Treatment of newly diagnosed severe aplastic anemia in children: Evidence-based recommendations.

Severe aplastic anemia (SAA) is a rare potentially fatal hematologic disorder. Although overall outcomes with treatment are excellent, there are variations in management approach, including differences in treatment between adult and pediatric patients. Certain aspects of treatment are under active investigation in clinical trials. Because of the rarity of the disease, some pediatric hematologists may have relatively limited experience with the complex management of SAA. The following recommendations reflect an up-to-date evidence-based approach to the treatment of children with newly diagnosed SAA.

Treatment of relapsed/refractory severe aplastic anemia in children: Evidence-based recommendations.

Severe aplastic anemia (SAA) is a rare potentially fatal hematologic disorder. Although overall outcomes with treatment are excellent, there are variations in management approach, including differences in treatment between adult and pediatric patients. Certain aspects of treatment are under active investigation in clinical trials. Because of the rarity of the disease, some pediatric hematologists may have relatively limited experience with the complex management of SAA. The following recommendations reflect an up-to-date evidence-based approach to the treatment of children with relapsed or refractory SAA.

Primary targeted medical therapy for management of bilateral head and neck lymphatic malformations in infants.

OBJECTIVES: Patients born with bilateral head and neck lymphatic malformations (BHNLMs) often require multiple invasive treatments, including tracheostomy. We hypothesized that primary targeted medical therapy (pTMT) with diagnostic needle aspiration reduces the need for invasive therapy such as surgical resection and/or sclerotherapy. METHODS: Retrospective case review was performed of infants with BHNLMs (Grade 2 or De Serres stage IV and V) treated only at our institution from 2000 to 2021. Patients were divided into two cohorts: those managed with pTMT and those managed with observation, sclerotherapy, or surgical intervention (non-pTMT). Data regarding interventions, clinical outcomes, morbidity, and mortality were analyzed with descriptive statistics. RESULTS: Nine children with BHNLMs met inclusion criteria. Three (33%) were in the pTMT cohort and six (66%) were non-pTMT. Eight (89%) malformations were genotyped, and all demonstrated hotspot PIK3CA variants. All pTMT patients had sirolimus initiated in the first month of life and underwent needle aspiration of malformation cyst fluid for cell-free DNA samples. All pTMT patients tolerated medical therapy. For the non-pTMT cohort, primary treatment included none (deceased, n = 1, 17%), observation with needle aspiration (n = 1, 17%), surgical resection (n = 2, 33%), or combination surgery and sclerotherapy (n = 2, 33%). Intubation duration, intensive care and initial hospital length of stay were not different between cohorts. Four non-pTMT patients (67%) required tracheostomy, and two (33%) died prior to discharge. All pTMT patients survived and none required tracheostomy. Non-pTMT patients required a median of two invasive therapies prior to discharge (IQR 1-4) and a mean total of 13 over the course of their lifetime (IQR 1-16), compared to the pTMT group who did not require any lifetime invasive therapy, even after initial pTMT and discharge home. CONCLUSION: This study compares patients with BHNLMs (Grade 2) treated with pTMT versus those treated with observation or invasive therapy. Patients treated with pTMT required no surgical or invasive procedural treatment of their malformations, no tracheostomy placement, no unplanned readmissions after discharge, and had no mortalities. Needle aspiration was useful as a therapeutic adjunct for cell-free DNA diagnosis of PIK3CA variants, which guided TMT.

Hematologic complications with age in Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure syndrome with leukemia predisposition. An understanding of the hematologic complications of SDS with age could guide clinical management, but data are limited for this rare disease. We conducted a cohort study of 153 subjects from 143 families with confirmed biallelic SBDS mutations enrolled on the North American Shwachman Diamond Registry or Bone Marrow Failure Registry. The SBDS c.258 + 2T>C variant was present in all but 1 patient. To evaluate the association between blood counts and age, 2146 blood counts were analyzed for 119 subjects. Absolute neutrophil counts were positively associated with age (P < .0001). Hemoglobin was also positively associated with age up to 18 years (P < .0001), but the association was negative thereafter (P = .0079). Platelet counts and marrow cellularity were negatively associated with age (P < .0001). Marrow cellularity did not correlate with blood counts. Severe marrow failure necessitating transplant developed in 8 subjects at a median age of 1.7 years (range, 0.4-39.5), with 7 of 8 requiring transplant prior to age 8 years. Twenty-six subjects (17%) developed a myeloid malignancy (16 myelodysplasia and 10 acute myeloid leukemia) at a median age of 12.3 years (range, 0.5-45.0) and 28.4 years (range, 14.4-47.3), respectively. A lymphoid malignancy developed in 1 patient at the age of 16.9 years. Hematologic complications were the major cause of mortality (17/20 deaths; 85%). These data inform surveillance of hematologic complications in SDS.

Diagnostic work-up for severe aplastic anemia in children: Consensus of the North American Pediatric Aplastic Anemia Consortium.

The North American Pediatric Aplastic Anemia Consortium (NAPAAC) is a group of pediatric hematologist-oncologists, hematopathologists, and bone marrow transplant physicians from 46 institutions in North America with interest and expertise in aplastic anemia, inherited bone marrow failure syndromes, and myelodysplastic syndromes. The NAPAAC Bone Marrow Failure Diagnosis and Care Guidelines Working Group was established with the charge of harmonizing the approach to the diagnostic workup of aplastic anemia in an effort to standardize best practices in the field. This document outlines the rationale for initial evaluations in pediatric patients presenting with signs and symptoms concerning for severe aplastic anemia.

Infantile Myelofibrosis and Myeloproliferation with CDC42 Dysfunction.

Studies of genetic blood disorders have advanced our understanding of the intrinsic regulation of hematopoiesis. However, such genetic studies have only yielded limited insights into how interactions between hematopoietic cells and their microenvironment are regulated. Here, we describe two affected siblings with infantile myelofibrosis and myeloproliferation that share a common de novo mutation in the Rho GTPase CDC42 (Chr1:22417990:C>T, p.R186C) due to paternal germline mosaicism. Functional studies using human cells and flies demonstrate that this CDC42 mutant has altered activity and thereby disrupts interactions between hematopoietic progenitors and key tissue microenvironmental factors. These findings suggest that further investigation of this and other related disorders may provide insights into how hematopoietic cell-microenvironment interactions play a role in human health and can be disrupted in disease. In addition, we suggest that deregulation of CDC42 may underlie more common blood disorders, such as primary myelofibrosis.

HLA-Haploidentical Hematopoietic Cell Transplantation for Treatment of Nonmalignant Diseases Using Nonmyeloablative Conditioning and Post-Transplant Cyclophosphamide.

Allogeneic hematopoietic cell transplant (HCT) is often the only curative therapy for patients with nonmalignant diseases; however, many patients do not have an HLA-matched donor. Historically, poor survival has been seen after HLA-haploidentical HCT because of poor immune reconstitution, increased infections, graft-versus-host disease (GVHD), and graft failure. Encouraging results have been reported using a nonmyeloablative T cell-replete HLA-haploidentical transplant approach in patients with hematologic malignancies. Here we report the outcomes of 23 patients with various nonmalignant diseases using a similar approach. Patients received HLA-haploidentical bone marrow (n = 17) or granulocyte colony-stimulating factor-mobilized peripheral blood stem cell (n = 6) grafts after conditioning with cyclophosphamide 50 mg/kg, fludarabine 150 mg/m2, and 2 or 4 Gy total body irradiation. Postgrafting immunosuppression consisted of cyclophosphamide, mycophenolate mofetil, tacrolimus, ± sirolimus. Median patient age at HCT was 10.8 years. Day 100 transplant-related mortality (TRM) was 0%. Two patients died at later time points, 1 from intracranial hemorrhage/disseminated fungal infection in the setting of graft failure and 1 from infection/GVHD. The estimated probabilities of grades II to IV and III to IV acute GVHD at day 100 and 2-year National Institutes of Health consensus chronic GVHD were 78%, 26%, and 42%, respectively. With a median follow-up of 2.5 years, the 2-year overall and event-free rates of survival were 91% and 78%, respectively. These results are encouraging and demonstrate favorable disease-specific lineage engraftment with low TRM in patients with nonmalignant diseases using nonmyeloablative conditioning followed by T cell-replete HLA-haploidentical grafts. However, additional strategies are needed for GVHD prevention to make this a viable treatment approach for patients with nonmalignant diseases.

Immunosuppressive therapy for pediatric aplastic anemia: a North American Pediatric Aplastic Anemia Consortium study.

Quality of response to immunosuppressive therapy and long-term outcomes for pediatric severe aplastic anemia remain incompletely characterized. Contemporary evidence to inform treatment of relapsed or refractory severe aplastic anemia for pediatric patients is also limited. The clinical features and outcomes for 314 children treated from 2002 to 2014 with immunosuppressive therapy for acquired severe aplastic anemia were analyzed retrospectively from 25 institutions in the North American Pediatric Aplastic Anemia Consortium. The majority of subjects (n=264) received horse anti-thymocyte globulin (hATG) plus cyclosporine (CyA) with a median 61 months follow up. Following hATG/CyA, 71.2% (95%CI: 65.3,76.6) achieved an objective response. In contrast to adult studies, the quality of response achieved in pediatric patients was high, with 59.8% (95%CI: 53.7,65.8) complete response and 68.2% (95%CI: 62.2,73.8) achieving at least a very good partial response with a platelet count ≥50×109L. At five years post-hATG/CyA, overall survival was 93% (95%CI: 89,96), but event-free survival without subsequent treatment was only 64% (95%CI: 57,69) without a plateau. Twelve of 171 evaluable patients (7%) acquired clonal abnormalities after diagnosis after a median 25.2 months (range: 4.3-71 months) post treatment. Myelodysplastic syndrome or leukemia developed in 6 of 314 (1.9%). For relapsed/refractory disease, treatment with a hematopoietic stem cell transplant had a superior event-free survival compared to second immunosuppressive therapy treatment in a multivariate analysis (HR=0.19, 95%CI: 0.08,0.47; P=0.0003). This study highlights the need for improved therapies to achieve sustained high-quality remission for children with severe aplastic anemia.

Bone Marrow Morphology Associated With Germline RUNX1 Mutations in Patients With Familial Platelet Disorder With Associated Myeloid Malignancy.

Germline mutations in RUNX1 result in autosomal dominant familial platelet disorder with associated myeloid malignancy (FPDMM). To characterize the hematopathologic features associated with a germline RUNX1 mutation, we reviewed a total of 42 bone marrow aspirates from 14 FPDMM patients, including 24 cases with no cytogenetic clonal abnormalities, and 18 with clonal karyotypes or leukemia. We found that all aspirate smears had ≥10% atypical megakaryocytes, predominantly characterized by small forms with hypolobated and eccentric nuclei, and forms with high nuclear-to-cytoplasmic ratios. Core biopsies showed variable cellularity and variable numbers of megakaryocytes with similar features to those in the aspirates. Granulocytic and/or erythroid dysplasia (≥10% cells per lineage) were present infrequently. Megakaryocytes with separate nuclear lobes were increased in patients with myelodysplastic syndrome (MDS) and acute leukemia. Comparison to an immune thrombocytopenic purpura cohort confirms increased megakaryocytes with hypolobated eccentric nuclei in FPDMM patients. As such, patients with FPDMM often have atypical megakaryocytes with small hypolobated and eccentric nuclei even in the absence of clonal cytogenetic abnormalities; these findings are related to the underlying RUNX1 germline mutation and not diagnostic of MDS. Isolated megakaryocytic dysplasia in patients with unexplained thrombocytopenia should raise the possibility of an underlying germline RUNX1 mutation.

Medical Management of Vascular Anomalies.

PURPOSE OF REVIEW: This chapter will summarize the most recent literature regarding the current state of medical treatment for vascular anomalies. RECENT FINDINGS: Research into the biology of these anomalies has strengthened our understanding of each anomaly and has helped to pave the way for more tailored treatment options involving molecular and/or genetic targets. SUMMARY: While there is still a role for surgical intervention, medical therapies that target the etiology of vascular anomalies may represent an alternative or adjunctive approach in the management of these lesions.

Evaluation of the Utility of Bone Marrow Morphology and Ancillary Studies in Pediatric Patients Under Surveillance for Myelodysplastic Syndrome.

OBJECTIVES: To evaluate the utility of flow cytometry, karyotype, and a fluorescence in situ hybridization (FISH) panel in screening children for myelodysplastic syndrome (MDS). METHODS: Bone marrow morphology, flow cytometry, karyotype, and FISH reports from 595 bone marrow specimens (246 patients) were analyzed. RESULTS: By morphology, 8.7% of cases demonstrated at least unilineage dysplasia and/or increased blasts. Flow cytometry identified definitive abnormalities in 2.8% of cases, all of which had abnormal morphology. Of the 42 cases (7.2%) with acquired karyotypic abnormalities, 26 had no morphologic dysplasia. With a 98.2% concordance between karyotype and MDS FISH, FISH only identified two additional cases, both with low-level (<4%) abnormalities. Peripheral blood count evaluation only identified the absence of thrombocytopenia to correlate with an absence of abnormal ancillary tests. CONCLUSIONS: The combination of morphologic evaluation and karyotype with judicious use of flow cytometry and MDS FISH is sufficient to detect abnormalities for these indications.

Allogeneic Hematopoietic Cell Transplantation Using Treosulfan-Based Conditioning for Treatment of Marrow Failure Disorders.

Hematopoietic cell transplantation (HCT) is effective in the treatment of inherited marrow failure disorders and other nonmalignant diseases. Conventional myeloablative conditioning regimens have been associated with high transplant-related mortality, particularly in patients with comorbid conditions. Here we report on 14 patients with marrow failure disorders (Shwachman-Diamond syndrome, n = 3; Diamond Blackfan anemia, n = 4; GATA2 deficiency, n = 2; paroxysmal nocturnal hemoglobinuria, n = 4; and an undefined marrow failure disorder, n = 1) who underwent HCT on a prospective, phase II, multicenter clinical trial. Patients were given HLA-matched related (n = 2) or unrelated (n = 12) grafts after conditioning with treosulfan (42 g/m2), fludarabine (150 mg/m2), ± thymoglobulin (n = 11; 6 mg/kg). All patients engrafted. At a median follow-up of 3 years, 13 patients are alive with complete correction of their underlying disease. These results indicate that the combination of treosulfan, fludarabine, and thymoglobulin is effective at establishing donor engraftment with a low toxicity profile and excellent disease-free survival in patients with marrow failure disorders.

Congenital amegakaryocytic thrombocytopenia.

Congenital amegakaryocytic thrombocytopenia (CAMT) is clinically characterized by thrombocytopenia presenting at birth in a child without congenital or skeletal malformations, reduced or absent bone marrow megakaryocytes, and eventual progression to bone marrow failure. Molecular studies in most cases confirm homozygous or compound heterozygous mutations in the thrombopoietin receptor c-Mpl. In addition to the clinical importance of recognizing this disorder, characterization of mutations identified in patients with CAMT has led to insights into thrombopoietin receptor structure and function. This review will summarize the diagnosis, pathophysiology, and management of CAMT.

Megakaryopoiesis.

The process of megakaryopoiesis and platelet production is complex, with the potential for regulation at multiple stages. Megakaryocytes are derived from the hematopoietic stem cell through successive lineage commitment steps, and they undergo a unique maturation process that includes polyploidization, development of an extensive internal demarcation membrane system, and finally formation of pro-platelet processes. Platelets are shed from these processes into vascular sinusoids within the bone marrow. Megakaryocyte differentiation is regulated both positively and negatively by transcription factors and cytokine signaling. Thrombopoietin (TPO) is the most important hematopoietic cytokine for platelet production. Clinically, acquired and inherited mutations affecting megakaryocytic transcription factors and thrombopoietin signaling have been identified in disorders of thrombocytopenia and thrombocytosis.

F104S c-Mpl responds to a transmembrane domain-binding thrombopoietin receptor agonist: proof of concept that selected receptor mutations in congenital amegakaryocytic thrombocytopenia can be stimulated with alternative thrombopoietic agents.

OBJECTIVE: To determine whether specific c-Mpl mutations might respond to thrombopoietin receptor agonists. MATERIALS AND METHODS: We created cell line models of type II c-Mpl mutations identified in congenital amegakaryocytic thrombocytopenia. We selected F104S c-Mpl for further study because it exhibited surface expression of the receptor. We measured proliferation of cell lines expressing wild-type or F104S c-Mpl in response to thrombopoietin receptor agonists targeting the extracellular (m-AMP4) or transmembrane (LGD-4665) domains of the receptor by 1-methyltetrazole-5-thiol assay. We measured thrombopoietin binding to the mutant receptor using an in vitro thrombopoietin uptake assay and identified F104 as a potentially critical residue for the interaction between the receptor and its ligand by aligning thrombopoietin and erythropoietin receptors from multiple species. RESULTS: Cells expressing F104S c-Mpl proliferated in response to LGD-4665, but not thrombopoietin or m-AMP4. Compared to thrombopoietin, LGD-4665 stimulates signaling with delayed kinetics in both wild-type and F104S c-Mpl-expressing cells. Although F104S c-Mpl is expressed on the cell surface in our BaF3 cell line model, the mutant receptor does not bind thrombopoietin. Comparison to the erythropoietin receptor suggests that F104 engages in hydrogen-bonding interactions that are critical for binding to thrombopoietin. CONCLUSIONS: These findings suggest that a small subset of patients with congenital amegakaryocytic thrombocytopenia might respond to treatment with thrombopoietin receptor agonists, but that responsiveness will depend on the type of mutation and agonist used. We postulate that F104 is critical for thrombopoietin binding. The kinetics of signaling in response to a transmembrane domain-binding agonist are delayed in comparison to thrombopoietin.

Ubiquitination and degradation of the thrombopoietin receptor c-Mpl.

Regulation of growth factor and cytokine signaling is essential for maintaining physiologic numbers of circulating hematopoietic cells. Thrombopoietin (Tpo), acting through its receptor c-Mpl, is required for hematopoietic stem cell maintenance and megakaryopoiesis. Therefore, the negative regulation of Tpo signaling is critical in many aspects of hematopoiesis. In this study, we determine the mechanisms of c-Mpl degradation in the negative regulation of Tpo signaling. We found that, after Tpo stimulation, c-Mpl is degraded by both the lysosomal and proteasomal pathways and c-Mpl is rapidly ubiquitinated. Using site-directed mutagenesis, we were able to determine that c-Mpl is ubiquitinated on both of its intracellular lysine (K) residues (K(553) and K(573)). By mutating these residues to arginine, ubiquitination and degradation were significantly reduced and caused hyperproliferation in cell lines expressing these mutated receptors. Using short interfering RNA and dominant negative overexpression, we also found that c-Cbl, which is activated by Tpo, acts as an E3 ubiquitin ligase in the ubiquitination of c-Mpl. Our findings identify a previously unknown negative regulatory pathway for Tpo signaling that may significantly impact our understanding of the mechanisms affecting the growth and differentiation of hematopoietic stem cells and megakaryocytes.