Infant Acute Lymphoblastic Leukemia Research Articles

Blinatumomab as a Curative Therapy Option for Relapsed/Refractory Infant Acute Lymphoblastic Leukemia Post-Hematopoietic Stem Cell Transplantation - Case Report

Blinatumomab as a Curative Therapy Option for Relapsed/Refractory Infant Acute Lymphoblastic Leukemia Post-Hematopoietic Stem Cell Transplantation - Case Report

Infant Acute Lymphoblastic Leukemia-New Therapeutic Opportunities.

Infant acute lymphoblastic leukemia (Infant ALL) is a kind of pediatric ALL, diagnosed in children under 1 year of age and accounts for less than 5% of pediatric ALL. In the infant ALL group, two subtypes can be distinguished: KMT2A-rearranged ALL, known as a more difficult to cure form and KMT2A- non-rearranged ALL with better survival outcomes. As infants with ALL have lesser treatment outcomes compared to older children, it is pivotal to provide novel treatment approaches. Progress in the development of molecularly targeted therapies and immunotherapy presents exciting opportunities for potential improvement. This comprehensive review synthesizes the current literature on the epidemiology, clinical presentation, molecular genetics, and therapeutic approaches specific to ALL in the infant population.

Clinical characteristics and prognosis of 28 cases of infant acute lymphoblastic leukemia

Objective: To analyze the clinical characteristics and prognosis of patients with infant acute lymphoblastic leukemia (IALL). Methods: A retrospective cohort study.Clinical data, treatment and prognosis of 28 cases of IALL who have been treated at Beijing Children's Hospital, Capital Medical University and Baoding Children's Hospital from October 2013 to May 2023 were analyzed retrospectively. Based on the results of fluorescence in situ hybridization (FISH), all patients were divided into KMT2A gene rearrangement (KMT2A-R) positive group and KMT2A-R negative group. The prognosis of two groups were compared. Kaplan-Meier method and Log-Rank test were used to analyze the survival of the patients. Results: Among 28 cases of IALL, there were 10 males and 18 females, with the onset age of 10.9 (9.4,11.8) months. In terms of immune classification, 25 cases were B-ALL (89%), while the remaining 3 cases were T-ALL (11%). Most infant B-ALL showed pro-B lymphocyte phenotype (16/25,64%). A total of 22 cases (79%) obtained chromosome karyotype results, of which 7 were normal karyotypes, no complex karyotypes and 15 were abnormal karyotypes were found. Among abnormal karyotypes, there were 4 cases of t (9; 11), 2 cases of t (4; 11), 2 cases of t (11; 19), 1 case of t (1; 11) and 6 cases of other abnormal karyotypes. A total of 19 cases (68%) were positive for KMT2A-R detected by FISH. The KMT2A fusion gene was detected by real-time PCR in 16 cases (57%). A total of 24 patients completed standardized induction chemotherapy and were able to undergo efficacy evaluation, 23 cases (96%) achieved complete remission through induction chemotherapy, 4 cases (17%) died of relapse. The 5-year event free survival rate (EFS) was (46±13)%, and the 5-year overall survival rate (OS) was (73±10)%.The survival time was 31.3 (3.3, 62.5) months. There was no significant statistical difference in 5-year EFS ((46±14)% vs. (61±18)%) and 5-year OS ((64±13)% vs. (86±13)%) between the KMT2A-R positive group (15 cases) and the KMT2A-R negative group (9 cases) (χ2=1.88, 1.47, P=0.170, 0.224). Conclusions: Most IALL patients were accompanied by KMT2A-R. They had poor tolerance to traditional chemotherapy, the relapse rate during treatment was high and the prognosis was poor.

Abstract B110: Race/ethnicity, socioeconomic status, and early death among children enrolled in clinical trials for acute lymphoblastic leukemia

Abstract Introduction: Prior work indicates that Hispanic and Black children diagnosed with leukemia may experience a higher risk of death in the period shortly after diagnosis, but no work has examined these disparities taking into account leukemia subtypes and the role socioeconomic status (SES) may play. We investigated reported race/ethnicity (RRE), health insurance status, neighborhood SES, and early death among pediatric acute lymphoblastic leukemia (ALL) patients enrolled on clinical trials. Methods: Using data from the Childhood Cancer Research Network as well as Children’s Oncology Group therapeutic trials, we estimated the associations between RRE (Non-Hispanic [NH] White, NH Black, NH Asian/Pacific Islander [PI], or Hispanic/Native American), health insurance status, neighborhood SES using the Yost Index, and death within 60 days of diagnosis using multivariable logistic regression. All analyses were adjusted for age at diagnosis, sex, and ALL subtype (Standard Risk B-Cell, High Risk B-Cell, Infant ALL, T-Cell). We also estimated associations among non-infant patients diagnosed with B-Cell ALL, additionally adjusting for standard or high-risk disease. All associations were estimated as odds ratios (OR) with 95% confidence intervals (CI). Results: Overall, we analyzed data from 8,634 pediatric ALL cases (7,559 B-Cell; 122 Infant ALL; 953 T-Cell) enrolled across 7 clinical trials (AALL0232, AALL0331, AALL0434, AALL0631, AALL0932, AALL1131, AALL1231). In total, there were 29 deaths within 60 days of diagnosis, including 22 among B-Cell, 4 among infant ALL, and 3 among T-Cell patients. In the full analysis, infant ALL subtype was very strongly associated with early death compared to Standard Risk B-Cell patients (OR: 37.8 [95% CI: 9.22, 137]). We also observed a suggestive association between NH Asian/PI RRE and higher odds of early death compared to NH Whites (OR: 3.10 [95% CI: 0.71, 9.63]), which remained after limiting the analysis to B-Cell patients (OR: 5.63 [95% CI: 1.24, 18.7]). No association was apparent between NH Black or Hispanic/Native American RRE and odds of early death. Neighborhood SES was not significantly associated with early death, though point estimates suggested a potential protective effect of increasing SES. Lack of health insurance was also suggestively associated with greater odds of early death (OR: 2.78 [95% CI: 0.60, 9.47]). Conclusion: Unlike prior work, we observed increased odds of death soon after diagnosis for NH Asian/PI children. Future work incorporating more detailed treatment and cytogenetic subtype information is needed to identify the factors contributing to this disparity. Citation Format: Sarah Lucht, Jeannette M. Sample, Logan Spector, Jenny N. Poynter, Lucie Turcotte, Erin L. Marcotte. Race/ethnicity, socioeconomic status, and early death among children enrolled in clinical trials for acute lymphoblastic leukemia [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B110.

P31 - INFANT WITH SAMDL9 MUTATION, MONOSOMY 7 AND ACUTE LYMPHOBLASTIC LEUKEMIA

P31 - INFANT WITH SAMDL9 MUTATION, MONOSOMY 7 AND ACUTE LYMPHOBLASTIC LEUKEMIA

Pre-Clinical Developmental Hematopoiesis Demonstrates the Heterogeneity of KMT2A-Rearranged Infant Acute Lymphoblastic Leukemic Populations

KMT2A-rearranged (KMT2A-r) infant acute lymphoblastic leukemia (ALL) has a poor prognosis with increased risk of relapse when diagnosed at younger ages. Understanding the origin of these leukemic cells is crucial to comprehend disease development and prognosis differences. The unclear delineation of the identity of early blood populations in humans is a challenge in defining the cell type(s) driving this infant ALL. Recent pre-clinical studies revealed that definitive hematopoietic stem cells (HSCs) have minimal contribution to embryonic hematopoiesis, while independently arising embryonic hematopoietic progenitors (HPCs) that possess more limited self-renewal and/or multilineage differentiation capacities, are both necessary and sufficient to sustain the developing embryo. Here, we aim to leverage pre-clinical knowledge on the markers that distinguish HSCs from HPCs to investigate the potential contribution of HPCs in driving prenatal and infant leukemias. We defined transcriptional signatures from developmental HSC and HPC populations using thirteen single cell RNA sequencing (scRNA-seq) datasets from zebrafish, eight datasets generated by our research group and five from independent published studies. We then employed a neural network approach to define the orthologous cell type signatures most likely correlating with human healthy and blast cells from publicly available data of 18 KMT2A-r infant ALL patients. In accordance with previous literature, HSC and lymphomyeloid progenitor signatures are both highly represented in the leukemic blast populations. Additionally, we found subpopulation differences in gene expression within leukemic blasts cells including lymphoid progenitor, granulocytic, and T cell signatures, possibly suggesting heterogeneity in either their cell type of origin or developmental differentiation potential. Our findings support the ability to use orthologous scRNA-seq to decipher new understanding on the role of HSCs and HPCs in hematological malignances affecting infants and children.

Delayed Intensification Including Venetoclax and Bortezomib Prolongs Survival in Very High Risk Acute Lymphoblastic Leukaemia

Modern multi-drug risk-stratified response-adapted chemotherapy protocols lead to >85% cure rates in childhood acute lymphoblastic leukaemia (ALL). Between 2013-2022, 707 children (0-18 years) with acute lymphoblastic leukaemia (ALL) treated systematically on the ICiCLe-ALL-14 risk-adapted protocol achieved a 3-year event-free survival (EFS) of 73% (95% confidence interval, CI 69-77). A very high risk (VHR) subgroup (N = 107) was identified with poor outcome (3-year EFS, 38% [95% CI 25-51]). This included patients with induction failure, persistent minimal residual disease (MRD) and/or select high risk cytogenetic subtypes ( BCR::ABL1 other ABL-class fusion ALL, TCF3::HLF, low hypodiploidy, IKZF1plus with high end-induction MRD, KMT2A-rearranged infant ALL). Co-culture based high-throughput drug response profiling (DRP) was performed to identify alternate chemotherapeutic drugs for these VHR patients. Primary ALL blasts extracted by density centrifugation, were seeded on immortalized human bone marrow stromal cells and treated with a 14-drug panel: prednisolone; vincristine (VCR), daunorubicin; asparaginase (ASNase), bortezomib (BZM), venetoclax (VEN), panobinostat (PNB), mitoxantrone, cyclophosphamide, cytarabine, selinexor, dasatinib, 6-thioguanine and idarubicin. Seventy-four patients (25 VHR, 37 high-risk, 7 intermediate-risk, and 5 standard-risk,) were analysed. Venetoclax, BZM and PNB were identified as active agents (IC 50 <100nM for VEN and PNB, <10nM for BZM) in 71%, 42% and 75% of VHR samples, respectively. Based on drug-sensitivity patterns of VHR ALL, a pilot study was initiated to prospectively evaluate the addition of VEN and BZM in the frontline delayed intensification (DI) phase of VHR ALL patients (N=24) after informed consent (Figure 1). Response was assessed before and after VHR-DI in 46% (11/24) patients. Good response, i.e. decrease in MRD ≥ 1 log after VHR-DI, was noted in 82% (9/11) patients. Landmark analysis comparing survival of patients (from start of DI) treated on standard DI versus VHR-DI showed significant improvement in 2-year EFS from 54% (95% CI 40-66) to 76% (95% CI 51-89) ( p=0.037, log-rank test). Overall, VHR-DI was tolerated well. No significant difference was noted in grade 4 and 5 toxicities (Common Terminology Criteria for Adverse Events v4.0) between standard DI and VHR-DI ( p= 0.053 & p=0.988 respectively, chi-square test). Fever-neutropenia and sepsis were the most frequent toxicities with VHR-DI. To identify potential mechanisms of resistance in poor-responders, transcriptome analysis was performed on diagnostic blast cells from VHR patients with good (N=5) or poor (N=5) response to VHR-DI and compared with highly sensitive SR patients (N=5). Upregulation of oxidative phosphorylation and ubiquitin-mediated proteolysis were found in good responders while non-responders showed upregulation of amino acid metabolic and RNA splicing pathways. In conclusion, drug response profiling identifies alternative combination therapies for VHR ALL. Our pilot study provides proof-of-concept of effectiveness and tolerability of inclusion of VEN and BZM in the post-induction treatment of patients with poor-response disease.

Fetal MLL-ENL Initiation Induces Developmental Stage-Specific Programs That Restrict Transformation and Depend on MLL3

Infant leukemias present as either acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML) and are commonly driven by KMT2A/ MLL rearrangements (MLLr). MLLr account for ~70% of infant ALL and ~50% of infant AML. Most of the MLLr that drive infant leukemias occur before birth and require very few cooperating mutations. These observations suggest that MLLr transform fetal/neonatal progenitors with great efficiency. However, this model introduces important paradoxes. First, the incidence of MLLr infant leukemia is ~20-fold lower than the incidence of adult MLLr leukemias. Second, fetal leukemias are exceedingly rare despite the fact that MLLr can arise before birth and require a low secondary mutation threshold for transformation. Third, even in mouse models that demonstrate efficient leukemogenesis, the fraction of MLLr progenitors that actually give rise to AML is quite small. Fourth, in our prior studies, we developed a doxycycline (DOX) inducible MLL-ENL mouse model (Tet-ME) to activate MLL-ENL and found that transformation efficiency is higher when MLL-ENL is induced shortly after birth rather than before birth (PMID: 31405949). These observations raise the question of whether fetal/neonatal progenitors carry intrinsic protection against transformation. To test whether fetal identity conveys protection against MLLr leukemia initiation, we activated MLL-ENL at embryonic day 10.5 (E10.5) or postnatal day 14 (P14), by removing DOX. We then assessed the leukemogenic potential of postnatal progenitors in transplantation assays. Strikingly, progenitors that expressed MLL-ENL from E10.5 onward could engraft efficiently but did not give rise to AML in recipient mice. In contrast, progenitors that expressed MLL-ENL induced from P14 onward gave rise to rapid, fully penetrant AML in recipient mice (Figure 1A). Our data show that MLL-ENL induces heritable epigenetic changes in fetal progenitors, that limit transformation potential after transplantation. To assess the unique response of fetal progenitors to MLL-ENL activation, we used our Tet-ME model and induced MLL-ENL before or after birth. We isolated P28 progenitors, repeated transplantation/survival assays and performed Cellular Indexing of Transcriptomes and Epitopes (CITE-seq), in parallel, to test whether MLL-ENL differentially reprograms pre- and post-natal progenitors (i.e., do P28 progenitors have distinct expression programs depending on when MLL-ENL is induced?). Hematopoietic progenitors had similar differentiation trajectories by pseudotime analysis, but distinct gene expression profiles after fetal and postnatal MLL-ENL induction. Some known MLL-ENL targets, including Hoxa9 and Pim1, were maintained at similar levels following fetal and postnatal induction. Other targets, including Meis1, Pbx1 and Sox4, were only expressed in the HSC/MPP cluster following postnatal induction. Metabolic and differentiation states also differed. As in the initial survival assays, MLL-ENL expressing progenitors only gave rise to AML following postnatal induction. We next sought to identify epigenetic regulators that enforce non-malignant cell fates following fetal MLL-ENL induction. We focused our screen on epigenetic factors with known tumor suppressor roles and identified the SET methyltransferase MLL3 as a candidate effector of fetal protection. Specifically, heterozygous Kmt2c deletions greatly accelerated AML initiation after fetal MLL-ENL induction, but not postnatal induction (Figure 1B). Notably, the MLL3/4 COMPASS-like complex was recently shown to antagonize the KMT2A-MENIN interaction and promote differentiation in AML cells. Our data reinforce the notion that there are ontological windows that permit or repress leukemic transformation. Quite surprisingly, fetal progenitors appear to be more resistant to transformation than neonatal progenitors, potentially accounting for the paucity of fetal MLLr leukemias. Infant leukemias that do occur must bypass heritable protective mechanisms, either via mutations or epigenetic reprogramming.

Outcome of Infants Treated on Total Therapy for Infants with Acute Lymphoblastic Leukemia I: Results from a Non-Randomized Multi-Center Study

Infants with KMT2A rearranged (KMT2Ar) acute lymphoblastic leukemia (ALL) have a poor prognosis with an event-free-survival (EFS) of 33.6-36.9% on recent Children's Oncology Group and Interfant trials. In preclinical studies, we identified bortezomib and vorinostat as novel active agents in primary KMT2Ar infant ALL specimens that have not been included in infant regimens to date. Total therapy for infants with acute lymphoblastic leukemia I (TINI) evaluated this novel combination in a multi-institutional pilot trial for infants with de novo ALL. In this study, bortezomib and vorinostat were incorporated into an ALL chemotherapy backbone containing dexamethasone, mitoxantrone, and pegasparaginase during induction and reinduction chemotherapy cycles. A run-in dose escalation evaluated 3 dose levels of vorinostat which was then followed by a dose expansion phase. The primary endpoint of the study was tolerability; secondary endpoints included minimal residual disease (MRD), 3-year EFS and overall survival (OS). Fifty patients enrolled between 2017-2021. Median follow up was 3.4 years (range 1.6-6.8 years). The six patients in the dose escalation phase had no dose-limiting toxicities. Forty-four patients received vorinostat at the third dose level (DL3) of 180 mg/m 2/dose. 68% of patients treated at DL3 had CNS involvement at diagnosis. 68% of DL3 patients carried a KMT2Ar. The most frequent KMT2A partner genes were AFF1 (40%), MLLT1 (20%), and MLLT3 (13%). The most frequent grade 3-4 non-hematologic adverse events during induction include neutropenic fever (45%), hypertension (43%), infection (40%), anorexia (32%), diarrhea (18%) and perineal skin ulceration (16%). There were 3 episodes of sepsis and 1 induction death due to parainfluenza pneumonia. Fewer events and no deaths or proven sepsis occurred during reinduction, with only grade 3 hypertriglyceridemia exceeding 10% of patients affected (18%). 79% of all patients and 69% of KMT2Ar patients became MRD negative by flow cytometry on day 22 of induction following our investigational block. Following induction intensification with cyclophosphamide, cytarabine, and mercaptopurine, 77% and 66% of all patients and KMT2Ar patients respectively were MRD negative at the time of count recovery. Eight of the forty-four patients, all KMT2Ar, underwent stem cell transplant in first remission due to persistent MRD positivity at the end of consolidation. The 3-year EFS and OS probabilities (SE) of all forty-four DL3 patients were 56.5% (8.0) and 70.5% (7.3), respectively. The 3-year cumulative incidence of relapse (CIR) with death considered a competing event was 29.3% (6.9) and the cumulative incidence of death (CID) as a first event was 8.9% (4.5). Patients lacking a KMT2Ar had superior outcomes with 85.7% (9.4) EFS and 92.9% (6.9) OS. The EFS and OS of KMT2Ar patients were 44.8% (9.5) and 62% (9.1) respectively. CIR and CID for KMT2Ar patients was 43.5% (9.6) and 7.2% (5) respectively. KMT2Ar patients ≥90 days at diagnosis had superior outcomes compared to their younger counterparts with 59.1% (10.5) EFS and 77.3% (8.9) OS. End of induction MRD was predictive of an event in this subgroup, with 85.6% (9.4) EFS for MRD negative patients and 14.3% (13.2) EFS for MRD positive patients. In contrast, KMT2Ar patients < 90 days of age at diagnosis had poor outcomes irrespective of MRD with 12.5% (11.7) EFS and 25% (15.3) OS. Notably KMT2Ar patients < 90 days who were MRD negative at the end of induction had a median time to relapse of 600 days (range 282-969 days), a late time point for this malignancy that typically relapses within a year from initial diagnosis. This suggests the regimen effectively reduced tumor burden leading to an extended duration of remission but failed to sufficiently eradicate disease to prevent relapse. We conclude that the incorporation of bortezomib and vorinostat for treatment of infants with ALL was well tolerated. MRD responses in KMT2Ar patients were robust. Although the study was not powered to evaluate outcomes, EFS and OS in KMT2Ar patients suggest a clinical signal worth pursuing given the low proportion of patients transplanted in first remission. The successor study, TINI 2 (NCT05848687), will build upon the bortezomib/vorinostat backbone by incorporating 2 cycles of blinatumomab and the menin inhibitor ziftomenib in combination with chemotherapy during reinduction.

Combining Menin and MEK Inhibition to Target Poor Prognostic KMT2A-Rearranged RAS Pathway-Mutant Acute Leukemia

Approximately 10% of all acute leukemias harbor a chromosomal translocation involving the Mixed Lineage Leukemia 1 ( MLL1/ KMT2A) gene locus with over 90 fusion partners. These fusions are especially prevalent in pediatric acute leukemias. The resulting KMT2A fusion proteins (KMT2A-r) drive leukemogenic gene expression through an interaction with a chromatin complex that includes the scaffold protein menin, giving rise to aggressive acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL). In infant ALL, frequently characterized by KMT2A-r, the presence of RAS pathway mutations has been associated with a poor prognosis. The prognostic impact of RAS pathway mutations in pediatric KMT2A-r AML is less established. We thus analyzed data from 1605 pediatric patients with AML treated on the Children's Oncology Group (COG) AAML0531 and AAML1031 studies, with 336 of these having KMT2A-r. 51% (170/336) of these patients had mutations in NRAS, KRAS or PTPN11. The combination of KMT2A-r with RAS pathway mutations was associated with worse EFS and OS, HR=1.71 (p<0.001) for EFS and OS multivariable models. Given the prevalence of RAS pathway mutations co-occurring with KMTA-r, and the high-risk leukemia associated with this mutational profile, we sought to identify efficacious targeted drug combinations for this subset of childhood leukemia. Early clinical trial data for the menin inhibitor revumenib (SNDX-5613) demonstrated efficacy in KMT2A-r leukemias. Responding patients, however, typically develop progressive disease with a subset having mutations in MEN1 the gene encoding menin. Effective combination treatments for full efficacy of this targeted therapy are needed. We evaluated the combination of RAS/MAPK targeting using the MEK1/2 inhibitor selumetinib with VTP-50469, a close analog of revumenib, in pediatric leukemias harboring both KMT2A-r and RAS mutations. Treatment of AML and ALL cell lines and cells from an AML patient-derived xenograft (PDX) model in vitro with this drug combination resulted in a synergistic decrease in viability, a G0/G1 cell cycle arrest, and an increase in apoptosis. The combination also resulted in decreased AML colony formation. We next explored potential mechanisms by which the combination treatment had a synergistic anti-leukemic effect. Both selumetinib and VTP-50469 treatment individually reduced phosphorylated ERK levels, but the combination had the most complete inhibitory effect. The combination treatment also resulted in loss of stem-like gene expression signatures, an increase in myeloid differentiation programs, and most strikingly, downregulation of targets of MYC signaling. Both MYC transcript and protein levels were reduced by each drug alone and even more effectively with the combination. Next, we evaluated the in vivo efficacy of this drug combination in three orthotopic pediatric AML and ALL pediatric PDX models harboring different KMT2A-r and RAS mutations (AML: KMT2A- AF10 with KRAS, KMT2A- PICALM with NRAS, ALL: MLL- AF4 with NRAS). NSG mice engrafted with PDX models via tail vein injection were assigned to four treatment groups: vehicle, chow containing revumenib (0.03%), selumetinib (50 mg/kg/day, 5 days a week), or the combination. After treatment for 18-28 days leukemia burden in bone marrow, spleen and peripheral blood was markedly decreased with the combination compared to the single drug-treated cohorts in 2 models with RAS mutations. In one of the AML models, single agent treatment with revumenib or selumetinib was highly efficacious at eradicating disease at the measured time point. We also found a striking decrease in levels of MYC, P-ERK and MEIS1 by western immunoblotting, highlighting on-target activity of the drugs and supporting our in vitro data for a role for MYC in the synergistic effect observed with the combination. In conclusion, new clinical data from a large cohort of COG patients demonstrates that KMT2A-r and co-occurring RAS pathway mutations portend a distinctively worse outcome for children with AML. The findings in our preclinical study suggest a promising, readily translatable targeted treatment for this patient population. Mechanistically, enhanced downregulation of both MYC signaling and the RAS/MAPK pathway with the combinatorial therapy is likely a strong contributor to the observed efficacy.

Transcriptomic without Clinical Response to Menin Inhibition As a Mechanism of Upfront Resistance in Samples from Mutliply Relapsed Patients with KMT2A-Rearranged Leukemia

Inhibitors of the Menin-KMT2A interaction are emerging as promising agents for the treatment of KMT2A-rearranged ( KMT2A-r) acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Menin is required for the recruitment of the KMT2A-fusion to a subset of its target genes. Early phase clinical trials have shown highly encouraging results in patients with relapsed and refractory leukemias with KMT2A rearrangements and NPM1 mutations. In a recently reported early phase clinical trial evaluating the Menin inhibitor revumenib, about 50% of patients failed to show a clinical response despite robust downregulation of known Menin:KMT2A target genes, suggesting on target activity of the drug. This was the most common pattern of upfront resistance. We sought to understand upfront resistance (in contrast to the reported induced resistance via MEN1 mutations) in patient derived xenograft models. To this end, we evaluated the efficacy of Menin-inhibition in KMT2A-r leukemias with high-risk features: high-risk KMT2A fusion partners in AML ( MLLT10, MLLT4), infant ALL, and serial samples from patients with multiply relapsed ALL. We found that AML samples with high-risk fusion partners banked at initial diagnosis were sensitive to Menin-inhibition in xenografts. In contrast, three highly pretreated samples from patients with KMT2A-AFF1 ALL showed only a slight, or no decrease in latentcy in the Menin-inhibitor arm. Interestingly, Menin inhibition showed excellent in vivo efficacy against leukemia samples obtained from the same three patients earlier in their disease course. Therefore, prior chemotherapy (+/- immunotherapy) selects for Menin-inhibitor resistant cells without prior exposure to Menin inhibitor. Transcriptomic analysis (RNA-Seq) documents sustained on-target efficacy of the Menin inhibitor on a transcriptional level in resistant cells. None of the samples contained a MEN1 mutation at any time point. Targeted panel sequencing at initial diagnosis and relapse documented clonal evolution and the emergence of multiple co-mutations (particularly RAS pathway mutations and TP53 mutations), however, all co-mutations tested failed to induce resistance as a single alteration. It is possible that the cooperation of multiple emerging mutations (rather than a single mutation) is sufficient to drive AML in a KMT2A-fusion independent manner, thus mediating Menin resistance. A transcriptomic response (i.e. downregulation of KMT2A-fusion target genes) without a clinical response was the most common pattern of upfront resistance in the recently reported phase I/II clinical trial of the Menin inhibitor revumenib. We conclude that it will be critical to use Menin-inhibitors upfront or in early lines of therapy before substantial clonal evolution has occurred.

Adrenal Suppression in Children During the Treatment for Acute Lymphoblastic Leukemia Beyond Induction.

Adrenal suppression (AS) is an iatrogenic, life-threatening condition that can occur after glucocorticoid exposure. Despite recognition that AS occurs after induction phase treatment in children with acute lymphoblastic leukemia (ALL), the risk of AS in phases beyond induction is unknown. We conducted a pilot study in pediatric patients with ALL to ascertain whether the risk of AS persists in post-induction phases of treatment. Patients diagnosed between 12 months to younger than 18 years with B or T-ALL and starting any new phase of treatment were eligible for the study. Relapsed or infant ALL were excluded. Low dose ACTH stimulation testing (LDST), measurement of albumin and cortisol binding globulin were performed in all patients. Screening for symptoms of AS was done. Twenty-four patients enrolled in the study. One was diagnosed with clear AS. Five others had a borderline cortisol peak, representing possible mild AS. Symptoms were nonspecific and did not help distinguish patients with normal LDST from those with borderline or abnormal results. Patients on treatment for ALL continue to be at risk of AS beyond induction treatment. Although this risk appears small, physicians must be vigilant as patients may be asymptomatic but could develop adrenal crisis during treatment.

B-389 Validation of One-step In-house RT-qPCR Method for Detecting MLL::AF4 Fusion

Abstract Background Rearrangements of mixed lineage leukemia gene (MLL) with 1 of >130 alternative partner genes is a recurrent cytogenetic finding in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) and is generally associated with a poor prognosis. Among the most common MLL rearrangement is t(4;11)(q21;q23), forming the MLL::AF4 (also known as KMT2A::AFF1) fusion gene. Unique among MLL rearrangements, MLL::AF4 is almost exclusively associated with pro-B cell ALL and is prototypical of infant ALL, where it carries a very poor prognosis. At least 10 different MLL-AF4 fusion transcripts have been found, due to break-points in different introns, some transcripts being more frequent in either adult or infant ALL. Fast and reliable diagnostic tests represent an important tool in clinical laboratories to detect these rearrangements and . In this context, this work aimed to validate a qualitative method to detect the main MLL::AF4 fusion isoforms. Methods We evaluated the performance of the Hs03043614_ft (e10e4, e9e4 and e9e5) and Hs03024412_ft (e11e4) inventoried TaqMan assays (Thermo Fisher Scientific). Total mRNA from negative and positive samples for each fusion was extracted by the TRIzol reagent according to the manufacturer’s instructions. All assays were performed using TaqMan RNA-to-Ct 1-Step kit (Thermo Fisher Scientific). The inventoried ABL1 assay (Thermo Fisher Scientific) was used as endogenous control. Using positive samples, negative samples and controls of each fusion were performed: sequence verification tests, standardization of RNA input, evaluation of reaction efficiency and determination of the detection limit (LOD). The performance of the assays was evaluated with ABL1 control separately from the fusions. Results Our results showed that both assays worked well for the detection of specific fusions. The best RNA input for the reactions was 200 ng. The efficiencies reactions were: a) with ABL1 in separate reactions: 92.6% and 110.0% for Hs03043614_ft and Hs03024412_ft assays, respectively; The literature recommends the limit of efficiency was between 90% and 110%. The LODs with ABL1 in separate reactions were 10−3 for both assays, respectively. There was no unspecific amplification in negative samples. Conclusion The TaqMan assays with RT one-step protocol evaluated are highly sensitive, fast, and reliable tools for detecting the most frequent MLL::AF4 fusion transcripts.

Constitutively Synergistic Multiagent Drug Formulations Targeting MERTK, FLT3, and BCL-2 for Treatment of AML.

Although high-dose, multiagent chemotherapy has improved leukemia survival rates, treatment outcomes remain poor in high-risk subsets, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) in infants. The development of new, more effective therapies for these patients is therefore an urgent, unmet clinical need. The dual MERTK/FLT3 inhibitor MRX-2843 and BCL-2 family protein inhibitors were screened in high-throughput against a panel of AML and MLL-rearranged precursor B-cell ALL (infant ALL) cell lines. A neural network model was built to correlate ratiometric drug synergy and target gene expression. Drugs were loaded into liposomal nanocarriers to assess primary AML cell responses. MRX-2843 synergized with venetoclax to reduce AML cell density in vitro. A neural network classifier based on drug exposure and target gene expression predicted drug synergy and growth inhibition in AML with high accuracy. Combination monovalent liposomal drug formulations delivered defined drug ratios intracellularly and recapitulated synergistic drug activity. The magnitude and frequency of synergistic responses were both maintained and improved following drug formulation in a genotypically diverse set of primary AML bone marrow specimens. We developed a nanoscale combination drug formulation that exploits ectopic expression of MERTK tyrosine kinase and dependency on BCL-2 family proteins for leukemia cell survival in pediatric AML and infant ALL cells. We demonstrate ratiometric drug delivery and synergistic cell killing in AML, a result achieved by a systematic, generalizable approach of combination drug screening and nanoscale formulation that may be extended to other drug pairs or diseases in the future.

The Outcome of Acute Lymphoblastic Leukemia in Infants at National Cancer Institute Egypt: 10 Years Experience

The Outcome of Acute Lymphoblastic Leukemia in Infants at National Cancer Institute Egypt: 10 Years Experience

Minimal residual disease predicts outcomes in KMT2A-rearranged but not KMT2A-germline infant acute lymphoblastic leukemia: Report from Children's Oncology Group study AALL0631.

We measured minimal residual disease (MRD) by multiparameter flow cytometry at three time points (TP) in 117 infants with KMT2A (lysine [K]-specific methyltransferase 2A)-rearranged and 58 with KMT2A-germline acute lymphoblastic leukemia (ALL) on Children's Oncology Group AALL0631 study. For KMT2A-rearranged patients, 3-year event-free survival (EFS) by MRD-positive (≥0.01%) versus MRD-negative (<0.01%) was: TP1: 25% (±6%) versus 49% (±7%; p=.0009); TP2: 21% (±8%) versus 47% (±7%; p<.0001); and TP3: 22% (±14%) versus 51% (±6%; p=.0178). For KMT2A-germline patients, 3-year EFS was: TP1: 88% (±12%) versus 87% (±5%; p=.73); TP2: 100% versus 88% (±5%; p=.24); and TP3: 100% versus 87% (±5%; p=.53). MRD was a strong independent outcome predictor in KMT2A-rearranged, but not KMT2A-germline infant ALL.

Blinatumomab Is Safe and Efficacious in Infant Acute Lymphoblastic Leukemia.

No toxic effects that led to treatment discontinuation or death were observed with blinatumomab.

Abstract 4937: Immunoproteasome inhibitors for the treatment of t(4;11)-driven ALL

Abstract The t(4;11)(q21;q23) chromosomal translocation that creates the MLL-AF4 fusion protein, confers a poor prognosis in infant acute lymphoblastic leukemia (ALL). This translocation also sensitizes cells to proteasome inhibitors bortezomib and carfilzomib, which are approved by the FDA for the treatment of multiple myeloma. Clinical activity of bortezomib in combination with standard chemotherapy has been documented in several clinical trials of ALL patients, and a case of a single-agent activity against relapsed leukemia driven by the MLL-AF4 translocation has been described. However, toxicities of bortezomib and carfilzomib may be unacceptable to pediatric patients. We found that the overwhelming majority of proteasomes in this subtype of ALL are lymphoid tissue specific immunoproteasomes. Cells with MLL-AF4 translocations were sensitive to pharmacologically relevant concentrations of specific immunoproteasome inhibitors ONX-0914 and M3258. Furthermore, both compounds dramatically delayed growth of orthotopic xenograft tumors in mice. Thus, immunoproteasomes are therapeutic targets in ALL and replacing bortezomib and carfilzomib with immunoproteasome inhibitors in ALL should reduce toxicities associated with inhibition of the proteasomes in non-lymphoid tissues. Citation Format: Alexei F. Kisselev, Tyler W. Jenkins, Elise Fitzerald, Andrey V. Maksimenko, Peter Panizzi, Jennifer L. Fields, Steven N. Fiering, Amit K. Mitra. Immunoproteasome inhibitors for the treatment of t(4;11)-driven ALL. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4937.

Proteasome inhibition targets the KMT2A transcriptional complex in acute lymphoblastic leukemia

Rearrangments in Histone-lysine-N-methyltransferase 2A (KMT2Ar) are associated with pediatric, adult and therapy-induced acute leukemias. Infants with KMT2Ar acute lymphoblastic leukemia (ALL) have a poor prognosis with an event-free-survival of 38%. Herein we evaluate 1116 FDA approved compounds in primary KMT2Ar infant ALL specimens and identify a sensitivity to proteasome inhibition. Upon exposure to this class of agents, cells demonstrate a depletion of histone H2B monoubiquitination (H2Bub1) and histone H3 lysine 79 dimethylation (H3K79me2) at KMT2A target genes in addition to a downregulation of the KMT2A gene expression signature, providing evidence that it targets the KMT2A transcriptional complex and alters the epigenome. A cohort of relapsed/refractory KMT2Ar patients treated with this approach on a compassionate basis had an overall response rate of 90%. In conclusion, we report on a high throughput drug screen in primary pediatric leukemia specimens whose results translate into clinically meaningful responses. This innovative treatment approach is now being evaluated in a multi-institutional upfront trial for infants with newly diagnosed ALL.

Successful Retransplantation With Killer Cell Immunoglobulin-like Receptor Ligand-mismatched Cord Blood in Infant Acute Lymphoblastic Leukemia That Relapsed After Transplantation.

The prognosis of children with KMT2A -rearranged ( KMT2A -r) acute lymphoblastic leukemia (ALL) remains dismal. This report describes the successful retransplantation of a patient with infant ALL who relapsed both bone marrow and central nervous system. The patient received HLA-matched cord blood transplantation (CBT) and relapsed 18 months later. After achieving the second remission, the patient received a killer cell immunoglobulin-like receptor ligand-mismatched CBT with a reduced-intensity conditioning regimen and has been in remission for 52 months. Thus, killer cell immunoglobulin-like receptor ligand-mismatched CBT with reduced-intensity conditioning might be a treatment option for patients with KMT2A- r ALL who relapsed after transplantation, even with extramedullary relapse.