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PURPOSE CD19-targeted chimeric antigen receptor T cells (CD19-CAR) and blinatumomab effectively induce remission in relapsed or refractory B-cell acute lymphoblastic leukemia (ALL) but are also associated with CD19 antigen modulation. There are limited data regarding the impact of prior blinatumomab exposure on subsequent CD19-CAR outcomes. PATIENTS AND METHODS We conducted a multicenter, retrospective review of children and young adults with relapsed or refractory ALL who received CD19-CAR between 2012 and 2019. Primary objectives addressed 6-month relapse-free survival (RFS) and event-free survival (EFS), stratified by blinatumomab use. Secondary objectives included comparison of longer-term survival outcomes, complete remission rates, CD19 modulation, and identification of factors associated with EFS. RESULTS Of 420 patients (median age, 12.7 years; interquartile range, 7.1-17.5) treated with commercial tisagenlecleucel or one of three investigational CD19-CAR constructs, 77 (18.3%) received prior blinatumomab. Blinatumomab-exposed patients more frequently harbored KMT2A rearrangements and underwent a prior stem-cell transplant than blinatumomab-naïve patients. Among patients evaluable for CD19-CAR response (n = 412), blinatumomab nonresponders had lower complete remission rates to CD19-CAR (20 of 31, 64.5%) than blinatumomab responders (39 of 42, 92.9%) or blinatumomab-naive patients (317 of 339, 93.5%), P < .0001. Following CD19-CAR, blinatumomab nonresponders had worse 6-month EFS (27.3%; 95% CI, 13.6 to 43.0) compared with blinatumomab responders (66.9%; 95% CI, 50.6 to 78.9; P < .0001) or blinatumomab-naïve patients (72.6%; 95% CI, 67.5 to 77; P < .0001) and worse RFS. High-disease burden independently associated with inferior EFS. CD19-dim or partial expression (preinfusion) was more frequently seen in blinatumomab-exposed patients (13.3% v 6.5%; P = .06) and associated with lower EFS and RFS. CONCLUSION With the largest series to date in pediatric CD19-CAR, and, to our knowledge, the first to study the impact of sequential CD19 targeting, we demonstrate that blinatumomab nonresponse and high-disease burden were independently associated with worse RFS and EFS, identifying important indicators of long-term outcomes following CD19-CAR.

PURPOSE Children's Oncology Group (COG) AALL0331 tested whether pegaspargase intensification on a low-intensity chemotherapy backbone would improve the continuous complete remission (CCR) rate in a low-risk subset of children with standard-risk B-acute lymphoblastic leukemia (ALL). METHODS AALL0331 enrolled 5,377 patients with National Cancer Institute standard-risk B-ALL (age 1-9 years, WBC < 50,000/μL) between 2005 and 2010. Following a common three-drug induction, a cohort of 1,857 eligible patients participated in the low-risk ALL random assignment. Low-risk criteria included no extramedullary disease, < 5% marrow blasts by day 15, end-induction marrow minimal residual disease < 0.1%, and favorable cytogenetics ( ETV6-RUNX1 fusion or simultaneous trisomies of chromosomes 4, 10, and 17). Random assignment was to standard COG low-intensity therapy (including two pegaspargase doses, one each during induction and delayed intensification) with or without four additional pegaspargase doses at 3-week intervals during consolidation and interim maintenance. The study was powered to detect a 4% improvement in 6-year CCR rate from 92% to 96%. RESULTS The 6-year CCR and overall survival (OS) rates for the entire low-risk cohort were 94.7% ± 0.6% and 98.7% ± 0.3%, respectively. The CCR rates were similar between arms (intensified pegaspargase 95.3% ± 0.8% v standard 94.0% ± 0.8%; P = .13) with no difference in OS (98.1% ± 0.5% v 99.2% ± 0.3%; P = .99). Compared to a subset of standard-risk study patients given identical therapy who had the same early response characteristics but did not have favorable or unfavorable cytogenetics, outcomes were significantly superior for low-risk patients (CCR hazard ratio 1.95; P = .0004; OS hazard ratio 5.42; P < .0001). CONCLUSION Standard COG therapy without intensified pegaspargase, which can easily be given as an outpatient with limited toxicity, cures nearly all children with B-ALL identified as low-risk by clinical, early response, and favorable cytogenetic criteria.

Secondary acute myeloid leukemia (sAML) is biologically and clinically distinct from de novo AML and shares specific genetic mutations with myelodysplastic syndromes (MDS). We retrospectively analy...

PURPOSE Nelarabine is effective in inducing remission in patients with relapsed and refractory T-cell acute lymphoblastic leukemia (T-ALL) but has not been fully evaluated in those with newly diagnosed disease. PATIENTS AND METHODS From 2007 to 2014, Children’s Oncology Group trial AALL0434 (ClinicalTrials.gov identifier: NCT00408005 ) enrolled 1,562 evaluable patients with T-ALL age 1-31 years who received the augmented Berlin-Frankfurt-Muenster (ABFM) regimen with a 2 × 2 pseudo-factorial randomization to receive escalating-dose methotrexate (MTX) without leucovorin rescue plus pegaspargase (C-MTX) or high-dose MTX (HDMTX) with leucovorin rescue. Intermediate- and high-risk patients were also randomly assigned after induction to receive or not receive six 5-day courses of nelarabine that was incorporated into ABFM. Patients who experienced induction failure were nonrandomly assigned to HDMTX plus nelarabine. Patients with overt CNS disease (CNS3; ≥ 5 WBCs/μL with blasts) received HDMTX and were randomly assigned to receive or not receive nelarabine. All patients, except those with low-risk disease, received cranial irradiation. RESULTS The 5-year event-free and overall survival rates were 83.7% ± 1.1% and 89.5% ± 0.9%, respectively. The 5-year disease-free survival (DFS) rates for patients with T-ALL randomly assigned to nelarabine (n = 323) and no nelarabine (n = 336) were 88.2% ± 2.4% and 82.1% ± 2.7%, respectively ( P = .029). Differences between DFS in a four-arm comparison were significant ( P = .01), with no interactions between the MTX and nelarabine randomizations ( P = .41). Patients treated with the best-performing arm, C-MTX plus nelarabine, had a 5-year DFS of 91% (n = 147). Patients who received nelarabine had significantly fewer isolated and combined CNS relapses compared with patients who did not receive nelarabine (1.3% ± 0.63% v 6.9% ± 1.4%, respectively; P = .0001). Toxicities, including neurotoxicity, were acceptable and similar between all four arms. CONCLUSION The addition of nelarabine to ABFM therapy improved DFS for children and young adults with newly diagnosed T-ALL without increased toxicity.

PURPOSE The Children’s Oncology Group (COG) protocol AALL0434 evaluated the safety and efficacy of multi-agent chemotherapy with Capizzi-based methotrexate/pegaspargase (C-MTX) in patients with newly diagnosed pediatric T-cell lymphoblastic lymphoma (T-LL) and gained preliminary data using nelarabine in high-risk patients. PATIENTS AND METHODS The trial enrolled 299 patients, age 1-31 years. High-risk (HR) patients had ≥ 1% minimal detectable disease (MDD) in the bone marrow at diagnosis or received prior steroid treatment. Induction failure was defined as failure to achieve a partial response (PR) by the end of the 4-week induction. All patients received the augmented Berlin-Frankfurt-Muenster (ABFM) C-MTX regimen. HR patients were randomly assigned to receive or not receive 6 5-day courses of nelarabine incorporated into ABFM. Patients with induction failure were nonrandomly assigned to ABFM C-MTX plus nelarabine. No patients received prophylactic cranial radiation; however, patients with CNS3 disease (CSF WBC ≥ 5/μL with blasts or cranial nerve palsies, brain/eye involvement, or hypothalamic syndrome) were ineligible. RESULTS At end-induction, 98.8% of evaluable participants had at least a PR. The 4-year event-free survival (EFS) and overall survival (OS) were 84.7% ± 2.3% and 89.0% ± 2.0%. The 4-year disease-free survival (DFS) from end-induction was 85.9% ± 2.6%. There was no difference in DFS observed between the HR and standard-risk groups ( P = .29) or by treatment regimen ( P = .55). Disease stage, tumor response, and MDD at diagnosis did not demonstrate thresholds that resulted in differences in EFS. Nelarabine did not show an advantage for HR patients. CNS relapse occurred in only 4 patients. CONCLUSION COG AALL0434 produced excellent outcomes in one of the largest trials ever conducted for patients with newly diagnosed T-LL. The COG ABFM regimen with C-MTX provided excellent EFS and OS without cranial radiation.

Background The recently updated European LeukemiaNet risk stratification guidelines combine cytogenetic abnormalities and genetic mutations to provide the means to triage patients with acute myeloid leukemia for optimal therapies. Despite the identification of many prognostic factors, relatively few have made their way into clinical practice. Methods In order to assess and improve the performance of the European LeukemiaNet guidelines, we developed novel prognostic models using the biomarkers from the guidelines, age, performance status and select transcript biomarkers. The models were developed separately for mononuclear cells and viable leukemic blasts from previously untreated acute myeloid leukemia patients (discovery cohort, N = 185) who received intensive chemotherapy. Models were validated in an independent set of similarly treated patients (validation cohort, N = 166). Results Models using European LeukemiaNet guidelines were significantly associated with clinical outcomes and, therefore, utilized as a baseline for comparisons. Models incorporating age and expression of select transcripts with biomarkers from European LeukemiaNet guidelines demonstrated higher area under the curve and C-statistics but did not show a substantial improvement in performance in the validation cohort. Subset analyses demonstrated that models using only the European LeukemiaNet guidelines were a better fit for younger patients (age ASXL1, CEBPA, RUNX1 and TP53, demonstrated that these mutations provide a limited overall contribution to risk stratification across the entire population, given the low frequency of mutations and confounding risk factors. Conclusions While European LeukemiaNet guidelines remain a critical tool for triaging patients with acute myeloid leukemia, the findings illustrate the need for additional prognostic factors, including age, to improve risk stratification.

Association of blinatumomab treatment with myelosuppression was examined in this study. Peripheral blood counts were assessed prior to, during, and after blinatumomab treatment in patients with relapsed/refractory Philadelphia chromosome-negative (Ph−) B-cell precursor (BCP) acute lymphoblastic leukemia (ALL, n = 267) and Ph+ BCP-ALL (n = 45) from the TOWER and ALCANTARA studies, respectively, or chemotherapy in patients with Ph− BCP-ALL (n = 109) from the TOWER study, all the patients with relapsed/refractory BCP-ALL and responders achieving complete remission (CR) or CR with partial/incomplete hematological recovery (CRh/CRi) were evaluated. Event-free survival (EFS) and overall survival (OS) were assessed in patients achieving CR and CRh/CRi. Median leukocyte, neutrophil, and platelet counts increased during two blinatumomab cycles but remained low longer after chemotherapy. Among the responders, there was a trend that a greater proportion of patients achieved CR with blinatumomab (Ph−, 76.5%, Ph+, 77.8%) versus with chemotherapy (Ph−, 63.6%). In the TOWER study, the survival prognosis for patients achieving CRh/CRi versus CR with blinatumomab was more similar (median OS, 11.9 (95% CI, 3.9–not estimable (NE)) vs. 15.0 (95% CI, 10.4–NE) months, p = 0.062) than with chemotherapy (5.2 (95% CI, 1.6–NE) vs. 18.9 (95% CI, 9.3–NE) months, p = 0.013). Blinatumomab treatment, with only temporary and transient myelosuppression, resulted in a greater survival benefit than chemotherapy.

Adolescent and young adult (AYA) patients 16–30 years old with high-risk acute lymphoblastic leukemia (HR-ALL) have inferior outcomes compared to younger HR-ALL patients. AALL0232 was a Phase 3 randomized Children’s Oncology Group trial for newly diagnosed HR B-ALL (1–30 years). Between 2004 and 2011, 3154 patients enrolled with 3040 eligible and evaluable for induction. AYA patients comprised 20% of patients (16–21 years, n = 551; 22–30 years, n = 46). 5-year event-free survival and overall survival was 65.4 ± 2.2% and 77.4 ± 2.0% for AYA patients compared to 78.1 ± 0.9% and 87.3 ± 0.7% for younger patients (p

PURPOSE Children’s Oncology Group (COG) AALL0331 tested whether intensified postinduction therapy that improves survival in children with high-risk B-cell acute lymphoblastic leukemia (ALL) would also improve outcomes for those with standard-risk (SR) ALL. PATIENTS AND METHODS AALL0331 enrolled 5,377 patients between 2005 and 2010. All patients received a 3-drug induction with dexamethasone, vincristine, and pegaspargase (PEG) and were then classified as SR low, SR average, or SR high. Patients with SR-average disease were randomly assigned to receive either standard 4-week consolidation (SC) or 8-week intensified augmented Berlin-Frankfurt-Münster (BFM) consolidation (IC). Those with SR-high disease were nonrandomly assigned to the full COG-augmented BFM regimen, including 2 interim maintenance and delayed intensification phases. RESULTS The 6-year event-free survival (EFS) rate for all patients enrolled in AALL0331 was 88.96% ± 0.46%, and overall survival (OS) was 95.54% ± 0.31%. For patients with SR-average disease, the 6-year continuous complete remission (CCR) and OS rates for SC versus IC were 87.8% ± 1.3% versus 89.1% ± 1.2% ( P = .52) and 95.8% ± 0.8% versus 95.2% ± 0.8% ( P = 1.0), respectively. Those with SR-average disease with end-induction minimal residual disease (MRD) of 0.01% to < 0.1% had an inferior outcome compared with those with lower MRD and no improvement with IC (6-year CCR: SC, 77.5% ± 4.8%; IC, 77.1% ± 4.8%; P = .71). At 6 years, the CCR and OS rates among 635 nonrandomly treated patients with SR-high disease were 85.55% ± 1.49% and 92.97% ± 1.08%, respectively. CONCLUSION The 6-year OS rate for > 5,000 children with SR ALL enrolled in AALL0331 exceeded 95%. The addition of IC to treatment for patients with SR-average disease did not improve CCR or OS, even in patients with higher MRD, in whom it might have been predicted to provide more value. The EFS and OS rates are excellent for this group of patients with SR ALL, with particularly good outcomes for those with SR-high disease.

B-cell maturation antigen (BCMA) is a validated target for chimeric antigen receptor (CAR) T-cell therapy in multiple myeloma (MM). Despite promising objective response rates, most patients relapse, and low levels of BCMA on a subset of tumor cells has been suggested as a probable escape mechanism. BCMA is actively cleaved from the tumor cell surface by the ubiquitous multisubunit γ-secretase (GS) complex, which reduces ligand density on tumor cells for CAR T-cell recognition and releases a soluble BCMA (sBCMA) fragment capable of inhibiting CAR T-cell function. Sufficient sBCMA can accumulate in the bone marrow of MM patients to inhibit CAR T-cell recognition of tumor cells, and potentially limit efficacy of BCMA-directed adoptive T-cell therapy. We investigated whether blocking BCMA cleavage by small-molecule GS inhibitors (GSIs) could augment BCMA-targeted CAR T-cell therapy. We found that exposure of myeloma cell lines and patient tumor samples to GSIs markedly increased surface BCMA levels in a dose-dependent fashion, concurrently decreased sBCMA concentrations, and improved tumor recognition by CAR T cells in vitro. GSI treatment of MM tumor-bearing NOD/SCID/γc(−/−) mice increased BCMA expression on tumor cells, decreased sBCMA in peripheral blood, and improved antitumor efficacy of BCMA-targeted CAR T-cell therapy. Importantly, short-term GSI administration to MM patients markedly increases the percentage of BCMA(+) tumor cells, and the levels of BCMA surface expression in vivo. Based on these data, a US Food and Drug Administration (FDA)-approved clinical trial has been initiated, combining GSI with concurrent BCMA CAR T-cell therapy. This trial was registered at www.clinicaltrials.gov as #NCT03502577.

In this review, we discuss applications of flow cytometric immunophenotyping (FCI) in the diagnostic evaluation and posttreatment monitoring of B and T lymphoblastic leukemia/lymphoma. We describe practical approaches to FCI at the time of diagnosis, with an emphasis on blast identification, lineage assignment, and distinction of B and T lymphoblastic leukemia/lymphoma from their morphologic and immunophenotypic mimics. We further review flow cytometric assays for the detection of minimal or measurable residual disease (MRD) after treatment, and illustrate both standard approaches, and newer strategies for improving sensitivity and circumventing the loss of immunophenotypic targets after immunotherapy. © 2019 International Clinical Cytometry Society.

Background: Patients with late, occurring ≥18 months post-diagnosis, isolated central nervous relapse (iCNS-R) of B-acute lymphoblastic leukemia (ALL) have excellent outcomes with chemotherapy plus cranial radiotherapy, with 5-yr overall survival (OS) approaching 80% in POG 9412. Subsequent relapse and radiation-related morbidity remain the causes of treatment failure and long-term sequelae. COG AALL02P2 aimed to maintain outcomes in patients with late iCNS-R using intensified chemotherapy and a decrease in cranial irradiation from 1800 to 1200 cGy. Procedures: COG AALL02P2 enrolled 118 eligible patients with B-ALL and early iCNS-R who received intensified systemic therapy, triple intrathecal chemotherapy and 1200 cGy cranial irradiation delivered at 12 months, with maintenance chemotherapy continuing until104 weeks post-diagnosis. Results: The 3-yr event-free and overall survival (EFS) and OS were 64.3±4.5% and 79.6±3.8%, with 46.1% (18/39) of relapses including the CNS. Of the 112 patients who completed therapy, 78 received protocol-specified radiation. Study enrollment was closed after interim monitoring analysis showed inferior EFS compared to POG 9412. Patients with initial NCI standard risk classification fared better than high risk patients. Conclusions: COG AALL02P2 showed inferior EFS but similar OS compared to POG 9412. Limitations included a small sample size, more intensive prior therapies, and a significant number of patients (34/118, 29%) who did not receive protocol-directed radiation due to early relapse prior to 1 year or did not otherwise follow the treatment plan. New approaches are needed to improve outcome for these patients and determine the optimal timing and dose of cranial radiation in the treatment of iCNS-R.

Purpose: AALL0932 evaluated two randomized maintenance interventions to optimize disease-free survival (DFS) while reducing the burden of therapy in children with newly diagnosed NCI standard-risk (SR) B-acute lymphoblastic leukemia (B-ALL). Methods: AALL0932 enrolled 9,229 patients with B-ALL; 2,364 average-risk (AR) patients were randomly assigned (2 × 2 factorial design) at the start of maintenance therapy to vincristine/dexamethasone pulses every 4 (VCR/DEX4) or every 12 (VCR/DEX12) weeks, and a starting dose of weekly oral methotrexate of 20 mg/m2 (MTX20) or 40 mg/m2 (MTX40). Results: Five-year event-free survival and overall survival (OS) from enrollment (with 95% CIs), for all eligible and evaluable SR B-ALL patients (n = 9,226), were 92.0% (91.1% and 92.8%) and 96.8% (96.2% and 97.3%), respectively. The 5-year DFS and OS from the start of maintenance for randomly assigned AR patients were 94.6% (93.3% and 95.9%) and 98.5% (97.7% and 99.2%), respectively. The 5-year DFS and OS for patients randomly assigned to receive VCR/DEX4 (n = 1,186) versus VCR/DEX12 (n = 1,178) were 94.1% (92.2% and 96.0%) and 98.3% (97.2% and 99.4%) v 95.1% (93.3% and 96.9%) and 98.6% (97.7% and 99.6%), respectively ( P = .86 and .69). The 5-year DFS and OS for AR patients randomly assigned to receive MTX20 versus MTX40 were 95.1% (93.3% and 96.8%) and 98.8% (97.9% and 99.7%) v 94.2% (92.2% and 96.1%) and 98.1% (97.0% and 99.2%), respectively ( P = .92 and .89). Conclusions: The 0NCI-SR AR B-ALL who received VCR/DEX12 had outstanding outcomes despite receiving one third of the vincristine/dexamethasone pulses previously used as standard of care on Children's Oncology Group (COG) trials. The higher starting dose of MTX of 40 mg/m2/week did not improve outcomes when compared with 20 mg/m2/week. The decreased frequency of vincristine/dexamethasone pulses has been incorporated into frontline COG B-ALL trials to decrease the burden of therapy for patients and their families.

Measurable residual disease (MRD) is an important biomarker in acute myeloid leukemia (AML) that is used for prognostic, predictive, monitoring, and efficacy-response assessments. The European LeukemiaNet (ELN) MRD Working Party evaluated standardization and harmonization of MRD in an ongoing manner and has updated the 2018 ELN MRD recommendations based on significant developments in the field. New and revised recommendations were established during in-person and online meetings, and a 2-stage Delphi poll was conducted to optimize consensus. All recommendations are graded by levels of evidence and agreement. Major changes include technical specifications for next-generation sequencing-based MRD testing and integrative assessments of MRD irrespective of technology. Other topics include use of MRD as a prognostic and surrogate end point for drug testing; selection of the technique, material, and appropriate time points for MRD assessment; and clinical implications of MRD assessment. In addition to technical recommendations for flow- and molecular-MRD analysis, we provide MRD thresholds and define MRD response, and detail how MRD results should be reported and combined if several techniques are used. MRD assessment in AML is complex and clinically relevant, and standardized approaches to application, interpretation, technical conduct, and reporting are of critical importance.

How conditioning intensity is related to outcomes of AML patients undergoing allografting in morphologic remission is an area of great ongoing interest. We studied 743 patients in morphologic remission and known pre-transplant measurable residual disease (MRD) status determined by multiparameter flow cytometry (MFC) who received a first allograft after myeloablative, reduced intensity, or nonmyeloablative conditioning (MAC, RIC, and NMA). Overall, relapse-free survival (RFS) and overall survival (OS) were longer after MAC than RIC or NMA conditioning, whereas relapse risks were not different. Among MRDpos patients, 3-year estimates of relapse risks and survival were similar across conditioning intensities. In contrast, among MRDneg patients, 3-year RFS and OS were longer for MAC (69% and 71%) than RIC (47% and 55%) and NMA conditioning (47% and 52%). Three-year relapse risks were lowest after MAC (18%) and highest after NMA conditioning (30%). Our data indicate an interaction between conditioning intensity, MFC-based pre-transplant MRD status, and outcome, with benefit of intensive conditioning primarily for patients transplanted in MRDneg remission. Differing from recent findings from other studies that indicated MAC is primarily beneficial for some or all patients with MRDpos pre-HCT status, our data suggest MAC should still be considered for MRDneg AML patients if tolerated.

The 5-year disease-free survival (DFS) of National Cancer Institute (NCI) high-risk (HR) B-lymphoblastic leukemia (B-ALL) patients with end of induction (EOI) minimal residual disease (MRD) ≥0.1% and end of consolidation (EOC) MRD ≥0.01% is 39 ± 7%, warranting consideration of hematopoietic stem cell transplant (HSCT). However, the impact of EOC MRD in NCI standard-risk (SR) B-ALL patients using COG regimens is unknown. We found that SR patients with MRD ≥0.01% at both EOI and EOC have a 4-year DFS/overall survival (OS) of 72.9 ± 19.0%/91.7 ± 10.8% versus 90.7 ± 2.9%/95.5 ± 2.0% (p = .0019/.25) for those with EOI MRD ≥0.01% and EOC MRD

PURPOSE The high-risk stratum of Children’s Oncology Group Study AALL1131 was designed to test the hypothesis that postinduction CNS prophylaxis with intrathecal triple therapy (ITT) including methotrexate, hydrocortisone, and cytarabine would improve the postinduction 5-year disease-free survival (DFS) compared with intrathecal methotrexate (IT MTX), when given on a modified augmented Berlin-Frankfurt-Münster backbone. PATIENTS AND METHODS Children with newly diagnosed National Cancer Institute (NCI) high-risk B-cell acute lymphoblastic leukemia (HR B-ALL) or NCI standard-risk B-ALL with defined minimal residual disease thresholds during induction were randomly assigned to receive postinduction IT MTX or ITT. Patients with CNS3-status disease were not eligible. Postinduction IT therapy was given for a total of 21 to 26 doses. Neurocognitive assessments were performed during therapy and during 1 year off therapy. RESULTS Random assignment was closed to accrual in March 2018 after a futility boundary had been crossed, concluding that ITT could not be shown to be superior to IT MTX. The 5-year postinduction DFS and overall survival rates (± SE) of children randomly assigned to IT MTX versus ITT were 93.2% ± 2.1% v 90.6% ± 2.3% ( P = .85), and 96.3% ± 1.5% v 96.7% ± 1.4% ( P = .77), respectively. There were no differences in the cumulative incidence of isolated bone marrow relapse, isolated CNS relapse, or combined bone marrow and CNS relapse rates, or in toxicities observed for patients receiving IT MTX compared with ITT. There were no significant differences in neurocognitive outcomes for patients receiving IT MTX compared with ITT. CONCLUSION Postinduction CNS prophylaxis with ITT did not improve 5-year DFS for children with HR B-ALL. The standard of care for CNS prophylaxis for children with B-ALL and no overt CNS involvement remains IT MTX.

Allogeneic hematopoietic cell transplantation (HCT) is often unsuccessful for monosomal karyotype (MK) acute myeloid leukemia (AML). To what degree failures are associated with pretransplant measurable residual disease (MRD)—a dominant adverse-risk factor—is unknown. We therefore studied 606 adults with intermediate- or adverse-risk AML in morphologic remission who underwent allogeneic HCT between 4/2006 and 1/2019. Sixty-eight (11%) patients had MK AML, the majority of whom with complex cytogenetics. Before HCT, MK AML patients more often tested MRDpos by multiparameter flow cytometry (49 vs. 18%; P < 0.001) and more likely had persistent cytogenetic abnormalities (44 vs. 13%; P < 0.001) than non-MK AML patients. Three-year relapse/overall survival estimates were 46%/43% and 72%/15% for MRDneg and MRDpos MK AML patients, respectively, contrasted to 20%/64% and 64%/38% for MRDneg and MRDpos non-MK AML patients, respectively. After multivariable adjustment, MRDpos remission status but not MK remained statistically significantly associated with shorter survival and higher relapse risk. Similar results were obtained in several patient subsets. In summary, while our study confirms higher relapse rates and shorter survival for MK-AML compared with non-MK AML patients, these outcomes are largely accounted for by the presence of other adverse prognostic factors, in particular higher likelihood of pre-HCT MRD.

BACKGROUND Abnormalities involving chromosome 7 are one of the most frequent chromosomal aberrations in myeloid neoplasms including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) and are associated with an adverse prognosis. Immunophenotyping by flow cytometry provides data that can assist in the diagnosis and classification of myeloid neoplasms. The immunophenotypic features of myeloid neoplasms with monosomy 7 or del(7q) have not been previously described in a comprehensive fashion. METHODS We retrospectively analyzed flow cytometric data of myeloid neoplasms with monosomy 7 or del(7q) and summarized associated immunophenotypic features. RESULTS Myeloid neoplasms with monosomy 7 typically demonstrate multiple immunophenotypic abnormalities on myeloid blasts and maturing myelomonocytic cells. Increased CD14 expression on maturing granulocytic cells was characteristically seen in myeloid neoplasms with monosomy 7. This abnormality was significantly more frequent in myeloid neoplasms with monosomy 7 than in those with del(7q) (92.9% vs. 8.2%, P

The identification of residual leukemia following therapy, termed minimal or measurable residual disease (MRD), has emerged as one of the most important prognostic factors for patients with acute leukemia, including acute myeloid leukemia (AML). Flow cytometry is a preferred method for MRD detection due to its general applicability and the rapid results that it makes available. In this article, the basic protocol outlines a simple and efficient method for the labeling of hematopoietic cells from bone marrow or peripheral blood with a panel of monoclonal antibodies designed both to highlight patterns of normal maturation and allow identification of neoplastic hematopoietic progenitor populations with a high degree of sensitivity and specificity. The method was developed in a clinical laboratory setting for the diagnosis of myeloid stem cell disorders and neoplasms, and has been extensively validated both technically and clinically for the detection of MRD in AML. © 2020 The Authors. Basic Protocol: Staining and flow cytometry for AML minimal residual disease detection Support Protocol: Analysis and interpretation of data for AML minimal residual disease detection.

BackgroundMinimal residual disease (MRD) after induction therapy is one of the strongest prognostic factors in childhood acute lymphoblastic leukemia (ALL), and MRD-directed treatment intensification improves survival. Little is known about the effects of inherited genetic variants on interpatient variability in MRD.MethodsA genome-wide association study was performed on 2597 children on the Children’s Oncology Group AALL0232 trial for high-risk B-cell ALL. Association between genotype and end-of-induction MRD levels was evaluated for 863 370 single nucleotide polymorphisms (SNPs), adjusting for genetic ancestry and treatment strata. Top variants were further evaluated in a validation cohort of 491 patients from the Children’s Oncology Group P9905 and 6 ALL trials. The independent prognostic value of single nucleotide polymorphisms was determined in multivariable analyses. All statistical tests were 2-sided.ResultsIn the discovery genome-wide association study, we identified a genome-wide significant association at the GATA3 locus (rs3824662, odds ratio [OR] = 1.58, 95% confidence interval [CI] = 1.35 to 1.84; P = 1.15 × 10-8 as a dichotomous variable). This association was replicated in the validation cohort (P = .003, MRD as a dichotomous variable). The rs3824662 risk allele independently predicted ALL relapse after adjusting for age, white blood cell count, and leukemia DNA index (P = .04 and .007 in the discovery and validation cohort, respectively) and remained prognostic when the analyses were restricted to MRD-negative patients (P = .04 and .03 for the discovery and validation cohorts, respectively).ConclusionInherited GATA3 variant rs3824662 strongly influences ALL response to remission induction therapy and is associated with relapse. This work highlights the potential utility of germline variants in upfront risk stratification in ALL.

With modern chemotherapy, approximately 90% of patients with pediatric acute lymphoblastic leukemia are now cured. However, subsets of patients can be identified who remain at very high risk of relapse with expected 4-year disease-free survival rates

Background The presence of measurable residual disease after therapy is a significant risk factor of relapse in patients with acute myeloid leukemia (AML). By detecting cells with leukemia-associated immunophenotype (LAIP), multiparameter flow cytometry (MFC) can detect residual leukemia at a level significantly lower than that detected by morphology. However, changes in LAIPs during or after therapy may pose a challenge to MRD testing. AML with mutated NPM1 represents the largest subtype of AML sharing a common leukemogenic mechanism and similar LAIPs. Here, we identified a common pattern of LAIPs in myeloid blasts with mutated NPM1, and studied its stability and limit of detection after therapy. Methods We summarized aberrancies of leukemic blasts with mutated NPM1 at diagnosis in 61 patients and paired relapse in 25 patients. In addition, we examined the detection of leukemic blasts in 590 specimens collected from 152 patients in complete remission after induction for AML/MDS-EB with mutated NPM1. Results Our findings demonstrate myeloid blasts with mutated NPM1 have a characteristic pattern of LAIPs that is present in nearly all cases of AML/MDS-EB with mutated NPM1 at initial diagnosis and relapse, regardless of morphologic variations, FLT3 ITD status, or karyotype abnormality. The myeloid blasts with mutated NPM1 can be detected at an approximate level of 0.1% of total leukocytes in morphologic remission with high specificity validated by clinical outcome. Conclusion The characteristic pattern of LAIPs of myeloid blasts with mutated NPM1 is common and stable, and allows sensitive and specific detection of AML or MDS with mutated NPM1 after therapy. © 2018 International Clinical Cytometry Society.

Early response to induction chemotherapy is an important prognostic factor in B-lymphoblastic leukemia (B-ALL). Here, we compare high-throughput sequencing (HTS) of IGH and TRG genes vs flow cytometry (FC) for measurable residual disease (MRD) detection at the end of induction chemotherapy in pediatric patients with newly diagnosed B-ALL. Six hundred nineteen paired pretreatment and end-of-induction bone marrow samples from Children's Oncology Group studies AALL0331 (clinicaltrials.gov #NCT00103285) (standard risk [SR]; with MRD by FC at any level) and AALL0232 (clinicaltrials.gov #NCT00075725) (high risk; with day 29 MRD

Measurable residual disease (MRD; previously termed minimal residual disease) is an independent, postdiagnosis, prognostic indicator in acute myeloid leukemia (AML) that is important for risk stratification and treatment planning, in conjunction with other well-established clinical, cytogenetic, and molecular data assessed at diagnosis. MRD can be evaluated using a variety of multiparameter flow cytometry and molecular protocols, but, to date, these approaches have not been qualitatively or quantitatively standardized, making their use in clinical practice challenging. The objective of this work was to identify key clinical and scientific issues in the measurement and application of MRD in AML, to achieve consensus on these issues, and to provide guidelines for the current and future use of MRD in clinical practice. The work was accomplished over 2 years, during 4 meetings by a specially designated MRD Working Party of the European LeukemiaNet. The group included 24 faculty with expertise in AML hematopathology, molecular diagnostics, clinical trials, and clinical medicine, from 19 institutions in Europe and the United States.

Minimal residual disease (MRD) after initial therapy is integral to risk stratification in B-precursor and T-precursor acute lymphoblastic leukemia (B-ALL, T-ALL). Although MRD determines depth of remission, remission remains defined by morphology. We determined the outcomes of children with discordant assessments of remission by morphology vs. flow cytometry using patients age 1-30.99 years enrolled on Children's Oncology Group ALL trials who underwent bone marrow assessment at the end of induction (N = 9350). Morphologic response was assessed locally as M1 (5% lymphoblasts; remission), M2 (5-25%), or M3 (25%). MRD was centrally measured by flow cytometry. Overall, 19.8% of patients with M2/M3 morphology had MRD 5%. M1 with MRD ≥ 5% was less common in B-ALL (0.9%) than T-ALL (6.9%; p 0.0001). In B-ALL, M1/MRD ≥ 5% was associated with superior 5-year event-free survival (EFS) than M2/MRD ≥ 5% (59.1% ± 6.5% vs. 39.1% ± 7.9%; p = 0.009), but was inferior to M1/MRD 5% (87.1% ± 0.4%; p 0.0001). MRD levels were higher in M2/MRD ≥ 5% than M1/MRD ≥ 5% patients. In T-ALL, EFS was not significantly different between M1/MRD ≥ 5% and M2/MRD ≥ 5%. Patients with morphologic remission but MRD ≥ 5% have outcomes similar to those who fail to achieve morphological remission, and significantly inferior to those with M1 marrows and concordant MRD, suggesting that flow cytometry should augment the definition of remission in ALL.

Purpose Germline TP53 variation is the genetic basis of Li-Fraumeni syndrome, a highly penetrant cancer predisposition condition. Recent reports of germline TP53 variants in childhood hypodiploid acute lymphoblastic leukemia (ALL) suggest that this type of leukemia is another manifestation of Li-Fraumeni syndrome; however, the pattern, prevalence, and clinical relevance of TP53 variants in childhood ALL remain unknown. Patients and Methods Targeted sequencing of TP53 coding regions was performed in 3,801 children from the Children’s Oncology Group frontline ALL clinical trials, AALL0232 and P9900. TP53 variant pathogenicity was evaluated according to experimentally determined transcriptional activity, in silico prediction of damaging effects, and prevalence in non-ALL control populations. TP53 variants were analyzed for their association with ALL presenting features and treatment outcomes. Results We identified 49 unique nonsilent rare TP53 coding variants in 77 (2.0%) of 3,801 patients sequenced, of which 22 variants were classified as pathogenic. TP53 pathogenic variants were significantly over-represented in ALL compared with non-ALL controls (odds ratio, 5.2; P < .001). Children with TP53 pathogenic variants were significantly older at ALL diagnosis (median age, 15.5 years v 7.3 years; P < .001) and were more likely to have hypodiploid ALL (65.4% v 1.2%; P < .001). Carrying germline TP53 pathogenic variants was associated with inferior event-free survival and overall survival (hazard ratio, 4.2 and 3.9; P < .001 and .001, respectively). In particular, children with TP53 pathogenic variants were at a dramatically higher risk of second cancers than those without pathogenic variants, with 5-year cumulative incidence of 25.1% and 0.7% ( P < .001), respectively. Conclusion Loss-of-function germline TP53 variants predispose children to ALL and to adverse treatment outcomes with ALL therapy, particularly the risk of second malignant neoplasms.

Current prognostic models for acute myeloid leukemia (AML) are inconsistent at predicting clinical outcomes for individual patients. Variability in the quality of specimens utilized for biomarker discovery and validation may contribute to this prognostic inconsistency.We evaluated the impact of sample heterogeneity on prognostic biomarkers and methods to mitigate any adverse effects of this heterogeneity in 240 cryopreserved bone marrow and peripheral blood specimens from AML patients enrolled on SWOG (Southwest Oncology Group) trials.Cryopreserved samples displayed a broad range in viability (37% with viabilities ≤60%) and nonleukemic cell contamination (13% with lymphocyte percentages20%). Specimen viability was impacted by transport time, AML immunophenotype, and, potentially, patients' age. The viability and cellular heterogeneity in unsorted samples significantly altered biomarker results. Enriching for viable AML blasts improved the RNA quality from specimens with poor viability and refined results for both DNA and RNA biomarkers. For example, FLT3-ITD allelic ratio, which is currently utilized to risk-stratify AML patients, was on average 1.49-fold higher in the viable AML blasts than in the unsorted specimens.To our knowledge, this is the first study to provide evidence that using cryopreserved specimens can introduce uncontrollable variables that may impact biomarker results and enrichment for viable AML blasts may mitigate this impact.

HyperCVAD is a commonly-used regimen for adults with newly-diagnosed acute lymphoblastic leukemia (ALL). However, relatively little is known about the application of minimal residual disease (MRD) detection with this treatment. To address this, we studied 142 adults with ALL treated with hyperCVAD over a 10-year period who had MRD assessed by either multi-parameter flow cytometry or (for patients with Philadelphia chromosome positive ALL) reverse transcriptase polymerase chain reaction for the BCR-ABL1 translocation. In a multivariate analysis, patients who achieved MRD negativity (MRDNeg ) at any point had significantly better overall survival (OS; hazard ratio [HR] 0.43; P = .01) and event-free survival (EFS; HR 0.27; P < .01). Of 121 patients with MRD assessed at various points within 90 days of starting hyperCVAD, 50% (n = 61) had achieved MRDNeg . Among those that became MRDNeg , the median time to MRDNeg was 68 days. Time to MRDNeg was significantly associated with EFS (P = .009), but not OS (P = .19), implying increasingly better EFS the earlier MRDNeg is achieved. These data add to our understanding of MRD assessment during treatment with hyperCVAD, aide clinicians with predicting relapse risk, and provide additional historical data on which future clinical trials can be designed.

Background: The presence of measurable residual disease (MRD) is strongly associated with treatment outcomes in acute myeloid leukemia (AML). However, > 20% of MRD negative cases (as assessed by flow cytometry) subsequently relapse. We sought to determine if ultrasensitive Duplex Sequencing (DS), which relies on double-stranded consensus-making to achieve an error rate below one-in-ten-million, yields better prognostic performance via molecular MRD detection. Methods: Retrospective targeted DNA sequencing of 29 genes recurrently mutated in adult AML was performed on paired diagnostic and remission bone marrow samples from patients enrolled on the SWOG trial S0106 (randomized 7+3 versus 7+3 + gemtuzumab ozogamicin (GO)). Patients were selected if they had remission samples with flow cytometry results (n=67). Non-error corrected sequencing was performed on diagnostic samples (average depth 279x) and DS was performed on remission samples (average duplex molecular depth 27,002x). For each patient, potential germline variants were identified and excluded from the analysis if the variant allele fraction (VAF) was ≥ 35% at both diagnosis and remission, or ≥ 40% at either time point and a gnomAD allele frequency ≥ 0.05. Somatic variants present at diagnosis were classified as potentially deleterious if computationally predicted as such and with a VAF ≥ 5% (≥ 1% for FLT3-ITD/NPM1 insertions). For analysis of residual disease in remission, we evaluated the following outcomes (events): overall survival (OS; death), relapse-free survival (RFS) and time to relapse (TTR; relapse with death a competing event). All outcomes were measured from date of morphologic remission to date of event, with patients without event censored at date of last contact. Associations between residual disease and outcomes were assessed using Cox regression models (cause-specific model for TTR). Results: The median age was 48 years (range 8-60). 32 patients were randomized to 7+3 and 30 to 7+3+GO. A total of 172 potentially deleterious variants were identified in the diagnostic samples. Variants had an average VAF of 31% (range 1.4-91.5%) at diagnosis and were detected in 23 of the 29 genes, with FLT3 being the most frequently mutated. Of the 67 patients analyzed, 93% (n=62) had at least one variant detected at diagnosis (median 2, range 0-9) and 68% (n=42) had at least one residual diagnostic variant also found in the remission sample. We defined the presence of DS MRD as non-DTA (DNMT3A, TET2, ASXL1) time-of-diagnosis mutations identified at the remission time point with a VAF > 0.1% and/or an NPM1 VAF > 0.01% (PMID:31860405). DS MRD was strongly associated with all outcomes, with hazard ratios (and 95% CI) for TTR: 7.1 (2.7-18.9); RFS: 4.9 (2.2-10.9) and OS: 5.1 (2.1-12.3). As a comparator, we correlated treatment outcomes with the results of a flow cytometry MRD assay previously carried out on the same samples during the S0106 trial. The prognostic association of flow MRD with TTR, RFS and OS was less strong (i.e., a smaller hazard ratio) than DS MRD, with TTR: 2.5 (0.9-6.7); RFS: 2.2 (0.9-5.4) and OS: 2.4 (1.0-6.1). RFS and TTR for DS MRD and flow cytometry are plotted below for the 62 patients with a variant detected at diagnosis. Comparing DS MRD with flow cytometry, discordance was found in 20 cases: 15 cases where DS was positive and flow negative, and 5 cases in the opposite direction. Among the 15 discordant cases with DS positive and flow negative, 9 relapsed and 2 died without relapse and 4 were alive without relapse at last contact. Among the 5 discordant cases with DS negative and flow positive, 1 relapsed, 1 died without relapse, and 3 were alive without relapse at last contact. Conclusions: Among the 67 patients evaluated in this prospectively collected study, the presence of MRD defined by DS was strongly associated with adverse disease outcomes. The vast majority of patients had at least one time-of-diagnosis mutation that could be tracked as a measure of MRD. When compared with flow cytometry, DS exhibited superior negative and positive predictive values for foretelling future relapse, although this could potentially reflect the historical version of the flow cytometry assay used. These findings suggest that DS is a powerful tool that could be used in patient management and for early treatment assessment in clinical trials. Figure 1 Figure 1. Disclosures Hourigan: Sellas: Research Funding. Radich: Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees.

Background: The CD22-directed antibody-drug conjugate inotuzumab ozogamicin (InO) is FDA-approved for adults with relapsed/refractory (R/R) CD22+ B-ALL and highly active in children with multiply R/R CD22+ B-ALL. Notable toxicities include cytopenias and hepatic toxicity, including sinusoidal obstruction syndrome (SOS) occurring primarily after subsequent hematopoietic stem cell transplantation (HSCT). The incorporation of InO into frontline ALL therapy for patients (pts) with CD22+ high-risk B-ALL who are not anticipated to require HSCT in first remission may optimize use of this effective agent without incurring significant SOS risk by minimizing relapse and therefore potential need for future HSCT. Methods: Children's Oncology Group (COG) AALL1732 is a phase 3 trial for pts 1-24 years of age with B-ALL who have high-risk features based on age, white blood cell count, or presence of extramedullary disease at diagnosis (Figure 1). Pts receive a 4-drug induction followed by augmented BFM consolidation. Those with surface CD22 expression on >20% of blasts at diagnosis and bone marrow minimal residual disease Results: Forty-eight pts continued on therapy after randomization, (Arm A: 25, Arm B: 23), with median age 12 years and body surface area 1.4m 2 on each arm (Table 1). There were no statistically significant differences in grade ≥3 ALT or bilirubin levels between Arms A and B through day 1 of DI. During DI, 4 pts on Arm B had grade 3 hyperbilirubinemia compared to none on Arm A, all in the setting of concurrent infection. The rates of SOS did not cross protocol-defined safety thresholds and were not statistically different between arms; on Arm A, 1/25 pts (4%, 1 grade 3 event) and on Arm B, 3/23 pts developed SOS (13%, 1 grade 4, 2 grade 2). The patient with grade 4 SOS discontinued protocol therapy. The duration of the IM1 block of therapy was significantly longer for patients on Arm B compared to those on Arm A (mean duration of 90 days (sd 15.3) versus 75.5 days (sd 14.4), p=0.002). Day 1 HD-MTX clearance to 14-day delay in the start of the second (Day 15) HD-MTX in 4/23 pts (17.4%) on Arm B compared to 0/25 on Arm A, p=0.046. In contrast, time to MTX clearance and rate of >96-hour clearance were not significantly different between the arms for the other HD-MTX doses (Days 15, 29, 43). Omission of oral 6MP occurred at a significantly higher frequency on Arm B compared to Arm A at several time points in IM1, with an association between HD-MTX clearance of >96 hours and the need to hold oral 6MP (p=0.011, p=0.018, and p=0.042 for days 1, 15, and 43, respectively). During DI, sepsis occurred more frequently in pts on Arm B. There were 6 (27%) Grade 3 events and 6 (27%) ≥Grade 4 events among 22 Arm B patients, including 3 pts with multiorgan failure and one death. In contrast, among 25 pts on Arm A, there were 2 (8%) Grade 3 sepsis events and no ≥Grade 4 events. The rate of sepsis was significantly higher on Arm B (12 of 22, 54%) compared to Arm A (2 of 25, 8%, p= 0.001). The majority of sepsis events were due to bacterial infections, and 8 of 12 events on Arm B occurred in the second half of DI. Conclusion: Planned safety phase analysis of the first 50 randomized pts demonstrated increased rates of delayed MTX clearance following the first HD-MTX infusion in IM1 and more sepsis events due to bacterial infections during DI in Arm B patients. Hepatic toxicity and SOS occurred infrequently and did not differ significantly between arms. We plan to amend AALL1732 to decrease the dose of InO by 20% and increase hydration for the first HD-MTX infusion. We will recommend prophylaxis with broad-spectrum antibiotics during periods of neutropenia in DI as well as monitoring of serum IgG levels, with replacement for IgG Figure 1 Figure 1. Disclosures McNeer: Jazz Pharmaceuticals: Other: Advisory Board Member. O'Brien: Jazz: Honoraria; Pfizer: Honoraria, Research Funding. Wood: Juno, Pfizer, Amgen, Seattle Genetics: Other: Laboratory Services Agreement; Pfizer, Amgen, Seattle Genetics: Honoraria. Raetz: Pfizer: Research Funding; Celgene: Other: DSMB member. Loh: MediSix therapeutics: Membership on an entity's Board of Directors or advisory committees.

Tyrosine kinase inhibitors (TKIs) have improved the outcomes of patients with Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL). Many older patients (pts) are not good candidates for intensive chemotherapy and are treated with TKIs plus corticosteroids or low intensity chemotherapy. Although the remission rates with this approach have been high, the median disease-free survival (DFS) has been short. Therefore, novel treatment strategies are needed. This trial evaluated the feasibility of combining the TKI dasatinib with prednisone and blinatumomab in older pts with Ph+ ALL. Methods: This trial was activated through the NCTN in January 2015 and closed to accrual in April 2021. Pt eligibility included: age ≥ 65 years; Ph+ or Ph-like ALL (with dasatinib-sensitive fusions or mutations); newly diagnosed or relapsed/ refractory; no evidence of central nervous system (CNS) disease; and adequate organ function. Treatment: For induction, pts received dasatinib 140 mg/d orally (PO) Days 1-56 along with prednisone 60 mg/m 2/d PO Days 1-24. Pts achieving complete remission (CR) or CR with incomplete count recovery (CRi) (Day 28 or Day 56) continued dasatinib until Day 84 followed by 3 cycles of post-remission therapy (PRT) with blinatumomab/ dasatinib. Pts not achieving CR or CRi by Day 56 received re-induction with blinatumomab. Response was assessed at Day 35 of blinatumomab. Pts not achieving CR/ CRi could receive a second cycle of blinatumomab. This was followed by 3 cycles of PRT with blinatumomab/ dasatinib. Maintenance therapy consisted of prednisone 60 mg/m 2/d x 5 days every 28 days for a total of 18 cycles along with dasatinib 140 mg po qd indefinitely. CNS prophylaxis included intrathecal (IT) methotrexate every 4-6 weeks x 8 doses. IT methotrexate was given at least 2 days apart from blinatumomab. Response was assessed at Days 28, 56, 84 and additional time points were dependent on response. Minimal residual disease (MRD) was assessed centrally by multi-color flow cytometry at Day 28. Statistics: 9 eligible/ evaluable pts receiving PRT were to be evaluated before enrolling additional pts. Dose-limiting toxicities (DLTs) were defined as: > Grade 3 non-hematologic toxicities with the exception of nausea, vomiting, or diarrhea (if manageable with supportive care measures) or Grade 4 neutropenia lasting > 42 days with possible relationship to dasatinib or blinatumomab. If due to unexpected accrual, 12 pts were evaluable for DLT, the following rules would apply. If > 4 of the 12 pts experienced a DLT, the study would be temporarily closed pending review. Upon re-opening, 8 additional eligible and evaluable pts would be enrolled for a total of 20 pts receiving blinatumomab. Results: Due to rapid accrual, 16 pts enrolled before accrual was paused to assess feasibility and safety. Twelve pts were evaluable for DLT and 4 experienced a DLT: Grade 3 dyspnea and gastrointestinal pain (n=1), Grade 3 hypertension (n=1), Grade 3 dyspnea (n=1), Grade 3 hyperglycemia (n=1). These adverse events were deemed acceptable by the NCI and FDA and the study re-opened. A total of 25 eligible pts were accrued. The median age was 73 years (range 62-87). All pts were newly diagnosed. One pt was Ph-like, with the remainder being Ph+; 89% of Ph+ pts had additional cytogenetic abnormalities. During induction, 2 pts experienced treatment-related non-hematologic Grade 4 toxicities. No Grade 4 or higher treatment-related non-hematologic toxicities occurred during post-remission therapy or maintenance. Twenty-three out of 25 pts (92%) achieved a CR during dasatinib-based and prednisone induction therapy. Four did not receive PRT (2 due to adverse events, 1 to receive transplant, 1 because of insurance issues). Sixteen pts who achieved CR had MRD data. Five out of 16 pts (31%) were MRD negative at Day 28. One pt remains on PRT and 10 are on maintenance. The median follow up for pts who are alive is 1.7 years. The median overall survival (OS) and DFS have not been reached as of July 8, 2021. Kaplan-Meier 3-year estimates of OS and DFS are 85% (95% CI 58%-95%) and 80% (95% CI 55%-92%), respectively (Figure). Conclusions: This trial demonstrates the feasibility of combining dasatinib and blinatumomab in Ph+ ALL. In addition, with 1.7 years of follow up, outcomes are encouraging with high estimated 3-year DFS and OS. Longer follow up will be needed to determine the durability of these results. Figure 1 Figure 1. Disclosures Advani: Kite Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Glycomimetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Immunogen: Research Funding; OBI: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Research Funding; Macrogenics: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Moseley: BioSight Ltd: Consultancy. Wood: Pfizer, Amgen, Seattle Genetics: Honoraria; Juno, Pfizer, Amgen, Seattle Genetics: Other: Laboratory Services Agreement. Park: Intellia: Consultancy; Kura Oncology: Consultancy; BMS: Consultancy; Affyimmune: Consultancy; Curocel: Consultancy; Novartis: Consultancy; Artiva: Consultancy; PrecisionBio: Consultancy; Servier: Consultancy; Kite Pharma: Consultancy; Innate Pharma: Consultancy; Minerva: Consultancy; Autolus: Consultancy; Amgen: Consultancy. Wieduwilt: Gilead: Membership on an entity's Board of Directors or advisory committees; Reata: Current holder of stock options in a privately-held company. Jeyakumar: Jazz: Research Funding; Pfizer: Research Funding. Atallah: Abbvie: Consultancy, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Takeda: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy. Gerds: PharmaEssentia Corporation: Consultancy; CTI BioPharma: Research Funding; Sierra Oncology: Consultancy; AbbVie: Consultancy; Celgene/Bristol Myers Squibb: Consultancy; Constellation: Consultancy; Novartis: Consultancy. O'Brien: Amgen, Astellas, Celgene, GlaxoSmithKline, Janssen Oncology, Aptose Biosciences Inc., Vaniam Group LLC, AbbVie, Alexion, Verastem, Juno Therapeutics, Vida Ventures, Autolus, Johnson and Johnson, Merck, Bristol Myers Squibb, NOVA Research Company, Eli Lill: Consultancy; Kite, Regeneron, Acerta, Caribou, Gilead, Pharmacyclics, TG Therapeutics, Pfizer, Sunesis: Research Funding. Othus: Celgene: Other: Data safety monitoring board; Merck: Consultancy; Biosight: Consultancy; Glycomimetics: Other: Data safety monitoring board; Daiichi Sankyo: Consultancy. Litzow: Jazz: Other: Advisory Board; Pluristem: Research Funding; Amgen: Research Funding; Actinium: Research Funding; Omeros: Other: Advisory Board; Astellas: Research Funding; AbbVie: Research Funding; Biosight: Other: Data monitoring committee. Stone: Actinium: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy; Arog: Consultancy, Research Funding; Astellas: Membership on an entity's Board of Directors or advisory committees; BerGen Bio: Membership on an entity's Board of Directors or advisory committees; Boston Pharmaceuticals: Consultancy; Bristol Myers Squibb: Consultancy; Elevate Bio: Membership on an entity's Board of Directors or advisory committees; Foghorn Therapeutics: Consultancy; Gemoab: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy; Innate: Consultancy; Janssen: Consultancy; Jazz: Consultancy; Novartis: Consultancy, Research Funding; Onconova: Consultancy; Syndax: Membership on an entity's Board of Directors or advisory committees; Syntrix/ACI: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Aprea: Consultancy; Amgen: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy; Agios: Consultancy, Research Funding; Celgene: Consultancy; Macrogenics: Consultancy. Erba: AbbVie Inc; Agios Pharmaceuticals Inc; ALX Oncology; Amgen Inc; Daiichi Sankyo Inc; FORMA Therapeutics; Forty Seven Inc; Gilead Sciences Inc; GlycoMimetics Inc; ImmunoGen Inc; Jazz Pharmaceuticals Inc; MacroGenics Inc; Novartis; PTC Therapeutics: Research Funding; AbbVie Inc; Agios Pharmaceuticals Inc; Bristol Myers Squibb; Celgene, a Bristol Myers Squibb company; Incyte Corporation; Jazz Pharmaceuticals Inc; Novartis: Speakers Bureau; AbbVie Inc: Other: Independent review committee; AbbVie Inc; Agios Pharmaceuticals Inc; Astellas; Bristol Myers Squibb; Celgene, a Bristol Myers Squibb company; Daiichi Sankyo Inc; Genentech, a member of the Roche Group; GlycoMimetics Inc; Incyte Corporation; Jazz Pharmaceuticals Inc; Kura Oncology; Nov: Other: Advisory Committee.

Introduction: Despite outstanding cure rates for newly diagnosed patients with acute lymphoblastic leukemia (ALL), outcomes for pediatric patients with relapsed or refractory (R/R) disease remain unsatisfactory. Inotuzumab ozogamicin (InO), a CD22-directed antibody-drug conjugate, has shown remarkable activity; however, predictors of response to InO beyond CD22 expression and site density have not yet been reported. Methods: In order to identify better predictors of response to InO, we analyzed 68 samples from 28 patients with multiply R/R ALL enrolled in Children's Oncology Group trial AALL1621 (NCT02981628). Samples were collected before and after treatment with InO. We performed a B-ALL-centric protein profiling approach for which we designed a custom CyTOF panel, encompassing 35 rationally selected proteins (antigens) as potential predictors of InO treatment. Those proteins included surface markers (such as CD10/19/20/22 expressed in B cells and CD3/CD14, CD33 expressed in non B cells), intracellular signaling pathways (such as MAPK, PI3K, JAK/STATs), survival pathways (such as BCL-2 family members) and pathways reported to be involved in resistance to various treatments (such as p53/pCHK1 pathway, ABC drug efflux transporters, and NF-kB). This approach allowed measurement of protein levels of all those antigens. We then designed a high-dimensional tumor cell-community clustering algorithm providing dimensionality reduction to determine the extent of antigen expression across all patients at diagnosis and end of cycle 1 and cycle 2 of treatment. Results: high-dimensional clustering analysis depicted distinct tumor profiles between complete responder and partial responder patient groups (Figure 1). While this clustering measured many antigens, those that contributed the most to identification of the groups were Bcl-xl, Bcl-2, p21, CD22, Mcl1, pCHK1 and pGP whereas those contributing the least were BAD, BIM, p4EBP1 and p53. Our analyses identified the presence of CD22 high cells and CD22 low/Bcl-2 high cells as predictors of good and poor response, respectively. Furthermore, analysis of residual leukemia cells at the end of cycle 1 or 2 showed persistent high expression of Bcl-2 family members. Conclusions: We have shown for the first time that a novel strategy for multidimensional single cell phenotyping of lymphoblasts can reliably identify a predictive dual-marker signature with CD22 high expression predicting good response to InO versus CD22 low/Bcl-2 high expression predicting poor response. This approach also identifies dynamic changes of tumor composition following InO treatment. These data provide new insights into predictors of response to InO and suggest that Bcl-2 inhibition may have the potential for synergistic clinical efficacy, allowing for a potential patient-stratified approach for InO combinatorial treatments. Given that this experimental approach can be readily done in any laboratory equipped with flow or mass cytometry, we expect that our findings may be further replicated and validated in other studies using InO. Acknowledgements This work was supported by U10CA180886 and U10CA180899, the St. Baldrick's Foundation (MLL), a Pfizer ASPIRE award (MLL, EDF, AW, MO, SZ, AT), the German Cancer Aid (AW) and Uehara Memorial Foundation (KI). MLL is the Benioff Chair of Children's Health and the Deborah and Arthur Ablin Chair of Pediatric Molecular Oncology. Figure 1 Figure 1. Disclosures Wood: Juno, Pfizer, Amgen, Seattle Genetics: Other: Laboratory Services Agreement; Pfizer, Amgen, Seattle Genetics: Honoraria. O'Brien: Jazz: Honoraria; Pfizer: Honoraria, Research Funding. Loh: MediSix therapeutics: Membership on an entity's Board of Directors or advisory committees.

Background: MRD testing in BCP-ALL is critical for appropriate patient management, but little is known regarding sample acquisition and testing heterogeneity across clinical practice settings. These factors may impact the quality and reliability of MRD assessment. Methods: Thirty-minute online surveys were conducted in May 2021 with hematologists/oncologists (HEME/ONCs) in the United States in both academic (acad) and community (comm) settings. Respondents were licensed physicians board certified in oncology and/or hematology who treated ≥2 BCP-ALL patients/year or ≥10 patients in the past 5 years, with over 25% of time spent in the clinical setting; pediatric HEME/ONCs were excluded. Survey enrollment is ongoing, with interim results presented here; a related survey for pathologists (PATHs) is underway. Results: HEME/ONC respondents (acad n=40, comm n=57, from 29 states) had been practicing as specialists for a median of between 11-15 years (choices were ranges, eg 6-10, 11-15, min-max was 1-34 years), and typically spent over 75% of their time in the clinic; 94% of respondents had ≥5 BCP-ALL patients/year and 92% ordered MRD tests for ≥5 patients/year. Typical timepoints for MRD testing included the end of induction/suspected complete remission, the end of consolidation, and at suspected disease progression; testing after the end of consolidation was infrequent in both groups (Table). Testing for MRD at the end of consolidation was notably more frequent in the academic setting. In both settings, the HEME/ONC ordering the MRD test generally also performed the bone marrow collection procedure (acad: 78%, comm: 56%). Resources consulted on bone marrow collection best practices included UpToDate (21%), ASH and ASCO (13%), NCCN guidelines (13%), and hematology/oncology journals. About half of practices had defined institutional protocols for bone marrow collection (acad: 55%, comm: 47%), nearly all of which were developed internally. The amount of bone marrow sample collected showed high variability, ranging from 1-10 draws (median=3) and 1-30 mL sample per draw (median=5 mL). While 49% of HEME/ONCs performed 5 draws and/or >6 mL per draw. In both settings, the first pull was identified and labeled in 35% of procedures; in those cases, the first-pull samples were used primarily for MRD testing in 60% of cases as recommended by NCCN guidelines (vs for morphology assessment and cytogenetic studies). HEME/ONCs typically relied on the expertise of pathologists to choose MRD testing methodology.Survey results indicate that external labs (both national clinical reference labs and commercial labs) were most commonly used for MRD assessments (63%); comm HEME/ONCs were more likely to use external reference labs and acad HEME/ONCs were more likely to use in-house labs. When asked to estimate the frequency with which different MRD methods were used, mean responses were 54% flow cytometry and 40% next-generation sequencing. While all HEME/ONCs indicated that MRD results were presented clearly in lab reports, there was a desire to include more guideline information about MRD interpretation and BCP-ALL treatment. Conclusion: Interim results identified broad heterogeneity in clinical practices affecting sample collection for MRD assessment in Ph- BCP-ALL in the US, indicating several opportunities for harmonization of routine MRD assessment in BCP-ALL. These opportunities include optimization of bone marrow sample collection techniques (volume/draw and identification/use of first pull for MRD), timing/frequency of specimen collection, serial MRD surveillance after consolidation, MRD method chosen, and standardizing reports to include guideline information. There were gaps in awareness of FDA-approved methods of MRD testing for BCP-ALL. Initiatives supporting provider education and harmonization of best practices from professional guideline committees/organizations are needed to optimize outcomes of BCP-ALL patients. Figure 1 Figure 1. Disclosures Hidalgo-Lopez: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Roboz: Janssen: Research Funding; Daiichi Sankyo: Consultancy; MEI Pharma - IDMC Chair: Consultancy; Actinium: Consultancy; AbbVie: Consultancy; Mesoblast: Consultancy; Bayer: Consultancy; Blueprint Medicines: Consultancy; Jazz: Consultancy; Janssen: Consultancy; Astex: Consultancy; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Agios: Consultancy; Astellas: Consultancy; Jasper Therapeutics: Consultancy; Helsinn: Consultancy; Glaxo SmithKline: Consultancy; Novartis: Consultancy; Amgen: Consultancy; AstraZeneca: Consultancy; Otsuka: Consultancy; Pfizer: Consultancy; Roche/Genentech: Consultancy. Wood: Pfizer, Amgen, Seattle Genetics: Honoraria; Juno, Pfizer, Amgen, Seattle Genetics: Other: Laboratory Services Agreement. Borowitz: Amgen, Blueprint Medicines: Honoraria. Jabbour: Amgen, AbbVie, Spectrum, BMS, Takeda, Pfizer, Adaptive, Genentech: Research Funding. Velasco: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Elkhouly: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Adedokun: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Zaman: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Iskander: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Logan: Amgen, Pfizer, AbbVie: Consultancy; Pharmacyclics, Astellas, Jazz, Kite, Kadmon, Autolus, Amphivena: Research Funding.

Background: The prognosis for patients (pts) with relapsed T-ALL and T-LL is dismal. The primary goal of T-ALL/T-LL treatment is to prevent relapse. In the phase 3 Children's Oncology Group (COG) clinical trial AALL1231 (NCT02112916), children, adolescents and young adults (age 1-30 years) with T-ALL and T-LL were treated with a modified augmented BFM (aBFM) backbone that used dexamethasone as the only corticosteroid and included two (rather than one) doses of pegaspargase during induction and delayed intensification. Pts were stratified as standard (SR), intermediate (IR), or very high risk (VHR), primarily based on disease response: morphology, minimal residual disease (MRD) performed by multiparameter flow cytometry at a central reference laboratory) at end of induction and consolidation (T-ALL), and radiographic response for T-LL. Pts were randomized 1:1 to receive/not receive bortezomib during induction and delayed intensification (1.3mg/m 2 x 4 doses per block). VHR T-ALL pts were defined as having day 29 M3 marrow (>25% blasts) or end of consolidation (EOC) MRD >0.1%. 10-15% of T-ALL pts were predicted to be VHR based on COG AALL0434. Pts with induction failure (M3 marrow by morphology) or EOC MRD >0.1% were expected to have 4-yr event-free survival (EFS) of ~66+/-16%. Following consolidation, VHR pts received 3 BFM-based intensification blocks in lieu of interim maintenance (IM). Detectable MRD following the intensification blocks was considered an event and these pts were removed from protocol therapy. VHR ALL pts who had undetectable MRD continued protocol therapy, received delayed intensification, an IM phase with Capizzi escalating methotrexate plus pegaspargase, and maintenance. A secondary aim of AALL1231 was to compare survival in VHR T-ALL pts with EOC MRD ≥ 0.1% but undetectable MRD after intensification of chemotherapy with those who continued to have detectable MRD and were eligible for other treatment strategies, including hematopoietic stem cell transplant (HSCT). This study also analyzed outcomes for pts with M3 marrow at the end of induction. Results: AALL1231 accrued 847 pts (824 eligible and evaluable) of 1400 anticipated from 2014 until early closure. The 3-year EFS for the bortezomib randomization for the SR and IR groups has been reported previously (Teachey, et. al ASH 2020). Because only 2 of 209 T-LL pts were VHR; this report focuses on the outcomes of the 5.2% (32/615) of T-ALL pts who were VHR. In total, 25 VHR T-ALL pts were EOC MRD >0.1%, and 18 of these had MRD sent at the end of HR intensification. Of the 8 pts who became MRD undetectable and continued protocol therapy, only 2 survived (3-year overall survival [OS] 25+15.3%). In contrast, 10 pts who had detectable MRD were taken off protocol and underwent HSCT. Of these 10, only one relapsed (3-year OS 90+12.7%). The 3-year OS for the 10 pts who were M3 at Day 29 was 60.0±17.0%. As there were not enough pts to assess the impact of EOC MRD on pts who were M3 at Day 29, we assessed the impact of EOC MRD on outcomes in M2 (5-25% blasts at Day 29; n = 24) and M3 pts, which defines induction failure in other cooperative groups. M2+M3 T-ALL who were EOC MRD 0.1% (n = 12) pts. Conclusions: T-ALL pts treated on AALL1231 who are EOC MRD ≥0.1% with undetectable MRD after 3 BFM-based intensification blocks had a very poor outcome when treated with standard cytotoxic chemotherapy. In contrast, while patient numbers are small, those pts that remained MRD-positive after 3 intensification blocks and underwent HSCT had an excellent outcome. These data not only impact the recommended treatment for T-ALL pts who are induction and consolidation failures, but also support the importance of the graft-versus-leukemia (GVL) effect in refractory T-ALL. Disclosures Hayashi: Magenta Therapeutics: Consultancy. August: Jazz: Membership on an entity's Board of Directors or advisory committees. Hermiston: Sobi: Consultancy; Novartis: Consultancy. Bollard: Cabeletta Bio: Membership on an entity's Board of Directors or advisory committees; Catamaran Bio and Mana Therapeutics: Other: member and cofounder; SOBI: Other: DSMB. Loh: MediSix therapeutics: Membership on an entity's Board of Directors or advisory committees. Raetz: Pfizer: Research Funding; Celgene: Other: DSMB member. Teachey: BEAM Therapeutics: Consultancy, Research Funding; NeoImmune Tech: Research Funding; Sobi: Consultancy; Janssen: Consultancy.

Minimal residual disease (MRD) is the most significant independent prognostic factor in acute lymphocytic leukemia (ALL). Monitoring MRD using sensitive techniques, including multiparametric flow cytometry (MFC) and quantitative polymerase chain reaction (qPCR)-based methods, has improved the assessment of treatment response and risk stratification for clinical management. New molecular methods, such as high-throughput next-generation sequencing (NGS), have evolved into routine laboratory tools to improve the sensitivity and specificity of MRD detection. It is essential to establish standardized protocols as to the timing of assessment and methodology used, to limit inter-laboratory variability. MRD has demonstrated utility for the identification of patients with suboptimal initial response to therapy who may benefit from more intensive or novel therapies, in addition to identifying patients with an excellent response to initial therapy who may be candidates for therapeutic reduction to limit toxicity. Herein, we review the methodological approaches to MRD detection in ALL and discuss the clinical implication of MRD in risk-directed therapy and practical issues.

Purpose To determine the prognostic significance of blasts, and of white and red blood cells, in CSF samples at diagnosis of acute lymphoblastic leukemia (ALL), a uniform CSF and risk group classification schema was incorporated into Children’s Oncology Group B-cell ALL (B-ALL) clinical trials. Methods CSF status was designated as follows: CNS1, no blasts; CNS2a to 2c, < 5 WBCs/μL and blasts with/without ≥ 10 RBCs/μL or ≥ 5 WBCs/μL plus blasts, with WBCs ≥ 5 times the number of RBCs; CNS3a to 3c, ≥ 5 WBCs/μL plus blasts with/without ≥ 10 RBCs/μL or clinical signs of CNS disease. CNS2 status did not affect therapy; patients with CNS3 status received two extra intrathecal treatments during induction and augmented postinduction therapy with 18 Gy of cranial radiation. Results Among 8,379 evaluable patients enrolled from 2004 to 2010, 7,395 (88.3%) had CNS1 status; 857 (10.2%), CNS2; and 127 (1.5%), CNS3. The 5-year event-free and overall survival rates were, respectively, 85% and 92.7% for CNS1, 76% and 86.8% for CNS2, and 76% and 82.1% for CNS3 ( P < .001). In multivariable analysis that included age, race/ethnicity, initial WBC, and day-29 minimal residual disease < 0.1%, CSF blast, regardless of cell count, was an independent adverse predictor of outcome for patients with standard- or high-risk disease according to National Cancer Institute criteria. The EFS difference reflected a significant difference in the incidence of CNS, not marrow, relapse in patients with CNS1 versus CNS2 and/or CNS3 status. Conclusion Low levels of CNS leukemia, regardless of RBCs, predict inferior outcome and higher rates of CNS relapse. These data suggest that additional augmentation of CNS-directed therapy is warranted for CNS2 disease.

Background Minimal residual disease (MRD) in B lymphoblastic leukemia (B-ALL) by flow cytometry is an established prognostic factor used to adjust treatment in most pediatric therapeutic protocols. MRD in B-ALL has been standardized by the Children's Oncology Group (COG) in North America, but not routine clinical labs. The Foundation for National Institutes of Health sought to harmonize MRD measurement among COG, oncology groups, academic, community and government, laboratories. Methods Listmode data from post-induction marrows were distributed from a reference lab to seven different clinical FCM labs with variable experience in B-ALL MRD. Labs were provided with the COG protocol. Files from 15 cases were distributed to the seven labs. Educational sessions were implemented, and 10 more listmode file cases analyzed. Results Among 105 initial challenges, the overall discordance rate was 26%. In the final round, performance improved considerably; out of 70 challenges, there were five false positives and one false negative (9% discordance), and no quantitative discordance. Four of six deviations occurred in a single lab. Three samples with hematogones were still misclassified as MRD. Conclusions Despite the provision of the COG standardized analysis protocol, even experienced laboratories require an educational component for B-ALL MRD analysis by FCM. Recognition of hematogones remains challenging for some labs when using the COG protocol. The results from this study suggest that dissemination of MRD testing to other North American laboratories as part of routine clinical management of B-ALL is possible but requires additional educational components to complement standardized methodology. © 2017 Clinical Cytometry Society

BACKGROUND Blinatumomab, a bispecific monoclonal antibody construct that enables CD3-positive T cells to recognize and eliminate CD19-positive acute lymphoblastic leukemia (ALL) blasts, was approved for use in patients with relapsed or refractory B-cell precursor ALL on the basis of single-group trials that showed efficacy and manageable toxic effects. METHODS In this multi-institutional phase 3 trial, we randomly assigned adults with heavily pre-treated B-cell precursor ALL, in a 2: 1 ratio, to receive either blinatumomab or standardof- care chemotherapy. The primary end point was overall survival. RESULTS Of the 405 patients who were randomly assigned to receive blinatumomab (271 patients) or chemotherapy (134 patients), 376 patients received at least one dose. Overall survival was significantly longer in the blinatumomab group than in the chemotherapy group. The median overall survival was 7.7 months in the blinatumomab group and 4.0 months in the chemotherapy group (hazard ratio for death with blinatumomab vs. chemotherapy, 0.71; 95% confidence interval [CI], 0.55 to 0.93; P = 0.01). Remission rates within 12 weeks after treatment initiation were significantly higher in the blinatumomab group than in the chemotherapy group, both with respect to complete remission with full hematologic recovery (34% vs. 16%, P

Minimal residual disease (MRD) after therapy has unequivocal prognostic value in acute leukemia. Over the past 20years, a number of techniques have evolved into routine laboratory tools to detect MRD, most notably, multiparametric flow cytometry (MFC) and quantitative polymerase chain reaction (PCR)-based molecular methods. There is growing evidence that the presence of MRD detected by MFC or molecular methods provides independent prognostic information and is associated with an increased risk of relapse and shortened survival. However, the predictive value of MRD may be affected by a lack of consensus as to the timing for assessment, the methodology used, and interlaboratory variation in test performance. Herein, we review the methodological principles of MRD assays, discuss the clinical implications of monitoring MRD for risk stratification and directing further therapy, and suggest potential areas for future investigation.

Current risk stratification for COG ALL patients (pts) relies on National Cancer Institute (NCI) risk group (RG) at diagnosis, somatic genetics, and early response to therapy as measured by specific thresholds of minimal residual disease (MRD) using flow cytometry on day 8 peripheral blood (D8 PB) and day 29 bone marrow (D29 BM). NCI RG is defined as age 1-10 years (yrs) and white blood cell count (WBC) We first log transformed WBC, D8 and D29 MRD and displayed these by treatment protocol, NCI RG, and FRG/URG (separating out Ph-like independently). Age and WBC followed the normal expected distribution with the median age of SR pts 4.0 yrs (range 1-9) and HR 12 yrs (range 1-30). Transformed MRD was displayed as a variable t(MRD), corresponding to the negative log; max t(D29 MRD) was 13.82, corresponding to MRD We next conducted a univariate analysis for risk factors for relapse, including sex, age, WBClog, CNS status, protocol-defined rapid early response status, FRG, URG, t(D8 MRD), and t(D29 MRD); all variables except CNS status were significant p < 0.0001). Multivariable modeling showed that WBClog, FRG, URG, t(D8 MRD), t(D29 MRD) retained significance (p < 0.0001). Finally, we applied the UK Prognostic Index (PIUKALL) equation [t(d29 MRD) x -0.218 + CYTO-GR x -0.440 + CYTO-HR x 1.066 + WBClog x 0.138] to the COG data using protocol, NCI RG, FRG, URG, IRG, or Ph-like RG in the model and validated the trends for relapse-free survival (RFS), which were similar in our groups with an overall median PIUKALL of -2.63 (mean -2.32, SD -.90, min -3.54, max 1.79). We next added in t(D8 MRD) to define a PICOG and determined that D8 PB MRD added significantly to the model, mostly through discriminating between the hazard ratios of the FRG and the URG RGs. Importantly, the D8 PB MRD led to a qualitatively more distinctive group with a potentially lower predicted RFS in NCI SR pts, a group that has been more difficult to predict in the past, and yet comprises nearly half of all relapse events. Our analyses of 21,178 COG B-ALL pts confirm and extend the utility of integrating WBC and MRD as continuous rather than categorical values to refine risk stratification for patient treatment and trial design. Disclosures Loh: Pfizer: Other: Institutional Research Funding; Medisix Therapeutics: Membership on an entity's Board of Directors or advisory committees. Borowitz:Amgen: Honoraria. Zweidler-McKay:ImmunoGen, Inc.: Current Employment. Mattano:Melinta Therapeutics: Consultancy; Novartis: Consultancy; Pfizer: Consultancy. Hunger:Amgen: Current equity holder in publicly-traded company, Honoraria; Novartis: Consultancy. Raetz:Celgene/BMS: Other; Pfizer: Research Funding. Mullighan:Illumina: Consultancy, Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; AbbVie, Inc.: Research Funding.

Background: Patients with Down syndrome (DS) have an approximately 10-fold increased risk of developing ALL, and the spectrum of genetic alterations differs from that of non-DS ALL. Rearrangement and/or overexpression of cytokine receptor-like factor 2 (CRLF2+) occurs in 50% of DS ALL, compared to only 5-10% CRLF2+ cases in non-DS children and adolescents. JAK2 or JAK1 mutations co-occur in about half of CRLF2+ cases in both DS and non-DS ALL. The prognostic significance of CRLF2+ ALL also appears to differ in the limited data reported to date, with less adverse impact in patients with DS compared to non-DS ALL. Here, we report the clinical characteristics and prognostic significance of B-ALL with CRLF2 overexpression and JAK alterations in children and adolescents/young adults (AYA) with DS who were treated on Children's Oncology Group (COG) clinical trials from 2003-2016. Methods: We analyzed clinical, laboratory, and outcome data for 317 patients with DS B-ALL treated on standard risk (SR) trials AALL0331 and AALL0932 and high risk (HR) trials AALL0232 and AALL1131, for whom CRLF2 status and rearrangement partners (IGH or P2RY8) were ascertained by flow cytometric assessment of surface expression; fluorescence in situ hybridization; and/or polymerase chain reaction (PCR) testing. JAK mutations were ascertained in a subset by PCR and sequencing. Minimal residual disease (MRD) was assessed by flow cytometry at the end of induction (EOI) and at end of consolidation (EOC) for a subset of EOI MRD+ patients. Results: We identified 168/317 (53.0%) CRLF2+ cases, and among those assessed for CRLF2 partner, 17/73 (23.3%) were IGH-CRLF2 and 56/73 (76.7%) were P2RY8-CRLF2. In the subset of 165 cases tested for JAK mutations (85 CRLF2- and 80 CRLF2+), 42/165 (25.4%) had JAK mutations, all of which co-occurred in CRLF2+ cases. CRLF2 positivity was significantly associated with younger age at diagnosis: 140/168 (83.3%) of CRLF2+ cases were under 10 years old, versus 106/149 (71.1%) of CRLF2- cases, p Discussion: Whereas CRLF2 and JAK alterations are associated with higher MRD, poorer survival, and increased CIR in patients with high-risk ALL without DS, these alterations do not demonstrate strong adverse prognostic impact in children and AYAs with DS-ALL treated on recent frontline COG trials, although larger sample sizes are needed to adequately assess for possible poorer prognoses associated with the CRLF2+/JAK+ and IGH-CRLF2 subgroups. Regardless, given the frequency of these targetable lesions and the increased risk of relapse and chemotherapy-associated toxicities in patients with DS-ALL, targeted therapies currently under investigation for these genetic lesions may be beneficial to replace some intensive blocks of therapy in DS-ALL with CRLF2 and/or JAK alterations, both to enhance anti-leukemic efficacy and decrease intensive chemotherapy-associated toxicities. Figure 1 Disclosures Tasian: Gilead Sciences: Research Funding; Incyte Corporation: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Borowitz:Amgen: Honoraria. Mullighan:Illumina: Consultancy, Honoraria, Speakers Bureau; AbbVie, Inc.: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau. Raetz:Celgene: Other: DSMB member; Pfizer: Other: Institutional research funding. Loh:Pfizer: Other: Institutional Research Funding; Medisix Therapeutics: Membership on an entity's Board of Directors or advisory committees. Hunger:Novartis: Consultancy; Amgen: Current equity holder in publicly-traded company, Honoraria.