Early B-cell development and B-cell maturation are impaired in patients with active hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by hyperinflammation and multiorgan dysfunction. Infections, including the reactivation of viruses, contribute to significant disease mortality in HLH. Although T-cell and natural killer cell-driven immune activation and dysregulation are well described, limited data exist on the status of B-cell compartment and humoral immune function in HLH. We noted marked suppression of early B-cell development in patients with active HLH. In vitro B-cell differentiation studies after exposure to HLH-defining cytokines, such as interferon gamma (IFN-γ) and tumor necrosis factor, recapitulated B-cell development arrest. Messenger RNA sequencing of human CD34+ cells exposed to IFN-γ demonstrated changes in genes and pathways affecting B-cell development and maturation. In addition, patients with active HLH exhibited a marked decrease in class-switched memory B (CSMB) cells and a decrease in bone marrow plasmablast/plasma cell compartments. The decrease in CSMB cells was associated with a decrease in circulating T follicular helper (cTfh) cells. Finally, lymph node and spleen evaluation in a patient with HLH revealed absent germinal center formation and hemophagocytosis with associated lymphopenia. Reassuringly, the frequency of CSMB and cTfh improved with the control of T-cell activation. Taken together, in patients with active HLH, these changes in B cells may affect the humoral immune response; however, further immune studies are needed to determine its clinical significance.

mTOR inhibition attenuates cTfh cell dysregulation and chronic T-cell activation in multilineage immune cytopenias.

mTOR inhibitors such as sirolimus are increasingly used in the management of multilineage immune cytopenia (m-IC) in children. Although sirolimus is effective in improving IC, it is unclear how sirolimus affects the broader immune dysregulation associated with m-IC. We profiled T- and B-cell subsets longitudinally and measured cytokines and chemokines before and after sirolimus treatment. Eleven of the 12 patients with m-IC who tolerated sirolimus were followed for a median duration of 17 months. All patients had an improvement in IC, and sirolimus therapy did not result in significant decreases in T-, B- and NK-cell numbers. However, the expansion and activation of circulating T follicular helper and the Th1 bias noted before the initiation of sirolimus were significantly decreased. Features of chronic T-cell activation and exhaustion within effector memory compartments of CD4+ and CD8+ T cells decreased with sirolimus therapy. Corresponding to these changes, plasma levels of CXCL9 and CXCL10 also decreased. Interestingly, no significant improvement in the proportion of class-switched memory B cells or frequencies of CD4+ naive T cells were noted. Longer follow-up and additional studies are needed to validate these findings and evaluate the effect of sirolimus on B-cell maturation.

Frequency of HLA-DR+CD38hi T cells identifies and quantifies T-cell activation in hemophagocytic lymphohistiocytosis, hyperinflammation, and immune regulatory disorders.

BACKGROUND: Quantifying T-cell activation is essential for the diagnosis and evaluation of treatment response in various hyperinflammatory and immune regulatory disorders, including hemophagocytic lymphohistiocytosis. Plasma soluble IL-2 receptor (sIL-2R) is a well-established biomarker for evaluating systemic T-cell activation. However, the limited availability of sIL-2R testing could result in delayed diagnosis. Furthermore, high sIL-2R levels may not always reflect T-cell activation. OBJECTIVES: To address these limitations, this study investigated whether cell surface markers of T-cell activation, HLA-DR, and CD38, as assessed by flow cytometry, could be used to quantify systemic T-cell activation in a variety of inflammatory disease states and examine its correlation with sIL-2R levels. METHODS: Results for sIL-2R, CXCL9, and ferritin assays were obtained from patient's medical records. Frequency of HLA-DR+CD38high(hi) T-cells was assessed in different T-cell subsets using flow cytometry. RESULTS: In this study's cohort, activation in total CD8+ T (r = 0.65; P < .0001) and CD4+ (r = 0.42; P < .0001) T-cell subsets significantly correlated with plasma sIL-2R levels. At the disease onset, the frequency of HLA-DR+CD38hi T cells in CD8+ T (r = 0.65, P < .0001) and CD4+ T (r = 0.77; P < .0001) effector memory (TEM) compartments correlated strongly with sIL-2R levels. Evaluation of T-cell activation markers in follow-up samples also revealed a positive correlation for both CD4+ TEM and CD8+ TEM activation with sIL-2R levels; thus, attesting its utility in initial diagnosis and in evaluating treatment response. The frequency of HLA-DR+CD38hi T-cells in the CD8+ TEM compartment also correlated with plasma CXCL9 (r = 0.42; P = .0120) and ferritin levels (r = 0.32; P = .0037). CONCLUSIONS: This study demonstrates that flow cytometry-based direct T-cell activation assessed by HLA-DR+CD38hi T cells accurately quantifies T-cell activation and strongly correlates with sIL-2R levels across a spectrum of hyperinflammatory and immune dysregulation disorders.

T-follicular helper cell expansion and chronic T-cell activation are characteristic immune anomalies in Evans syndrome.

Pediatric Evans syndrome (pES) is increasingly identified as the presenting manifestation of several inborn errors of immunity. Despite an improved understanding of genetic defects in pES, the underlying immunobiology of pES is poorly defined, and characteristic diagnostic immune parameters are lacking. We describe the immune characteristics of 24 patients with pES and compared them with 22 patients with chronic immune thrombocytopenia (cITP) and 24 healthy controls (HCs). Compared with patients with cITP and HC, patients with pES had increased circulating T-follicular helper cells (cTfh), increased T-cell activation, and decreased naïve CD4+ T cells for age. Despite normal or high immunoglobulin G (IgG) in most pES at presentation, class-switched memory B cells were decreased. Within the cTfh subset, we noted features of postactivation exhaustion with upregulation of several canonical checkpoint inhibitors. T-cell receptor ß chain (TCR-ß) repertoire analysis of cTfh cells revealed increased oligoclonality in patients with pES compared with HCs. Among patients with pES, those without a known gene defect had a similar characteristic immune abnormality as patients with defined genetic defects. Similarly, patients with pES with normal IgG had similar T-cell abnormalities as patients with low IgG. Because genetic defects have been identified in less than half of patients with pES, our findings of similar immune abnormalities across all patients with pES help establish a common characteristic immunopathology in pES, irrespective of the underlying genetic etiology.

Baseline Quality of Life in Pediatric Patients With Low-flow Vascular Malformations.

OBJECTIVE: The aim of this study was to quantify and describe baseline patient and parent-proxy health-related quality of life scores in patients with low-flow vascular malformations at a single, tertiary-care vascular anomalies clinic. STUDY DESIGN: This is a retrospective study of data collected on patients with low-flow vascular malformations between the ages of 2 to 25 who were seen at a single, tertiary-care center vascular anomalies clinic. A total of 266 patients are included in this study. RESULTS: Patients with lymphatic malformations report decreased quality of life scores as compared with venous malformations in the emotional, psychological, school, and social domains. Patients with lower extremity malformation report decreased quality of life scores as compared with head/neck, trunk, upper extremity, and multifocal malformations; most notably in the physical domain. CONCLUSIONS: Treatment of low-flow vascular malformations should aim to improve patient quality of life. The use of standardized health-related quality of life measures in this study quantifies baseline quality of life scores among patients with low-flow vascular malformations.

Distinguishing immune activation and inflammatory signatures of multisystem inflammatory syndrome in children (MIS-C) versus hemophagocytic lymphohistiocytosis (HLH).

BACKGROUND: Multisystem inflammatory syndrome in children (MIS-C) is a potentially life-threatening sequela of severe acute respiratory syndrome coronavirus 2 infection characterized by hyperinflammation and multiorgan dysfunction. Although hyperinflammation is a prominent manifestation of MIS-C, there is limited understanding of how the inflammatory state of MIS-C differs from that of well-characterized hyperinflammatory syndromes such as hemophagocytic lymphohistiocytosis (HLH). OBJECTIVES: We sought to compare the qualitative and quantitative inflammatory profile differences between patients with MIS-C, coronavirus disease 2019, and HLH. METHODS: Clinical data abstraction from patient charts, T-cell immunophenotyping, and multiplex cytokine and chemokine profiling were performed for patients with MIS-C, patients with coronavirus disease 2019, and patients with HLH. RESULTS: We found that both patients with MIS-C and patients with HLH showed robust T-cell activation, markers of senescence, and exhaustion along with elevated TH1 and proinflammatory cytokines such as IFN-γ, C-X-C motif chemokine ligand 9, and C-X-C motif chemokine ligand 10. In comparison, the amplitude of T-cell activation and the levels of cytokines/chemokines were higher in patients with HLH when compared with patients with MIS-C. Distinguishing inflammatory features of MIS-C included elevation in TH2 inflammatory cytokines such as IL-4 and IL-13 and cytokine mediators of angiogenesis, vascular injury, and tissue repair such as vascular endothelial growth factor A and platelet-derived growth factor. Immune activation and hypercytokinemia in MIS-C resolved at follow-up. In addition, when these immune parameters were correlated with clinical parameters, CD8+ T-cell activation correlated with cardiac dysfunction parameters such as B-type natriuretic peptide and troponin and inversely correlated with platelet count. CONCLUSIONS: Overall, this study characterizes unique and overlapping immunologic features that help to define the hyperinflammation associated with MIS-C versus HLH.

Coagulopathy and related complications following sclerotherapy of congenital venous malformations.

BACKGROUND: Congenital venous malformations (VMs) are low-flow vascular anomalies that can cause coagulation abnormalities. This phenomenon, referred to as localized intravascular coagulopathy (LIC), is characterized by elevated D-dimer, hypofibrinogenemia, and/or thrombocytopenia. Increased risk for LIC includes patients with an extensive VM, multifocal VM, and Klippel-Trenaunay and CLOVES syndromes. Peri-procedural anticoagulation has been given to prevent complications from LIC in patients undergoing surgical/interventional procedures; however, the rate of clinically relevant complications from sclerotherapy is largely unknown. The purpose of this study is to describe a single-institution's incidence of LIC in patients with VMs and coagulopathy-related complications following sclerotherapy. DESIGN/METHODS: Retrospective chart review of patients, 0-21 years of age, with VM who underwent sclerotherapy without peri-procedural anticoagulation and had coagulation profiles evaluated within 1 month prior to sclerotherapy. DATA COLLECTED: diagnosis type (high vs. low risk for LIC), coagulation profile (including PT/PTT, D-dimer, fibrinogen, and platelet count), sclerosant used, and post-procedure outcomes. Coagulopathy-related complications included clinically relevant bleeding, deep vein thrombosis (DVT), and pulmonary embolism (PE). RESULTS: N = 138 patients; 59.4% were female. The most common location of VM was the lower extremity (47.8%; 66/138). Of patients with high-risk VMs (29/138 [21%]), 11/29 (37.9%) had laboratory values consistent with LIC, whereas 5% (5/109) of low-risk VMs had LIC. In sum, 492 sclerotherapy procedures were performed with no complications of bleeding, DVT, or PE. CONCLUSION: Patients undergoing sclerotherapy for VM with abnormal coagulation profiles may not require peri-procedural low molecular weight heparin (LMWH). Further studies are needed to precisely define which patients would benefit from anticoagulation.

How we established a multidisciplinary program for vascular anomalies.

The care of patients with vascular anomalies is quickly becoming a complex field requiring high-quality, coordinated multidisciplinary care. In this article, we review the history of multidisciplinary care in this field, discuss the benefits of this model of care, and outline some of the essential components and structure of a successful vascular anomalies team. We provide an overview of two example programs and a roadmap for other centers to develop their own multidisciplinary vascular anomalies teams.

Clinical features and neurological outcomes in pediatric immune-mediated thrombotic thrombocytopenic purpura: A report from a large pediatric hematology center.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is a potentially life-threatening disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia, and severely reduced or absent ADAMTS13 (A disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13) activity, with varying degrees of organ dysfunction. As TTP is rare in pediatrics, most of the medical and scientific literature has largely reported on adult patients. As a result, limited data exist regarding the clinical features, comorbidities, treatment response, and long-term outcomes in pediatric patients with immune-mediated TTP. METHODS: A single-center retrospective cohort study was conducted of all children and adolescents presenting to Children's Healthcare of Atlanta, Atlanta, Georgia, between the years 2001 and 2021 with immune-mediated TTP (iTTP). Clinical features, treatments, and outcomes, including long-term neurocognitive function, were analyzed. RESULTS: Eighteen individuals were identified, six of whom had a total of 10 relapses, amounting to 28 episodes overall. Thirty-eight percent of the patients experienced exacerbations but, ultimately, 85% achieved a clinical response and clinical remission. Only one in-hospital death occurred (mortality rate 5.5%). Seventy-three percent of analyzed patients demonstrated long-term neurocognitive abnormalities, including cognitive delay, learning difficulties, and severe depression. CONCLUSIONS: Children and adolescents recovering from iTTP are at high risk for neurocognitive deficits from initial and possibly ongoing microvascular disease. Due to risk for long-term neurological deficits, we recommend neuropsychological testing in addition to monitoring of other organ functions in all children with TTP, as well as long-term surveillance of ADAMTS13 activity during remission to detect and promptly treat early relapse.

SLC25A38 congenital sideroblastic anemia: Phenotypes and genotypes of 31 individuals from 24 families, including 11 novel mutations, and a review of the literature.

The congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited disorders of erythropoiesis characterized by pathologic deposits of iron in the mitochondria of developing erythroblasts. Mutations in the mitochondrial glycine carrier SLC25A38 cause the most common recessive form of CSA. Nonetheless, the disease is still rare, there being fewer than 70 reported families. Here we describe the clinical phenotype and genotypes of 31 individuals from 24 families, including 11 novel mutations. We also review the spectrum of reported mutations and genotypes associated with the disease, describe the unique localization of missense mutations in transmembrane domains and account for the presence of several alleles in different populations.

T Cell-Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis (HLH) Occurs in Non-Asians and Is Associated with a T Cell Activation State that Is Comparable to Primary HLH.

PURPOSE: T cell-Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (T cell-EBV-HLH) is prevalent in East Asia and has poor prognosis. Understanding of this disease is limited, and literature regarding prevalence in North America is scarce. Herein, we summarize our experience. METHODS: A retrospective analysis of T cell-EBV-HLH patients admitted to Children's Healthcare of Atlanta (GA, USA) from 2010 to 2020 was conducted. Additional immune studies were completed in a subset of patients. RESULTS: We report 15 patients (10 months-19 years of age) diagnosed with T cell-EBV-HLH. Nine patients were Hispanic, and the majority did not have primary HLH (p-HLH) gene defects. Soluble interleukin-2 receptor levels in T cell-EBV-HLH were significantly higher than other forms of secondary-HLH but comparable to p-HLH, and it correlated with disease severity at presentation. Natural killer cell function was decreased in most patients despite a negative workup for p-HLH. Depending on disease severity, initial therapy included dexamethasone or dexamethasone and etoposide. Refractory patients were managed with blended regimens that included one or more of the following therapies: combination chemotherapy, alemtuzumab, emapalumab, and nivolumab. Rituximab did not appreciably decrease EBV viremia in most patients. Non-critically ill patients responded well to immunosuppressive therapy and are long-term survivors without undergoing allogeneic hematopoietic stem cell transplantation (HSCT). Alemtuzumab resulted in inflammation flare in two of the three patients. Three patients underwent allogeneic HSCT, with disease relapse noted in one. At a median follow-up of 3 years, 10 of the 15 patients are alive. CONCLUSION: T cell-EBV-HLH occurs in the USA among the non-Asian populations, especially in those who are Hispanic.

Impact of respiratory viral panel testing on length of stay in pediatric cancer patients admitted with fever and neutropenia.

BACKGROUND: Polymerase chain reaction (PCR) respiratory viral panel (RVP) testing is often used in evaluation of pediatric cancer patients with febrile neutropenia (FN), but correlation with adverse outcomes has not been well characterized. PROCEDURE: A retrospective cohort of all children ages 0-21 years with cancer admitted to Children's Healthcare of Atlanta for FN from January 2013 to June 2016 was identified. Patient demographic and clinical variables such as age, RVP results, length of stay (LOS), and deaths were abstracted. Relationship between RVP testing and positivity and LOS, highest temperature (Tmax), hypotension and intensive care unit (ICU) admission were compared using Wilcoxon rank sums, chi-square, or Fisher's exact tests adjusting for age, sex, bacteremia, and diagnosis. RESULTS: The 404 patients identified had 787 total FN admissions. RVPs were sent in 38% of admissions and were positive in 59%. Patients with RVPs sent were younger (median 5.5 vs 8.0 years, P < .0001) with higher Tmax (39.2° vs 39.1°, P = .016). The most common virus identified was rhinovirus/Enterovirus (61%). There were no significant differences in highest temperature or lowest blood pressure based on RVP positivity. Patients admitted to the ICU were more likely to have RVPs sent (odds ratio [OR] = 3.19, P < .002); however, neither having RVP testing nor RVP positivity were significantly associated with increased LOS or death. Coinfection with bacteremia and a respiratory virus was identified in 9.1% of patients. CONCLUSIONS: These data raise the question of the utility of sending potentially costly RVP testing as RVP positivity during febrile neutropenia does not impact LOS, degree of hypotension, or ICU admission.

Congenital Limb Overgrowth Syndromes Associated with Vascular Anomalies.

Congenital limb length discrepancy disorders are frequently associated with a variety of vascular anomalies and have unique genetic and phenotypic features. Many of these syndromes have been linked to sporadic somatic mosaicism involving mutations of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway, which has an important role in tissue growth and angiogenesis. Radiologists who are aware of congenital limb length discrepancies can make specific diagnoses based on imaging findings. Although genetic confirmation is necessary for a definitive diagnosis, the radiologist serves as a central figure in the identification and treatment of these disorders. The clinical presentations, diagnostic and imaging workups, and treatment options available for patients with Klippel-Trenaunay syndrome, CLOVES (congenital lipomatous overgrowth, vascular anomalies, epidermal nevi, and scoliosis/spinal deformities) syndrome, fibroadipose vascular anomaly, phosphatase and tensin homolog mutation spectrum, Parkes-Weber syndrome, and Proteus syndrome are reviewed. ©RSNA, 2019.

Volumetric Assessment of Pediatric Vascular Malformations Using a Rapid, Hand-Held Three-Dimensional Imaging System.

The effect of percutaneous, surgical, and medical therapies for vascular malformations (VMs) is often difficult to quantify volumetrically using cross-sectional imaging. Volumetric measurement is often estimated with serial, expensive MRI examinations which may require sedation or anesthesia. We aim to explore whether a portable 3D scanning device is capable of rapid, accurate volumetric analysis of pediatric VMs. Using an iPad-mounted infrared scanning device, 3D scans of patient faces, arms, and legs were acquired over an 8-month study period. Proprietary software was use to perform subsequent volumetric analysis. Of a total of 30 unilateral VMs involving either the face, arms, or legs, 26 (86.7%) VMs were correctly localized by discerning the larger volume of the affected side compared to the normal contralateral side. For patients with unilateral facial VMs (n = 10), volume discrepancy between normal and affected sides differed compared with normal controls (n = 19). This was true for both absolute (60 cc ± 55 vs 15 cc ± 8, p = 0.03) as well as relative (18.1% ± 13.2 vs 4.0% ± 2.1, p = 0.008) volume discrepancy. Following treatment, two patients experienced change in leg volume discrepancy ranging from - 17.3 to - 0.4%. Using a portable 3D scanning device, we were able to rapidly and noninvasively detect and quantify volume discrepancy resulting from VMs of the face, arms, and legs. Preliminary data suggests this technology can detect volume reduction of VMs in response to therapy.

Single-platelet nanomechanics measured by high-throughput cytometry.

Haemostasis occurs at sites of vascular injury, where flowing blood forms a clot, a dynamic and heterogeneous fibrin-based biomaterial. Paramount in the clot's capability to stem haemorrhage are its changing mechanical properties, the major drivers of which are the contractile forces exerted by platelets against the fibrin scaffold. However, how platelets transduce microenvironmental cues to mediate contraction and alter clot mechanics is unknown. This is clinically relevant, as overly softened and stiffened clots are associated with bleeding and thrombotic disorders. Here, we report a high-throughput hydrogel-based platelet-contraction cytometer that quantifies single-platelet contraction forces in different clot microenvironments. We also show that platelets, via the Rho/ROCK pathway, synergistically couple mechanical and biochemical inputs to mediate contraction. Moreover, highly contractile platelet subpopulations present in healthy controls are conspicuously absent in a subset of patients with undiagnosed bleeding disorders, and therefore may function as a clinical diagnostic biophysical biomarker.

Sirolimus-induced pulmonary hemorrhage of an infant with massive cervicofacial lymphatic malformation: A case report.

Lymphatic malformations (LMs) are uncommon congenital abnormalities of the lymphatic system. As more than half of these lesions develop in the head and neck, LMs can be life-threatening if associated with airway involvement. LMs necessitate a multidisciplinary treatment approach, frequently including surgery and sclerotherapy. We present a case report of a 32-week pre-term male infant with a massive cervicofacial LM necessitating delivery via ex-utero intrapartum treatment (EXIT). The patient was treated with numerous rounds of sclerotherapy, systemic sirolimus, and surgical debulking, but ultimately died at 4 months of age due to acute pulmonary hemorrhage, which may have been related to sirolimus due to the absence of any other associable organ involvement or derangement. We document the patient's clinical course and treatment regimen, highlighting the myriad modalities employed to treat these challenging lesions, and describe a potentially lethal complication of sirolimus therapy not previously described in the treatment of pediatric LM.

Two mutations in the KINDLIN3 gene of a new leukocyte adhesion deficiency III patient reveal distinct effects on leukocyte function in vitro.

In the disorder leukocyte adhesion deficiency III (LAD-III), integrins on platelets and leukocytes are expressed but fail to function and this leads to severe bleeding and infections at an early age. Mutation in the KINDLIN3 (FERMT3) gene is the cause of LAD-III in patients from the Middle East, Malta, and Turkey. We describe 2 novel homozygous mutations in the KINDLIN3 gene of a new African-American patient that destabilize KINDLIN3 mRNA leading to loss of kindlin-3 protein. Transfection of wild-type (WT) KINDLIN3 cDNA restored integrin-related adhesion and migration in the LAD-III patient's T and B lymphocytes. We analyzed the individual mutations separately in vitro to learn more about the function of the kindlin-3 protein. The first G>A mutation gives rise to a Gly308Arg change at the end of FERM (protein 4.1, ezrin, radixin, moesin) subdomain 2, and the second mutation is a base deletion causing early termination within the pleckstrin homology (PH) domain. This second mutation prevented membrane association of kindlin-3 and did not restore either adhesion or migration, whereas the FERM subdomain 2 mutation affected only migration. Thus, these LAD-III patient mutations have highlighted functionally important regions of kindlin-3 that alter leukocyte integrin-dependent function in 2 distinct ways.

Neonatal Vascular Tumors.

Vascular tumors are a part of the vascular anomalies spectrum. Vascular malformations are congenital vascular lesions, originating from a mesenchymal stem cell defect and distinguished from vascular tumors by their low cell turnover and lack of invasiveness. They tend to grow in proportion to the child. Vascular tumors are proliferative and range from benign proliferation to malignant tumors. The appropriate differential diagnosis is imperative. Infantile hemangioma can be diagnosed clinically and rarely requires therapy; more rare tumors are difficult to diagnose and treat. This review provides an overview of vascular tumors seen in the neonatal period and summarizes treatment options.