The effect of CR vs CRi was smaller post C2 although outcomes were still worst in CRi/MRD+ve patients.Of 204 NPM1 wt standard risk patients with C2 MRD data 83 had an allograft (44 in CR1: 29 MRD-, 15 MRD+). In Mantel-Byar analyses, although numbers were small, there was a suggestion that transplant could be considered in MRD +ve (HR 0.72 (0.31-1.69)) but not MRD -ve patients (HR 1.68 (0.75-3.85); p-value for interaction p=0.16).ConclusionMRD status by MFC refines response criteria at induction time-points to differentiate NPM1 wild type standard risk patients with poor outcome and helps define a group of patients who may benefit from SCT in CR1. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.