Faculty Bibliography

Background Acute Lymphoblastic Leukemia (ALL) is the most common pediatric cancer and while curable in the majority of cases, 15%-20% of children will relapse with only 50% surviving long-term. Treatment failures arise from the outgrowth of pre-existing or acquired sub-clones that are genetically or epigenetically primed to resist treatment. In addition, the bone marrow microenvironment is known to provide a protective niche. We performed the first mapping of the human B-cell ALL (B-ALL) immune bone marrow (BM) microenvironment at single cell resolution at diagnosis, remission and relapse (Witkowski, 2020). We uncovered a striking rewiring of the myeloid compartment during B-ALL progression with significant over-representation of a leukemia-associated monocyte subpopulation expressing high levels of the Macrophage Colony Stimulating Factor Receptor (M-CSFR/CSF1R). Using both peripheral blood (PB) complete blood count analysis and RNA-seq data, we demonstrated that high monocyte abundance at B-ALL diagnosis is predictive of inferior pediatric and adult overall survival in two large independent clinical cohorts. To determine the association of non-classical monocyte abundance in BM and PB with risk of relapse, we examined a cohort of clinical samples from children enrolled on Children's Oncology Group (COG) protocols. Methods Using an unmatched case-control design, we established a preliminary cohort of PB and BM samples collected at diagnosis from 24 B-ALL patients with eventual relapse and 24 patients in long-term remission. Four remission samples from an NYU Langone cohort were used to validate the expansion of this population in the presence of B-ALL. We applied a customized flow cytometry based assay to identify CD115-expressing human monocyte subsets: classical (CD45 ⁺CD56 ^-CD14 ⁺CD16 ^-), non-classical (CD45 ⁺CD56 ^-CD14 ^-CD16 ⁺), as well as B-cells (CD19, CD22, CD10) and T/NK cells (CD3, CD56). We then performed univariate and multivariable analysis of outcome (relapse versus long-term remission) compared to monocyte subset abundance, adjusting for potential confounding factors (age, gender, CNS status, NCI risk, genetic subtype, WBC at diagnosis, and end of induction minimal residual disease). Results We observed a significantly higher percentage of non-classical monocytes in the diagnostic BM from the COG cohort when compared to remission samples (COG diagnostic B-ALL BM non-classical percentage mean 52.19% vs NYU B-ALL remission BM non-classical percentage mean 1.775%, P = 0.0001). We also observed a strong correlation between the percentage of non-classical monocytes in the PB when compared to their matched BM specimens (r = 0.6, P = 0.0001). Multivariable analysis revealed a significant association between PB non-classical monocyte percentage at diagnosis and patient outcome (remission cohort non-classical monocyte percentage [mean, range]: 52.4%, 33.3-68.1%, n = 24, relapse cohort non-classical monocyte percentage: 65.9%, 56.4-84.7%, n = 24, P = 0.021). Similarly, a strong trend was observed in the BM, although it did not reach statistical significance. Flow cytometric analysis confirmed CD115 (M-CSFR/CSF1R) expression in this non-classical monocyte population, thereby validating a novel target for intervention. Conclusions These findings validate the presence of a unique monocyte subpopulation associated with childhood B-ALL and suggests that assessing this population in PB at diagnosis may be of prognostic significance. The availability of small molecule inhibitors and monoclonal antibodies targeting CSF1R-expressing monocytes may offer a novel approach to treating B-ALL. [Formula presented] Disclosures: Raetz: Pfizer: Research Funding; Celgene: Other: DSMB member. Teachey: Sobi: Consultancy; NeoImmune Tech: Research Funding; Janssen: Consultancy; BEAM Therapeutics: Consultancy, Research Funding. Aifantis: AstraZeneca: Research Funding; Foresite (FL2020-010) LLC: Consultancy.
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We conducted the first HLA allele and genome wide association study to identify loci associated with hypersensitivity reactions exclusively to the PEGylated preparation of asparaginase (pegaspargase) in racially diverse cohorts of pediatric leukemia patients: St. Jude Children's Research Hospital's Total XVI (TXVI, n = 598), Children's Oncology Group AALL0232 (n = 2472) and AALL0434 (n = 1189). Germline DNA was genotyped using arrays. Genetic variants not genotyped directly were imputed. HLA alleles were imputed using SNP2HLA or inferred using BWAkit. Analyses between genetic variants and hypersensitivity were performed in each cohort first using cohort-specific covariates and then combined using meta-analyses. Nongenetic risk factors included fewer intrathecal injections (P = 2.7x10^-5 in TXVI) and male sex (P = 0.025 in AALL0232). HLA alleles DQB1*02:02, DRB1*07:01, and DQA1*02:01 had the strongest associations with pegaspargase hypersensitivity (P < 5.0x10^-5 ) in patients with primarily European ancestry (EA), with the three alleles associating in a single haplotype. The top allele HLA-DQB1*02:02 was tagged by HLA-DQB1 rs1694129 in EAs (r² = 0.96) and less so in non-EAs. All single nucleotide polymorphisms associated with pegaspargase hypersensitivity reaching genome-wide significance in EAs were in class II HLA loci, and were partially replicated in non-EAs, as is true for other HLA associations. The rs9958628 variant, in ARHGAP28 (previously linked to immune response in children) had the strongest genetic association (P = 8.9x10^-9 ) in non-EAs. The HLA-DQB1*02:02-DRB1*07:01-DQA1*02:01 associated with hypersensitivity reactions to pegaspargase is the same haplotype associated with reactions to non-PEGylated asparaginase, even though the antigens differ between the two preparations.

Early T-cell precursor phenotype acute lymphoblastic leukemia (ETP-ALL) is a subtype of T-ALL with a unique immunophenotype and genetic abnormalities distinct from conventional T-ALL. A subset of T lymphoblastic lymphoma (T-LLy) also demonstrates the early T-cell precursor immunophenotype and may be a counterpart of ETP-ALL. Unlike ETP-ALL, the incidence, clinical features, and genomic features of ETP-LLy are unknown. We reviewed the immunophenotyping data of 218 T-LLy patients who enrolled in the Children's Oncology Group AALL0434 clinical trial and identified 9 cases (4%) exhibiting a definitive ETP immunophenotype. We performed single-nucleotide polymorphism array profiling on 9 ETP-LLy and 15 non-ETP T-LLy cases. Compared with non-ETP T-LLy, ETP-LLy showed less frequent deletion of 9p (CKDN2A/B), more frequent deletion of 12p (ETV6) and 1p (RPL22), and more frequent absence of biallelic T-cell receptor γ deletions. Recurrent abnormalities previously described in ETP-ALL such as deletions of 5q and 13q and gain of 6q were not observed in ETP-LLy cases. There were no failures of therapy among the ETP-LLy subtype with a 4-year event-free survival of 100%. Overall, ETP-LLy does not exhibit unifying genetic alterations but shows some distinct genomic features from non-ETP T-LLy suggesting that ETP-LLy may be a distinct entity from non-ETP T-LLy.

BACKGROUND:Patients with late, ≥18 months postdiagnosis, isolated central nervous relapse (iCNS-R) of B-acute lymphoblastic leukemia (ALL) have excellent outcomes with chemotherapy plus cranial radiotherapy, with 5-year 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/METHODS:COG AALL02P2 enrolled 118 eligible patients with B-cell ALL (B-ALL) and late iCNS-R who received intensified systemic therapy, triple intrathecal chemotherapy, and 1200 cGy cranial irradiation delivered at 12 months, with maintenance chemotherapy continuing until 104 weeks postdiagnosis. RESULTS:The 3-year event-free survival (EFS) and OS were 64.3% ± 4.5% and 79.6% ± 3.8%, with 46.1% (18/39) of second 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.

Genetic alterations in the RUNX1 gene are associated with benign and malignant blood disorders, particularly of megakaryocyte and myeloid lineages. The role of RUNX1 in acute lymphoblastic leukemia (ALL) is less clear, particularly how germline genetic variation influences the predisposition to this type of leukemia. Sequencing 4,836 children with B-ALL and 1,354 cases of T-ALL, we identified 31 and 18 germline RUNX1 variants, respectively. RUNX1 variants in B-ALL consistently showed minimal damaging effects. By contrast, 6 T-ALL-related variants result in drastic loss of RUNX1 activity as a transcription activator in vitro. Ectopic expression of dominant-negative RUNX1 variants in human CD34+ cells repressed differentiation into erythroid, megakaryocytes, and T cells, while promoting myeloid cell development. Chromatin immunoprecipitation sequencing of T-ALL models showed distinctive patterns of RUNX1 binding by variant proteins. Further whole genome sequencing identified JAK3 mutation as the most frequent somatic genomic abnormality in T-ALL with germline RUNX1 variants. Co-introduction of RUNX1 variant and JAK3 mutation in hematopoietic stem and progenitor cells in mice gave rise to T-ALL with early T-cell precursor phenotype. Taken together, these results indicated that RUNX1 is an important predisposition gene for T-ALL and pointed to novel biology of RUNX1-mediated leukemogenesis in the lymphoid lineages.

Infants with KMT2A-rearranged acute lymphoblastic leukemia (KMT2A-r ALL) have a poor prognosis. KMT2A-r ALL overexpresses FLT3, and the FLT3 inhibitor (FLT3i) lestaurtinib potentiates chemotherapy-induced cytotoxicity in preclinical models. Children's Oncology Group (COG) AALL0631 tested whether adding lestaurtinib to post-induction chemotherapy improved event-free survival (EFS). After chemotherapy induction, KMT2A-r infants received either chemotherapy only or chemotherapy plus lestaurtinib. Correlative assays included FLT3i plasma pharmacodynamics (PD), which categorized patients as inhibited or uninhibited, and FLT3i ex vivo sensitivity (EVS), which categorized leukemic blasts as sensitive or resistant. There was no difference in 3-year EFS between patients treated with chemotherapy plus lestaurtinib (n = 67, 36 ± 6%) vs. chemotherapy only (n = 54, 39 ± 7%, p = 0.67). However, for the lestaurtinib-treated patients, FLT3i PD and FLT3i EVS significantly correlated with EFS. For FLT3i PD, EFS for inhibited/uninhibited was 59 ± 10%/28 ± 7% (p = 0.009) and for FLTi EVS, EFS for sensitive/resistant was 52 ± 8%/5 ± 5% (p < 0.001). Seventeen patients were both inhibited and sensitive, with an EFS of 88 ± 8%. Adding lestaurtinib did not improve EFS overall, but patients achieving potent FLT3 inhibition and those whose leukemia blasts were sensitive FLT3-inhibition ex vivo did benefit from the addition of lestaurtinib. Patient selection and PD-guided dose escalation may enhance the efficacy of FLT3 inhibition for KMT2A-r infant ALL.

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and advances in its clinical and laboratory biology have grown exponentially over the last few decades. Treatment outcome has improved steadily with over 90% of patients surviving 5 years from initial diagnosis. This success can be attributed in part to the development of a risk stratification approach to identify those subsets of patients with an outstanding outcome that might qualify for a reduction in therapy associated with fewer short and long term side effects. Likewise, recognition of patients with an inferior prognosis allows for augmentation of therapy, which has been shown to improve outcome. Among the clinical and biological variables known to impact prognosis, the kinetics of the reduction in tumor burden during initial therapy has emerged as the most important prognostic variable. Specifically, various methods have been used to detect minimal residual disease (MRD) with flow cytometric and molecular detection of antigen receptor gene rearrangements being the most common. However, many questions remain as to the optimal timing of these assays, their sensitivity, integration with other variables and role in treatment allocation of various ALL subgroups. Importantly, the emergence of next generation sequencing assays is likely to broaden the use of these assays to track disease evolution. This review will discuss the biological basis for utilizing MRD in risk assessment, the technical approaches and limitations of MRD detection and its emerging applications.

PURPOSE/OBJECTIVE: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: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:< .0001). CONCLUSION/CONCLUSIONS: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.