Mutations in growth factor independent-1 associated with human neutropenia block murine granulopoiesis through colony stimulating factor-1.

Severe congenital neutropenia (SCN) is characterized by a deficiency of mature neutrophils, leading to recurrent bacterial and fungal infections. Although mutations in Elastase-2, neutrophil (ELA2) predominate in human SCN, mutation of Ela2 in mice does not recapitulate SCN. The growth factor independent-1 (GFI1) transcription factor regulates ELA2. Mutations in GFI1 are associated with human SCN, and genetic deletion of Gfi1 results in murine neutropenia. We examined whether human SCN-associated GFI1N382S mutant proteins are causal in SCN and found that GFI1 functions as a rate-limiting granulopoietic molecular switch. The N382S mutation inhibited GFI1 DNA binding and resulted in a dominant-negative block to murine granulopoiesis. Moreover, Gfi1N382S selectively derepressed the monopoietic cytokine CSF1 and its receptor. Gfi1N382S-expressing Csf1-/- cells formed neutrophils. These results reveal a common transcriptional program that underlies both human and murine myelopoiesis, and that is central to the pathogenesis of SCN associated with mutations in GFI1. This shared transcriptional pathway may provide new avenues for understanding SCN caused by mutations in other genes and for clinical intervention into human neutropenias.

Long-term Erythrocytapheresis Is Associated With Reduced Liver Iron Concentration in Sickle Cell Disease.

BACKGROUND: Erythrocytapheresis procedures are increasingly used in sickle cell disease. Serum ferritin and noninvasive magnetic resonance imaging measurements of liver iron concentration (LIC) are frequently used to monitor iron overload secondary to hypertransfusion. There is a paucity of data describing the impact of long-term erythrocytapheresis (LTE) on LIC. MATERIALS AND METHODS: We measured magnetic resonance imaging liver and cardiac iron on LTE subjects and stratified them into 2 groups: higher LIC (>3 mg/g) and lower LIC (<3 mg/g). χ(2) and t test were used to test for differences between the 2 groups. Logistic regression and generalized linear mixed-effects models were used to test what impacted LIC. RESULTS: None of 29 sickle cell disease subjects maintained on LTE had high cardiac iron concentration. LIC was associated with serum ferritin (r=0.697, P<0.001) but was not associated with the total number of LTE procedures (r=-0.088, P=0.656) or total number of simple transfusions (r=0.316, P=0.108). The total number of LTE procedures was not associated with serum ferritin (r=0.040, P=0.838), the total number of simple transfusions (r=-0.258, P=0.184), or LIC group (r=-0.111, P=0.566). CONCLUSION: There was no significant correlation between duration of LTE maintenance and LIC.

The evaluation of a new apheresis device for automated red blood cell exchange procedures in patients with sickle cell disease.

BACKGROUND: The Spectra Optia apheresis system (SO), a blood component separator, can be used to perform red blood cell exchange (RBCX) procedures for the transfusion management of sickle cell disease (SCD) in adults and children. This study was designed to evaluate the performance of the SO RBCX protocols (exchange and depletion/exchange) in patients with SCD. STUDY DESIGN AND METHODS: Patients with SCD and a need for an RBCX procedure as part of a chronic program or as a single procedure were enrolled in this multicenter, single-arm, open-label study. The primary goal of the study was to confirm that the predicted percentage of the patient's original RBCs remaining at the end of the procedure (FCRp) reflects the actual cell fraction remaining, as measured by %HbS (FCRa). Secondary endpoints included ability of the SO to achieve the desired final hematocrit (Hct) and device-related serious adverse events (SAEs). RESULTS: Seventy-two patients 12 years of age or older were enrolled in the study; 60 were evaluable. The ratio of FCRa to FCRp after the RBCX procedure was 0.90, well within the prespecified range of 0.75 to 1.25. The SO was able to achieve the desired final Hct in the evaluable population. The safety profile was favorable, and no patients had an SAE or unexpected adverse device effect or withdrew from the procedure or treatment due to an adverse event. CONCLUSION: The SO performed effectively and safely for both the RBCX procedure and the RBCX depletion/exchange procedure.

Impact of long-term erythrocytapheresis on growth and peak height velocity of children with sickle cell disease.

BACKGROUND: Children with sickle cell disease (SCD) lag in weight and height and have a delayed growth spurt compared to normal children. We studied the effect of long-term erythrocytapheresis (LTE) on the growth of children with SCD and the age at which they attained peak height velocity. PROCEDURE: A retrospective chart review was performed recording weight, height, and body mass index (BMI) measurements of 36 patients with SCD who received LTE every 3-5 weeks for an average duration of 5 years. The z-scores for weight, height, and BMI of these patients were compared with that of patients with SCD from the Cooperative Study of Sickle Cell Disease (CSSCD) and a sub-set of 64 controls matched for age, sex, and initial growth parameter z-scores at the start of LTE. RESULTS: The z-scores for all parameters improved significantly for our patients on LTE compared to match controls from CSSCD and the entire pediatric CSSCD cohort (P-value: <0.01). Peak height velocity was achieved 2 months earlier for females (P-value: 0.94) and 11 months earlier for males (P-value: 0.02), who started LTE before 14 years of age, compared to matched CSSCD controls. The study subjects who had not been on regular simple transfusions prior to starting LTE had a mean serum ferritin of 681 ng/ml after LTE for an average duration of 63 months. CONCLUSION: LTE improves the growth of children with SCD without the risk of iron overload.

Intensive induction chemotherapy followed by myeloablative chemotherapy with autologous hematopoietic progenitor cell rescue for young children newly-diagnosed with central nervous system atypical teratoid/rhabdoid tumors: the Head Start III experience.

BACKGROUND: Atypical teratoid/rhabdoid tumor (AT/RT) of the central nervous system (CNS) is a rare embryonal neoplasm of early childhood with dismal outcome and no current uniformly accepted treatment. Given its highly aggressive nature and predilection for dissemination at diagnosis, intensive multimodal therapy is required. MATERIALS AND METHODS: Nineteen children with newly diagnosed CNS AT/RT were treated on the head start (HS) III protocol. Treatment consisted of surgical resection, 5 cycles of induction chemotherapy, followed by consolidation with myeloablative chemotherapy and autologous hematopoietic progenitor cell rescue (AuHCR). Irradiation was given following recovery from consolidation based on patient age, disease extent at diagnosis, and treatment response to induction. RESULTS: Nineteen children (median age of 14 months) were treated on HS III between 2003 and 2009. Only four finished induction and three proceeded to consolidation. There are presently four survivors at 40, 42, 46, and 79 months from study enrollment. Eleven patients experienced tumor progression at a median time to progression of 4.1 months of whom 10 died with a median time from progression to death of 2.6 months. Five toxic deaths occurred, three of them while on the study. The 3-year event-free survival (EFS) and overall survival (OS) for the whole group was 21 ± 9% and 26 ± 10%, respectively. Five patients received irradiation at progression with only one long-term survivor. CONCLUSION: A minority of children with CNS AT/RT treated on HS III may be long-term survivors without irradiation. More effective therapies are desperately needed.

Absolute lymphocyte counts as prognostic indicators for immune thrombocytopenia outcomes in children.

BACKGROUND: Recent studies reviewing immune mechanisms of immune thrombocytopenia (ITP) have suggested acute and chronic forms may represent distinct immunopathological disorders. This study evaluated absolute lymphocyte counts (ALCs) as predictors for ITP outcomes. PROCEDURE: CBCs with differential counts were ascertained at presentation, 3, 6, and 12 months for 204 patients. Receiver operating characteristic (ROC) curves were used to determine cutoff values. Logistic regression models and recursive partitioning were used to evaluate which variables were significantly associated with outcomes. RESULTS: ALC values at presentation were not independently predictive of disease duration. However, ALC values at 3 months were significant predictors. Sixty-eight percent (40/59) of patients >8 years of age and 43% (20/46) of patients ≤ 8 years who had an ALC ≤ 3,000/µl at 3 months developed chronic ITP. This compares to chronic rates of only 25% (3/12) and 2% (2/87) of patients >8 and ≤ 8 years, respectively, with an ALC > 3,000/µl at 3 months. Further, 92% (60/65) of patients who developed chronic ITP had a 3-month ALC ≤ 3,000/µl. An ALC > 3,000/µl at 3 months is a strong predictor for platelet recovery as only 5% (5/99) of these patients developed chronic ITP. CONCLUSION: This study suggests progression to lower lymphocyte counts over the first few months of disease is a strong predictor for chronic ITP, allowing for risk stratification of patients, particularly when used in conjunction with other known predictors. Further research is needed to confirm these findings and to fully investigate the pathophysiological mechanisms responsible for this association.

Cerebral toxoplasmosis after tandem high-dose chemotherapy and autologous hematopoietic cell transplant for neuroblastoma.

Toxoplasmosis is a well-recognized life-threatening complication of hematopoietic cell transplantation (HCT). This report describes a pediatric patient with stage 4 neuroblastoma who developed cerebral toxoplasmosis after tandem high-dose chemotherapy with autologous HCT. Toxoplasmosis is rare in patients undergoing autologous HCT; however, tandem autologous HCT is more immunosuppressive than a single autologous HCT. Toxoplasmosis is a potential complication in autologous as well as allogeneic transplants, and should be considered in any post-HCT patient with neurological dysfunction. Rapid diagnosis and immediate antimicrobial treatment are crucial to avoid morbidity and mortality. Evaluation of toxoplasma serology should be standard in all patients undergoing tandem autologous HCT and seropositive patients should be started on appropriate prophylactic therapy.

Recurrent neutropenic enterocolitis in a pediatric patient.

Management of an 8-year-old boy with Hodgkin lymphoma is presented. The patient had several recurrences of neutropenic enterocolitis and eventually required ileocecectomy. A review of the literature on this difficult problem affecting pediatric oncology patients is presented.

Extracorporeal photopheresis for the treatment of severe, refractory steroid dependent pediatric Crohn's Disease.

Crohn's disease is a chronic, inflammatory disease of the gastrointestinal tract, affecting both children and adults. Extracorporeal photopheresis (ECP) has been used in steroid dependent adults with moderate to severely active Crohn's disease, with response rates up to 50%, with up to 25% complete responses. A 12-year-old male patient had severe unremitting Crohn's disease for one year, despite treatment with anti-inflammatory, immunosuppressive, and biologic agents. He failed elemental enteral nutrition and required total parenteral nutrition (TPN). A diverting colostomy for perforation was required. He required frequent hospitalizations and required homebound schooling. Endoscopy revealed severe inflammation and ulcerations of the entire colon. ECP was begun twice weekly for 4 weeks, then twice per week every 14 days for a total of 28 weeks. ECP was well tolerated and prednisone was gradually discontinued. He continued daily azathioprine and infliximab at 6 week intervals. TPN was weaned as enteral intake improved. Disease abatement allowed a return to school and normal activities. Endoscopy at completion of ECP course revealed normal upper tract, normal ano-rectum, and decreased, although significant, colonic disease. This response has continued for at least 16 months since completion of ECP. We conclude that ECP is useful for pediatric patients with steroid dependent Crohn's disease and prospective evaluation is warranted.

Mutations in proto-oncogene GFI1 cause human neutropenia and target ELA2.

Mice lacking the transcriptional repressor oncoprotein Gfi1 are unexpectedly neutropenic. We therefore screened GFI1 as a candidate for association with neutropenia in affected individuals without mutations in ELA2 (encoding neutrophil elastase), the most common cause of severe congenital neutropenia (SCN; ref. 3). We found dominant negative zinc finger mutations that disable transcriptional repressor activity. The phenotype also includes immunodeficient lymphocytes and production of a circulating population of myeloid cells that appear immature. We show by chromatin immunoprecipitation, gel shift, reporter assays and elevated expression of ELA2 in vivo in neutropenic individuals that GFI1 represses ELA2, linking these two genes in a common pathway involved in myeloid differentiation.

Utility of impedance cardiography for the detection of hemodynamic changes in stable patients with sickle cell disease.

OBJECTIVES: The study sought to assess the potential utility of impedance cardiography (ICG) to detect hemodynamic changes after erythrocytapheresis in stable children with sickle cell disease (SCD). METHODS: We prospectively monitored cardiac index, systemic vascular resistance index, heart rate, and blood pressure using ICG before and after erythrocytapheresis in 26 stable children with SCD. Echocardiography was carried out in all patients to evaluate left ventricular systolic function. Hemoglobin (Hb), sickle cell hemoglobin (HbS), and ferritin levels were also measured. RESULTS: Of a total of 78 erythrocytapheresis procedures in 26 children with SCD, 22 (28.2%) had hypotensive episodes defined as a decrease in systolic, diastolic, or mean blood pressure by 10 mmHg. Risk factors for developing hypotension during erythrocytapheresis were identified with logistic regression analysis: lower-body surface area and decrease in cardiac index. In contrast, age, prepheresis Hb and HbS, serum ferritin levels, and left ventricular function at baseline were not associated with hypotension. CONCLUSIONS: This study demonstrates the feasibility of the ICG technique to detect the hemodynamic changes in children with SCD after an erythrocytapheresis procedure.

Antibody development in pediatric sickle cell patients undergoing erythrocytapheresis.

BACKGROUND: Erythrocytapheresis, or red blood cell exchange transfusion (RBCX), with donor red blood cell (RBC) units is now increasingly used in the treatment of acute and chronic complications of sickle cell disease (SCD). As in all transfusions, RCBX carries a risk of immunization against foreign antigen on transfused cells. However, by selecting donor units with RBC phenotypes similar to the patient, the risk of allo- and autoimmunization can be reduced. PROCEDURE: The formation of RBC alloantibodies and/or autoantibodies in 32 multitransfused pediatric SCD patients undergoing monthly RBCX over a 11-year period (12/1998 to 12/2009) was evaluated utilizing a retrospective patient chart review at Kosair Children's Hospital, Louisville, Kentucky. RESULTS: After starting C, E, K antigen-matched RBCX, the rate of clinically significant allo-immunization decreased from 0.189/100 to 0.053/100 U, with a relative risk of 27.9%. Likewise, the rate of autoimmunization decreased from 0.063/100 to 0.035/100 U, with a relative risk of 55.9%. CONCLUSION: After controlling for clinically insignificant antibodies, our auto- and alloimmunization rate was much less than previously reported values. In addition, the incidence of clinically significant allo- and autoimmunization decreased in our patient population after starting minor antigen-matched RBCX. These results suggest that by matching selected RBC phenotypes, there may be an association in the risk of allo- and autoimmunization of multi-transfused SCD patients.

Pediatric dosing of rituximab revisited: serum concentrations in opsoclonus-myoclonus syndrome.

To longitudinally assess serum concentrations of rituximab, it was administered intravenously to 25 children with opsoclonus-myoclonus syndrome at 375 mg/m2 on each of 4 consecutive weeks with (Group I and II) or without (Group III) conventional immunotherapy. Serum rituximab levels, drawn before and after each infusion and at later intervals, were analyzed by enzyme-linked immunosorbent assay. Rituximab concentration increased stepwise with each infusion, dropping by the next infusion, thereby forming 4 discrete peaks (Cmax) and troughs (Cmin). It then fell precipitously to trace levels at 4 months. However, Cmax and Cmin curves differed significantly between groups. Compared with the youngest children (Group I), the oldest (Group III) had a 34% lower rituximab concentration at the fourth infusion, 45% less IgM depletion 1 month later, and received 20% less rituximab when the dose was recalculated as mg/kg. Serum IgM and rituximab levels were negatively correlated. Peak rituximab concentration did not correlate with adrenocorticotropic hormone dose. These results indicate that the degree of serum IgM depletion is a useful indicator for rituximab dose equivalency in children of different ages. They also suggest that pediatric rituximab dosing should be based on body weight, not surface area. (ClinicalTrials.gov NCT00244361).

Clinical application of microfluidic leukocyte enrichment protocol in mild phenotype sickle cell disease (SCD).

Nucleated cell populations, including leukocytes and circulating endothelial cells, provide an ideal sample for studies seeking to understand the pathogenesis of diseases for development of drugs and treatments. Conventional leukocyte enrichment protocols have limitations with respect to selective cell loss and artifactual activation. An automated microfluidic device was developed for leukocyte enrichment from peripheral blood to ensure enumeration of high quality sample without cell loss or artifactual activation. Pre-clinical trials have shown the efficiency of the device to maximize cell yield and minimize artifactual activation in comparison to conventional techniques. Clinical validation and the ability of the microfluidic technique to enrich leukocyte samples to understand disease processes was accomplished in this study by quantifying circulating nucleated cells and their activation status in healthy controls and mild phenotype sickle cell disease (SCD) patients. Results confirm the clinical effectiveness of this technique to accurately characterize immune and inflammatory status.

Neonatal alloimmune thrombocytopenia due to HPA-9b incompatibility.

Neonatal alloimmune thrombocytopenia (NAIT) is one of the most frequent causes of both severe thrombocytopenia and intracranial hemorrhage (ICH) in fetuses and term neonates. The diagnosis is established by demonstrating antibodies against human platelet antigens (HPA) and discordance in platelet antigen typing between parents or between the mother and neonate. We report a case of NAIT that was likely due to maternal sensitization to HPA-9b (Max(a)), a recently recognized, rare platelet-specific antigen.

Leukemia inhibitory factor: a newly identified metastatic factor in rhabdomyosarcomas.

Rhabdomyosarcoma frequently infiltrates bone marrow and this process involves the stromal-derived factor-1 (SDF-1)-CXCR4 axis. Because leukemia inhibitory factor (LIF), like SDF-1, is secreted by bone marrow stroma and directs the regeneration of skeletal muscles, we examined whether the LIF-LIF receptor (LIF-R) axis affects the biology of rhabdomyosarcoma cells. We found that in rhabdomyosarcoma cells, LIF stimulates the following: (a) phosphorylation of mitogen-activated protein kinase p42/44, AKT, and signal transducers and activators of transcription 3, (b) adhesion and chemotaxis, and (c) increased resistance to cytostatics. To compare the biological effects of LIF versus SDF-1, we examined the RH30 cell line, which is highly responsive to both ligands, and found that the chemotaxis of these cells is significantly reduced when the inhibitors of both receptors (T140 for CXCR4 and gp190 blocking antibody for LIF-R) are added simultaneously. Subsequently, by using repetitive chemotaxis to LIF or SDF-1, we selected from the RH30 line subpopulations of cells that respond to LIF but not SDF-1 (RH30-L) or to SDF-1 but not LIF (RH30-S). We found that (a) RH30-L cells seed better to the bone marrow, liver, and lymph nodes of immunodeficient mice than RH30-S cells and (b) mice inoculated i.m. with the RH30-L cells had more rhabdomyosarcoma cells in the bone marrow and lung after 6 weeks. Thus, we present the first evidence that the LIF-LIF-R axis may direct rhabdomyosarcoma metastasis. Further, because we showed that the in vivo metastasis of RH30 cells is inhibited by small interfering RNA against LIF-R, molecular targeting of this axis could become a new strategy to control the metastasis of rhabdomyosarcoma.

Middle Cerebral Artery Stroke as Amusement Park Injury: Case Report and Review of the Literature.

Strokes as amusement park injuries are rare, but have been reported in the literature. Only about 20 cases of cerebrovascular accidents after amusement park visits have been described. We report a healthy 12-year-old boy who presented with facial droop, slurred speech, and inability to use his right arm after riding roller coasters at a local amusement park. He was evaluated and found to have a left middle cerebral artery (MCA) infarction. The patient was treated with anticoagulants and has recovered with no major residual symptoms. It is likely that his neurological symptoms occurred due to the high head accelerations experienced on the roller coasters, which are more detrimental to children due to immature cervical spine development and muscle strength. Early diagnosis of dissection and stroke results in a favorable prognosis. Providers and parents should be aware of the potential risk of roller coasters and act quickly on neurologic changes in children that have recently been to an amusement park.

Cerebral oximetry improves detection of sickle cell patients at risk for nocturnal cerebral hypoxia.

We previously used cerebral oximetry to identify low cerebral venous oxygen saturation in waking children with sickle cell disease (SCD). Because arterial oxyhemoglobin desaturation is common during sleep in SCD patients, this study compared both waking and sleeping systemic arterial and cerebral venous oxygenation dynamics in children with and without SCD. Seventeen African-American (AA) children with homozygous SCD [8 (4-15) years; 29% male; normal transcranial Doppler velocities] were compared with a control cohort (CON) comprised of six healthy AA children [9 (4-16) years, 33% male]. Standard all-night polysomnographic recordings were performed, including measurement of arterial oxygen saturation by pulse oximetry (SpO(2)). Regional cerebral oxygen saturation (rSO(2)) was measured non-invasively with cerebral oximetry. Intra-cohort comparisons examined the influence of sleep on SpO(2) and rSO(2) in the subjects. Inter-cohort comparisons of SpO(2), rSO(2,) and the rSO(2)/SpO(2) ratio assessed the impact of SCD on systemic and cerebral oxygenation during wakefulness and sleep. Cohort differences in SpO(2) were not statistically significant in either wakefulness or sleep. However, only in the SCD cohort was the magnitude of SpO(2) change statistically significant (P = 0.002). In contrast, both waking and sleep rSO(2) cohort median values did differ significantly [awake: CON 76 (67-86) vs. SCD 62 (58-71), P = 0.01; sleep: CON 65 (60-77) vs. SCD 55 (48-61), P = 0.01)]. The waking rSO(2)/SpO(2) ratio was also significantly lower in the SCD group [CON 0.78 (0.68-0.88) vs. SCD of 0.66 (0.61-0.72); P = 0.015]. During sleep, the ratio was also significantly lower in the SCD group [CON 0.71 (0.66-0.81) vs. SCD 0.59 (0.52-0.65); P = 0.011]. Our findings suggest that SCD patients may be at increased risk of cerebral hypoxia during both wakefulness and sleep.

Spinal Cord Infarction in Hemoglobin SC Disease as an Amusement Park Accident.

Spinal cord infarction (SCI) is extremely rare in children, and only 2 other reports have described the occurrence of SCI in patients with hemoglobin SC disease (HbSC). Amusement park accidents are serious injuries. Patients with preexisting conditions, such as hypertension, cardiac disease, and recent back or neck injuries, may be at an increased risk. We report the case of a 12-year-old girl with HbSC with a past history of only 2 admissions for pain crises, who presented to the emergency department with symptoms of SCI after riding a roller coaster. Fibrocartilaginous embolism (FCE) is an increasingly recognized cause of SCI after events that put strain on the axial skeleton, such as many amusement park rides. Although radiologic criteria for FCE have been proposed, FCE remains a diagnosis of exclusion. To the best of our knowledge, this is the first documented case of SCI in a patient with HbSC and the first case of FCE after an amusement park accident. This case report highlights that HbSC may confound the differential diagnosis of SCI and aims to document an association with FCE in pediatric patients.

Intensification of therapy for children with lower-risk acute lymphoblastic leukemia: long-term follow-up of patients treated on Children's Cancer Group Trial 1881.

PURPOSE: From December 1988 through December 1992, the Children's Cancer Group (CCG) conducted a randomized trial (CCG-1881) designed to evaluate the impact of adding a single delayed intensification phase of therapy to standard therapy for patients with newly diagnosed low-risk acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Patients (n = 778) with newly diagnosed ALL, 2 to 9 years of age at diagnosis with an initial WBC count less than 10,000/microL, were eligible for this protocol. All patients received induction, consolidation, and interim maintenance phases of therapy over the first 16 weeks. At week 16, patients remaining in remission were randomly assigned to receive or not receive a single 7-week delayed intensification (DI) phase of therapy. Maintenance therapy was given in lieu of or after DI, with total duration of therapy approximately 3 years for boys and 2 years for girls. RESULTS: Patients randomized to receive DI experienced fewer relapse events in all categories. Kaplan-Meier life-table estimates for continuous complete remission (CCR) at 7 years for the randomized regimens were 77% (SE, 2.4%) for the standard regimen and 83% (SE, 2.7%) for the DI regimen (P =.072). The only prognostic factor of significance post-randomization in this selected low-risk population was the day 14 marrow response (P =.0001). CONCLUSION: The addition of a single DI phase of therapy was well tolerated and augmented 7-year CCR by 6% (SE of the difference, 3.3%), resulting in 26% fewer adverse events. Overall survival for eligible patients at 7 years is 90% (SE, 1.2%).