Flow Cytometry Minimal Residual Disease 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.

Efficacy and safety of chimeric antigen receptor T-cell therapy followed by allogeneic hematopoietic stem cell transplantation in 21 patients with Ph-like acute lymphoblastic leukemia

Objective: To evaluate the efficacy and safety of chimeric antigen receptor T-cell (CAR-T) therapy followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with Ph-like acute lymphoblastic leukemia (Ph-ALL) . Methods: Patients with Ph-ALL who underwent CAR-T therapy followed by allo-HSCT from March 2018 to August 2023 at the First Affiliated Hospital of Soochow University were included, and their clinical data were retrospectively analyzed. Results: Of the 21 patients, 14 were male and 7 were female. The median age at the time of CAR-T therapy was 22 (6-50) years. Seven patients had ABL1-like rearrangements, and 14 had JAK-STAT rearrangements. Prior to CAR-T therapy, 12 patients experienced hematologic relapse; 7 were multiparameter flow cytometry minimal residual disease (MFC-MRD) -positive and 2 were MFC-MRD-negative. CAR-T cells were derived from patients' autologous lymphocytes. Nine patients were treated with CD19 CAR-T cells, and 12 were treated with CD19/CD22 CAR-T cells. After assessment on day 28 after CAR-T therapy, 95.2% of the patients achieved complete remission, with an MRD-negative remission rate of 75%. Nineteen patients developed grade 0-2 cytokine release syndrome (CRS) and 2 patients suffered grade 3 CRS, all cases of which resolved after treatment. All patients underwent allo-HSCT after CAR-T therapy. The median time from CAR-T therapy to allo-HSCT was 63 (38-114) days. Five patients experienced relapse after CAR-T therapy, including four with hematologic relapse and one with molecular relapse. The 3-year overall survival (OS) rates in the ABL1 and JAK-STAT groups were (83.3±15.2) % and (66.6±17.2) %, respectively (P=0.68) . The 3-year relapse-free survival (RFS) rates were (50.0±20.4) % and (55.6±15.4) % in the ABL1 and JAK-STAT groups, respectively. There was no significant difference in 3-year OS or RFS between the two groups. Conclusions: CAR-T therapy followed by allo-HSCT leads to rapid remission in most patients with Ph-ALL and prolongs leukemia-free survival.

Prognostic significance of multiparametric flow cytometry minimal residual disease at two time points after induction in pediatric acute myeloid leukemia

BackgroundPrompt response to induction chemotherapy is a prognostic factor in pediatric acute myeloid leukemia. In this study, we aimed to evaluate the prognostic significance of multiparametric flow cytometry-minimal residual disease (MFC-MRD), assessed at the end of the first and second induction courses.MethodsMFC-MRD was performed at the end of the first induction (TP1) in 524 patients and second induction (TP2) in 467 patients who were treated according to the modified Medical Research Council (UK) acute myeloid leukemia 15 protocol.ResultsUsing a 0.1% cutoff level, patients with MFC-MRD at the two time points had lower event-free survival and overall survival. Only the TP2 MFC-MRD level could predict the outcome in a separate analysis of high and intermediate risks based on European LeukemiaNet risk stratification and KMT2A rearrangement. The TP2 MFC-MRD level could further differentiate the prognosis of patients into complete remission or non-complete remission based on morphological evaluation. Multivariate analysis indicated the TP2 MFC-MRD level as an independent adverse prognostic factor for event-free survival and overall survival. When comparing patients with MFC-MRD ≥ 0.1%, those who underwent hematopoietic stem cell transplant during the first complete remission had significantly higher 5-year event-free survival and overall survival and lower cumulative incidence of relapse than those who only received consolidation chemotherapy.ConclusionsThe TP2 MFC-MRD level can predict the outcomes in pediatric patients with acute myeloid leukemia and help stratify post-remission treatment.

Combination of minimal residual disease on day 15 and copy number alterations results in BCR-ABL1-negative pediatric B-ALL: A powerful tool for prediction of induction failure

The current genomic abnormalities provide prognostic value in pediatric Acute Lymphoblastic Leukemia (ALL). Furthermore, Copy Number Alteration (CNA) has recently been used to improve the genetic risk stratification of patients. This study aimed to evaluate CNA profiles in BCR-ABL1-negative pediatric B-ALL patients and correlate the data with Minimal Residual Disease (MRD) results after induction therapy. We examined 82 bone marrow samples from pediatric BCR-ABL1-negative B-ALL using the MLPA method for the most common CNAs, including IKZF1, CDKN2A/B, PAX5, RB1, BTG1, ETV6, EBF1, JAK2, and PAR1 region. Subsequently, patients were followed-up by multiparameter Flow Cytometry for MRD (MFC-MRD) assessment on days 15 and 33 after induction. Data showed that 58.5 % of patients carried at least one gene deletion, whereas 41.7 % of them carried more than one gene deletion simultaneously. The most frequent gene deletions were CDKN2A/B, ETV6, and IKZF1 (30.5 %, 14.6 %, and 14.6 %, respectively), while the PAR1 region showed predominantly duplication (30.5 %). CDKN2A/B and IKZF1 were related to positive MRD results on day 15 (p = 0.003 and p = 0.007, respectively). The simultaneous presence of more than one deletion was significantly associated with high induction failure (p = 0.001). Also, according to the CNA profile criteria, the CNA with poor risk (CNA-PR) profile was statistically associated with older age and positive MRD results on day 15 (p = 0.014 and p = 0.013, respectively). According to our results, the combined use of CNAs with MRD results on day 15 can predict induction failure and be helpful in ameliorating B-ALL risk stratification and treatment approaches.

Centralized MRD Assessment at Early Timepoints in Gimema AML1718 Trial, a Phase 2 Open-Label Study of Venetoclax, Fludarabine, Idarubicin and Cytarabine (V-FLAI) in the Induction Therapy of Non Low-Risk AML

Background: New therapeutic strategies are strongly needed to improve the prognosis of high risk Acute Myeloid Leukemia (AML) patients, such as combination of novel agents and conventional chemotherapy. The Italian GIMEMA AML1718 trial (NCT03455504) investigate the safety and efficacy of the BCL-2 inhibitor venetoclax (VEN) in combination with intensive fludarabine-based induction (FLAI) [fludarabine 30 mg/sqm from day 1 to day 5, cytarabine 2000 mg/sqm from day 1 to day 5, and idarubicin 8 mg/sqm on days 1, 3 and 5] as first-line therapy for newly diagnosed non low-risk ELN AML patients. Results from the Planned Interim Analysis of Safety Run-in and Part 1 (early expansion cohorts) were presented at the last 2022 ASH Meeting. Median overall survival (OS) was not reached; probability of 12-month OS was 76%. Median disease-free survival was not reached. With a median follow-up of 10.5 months, 28 patients (49%) received allogeneic stem cell transplantation (HSCT) in first complete remission (CR). Centralized multicolour flow cytometry minimal residual disease (MFC-MRD) assessment was planned during the phase 2, part 2 of the study (confirmatory cohort) where the lower effective dose of VEN (400 mg/day) was administered in association with FLAI. Here we report results from early time-points (TPs) MRD analysis with the aim of identify the most informative TPs for MRD assessment and prognostic correlations. Methods: Erythrocyte-lysed whole bone marrow (BM) samples obtained at diagnosis from patients enrolled in the confirmatory cohort were centralized and analysed with a broad panel of monoclonal antibodies to identify the leukemia-associated phenotype (LAIP) which was used to track residual leukemic cells during follow-up. Eight color flow cytometry analysis was performed at pre-defined TPs (TP1: post-induction I, TP2: post induction II/consolidation I, following TPs: post consolidation/pre-transplantation) (FACSCantoII; BD Facs Diva Software V6.1.3). A positive flow MRD was defined by the presence of no less than 10 clustered leukemic cells/10^4 total events. Enrollment closed on January 2023. Collection and analysis of later TPs is ongoing. Results: Sixty-seven patients from 11 centers were enrolled in the phase 2, part 2. Risk stratification according to ELN 2017 was intermediate in 46% of patients and high risk in 54%. Fifty-eight/67 patients (87%) obtained CR after induction I. In the centralized MRD analysis, 170 samples has been collected and analysed so far (60 baseline samples, 110 MRD samples). Seven patients lacking baseline sample for LAIP identification were excluded from the analysis. TP1 was available in 57/59 patient achieving CR (97%), and TP2 was available in 29 patients, so far. MFC-MRD negativity was obtained in 31/57 (54%) at TP1 (post V-FLAI, Fig. 1). An increase in MFC-MRD negativity rate was observed at TP2 (post Induction II or consolidation I) with 20 MFC-MRD negative patients/29 available samples (69%, Fig. 1). Thirteen/110 follow up samples (8%), albeit resulting MRD negative, were considered inadequate for the analysis due to haemodilution, 9 from TP1 and 4 from TP2 (Fig. 1). TP1 and TP2 MRD assessment were scheduled at day 28 of Induction I and Induction II/consolidation, respectively. However, in most cases, delayed haematological recovery was observed after V-FLAI, thus resulting in suboptimal samples for MRD evaluation. Conclusions: Preliminary results from centralized MRD analysis confirm that the combination therapy is able to induce high-quality remissions with a very high percentage of MFC-MRD negativity in a difficult cohort of patients, with a higher percentage of MFC-MRD-negative after the completion of the second course of therapy. Delayed haematological recovery may impact on reliability of MRD assessment due to hypocellular and regenerative marrow samples, suggesting that in those cases MRD analysis should be postponed. Correlation with survival will be performed as the data collection will be complete.

Early Peripheral Blood Blast Clearance As a Prognostic Marker for Early Treatment Response in Pediatric Acute Myeloid Leukemia

Background and Significance The cure rate of pediatric acute myeloid leukemia (AML) has improved significantly; however, relapses still occur in up to 30% of the total patient population in high-income countries (HICs), and in an even higher percentage of patients in low- and middle-income countries (LMICs). Early treatment response, cytogenetics and minimal residual disease (MRD) are well-known prognostic factors for treatment outcome; however, sophisticated techniques for identifying prognostic factors are often lacking in LMICs, and are sometimes unsuccessful in HICs. Early treatment response assessed by a bone marrow (BM) aspirate on day 15 or day 22 (D22) (depending on the applicable protocol) after initiation of chemotherapy is a known prognostic factor for outcome in patients with pediatric AML, but does not allow modifications of the first induction chemotherapy course (Creutzig 2014, Abrahamsson 2011, Kern 2003). In both pediatric acute lymphoid leukemia (ALL) and adult AML, early peripheral blood blast clearance (PBBC) is a known early indicator of treatment response (Manabe 2008, Arellano 2012), whereas to the best of our knowledge, this has not yet been reported in pediatric AML. In this study, we evaluated the significance of time to complete PBBC, and rate of PBBC after initiation of chemotherapy, in relation to disease status in BM D22, as measure of early treatment response that has prognostic significance. Study Design and Methods Pediatric patients with AML, aged 0-18 years, that were treated according to the NOPHO-DBH AML-2012 protocol were included. The first 5 days of induction therapy consisted of etoposide, followed by 7 days of low-dose cytarabine and mitoxantrone or liposomal daunorubicin. Peripheral blood (PB) was assessed by morphology at the time of diagnosis, daily in the first week, and thrice weekly until D22 or until occurrence of complete clearance of blasts. BM D22 was assessed by morphology and flow cytometry. A receiver operating characteristic (ROC) analysis was performed to evaluate the predictive power of days from starting chemotherapy to complete PBBC. The correlation between time to complete PBBC and BM D22 results was analyzed using a logistic regression model. Multivariable logistic regression models were applied for the association after adjusting for effects of other covariates. Rate of PBBC was defined as the percentage of the absolute PB blast count on the day of diagnosis that cleared with each day of therapy, on average, until D22 or the day of complete PBBC. Results In total, 319 patients were included, out of whom 241 were eligible for analysis, and 78 not eligible due to missing or incomplete data. Day 9 after initiation of chemotherapy was identified as the most discriminating cut-off point for predicting BM D22 response. Patients were categorized into early PBBC (≤9 days; n=159) and delayed PBBC (>9 days; n=82) groups. Early PBBC was associated with having <5% AML cells in BM D22 as assessed by both morphology (OR 3.11; P=0.005) and flow cytometry (OR 2.73; P=0.041). Early PBBC also showed a much lower likelihood of having >15% AML cells in BM D22 by morphology (OR 0.08; P=0.002). None of the early PBBC patients had >15% AML cells in BM D22 by flow cytometry. For 217 patients (selection due to missing data [n=24]), the rate of PBBC could be calculated, and a discriminative cut-off value of 11% was identified. Patients were categorized into high rate of PBBC (>11%; n=132) and low rate of PBBC (≤11%; n=85). High rate of PBBC was associated with having <5% AML cells in BM D22 by flow cytometry (OR 4.30; P=0.007) and negative MRD status (OR 2.02; P=0.004), while also showing a strong association with a lower likelihood of >15% AML cells in BM D22 (OR 0.11; P=0.007) by morphology. None of the high rate PBBC patients had >15% AML cells in BM D22 by flow cytometry. Conclusion Both early complete PBBC and rate of PBBC after initiation of chemotherapy holds promise as prognostic marker for clinical outcome in pediatric AML, as it correlates significantly with BM D22 response. These findings may offer an early and easily accessible prognostic factor, particularly beneficial for LMICs, as well as for patients with unsuccessful MRD analyses.

Analysis of 7 cases of pediatric acute myeloid leukemia with DEK-NUP214 fusion gene

Objective: To investigate the clinical features, treatment regime, and outcome of pediatric acute myeloid leukemia (AML) with DEK-NUP214 fusion gene. Methods: The clinical data, genetic and molecular results, treatment process and survival status of 7 cases of DEK-NUP214 fusion gene positive AML children admitted to the Pediatric Blood Diseases Center of Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences from May 2015 to February 2022 were analyzed retrospectively. Results: DEK-NUP214 fusion gene positive AML accounted for 1.02% (7/683) of pediatric AML diagnosed in the same period, with 4 males and 3 females. The age of disease onset was 8.2 (7.5, 9.5) years. The blast percentage in bone marrow was 0.275 (0.225, 0.480), and 6 cases were M5 by FAB classification. Pathological hematopoiesis was observed in all cases except for one whose bone marrow morphology was unknown. Three cases carried FLT3-ITD mutations, 4 cases carried NRAS mutations, and 2 cases carried KRAS mutations. After diagnosis, 4 cases received IAE induction regimen (idarubicin, cytarabine and etoposide), 1 case received MAE induction regimen (mitoxantrone, cytarabine and etoposide), 1 case received DAH induction regimen (daunorubicin, cytarabine and homoharringtonine) and 1 case received DAE induction regimen (daunorubicin, cytarabine and etoposide). Complete remission was achieved in 3 cases after one course of induction. Four cases who did not achieved complete remission received CAG (aclarubicin, cytarabine and granulocyte colony-stimulating factor), IAH (idarubicin, cytarabine and homoharringtonine), CAG combined with cladribine, and HAG (homoharringtonine, cytarabine and granulocyte colony-stimulating factor) combined with cladribine reinduction therapy, respectively, all 4 cases reached complete remission. Six patients received hematopoietic stem cell transplantation (HSCT) after 1-2 sessions of intensive consolidation treatment, except that one case was lost to follow-up after complete remission. The time from diagnosis to HSCT was 143 (121, 174) days. Before HSCT, one case was positive for flow cytometry minimal residual disease and 3 cases were positive for DEK-NUP214 fusion gene. Three cases accepted haploid donors, 2 cases accepted unrelated cord blood donors, and 1 case accepted matched sibling donor. The follow-up time was 20.4 (12.9, 53.1) months, the overall survival and event free survival rates were all 100%. Conclusions: Pediatric AML with DEK-NUP214 fusion gene is a unique and rare subtype, often diagnosed in relatively older children. The disease is characterized with a low blast percentage in bone marrow, significant pathological hematopoiesis and a high mutation rate in FLT3-ITD and RAS genes. Low remission rate by chemotherapy only and very high recurrence rate indicate its high malignancy and poor prognosis. Early HSCT after the first complete remission can improve its prognosis.

Graft Versus Leukaemia (GvL): Identification & Characterisation of Alloreactive Antigens and Cognate T Cell Responses in Acute Myeloid Leukemia

Allogeneic stem cell transplantation (allo-SCT) is the most established, curative T cell cancer immunotherapy. The curative effect of allo-SCT relies on a graft-versus-leukemia (GvL) effect; but this can be difficult to dissociate from graft-versus-host disease (GvHD). For decades, the key unsolved challenge has been to identify common alloreactive peptide-human leukocyte antigen (HLA) complexes targeted by T cells that mediate GvL but not GvHD. We developed a workflow for unbiased identification of alloreactive antigens and analysis of cognate donor alloreactive T cells and their T cell receptors (TCRs; Fig 1). We studied sequential bone marrow and peripheral blood samples, collected over 15 years, from 12 high-risk AML patients who remain in continuous complete remission following allo-SCT (all >2 years). All patients had relapsed at least once pre-allo-SCT and/or were AML flow cytometry minimal residual disease (MRD)+ pre-allo-SCT. 7 had received donor lymphocyte infusions. No patients developed >1 grade GvHD. We performed whole exome sequencing of DNA from isolated patient T cells (patient germline), AML blasts and donor DNA to identify: (i) AML-specific coding variants; and (ii) germline coding single nucleotide polymorphisms (SNPs), mismatched between patient and donor. 98.7% of the mismatched variants were germline. More variants were identified in unrelated compared with sibling donors (mean: 5744 vs 3253 variants). To narrow the list of variants taken forward for functional studies, we applied two sequential filtering steps: (i) we restricted analysis to variants expressed in the patients' AML cells (determined by RNA-Seq). 28.7% of all mismatched coding variants were expressed in AML cells; and (ii) we used in silico prediction of HLA-binding affinities of peptides spanning variant amino acids to patient-specific HLA-I and -II using NetMHCpan 4.0 and NetMHCIIpan 3.0. To functionally identify alloreactive antigens, we then screened ~200-900 peptides per patient-donor pair (total 5300 peptides) for recognition by T cells present in patient peripheral blood collected 2-7 years after allo-SCT. This enriches for the identification of long-lived anti-AML responses at a time when patients were both infection- and GvHD- free. Response screening, using IFNγ ELISPOT assays, identified T cell responses to 24 germline, mismatched epitopes. In all cases, responses were only observed when the amino acid encoded by the patient SNP, but not the donor allele, was present in the peptide. The phenotype of the epitope-specific T cells was established by intracellular cytokine staining (ICS) and flow cytometry analysis, and the HLA restriction of each T cell response was determined using HLA-I, -DR, -DP and -DQ blocking antibodies. Of the 24 alloreactive peptides,19 elicited CD4+ HLA class II restricted responses and 3 were targeted by CD8+ class I restricted T cells. Two remain to be characterised (Fig 2). The population allele frequency of the germline variant SNPs targeted varies from 0.5% to 72% in the Caucasian population. Likewise, some of the restricting HLA alleles are very common (e.g. a HLA-DP allele presenting 3 epitope peptides is present in ~66% of the Caucasian population). ICS showed that epitope-specific CD4+ T cells produce Th1 cytokines (IFNg, TNFa) and degranulate (express CD107a) in response to peptide stimulation (Fig 2). Next, we performed single cell 5'RNA-seq with TCR-seq of epitope-restricted T cells (isolated via IFN-g catch assay) for all CD4+ epitope-specific T cells. Alloreactive CD4+ T cells have cytokine-producing and cytotoxic effector potential, expressing MIP1a and b, IFNg, granzyme B and FasL (Fig 2). Finally, lentiviral-based gene transfer of one of the candidate alloreactive epitope-specific TCRs into third party primary T cells confers the capacity to generate IFNg and TNFa in response to peptide stimulation, and to recognise primary AML cell-derived antigen in the presence of monocyte-derived dendritic cells (Fig 2). In vitro and in vivo killing assays are in progress. The high proportion of CD4+ T Cell responses identified by our pipeline is concordant with downregulation of HLA II expression on AML blasts at relapse post-allo-SCT, which is a common mechanism of immune evasion, found in one-third of patients. Our approach dissects the biology of alloreactive anti-AML GvL and GvHD responses and to manipulate them for therapeutic purposes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pro-Active 4SCAR2.0 Therapy with NGS-MRD Disease Monitoring in High-Risk B Cell Leukemia Management

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HCT) is a standard treatment for high-risk relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, about 30-40% of patients still relapse after HCT. Chimeric antigen receptor-modified T cell (CAR-T) therapy holds great promise as a salvage approach. Although CD19-targeted CAR-Ts have demonstrated a rapid and significant anti-tumor effect in patients with B-cell malignancies, many still suffer disease relapse due to the decrease or loss of CD19 antigen. Therefore, novel curative approaches are needed. PURPOSE: The aim of this study is to determine the best timing for applying CAR-T therapy in high-risk B-ALL patients based on high-sensitivity analysis of tumor cells by next-generation sequencing (NGS) detecting minimal residual disease (MRD). An innovative CAR-T therapy concept, CAR2.0, has been proposed, which uses multi-antigen-targeted CAR-Ts to prevent antigen escape and prolong remission. Based on NGS-MRD detection, patients with potential tumor relapse can be identified early and managed accordingly. On the other hand, if extended negative NGS-MRD is found, the patient is deemed at low risk and no further treatment is needed. The study proposes to treat high-risk B-ALL patients in the first remission period with CAR2.0 followed by continued disease monitoring by NGS-MRD to avoid HCT. METHODS: B-ALL patients who have high tumor burden and high-risk genetic indication is enrolled with the understanding that CAR-T therapy will be applied in the first remission and disease will be continuously monitored by NGS-MRD. The tumor cells from initial diagnosis will be analyzed for target antigens by immunostaining and to detect clonally rearranged immunoglobulin gene sequences by NGS-MRD with sensitivity ≤10-6. Patients will receive 4SCAR19 (CD19scFv-CD28/CD27/CD3ζ-iCasp9) therapy in the first remission and will be followed up for CAR-Ts, B cells and IgG/IgA/IgM in the blood, and bi-monthly NGS-MRD for residual tumor cells in the bone marrow (BM). A booster CAR-T treatment targeting additional tumor antigen such as CD22 will be applied 2 months later. A consolidation CAR-T treatment based on known target antigens will be applied 6 months after the first CAR-T infusion. The cyclophosphamide/fludarabine chemo-conditioning will be included prior to the first CAR-T treatment only. The CAR-T infusion dose is 1-5x106 CAR-Ts/kg. The quality of apheresis cells, efficiency of gene transfer, T cell proliferation, CAR-T infusion dose and blood CAR copies are quantitatively documented. The end point for this study is the establishment of NGS-MRD negativity for 1 year, with continued follow-up for 5 years. RESULTS: A 20 years old female was diagnosed with BCR/ABL1 P190 positive B-ALL, with 82.52% tumor blasts in the BM. NGS identified two high-frequency IgH clones at 66.29% and 30.41%. The patient received induction chemotherapy (VTCP, CAM, VP+MTX) combined with TKI therapy (Nilotinib) and achieved complete remission (CR) after the first round of treatment based on both flow cytometry and NGS MRD analyses. During the fourth round of chemotherapy (Methotrexate-Cytarabine-Dexamethasone), the patient suffered severe oral and intestinal mucositis, along with kidney and liver toxicity and required week-long dialysis. A decision was made to stop chemotherapy and start the first 4SCAR19 therapy of 3.53x106 CAR-Ts/kg, while maintaining the TKI therapy. The CAR-T cells in blood, the B cell count and Ig levels were monitored, and a booster CD22/CD19 dual CAR-Ts (4SCAR2219) was given at a dose of 3.35 x106 CAR-Ts/kg on day 96 (see figure). Consistent with 4SCART safety profile, the patient experienced no greater than grade 1 CRS. NGS-MRD detection of tumor cell clones remained negative on day 96 after the first CAR-T infusion and at 6 months into the remission. In summary, the 4SCAR2.0 therapy regimen appeared safe and effective in the innovative NGS-MRD/4SCAR2.0 B-ALL study regimen with in vivo persistency for more than three months. The simultaneous expansion of CD19 mono-specific and CD22/CD19 bi-specific CAR-Ts supports a stable maintenance treatment with good life quality as compared with conventional chemotherapy. CONCLUSIONS: The combination of 4SCAR2.0 with NGS-MRD detection of tumor clones in the management of high-risk B-ALL appears to be safe and may be a logical option for patients not elected for HCT therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Real-World Study of the Effect of rhG-CSF on Clinical Efficacy and Flow Cytometry MRD after Initial Induction Therapy for Acute Myeloid Leukemia

To investigate the effect of recombinant human granulocyte colony stimulating factor (rhG-CSF) on the clinical efficacy and flow cytometry (FCM) minimal residual disease (MRD) of patients with acute myeloid leukemia (AML) after initial induction therapy in the real world. The clinical data of 44 AML patients who were diagnosed for the first time in the Department of Hematology, The Second Hospital of Anhui Medical University, and received the initial induction therapy were retrospectively analyzed. According to whether rhG-CSF was used after treatment, these patients were divided into control group and therapy group. The complete remission (CR) rate, duration of neutropenia, incidence of infection, duration of fever, cost of antibiotics drugs, length of hospital stay, FCM MRD, and relapse-free survival (RFS) time were compared between the two groups. The CR rate in the control group was 60%, and 74% in the therapy group (P=0.3429). The duration of neutropenia was (21.28±7.91) days in the control group and (14.79±3.07) days in the therapy group (P=0.0016). The duration of fever was (12.80±7.31) days in the control group and (9.11±7.48) days in the therapy group (P=0.0136). While, there were no statistically significant differences between the two groups in the incidence of infection, cost of antibacterial drugs, length of hospital stay and RFS time (all P>0.05). In addition, it is particularly noteworthy that among the patients who finally obtained CR in the therapy group, 66% of them had myeloid precursor cells detected by peripheral blood FCM (accounting for 2.25%±0.99%) at the time of the first release of neutropenia, which was easy to be misdiagnosed as MRD positive. rhG-CSF not only don't affect the clinical remission rate after the initial induction treatment of AML, but also significantly shortens the time of duration of neutropenia and fever, however, it may affect the analysis of peripheral blood FCM MRD detection results when the neutropenia is released for the first time.

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 2793: Minimal residual disease (MRD) as a predictive biomarker/endpoint for novel drug development

Abstract MRD is defined as the continual presence of cancer cells in patients post therapy that is below the threshold of detection by conventional morphologic or radiologic assessments. Monitoring patient status clinically as MRD negative or MRD positive has become the basis for predicting outcomes, remission or reoccurrence, as well as determining therapeutic options. The three primary methods to assess MRD status in patients include Flow Cytometry (FC), Polymerase Chain Reaction (PCR) and Next-Generation Sequencing (NGS). All three are highly quantitative measurements with a high degree of sensitivity to detect “new” circulating tumor cells or ctDNA that can potentially indicate disease recurrence before it happens. The choice of MRD methodology usually depends on the tumor type, liquid or solid. Flow Cytometry has been widely used to monitor MRD status in patients with hematologic malignancies. Specifically designed MRD panels have been used clinically in CLL, MM, CML and AML patients. In addition to its prognostic/diagnostic relevance, recent guidance by the FDA is now driving the use of MRD testing as a predictive biomarker/endpoint for novel cancer drug development. NeoGenomics Pharma Services has provided flow cytometry MRD expertise to multiple sponsors conducting clinical trials to evaluate the effectiveness of novel cancer therapy. In a CLL study, NeoGenomics employed the ERIC MRD assessment to monitor patient status during therapeutic intervention at various points throughout treatment. Representative patient data show progression from full, active disease prior to therapy to non-detectable disease levels below 0.01%MRD. These examples show patient progression through multiple cycles of treatment to full remission. Clearly, this is an example of how MRD can serve as the primary “biomarker” to evaluate a patient’s response to novel drug therapy in clinical trials. This presentation will further discuss the data and the relevance of MRD testing to support new drug development. Citation Format: Nicholas Jones, Brian Ngo, Floyd Davis, Benjamin Fancke. Minimal residual disease (MRD) as a predictive biomarker/endpoint for novel drug development [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 2793.

Post-Transplantation Day +100 Minimal Residual Disease Detection Rather Than Mixed Chimerism Predicts Relapses after Allogeneic Stem Cell Transplantation for Intermediate-Risk Acute Myelogenous Leukemia Patients Undergoing Transplantation in Complete Remission

Chimerism and minimal residual disease (MRD) are suggested to be prognostic for post-transplantation relapse in patients with acute myelogenous leukemia (AML). Nevertheless, the predictive value of both approaches in homogeneous populations remains underinvestigated. Here we suggest that MRD may have greater predictive value for relapse than mixed chimerism (MC) in intermediate-risk AML patients. Seventy-nine patients with intermediate-risk AML (40 males; median age, 57 years [range, 19 to 77 years]) were included. MRD detection on day +100 was performed in bone marrow via multiparameter flow cytometry (MFC) and quantitative real-time PCR (qPCR) for patients with an NPM1 mutation. Chimerism analysis was performed in peripheral blood. MC was defined as the persistence of <99.9% of donor alleles. The area under the receiver operating characteristic curve was highest for qPCR-MRD (.93), followed by MFC-MRD (.80) and MC (.65). The highest rate of relapse at 3 years was observed in day +100 qPCR-MRD-positive patients (100%), followed by MFC-MRD-positive patients (55%; P < .001). No patients with MC and without detectable MRD experienced relapse. The median 3-year overall survival (OS) and leukemia-free survival (LFS) for patients with MC without detectable MRD were both 86% (range, 61% to 96%), significantly higher than the values in day +100 MFC-MRD-positive patients (OS, 61% [range, 36% to 84%]; LFS: 30% [range, 11% to 59%]) and with day +100 qPCR-MRD-positive patients (OS: 17% [range, 3% to 56%], P = .001; LFS: 0%, P < .001). In patients with intermediate-risk AML, the qPCR-MRD on day +100 was highly predictive for relapse and long-term survival after allogeneic stem cell transplantation, followed closely by MFC-MRD. In contrast, chimerism status had limited predictive potential. Thus, molecular and flow cytometry MRD monitoring rather than MC in the first several months post-transplantation can identify patients at increased risk of relapse who may benefit from early post-transplantation preemptive intervention.

Post-Transplantation Multicolored Flow Cytometry–Minimal Residual Disease Status on Day 100 Predicts Outcomes for Patients With Refractory Acute Myeloid Leukemia

Patients with relapsed/refractory acute myeloid leukemia (AML) have a dismal prognosis. Allogeneic stem cell transplantation (allo-SCT) provides a curative approach; however, the overall survival (OS) remains low (20% to 40%). In this setting, although some effective approaches have been evaluated in recent years, the management of such patients still remains challenging. In this study we evaluated the predictive role of post-transplant day 100 minimal residual disease (MRD) detection for post-transplantation outcomes for patients with refractory AML. Fifty-six adult patients with refractory AML (median age 58, range 20-76; male, 61%) who underwent allo-SCT were included in this retrospective monocentric study. Twenty-nine patients (52%) received fludarabine, amsacrine, and cytarabine (FLAMSA)–based conditioning. MRD was assessed using multicolored flow cytometry (MFC) according to European Leukemia Net guidelines (“different from normal” and leukemia-associated phenotype). The sensitivity of the method was 10−4 to 10−5. The median marrow blast count at allo-SCT was 25% (range 6% to 91%). At day 100 after transplantation, 40 patients (71%) experienced MFC-MRD negativity, and 16 patients (29%) were MRD positive. All included patients survived at least 100 days after transplantation without relapse. Univariate and multivariate analysis based on Kaplan-Meier and Cox proportional hazards method were performed. The median follow-up was 16 months (range 3 to 66). The post-transplantation day 100 MRD-negative patients instead received 2 allografts (27% versus 6%, P = .08). In multivariate analysis, day 100 MRD status (negative versus positive) (OS: 0.23 [0.1 to 0.54], P =0.001; relapses: 0.20 [0.1 to 0.49], P = .0005) and FLAMSA versus other regimens (0.34 [0.1 to 0.83], P = .018; relapses: 0.43 [0.17 to 1.1], P = .07) independently impacted post-transplantation survival. We suggest that post-transplantation day 100 MFC-MRD detection plays predictive role in refractory AML patients and may help to define possible candidates for early post-transplantation interventions aiming to decrease the relapse risk and improve survival.

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.

Performance of Next Generation Sequencing for Minimal Residual Disease Detection for Pediatric Patients with Acute Lymphoblastic Leukemia: Results from the Prospective Clinical Trial DFCI 16-001

Performance of Next Generation Sequencing for Minimal Residual Disease Detection for Pediatric Patients with Acute Lymphoblastic Leukemia: Results from the Prospective Clinical Trial DFCI 16-001

ALL Maintenance Treatment for Early Loss of B-Cell Aplasia after Tisagenlecleucel Therapy

Chimeric antigen receptor (CAR) T-cell therapy is a new, effective treatment for patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukaemia (ALL). Tisagenlecleucel achieved a complete remission (CR) rate and minimal residual disease (MRD) negativity of 81% at 3 months in the pivotal study; overall survival (OS) was 76% at 12 months (Maude et al, 2018). Real world data confirmed similar outcomes, with 1-year OS of 77% and event free survival (EFS) of 52% (Pasquini et al, 2020). Relapse can occur in the form of CD19 negative or CD19 positive ALL. The latter is associated with lack of persistence of the CAR T product. B-cell aplasia (BCA) is an indirect measure of CAR T presence. Early (<6 months from infusion) loss of BCA is associated with high relapse risk (Pillai et al, 2019); therefore, allogeneic stem cell transplantation (SCT) is often considered. However, SCT is associated with therapy-related morbidity and mortality and not all patients will find a suitable donor. Therefore, the optimal management of patients with loss of BCA is yet to be defined.In our centre, we administered maintenance therapy to a cohort of children with early loss of BCA. When compared to UK patients undergoing SCT for the same indication, we noted promising early outcomes. We report the findings here.We collected data on children with r/r ALL treated with tisagenlecleucel at Great Ormond Street Hospital (GOSH) from January 2018 to January 2021 who presented loss of BCA without evidence of disease (negative molecular or flow cytometry MRD) within 12 months from infusion. Loss of BCA was defined as peripheral B-cell count ≥0.10 x 10^9/L or bone marrow (BM) CD19+ events ≥0.1%. We compared outcomes of children who received maintenance as per UKALL 2011 protocol at GOSH to those who received SCT for the same indication from all UK paediatric centres.Fourteen patients from GOSH met the inclusion criteria. Four had loss of BCA after 6 months from CAR T infusion, none of them received additional therapy and they are all alive and in CR at a median of 535 days after CAR T infusion (Figure 1, A and B). Ten patients recovered B cells at <6 months: 3 proceeded to SCT, 6 started on maintenance therapy, 1 received other treatment. In 2 cases, maintenance was commenced after a second CAR T infusion. Two patients from the UK cohort met the inclusion criteria for the SCT group. Analysis was performed on 6 children who received maintenance and 5 who had SCT. Baseline characteristics of the 2 groups were similar (male/female ratio, median age at infusion, cytogenetics). Time from infusion to loss of BCA did not differ: the median was 80 days (range 28-168) in children who had maintenance vs 93 days (range 28-150) in those who had SCT. At a median follow up of 511 days (range 222-812), 3/6 children who received maintenance relapsed at median 210 days after infusion and proceeded to further treatment, no patient relapsed post SCT. One child died of disease in the maintenance group 237 days after infusion, 2 children died of transplant related mortality in the SCT group at 222 and 422 days post infusion. OS and EFS did not differ statistically between the 2 groups, as shown in Figure 1 (C and D). We observed that outcome for patients who presented loss of BCA within or at 2 months from infusion was poor regardless of the intervention (maintenance or SCT).Management of patients who experience early loss of BCA after CAR T is challenging and there are little data to support optimal treatment. In our experience, maintenance therapy compared favourably with SCT with similar rate of OS and EFS. Of note, 2/5 patients died of TRM in the SCT group highlighting the toxicity of this approach in such heavily pre-treated patients. On the other hand, maintenance is a well tolerated, low-cost treatment which can be easily delivered on an out-patient basis. Our preliminary data support investigation of this strategy in larger, prospectively-recruited cohorts of patients. Moreover, our preliminary data suggest that the time of loss of BCA is a crucial clinical parameter, as children who developed it within 2 months from infusion had the worst outcome, possibly reflecting prior therapy intensity and its impact on autologous T cells. [Display omitted] DisclosuresAmrolia: ADC Therapeutics: Other: Named inventor on a patent which is being transferred to ADCT.; Autolus: Patents & Royalties. Ghorashian: UCLB: Patents & Royalties: CARPALL; Novartis: Honoraria.

Outcomes of Venetoclax and Hypomethylating Agents (HMA) in Adult Patients with KMT2A-Rearranged Leukemias

BackgroundRearrangements of lysine methyltransferase 2A (KMT2A) gene, previously known as the MLL gene, occur in 3 to 5% of adult patients with de novo AML and are enriched in therapy-related disease after treatment with topoisomerase II inhibitors (Bill, M et al. PNAS. 2020). KMT2A encodes a histone H3 lysine 4 methyltransferase and KMT2A rearrangements result in fusion proteins that induce aberrant Hox gene expression. (Armstrong, SA et al. Nature. 2002) Among patients with KMT2A rearranged AML (rAML) receiving intensive chemotherapy, the fusion partner impacts prognosis, and KMT2A-MLLT3 is associated with an intermediate risk while other KMT2A rearrangements are associated with adverse risk in the ELN classification. (Dohner, H et al. Blood. 2017) Here we evaluate the outcomes of patients with newly diagnosed and relapsed or refractory (R/R) AML with KMT2A rearrangements receiving venetoclax and hypomethylating agents (HMA).MethodsMedical records of 333 patients with newly diagnosed or R/R AML receiving venetoclax in combination with HMAs at City of Hope National Medical Center between 11/1/2015 and 4/15/2020 were reviewed. Criteria for inclusion were a pathologically confirmed diagnosis of AML, age > 18 years, treatment with either decitabine or azacitidine in combination with venetoclax. KMT2A rearrangements were detected by karyotype and confirmed by FISH or RNA sequencing. Responses were evaluated per the ELN criteria (Dohner, H et al. Blood. 2017) Minimal residual disease flow cytometry was performed at the University of Washington. Patient characteristics were summarized by frequency and associations between overall response and patient and disease characteristics were tested by Fisher's exact test. OS was evaluated by the Kaplan-Meier method and the difference between groups was determined by log-rank test. All statistical analyses were performed using SPSS and Prism.ResultsWe identified 18 patients (5.4%) with KMT2A rAML who met criteria for inclusion. MLLT3 was the predominant fusion partner, occurring in nine patients followed by ELL (n=2), AFDN (n=2), MLLT6 (n=1), MLLT10 (n=1), AFF1 (n=1), CBL (n=1), and TET1 (n=1). The cohort included both newly diagnosed (n=10) and R/R (n=8) AML patients. 44% had therapy-related or secondary AML. NRAS or KRAS mutations occurred in 4 out of 13 patients (31%) with available sequencing prior to treatment. Decitabine was the predominant HMA used in combination with venetoclax and 56% of all patients received 10-day dosing during the first cycle. For the total cohort, 9 patients achieved an overall response (ORR 50%), including 8 patients with a complete remission (CR/CRi 44%) and 1 (6%) patient with a morphologic leukemia free state, Figure 1. All six of the responders who were tested for MRD were negative. For treatment naïve patients, we observed a CR/CRi rate of 70% and a median survival of 11 months. On univariate analysis, R/R disease was the only factor associated with a significant decrease in response (ORR 12.5% vs. 80%, p=0.015, Table 1). With a median follow-up of 14.4 months for responding patients, median OS for the cohort was 6.59 months and 19.15 months for responding patients (Figure 2). The presence of NRAS or KRAS mutations was the only factor significantly impacting survival (HR 6.04, log rank 0.05, Table 1). Notably, the KMT2A fusion partner type did not impact response or survival. Allogeneic stem cell transplant was performed in 4 out of 9 (44%) responding patients.ConclusionHere, we show that venetoclax in combination with HMA led to a high rate of response and prolonged survival in a high-risk KMT2A rAML population. The outcomes of newly diagnosed KMT2A rAML patients after treatment with venetoclax and HMA in this study are similar to chemotherapy outcomes in patients aged < 60 years (CR 68% and median OS 0.9 months; Bill, M et al. PNAS. 2020). Consistent with previous studies, we found that MLLT3 was the most common fusion partner, occurring in 50% of patients and that RAS mutations were also common (~30%). In contrast to what has been reported for chemotherapy outcomes and in the ELN classification, the KMT2A-MLLT3 translocation was not associated with improved outcomes when compared to other KMT2A translocations. While this study was limited in being retrospective and having a small and heterogeneous population, our findings suggest that venetoclax and HMA are effective in KMT2A rAML and warrant further investigation. [Display omitted] DisclosuresKoller: Novartis: Consultancy. Al Malki: Hansa Biopharma: Consultancy; Rigel Pharma: Consultancy; Neximmune: Consultancy; Jazz Pharmaceuticals, Inc.: Consultancy; CareDx: Consultancy. Aribi: Seagen: Consultancy. Ali: Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Speakers Bureau; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees. Marcucci: Novartis: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings. Pullarkat: AbbVie, Amgen, Genentech, Jazz Pharmaceuticals, Novartis, Pfizer, and Servier: Membership on an entity's Board of Directors or advisory committees; Amgen, Dova, and Novartis: Consultancy, Honoraria.

Response Adaptive Salvage Treatment with Carfilzomib-Thalidomide-Dexamethasone for Newly Diagnosed Transplant Eligible Multiple Myeloma Patients Failing Front-Line Bortezomib-Based Induction Therapy - Final Analysis from the Australasian Leukemia and Lymphoma Group (ALLG) MM17 Trial

Response Adaptive Salvage Treatment with Carfilzomib-Thalidomide-Dexamethasone for Newly Diagnosed Transplant Eligible Multiple Myeloma Patients Failing Front-Line Bortezomib-Based Induction Therapy - Final Analysis from the Australasian Leukemia and Lymphoma Group (ALLG) MM17 Trial

Molecular and MRD-Based Characterization of Acute Lymphoblastic Leukemia in Mexico: Experience from the Mexico in Alliance with St. Jude “Bridge Project”

▪Background: Cancer is the second leading cause of death in Mexico among children 5-14 years. In 2017, Mexico in Alliance with St. Jude (MAS), a multi-site intersectoral collaboration, explored reasons for suboptimal outcomes for children with acute lymphoblastic leukemia (ALL). Results showed 82% of patients to be classified as high-risk and 30% of patients be missing standard molecular or minimal residual disease (MRD) studies needed to inform risk- group stratification. Making sense of the molecular characteristics was challenging in the context of variable access and lack of common denominators. These findings led to the development of the “Bridge Project”; a prospective quality improvement demonstration project aiming to bridge this access and quality gap through multi-site collaboration, centralization, and standardization.Methods: Six MAS collaborating hospitals, located in six different states (Mérida, Guadalajara, Sinaloa, Baja California, Chiapas, and Veracruz), have sent diagnostic samples for children 0-18 years old with suspected ALL to Hospital Infantil Teletón de Oncología (HITO), in Queretaro, which serves as the centralized laboratory for the MAS cooperative group. Access to the diagnostic panel is secured upfront through dedicated funding obtained from a local foundation. The first sample was shipped in July 2019. The consensus-derived diagnostic panel includes morphology, immunophenotype, karyotype, fluorescent in situ hybridization (FISH), molecular biology (RT-PCR) and flow cytometry (FC) MRD at two time points. Protocols vary by institution and MRD evaluation is only done if the institutional protocol incorporates MRD-evaluation into the risk-group stratification. Hospitals send several empty boxes prior to shipping patient samples to assess their regional shipping vendors and timelines. The also standardize data collection processes and use PDSA cycles to monitor sample quality and address issues with sample quality. HITO produces FC results (for diagnosis or MRD evaluation) and FISH/cytogenetic results within 3 to 21 days of sample arrival.Results: As of July 2021, the centralized lab has received samples from 217 patients with suspected ALL through this project and 93% of these samples have arrived within the 48hr target. Of these, 176 (81.1%) had confirmed ALL and 14 (6.4%) had acute myeloid leukemia (AML), 2 (0.9%) cases had other malignant conditions, and in 25 (11.5%) of the cases leukemia was ruled out. Among the 176 cases of confirmed ALL, 162 (92%) had B-cell lineage and 14 (8%) had T-cell lineage. FISH was reported for 172 (97.7%) patients and positive in 126 (73.2%) cases; 46 (26.7%) cases had reported gains, 34 (19.7%) detected t(12;21), 18 (10.4%) detected iAMP cr21, 8 (4.7%) detected breaks of the MLL gene t(4;11), 7 (4%) detected t(1;19), and 6 (3.4%) detected t(9;22). Four T-cell ALL cases had breaks CDKN2A del(9)(p21) and three cases had TRA/D rearrangement (14) (q11.2). Karyotype alterations were detected in 90 (52.3%) of the samples, of which 41 (23.8%) showed hyperdiploidy and 13 (7%) showed complex karyotype. One case of hypodiploid was identified. Day 15 MRD was assessed in 131 (80%) patients with B-cell lineage, Day 84 MRD was assessed in 99 (61.1%) patients with B-cell lineage, and Day 29 MRD in 8 (57.1%) patients with T-cell lineage whose treatment schema utilizes MRD-based stratification. Among B-lineage patients, 84% had MRD <1% at Day 15 and 94.9% MRD <0.01% at Day 84. Among T-cell lineage patients, 75% had MRD <0.01% at Day 29 and 80% MRD <0.01% at Day 84, but numbers for T-cell lineage were small.Conclusions: Given proper structural and financial supports, patients with suspected childhood ALL in Mexico can access a comprehensive diagnostic panel following a centralized laboratory approach. Preliminary results from the Bridge Project allow characterizing childhood ALL to a degree that has not been previously possible in Mexico. In this cohort, favorable characteristics such a t(12;21), gains, and hyperdiploidy are observed in frequencies similar to those reported in Hispanic cohorts in high-income countries. MRD results are for B-cell lineage are also consistent with the literature. Continued engagement in this project from hospitals in diverse geographic settings and with diverse patient populations will allow the MAS cooperative group to continue to improve the characterization of childhood ALL in Mexico. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.