Azacitidine as epigenetic priming for chemotherapy is safe and well-tolerated in infants with newly diagnosed KMT2A-rearranged acute lymphoblastic leukemia: Children's Oncology Group trial AALL15P1.

Infants less than 1 year old diagnosed with KMT2A-rearranged (KMT2A-r) acute lymphoblastic leukemia (ALL) are at high risk of remission failure, relapse, and death due to leukemia, despite intensive therapies. Infant KMT2A-r ALL blasts are characterized by DNA hypermethylation. Epigenetic priming with DNA methyltransferase inhibitors increases the cytotoxicity of chemotherapy in preclinical studies. The Children's Oncology Group trial AALL15P1 tested the safety and tolerability of five days of azacitidine immediately prior to the start of chemotherapy on day six, in four post-induction chemotherapy courses for infants with newly diagnosed KMT2A-r ALL. The treatment was welltolerated, with only two of 31 evaluable patients (6.5%) experiencing dose-limiting toxicity. Whole genome bisulfite sequencing of peripheral blood mononuclear cells (PBMCs) demonstrated decreased DNA methylation in 87% of samples tested following five days of azacitidine. Event-free survival was similar to prior studies of newly diagnosed infant ALL. Azacitidine is safe and results in decreased DNA methylation of PBMCs in infants with KMT2A-r ALL, but the incorporation of azacitidine to enhance cytotoxicity did not impact survival. Clinicaltrials.gov identifier: NCT02828358.

Nivolumab Plus 5-Azacitidine in Pediatric Relapsed/Refractory Acute Myeloid Leukemia (AML): Phase I/II Trial Results from the Therapeutic Advances in Childhood Leukemia and Lymphoma (TACL) Consortium.

Improvements in survival have been made over the past two decades for childhood acute myeloid leukemia (AML), but the approximately 40% of patients who relapse continue to have poor outcomes. A combination of checkpoint-inhibitor nivolumab and azacitidine has demonstrated improvements in median survival in adults with AML. This phase I/II study with nivolumab and azacitidine in children with relapsed/refractory AML (NCT03825367) was conducted through the Therapeutic Advances in Childhood Leukemia & Lymphoma consortium. Thirteen patients, median age 13.7 years, were enrolled. Patients had refractory disease with multiple reinduction attempts. Twelve evaluable patients were treated at the recommended phase II dose (established at dose level 1, 3 mg/kg/dose). Four patients (33%) maintained stable disease. This combination was well tolerated, with no dose-limiting toxicities observed. Grade 3-4 adverse events (AEs) were primarily hematological. Febrile neutropenia was the most common AE ≥ grade 3. A trend to improved quality of life was noted. Increases in CD8+ T cells and reductions in CD4+/CD8+ T cells and demethylation were observed. The combination was well tolerated and had an acceptable safety profile in pediatric patients with relapsed/refractory AML. Future studies might explore this combination for the maintenance of remission in children with AML at high risk of relapse.

Single-cell analysis reveals altered tumor microenvironments of relapse- and remission-associated pediatric acute myeloid leukemia.

Acute myeloid leukemia (AML) microenvironment exhibits cellular and molecular differences among various subtypes. Here, we utilize single-cell RNA sequencing (scRNA-seq) to analyze pediatric AML bone marrow (BM) samples from diagnosis (Dx), end of induction (EOI), and relapse timepoints. Analysis of Dx, EOI scRNA-seq, and TARGET AML RNA-seq datasets reveals an AML blasts-associated 7-gene signature (CLEC11A, PRAME, AZU1, NREP, ARMH1, C1QBP, TRH), which we validate on independent datasets. The analysis reveals distinct clusters of Dx relapse- and continuous complete remission (CCR)-associated AML-blasts with differential expression of genes associated with survival. At Dx, relapse-associated samples have more exhausted T cells while CCR-associated samples have more inflammatory M1 macrophages. Post-therapy EOI residual blasts overexpress fatty acid oxidation, tumor growth, and stemness genes. Also, a post-therapy T-cell cluster associated with relapse samples exhibits downregulation of MHC Class I and T-cell regulatory genes. Altogether, this study deeply characterizes pediatric AML relapse- and CCR-associated samples to provide insights into the BM microenvironment landscape.

BCMA CAR-T induces complete and durable remission in refractory plasmablastic lymphoma.

Plasmablastic lymphoma (PBL) is a rare subtype of aggressive large B-cell lymphoma, with a dismal prognosis despite aggressive therapies. New approaches are needed for those with refractory disease. PBL expresses antigens similar to multiple myeloma (MM), including B-cell maturation antigen (BCMA). Chimeric antigen receptor T-cell (CAR-T) therapy directed against BCMA has shown efficacy for the treatment of heavily pretreated MM with low rates of grades 3 and 4 cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) in a phase Ib/II trial (A Study of JNJ-68284528, a CAR-T Directed Against BCMA in Participants With Relapsed or Refractory Multiple Myeloma (CARTITUDE-1), NCT03548207). However, data for the use of BCMA CAR-T for treating PBL are lacking.We report a challenging case of multiple refractory PBL that emerged from B-cell acute lymphoblastic leukemia in an adolescent who failed to respond to an allogeneic hematopoietic cell transplant. The patient developed rapidly advancing disease despite withdrawal of immunosuppression, treatment with etoposide, ibrutinib, and daratumumab, prompting consideration of BCMA CAR-T (under emergency investigational new drug (eIND)). The patient achieved a complete remission (CR), without recurrent acute graft versus host disease (GVHD), CRS or ICANS after BCMA CAR-T therapy. BCMA CAR-T expansion was detected in vivo, peaking on day 15. The patient remains in CR for more than a year post CAR-T therapy, supporting consideration of immunotherapy for future patients with refractory PBL, a disease with few treatment options.

Mismatched Unrelated Donor Hematopoietic Stem Cell Transplantation Rescues Marrow Failure From Acute Leukemia Therapy in a Patient With Previously Undiagnosed Ligase IV Syndrome.

Patients with DNA double-strand breakage repair disorders are at increased risk of malignancy which is often difficult to treat given underlying sensitivity to chemotherapy and radiotherapy, lending an important role to hematopoietic stem cell transplantation. The choice of conditioning regimen used must balance reducing risk of rejection with minimizing excessive toxicity from myeloablative chemotherapy or ionizing radiation. We describe successful engraftment following a nonmyeloablative hematopoietic stem cell transplantation in a patient with Ligase IV syndrome and numerous pretransplant complications including malignancy, cardiac failure, and secondary hemophagocytic lymphohistiocytosis. Congruent with prior reports, a reduced intensity regimen appears efficacious in Ligase IV syndrome patients.

Phase 1b study of carfilzomib with induction chemotherapy in pediatric relapsed/refractory acute lymphoblastic leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in childhood. Survival for patients following relapse remains poor, and achieving complete remission (CR) after relapse is the first critical step to cure. Carfilzomib is a proteasome inhibitor with an acceptable safety profile and clinical activity in adults with multiple myeloma but has not been assessed in children. The primary objective of this phase 1b study was to assess the safety and tolerability of carfilzomib combined with vincristine, dexamethasone, asparaginase, and daunorubicin (VXLD) in children with relapsed and/or refractory ALL. METHODS: Patients aged 1-21 years (n = 24) received 4-week induction therapy with carfilzomib at dose levels of 27 mg/m2 (n = 3), 36 mg/m2 (n = 7), 45 mg/m2 (n = 4), and 56 mg/m2 (n = 10) in combination with VXLD. Patients achieving stable disease were offered further consolidation chemotherapy. Analyses were based on the safety evaluable population. RESULTS: Following dose escalation of carfilzomib, the recommended phase 2 carfilzomib dose was identified as 56 mg/m2 . Grade ≥3 hematological adverse events were common (83%, 20/24 patients), and serious treatment-emergent adverse events occurred in 58% (14/24) of patients. At the end of induction, CR/CR with incomplete platelet recovery (CRp)/CR with incomplete blood count recovery (CRi) was identified in 50% of patients (n = 12/24). By the end of consolidation, cumulative CR/CRp/CRi was identified in 58% of patients (n = 14/24). CONCLUSION: These data support the use of carfilzomib in pediatric patients with relapsed and/or refractory ALL.

Comprehensive Genomic Profiling of High-Risk Pediatric Cancer Patients Has a Measurable Impact on Clinical Care.

PURPOSE: Profiling of pediatric cancers through deep sequencing of large gene panels and whole exomes is rapidly being adopted in many clinical settings. However, the most impactful approach to genomic profiling of pediatric cancers remains to be defined. METHODS: We conducted a prospective precision medicine trial, using whole-exome sequencing of tumor and germline tissue and whole-transcriptome sequencing (RNA Seq) of tumor tissue to characterize the mutational landscape of 127 tumors from 126 unique patients across the spectrum of pediatric brain tumors, hematologic malignancies, and extracranial solid tumors. RESULTS: We identified somatic tumor alterations in 121/127 (95.3%) tumor samples and identified cancer predisposition syndromes on the basis of known pathogenic or likely pathogenic germline mutations in cancer predisposition genes in 9/126 patients (7.1%). Additionally, we developed a novel scoring system for measuring the impact of tumor and germline sequencing, encompassing therapeutically relevant genomic alterations, cancer-related germline findings, recommendations for treatment, and refinement of risk stratification or prognosis. At least one impactful finding from the genomic results was identified in 108/127 (85%) samples sequenced. A recommendation to consider a targeted agent was provided for 82/126 (65.1%) patients. Twenty patients ultimately received therapy with a molecularly targeted agent, representing 24% of those who received a targeted agent recommendation and 16% of the total cohort. CONCLUSION: Paired tumor/normal whole-exome sequencing and tumor RNA Seq of de novo or relapsed/refractory tumors was feasible and clinically impactful in high-risk pediatric cancer patients.

Matched Targeted Therapy for Pediatric Patients with Relapsed, Refractory, or High-Risk Leukemias: A Report from the LEAP Consortium.

Despite a remarkable increase in the genomic profiling of cancer, integration of genomic discoveries into clinical care has lagged behind. We report the feasibility of rapid identification of targetable mutations in 153 pediatric patients with relapsed/refractory or high-risk leukemias enrolled on a prospective clinical trial conducted by the LEAP Consortium. Eighteen percent of patients had a high confidence Tier 1 or 2 recommendation. We describe clinical responses in the 14% of patients with relapsed/refractory leukemia who received the matched targeted therapy. Further, in order to inform future targeted therapy for patients, we validated variants of uncertain significance, performed ex vivo drug-sensitivity testing in patient leukemia samples, and identified new combinations of targeted therapies in cell lines and patient-derived xenograft models. These data and our collaborative approach should inform the design of future precision medicine trials. SIGNIFICANCE: Patients with relapsed/refractory leukemias face limited treatment options. Systematic integration of precision medicine efforts can inform therapy. We report the feasibility of identifying targetable mutations in children with leukemia and describe correlative biology studies validating therapeutic hypotheses and novel mutations.See related commentary by Bornhauser and Bourquin, p. 1322.This article is highlighted in the In This Issue feature, p. 1307.

Germline mutations in the bone marrow microenvironment and dysregulated hematopoiesis.

The relationship between the hematopoietic stem cell (HSC) population and its surrounding bone marrow microenvironment is a rapidly evolving area of research. Normal HSC processes rely heavily on a complex communication network involving various marrow niches. Although leukemogenesis largely results from abnormal genetic activity within the leukemia stem cell itself, mounting evidence indicates a significant contributory role played by marrow niche dysregulation. Furthermore, numerous instances of activating or inactivating germline mutations within marrow microenvironment cells have been shown to be sufficient for development of myelodysplastic syndrome, myeloproliferative neoplasm, and acute myeloid leukemia, even in the context of wild-type HSCs. Recent evidence suggests that targeting aberrant chemokine production from germline-mutated marrow stromal cells can potentially reverse the process of leukemogenesis. This elaborate interplay between the HSC population and the marrow microenvironment allows for a number of unique clinical possibilities in efforts to induce remission, enhance chemosensitivity, manage relapsed disease, and prevent leukemia development, both in de novo and germline mutation-associated leukemias, including the use of targeted cytokine/chemokine inhibitors, immune checkpoint blockade, CXCR4/CXCL12 axis antagonists, and combined allogeneic HSC and mesenchymal stem cell transplantation. In this review, we discuss the pathways underlying normal and abnormal bone marrow niche functioning, the relationship between germline mutations in the stem cell microenvironment and dysregulated hematopoiesis, and future clinical perspectives that may be particularly applicable to prevention and treatment of germline-associated leukemias.

Phase I trial of the mTOR inhibitor everolimus in combination with multi-agent chemotherapy in relapsed childhood acute lymphoblastic leukemia.

BACKGROUND: We sought to determine the feasibility of co-administering everolimus with a four-drug reinduction in children and adolescents with acute lymphoblastic leukemia (ALL) experiencing a first marrow relapse. PROCEDURE: This phase I study tested everolimus with vincristine, prednisone, pegaspargase and doxorubicin in patients with marrow relapse occurring >18 months after first complete remission (CR). The primary aim was to identify the maximum tolerated dose of everolimus. Three dose levels (DLs) were tested during dose escalation (2, 3, and 5 mg/m2 /day). Additional patients were enrolled at the 3- and 5 mg/m2 /day DLs to further evaluate toxicity (dose expansion). RESULTS: Thirteen patients enrolled during dose escalation and nine during dose expansion. During dose escalation, one dose-limiting toxicity occurred (grade 4 hyperbilirubinemia) in six evaluable patients at DL3 (5 mg/m2 /day). The most common grade ≥3 adverse events were febrile neutropenia, infections, transaminitis, hyperbilirubinemia, and hypophosphatemia. Two of the 12 patients treated at DL3 developed Rothia mucilaginosa meningitis. Nineteen patients (86%) achieved a second CR (CR2). Of those, 13 (68%) had a low end-reinduction minimal residual disease (MRD) level (≤10-3 by polymerase chain reaction-based assay). The CR2 rate for patients with B-cell ALL treated at DL3 (n = 12) was 92%; 82% of these patients had low MRD. CONCLUSIONS: Everolimus combined with four-drug reinduction chemotherapy was generally well tolerated and associated with favorable rates of CR2 and low end-reinduction MRD. The recommended phase 2 dose of everolimus given in combination with a four-drug reinduction is 5 mg/m2 /day. This promising combination should be further evaluated in a larger patient cohort.

Hyperleukocytosis in infant acute leukemia: a role for manual exchange transfusion for leukoreduction.

BACKGROUND: Hyperleukocytosis is a serious, life-threatening complication of pediatric acute leukemia that can cause neurologic injury, pulmonary leukostasis, metabolic derangements, and coagulopathy. Acute leukemia has the highest risk of mortality and morbidity at presentation when associated with hyperleukocytosis. Infant leukemia presents unique challenges and treatment considerations due to the disease itself and size and overall health of the patient. While medical management of hyperleukocytosis in older patients with acute leukemia has been described, including cytoreductive procedures with automated leukapheresis (AL) or manual whole blood (WB) exchange transfusion, very little data exist for standardized management of hyperleukocytosis in infant leukemia patients. CASE REPORTS: We describe four cases of infant acute leukemia presenting with hyperleukocytosis and leukostasis who each received manual WB exchange transfusions in conjunction with induction chemotherapy and review the existing literature on the use of procedural leukoreduction in infants with hyperleukocytosis. Special attention is given to challenges and technical aspects of leukapheresis in infants: when to perform manual WB exchange versus AL, optimal vascular access, blood product selection, exchange rates, and the monitoring for complications. Using published cases, we outline benefits versus risks of manual WB exchange and AL in infants less than 10 kg. CONCLUSION: If providers perform procedural leukoreduction, the literature and our experience demonstrate manual WB exchange transfusion is favored over AL in infants less than 10 kg because of technical and complication risks associated with AL. Additional studies are needed to understand the impact of cytoreduction on long-term outcomes.

Outcomes of adults and children with primary mediastinal B-cell lymphoma treated with dose-adjusted EPOCH-R.

Treatment with dose-adjusted EPOCH (etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone) chemotherapy and rituximab (DA-EPOCH-R) has become the standard of care for primary mediastinal B-cell lymphoma (PMBCL) at many institutions despite limited data in the multi-centre setting. We report a large, multi-centre retrospective analysis of children and adults with PMBCL treated with DA-EPOCH-R to characterize outcomes and evaluate prognostic factors. We assessed 156 patients with PMBCL treated with DA-EPOCH-R across 24 academic centres, including 38 children and 118 adults. All patients received at least one cycle of DA-EPOCH-R. Radiation therapy was administered in 14·9% of patients. With median follow-up of 22·6 months, the estimated 3-year event-free survival (EFS) was 85·9% [95% confidence interval (CI) 80·3-91·5] and overall survival was 95·4% (95% CI 91·8-99·0). Outcomes were not statistically different between paediatric and adult patients. Thrombotic complications were reported in 28·2% of patients and were more common in paediatric patients (45·9% vs. 22·9%, P = 0·011). Seventy-five per cent of patients had a negative fluorodeoxyglucose positron emission tomography (FDG-PET) scan at the completion of DA-EPOCH-R, defined as Deauville score 1-3. Negative FDG-PET at end-of-therapy was associated with improved EFS (95·4% vs. 54·9%, P < 0·001). Our data support the use of DA-EPOCH-R for the treatment of PMBCL in children and adults. Patients with a positive end-of-therapy FDG-PET scan have an inferior outcome.

Leukaemogenic effects of Ptpn11 activating mutations in the stem cell microenvironment.

Germline activating mutations of the protein tyrosine phosphatase SHP2 (encoded by PTPN11), a positive regulator of the RAS signalling pathway, are found in 50% of patients with Noonan syndrome. These patients have an increased risk of developing leukaemia, especially juvenile myelomonocytic leukaemia (JMML), a childhood myeloproliferative neoplasm (MPN). Previous studies have demonstrated that mutations in Ptpn11 induce a JMML-like MPN through cell-autonomous mechanisms that are dependent on Shp2 catalytic activity. However, the effect of these mutations in the bone marrow microenvironment remains unclear. Here we report that Ptpn11 activating mutations in the mouse bone marrow microenvironment promote the development and progression of MPN through profound detrimental effects on haematopoietic stem cells (HSCs). Ptpn11 mutations in mesenchymal stem/progenitor cells and osteoprogenitors, but not in differentiated osteoblasts or endothelial cells, cause excessive production of the CC chemokine CCL3 (also known as MIP-1α), which recruits monocytes to the area in which HSCs also reside. Consequently, HSCs are hyperactivated by interleukin-1ß and possibly other proinflammatory cytokines produced by monocytes, leading to exacerbated MPN and to donor-cell-derived MPN following stem cell transplantation. Remarkably, administration of CCL3 receptor antagonists effectively reverses MPN development induced by the Ptpn11-mutated bone marrow microenvironment. This study reveals the critical contribution of Ptpn11 mutations in the bone marrow microenvironment to leukaemogenesis and identifies CCL3 as a potential therapeutic target for controlling leukaemic progression in Noonan syndrome and for improving stem cell transplantation therapy in Noonan-syndrome-associated leukaemias.

Maintenance Treatment With Low-Dose Mercaptopurine in Combination With Allopurinol in Children With Acute Lymphoblastic Leukemia and Mercaptopurine-Induced Pancreatitis.

Mercaptopurine (6-mercaptopurine, 6MP) is a mainstay of curative therapy in childhood acute lymphoblastic leukemia (ALL), and contributes to its 90% overall survival rate. We present two patients with ALL who suffered with severe pancreatitis secondary to 6MP. Through the use of allopurinol in conjunction with reduced dose 6MP, we were able to continue 6MP without further pancreatitis. This report contributes to the small body of literature on 6MP associated pancreatitis in childhood ALL and describes a novel approach to continued use of 6MP during therapy.